An addendum to this report has been issued and can be found at <https://www.cdpr.ca.gov/docs/registration/
reevaluation/chemicals/addendum_neonicotinoid_risk_determination.pdf>

California Department of Pesticide Regulation
Pesticide Registration Branch

California Neonicotinoid Risk Determination

July 2018

Prepared by:John Troiano, Ph.D., Research Scientist III
Brigitte Tafarella, Environmental Scientist
Alexander Kolosovich, Senior Environmental Scientist (Specialist)
Rochelle Cameron, Environmental Scientist
Denise Alder, Senior Environmental Scientist (Specialist)
Russell Darling, Senior Environmental Scientist (Specialist)
Approved by:
Marylou Verder-Carlos, DVM, MPVM., Assistant Director
Ann Prichard, Environmental Program Manager II
Margaret Reiff, Environmental Program Manager I

California Environmental Protection Agency
California Department of Pesticide Regulation
Pesticide Registration Branch1001 I Street, P.O. Box 4015
Sacramento, California 95812

Contributors and Acknowledgments
DPR acknowledges the contributions and efforts of the following individuals and organizations:Contributors
Richard Bireley (former Senior Environmental Scientist (Specialist) with the California Department of
Pesticide Regulation), Senior Environmental Scientist (Specialist), California Department of Fish andWildlife
Baktazh Azizi, Scientific Aid, California Department of Pesticide Regulation
Joseph Sullivan, Certified Wildlife Biologist, Ardea Consulting

Acknowledgments
The U.S. Environmental Protection Agency’s Office of Pesticide Programs
Health Canada’s Pest Management Regulatory Agency

On the cover: Photo of a bee on honeycomb. Photograph by DPR staff.
iTable of Contents
1.0 Executive Summary……………………………………………………………….

1

2.0 Background………………………………………………………………………..

3

3.0 Scope………………………………………………………………………………

4

3.1 Pesticide Type, Class, And Mode of Action………………………………

4

3.2 Use Characterization……………………………………………………....

4

3.3 Environmental Fate and Transport………………………………………...

5

3.4 Potential for Effects on Pollination Activity………………………………

6

3.5 Colony Level Exposure and Effects………………………………………

6

4.0 Risk Characterization Methodology………………………………………………

7

4.1 Overview of Risk Determination Process…………………………………

7

4.2 Effects Characterization…………………………………………………...

8

4.3 Exposure Characterization………………………………………………...

11

4.4 Risk Determination Categories……………………………………………

13

5.0 Risk Characterization……………………………………………………………..

13

6.0 Conclusions……………………………………………………………………….

18

6.1 Overview by Crop Grouping……………………………………………...

18

6.2 Seed Treatments and Tree Injection Applications………………………...

21

7.0 Considerations for Mitigation……………………………………………………..

21

8.0 Risk Appraisal …………………………………………………………………….

22

Bibliography ………………………………………………………………………….

28

Appendix 1. Reevaluation Letter Initiating the Reevaluation of Imidacloprid,
Clothianidin, Thiamethoxam, and Dinotefuran……………………………………….

30

Appendix 2. California Food and Agricultural Code (FAC) section (§) 12838………

33

Appendix 3. California Registered Agricultural Uses of Imidacloprid,
Thiamethoxam, Clothianidin, and Dinotefuran……………………………………….

35

Appendix 4. 40 CFR 180.41 Crop Group..…………………………………………....

58

ii

Appendix 5. Physicochemical Properties of Imidacloprid, Clothianidin,
Thiamethoxam, and Dinotefuran……………………………………………………..

82

Appendix 6. Tier I Toxicity Values for Imidacloprid, Clothianidin, Thiamethoxam,
and Dinotefuran……………………………………………………………………….

83

Appendix 7. Open Literature References Considered for Use in this Risk
Determination Document……………………………………………………………..

84

Appendix 8. Data Evaluations for the Colony Feeding Studies that were Included inthis Risk Determination Document……………………………………………………

88

Appendix 9. Foliar and Soil Residue Studies Considered for Use in this Risk
Determination Document……………………………………………………………...

600

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination
Document……………………………………………………………………………...

613

Appendix 11. Descriptive Statistics Derived from the Residue Studies Included inthis Risk Determination Document…………………………………………………… 1133
Appendix 12. Letters Notifying Registrants of Reevaluation………………………… 1166

iii

1.0

Executive Summary

California leads the nation in cash farm receipts, and its agricultural production includes morethan 400 commodities representing over a third of the country’s vegetables and two-thirds of thecountry’s fruits and nuts. Many of these agricultural commodities rely on pollination by bees for
optimal production. Today, more than 2.5 million honey bee colonies in the United States
pollinate an estimated $15 billion of crops each year, ranging from almonds to zucchini. Of
these, approximately 1.8 million colonies are used in the pollination of California’s almond crops
alone.
Colony losses of these critical natural and managed pollinators have triggered worldwide
concern in recent years. Multiple factors may contribute to colony losses and other risks topollinator and hive health, including possible effects of neonicotinoid pesticides. This risk
determination report, prepared by the Department of Pesticide Regulation (DPR) in response to
California Food and Agriculture Code Section 12838, assesses those potential effects.
Neonicotinoid insecticides are systemic pesticides that kill insects by attacking their central
nervous system. These insecticides are absorbed into plants and distributed throughout theirtissues to their stems, leaves, roots, fruits and flowers. Neonicotinoids play an important role inthe control of agricultural insect pests. Some examples include: Aphids that transmit citrus tristeza virus to citrus affecting the roots, leaves, and fruit
causing a rapid decline in tree growth leading to death; The glassy-winged sharpshooter that transmits Pierce’s disease to grapevines, abacterium that blocks the movement of water within the plant, killing the vines within 1-3
years; and The Asian citrus psyllid that transmits huanglongbing disease to citrus trees causing ayellowing of tree shoots, asymmetrical and bitter fruit, and tree death in 5-8 years.
All of these diseases are known to spread rapidly and have the potential to cause massive
destruction to the crops affected.
Neonicotinoids are insecticides developed as alternatives to organophosphates and carbamatesthat have a greater potential to affect human health (Cimino et al., 2017). Pesticide use reports
received by DPR from the County Agricultural Commissioners across the state between 2007
and 2016 show that the use of neonicotinoids (imidacloprid, thiamethoxam, clothianidin, anddinotefuran) increased by 69.6%, while organophosphate and carbamate use decreased by 41.5%
and 20.9%, respectively. California requires the monthly reporting of agricultural pesticide use toCounty Agricultural Commissioners, who in turn, report the data to DPR.
DPR was advised of the potentially harmful effects of neonicotinoids on pollinators in 2008.
Studies of imidacloprid on ornamental plants revealed high levels of the insecticide in leaves andblossoms of treated plants, as well as increased imidacloprid residue levels in leaves andblossoms over time, indicating potential threats to pollinator health. In response to the disclosure,
DPR placed pesticide products containing imidacloprid and the related neonicotinoid active
ingredients thiamethoxam, clothianidin, and dinotefuran, into reevaluation on February 27, 20091

to assess the magnitude of their residues in the pollen and nectar of agricultural crops and thecorresponding levels of risk to honey bee colonies. The reevaluation covered 50 registrants and282 pesticide products with formulations or applications likely to move into plants that bloom orserve as a foraging source for honey bees and other pollinators (Appendix 1). In 2014, theLegislature adopted AB 1789 (Chapter 578, Statutes of 2014) requiring DPR to issue adetermination with respect to its reevaluation of neonicotinoids by July 1, 2018, and adopt
control measures necessary to protect pollinator health within two years after making thedetermination (Appendix 2).
This risk determination report documents the results of the DPR’s neonicotinoid reevaluation andits first ecologically-based risk assessment. As part of this assessment, the department partneredwith scientists at the U.S. Environmental Protection Agency’s (U.S. EPA) Office of Pesticide
Programs and the Health Canada Pest Management Regulatory Agency to develop the methods
and procedures used to conduct ecologically-based studies on the effects of neonicotinoids. DPR
followed the methods established by the group to assess the risks of exposure to bee colonies
foraging on nectar and pollen in crops treated with the subject neonicotinoids, comparing thelevels of neonicotinoid residues to concentrations that cause colony-level effects such as
decreased colony strength and decreased stores of honey in honeycombs.
DPR based its risk determination on a series of studies that exposed bee colonies to four types ofneonicotinoids (imidacloprid, thiamethoxam, clothianidin and dinotefuran) to establish residue
levels in pollen and nectar that produced no observed effects on the colonies (No Observed
Effect Concentrations, or NOECs). The department compared those NOEC values to residue
levels found on selected agricultural crops in the field. DPR scientists then determined risk levels
for combinations of specific crop groups and pesticide application methods (e.g., foliar [applied
to leaves] or soil). Crop-application combinations with pollen or nectar residue levels thatexceeded the NOEC values were determined to present a risk. Crop-application combinations
with residue levels below the NOEC values were determined to be low risk. These risk
determinations were based on the maximum allowed annual application rates in California foreach agricultural crop group for each of the neonicotinoids listed above, and therefore represent“worst-case” scenarios (Appendix 3). Actual annual application rates may present less risk.
Crop groups considered to present a risk at maximum annual application levels of at least one of
the neonicotinoids listed above include fruiting vegetables (e.g., cucumbers, tomatoes), berries,
citrus, and tree nuts. Among the crop groups for which maximum application levels are
considered a low risk are root and tuber vegetables (e.g., potatoes, turnips), bulb vegetables (e.g.,
onions, garlic), leafy vegetables and legumes. Again, these are conservative assessments basedon maximum allowable application rates, and vary according to the neonicotinoid applied.
Additional information on crop group risk may be found in Table 6.
Going forward, DPR will consider mitigation measures for neonicotinoid applications to crops
characterized as at risk to reduce residues to levels below the respective NOEC. Such measures
could include modifying application rates or the times at which applications may occur. This
mitigation process will likely take two years to complete and will include continued research,
consultation with experts, other stakeholders, and the use of technology designed to predictmeasures necessary to ensure bee colony health.

2

2.0

Background

On February 27, 2009, DPR placed certain pesticide products containing the neonicotinoid active
ingredients, imidacloprid, thiamethoxam, clothianidin, and dinotefuran, into reevaluation(Appendix 1). DPR initiated the reevaluation based on submitted adverse effects disclosure data
involving the active ingredient imidacloprid. DPR’s Ecotoxicology unit evaluated the adverse
effects data and noted high concentrations of imidacloprid in leaves and blossoms of treatedornamental plants, with an increase in measured concentrations over time. These observations ofresidues in treated plants led to a concern over potential exposure of honey bee colonies used forpollination services where hives are purposely placed around agricultural fields. Thiamethoxam,
clothianidin, and dinotefuran are in the same chemical class as imidacloprid, known as thenitroguanidine-substituted neonicotinoid insecticides, and have similar physicochemical
properties (e.g., soil mobility, half-lives, and toxicity to honey bees; Appendix 5). Thus, DPR
included these active ingredients in the reevaluation. The purpose of this reevaluation is toprovide DPR with a better understanding of the magnitude of neonicotinoid residues in pollen
and nectar of agricultural commodities resulting from legal pesticide applications and theresulting level of risk to honey bee colonies. These data are necessary to provide a credible
scientific basis for potential regulatory action to mitigate any significant adverse effects onhoney bee health resulting from the use of neonicotinoid insecticides. DPR exempted from thereevaluation products formulated as a gel or impregnated in a strip, termiticides, flea control
products combined with rodenticides, pet spot products, ant and roach baits, premise applications
for control of nuisance pests, and manufacturing use only products because as formulated orapplied, it is unlikely that the neonicotinoid in such products will move into plants that bloom oris a source of forage for honey bees and pollinators.
As part of the reevaluation, DPR required pesticide manufacturers to provide additional data thatwould allow DPR scientists to conduct a scientific determination of risk. DPR’s reevaluation
focused on gathering data on residue concentrations in the nectar and pollen of certain
neonicotinoid-treated orchard and row crops. On September 15, 2009, DPR issued letters to theregistrants of the four pesticide active ingredients describing the objectives and basic design ofthe studies to be conducted. Sampling was to be conducted in a minimum of three agriculturalsites over two consecutive years. When possible, the agricultural sites were selected based on
soil texture with three replicates in sandy, coarse-textured soils, three replicates in loamy,
medium-textured soils, and three replicates in clayey, fine-textured soils. DPR used the Pesticide
Use Reporting database to determine the crops of focus for each active ingredient (DPR, 2018b).
On March 12, 2012, DPR modified its residue study strategy to require applications at thehighest maximum annual application rate for two consecutive years.
DPR partnered with scientists at the U.S. Environmental Protection Agency’s (U.S. EPA) Officeof Pesticide Programs and Health Canada Pest Management Regulatory Agency (PMRA) toensure that required data on the effects of neonicotinoids would provide useful and reliable
information across the board for all three agencies to use in guiding their regulatory actions. On
June 20, 2014, a Presidential Memorandum creating a federal strategy to promote the health ofhoney bees and other pollinators was signed. Subsequently, DPR, U.S. EPA, and PMRA
published a collaborative document titled, Guidance for Assessing Pesticide Risks to Bees

3

(U.S. EPA, PMRA, and DPR, 2014), which established a tiered approach to data collection andrisk assessment.
In January 2016, U.S. EPA, in collaboration with DPR, issued a preliminary pollinator risk
assessment for imidacloprid. In January of 2017, U.S. EPA issued preliminary pollinator risk
assessments for thiamethoxam, clothianidin, and dinotefuran. U.S. EPA’s preliminary pollinatorrisk assessments include Tier I (acute toxicity) assessments based on model-generated estimatesof exposure and laboratory toxicity data at the individual bee level, for all four active ingredients.
The Tier I assessments indicate that there is potential risk to honey bees for all crops andapplication methods where there is a potential for on-field exposure (U.S. EPA and DPR, 2016;
U.S. EPA, 2017a; U.S. EPA, 2017b). In accordance with the Guidance for Assessing Pesticide
Risks to Bees (U.S. EPA, PMRA, and DPR, 2014), U.S. EPA conducted Tier II assessments for
imidacloprid, thiamethoxam and clothianidin, and a Tier I assessment on dinotefuran using
available data. Tier II assessments compare residue data to colony-level effects data.
A refined Tier II assessment is the focus of DPR’s risk determination document. DPR’s
determination starts with U.S. EPA’s preliminary pollinator assessments and includes new datasubmitted to DPR for all four active ingredients since the issuance of U.S. EPA’s preliminary
pollinator assessments. This risk determination document meets the requirements of
FAC §12838 (a) which states, “On or before July 1, 2018, the department shall issue adetermination with respect to its reevaluation of neonicotinoids” (Appendix 2).
3.0

Scope

3.1

Pesticide Type, Class, and Mode of Action

Neonicotinoid insecticides are systemic pesticides that target nicotinic acetylcholine receptors in
the central nervous system of insects. DPR’s neonicotinoid reevaluation focuses on thenitroguanidine-substituted neonicotinoids (imidacloprid, clothianidin, thiamethoxam, anddinotefuran) as all four active ingredients share similar physicochemical characteristics andtoxicity to honey bees. Neonicotinoids are systemic compounds and readily move through thevascular system, xylem and phloem, of plants which then translocate into various plant tissues.
Neonicotinoids can be applied using several different application methods including foliar
application by aerial or ground spray equipment, soil drench, chemigation, or seed treatment(U.S. EPA and DPR, 2016; U.S. EPA, 2017a; U.S. EPA, 2017b).
3.2

Use Characterization

DPR first registered a pesticide product containing imidacloprid for sale and use in the State ofCalifornia in 1994. Approximately ten years later, DPR registered the first pesticide products
containing dinotefuran, clothianidin, and thiamethoxam (DPR, 2018a). Neonicotinoids arewidely used pesticides with a variety of uses ranging from agricultural and residential
insecticides, pet products, termiticides, ant and roach baits, and premise application products fornuisance pests. Neonicotinoids are currently registered for use on a diverse array of crops in
California such as, but not limited to: citrus fruits, oilseed crops (e.g., cotton), cucurbit
vegetables, fruiting vegetables, pome fruits, stone fruits, cereal grains, tree nuts, Brassica (Cole)

4leafy vegetables, root and tuber vegetables, leafy vegetables, legume vegetables, and bulb
vegetables. For more information on registered agricultural use sites and specific application
rates for each of the neonicotinoid active ingredients, refer to Appendix 3.
Neonicotinoids were developed as alternatives to organophosphates and carbamates (Cimino etal., 2017). Neonicotinoids play an important role in the integrated control of agricultural insect
pests such as: aphids that transmit citrus tristeza virus to citrus; the glassy-winged sharpshooter
that transmits Pierce’s disease to grapevines; and the Asian citrus psyllid that transmits
huanglongbing disease to citrus trees.
Pesticide use reports (PUR) between 2007 and 2016 indicate that use of neonicotinoids(imidacloprid, thiamethoxam, clothianidin, and dinotefuran) increased by 69.6% (131,168 lbs.
neonicotinoid active ingredients used in 2007; 431,132 lbs. neonicotinoid active ingredients usedin 2016) while organophosphate and carbamate use decreased by 41.5% (3,775,011 lbs.
organophosphate active ingredients (listed below) used in 2007; 2,209,448 lbs. active ingredientsused in 2016) and 20.9% (666,035 lbs. carbamate active ingredients (listed below) used in 2007;
526,677 lbs. active ingredients used in 2016), respectively. In 2016, organophosphates were
frequently applied to oranges, almonds, walnuts, lettuce, and cotton while carbamates were
frequently applied to oranges, corn, lettuce, tomatoes, and alfalfa. The most frequentneonicotinoid use sites in 2016 include grapes, tomatoes, oranges, tangerines, and pistachios.
The inquiry into the PUR database for the organophosphate chemical group included the active
ingredients acephate, bensulide, chlorpyrifos, diazinon, DDVP, dimethoate, fosthiazate
malathion, ethoprop, naled, phorate, phosmet, tetrachlorvinphos, tribufos, disulfoton, ethoprop,
fenamiphos, methamidophos, methidathion, oxydemeton-methyl, and profenofos while thecarbamate group included the active ingredients formetanate HCI, methiocarb, methomyl,
oxamyl, propoxur, thiodicarb, aldicarb, carbofuran, and carbaryl. Other chemicals that belong
within the organophosphate and carbamate chemical group are not currently registered in theState of California.
3.3

Environmental Fate and Transport

Since neonicotinoids are systemic insecticides, they are transported through the vascular systemof plants to all tissues, including leaves, nectar and pollen. Both foliar and soil applications ofneonicotinoids have resulted in detectable residues in both nectar and pollen following
absorption by the foliage, roots, or stems of plants (U.S. EPA and DPR, 2016; U.S. EPA, 2017a;
U.S. EPA, 2017b). Physicochemical characteristics consistent among the four neonicotinoid
active ingredients include a low organic carbon normalized soil adsorption coefficient (Koc)value, low volatility, longevity in soil after application, and relatively high water solubility(Appendix 5). These properties contribute to the pesticides being highly available for uptake byplant roots. Moreover, neonicotinoids have two main routes of degradation through aquatic
photolysis and aerobic soil metabolism (U.S. EPA and DPR, 2016; U.S. EPA, 2017a; U.S. EPA,
2017b). Degradation produces a variety of breakdown products known as metabolites. Refer toAppendix 5 for the specific physicochemical properties and environmental fate of each active
ingredient.

5This risk determination document includes measurements of metabolite concentrations identified
as having similar or greater toxicity to honey bees than the parent compound. For imidacloprid,
the evaluation includes the parent and two metabolites, imidacloprid-olefin (IMI-olefin) andimidacloprid-5-hydroxy (5-OH-IMI), since all three compounds have a similar toxicity to honeybees (U.S. EPA and DPR, 2016). Other metabolites do not have a similar toxicity (e.g. 6chloronicotinic acid, 6-chloro-picolylalcohol, nitrosamine and urea). The risk determination willrefer to total imidacloprid, which is the summation of residues of the parent imidacloprid, andthe metabolites IMI-olefin and 5-OH-IMI.
The metabolite of concern for thiamethoxam is CGA-322704 (i.e., clothianidin), which itself isan active ingredient in registered pesticide products. As both compounds are toxic to honey bees
(U.S. EPA, 2017b), concentrations of total residues for parent (thiamethoxam) and CGA-322704
will be reported and assessed. For clothianidin, the metabolites, N-(2-chloro-5-thiazolylmethyl)N’-methylurea (TZMU) and N-(2-chloro-5-thizolylmethyl)-N’-nitroguanidine (TZNG) areroutinely measured in the plant residue studies. Based on acute toxicity data, TZMU and TZNG
are orders of magnitude less toxic to honey bees than the parent clothianidin (U.S. EPA, 2017b).
As a result, DPR did not include these metabolites in the risk determination and all references toclothianidin refer to the parent molecule alone.
Dinotefuran metabolites measured in plant tissues include 1-methyl-2-nitro-3-(tetrahydro-3furylmethyl) guanidine (UF) and 1-methyl-3-(tetrahydro-3-furylmethyl) guanidinium dihydrogen
phosphate (DN). Toxicity data submitted to DPR indicate the UF and DN metabolites are lesstoxic to honeybees, so those metabolites are not included in DPR’s risk determination and allreferences to dinotefuran refer to the parent molecule alone (U.S. EPA, 2017a).
3.4

Potential for Effects on Pollination Activity

This risk determination focuses on potential effects of neonicotinoid exposure on honey bees
(Apis mellifera) after feeding on nectar and pollen containing neonicotinoid residues. Honey bees
are purposefully situated around agricultural sites during bloom to pollinate various crops. As aresult, foraging bees could be exposed to residues of these four neonicotinoids from applications
made prior to bloom, during flowering, or post-bloom if the residues in bee-attractive matrices(e.g., pollen and nectar) persist for a sufficient duration. DPR’s reevaluation required that plant
residue studies be conducted using worst-case application scenarios (e.g., maximum application
rates, minimum reapplication intervals) found on currently registered pesticide labels. These
scenarios generally result in the highest realistic concentrations in the bee-attractive matrices.
Apis bees serve as a surrogate for other non-Apis species of bees (e.g., bumble bees) that may be
exposed under agricultural conditions. This surrogate approach is consistent with the Guidance
for Assessing Pesticide Risks to Bees (U.S. EPA, PMRA, and CDPR, 2014). As described in theguidance document, the husbandry, life cycles, and contribution of pollinator services of honeybees are well-studied.
3.5

Colony Level Exposure and Effects

DPR evaluated both registrant-submitted and open literature (i.e., peer-reviewed research studies
published in scientific journals) Tier II semi-field studies for this risk determination. The purpose

6

of Tier II studies is to evaluate possible colony-level effects on hive health through foraging on
nectar and pollen. DPR quantitatively evaluated oral consumption (e.g., consumption ofcontaminated nectar and pollen) as the primary exposure route for honey bees in thisdetermination. In Tier II studies denoted as colony feeding studies, honey bee colonies are
exposed to known concentrations of a compound in either surrogate nectar or pollen andmeasurements are taken that reflect the health of hives. Based on the observed responses fromthe colony feeding studies, No Observed Effects Concentrations (NOECs) are derived for eachactive ingredient. In this determination, DPR used the NOEC values to determine each active
ingredient’s potential to cause effects on hive health. The submitted colony feeding studies
measured several response variables including colony survival, the number of cells containing
various brood stages (eggs/larvae/pupae), the total population of adult bees per hive, and thenumber of cells containing food stores (pollen and nectar). Overall, the purpose of these studies
is to determine the concentration of each neonicotinoid that honey bees can safely consume overa six-week period with no significant adverse colony-level effects. NOEC values were
established for each of the four neonicotinoids in each of the two bee-attractive matrices (pollen
and nectar; Table 1 below). DPR scientists compared these values to neonicotinoid
concentrations in nectar and pollen collected from representative crops after worst-case scenario
applications. DPR also evaluated and considered adverse effects data submitted pursuant to
California Food and Agricultural Code (FAC) section 12825.5. However, those data did notprovide information pertinent to the scope of this risk determination.
4.0

Risk Characterization Methodology

4.1

Overview of Risk Determination Process

The risk determination process generally follows the methods of a Tier II assessment as detailed
in the Guidance for Assessing Pesticide Risks to Bees (U.S. EPA, PMRA, and CDPR, 2014). In
accordance with the tiered risk assessment process, risks to bees were determined by comparing
available exposure data to colony-level effects data. According to Tier I laboratory data,
nitroguanidine-substituted neonicotinoids are acutely toxic to individual bees through bothcontact and oral exposure (Appendix 6). Contact exposure may occur through dermal uptake ofresidues on plant surfaces or by direct spray deposition onto bees. Oral exposure mainly occurs
through the ingestion of contaminated pollen or nectar. Applications can be timed to avoid
contact by spray deposition. However, risks to honey bees from oral exposure are more complex
to regulate. Upon translocation of the systemic nitroguanidine-substituted neonicotinoids insideplant tissues, concentrations in pollen and nectar may persist, resulting in risks from oral
consumption and/or transfer of residues back to the hives.
This risk determination focuses on potential effects posed by oral consumption, so exposure data
were determined from measured residue concentrations of nitroguanidine-substituted
neonicotinoids and their bee-toxic metabolites in the nectar and pollen of agricultural crops
following worst-case scenario applications in compliance with product labels. The exposure data
were compared to effects data generated from exposure of honey bee colonies to nectar or pollen
spiked with known concentrations of imidacloprid, thiamethoxam, clothianidin, or dinotefuranwith various colony-level parameters measured over time. The Tier II data discussed in this risk
determination builds upon the preliminary pollinator risk assessments published by the U.S. EPA

7

(U.S. EPA and DPR, 2016; U.S. EPA, 2017a; U.S. EPA, 2017b) for the four neonicotinoid active
ingredients while also incorporating additional California-specific data.
DPR scientists made risk determinations for specific crop groups and application method
combinations (e.g., foliar, soil), and characterized them as either having a determination of risk
or low risk to honey bee colonies. A determination of risk resulted when residue concentrations
in nectar or pollen exceeded the colony-level NOEC for that matrix (e.g., pollen or nectar).
Conversely, a determination of low risk resulted when residue concentrations in pollen or nectar
did not exceed the respective colony-level effects concentration (e.g., the concentrations were
low enough that they would not result in any significant adverse effects to honey bee colonies).
The risk determinations are based on oral exposure (e.g., the consumption of contaminated nectar
and pollen). Methods used to generate the effects data and exposure data and their utilization in
the risk determinations are described in Section 4.2.
Risk determinations were only conducted for foliar and soil applications. Risks from seedtreatment applications were evaluated in the preliminary pollinator risk assessments published by
U.S. EPA (U.S. EPA and DPR, 2016; U.S. EPA, 2017a; U.S. EPA, 2017b). The preliminary
assessment for imidacloprid evaluated multiple seed treatment residue studies conducted on corn,
canola, and sunflower. These studies generally reported no residues in pollen and nectar above
the limit of detection. Values are well below their respective NOEC values, supporting theconclusion that imidacloprid seed treatments pose a low risk to honey bees. The preliminary
pollinator risk assessment for clothianidin and thiamethoxam evaluated multiple seed treatment
residue studies conducted on corn, sunflower, melon, canola, cotton, and soybean. The resulting
residue concentrations are all below the respective NOECs, supporting the conclusion thatclothianidin or thiamethoxam seed treatments pose a low risk to honey bee colonies. Dinotefuran
is not registered for any seed treatment applications. There have been issues in other states andcountries with contact exposure resulting from abraded seed coat dust at planting, but the U.S.
EPA has addressed this with best management practices (U.S. EPA and DPR, 2016). DPR has norecords of such incidents occurring in California.
4.2

Effects Characterization

Potential effects on honey bees were determined from Tier II studies, which assess effects of
exposure at the colony level. The complex nature of assessing hive dynamics and colony-level
effects necessitated multiagency collaboration to develop protocols that maximized theregulatory usefulness of such studies. Accordingly, study protocols were developed
collaboratively through the efforts of DPR, U.S. EPA, and PMRA scientists, and in consultation
with industry experts. This cooperative effort aimed at ensuring reproducibility of results andmaximizing statistical power to detect effects while minimizing uncertainties and potential
confounding factors, such as diseases, pests, or poor nutrition, which have each been
independently associated with declines in colony health.
In comparison to Tier I laboratory studies, which focus on individual bees, Tier II studies focus
on colony-level effects and assess a longer period of exposure under conditions that are more
representative of exposure in the field. These include semi-field studies such as tunnel studies
and colony feeding studies. Tunnel studies typically involve enclosing small bee colonies within8

a confined area of treated crops on which bees forage. In colony feeding studies, unconfinedcolonies are provided a food source, such as sucrose solution or pollen patties, that has been
spiked with a known and measured concentration of a specific pesticide. Multiple concentrations
are tested to produce a dose-response relationship between the concentrations tested and theobserved health of the hives. In colony feeding studies, bees are generally exposed to the test
feeding substances for six weeks. Measurements of hive health (i.e., Colony Condition
Assessments) are taken at multiple time points prior to, during, and after the exposure period.
Additionally, an overwintering component is typically included, with at least one additional
assessment after the overwintering period. Hive health is determined by measuring parameterssuch as the population of adult bees (i.e., colony strength), the number of cells containing various
brood stages (eggs, larvae, and pupae), and measuring hive resources in terms of honey and bee
bread production (U.S. EPA, PMRA, and CDPR, 2014).
The Tier II studies considered in this risk determination document were subject to thoroughevaluation for scientific acceptability. As part of this evaluation, DPR, U.S. EPA, and PMRA
scientists assessed registrant-submitted study protocols prior to study initiation to ensure that the
study designs were scientifically sound. Some examples of the types of requirements necessary
for a study design to be deemed scientifically sound include adequate replication andconfirmation of exposure by repeated sampling and analysis of spiked sugar solutions or spiked
pollen patties to ensure that the honey bee colonies are actually exposed to the neonicotinoid
concentrations as planned. Many of the open literature studies reviewed by DPR scientists(Appendix 7) lacked this level of replication and confirmation of exposure. In some cases, study
authors were reluctant to provide DPR statisticians with the raw data needed to conduct
independent statistical analyses. All colony-level NOEC values used in this assessment are basedon mean measured concentrations that resulted from analyses conducted in compliance withrigorous analytical quality control procedures. For scientifically acceptable studies, DPR, U.S.
EPA, and PMRA statisticians conducted independent statistical analysis of raw data on pertinent
endpoints. In its risk determination process, DPR used the measured concentrations in the sugar
solutions or spiked pollen patties associated with these regulatory endpoints. This results in alevel of accuracy and certainty that cannot be achieved using nominal concentrations that were
never confirmed analytically.
Although DPR considered both open literature and registrant-submitted studies, the registrantsubmitted studies were generally found to be more robust and comprehensive whencharacterizing colony-level effects. These studies had greater replication and confirmation of
exposure, and the raw data were available for independent statistical analysis. DPR, U.S. EPA,
and PMRA statisticians and biologists independently determined the NOEC values for eachactive ingredient from studies found to be scientifically acceptable. Refer to Table 1 below for
the NOEC concentrations determined for each active ingredient and matrix (i.e., nectar andpollen) combination (e.g. thiamethoxam in nectar). Utilizing only scientifically acceptablestudies in the risk determination process produced data gaps in colony-level effects data forpollen. Specifically, acceptable pollen colony feeding studies were not available forthiamethoxam or dinotefuran, necessitating the use of another neonicotinoid as a surrogate.
Accordingly, the NOEC value for clothianidin in pollen was bridged to thiamethoxam anddinotefuran. DPR found an acceptable colony feeding study conducted with pollen spiked with9

imidacloprid in the open literature (Dively et al., 2015). For a review of all the colony feedingstudies included in this document, refer to Appendix 8.
As indicated in Table 1, NOEC values are lower for nectar than for pollen. These differences
may be explained by the nature of these resources and how they are utilized within the hive. The
movement of nectar around the hive is rapid and has been described as a cascade effect where it
ultimately encounters most of the hive occupants and matrices. In addition, nectar is added topollen by hive bees to produce bee bread. In contrast, bees foraging for pollen bring the pollen
into the hive and pack it directly into pollen cells themselves. Bees consume less pollen thannectar, based on estimated food consumption rates for honey bees (U.S. EPA, PMRA, and
CDPR, 2014). The highest consumption rate of pollen is found in new worker bees that clean andcap cells within the hive. These bees consume only 1.3 – 12 mg/day of pollen compared toapproximately 60 mg/day of nectar. After 10 days, the new worker bees move to brood andqueen tending. During brood and queen tending, worker consumption of pollen remains thesame, whereas, nectar consumption more than doubles to 113 – 167 mg/day (U.S. EPA, PMRA,
and CDPR, 2014). All other adult bees consume less pollen per day than nectar. This tendency
for immediate exposure of residues in nectar brought back to hives, and the more limited
exposure to pollen within the hive, suggests that concentrations of a toxic substance in pollen
must be higher than concentrations in nectar to elicit a colony-level effect.

10

Table 1. Pollen and Nectar NOECs used in the Risk Determinations for
Imidacloprid, Thiamethoxam, Clothianidin, and Dinotefuran.

Active Ingredient
NOEC (µg/Kg)
Nectar – Colony Feeding Studies
Imidacloprid a
23b
Thiamethoxam
30c
Clothianidin
19dDinotefuran
71
Pollen – Colony Feeding Studies
97.5
Imidacloprid efThiamethoxam
372g
Clothianidin
372
f
372
Dinotefuran
All toxicity values derived from the following colony feeding studies:aBocksch, 2014.
Bocksch, 2015.
cLouque, 2016.
dBocksch, 2016.
eDively et al., 2015.
bf

Bridged from the registrant-submitted colony feeding study withclothianidin.

gBocksch and Werner, 2018.

4.3

Exposure Characterization

To determine the expected on-field exposure, measurements of imidacloprid, thiamethoxam,
clothianidin, and dinotefuran were taken in pollen and nectar from previously treated crops. Data
were generated for the worst-case scenarios (i.e., highest annual application rates, minimum
reapplication intervals, etc.) in compliance with product label directions to provide an estimate of
the highest concentrations expected for each active ingredient in nectar and pollen of agricultural
crops. The plants were treated under standard agricultural practices (e.g. foliar applications, soil
applications, or seed treatments along with irrigation, use of fertilizers, other maintenancechemicals, etc.) as indicated on product labels for crops under investigation. Nectar and pollen
samples were not available for all crops. For example, tomato flowers do not produce nectar. In
such instances, only pollen samples were available for inclusion in the risk determination.
Another exception can be seen with cotton, which produces extra-floral nectar in addition tofloral nectar. Cotton extra-floral nectar is known to be a highly attractive resource of forage forhoney bees and some beekeepers place their hives near cotton for honey production (McGregor,
1976; USDA, 2017). Accordingly, extra-floral nectar was included in this risk determination for11

applications to cotton crops. In the rare cases where floral pollen samples were not available foranalysis, measured residue concentrations in anthers served as a surrogate.
Statistical analyses were conducted on measured neonicotinoid concentrations in bee-relevant
matrices (e.g., pollen and nectar) for each acceptable residue study. DPR did not conduct
statistical analysis on seed treatment residue studies, as concentrations were always low, andoften below analytically detectable limits. Statistical analysis included the generation of the
cumulative empirical distributions of measured concentrations. The cumulative distributions
calculate a series of percentile values representing the proportion of samples that are below thatvalue. For estimation of exposure, the concentration chosen at a specified percentage of thesample is the value that represents the exposure value that would be compared to the NOEC
value derived from colony feeding studies to characterize potential risk.
For the risk determination, DPR scientists took many factors into consideration when
determining which percentile value to use for protection of honey bee colonies. Use of moderatestatistics, such as the mean or median, would not reflect the possible danger posed at the higher
end of measured distributions, and therefore, would not be protective for two reasons: First,
considering the extent of agricultural applications made for each crop in California, the amountof data collected is relatively small compared to the total population (e.g., 27 samples of nectar
collected from pumpkins might not be representative of all pumpkins grown in California). Thus,
the range in actual concentrations could be much greater and extreme residue values that appear
to be statistical outliers might not actually be outliers if more samples were available. Many of
the studies used in this risk determination have less than twenty total samples, even when all data
were combined from studies spanning two years. Second, concentrations measured in nectar insome of the studies were high enough to be of concern for acute toxicity to honey bees. Use ofmoderate statistics such as the mean concentration would not reflect the risks from these extreme
exposures.
On the other hand, use of maximum measured values in the risk determination could be overly
protective because they include outliers. Many samples taken for estimation of exposure
represented only one point in time, so it is unknown if the concentrations in pollen and nectar
were increasing or decreasing. Since these measured concentrations only provide a snapshot of
exposure, direct comparison of colony level NOEC values to maximum values measured in thepollen or nectar samples has a high degree of uncertainty because the duration of exposure toconcentrations that exceed the NOEC might be significantly shorter than the six-week durationof the colony feeding studies. This would vary for each crop and would depend on the durationof bloom. Based on the uncertainty associated with the duration of exposure, use of higher
percentiles could be unrealistic. Consequently, the 90th percentile value was determined to be apoint in the distribution where the value represented a realistic, yet protective approach todetermining risk.
DPR based this risk determination document on numerous residue studies submitted by theregistrants of neonicotinoid insecticide products. Descriptions of methods, results, andlimitations of these studies are available in Appendix 10. In addition, the cumulative empirical
distributions of measured concentrations for each residue study included in this document arepresented in Appendix 11. In many cases, residue data was only available for one or two crops

12within a specific crop group [as defined in Title 40 Code of Federal Regulations (40 CFR) §180.41; Appendix 4]. In cases where residue data was lacking for a specific crop, the worst-casescenario within the same crop group was used to represent the missing crop. Additionally, there
were cases in which there was no residue data available for an entire crop group. In such
instances, data from an appropriate surrogate was used, such as the same crop group from a study
utilizing a different nitroguanidine-substituted neonicotinoid active ingredient. In its reevaluation
letter, DPR notified registrants of its intention to bridge data from one active ingredient to any of
the other three active ingredients if no residue data were available for a given crop or crop group(Appendix 12).
4.4

Risk Determination Categories

DPR conducted risk determinations for agricultural uses registered in California with expected
worst-case on-field exposure to honey bees. Applications of neonicotinoid insecticides may
result in on-field exposure to honey bees when the crop is bee-attractive and harvested after
bloom. Crop groups with limited on-field exposure to honey bees are considered low-risk.
According to the United States Department of Agriculture document, Attractiveness ofAgricultural Crops to Pollinating Bees for the Collection of Nectar and/or Pollen (USDA,
2017), certain crops and crop groups, such as bulb vegetables, leafy vegetables (including
Brassica vegetables), and globe artichokes, are generally harvested before bloom, except whengrown for seed. Thus, the risk determinations for these crops and crop groups are classified as
low risk, except when grown for seed.
For crops associated with expected on-field exposure to honey bees, the 90th percentile wascalculated using residue data conducted at the maximum annual application rate and compared tocolony-level NOEC values to determine risk. Risk determinations were categorized as either
having a determination of risk, or low risk to honey bee colonies. Determinations of risk weremade for those crops or crop groups with 90th percentile pollen or nectar residue values thatexceed the appropriate NOEC value for the specific active ingredient and matrix. Conversely,
low risk determinations are those crops or crop groups with 90th percentile pollen or nectar
residue values that do not exceed the appropriate NOEC value. For more details, including
exceptions, refer to Tables 2-5.
It is important to note that determinations of risk in Table 6 were derived from worst-caseapplication scenarios. Crops with determinations of risk may be able to be mitigated bymodifying label use directions in a manner that will result in residues that are below therespective NOEC values. By definition, if the residues in pollen or nectar are below therespective colony-level NOEC values, then no significant colony effects are expected to occur.
The footnotes in Table 6 provide guidance on some potential adjustments to management
practices and label directions based on submitted data that could result in a determination of low
risks to honey bee colonies.
5.0

Risk Characterization

DPR made risk determinations for specific crops where crop-specific data was available (Tables
2, 3, 4, and 5 below). In most cases, residue data were only available for one or two crops within13

a specific crop group. In cases where there were no residue data for other crops in the group, theworst-case scenario (i.e., specific crop data that resulted in the highest residues in pollen andnectar) within the same crop group was used to represent all other crops in that crop group. For
example, for imidacloprid, the Berries Crop Group (Crop Group 13) includes both a strawberryand blueberry residue study (Table 2). The strawberry study resulted in higher residues than the
blueberry study. Thus, DPR used the risk determination based on the strawberry residue data to
represent all crops within Crop Group 13, with the exception of blueberries. The submission of
additional data can change these determinations on a crop by crop basis.
Similarly, if a given crop and active ingredient had more than one acceptable residue study, thestudy that resulted in the higher residues was used to represent that crop in the final risk
determination for that crop. One case in which this occurred was with thiamethoxam andcucumber. There are two acceptable cucumber residue studies. DPR used the study with thehighest residues in pollen and nectar to represent cucumbers in the overall risk determination.
This conservative approach is appropriate given the limitations of the residue data in terms of
relatively small sample sizes, environmental variability, and the various other factors (e.g., soil
texture, irrigation practices, use of fertilizers, temperature, etc.) that can influence how
representative these data sets are of the crops grown in various microclimates of California. If noacceptable residue data was available for a crop group, data from an appropriate surrogate wasused, such as data on the same crop group using a different nitroguanidine-substituted
neonicotinoid active ingredient.
Tables 2, 3, 4, and 5 below, show which crops had available residue data at the maximum
application rate. These tables compare the resulting residue concentrations to appropriate NOEC
values, state if the residues exceed the respective NOEC values, and make determinations of risk
based on 90th percentile residue values:

14

Table 2. Imidacloprid 90th percentile residue values and NOEC exceedances.
Crop Group

Crop

Residue StudyLab ID

Crop Group 8. Fruiting
Vegetables GroupTomatoEBNTN012

OrangeEBNTY007

Citrus a

EBNTL056-7

Apple

EBNTN014

Stone Fruit b

EBNTN013

CherryEBNTY008

Blueberry

EBNTY006

Strawberry

EBNTL056-04

Crop Group 10. Citrus
Fruit Group
Crop Group 11. Pome
Fruits Group
Crop Group 12. Stone
Fruits Group

Crop Group 13. Berries
Group

EBNTN011
Crop Group 20. OilseedGroup

CottonEBNTY010

Imidacloprid
Residue Residue
Application
Matrix(µg/Kg)1 Soil + 2
Pollen476.9
FoliarPollen3257.92 Foliar (PreBloom)
Nectar
267.1
1 SoilNectar25.0
Pollen58.5
1 Soil + 2
FoliarNectar3.5
Pollen136.2
1 Soil + 2
FoliarNectar9.5
Pollen393.8
5 Foliar
Nectar5.1
Pollen17.5
1 SoilNectar
4.6
1 SoilPollen247.0
Pollen182.2
1 Soil + 3
Floral
107.0
Foliar (AtNectarExtrafloral
Bloom)
578.6
NectarPollen6.6
Floral
5 Foliar (Pre18.4
NectarBloom)
Extrafloral
13.3
NectarNOEC(µg/Kg)Exceedance
(Y/N)

97.5

Y

97.5
23
23
97.5
23
97.5
23
97.5
23
97.5
23
97.5
97.5

Y
Y
Y
N
N
Y
N
Y
N
N
N
Y
Y

23

Y

23

Y

97.5

N

23

N

23

N

Risk

Notes:Red shading indicates soil or foliar applications that result in pollen or nectar residues that exceed the NOEC.
Green shading indicates soil or foliar applications that do not result in pollen or nectar residues that exceed the NOEC.
a

Residue study was conducted on multiple crops within the crop group, including orange, tangerine, grapefruit, tangelo, and lemon. However,data was not analyzed by individual crop due to limited replication.
b

Residue study was conducted on multiple crops within the crop group, including cherry, plum, apricot, and peach. However, data was notanalyzed by individual crop due to limited replication.

Table 3. Thiamethoxam 90th percentile residue values and NOEC exceedances.
Thiamethoxam
Residue Study
Residue
Application
Lab ID
MatrixCrop Group

Crop

Crop Group 6. Legume
Vegetables (Succulent or Dried)Group

Soybean

TK0250070

2 Foliar

TomatoTK0222531
TK0242072

2 Foliar
1 SoilPepper

TK0236306

1 Soil

TK0024668

1 Soil

TK0222532

2 Foliar

TK0222530

1 Soil

TK0222530

1 Soil

TK0242074

2 Foliar

SummerSquash

TK0222530

1 SoilCitrus a

TK0177221

1 SoilSweet
OrangeTK0250069

2 Foliar

Apple b

TK0250071

1 Foliar

Stone Fruit c

TK0177222

2 Foliar

Blueberry

TK0250072

3 Foliar

TK0177224

3 Foliar

TK0250068

1 SoilCranberry

TK0236307

3 Foliar

Corn

TK0258214

Crop Group 8. Fruiting
Vegetables Group

Muskmelon

Crop Group 11. Pome Fruits
Group
Crop Group 12. Stone Fruits
Group

Crop Group 13. Berries Group

Crop Group 15. Cereal Grains
Group
Crop Group 20. Oilseed Group

TK0177223

41.2

372

N

4.7

30

N

6519.7
157.2
259.9
180.9
10.8
13.2
1079.9
288.6
119.7
27.9
8.1
12.2
18.0
15.0
16.1
31.7
62.3
10.2
126.7
2.1
1954.7
225.4
1.6
133.2
836.4
613.0
7411.0
301.0
541.0
52.3
1226.4
921.9

372
372
372
30
372
30
372
30
372
30
372
30
372
30
372
30
372
30
372
30
372
30
372
30
372
30
372
30
372
30
372
30

Y
N
N
Y
N
N
Y
Y
N
N
N
N
N
N
N
Y
N
N
N
N
Y
Y
N
Y
Y
Y
Y
Y
Y
Y
Y
Y

Seed + 2
FoliarPollen

538.9

372

Y

2 Foliar

PollenNectarExtrafloral
Nectar

102.5
5.8

372
30

N
N

125.9

30

Y

Strawberry

CottonExceedance(Y/N)

PollenPollenPollenNectarPollenNectarPollenNectarPollenNectarPollenNectarPollenNectarPollenNectarPollenNectarPollenNectarPollenNectarPollenNectarPollenNectarPollenNectarPollenNectarPollenNectarPumpkin

Crop Group 10. Citrus Fruit
Group

NOEC
(µg/Kg)NectarCucumberCrop Group 9. Cucurbit
Vegetables Group

Anthers

Residue(µg/Kg)

Risk

Notes:Red shading indicates soil or foliar applications that result in pollen or nectar residues that exceed the NOEC.
Green shading indicates soil or foliar applications that do not result in pollen or nectar residues that exceed the NOEC.
a

Residue study was conducted on multiple crops within the crop group, including orange and lemon. However, data was not analyzed byindividual crop due to limited replication.

b
The residue study for this crop was not conducted at the maximum application rate allowed by the product label, therefore worst-case residues
are expected to be higher than reported in this table.
c

Residue study was conducted on multiple crops within the crop group, including peach, plum, cherry, and prune. However, data was notanalyzed by individual crop due to limited replication.

Table 4. Clothianidin 90th percentile residue values and NOEC exceedances.
Clothianidin
Residue
Application
MatrixCrop Group

Crop

Residue StudyLab ID

Crop Group 1. Root andTuber Vegetables Group

PotatoVP-38985

1 SoilCucumber

VP-38938

1 SoilMelon

VP-38938

1 SoilSquash

VP-38938

1 Soil

VP-38938

1 Soil

VP-38263

1 Soil (AtPlanting)

VP-38313

2 Foliar

VP-38971

1 Soil (PostEmergence)

Crop Group 9. Cucurbit
Vegetables GroupPumpkin

Crop Group 11. Pome
Fruits GroupApple aVP-38552

1 Foliar (PostBloom)

Crop Group 12. Stone
Fruits Group

Peach aVP-38563

2 Foliar (PostBloom)

Crop Group 13. Berries
Group

Grape

VP-38992

Crop Group 14. Tree Nuts
Almond aGroup

VP-38473

Crop Group 20. OilseedGroup

VP-38259

Cotton1 Soil
1 Foliar (PreBloom)
2 Foliar (PostBloom)
2 Foliar

Residue NOEC Exceedance(µg/Kg) (µg/Kg)(Y/N)

Pollen

113.9

372

N

Anthers
NectarAnthers
NectarPollenNectarPollenNectarPollenNectarPollenNectarPollenNectarPollenNectarPollenNectarPollen

32
39.6
18.7
14.6
10.7
4.4
21
6.6
17
6.3
71
5
20.3
9.9
57.4
0.71
10
0.3
157.3

372
19
372
19
372
19
372
19
372
19
372
19
372
19
372
19
372
19
372

N
Y
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N

Pollen1229.8372

Y

PollenNectarPollenNectarExtrafloral
Nectar

12.7
0.8
246
79.4

372
19
372
19

N
N
N
Y

647

19

Y

Risk

Notes:Red shading indicates soil or foliar applications that result in pollen or nectar residues that exceed the NOEC.
Green shading indicates soil or foliar applications that do not result in pollen or nectar residues that exceed the NOEC.
a

Data indicate that post-bloom applications on these crops are not associated with a determination of risk to honey bees; however, thesestudies do not represent the worst-case scenario for the crop group.

Table 5. Dinotefuran 90th percentile residue values and NOEC exceedances.
Dinotefuran
Residue Study
Residue
Application
Lab ID
MatrixCrop Group

Crop

Crop Group 1. Root andTuber Vegetables Group

Potato10934.4100

1 SoilBell
Pepper

S16-01167

2 SoilTomato10934.4103

2 Foliar
2 Soil

Pumpkin10934.4104

2 SoilCherry10934.4105

2 Foliar

Blueberry

10934.4107

2 Foliar

Cranberry

10934.4101

2 Foliar

Cotton43411B104

2 Foliar

Crop Group 8. Fruiting
Vegetables Group
Crop Group 9. Cucurbit
Vegetables Group
Crop Group 12. Stone
Fruits Group
Crop Group 13. Berries
Group

Crop Group 20. Oilseed
Group

Residue NOEC Exceedance(µg/Kg) (µg/Kg)(Y/N)

Anthers

56.9

372

N

PollenNectarPollenPollenPollenNectarPollenNectarPollenNectarPollenNectarPollenFloralNectarExtrafloral
Nectar183
4.46
10438.6
5532.4
88.3
39.0
130.5
12.5
468.9
470.8
763.5
780.9
6968

372
71
372
372
372
71
372
71
372
71
372
71
372

N
N
Y
Y
N
N
N
N
Y
Y
Y
Y
Y

81.6

71

Y

1660

71

Y

Risk

Notes:Red shading indicates soil or foliar applications that result in pollen or nectar residues that exceed the NOEC.
Green shading indicates soil or foliar applications that do not result in pollen or nectar residues that exceed the
NOEC.

6.0

CONCLUSIONS

6.1.

Overview by Crop GroupingIn summary, this risk determination document is based upon colony-level risks to honey bees
resulting from the consumption of nectar or pollen containing neonicotinoid residues that exceed
the colony-level NOEC values. DPR conducted risk determinations for the maximum annual
application rate of each agricultural crop group as found on currently registered imidacloprid,
clothianidin, thiamethoxam, and dinotefuran product labels (Appendix 3). DPR’s risk
determinations for soil and foliar applications on registered agricultural crop groupings for
imidacloprid, thiamethoxam, clothianidin, and dinotefuran are detailed below and in Table 6.
The risk determination process discussed previously states that crop groups are categorized aseither having a determination of risk or low risk to honey bee colonies. In Table 6 below, red
shading indicates a determination of risk for all crops in the crop group. Green shading indicatesa determination of low risk for the entire crop group. Yellow shading is to be considered ashaving a determination of risk for the crop group, with some crop-specific exceptions. Only cropgroups currently registered for agricultural use in California are included in this risk
determination, with crop-specific exceptions noted in Table 6.
For imidacloprid, using the 90th percentile as the expected exposure to honey bees, the following
crop groups have a determination of low risk: Root and Tuber Vegetables (Crop Group 1), Bulb
Vegetables (Crop Group 3), Leafy Vegetables (Except Brassica Vegetables) (Crop Group 4),
18

Brassica (Cole) Leafy Vegetables (Crop Group 5), Legume Vegetables (Succulent or Dried)(Crop Group 6), Pome Fruits (Crop Group 11), Herbs and Spices (Crop Group 19), and Globe
Artichoke. The following crop groups have a determination of risk for imidacloprid: Fruiting
Vegetables (Crop Group 8), Cucurbit Vegetables (Crop Group 9), Citrus Fruit (Crop Group 10),
Stone Fruits (Crop Group 12), Berries (Crop Group 13), Tree Nuts (Crop Group 14), Oilseed
Crops (Crop Group 20), Tropical and Subtropical Fruits with Inedible Peels (Crop Group 24),
Hops, Tobacco, and Coffee.
For thiamethoxam, using the 90th percentile as the expected exposure to honey bees, thefollowing crop groups have a determination of low risk: Root and Tuber Vegetables (Crop Group1), Bulb Vegetables (Crop Group 3), Leafy Vegetables (Except Brassica Vegetables) (CropGroup 4), Brassica (Cole) Leafy Vegetables (Crop Group 5), Legume Vegetables (Succulent orDried) (Crop Group 6), Citrus Fruit (Crop Group 10), Globe Artichoke, and Mint. The following
crop groups have a determination of risk for thiamethoxam: Fruiting Vegetables (Crop Group 8),
Cucurbit Vegetables (Crop Group 9), Pome Fruits (Crop Group 11), Stone Fruits (Crop Group12), Berries (Crop Group 13), Cereal Grains (Crop Group 15), Oilseed Crops (Crop Group 20),
Tropical and Subtropical Fruits with Inedible Peels (Crop Group 24), Hops, and Tobacco.
For clothianidin, using the 90th percentile as the expected exposure to honey bees, the following
crop groups have a determination of low risk: Root and Tuber Vegetables (Crop Group 1), Leafy
Vegetables (Except Brassica Vegetables) (Crop Group 4), Brassica (Cole) Leafy Vegetables(Crop Group 5), and Legume Vegetables (Succulent or Dried) (Crop Group 6). The following
crop groups have a determination of risk for clothianidin: Cucurbit Vegetables (Crop Group 9),
Citrus Fruit (Crop Group 10), Pome Fruits (Crop Group 11), Stone Fruits (Crop Group 12),
Berries (Crop Group 13), Tree Nuts (Crop Group 14), Cereal Grains (Crop Group 15), Oilseed
Crops (Crop Group 20), Tropical and Subtropical Fruits with Inedible Peels (Crop Group 24),
and Tobacco.
For dinotefuran, using the 90th percentile as the expected exposure to honey bees, the following
crop groups have a determination of low risk: Root and Tuber Vegetables (Crop Group 1), Bulb
Vegetables (Crop Group 3), Leafy Vegetables (Except Brassica Vegetables) (Crop Group 4),
Brassica (Cole) Leafy Vegetables (Crop Group 5), Cucurbit Vegetables (Crop Group 9), andStone Fruits (Crop Group 12). The following crop groups have a determination of risk fordinotefuran: Fruiting Vegetables (Crop Group 8), Berries (Crop Group 13), and Oilseed Crops(Crop Group 20).
There are crop- and application-specific exceptions for the risk determinations mentioned above.
Please refer to Tables 2-6 for more detail on exceptions.

19

Table 6. Risk determinations for foliar or soil applications of imidacloprid, thiamethoxam,
clothianidin, and dinotefuran at the maximum allowed annual application rate based on 90th
percentile residue values.
Crop Group

Imidacloprid
Risk
Notes

Thiamethoxam
Risk
Notes

Clothianidin
Risk
Notes

Dinotefuran
Risk
Notes

Crop Group 1. Root and Tuber
Vegetables Groupdd

Crop Group 3. Bulb Vegetables Group

c

c

c

c

c

c

c

c

c

c

Crop Group 4. Leafy Vegetables (Except
Brassica Vegetables) Group
Crop Group 5. Brassica (Cole) Leafy
Vegetables
Crop Group 6. Legume Vegetables(Succulent or Dried) Group
Crop Group 8. Fruiting Vegetables
Group
Crop Group 9. Cucurbit Vegetables
Group

b

c

b
k

b

h

j

Crop Group 10. Citrus Fruit Group
Crop Group 11. Pome Fruits Groupb, e

Crop Group 12. Stone Fruits Groupb, e

Crop Group 13. Berries Groupi

Crop Group 14. Tree Nuts Group

g

e, g

Crop Group 15. Cereal Grains Group
Crop Group 19. Herbs and SpicesbcCrop Group 20. Oilseed Group
Crop Group 24. Tropical and SubtropicalFruit, Inedible Peel Groupff

Globe Artichoke accHops aff

fc

Mint aTobacco af

Coffee afff

Notes:
Patterned gray shading indicates that the active ingredient is not currently registered for foliar or soil applications on thecrop group.
Red shading indicates a determination of risk for all crops in the crop group based on evaluated data.
Yellow shading indicates a determination of risk for the crop group; however, there were crop-specific or applicationspecific exceptions indicating low risk.
Green shading indicates a determination of low risk for the crop group based on evaluated data.
For additional detail on residue values, please see tables 3, 4, 5, and 6.
a

Not categorized into a general crop group, according to 40 CFR 180.41 crop group tables.
Risk determination category bridged from thiamethoxam.
c
No on-field exposure to honey bees expected unless grown for seed.
d
Risk determination category bridged from clothianidin.
eRisk except for post-bloom, pre-harvest applications.
f
In absence of tier II data and no similar crop groups from which to bridge, the crop group determination defaults to risk to honey bees.
g
Risk determination category bridged from thiamethoxam stone fruit, as tree nuts and stone fruits are taxonomically related.
hRisk except certain applications to pumpkin, muskmelon, and cucumber.
i
Risk except certain applications to blueberry.
j
Risk except certain applications to melon, pumpkin, and squash.
bkRisk except certain applications to bell pepper.

6.2

Seed Treatments and Tree Injection Applications

Risk determinations were only conducted for foliar and soil applications. Risks from seedtreatment applications were evaluated in the preliminary pollinator risk assessments published by
U.S. EPA (U.S. EPA and DPR, 2016; U.S. EPA, 2017a; U.S. EPA, 2017b). The preliminary
assessment for imidacloprid evaluated multiple seed treatment residue studies conducted on corn,
canola, and sunflower. These studies generally reported no residues in pollen and nectar above
the limit of detection. Values are well below their respective NOEC values, supporting theconclusion that imidacloprid seed treatments pose a low risk to honey bees. The preliminary
pollinator risk assessment for clothianidin and thiamethoxam evaluated multiple seed treatment
residue studies conducted on corn, sunflower, melon, canola, cotton, and soybean. The resulting
residue concentrations are all below the respective NOECs, supporting the conclusion thatclothianidin or thiamethoxam seed treatments pose a low risk to honey bee colonies. Dinotefuran
is not registered for any seed treatment applications. There have been issues in other states andcountries with contact exposure resulting from abraded seed coat dust at planting, but the U.S.
EPA has addressed this with best management practices (U.S. EPA and DPR, 2016). DPR has norecords of such incidents occurring in California.
DPR considered a single residue study testing a tree injection application in this risk
determination. This study measured residues of dinotefuran in pollen and nectar following tree
injection applications to cherry trees. Dinotefuran 20SG, EPA Reg. No. 86203-12, was injected
into the trunks of cherry trees late in the season (September), before leaf drop, at a rate of 2grams of product per inch of trunk diameter either at breast height or right below the first trunk
bifurcation. Samples of pollen and nectar were collected 165-243 days after the last application.
The maximum measured dinotefuran residues resulting from tree injection applications were
31,688 µg/Kg in pollen (201 days after application) and 17,484 µg/Kg in nectar (237 days after
application); the corresponding 90th percentile measured residues were 24,894 µg/Kg in pollen
and 16,241 µg/Kg in nectar (Lab Study ID 10934.4105; Louque, 2016). These are some of thehighest residues noted in pollen and nectar from any application methods on any crops.
Currently, no products containing the four neonicotinoids are registered with DPR that allow tree
injections to stone fruits or any other agricultural crops in California.
7.0

Considerations for Mitigation

The focus of this document is to identify risks to honey bees at the colony level following
applications of imidacloprid, clothianidin, thiamethoxam, and dinotefuran. As stated previously,
only worst-case application scenarios, as allowed by currently registered labels in California,
were included for analysis. Studies involving less frequent application intervals or lower
application rates were excluded from consideration in this document. However, these studies
contain valuable data to help inform future mitigation options. For instance, clothianidin residue
studies included data on post-bloom applications to several crops, such as peach, almond, andapple. Though these studies were not considered worst-case, and thus not included in the overallrisk determination, the resultant residues did not exceed the NOEC and would be categorized as
low risk. The information from the additional studies provides potential directions fordevelopment of management practices based on the number of applications, frequency ofreapplication, soil texture, timing of applications in relation to bloom, and application site.

21

Though outside the scope of this document, additional analysis of the submitted data would
likely provide further science-based mitigation options to reduce risks to honey bees fromagricultural applications of nitroguanidine-substituted neonicotinoid pesticides.
8.0

Risk Appraisal

The comparison of neonicotinoid concentrations measured in nectar and pollen of treated crops
to NOEC concentrations developed from colony feeding studies is not straightforward for several
reasons. First, the duration of exposure in the colony feeding studies was set at six weeks basedon bloom duration. Calculating a realistic duration of exposure for pollinating bees is difficult
because of differences in blooming periods of crops and commercial beekeeping management
practices. The flowering intervals for different crops can be relatively short, such as for early
flowering fruit and nut trees, or long, such as for cotton plants, where plants continuously flower
throughout the growing season. In addition, during the growing season, managed honey beecolonies are often transported from one flowering crop to another, which extends the duration of
exposure. In colony feeding studies, the spiked sugar solutions or pollen patties were regularly
replenished throughout the 6-week exposure to ensure the colonies were exposed to a consistentconcentration. As indicated by the data, concentrations measured in flowers can be variable, so
pollinating honey bee colonies are likely to be exposed to a range of concentrations. In addition,
the data presented in this document shows that concentrations measured in the nectar and pollenof certain plants could be orders of magnitude higher than the highest dose levels used in thecolony feeding studies. This could result in exposure to residues that are acutely toxic to worker
honey bees.
If distributional statistics at the lower to middle portion (i.e. 25th or 50th percentiles) of themeasured range in concentrations of treated crops are compared to the NOEC values derived
from the colony feeding studies, they could underestimate the potential risk to pollinating honeybee colonies. Conversely, if statistics at the upper end of the distribution are used, they could beoverly conservative. Tables 7 to 10 present a visual comparison for the range in potential
exceedances that would result from using the 50th, 75th, 90th or 100th (maximum) percentile
residue values for each crop and application scenario reviewed for imidacloprid, thiamethoxam,
clothianidin, and dinotefuran. The comparison between the 50th and 100th percentile to the NOEC
conforms to the observation that the 50th percentile would likely not be protective, whereas, the100th percentile is potentially overprotective.
The concentrations calculated for each of the percentiles are presented in Appendix 11. Of
significance is the rather large range in values that was measured in some of the treated crops.
Both nectar and pollen values at the highest percentiles were measured in the parts per million,
values that would cause acute toxicity.
In summary, the 50th percentile concentration would likely not be protective of honey beecolonies, especially in light of extremely high values that were measured for certain
combinations of crop and application methods. On the other hand, the maximum concentration
value would likely be overly protective because of complications in the comparison of total
exposure to NOEC values generated from the colony feeding studies. The uncertainty is causedby difficulties in calculating the total magnitude and duration of exposure, as there are22

potentially large differences in exposure durations between the bees in the colony feeding study
and those bees foraging on the flowers of the crops they are pollinating. Use of the 90th
percentile residue values indicate either a determination of risk or a determination of low risk forthe studies evaluated and appears to be a realistic, yet protective approach.

23

Table 7. Imidacloprid comparison of NOEC exceedances based on maximum and 50th, 75th, and 90th
percentile values for each crop and application scenario, based on acceptable data.
Imidacloprid
Crop Group

Crop

Crop Group 8. Fruiting Vegetables GroupTomatoOrangeCrop Group 10. Citrus Fruit Group
Crop Group 11. Pome Fruits Group
Crop Group 12. Stone Fruits Group
Crop Group 13. Berries Group
Crop Group 20. Oilseed Group

Application Type

1 Soil + 2 Foliar
2 Foliar (Pre-Bloom)
1 SoilCitrus aApple
1 Soil + 2 Foliar
1 Soil + 2 Foliar
Stone Fruit b
Cherry5 Foliar
Blueberry
1 SoilStrawberry
1 Soil
1 Soil + 3 Foliar (At Bloom)
Cotton5 Foliar (Pre-Bloom)
Crop Groups With Data Gaps

Crop Group 1. Root and Tuber Vegetables
Group

N/A

N/A

Crop Group 3. Bulb Vegetables Group

N/A

N/A

Crop Group 4. Leafy Vegetables (Except
Brassica Vegetables) Group

N/A

N/A

N/A

N/A

N/A

N/A

N/A
N/A
N/A

N/A
N/A
N/A

N/A

N/A

N/A

N/A

Hops

N/A

N/A

TobaccoN/A

N/A

CoffeeN/A

N/A

Crop Group 5. Brassica (Cole) Leafy
Vegetables
Crop Group 6. Legume Vegetables(Succulent or Dried) Group
Crop Group 9. Cucurbit Vegetables Group
Crop Group 14. Tree Nuts Group
Crop Group 19. Herbs and Spices
Crop Group 24. Tropical and SubtropicalFruit, Inedible Peel Group
Globe Artichoke

Exceedance Category
50% 75% 90% Max

Notes:Red shading indicates soil or foliar applications that result in pollen or nectar residues that exceed the
NOEC.
Green shading indicates soil or foliar applications that do not result in pollen or nectar residues thatexceed the NOEC.
a

Residue study was conducted on multiple crops within the crop group, including orange, tangerine, grapefruit, tangelos,
and lemon. However, data was not analyzed by individual crop due to limited replication.

b

Residue study was conducted on multiple crops within the crop group, including cherry, plum, apricot, and peach.
However, data was not analyzed by individual crop due to limited replication.

Table 8. Thiamethoxam comparison of NOEC exceedances based on maximum and 50th,
75th, and 90th percentile values for each crop and application scenario, based on acceptable
data.
Thiamethoxam
Crop Group

Crop

Application Type

Crop Group 6. Legume Vegetables(Succulent or Dried) Group

Soybean

2 Foliar

Crop Group 8. Fruiting Vegetables
Group

Crop Group 9. Cucurbit Vegetables
Group

Crop Group 10. Citrus Fruit Group
Crop Group 11. Pome Fruits Group
Crop Group 12. Stone Fruits Group

Crop Group 13. Berries Group

Crop Group 15. Cereal Grains Group
Crop Group 20. Oilseed Group

Exceedance Category
50% 75% 90% Max

2 Foliar
1 SoilPepper
1 Soil
1 SoilCucumber
2 Foliar
Muskmelon
1 Soil
1 Soil
Pumpkin
2 Foliar
Summer Squash
1 Soila
1 SoilCitrus
Sweet Orange
2 FoliarApple
1 Foliarb
2 Foliar
Stone Fruit
Blueberry
3 Foliar
3 Foliar
Strawberry
1 SoilCranberry
3 Foliar
Corn
Seed + 2 Foliar
Cotton2 Foliar
Crop Groups With Data Gaps
TomatoCrop Group 1. Root and Tuber
Vegetables Group

N/A

N/A

Crop Group 3. Bulb Vegetables Group

N/A

N/A

Crop Group 4. Leafy Vegetables(Except Brassica Vegetables) Group

N/A

N/A

Crop Group 5. Brassica (Cole) Leafy
Vegetables

N/A

N/A

Crop Group 24. Tropical and
Subtropical Fruit, Inedible Peel Group

N/A

N/A

Globe Artichoke
Hops
Mint
Tobacco

N/A
N/A
N/A
N/A

N/A
N/A
N/A
N/A

Notes:Red shading indicates soil or foliar applications that result in pollen or nectar residues thatexceed the NOEC.
Green shading indicates soil or foliar applications that do not result in pollen or nectar residues
that exceed the NOEC.
a

Residue study was conducted on multiple crops within the crop group, including orange and lemon. However, data
was not analyzed by individual crop due to limited replication.
b

Residue study was conducted on multiple crops within the crop group, including peach, plum, cherry, and prune.
However, data was not analyzed by individual crop due to limited replication.

Table 9. Clothianidin comparison of NOEC exceedances based on maximum and 50th, 75th, and90th percentile values for each crop and application scenario, based on acceptable data.
Clothianidin
Crop Group

Crop

Application Type

Crop Group 1. Root and Tuber
Vegetables Group

Potato1 SoilCrop Group 9. Cucurbit Vegetables
Group

Crop Group 13. Berries Group
Crop Group 20. Oilseed Group

Exceedance Category
50% 75% 90% Max

Cucumber
Melon
Squash

1 Soil
1 Soil
1 Soil
1 Soil
1 Soil (At Planting)
Pumpkin
2 Foliar
1 Soil (Post-Emergence)
1 SoilGrape
1 Foliar (Pre-Bloom)
Cotton2 Foliar
Crop Groups With Data Gaps

Crop Group 4. Leafy Vegetables (Except
Brassica Vegetables) Group

N/A

N/A

Crop Group 5. Brassica (Cole) Leafy
Vegetables

N/A

N/A

Crop Group 6. Legume Vegetables(Succulent or Dried) Group

N/A

N/A

Crop Group 11. Pome Fruits Group a
Crop Group 12. Stone Fruits Group b
Crop Group 14. Tree Nuts Group c
Crop Group 15. Cereal Grains Group

N/A
N/A
N/A
N/A

N/A
N/A
N/A
N/A

Crop Group 24. Tropical and SubtropicalFruit, Inedible Peel Group

N/A

N/A

TobaccoN/A

N/A

Notes:Red shading indicates soil or foliar applications that result in pollen or nectar residues thatexceed the NOEC.
Green shading indicates soil or foliar applications that do not result in pollen or nectar residues
that exceed the NOEC.
a

Risk except post-bloom foliar applications to apple.
Risk except post-bloom foliar applications to peach.
c
Risk except post-bloom foliar applications to almond.
bTable 10. Dinotefuran comparison of NOEC exceedances based on maximum and 50th, 75th, and90th percentile values for each crop and application scenario, based on acceptable data.
Dinotefuran
Crop Group

Crop

Application
Type

Crop Group 1. Root and Tuber
Vegetables Group

Potato1 SoilBell Pepper

2 Soil
2 Soil2 Foliar

Crop Group 8. Fruiting Vegetables
Group
Crop Group 9. Cucurbit Vegetables
Group

TomatoPumpkin

Exceedance Category
50% 75% 90% Max

2 SoilCrop Group 12. Stone Fruits Group

Cherry2 Foliar
Blueberry
2 Foliar
Crop Group 13. Berries Group
Cranberry
2 Foliar
Crop Group 20. Oilseed Group
Cotton2 Foliar
Crop Groups With Data Gaps
Crop Group 3. Bulb Vegetables Group
N/A
N/A
Crop Group 4. Leafy Vegetables(Except Brassica Vegetables) Group

N/A

N/A

Crop Group 5. Brassica (Cole) Leafy
Vegetables

N/A

N/A

Notes:Red shading indicates soil or foliar applications that result in pollen or nectar residues thatexceed the NOEC.
Green shading indicates soil or foliar applications that do not result in pollen or nectar
residues that exceed the NOEC.

Bibliography
1. Bocksch, S. (2014). Honey bee brood and colony level effects following Imidacloprid
intake via treated artificial diet in a field study in North Carolina: Final Report.
Unpublished study prepared by Eurofins Agroscience Services, Inc. 360p., Laboratory
Report Number S13-03176. MRID 49510001. CDPR Study ID 281556.
2. Bocksch, S. (2015). Thiamethoxam Technical - Honey Bee Brood and Colony Level
Effects Following Thiamethoxam Intake via Treated Sucrose Solution in a Field Study inNorth Carolina: Final Report. Unpublished study prepared by Eurofins Agroscience
Services EcoChem Gmbh. 468p., Laboratory Report Number S14-02633. MRID
49757201. CDPR Study ID 288917.
3. Bocksch, S. (2016). Honey Bee Brood and Colony Level Effects Following Dinotefuran
Intake via Treated Sucrose Solution in a Field Study in North Carolina: Final Report.
Unpublished study prepared by Eurofins Agroscience Services EcoChem GmbH, &Eurofins Agroscience Services Ecotox GmbH. 523p., Laboratory Report Number S1500102. MRID 50147001. CDPR Study ID 296826.
4. Bocksch, S., Werner, S. (2018). Clothianidin Technical - Honey Bee Brood and Colony
Level Effects Following Clothianidin Intake via Treated Pollen in a Field Study in North
Carolina - USA 2017. Unpublished study prepared by Eurofins Agroscience Services
EcoChem CmbH. 192p., Laboratory Report Number S17-02137. MRID 50478501.
CDPR Study ID 305901.
5. Cimino, A.M., Boyles, A.L., Thayer, K.A., & Perry, M.J. (2017). Effects ofneonicotinoid pesticide exposure on human health: a systematic review. Environ Health
Perspect 125(2),155–162. DOI:10.1289/EHP515.
6. Dively, G.P., Embrey, M.S., Kamel, A., Hawthorne, D.J., & J.S. Pettis. (2015).
Assessment of chronic sublethal effects of imidacloprid on honey bee colony health.
PLoS ONE, 10(3), e011874. DOI:10.1371/journal.pone.0118748.
7. DPR. (2018a). Pesticide Chemical Information Query. California Department of Pesticide
Regulation Internal Database. Accessed: May 18, 2018.
8. DPR. (2018b). Pesticide Use Reporting. California Pesticide Information Portal.
<http://calpip.cdpr.ca.gov/main.cfm>. Accessed: June 5, 2018.
9. Louque, J. (2016). Colony feeding study evaluating the chronic effects of clothianidinfortified sugar diet on honey bee (Apis mellifera) colony health under free foraging
conditions: Final Report. Unpublished study prepared by Smithers Viscient. 550p.,
Laboratory Report Number 13798.4143. MRID 49836101 CDPR Study ID TBD.

28

10. McGregor, S.E. (1976). Insect Pollination of Cultivated Crop Plants. Washinton, D.C.:
United States Department of Agriculture, Agricultural Research Services.
11. U.S. EPA, & DPR. (2016). Preliminary pollinator assessment to support the registration
review of imidacloprid. Report Number EPA-HQ-OPP-2008-0844-0140. Washington,
D.C.: U.S. EPA.
12. U.S. EPA, PMRA, & DPR. (2014). Guidance for Assessing Pesticide Risks to Bees.
Washington, D.C.: U.S. EPA.
13. U.S. EPA. (2017a). Draft assessment of the potential effects of dinotefuran on bees.
Report Number EPA-HQ-OPP-2011-0920-0014. Washington, D.C.: Author.
14. U.S. EPA. (2017b). Preliminary bee risk assessment to support the registration review ofclothianidin and thiamethoxam. Report Number EPA-HQ-OPP-2011-0865-0173.
Washington, D.C.: Author.
15. USDA. (2017). Attractiveness of agricultural crops to pollinating bees for the collectionof nectar and/or pollen. Washington, D.C.: Author.

29

Appendix 1. Reevaluation Letter Initiating the Reevaluation of Imidacloprid,
Clothianidin, Thiamethoxam, and Dinotefuran

Department of Pesticide Regulation
Mary-Ann Warmerdam
Director

Arnold Schwarzenegger

California Notice 2009-02

Governor

POST UNTIL March 31, 2009
NOTICE OF DECISION TO INITIATE REEVALUATION OF
CHEMICALS IN THE NITROGUANIDINE INSECTICIDE CLASS OFNEONICOTINOIDS.
Pursuant to Section 6220, et seq., Title 3. California Code of Regulations, the Director of the
Department of Pesticide Regulation (DPR) notices her decision to initiate a reevaluation ofcertain pesticide products within the nitroguanidine insecticide class of neonicotinoids andcontaining the following active ingredients: imidacloprid, clothianidin, dinotefuran, andthiamethoxam. Interested persons may comment on this decision up to and including the dateshown on the top-right corner of this notice to the Department of Pesticide Regulation, Pesticide
Registration Branch, 1001 I Street, P.O. Box 4015, Sacramento, California 95812-4015.
REEVALUATIONDPR is hereby commencing a reevaluation of chemicals in the nitroguanidine insecticide class ofneonicotinoids and containing the following active ingredients: imidacloprid, clothianidin,
dinotefuran, and thiamethoxam. This reevaluation involves 50 registrants and 282 pesticide
products. DPR determined that the number of products included in this reevaluation were too
numerous to list within this notice. A list of products included in the reevaluation is available
upon written request to the address listed above or on DPR’s Web site at:<http://www.cdpr.ca.gov/docs/registration/reevaluation/chemicals/neonicotinoids.htm>.
BASIS OF REEVALUATION
In 2008, DPR received an adverse effects disclosure pursuant to Federal Insecticide Fungicide
and Rodenticide Act (FIFRA) section 6(a)(2) and Food and Agricultural Codesection 12825.5 regarding the active ingredient imidacloprid. The disclosure included twelve
residue and two combination residue, honey, bumble bee studies of imidacloprid use on anumber of ornamental plants. DPR’s evaluation of the data noted two critical findings. One, highlevels of imidacloprid in leaves and blossoms of treated plants, and two, increases in residue
levels over time.
Imidacloprid levels in leaves and blossoms varied depending on the application rate and the typeof plant, but the data indicate that residues in some plants measured higher than 4 parts per
million (ppm). The data also indicate that when using soil application methods, imidacloprid
residues remained relatively low for the first six months after application, followed by a dramaticincrease that remained stable in some cases for more than 500 days after treatment. Where
imidacloprid was applied to the soil, no significant decline in residue levels was observed in anyof the studies, even in studies where residues were tested at 540 days after treatment. DPR foundthat the treatment rates used in the studies where high imidacloprid residue levels were found in
1001 I Street • P.O. Box 4015 • Sacramento, California 95812-4015 • www.cdpr.ca.gov
A Department of the California Environmental Protection Agency
Printed on recycled paper, 100% post-consumer--processed chlorine-free.

30

Appendix 1. Reevaluation Letter Initiating the Reevaluation of Imidacloprid,
Clothianidin, Thiamethoxam, and Dinotefuran

California Notice 2009-02
Page 2leaves and blossoms, were comparable to application rates found on currently registered labels
for orchards, assuming the orchards were planted at a density of 200 trees per acre or fewer. The
data indicate that use of imidacloprid on an annual basis may be additive, in that significant
residues from the previous use season appear to be available to the treated plant. DPR also
received preliminary information from a University of California at Riverside researcher who isinvestigating imidacloprid residues in eucalyptus nectar and pollen. The researcher’s preliminary
results indicate imidacloprid residues in eucalyptus nectar at levels of up to 550 parts per
billion (ppb).
Based upon data on file, DPR estimates the lethal concentration of imidacloprid needed to kill
50 percent of a test population (LC50) of honey bees is 185 ppb1. In their everyday foraging andpollination activities, honey bees collect both nectar and pollen from flowering plants. If theimidacloprid residue levels in a plant’s nectar and pollen are similar to those found in the leaves
and blossoms of the plants described in the adverse effects data, the levels are well above theestimated LC50 for honey bees. The levels found in some of the plants were more than twenty
times the estimated honey bee LC50 of 185 ppb.
All of the neonicotinoids share many of the same characteristics as imidacloprid. However, thethree other neonicotinoids included in this reevaluation, clothianidin, dinotefuran, andthiamethoxam, are in the same chemical family (nitroguanidines) as imidacloprid. These threeother active ingredients, in particular, have soil mobility characteristics and half-lives that arevery similar to imidacloprid. Based on available data, DPR scientists believe these active
ingredients would have the same potential residue concerns as imidacloprid. Data also indicatethat these active ingredients are similar to imidacloprid in toxicity to honey bees. Due to thechemical and toxicological similarities between imidacloprid and the other neonicotinoids, DPR
is providing those registrants with the option of generating data on their own chemicals orproviding/relying upon data generated using a surrogate nitroguanidine.
DPR exempted the following formulation categories and product types from the reevaluation:
1.
2.
3.
4.
5.
6.
7.

Formulated as a gel or impregnated in a strip;
Termiticide;
Flea control products combined with rodenticide;
Pet spot applications;
Ant and roach baits;
Premise application for control of nuisance pests; or,Manufacturing use only products.

1

The LC50 was estimated by converting the acute oral LD50 (the amount of a material that causes the death of
50 percent of a test population) to a concentration in nectar using the standard consumption model used in bee
feeding studies.

31Appendix 1. Reevaluation Letter Initiating the Reevaluation of Imidacloprid,
Clothianidin, Thiamethoxam, and Dinotefuran

California Notice 2009-02
Page 3
DPR exempted the above types of products from the reevaluation because the manner in which
the products are formulated or applied makes it unlikely that the neonicotinoid will move intoplants that bloom or be a source of forage for honey bees and pollinators.
DPR has not yet made a final decision as to the data it will require registrants to conduct
pursuant to this reevaluation. In general, DPR intends to require registrants to analyze residues
from the nectar and pollen of a representative number of crops grown in California in order tobetter understand the impact of neonicotinoids on honey bees. In addition, DPR plans to require
acute toxicity studies on various honey bee life stages.
DPR plans to work closely with the United States Environmental Protection Agency’s(U.S. EPA’s) Office of Pesticide Programs throughout the reevaluation process. U.S. EPA’s
registration review docket for imidacloprid<http://www.epa.gov/oppsrrd1/registration_review/imidacloprid/index.htm> opened in
December 17, 2008, and the docket for nithiazine is scheduled to be opened in March 2009. In
order to better ensure a “level playing field” for the neonicotinoid class as a whole, and to best
take advantage of new research as it becomes available, U.S. EPA has scheduled the docket
openings for the remaining neonicotinoids (acetamiprid, clothianidin, dinotefuran, thiacloprid,
and thiamethoxam) for fiscal year 2012.
For information regarding the reevaluation process, please contact either Ms. Denise Webster, by
e-mail at <dwebster@cdpr.ca.gov> or by telephone at (916) 324-3522, or Ms. Alveena Prasad,by e-mail at <aprasad@cdpr.ca.gov> or by telephone at (916) 324-3905.

Original signed by
Ann M. Prichard, Chief
Pesticide Registration Branch(916) 324-3931

February 27, 2009
Date

cc: Ms. Denise Webster, Program Specialist
Ms. Alveena Prasad, Environmental Scientist

32

Appendix 2. California Food and Agricultural Code (FAC) section (§) 12838

Assembly Bill No. 1789
CHAPTER 578
An act to add Section 12838 to the Food and Agricultural Code,relating to pesticides.
[Approved by Governor September 26, 2014. Filed with
Secretary of State September 26, 2014.]

Legislative counsel’s
digestAB 1789, Williams. Pesticides: neonicotinoids: reevaluation:
determination: control measures.
Existing law requires pesticides to be registered by the Department of
Pesticide Regulation. Existing law requires that a pesticide be thoroughly
evaluated prior to registration, and provides for the continued evaluationof registered pesticides.
This bill would require the department, by July 1, 2018, to issue adetermination with respect to its reevaluation of neonicotinoids. The billwould require the department, on or before 2 years after making thisdetermination, to adopt any control measures necessary to protect
pollinator health.
The bill would require the department to submit a report to theappropriate committees of the Legislature if the department is unable toadopt those control measures and to update the report annually until the
department adopts those control measures.
The people of the State of California do enact as follows:
SECTION 1. (a) The Legislature finds and declares all of the following:(1) Honey bees are vital to the pollination of many of California’s
crops, which are critical to our national food system and essential to theeconomy of the state.
(2) Annual colony losses from 2006 to 2011, inclusive, averaged
about 33 percent each year, which is more than double what is
considered sustainable according to the United States Department ofFood and Agriculture.
(3) Scientists now largely agree that a combination of factors is toblame for declining pollinator health, including lack of varied forage andnutrition, pathogens and pests such as the Varroa mite, and chronic andacute exposure to a variety of pesticides.
(4) Based on data submitted to the Department of Pesticide
Regulation showing a potential hazard to honey bees, thedepartment initiated a33

Appendix 2. California Food and Agricultural Code (FAC) section (§) 12838
Ch. 578—2—

reevaluation process for four neonicotinoid compounds in 2009:
imidacloprid, thiamethoxam, clothianidin, and dinotefuran.
(b) It is the intent of the Legislature to set a timeline for completion of
the reevaluation of neonicotinoid compounds to ensure that the Departmentof Pesticide Regulation completes a thorough, scientifically sound, andtimely analysis of the effects of neonicotinoids on pollinator health.
SEC. 2. Section 12838 is added to the Food and Agricultural Code, toread:
12838. (a) On or before July 1, 2018, the department shall issue adetermination with respect to its reevaluation of neonicotinoids.
(b) (1) Within two years after making the determination specified in
subdivision (a), the department shall adopt any control measures necessary
to protect pollinator health.
(2) If the department is unable to adopt necessary control measures within
two years as required in paragraph (1), the department shall submit a reportto the appropriate committees of the Legislature setting forth the reasons
the requirement of paragraph (1) has not been met.
(3) The department shall update the report submitted to the appropriate
committees of the Legislature pursuant to paragraph (2) every year until the
department adopts the necessary control measures specified in paragraph
(1).

34

Appendix 3. California Registered Agricultural Uses of Imidacloprid, Thiamethoxam, Clothianidin, andDinotefuran
The neonicotinoid informational use tables include crop groups that have been defined in Title 40 of the Code of Federal Regulations (40 CFR)
Part 180.41. In accordance with the risk determination, a single crop or a subset of a crop group could represent an entire crop group listed in thetables. Crop groups and use rates in the tables are representative of agricultural commodities that are currently registered for use in California.
40 CFR Part 180.41 does not categorize hops, globe artichoke, and peanuts into crop groups as these are seen as miscellaneous commodities.

Imidacloprid
Crop groups listed

Berry
and
Small
Fruit

Low Growing
Berry

Maximum single
application rate (soilor foliar)

Maximum annual orseasonal application
rate (soil or foliar)

Minimum
reapplicationinterval

0.047 lbs ai/A (foliar)

0.14 lbs ai/A (foliar)

5 days0.50 lbs ai/A (soil)

0.50 lbs ai/A (soil)

Restrictions•••

Bushberry

0.1 lbs ai/A (foliar)

0.5 lbs ai/A (foliar)

Caneberry

0.5 lbs ai/A (soil)
0.1 lbs ai/A (foliar)

0.5 lbs ai/A (foliar)
0.3 lbs ai/A (foliar)

7 days••Do not apply pre-bloom or during bloom
or when bees are foraging.

•

Apply with ground applicationequipment only.

7 days
0.5 lbs ai/A (soil)
Small fruit vine
climbing
subgroup except
fuzzy kiwifruit

0.05 lbs ai/A (foliar)

0.5 lbs ai/A (soil)
0.1 lbs ai/A (foliar)
14 days

0.5 lbs ai/A (soil)

0.5 lbs ai/A (soil)

35

When applied as a soil post-harvest
treatment, the maximum single
application rate the maximum annual
seasonal application rate is 0.38 lb ai/A.
Do not use both soil application methods
on the same crop in the same season.
Do not apply during bloom or within 10
days prior to bloom or when bees areforaging.
Do not apply pre-bloom or during bloom
or when bees are foraging.

Appendix 3. California Registered Agricultural Uses of Imidacloprid, Thiamethoxam, Clothianidin, andDinotefuran
Imidacloprid
Crop groups listed

Maximum single
application rate (soilor foliar)

Maximum annual orseasonal application
rate (soil or foliar)

Minimum
reapplicationinterval

Citrus Fruit

0.25 lbs ai/A (foliar)

0.50 lbs ai/A (foliar)

10 days•0.50 lbs ai/A (soil)

0.50 lbs ai/A (soil)

Do not apply during bloom or within 10
days prior to bloom or when bees areforaging.

0.0013 lbs ai/ft3 (soil;
containerized)

0.0037 lbs ai/plant(soil; containerized)

0.063 lbs ai/A (foliar)

0.31 lbs ai/A (foliar)

7 days•0.33 lbs ai/A (soil)

0.33 lbs ai/A (soil)

Regardless of formulation or method of
application, apply no more than 0.5 lb.
active ingredient per acre per year,
including seed treatment, soil, and foliar
uses.
Do not graze treated fields after any
application imidacloprid

Oilseed

0.5 lbs ai/100 lb seed
(seed treatment;
cotton)Restrictions•

1 lbs ai/100 lbs seed
(seed treatment;
canola, rapeseed,mustard seed, flax,
crambe, borage)
0.5 mg ai/seed (seedtreatment; safflower,
sunflower)
Cucurbit Vegetable

0.38 lbs ai/A (soil)

0.38 lbs ai/A (soil)

36•

Not for use on crops grown for seed
unless allowed by state-specific 24(c)labeling.

Appendix 3. California Registered Agricultural Uses of Imidacloprid, Thiamethoxam, Clothianidin, andDinotefuran
Imidacloprid
Crop groups listed

Maximum single
application rate (soilor foliar)

Maximum annual orseasonal application
rate (soil or foliar)

Minimum
reapplicationinterval

Fruiting Vegetable

0.075 lbs ai/A (foliar)

0.24 lbs ai/A (foliar)

5 days•0.38 lbs ai/A (soil)

0.38 lbs ai/A (soil)

Not for use on crops grown for seed
unless allowed by state-specific 24(c)labeling.

0.5 lbs ai/A (soil;
okra and peppers)

0.5 lbs ai/A (soil; okra
and peppers)

0.1 lbs ai/A (foliar)

0.5 lbs ai/A (foliar)

10 days•

Do not apply pre-bloom or during bloom
or when bees are foraging.

0.25 lbs ai/A (foliar;
pear)

0.5 lbs ai/A (foliar;
pear)

0.38 lbs ai/A (soil)

0.38 lbs ai/A (soil)

0.1 lbs ai/A (foliar)

0.5 lbs ai/A (foliar)

10 days•

0.1 lbs ai/A (foliar;
apricot, nectarine, andpeach)

0.3 lbs ai/A (foliar;
apricot, nectarine, andpeach)

7 days (apricot,
nectarine, andpeach)•The maximum annual foliar rate
allowed per year for apricot, nectarine,
and peach: 0.3 lb ai/A
Do not apply pre-bloom or during bloom
or when bees are foraging

0.38 lbs ai/A (soil)

0.38 lbs ai/A (soil)

Pome Fruit

Stone Fruit

37

Restrictions

Appendix 3. California Registered Agricultural Uses of Imidacloprid, Thiamethoxam, Clothianidin, andDinotefuran
Imidacloprid
Crop groups listed

Maximum single
application rate (soilor foliar)

Cereal Grains

0.53 lbs ai/100 lbs
seed (seed treatment;
field corn)

Maximum annual orseasonal application
rate (soil or foliar)

Minimum
reapplicationinterval

Restrictions••

0.94 lbs ai/100 lbs
seed (seed treatment;
wheat, barley, oats,rye, triticale)•

0.25 lbs ai/100 lbs
seed (seed treatment;sorghum, millet)
0.2 lbs ai/100,000 ofpelleted seed
(seed treatment;
sugar beet)
0.094 lbs ai/100,000
of raw seed (seedtreatment; sugar beet)
0.25 lbs ai/100 lbs
seed (seed treatment;
popcorn)0.25 lbs ai/100 lbs
seed (seed treatment;
sweet corn)38

Corn: Do not graze or feed livestock ontreated areas for 45 days after planting.
Wheat, barley, oats, rye, triticale,sorghum, and millet: Do not graze or
feed livestock on treated areas for 45days after planting.
The maximum application rate forimidacloprid (including seed treatments,
foliar applications, and soil applications)is 0.5 pound active ingredient per acre
per calendar year.

Appendix 3. California Registered Agricultural Uses of Imidacloprid, Thiamethoxam, Clothianidin, andDinotefuran
Imidacloprid
Crop groups listed

Maximum single
application rate (soilor foliar)

Maximum annual orseasonal application
rate (soil or foliar)

Minimum
reapplicationinterval

Tree Nut Group

0.1 lbs ai/A (foliar)

0.36 lbs ai/A (foliar)

6 days0.50 lbs ai/A (soil)

0.50 lbs ai/A (soil)

0.047 lbs ai/A (foliar)

0.24 lbs ai/A (foliar)

0.38 lbs ai/A (soil)

0.38 lbs ai/A (soli)

0.1 lbs/A (foliar)

Brassica (Cole) Leafy
Vegetable
Tropical and SubtropicalFruit, Inedible Peel Group

Root and Tuber Vegetables

Restrictions•

Do not apply to almonds•

Do not apply pre-bloom or during
bloom or when bees are foraging.

5 days•Not for use on crops grown for seed
unless allowed by state-specific 24(c)labeling.

0.5 lbs ai/A (foliar)

7 days•Do not apply pre-bloom or during bloom
or when bees are foraging.

0.1lbs ai/A (foliar;
pomegranate)

0.3 lbs ai/A (foliar;
pomegranate)

14 days(banana andplantain)

0.5 lbs ai/A (soil)
0.047 lbs ai/A (foliar;
potato)0.5 lbs ai/A (soil)
0.2 lbs ai/A (foliar;
potato)•0.044 lbs ai/A (foliar)

0.13 lbs ai/A (foliar)•

0.044 lbs ai/A (foliar;
radish)0.044 lbs ai/A (foliar;
radish)•0.31 lbs ai/A (soil;
potato)0.31 lbs ai/A (soil;
potato)Not for use on crops grown for seed
unless allowed by state-specific 24(c)labeling.
Side-dress no more than 0.3 fl oz/1000
row feet no later than 45 days after
planting.
Sugar beet: No not apply immediately
prior to bud opening or during bloom orwhen bees are foraging.

0.38 lbs ai/A (soil)
0.18 lbs ai/A (soil;
sugar beet)

0.38 lbs ai/A (soil)
0.18 lbs ai/A (soil;
sugar beet)

7 days (potato)
5 days39

Appendix 3. California Registered Agricultural Uses of Imidacloprid, Thiamethoxam, Clothianidin, andDinotefuran
Imidacloprid
Crop groups listed

Maximum single
application rate (soilor foliar)

Root and Tuber
Vegetables, continued

0.26 lbs ai/A (seedtreatment; potato)

Leafy Vegetable(Except Brassica
Vegetable)
Legume Vegetables(Succulent or Dried)

Maximum annual orseasonal application
rate (soil or foliar)

Minimum
reapplicationinterval

0.25 lbs ai/100 lbs
seed (seed treatment;
carrot)0.047 lbs ai/A (foliar)

0.24 lbs ai/A (foliar)

5 days•0.38 lbs ai/A (soil)
0.044 lbs ai/A (foliar)

0.38 lbs ai/A (soil)
0.13 lbs ai/A (foliar)

7 days

•0.047 lb ai/A (foliar;
soybean)

0.14 lbs ai/A (foliar;
soybean)

0.38 lbs ai/A (soil)

0.38 lbs ai/A (soil)••

0.125 lbs ai/100 lbs
seed (seed treatment;soybean)
Herbs and Spices

Bulb Vegetables

Restrictions

0.044 lbs ai/A (foliar)

0.13 lbs ai/A (foliar)

0.38 lbs ai/A (soil)

0.38 lbs ai/A (soil)

0.5 lbs ai/A (soil)

0.5 lbs ai/A (soil)

40

Not for use on crops grown for seed
unless allowed by state-specific 24(c)labeling.
Not for use on crops grown for seed
unless allowed by state-specific 24(c)labeling.
Foliar and soil application on Soybean
not permitted in California unlessotherwise directed by state specific 24(c)labeling.
Soybean: Do not graze or feed livestock
on soybean forage or hay.

5 days•Not for use on crops grown for seed
unless by state-specific 24(c) labeling.

Appendix 3. California Registered Agricultural Uses of Imidacloprid, Thiamethoxam, Clothianidin, andDinotefuran
Imidacloprid
Crop groups listed

Maximum single
application rate (soilor foliar)

Maximum annual orseasonal application
rate (soil or foliar)

Minimum
reapplicationinterval

Tobacco0.05 lbs ai/A (foliar)

0.28 lbs ai/A (foliar)

7 days

0.5 lbs ai/A (soil)

Coffee0.016 lbs ai/1,000
plants (soil)
0.1 lbs ai/A (foliar)

0.5 lbs ai/A (foliar)

7 daysHops

0.5 lbs ai/A (soil)
0.1 lbs ai/A (foliar)

0.5 lbs ai/A (soil)
0.3 lbs ai/A (foliar)

21 days

Globe Artichoke

0.3 lbs ai/A (soil)
0.125 lbs ai/A (foliar)

0.3 lbs ai/A (soil)
0.5 lbs ai/A (foliar)

14 days

41

Restrictions•

Do not apply pre-bloom or during bloom
or when bees are foraging.

Appendix 3. California Registered Agricultural Uses of Imidacloprid, Thiamethoxam, Clothianidin, andDinotefuran
Thiamethoxam
Crop groups listed

Cucurbit Vegetables

Citrus Fruit

Maximum single
application rate (soilor foliar)

Maximum annual orseasonal application
rate (soil or foliar)

Minimum
reapplicationinterval

0.086 lbs ai/A (foliar)
0.172lbs ai/A (soil)
0.75 mg ai/seed, Do
not exceed 0.164 lbs
ai/A (seed treatment)
0.086 lbs ai/A (foliar)

0.172 lbs ai/A (foliar)
0.172lbs ai/A (soil)

5 days••0.172 lbs ai/A (foliar)

7 days0.172lbs ai/A (soil)

0.172lbs ai/A (soil)• Thiamethoxam is highly toxic to bees
exposed to direct treatment on blooming
crops. Do not apply during pre-bloom or
during bloom when bees are actively
foraging. Do not apply thiamethoxam or
allow it to drift to blooming crops or
weeds if bees are foraging in for adjacentto the treatment area. This is especiallycritical if there are adjacent orchards thatare blooming. After a thiamethoxam
application, wait at least 5 days before
placing beehives in the treated field. Ifbees are foraging in the ground cover andit contains any blooming plants or weeds,
always remove flowers before making anapplication. This may be accomplished bymowing, disking, mulching, flailing, orapplying a labeled herbicide.

42

Restrictions

Refer to Pollinator Precautions section.
Refer to Resistance Management
section.

Appendix 3. California Registered Agricultural Uses of Imidacloprid, Thiamethoxam, Clothianidin, andDinotefuran
Thiamethoxam
Crop groups listedOilseedMaximum single
application rate (soilor foliar)

Maximum annual orseasonal application
rate (soil or foliar)

Minimum
reapplicationinterval

0.063 lbs ai/A (foliar)

0.125 lbs ai/A (foliar)

5 days

•0.375 mg ai/seed(Seed treatment;
Cotton)0.075 lbs ai/A (seedtreatment; cotton)•

Treated sunflower seed must be planted
at a minimum depth of one inch.

0.14 lbs ai/A (seedtreatment; safflower)

Do not apply aneonicotinoid
insecticide
within 45 daysof planting
seed treatedcotton seeds

To protect the Preble's Meadow Jumping
Mouse, sunflower seed treated with
Cruiser 5FS Alfalfa may not be plantedin Elbert or Weld Counties in Colorado.

0.172 lbs ai/A (foliar)

7 days•Refer to Pollinator Precautions section.

•

Refer to Resistance Management
section.

•Refer to Pollinator Precautions section.

•

Refer to Resistance Management
section.

0.25 mg ai/seed (seedtreatment; Sunflower)
0.039 lbs ai/100 lbs
seed (seed treatment;
safflower)
Stone Fruit

Berry and
Small Fruit

Small fruitvine
climbing
subgroup
exceptfuzzy
kiwifruit

0.086 lbs ai/A (foliar)

0.14 lbs ai/A (seedtreatment; sunflower)

0.055 lbs ai/A (foliar)

0.109 lbs ai/A (foliar)

0.266 lbs ai/A (soil)

0.266 lbs ai/A (soil)

43

14 days

Restrictions

Appendix 3. California Registered Agricultural Uses of Imidacloprid, Thiamethoxam, Clothianidin, andDinotefuran
Thiamethoxam
Crop groups listed

Maximum single
application rate (soilor foliar)

Maximum annual orseasonal application
rate (soil or foliar)

Minimum
reapplicationinterval

Berry and
Small Fruit,continued

Low
growing
berrysubgroup

0.063 lbs ai/A (foliar)

0.188 lbs ai/A (foliar)

10 days0.188 lbs ai/A (soil)

0.188 lbs ai/A (soil)

Bushberry

0.063 lbs ai/A (foliar)

0.188 lbs ai/A (foliar)

0.188 lbs ai/A (soil)

0.188 lbs ai/A (soil)

Fruiting Vegetables

0.086 lbs ai/A (foliar)

0.172 lbs ai/A (foliar)

0.172lbs ai/A (soil)

0.172 lbs ai/A (soil)

44

7 days

5 daysRestrictions•

Do not apply by air•

Refer to Pollinator Precautions section.

•

Refer to Resistance Management
section.

•Apply after bud-break, but prior to thebeginning of bloom (first open blooms)•

Refer to Pollinator Precautions section.

•

Refer to Resistance Management
section.

•Refer to Pollinator Precautions section.

•

Refer to Resistance Management
section.

Appendix 3. California Registered Agricultural Uses of Imidacloprid, Thiamethoxam, Clothianidin, andDinotefuran
Thiamethoxam
Crop groups listed

Legume Vegetables(Succulent or Dried)

Maximum single
application rate (soilor foliar)

Maximum annual orseasonal application
rate (soil or foliar)

Minimum
reapplicationinterval

0.031 lbs ai/A (foliar)

0.125 lbs ai/A (foliar)

7 days•Refer to Pollinator Precautionary section

Do not apply aneonicotinoid
insecticide
within 45 daysof planting
seed treatedwith Cruiser
5FS.

•

Refer to Resistance Management section

7 days•Refer to Pollinator Precautions section.

•

Refer to Resistance Management
section.

•Refer to Pollinator Precautions section.

•

Refer to Resistance Management
section.

0.05 lbs ai/100 lbs
seed, Do not exceed
0.075 lbs ai/A (seedtreatment)
0.05 lbs ai/100 lbs
seed, Do not exceed
0.083 lbs ai/A (seedtreatment; soybean)
Leafy Vegetables (Except
Brassica Vegetables)

0.086 lbs ai/A (foliar)

0.172 lbs ai/A (foliar)

0.172lbs ai/A (soil)

0.172lbs ai/A (soil)

Bulb Vegetables

0.266 lbs ai/A (seedtreatment)

Brassica (Cole) Leafy
Vegetables

0.086 lbs ai/A (foliar)

0.172 lbs ai/A (foliar)

0.172 lbs ai/A (soil)

0.172 lbs ai/A (soil)

45

7 daysRestrictions

Appendix 3. California Registered Agricultural Uses of Imidacloprid, Thiamethoxam, Clothianidin, andDinotefuran
Thiamethoxam
Crop groups listed

Maximum single
application rate (soilor foliar)

Maximum annual orseasonal application
rate (soil or foliar)

Minimum
reapplicationinterval

Root and
Tuber
Vegetables

0.047 lbs ai/A (foliar)

0.094 lbs ai/A (foliar)

7 days0.125 lbs ai/A (soil)

0.125 lbs ai/A (soil)

Tuberous
and Corm

0.125 lbs ai/A (seedtreatment)

Root
Vegetables

0.063 lbs ai/A (foliar)

0.125 lbs ai/A (foliar)

0.188 lbs ai/A (soil)

0.188 lbs ai/A (soil)

0.63 lbs ai/A (foliar;
radish)0.063 lbs ai/A (foliar;
radish)0.102 lbs ai/A (soil;
radish)0.102 lbs ai/A (soil;
radish)70 gram ai/100,000
seeds; Do not exceed
0.206 lbs ai/A (seedtreatment; Sugar
Beets)

46

7 daysRestrictions•

Refer to Pollinator Precautions section.

•

Refer to Resistance Management
section.

•

Do not use this thiamethoxam on potato
seed in Nassau or Suffolk County, NewYork.

Appendix 3. California Registered Agricultural Uses of Imidacloprid, Thiamethoxam, Clothianidin, andDinotefuran
Thiamethoxam
Crop groups listed

Maximum single
application rate (soilor foliar)

Maximum annual orseasonal application
rate (soil or foliar)

Minimum
reapplicationinterval

Pome Fruit

0.086 lbs ai/A (foliar)

0.258 lbs ai/A (foliar)

10 days

Tropical and SubtropicalFruit, Inedible Peel Group
Globe Artichoke

Peanuts

0.063 lbs ai/A (foliar)

0.047 lbs ai/A (foliar)

0.188 lbs ai/A (foliar)

0.094 lbs ai/A (foliar)

0.29 mg ai/seed; Do
not exceed 0.08 lbs
ai/A (seed treatment)

47

7 days7 daysRestrictions•

Refer to Pollinator Precautionary Section•

Refer to resistance management section•

Refer to Pollinator Precautionary Section•

Refer to resistance management section•

Refer to Pollinator Precautions section.

•

Refer to Resistance Management
section.

•

Do not use a thiamethoxam rate that will
result in more than 0.08 lbs ai/A (35.0
grams ai/A) per season, based on a
maximum seeding rate of 120,700
seeds/acre.

•

Do not use in hopper box, planter box,
slurry box, or other farmer applied
applications. Apply thiamethoxam seedtreatment in commercial seed treatment
facilities only.

Appendix 3. California Registered Agricultural Uses of Imidacloprid, Thiamethoxam, Clothianidin, andDinotefuran
Thiamethoxam
Crop groups listed

Cereal Grains

Maximum single
application rate (soilor foliar)

Maximum annual orseasonal application
rate (soil or foliar)

Minimum
reapplicationinterval

0.0625 lbs ai/A
(foliar; barley)

0.125 lbs ai/A (foliar;
barely)7 days

0.052 lbs ai/100 lbs
seeds, Do not exceed
0.52 lbs ai/A (seedtreatment; barley)
0.80 mg ai/kernel, Do
not exceed 0.165 lbs
ai/A (seed treatment;
corn)
0.03 mg ai/seed, Do
not exceed 0.17 lb
ai/A (seed treatment;
rice)0.093 mg ai/seed, Do
not exceed 0.03 lbs
ai/A (seed treatment;sorghum)
0.052 lbs ai/100 lbs
seeds, Do not exceed

48

Restrictions•

Refer to Pollinator Precautionary Section•

Refer to resistance management section•

For field, pop, seed and sweet corn, do
not use a cruiser rate that will result in
more than 0.21 lb ai/A based on a
maximum seeding rate for sweet corn of
75,000 seeds/acre.

•

Do not apply more than 215 gallons per
8 hour day for seed treatments utilizing aclosed system.

•

Do not apply more than 38 gallons ofthiamethoxam per 8 hour day for seedtreatments utilizing an open system. If it
is necessary to apply more than 28
gallons of cruiser per 8 hour day, aclosed system must be used•

A closed system must be used forcommercial treatment of sorghum seed

Appendix 3. California Registered Agricultural Uses of Imidacloprid, Thiamethoxam, Clothianidin, andDinotefuran
Thiamethoxam
Crop groups listed

Cereal Grains, continued

Maximum single
application rate (soilor foliar)

Maximum annual orseasonal application
rate (soil or foliar)

Minimum
reapplicationinterval

0.08 lbs ai/A (seedtreatment; wheat)

Restrictions•

Not for use in water seeded riceproduction. Do not plant or sow
thiamethoxam treated rice seed by aerial
application equipment. Do not use
treated fields for the aquaculture ofedible fish and crustacean.

0.052 lbs ai/100 lbs
seeds, Do not exceed
0.04 lbs ai/A (seedtreatment; buckwheat,
pearl millet, proso
millet, oats, rye,tesinte, triticale, andwild rice)
Hops

0.125 lbs ai/A (soil)

0.125 lbs ai/A (soil)

Tobacco0.047 lbs ai/A (foliar)

0.047 lbs ai/A (foliar)•

Refer to Pollinator Precautionary Section

0.43 oz/1,000 plants(soil)

0.125 lbs ai/A (soil)•

Refer to resistance management section

0.063 lbs ai/A (foliar)

0.188 lbs ai/A (foliar)•

Refer to Pollinator Precautionary Section•

Refer to resistance management section

Mint

49

14 daysAppendix 3. California Registered Agricultural Uses of Imidacloprid, Thiamethoxam, Clothianidin, andDinotefuran
Clothianidin
Crop groups listed

Maximum single
application rate (soilor foliar)

Maximum annual orseasonal application
rate (soil or foliar)

Minimum
reapplicationinterval

Restrictions

Tree Nuts

0.1 lbs ai/A (foliar)

0.2 lbs ai/A (foliar)

Do not apply
treatments lessthan 10 days
apart

•

Insecticide must not be applied during
bloom or when bees are foraging.

•

Do not feed or allow livestock to graze
on cover crops from treated orchards.

•

Regardless of the application method, do
not apply more than 0.2 lb active
ingredient clothianidin per acre per year.

•

Do not apply treatment between 50%
row closure and petal fall.

•

Do not make more than one application
per year prior to 50% row closure.

•

Regardless of the application method, do
not apply more than 0.2 lb active
ingredient clothianidin per acre per year.

•

Do not apply by air except for potato.

•

Regardless of application method (seedtreatment, soil, or foliar), do not apply
more than 0.2 lb active ingredient
clothianidin per acre per year.

Root and Tuber
Vegetables

Cereal Grains

0.05 lbs ai/A (foliar)

0.2 lbs ai/A (foliar)

0.2 lbs ai/A (soil)

0.2 lbs ai/A (soil)

0.075 lbs ai/A (foliar;
rice)

50

Do not apply
treatments lessthan 7 daysapartAppendix 3. California Registered Agricultural Uses of Imidacloprid, Thiamethoxam, Clothianidin, andDinotefuran
Clothianidin
Crop groups listed

Maximum single
application rate (soilor foliar)

Cereal Grains, continued

0.023 mg ai/seed (seedtreatment; except
corn)

Maximum annual orseasonal application
rate (soil or foliar)

1.25 mg ai/seed (seedtreatment; corn)

51

Minimum
reapplicationinterval

Restrictions•

For use only in commercial seedtreatment facilities. Not for use in hopper
box, planter box, slurry box, or other onfarm seed treatment applications except
for cereal grains and potato seed pieces•

Regardless of application method (seedtreatment, soil, or foliar), do not apply
more than 0.2 lb active ingredient
clothianidin per acre per year.

•

Rice: Do not apply Insecticide after third
tillering has initiated.

•

Rice: Do not apply Insecticide following
a clothianidin seed treatment application.

•

Rice: Do not use Insecticide treated rice
fields for the aquaculture of edible fish
and crustaceans.

•

Rice: Insecticide is not to be used on rice
crops that contain or support crawfish or
any form of aquaculture operation.

•

Rice: Insecticide is not to be used on rice
crops near fish farm, shrimp, prawn or
crab pond (or nursery) operations -Appendix 3. California Registered Agricultural Uses of Imidacloprid, Thiamethoxam, Clothianidin, andDinotefuran
Clothianidin
Crop groups listed

Maximum single
application rate (soilor foliar)

Maximum annual orseasonal application
rate (soil or foliar)

Minimum
reapplicationinterval

Restrictions

Cereal Grains, continued

Legume Vegetables(Succulent or Dried)particularly when weather conditions are
conducive to drift. Exercise caution with
air and ground applications near those
operations to avoid product drift.
0.1 lbs ai/A (foliar)

0.2 lbs ai/A (foliar)

0.13 mg ai/seed (seedtreatment)

Oilseed

0.083 lbs ai/A (foliar)

0.02 lbs ai/A (foliar)

0.018 mg ai/seed (seedtreatment; canola,rapeseed)52

Do not apply
foliartreatments lessthan 7 daysapart

One year•

Do not make foliar applications ofclothianidin in fields treated with aneonicotinoid insecticide seedtreatment(s) within 45 days after
planting.

•

Regardless of formulation or type of
application method, do not apply morethan 0.2 lb ai of clothianidin per acre per
year.

•

Do not graze or feed soybean forage andhay to livestock.

•

Do not make application after pinheadsquare formation.

Appendix 3. California Registered Agricultural Uses of Imidacloprid, Thiamethoxam, Clothianidin, andDinotefuran
Clothianidin
Crop groups listed

Cucurbit Vegetables

Brassica (Cole) Leafy
Vegetables

Leafy Vegetables (Except
Brassica Vegetables)

Tropical and SubtropicalFruit, Inedible Peel

Maximum single
application rate (soilor foliar)

Maximum annual orseasonal application
rate (soil or foliar)

Minimum
reapplicationinterval

Restrictions

0.067 lbs ai/A (foliar)

0.2 lbs ai/A (foliar)•

0.2 lbs ai/A (soil)

0.2 lbs ai/A (soil)

Do not apply
treatments lessthan 10 days
apart

Insecticide must not be applied during
bloom or when bees are foraging.

•

Do not make application after 4th trueleaf on main stem is unfolded•

Insecticide must not be applied during
bloom or when bees are foraging.
Do not use on crops grown for seedproduction•

Do not use on crops grown for seedproduction.

•

Insecticide must not be applied during
bloom or when bees are foraging.

•

Do not feed or allow livestock to graze
on cover crops from treated orchards.

•

Insecticide must not be applied during
bloom or when bees are foraging.

•

Post bloom applications only

0.067 lbs ai/A (foliar)

0.2 lbs ai/A (foliar)

0.2 lbs ai/A (soil)

0.2 lbs ai/A (soil)

Do not apply
treatments lessthan 10 days
apart

0.067 lbs ai/A (foliar)

0.2 lbs ai/A (foliar)

10 days

0.2 lbs ai/A (soil)

0.2 lbs ai/A (soil)

0.1 lb ai/A (foliar;
pomegranate)

0.2 lbs ai/A (foliar;
pomegranate)

53

Do not apply
treatments lessthan 14 days
apart

•

Appendix 3. California Registered Agricultural Uses of Imidacloprid, Thiamethoxam, Clothianidin, andDinotefuran
Clothianidin
Crop groups listed

Maximum single
application rate (soilor foliar)

Maximum annual orseasonal application
rate (soil or foliar)

Minimum
reapplicationinterval

Berry and
Small Fruit

Small fruitvine
climbing
subgroup
exceptfuzzy
kiwifruit

0.1 lbs ai/A (foliar)

0.2 lbs ai/A (foliar)

One year for
foliar

0.2 lbs ai/A (soil)

0.2 lbs ai/A (soil)

Tropical and SubtropicalFruit, Edible Peel Group

0.1 lbs ai/A (foliar;
fig)

0.2 lbs ai/A (foliar;
fig)

Stone Fruit

0.1 lbs ai/A (foliar;
peach)

0.2 lbs ai/A (foliar;
peach)

TobaccoRestrictions

For soil: do not
apply
treatments lessthan 14 days
apart

0.067 lbs ai/A (foliar)

0.2 lbs ai/A (foliar)

54

Do not apply
treatments
less than 14days apart.
Do not apply
treatments lessthan 10 days
apart.
Do not apply
treatments lessthan 7 days
apart.

•Do not feed or allow livestock to graze
on cover crops from treated orchards.

•

Do not feed or allow livestock to graze
on cover crops from treated orchards.

•

Insecticide must not be applied during
bloom or when bees are foraging.

Appendix 3. California Registered Agricultural Uses of Imidacloprid, Thiamethoxam, Clothianidin, andDinotefuran
Clothianidin
Crop groups listed

Maximum single
application rate (soilor foliar)

Maximum annual orseasonal application
rate (soil or foliar)

Pome Fruit

0.1 lbs ai/A (foliar)

0.2 lbs ai/A (foliar)

Minimum
reapplicationinterval

Restrictions•

Do not feed or allow livestock to graze
on cover crops from treated orchards.

•

Insecticide must not be applied during
bloom or when bees are foraging.

Dinotefuran
Crop groups listed

Maximum single
application rate (soilor foliar)

Maximum annual orseasonal application
rate (soil or foliar)

Minimum
reapplicationinterval

Oilseed0.134 lbs ai/A (foliar)

0.268 lbs ai/A (foliar)

7 days•Follow application instructions as
indicated in the Bee Hazard Direction
for Use.

Cucurbit Vegetable

0.179 lbs ai/A (foliar)

0.268 lbs ai/A (foliar)

7 days•0.33 lbs ai/A (soil)

0.536 lbs ai/A (soil)

Follow application instructions as
indicated in Bee Hazard Direction forUse.

•

Do not combine foliar applications withsoil applications, or vice versa. Only use
one application method.

55

Restrictions

Appendix 3. California Registered Agricultural Uses of Imidacloprid, Thiamethoxam, Clothianidin, andDinotefuran
Dinotefuran
Crop groups listed

Fruiting Vegetable

Root and Tuber
Vegetables

Brassica Head & Stem
Vegetables

Maximum single
application rate (soilor foliar)

Maximum annual orseasonal application
rate (soil or foliar)

Minimum
reapplicationinterval

0.179 lbs ai/A (foliar)

0.268 lbs ai/A (foliar)

7 days0.33 lbs ai/A (soil)

0.536 lbs ai/A (soil)

0.068 lbs ai/A (foliar)

0.203 lbs ai/A (foliar)

0.338 lbs ai/A (soil)

0.338 lbs ai/A (soil)

0.179 lbs ai/A (foliar)

0.268 lbs ai/A (foliar)

0.33 lbs ai/A (soil)

0.536 lbs ai/A (soil)

56

14 days

7 daysRestrictions•

Follow application instructions as
indicated in Bee Hazard Direction forUse.

•

Do not combine foliar applications withsoil applications, or vice versa. Only use
one application method.

•

Do not apply to vegetables grown for
seed.

•

Follow application instructions as
indicated in Bee Hazard Direction forUse.

•

Do not combine foliar applications withsoil applications, or vice versa. Only use
one application method.

•

Do not combine foliar applications withsoil applications, or vice versa. Only use
one application method.

•

Do not apply to vegetables grown forseed.

Appendix 3. California Registered Agricultural Uses of Imidacloprid, Thiamethoxam, Clothianidin, andDinotefuran
Dinotefuran
Crop groups listed

Maximum single
application rate (soilor foliar)

Maximum annual orseasonal application
rate (soil or foliar)

Minimum
reapplicationinterval

Leafy Vegetables (Except
Brassica Vegetables)

0.134 lbs ai/A (foliar)

0.268 lbs ai/A (foliar)

7 days0.180 lbs ai/A (foliar;
watercress)

0.360 lbs ai/A (foliar;
watercress)

0.33 lbs ai/A (soil)

0.536 lbs ai/A (soil)

0.180 lbs ai/A (foliar)

0.270 lbs ai/A (foliar)

0.270 lbs ai/A (soil)

0.270 lbs ai/A (soil)

0.135 lbs ai/A (foliar)

0.270 lbs ai/A (foliar)

0.338 lb ai/A (soil)

0.338 lb ai/A (soil)

Bulb Vegetables

Berry
and
Small
Fruit

Small fruit vine
climbing
subgroup except
fuzzy kiwifruit

Low Growing
Berry Subgroup,
exceptstrawberry

0.180 lbs ai/A (foliar)

0.360 lbs ai/A (foliar)

57

Restrictions•

Do not combine foliar applications withsoil applications, or vice versa. Only use
one application method.

•

Do not apply to vegetables grown forseed.

7 days•Regardless of application method, do not
exceed 0.383 lbs ai/A per crop season.

14 days•

Follow application instructions as
indicated in Bee Hazard Direction for
Use.

•

Regardless of application method, do not
apply more than a total of 0.540 lbs ai/A
per season of Dinoteufran 20 SG.

14 daysAppendix 4. 40 CFR 180.41 Crop Group Tables§ 180.41

40 CFR Ch. I (7-H 7 Edition)

the pre-existing crop group; however,
the revised crop group number will befollowed by a hyphen and the final two
digits of the year in which it was estab­lished (e.g., if Crop Group 1 is amended
in 2007, the· revised group will be des­ignated as Crop Group 1-07). If the pre­existing crop group had crop· sub­groups, these subgroups vti.11 be num­
bered in a similar fashion in the re­vised crop group. The name of the re­vised erop group will not be changed
from the pre-existing crop group unlessthe revision so changes the composi­tion of the crop group that the pre-ex­
lsUng name is no longer accurate, Oncea revised crop group is established,
EPA will no longer establisll tolerances
under the pre-existing crop group. At
appropriate times, EPA wtll amend tol­eranees for crop groups that have beensuperseded by revised crop groups toconform the pre-existing crop group to
the revised crop group, Once all of thetolerances for the pre-existing cropgroup have been updated, tlle pre-exist­
ing crop group will be removed fromthe OFR.
(k) Establishment of a tolerance does
not substitute for the additional needto register the pesticide under a com­panion law, the Federal Insecticide,
Fungicide, and Rodenticide Act. The
Registration Division of the Office ofPesticide Programs should be con-tacted concerning procedures for reg­istration of new uses of a pesticide.
[60 FR 26635, May 17, 1995, as amended at 70
FR 33363, June 8, 2006; 72 FR 69155, Dec. 7.
2007; 75 FR 56014, Sept. 15, 2010; Bl FR 26476,
May 3, 2016) ,§ 180.41 Crop group tables.
(a,) The tables in this section are to
be used in conjunction with §180,40 toestablish crop gToup tolerances,(b) Commodities not listed are riot
considered as inclnded in the groups forthe purposes of paragraph (b), and indi­vidual tolerances must be established,
Miscellaneous
comrhodities
inten­
tionally not included in any group i.n­clude globe artichoke, hops, peanut,
and water chestnut,(e) Each group is identified by agroup name and consists of. a list ofrepresentative commodities followed
by a list of all commodity members forthe group. If the group includes sub­groups·, each subgroup lists the sub­group name, the representative com­modity or commodities, and the mem­ber commodities for the subgroup. Sub­
groups, which are a subset of their as­sociated crop group, are established forsome but not all crops groups.
(1) Crop Group .1: Root and Tuber
Vegetables Group.
(i) Representative commodities. Carrot,
potato, radish, and sugai.' beet.
(ii) Table. The following table 1 lists
all the commodities included in Crop
Group 1 and identifies the related crop
subgroups.

TABLE 1-CROP GROUP 1: ROOT AND TUBER VEGETABLES
Related crop
subgroups

Commodities
Arracacha (Arracacfa xa.nthorrlllza)
Arrowroot (Maranta a.rundinacea)
Artichoke, Chinese (Stachys affinis) ...
Artichoke, Jerusalem (Heliaothus tuberosus)
Beet, garden (Beta vulgaris) .
Beet, sugar (Bera vu/garis)
Burdock, edible {Arctium lappa) .
Canna, edible (Queensland arrowroot) {Canna indica) ...
Carrot (Daucus ca.rota) .............. ".. .... ..........
Cassava1 bitter and swoot (Manihot a.w;ulentt.) ..
Celerlac (celery root) (Apium graveolens var. rapac1;,um) .
Chay<Jte (root} (Sechium edule) ..................
Chervil, turnip-rooted (Cha.erophyl/um bu/bosum).
Chicory (Cichonum intybus) ................
Chuta ( Cypen;s esculentus)
Dasheen (laro) {Colocasia escu/enta) ..
Ginger (Zingiber officinale)
Ginseng (Panax quinquefofius)
Horseradish (Armoracia rustioana) ..
Leren ( Calathea a!louia)

1C, 101C, 101C, 1D
1C, 101A, 181A
1A, 181C, 1D
1A, 181C, 1D
1A, 1B
1C, 1D
1A, 18
1A, 1B
1C, 1D,c, 1D
1C, 1D
1A, 18
1A, 18,c, 1D

490

58

Appendix 4. 40 CFR 180.41 Crop Group Tables

Environmental Protection Agency§180.41

TABLE 1-CROP GROUP 1: ROOT AND TUBER VEGETABLES-Continued
Related crop
subgroups

Commodities
Parsley, turnip-rooted (Petrosefinum crispum var. tuberosum) .....................................................
. ..
Parsnip {Pastinaca sativa)
Potato (So/anum tuberosum) . .
Radish (Raphanus sativus) ....
............. ......... .. .....
Radish, oriental (daikon) (Aaphanus sativus subvar. /ongipinnatus)
Rutabaga (Brassica campestris var. napobrasslca) .
Salsify (oyster plant) (Tragopogon porrifofius).
Salsify, black {Scorzonera hispanica)
Salsify, Spanish (Scolymus hispanicus) ...... .
Skirret (Sium sisarum) .
.
Sweet potato (/pomoea batatas) ..............
Tanier (cocoyam) (Xanthosoma sagiWfolium) ..... ..................................................................................................
Turmeric (CLJrcuma longa) .
............................................
Turnip (Brassica rapa var. rapa) ....
Yam bean (jicama, manoic pea)
(Pachyrhizus spp.) .
::······"··:::::::::::::::::::::::···············································
Yam, true (Dioscorea spp.): =: ::: ;.,.::•::•:• : :_...............................

:: ::: :: :;...................................................................................... .
··················:·:::::::::::::::::::::(iii) Table. The following table 2 iden­tifies the crop subgroups for Crop
Group 1, specifies the representative

1A, 18iA, 181C
1A, 18
1A, 18
1A, 18
1A, 18
1A, 18IA, 18
1A, 181C, 1D
1C, 1D
1C, 101A, 181C, 101C, 10commodity(ies) for each subgroup, andlists all the commodities included ineach subgroup,

TABLE 2-CROP GROUP 1 SUBGROUP LISTING
Commodities

Representative commodities
Crop Subgroup 1A. Root vegetables
subgroup.
Carrot, radish, and sugar beet.
Crop Subgroup 1B. Root vegetables (except
sugar beet) subgroup.
Carrot and radish. .
Crop Subgroup 1C. Tuberous and corm
vegetables subgroup.
Potato.
Crop Subgroup 1 D. Tuberous and corm
vegetables (except potato) subgroup.
Sweet potato.

Beet, garden; beet, sugar; burdock, edible; carrot; celeriac; cheivil, turnip-root­
ed; chicory; ginseng; horseradish; parsley, turnip-rooted; parsnip; radish; rad­ish, oriental; rutabaga; salsify; salsify, black; salsify, Spanish; skirret; turnip.
Beet, garden; burdock, edible; carrot; celeriac; chervil, turnip-rooted; chicory;ginseng; horseradish; parsley, turnip-rooted; parsnip; radish; radish, oriental;
rutabaga; salsify; salsify, black; salsify, Spanish; skirr�t; turnip.
Arracacha; arrowroot; artichoke, Chinese; artichoke, Jerusalem; canna, edible;
cassava, bitter and sweet; chayote (root); chufa; dasheen; ginger; leren; po­tato; sweet potato; tanier; turmeric; yam bean; yam, true.
Arracacha; arrowroot; artichoke, Chinese; artichoke, Jerusalem; canna, edible;
cassava, bitter and sweet; chayote (root); chufa; dasheen; ginger; leren;
sweet potato; tanier; turmeric; yam bean; yam, true.

(2) Crop Group 2. Leaves of Root andTuber Vegetables (Human Food or Ani­
mal Feed) Group (Human Food or Ani­
mal Feed) Group.
(i) Representative commodities. Turnip
and garden beet or sugar beet.
(ii) Commodities. The following is alist of all the commodities included in
Crop Group 2:
CROP GROUP 2: LEAVES OF ROOT AND TUBER
VEGETABLES (HUMAN FOOD OR ANIMAL
FEED) GROUP-COMMODTIIES

Beet, garden (Beta vulgaris)
Beet, sugar (Beta vulgaris)
Burdock, edible (Arctium Zappa)

Carrot (Daucus carota)

Cassava, bitter and sweet (Manihot esculenta)
Celeriac (celery root) (Apium graveolens var.

rapaceum)

Chervil,turnip-rooted(Chaerophyllum

bulbosum)
Chicory (Cichorium intybus)

Dasheen (taro) (Colocasia esculenta)
Parsnip (Pastinaca sativa)
Radish (Raphanus salivus)
Radish, oriental (daikon) (Raphanus sativus
subvar. longipinnatus)
var.
(Brassica
campestris
napobrassica)
Salsify, black (Scorzonera hispanioa)
Sweet potato (lpomoea batatas)
Tanier (cocoyam) (Xanthosoma sagittifolium)

Rutabaga

491

59Appendix 4. 40 CFR 180.41 Crop Group Tables

40 CFR Ch. I (7-1-17 Edition)§ 180.41
Turnip (Brassica rapa. var. rapa)
Yam, true (Diosc:orea spp.)

CROP GROUP 3: BULB VEGETABLE (Allium SPP.)GROUP-COMMODITIES-Continued(3) Crop Group 3. Bulb Vegetables(Allium spp.) Group.
(i) Representative commodities. Onion,

Leek (AllitJm ampeloprasum, A. panum, A. fricoccum)
Onion, dry bulb and green (Alfium cepa, A. ffstulosum)Onion, Welsh, (Allium fistulosum)
Shallot (AJ/Jum r:epa var. cepa}

green; and onion, dry bulb.
(H) Commodities. The following is alist of all the commodities in Crop
Group 3.
CROP GROUP 3: BULB VEGETABLE (A//ium SPP.)GROUP-COMMODITIES
Garlic, bulb (Allium sat/vum}

Garr!c, great headed, (elephant) (Allfum ampeloprasum vm.
ampeloprasum}(4) Crop Group 3-0'7. Bulb Vegetable
Group.
(i) Representative Commodities. Oni.on,
bulb and onJon, green.
(ii) Table. The following Table 1 lists
all the commodities listed in Crop
Group 3-07 and identifies the related
crop subgroups.

TABLE 1-CROP GROUP 3-07: BULB VEGETABLE GROUP 1
Commodities

Related crop subgroups

Chive, fresh leaves (Affium schoenoprasum L) ,.....
Chive, Chinese, fresh !eaves (Alllum tuberosum Rottier ex Spreng)
Daylily, bu!b (Hemerocallis fulva (L) L var. fulva)
Elegans hosta {Hosta Siebo!diana. (Hook.) Eng~
Frilil!aria, bulb (Frilil!aria. L. friti/Jary) .
Frltillarla, leaves (Fritiflan'a L. friti/lary) .........
............ .
Garlic, bufb (Allium satfvum L. var. sativum) (A. sativum Common Garlic Group) ........................... .
Garlic, great headed, bulb (Alfium ampeloprnsum L var. ampeloprasum) (A a.mpe/oprasum GreatHeaded Garlic Group)"·"".......................... .
Garlic, Serpent, bulb (Al!ium sativum var. ophloscorodon or A. sativum Ophioscorodon Group} .
Kurrat (Allium kurrat Schweinf. Ex. K. Krause or A. ampeloprasum Kurrat Group) .
Lady's leek (Allium cen1uum Roth) ... " ...................................................................
Leek A!Num porrum L. (syn: A. ampeloprasum L. var. porrum (L) J. Gay) (A.ampoloprasum Leek
Group) .

Leck, wild (Allium tricoccum Aiton) .
.. ...................................
Lily. bulb (Lilium spp. (Ulfum Lelchtffnii var. maximowiczii, Lilium /ancifofium))Onion, Beltsvnle bunching (Alffum x prol/femm (Moench) Schrad.) {syn: Al/ium tistufosum L. x A cepal.).. ,......

.. .................................. ..

3-07B
3-078
3-07A
3-078

3-07A
3-07B
3-07A

3-0?A
3-07A
3-07B
3-078

3-07B
3-07B
3-07A

3-078

Onion, bulb (Aflium cspa L. var. cepa)' (A. cepa Common Onion Group) ....
Onion, Chinese, bu!b (All/um chinense G. Don.) (syn: A bakeri Rege!)

3-07A
3-0?A

Onion, fresh (A/fium fistu/oswn L. var. cr:iespitosum Makino) .,3-078

Onion, green (A/Hum cepa L. var. cepa) (A. cepa Common Onion Group) .
. ....... .
Onion, macrostem (A/lium macrostemam Bunge) ........ ...............
Onion, pearl (Allium porrum var. sectlvum or A. ampeloprasum Pearl Onion Group) .
Onion, potato, bulb (A!fium cepa L. var. aggrega.tum G. Don.) (A. cepe. Aggregatum Group) .............. ..
Onion, tree, tops (Aflium x proliterom (Moench) Schrad. ex Willd.) (syn: A. cepa var. proliferum(Moench) Regel; A cepa L var. bu!biferum L.H. Balley; A. cepa L. var. vivfpamm (Metz.) Al.et.) ....
Onion, Welsh, tops (Alfium fistulosum L.) ......................... .
Shallot, bulb (Af!ium c~pa var. aggregatum G. Don.) .............. .
Shallot, fresh !eaves {A!fwm cepa var. aggregatum G. Don.} . '"" ......... "'" ........... " ..

3-07B
3-07B
3-07A

3·07A
3-07B

3-078
3-07A
3·07B

Cultlvars, va1'ieties, and/or hybrlds of thes·e.

(iii) Table. The following Table 2
identifies the crop suhgToups for Crop
Group 3-07, specifies the representative

commodities for each subgroup andUsts all the commodities included ineach subgroup.

TABLE 2-CROP GROUP 3-07: SUBGROUP LISTING
Representative commodities

Commodities

Crop subgroup 3-07A. Onion, bulb, sub­group.

Daylify, bulb; fritil!aria, bulb; garlic, bufb; garlic, great-headed, bulb; garllc, serpent,bulb; lily, bu!b; onion, bulb; onion, Chinese, bulb; onion, pearl; onion, potato.
bulb; shallot bulb; cultiv:;us, varieties, and/or hybrids of these.
Chive, fresh leaves; chive, Chinese, fresh leaves; elegans hosta; fritillaria, leaves;
k:Lirrat; lady's leek; leek; leek, wild; Onion, Beltsvme bunching; onion, fresh;
onion, green; onion, macrostem; onion, lree, tops; onion, Welsh, lops; shallot,
fresh leaves; cultivars, varieties, and/or hybrids of these.

Onion, bulb.
Crop subgroup 3-078. Onion, green. sub­group

Onion, green.

492

60

Appendix 4. 40 CFR 180.41 Crop Group Tables

Environmental Protection Agency§180.41(5) Crop Group 4. Leafy Vegetables(Except Brassica Vegetables) Group.
(i) Representative commodities. Celery,head lettuce, leaf lettuce, and spinach(ii) Table. The following table 1 lists
all the commodities included in Crop
Group 4 and identifies the related crop
subgroups.

(Spinacia oleracea).
TABLE 1-CROP GROUP 4: LEAFY VEGETABLES (EXCEPT BRASSICA. VEGETABLES) GROUP
Related crop
subgroups

Commodities
Amaranth (leafy amaranth, Chinese spinach, tampala) (Amaranthus spp.) .....
Arugula (Roquette) (Eruca sativa) .
Cardoon ( Cynara carduncu!us) .
Celery (Apium graveo/ens var. du/ce)
Celery, Chinese (Apfum graveofens var. secalinum) .
Celtuce (Lactuca sativa var. angustana) .
Chervil (Anthriscus cerefolium) .
Chrysanthemum, edible-leaved (Chrysanthemum coronarium var. coronarium) .
Chrysanthemum, garland (Chrysanthemum coronarium var. spatiosum) ...
Corn salad ( Va.lerianella locusta)
Cress, garden (Lepidium sat/vum) .
. ............... .
Cress, upland (yellow rocket, winter cress) (Barbarea vulgaris) .
Dandelion ( Taraxacum officina/e)Dock (sorrel) (Rumexspp.) .
Endive (escarole) (Cichorium endivia)......
Fennel, Florence (finochio) (Faenicu!um vulgare Azoricum Group) ....
Lettuce, head and leaf (Lactuca sativa) .
Orach (Atripfex hortensis) ..
Parsley (Petroselinum caspum) .
Purslane, garden (Portulaca aleracea)
Purslane 1 winter (Mantia perfoliata) ....
Radicchio (red chicory) (Cichorium intybus)
Rhubarb (Rheum rhabarbarum) .
Spinach ( Spinacia oleracea)
Spinach, New Zealand (Tetragon/a tetragonioides, T. expansa) .
Spinach, vine (Malabar spinach, Indian spinach) (Basel/a alba)
Swiss chard (Beta vu/garis var. cicfa) .

(iii) Table. The following table 2 iden­tifies the crop subgroups for Crop
Group 4, specifies the representative

4A
4A
4B
4B
4B
4B
4A
4A
4A
4A
4A
4A
4A
4A
4A
4B
4A
4A
4A
4A
4A
4A
4B
4A
4A
4A
4B

commodities for each subgroup, andlists all the commodities included ineach subgroup.

TABLE 2-CROP GROUP 4 SUBGROUP LISTING
Representative commodities

Crop Subgroup 4A. Leafy greens subgroup.
Head lettuce and leaf lettuce, and spinach(Spinacia o/eracea).

Crop Subgroup 4B. Leaf petioles subgroup.
Celery.

Commodities

Amaranth; arugula; chervil; chrysanthemum, edible-leaved; chrysanthemum,
garland; com salad; cress, garden; cress, upland; dandelion; dock; endive;lettuce; orach; parsley; purslane, garden; purslane, winter; radicchio (redchicory); spinach; spinach, New Zealand; spinach, vine.
Cardoon; celery; celery, Chinese; celtuce; fennel, Florence; rhubarb; Swiss
chard.

(6) Crop Group 4-16. Leafy Vegetable
Group.
(i) Representative commodities. Headlettuce, leaf lettuce, mustard greens,and spinach.

(ii) Commodities. The following Table
I lists all commodities included in Crop
Group 4-16.

TABLE 1-CROP GROUP 4-16: LEAFY VEGETABLE GROUP
Related crop sub·groupsCommodities
Amaranth, Chinese (Amaranthus tricolor L.) .

4-16A

493

61

Appendix 4. 40 CFR 180.41 Crop Group Tables§180.41

40 CFR Ch. I (7-1-17 Edition)
TABLE 1-CROP GROUP 4-16: LEAFiVEGETABLE GROUP-Continued
Related crop sub­groupsCommodities
Amaranth, leafy (Amaranthus spp.) .. .
Arugula (Eruca sativa Mill.) ...........................
Aster, Indian (Ka/imeris indica (L.) Sch. Bip.) .
. . ... .........
Blackjack (Bidens pi/osa L.) ...............................
Broccoli, Chinese (Brassica o/eracea var. albog/abra (L.H. Bailey) Musil) .
Broccoli raab (Brassica rovo L.H. Bailey) . .
...................... ...................
Cabbage, abyssinian (Brassica carinata A. Braun)
Cabbage, Chinese, bok choy (Brass/ca rapa subsp. chinensis (L) Hanel!) ..........................
Cabbage, seakale (Brassica oferacea L. var. cosrata DC.)
Cat's whiskers (Cleome gynandra L.) ..........
Cham-chwi (Doelfingeria scabra (Thunb.) Nees) .
Cham-na-mul (Pimpinel/a calycina Maxim)
Chervil, fresh leaves (Anthriscus ceretolium (L.) Hoffm.)
Chipilin (Crotalaria longirostrata Hook & Arn) .
Chrysanthemum, garland (Gfebionis coronaria (L) Cass. ex Spach. G/ebionis spp.) ........
Cilantro, fresh leaves (Coriandrum satlvum L.) .
Collards (Brassica oleracea L. var. viridis L.) ...
Corn salad (Valeriane/la spp.) ......... .
Cosmos (Cosmos caudatus Kunth)Cress, garden (Lepidium satfvum L.) .
Cress, upland (Barbarea vulgaris W.T. Aiton) .....
Dandelion, leaves (Taraxacum officinale F.H. Wigg. Aggr.)
Dang-gwi, leaves (Angelica gigas Nakai)
Dillweed (Anethum graveolens L.) .
Dock (Rumex patientia L.) . . ........................... .
Dol-nam-mul (Sedum sarmentosum Bunge) ..............
Ebola (Crassocephalum crepidioides (BE!nth.) S. Moore)
Endive (Cichorium endivia L.) . .
Escarole (Cichorium endivia L.) ...............
Fameflower (Talinum fruticosum (L) Juss.) .............. "...... . .. . .........................
Feather cockscomb (Glinus oppositifo!ius (L.) Aug. DC.)
Good King Henry (Chenopodium bonus-henricus L.) ........
.... .........................
Hanover salad (Brass/ca napus var. pabu!aria (DC.) Rchb.) .
Huauzontle (Chenopodium berlandieri Moq.) ....
Jute, leaves (Corchorus spp.) ......................
Kale (Brassica oleracea L. var. Sabellica L.) ..................
..............................
Lettuce, bitter (Launaea cornuta (Hochst. ex Oliv. & Hiern) C. Jeffrey) . .
.........
Lettuce, head (Lactuca sativa L.; including Lactuca sativa var. capitata L.) ..
Lettuce, leaf (Lactuca sativa L.; including Lactuca sativa var. /ongifolia Lam.; Lacruca sariva var. crispa L.) ..
Maca, leaves (Lepidium meyenii Walp.) .
.......
Mizuna (Brassica ra'pa L. subsp. nipposinica (L.H. Bailey) Hanel!)
Mustard greens (Brass/ca juncea subsp., including Brassica juncea {L.) Czern. subsp. integrifolia (H. West)
Thell., Brassica juncea (L.) Czern. var. tsatsai (T.L. Mao) Gladis) .......
.......................................
Orach (Atriplex hortensis L.) ........
... ... .................
Parsley, fresh leaves (Petroselinum crispum (Mill.) Fuss; Petroselinum crispum var. neapo/ifanum Danert) ...
Plantain, buckthorn (Plantago lanceolata L.) ....
Primrose, English (Primula vu/garis Huds.)
Purslane,.garden (Portulaca oleracea L.) ...... .... .................................
Purslane, winter (Claytonia perfofiata Donn ex Willd.) .
Radicchio (Cichorium intybus L.)................................. . ..................................... . . .. . ..................
Radish, leaves (Raphanus sativus L. var sativus, including Raphanus sativus L. var. mougri H. W. J. Helm......... . ..... .. ... .................................. ..
(Raphanus sativus L. var. oleiformis Pers) .
Rape greens (Brassica napus L. var. napus, including Brass/ca rapa subsp. trifocu!aris (Roxb.) Hanelt;
Brass/ca rapa subsp. dichotoma (Roxb.) Hanelt; Brass/ca rapa subsp. o/eifera Met) .
Rocket, wild (Diplotaxis tenuifolia (L.) DC.) .............
.................................. .... .
Shepherd's purse (Capselfa bursa-pastoris (L.) Medik) ..... ................... .
Spinach (Spinacia o!eracea L.)
Spinach, Malabar (Basel/a alba L.) .
. . .................. .
Spinach, New Zealand (Tetragonia tetragon/aides (Pall.) Kuntze)
Spinach, tanier (Xanthosoma brasi/iense (Desf.) Engl.) ..
Swiss chard (Beta vu!garis L. ssp. vu!garis)
Turnip greens (Brassica rapa L. ssp. rapa)
Violet, Chinese, leaves (Asystasia gangetica (L.) T. Anderson}
Watercress (Nasturlium officinale W.T. Aiton) .........
Cultivars, varieties, and hybrids of these commodities.

:T

494

62

4-1BA
4-16B
4-16A
4-1BA
4-168
4-168
4-16B
4-168
4-168
4-16A
4-16A
4-16A
4-16A
4-16A
4-1BA
4-16A
4-16B
4-16A
4-16A
4-16B
4-168
4-16A
4-16A
4-16A
4-1BA
4-1BA
4-16A
4-16A
4-1BA
4-1BA
4-16A
4-1BA
4-16B
4-16A
4-16A
4-168
4-16A
4-16A
4-16A
4-168
4-16B
4-16B
4-1BA
4-16A
4-16A
4-16A
4-16A
4-16A
4-16A
4-168
4-168
4-16B
4-168
4-1BA
4-16A
4-16A
4-16A
4-16A
4-168
4-16A
4-168

Appendix 4. 40 CFR 180.41 Crop Group Tables

Environmental Protection Agency§180.41(iii) Crop subgroups. The following
Table 2 identifies the crop subgroups
for Crop Group 4-16, specifies the rep-resentative commodities for each sub..
group, and lists all the commodities in­cluded in each subgroup.

TABLE 2-CROP GROUP 4-16: SUBGROUP LISTING
Representative commodities

Comfnodities

Crop Subgroup 4-16A. Leafy greens subgroup
Head lettuce, leaf lettuce, and spinach .. ........................ Amaranth, Chinese; amaranth, leafy; aster, Indian;, blackjack; cat's
whiskers; cham-chwi; cham-na-mul; chervil, fresh leaves; chipilin;chrysanthemum, garland; cilantro, fresh leaves; corn salad; cosmos;
dandelion, leaves; dang-gwi, leaves; dillweed; dock; dol-nam-mul;
ebolo; endive; escarole; fameflower; feather cockscomb; Good KingHenry; huauzontle; jute, leaves; lettuce, bitter; lettuce, head; lettuce,
leaf; orach; parsley, fresh leaves; plantain, buckhorn; primrose,
English; purslane, garden; purslane, winter; radicchio; spinach;spinach, Malabar; spinach, New Zealand; spinaph, tanier; Swiss
chard; violet, Chinese, leaves; cultivars, varieties, and hybrids ofthese commodities.
Crop Subgroup 4-16B. Brassies leafy greens subgroup

Mustard greens

Arugula; broccoli, Chinese; broccoli raab; cabbage, abyssinian; cab­bage, Chinese, bok choy; cabbage, seakale; collards; cress, gar­den; cress, upland; hanover salad; kale; maca, leaves; mizuna;mustard greens; radish, leaves; rape greens; rocket, wild; shep­
herd's purse; turnip greens; watercress; cultivars, varieties, and hy­
brids of these commodities.

(7) Crop Group 5. Brassica (Cole) Leafy
Vegetables Group.
(i) Representative commodities. Broc­
coli or cauliflower; cabbage; and mus­tard_greens.

(ii) Table. The following table 1 lists
all the commodities included in Crop
Group 5 and identifies the related crop
subgroups.

TABLE 1-CROP GROUP 5: Brassica (COLE) LEAFY VEGETABLES
Related crop
subgroups

Commodities
Broccoli (Brassica oleracea var. botrytis) ..
Broccoli, Chinese (gai Ion) (Brassica alboglabra) ......... ················"'"·····································
Broccoli raab (rapini) (Brassica campestris)
Brussels sprouts (Brassica oleracea var. gemrflifera)
Cabbage (Brassica oleracea) .
Cabbage, Chinese (bok choy) (Brassica chinensis)
Cabbage, Chinese (napa) (Brassica pekinensis)
Cabbage, Chinese mustard (gai choy) (Brassica campestris)
Cauliflower (Brassica o/era.cea var. botrytls) .
Cavaro broccolo (Brassica oleracea var. botrytis)
Collards (Brassica o/eracea var. acepha/a)Kale (Brassica o/era.cea var. acepha/a) ........
Kohlrabi (Brassica o/eracea var. gongy/odes)
Mizuna (Brassica rapa Japonica Group) .
Mustard greens (Brassica juncea) .
Mustard spinach (Brassica rapa Perviridis Group) . .
Rape greens (Brassica napus) ......................

(iii) Table. The following table 2 iden­tifies the crop subgroups for Crop
Group 5, specifies the representative

5A
5A
5B
5A
5A
5B
5A
5A
5A
5A
5B
5B
5A
58
5B
5B
58

commodity(ies) for each subgroup, andlists all the commodities included ineach subgroup.

495

63

Appendix 4. 40 CFR 180.41 Crop Group Tables§180.41

40 CFR Ch. I (7-1-17 Edition)
TABLE 2--CROP GROUP 5 SUBGROUP LISTING

Representative commodities
Crop Subgroup SA. Head and stem Brassica
subgroup
Broccoli or cauliflower; and cabbage ........
,-,-- greens
I
Crop Subgroup 58. Leafy Brassica
subgroup.
Mustard greens ..

Commodities

Broccoli; broccoli, Chinese; brussels sprouts; cabbage; cabbage, Chinese(napa); cabbage, Chinese mustard; cauliflower; cavalo broccolo; kohlrabi

Broccoli raab; cabbage, Chinese (bok choy); collards; kale; mizuna; mustard
greens; mustard spinach; rape greens(8) Crop Group 5-16. Brassica Head andStem Vegetable Group.
(i) Representative commodities. Broc­
coli or cauliflower and cabbage.
(ii) Commodities. The following List 1
contains all commodities included in
Crop Group 5-16.
LIST 1-CROP GROUP 5-16: BRASSICA HEAD
AND STEM VEGETABLE GROUP
Commodities
Broccoli (Brassica olera.cea L. var. italica Plenck)
Brussels sprouts (Brassica oleracea L. var. gemmifera(DC.) Zenker)
Cabbage (Brassica oleracea L. var. capitata L.)
Cabbage, Chinese, napa (Brassica rapa L. subsp.
pekinensis (Lour.) Hanelt)

LIST 1-CROP GROUP 5-16: 8RASS1CA HEAD
AND STEM VEGETABLE GROUP-Continued
Commodities
Cauliflower (Brassica oleracea L. var. capffata L)
Cultivars, varieties, and hybrids of these commodities.

(9) Crop Group 6. Legume Vegetables(Succulent or Dried) Group.
(i) Representative commodities. Bean
(Phaseolus spp.; one succulent cultivar
and one dried cultivar); pea (Pisum spp.:
one succulent cultivar and one dried
cultivar): and soybean.
(ii) Table. The following table 1 lists
all the commodities included in Crop
Group 6 and identifies the related crop
subgroups.

TABLE 1-CROP GROUP 6: LEGUME VEGETABLES (SUCCULENT OR DRIED)
Related crop
subgroups

Commodities
Bean {Lupinus spp.) (includes grain lupin, sweet lupin, white lupin, and white sweet lupin) . ..... ..... ........ "' .... .
Bean (Phaseo/us spp.) (includes field bean, kidney bean, lima bean, navy bean, pinto bean, runner bean,
snap bean, tepary bean, wax bean) .
.................................
Bean (Vigna spp.) (includes adzuki bean, asparagus bean, blElckeyed pea, catjang, Chinese longbean,
cowpea, Crowder pea, moth bean, mung bean, rice bean, southern pea, urd bean, yardlong bean) .............
...........................
Broad bean (lava bean) (Vicia faba) .................
Chickpea (garbanzo bean) (Cicer arietinum) ..... .
Guar (Cyamopsis tetragono/oba)
Jackbean (Canavafia ensiformis) ................................... . .. . . ......................
Lablab bean (hyacinth bean) (Lab/ab purpureus). . ...... .................................
Lentil (Lens escufenta) .
. . ........ ..........................
Pea (Pisum spp.) {includes dwarf pea, edible-pod pea, En glish pea, field pea, garden pea, green pea, snowpea, sugar snap pea) ...
Pigeon pea (Ca.janus cajan)
Soybean ( Gfycine max)..........
Soybean (immature seed) (Glycine max) .... ..
Sword bean (Canavafia g/adiata) .

(iii) Table. The following table 2 iden­. tifies the crop subgroups for Crop
Group 6. specifies the representative

6A, 68, BC
68, 6C
6C
6C
6A
6C
6C
6A, BB, 6C
6A, 68, BC
NIA
6A
6A

commodities for each subgroup, andlists all the commodities included ineach subgroup.

496

64

6C
6A, 68, 6C

Appendix 4. 40 CFR 180.41 Crop Group Tables

Environmental Protection Agency§180.41

TABLE 2-CROP GROUP 6 SUBGROUP LISTING
Commodities

Representative commodities

Crop Subgroup 6A. Edible-podded legume
vegetables subgroup.
Any one succulent cuttivar of edible-podded Bean (Phaseofus spp.) (includes runner !:Jean, snap bean, wax bean); bean
( Vigna spp.) (includes asparagus bean, Chinese longbean, moth bean,
bean (Phaseo!us spp.) and any one suc­yardlong bean); jackbean; pea (Pisum spp.) (includes dwarf pea, edible-pod
culent cultivar of edible-podded pea (Pisum
pea, snow pea, sugar snap pea); pigeon pea; soybean (immature seed);spp.) ..
sword bean.
Crop Subgroup 68. Succulent shelled pea
I•
and bean subgroup.
Any succulent shelled cultivar of bean Bean (Phaseo/1.1s spp.) (includes lima bean (green)); broad bean (succulent);
bean (Vigna spp.) (includes blackeyed pea, cowpea, southern pea); pea(Phaseolus spp.) and garden pea (Pisum(Pisum spp.) (includes English pea, garden pea, green pea); pigeon pea.
spp.) ..
Crop Subgroup 6C. Dried shelled pea andbean (except soybean) subgroup
Any one dtied cultivar of bean (Phaseo!us Dried cultivars of bean (Lupinus spp.) (includes grain lupin, Jw-eet lupin, white
lupin, and white sweet lupin); (Phaseo/1.1s spp.) (includes field bean, kidneyspp.); and any one dried cultivar of pea
bean, lima bean (dry), navy bean, pinto bean; tepary bean; bean (Vigna(Pisum spp.).
spp.) (includes adzuki bean, blackeyed pea, catjang, cowpea, Crowder pea,
moth bean, mung bean, rice bean, southern pea, urd bean); broad bean(dry); chickpea; guar; lablab bean; lentil; pea (Pisum spp.) (includes fieldpea); pigeon pea.

(10) Crop Group 7. Foliage of Legume
Vegetables Group.
(i) Representative commodities. Any
cultivar of bean (Phaseolus spp.), fieldpea (Pisum spp.), and soybean.

(ii) Table. The following table 1 lists
the commodities included in Crop
Group 7.

TABLE 1-CROP GROUP 7: FOLIAGE OF LEGUME VEGETABLES GROUP
Commodities

Representative commodities

Any cultivar at bean (Phaseo!us spp.) and field Plant parts of any legume vegetable included in the legume vegetables that willpea (Pisum spp.), and soybean (Glycine
be used as animal feed.
max).

(iii) ·Table. The following table 2 iden­tifies the crop subgroup for Crop Group
7 and specifies the representative com-modities for the subgroup, and lists all
the commodities included in the sub­group.

TABLE 2-CROP GROUP 7 SUBGROUP LISTING
Representative commodities

Commodities

Crop Subgroup 7A. Foliage of legume
vegetables (except soybeans) subgroup
Any cultivar of bean (Phaseo!us spp.), andfield pea (Pisum spp.).

Plant parts of any legume vegetable (except soybeans) included in the legume
vegetables group that will be used as animal feed.

(11) Crop Group 8. Fruiting Vegetables
Group.
(i) Representative commodities. To­
mato, bell pepper, and one cultivar ofnon-bell pepper.
(ii) Commodities. The following is alist of all the commodities included in
Crop Group 8:

CROP GROUP 8: FRUITING VEGETABLES(EXCEPT CUOURBITS)-COMMODITIES

Egg·plant (Solanum melongena)
Groundcherry (Physalis spp.)
Pepino (Solanum muricatum)
Pepper (Capsicum spp.) (includes bell pepper,
chili pepper, cooking pepper, pimento,
sweet pepper)
Tomatillo (Physalis ixocarpa)
Tomato (Lycopersicon esculentum)

497

65

Appendix 4. 40 CFR 180.41 Crop Group Tables

40 CFR Ch. I (7-1-17 Edition)§180.41(12) Crop Group 8-10. Fruiting Voge­
table Group.
(i) Representa.tive commodities. To­
mato. standard size, and one cultivar ofsmall tomato; bell pepper and one
cultivar of small nonbell pepper.
(ii) Commodities. The following is alist of all commodities included in the
Crop group 8--10.

TABLE 1-CROP GROUP 8-10: FRUITING VEGETABLE GROUP
Related crop sub~groupsCommodities------------------------------;--African eggplant, SDlanum macrocarpon L
8--10B, 8-10C
Bush tomato, Solanum centrale J.M. Black
Cocona, Solanum sessiliflorum Dunal .
Currant tomato. Lycopersicon pimplne/lifol/um L .

8-1DA
8-10A
8-10A
8-iOB, 8-10C
8-iOA
8-10A
8-iOA
8-10B, 8-10C
8-'IOA
8-108, 8-10C
8-iOB, 8-10C
8-10B, 8-10C
8-108
8-10B, 8-10C

Eggplant, Solanum melongena L ..... .
Garden huckleberry, Sofanum sca.brum MillGoji berry, Lycium barbarum L ............... , .......................................................................... ., .......... ,.. .
Groundcherry, Physalls alkekeng/ L., P. grisea (Watelf.) M. Martinez, P. peruviana L., P. pubescens
Martyn1a, Proboscidea lauisianioa {Milt.) Thell ..•.•.•t.

NaranJilla, Solanum q11itoense Lam "'Okra, Abelmoschus esculentus {L.} Moench ....
Pea eggplant, So!anum torvum Sw ... .

Pep!no, Solanum muricatum Aiton ..... .,. .................. ..
Pepper, bell, Capsicum annuum L. var. annuum, Capsjcum spp ...
Pepper, nonbell, CapsfculJ1 chinese Jacq., C. annuum L. var. anmwm, C. frutescens L., C. ba.ccatum L.,

C. pubesoens Ruiz & Pav., Capsicum spp.
Roselle, Hibiscus sa.bdariffa L
Scarlet eggp!ant, So/anum aethiop!cfJm L .... .
Sunbeny, So/anum retroflexum Dunal ............. ..
Tomatillo. Physalis philadelphic:a Lam ....................

8-108. S-10C... , 8-10B, 8-10C....................

Tomato, Solanum lycoperslcum L., Solanum lycoperslcum L. var. lycoperslcum.
Tree tomato, So!anum be-taceum Gav ...
Cultivars, vartGtics and/or hybtids of these .............

(iii) Table. The following Table 2
identifies the crop subg'!'oups for Crop
Group 8-10, specifies the representative

::

..

l'

!=l~!
8-10A

------

commodities for each subgroup andlists all the commodities included ineach subgroup.

TABLE 2-CROP GROUP 8-10. SUBGROUP LISTING
Representative commodities

Commodities

Crop subgroup 8-10A. Tomato subgroup
Tomato, standard size, and one oultivar of small tomato ...........

! Blish tomato; cocona: currant tomato; garden huckleberry; goji------------------+-------------·----------··

berry; groundcherry; naran]illa; sunberry; tomatlllo; tomato;
tree tomato; cultivars, varieties, and/or hybrids of these.

Crop subgroup 8-iOB. Pepper/Eggplant subgroup-1· Africa.n eggplant; bell pepper; eggplant; Ma.rtynla; nonbell pep·

Bell pepper and one curnvar of small nonball pepper .

par: okra; pea eggplant; peplno; rosella; scarlet eggplant;-cultivars, varieties, and/or hybrids of these.

jCrop subgroup 8-1 OC. Nonbe!I pepper/Eggplant subgroup1

One cultivac of small nonbell popper or ono cultivar of small
eggplant.

I African eggplant; eggplant; martynia; nonbell pepper, okra; pea
eggplant; pepino; roselle; scart.et eggplant: cu!tivars, vari­eties, and/or hybrids of these.

-----·-·-----·-··----······......

(13) Crop Group 9, Cucurbit Vegeta­
bles Group.
(i) Represenlalive commodities. Cucum­
ber, muskmelon, and summer squash.

(ii) Table. The follov;ing table 1 lists
all the commodities included in Crop
Group 9 and identifies the related sub­groups.

TABLE 1-CROP GROUP 9: CUCURBIT VEGETABLES- - - - - - - · - · · - · · · · - - - - -··---·---- ... ········--Commodities

Related crop

subgroups- - - - - - - - - - - - - - - - - - - - - - - --·------ ....------------Ch~yote (fruit) {Sechium edule) ....................... .

9B

498

66

Appendix 4. 40 CFR 180.41 Crop Group Tables

Environmental Protection Agency§180.41

TABLE 1-CROP GROUP 9: CUCURBIT VEGETABLES-Continued
Related crop
subgroups

Commodities
Chinese waxgourd {Chinese preserving melon) (Benincasa hisplda) ..
Citron melon (Citrul/us !anatus var. citroides)
Cucumber (Cucumis sativus) .
Gherkin (Cucumis anguria) ...... ........................ ......
... ...
Gourd, edible (Lagenaria spp.) (includes hyotan, cucuzza); (Luffa acutangu/a, L. cylindrica) (includes hechima,
Chinese okra) ....
. ... ............................................. .....................................
Momordica spp. (includes balsam apple, balsam pear, bitter melon, Chinese cucumber) ................ .
Muskmelon (hybrids and/or cultivars of Cucumis melo) (includes true cantaloupe, cantaloupe, casaba, cren­shaw melon, golden pershaw melon, honeydew melon, honey balls, mango melon, Persian melon, pine­
apple melon, Santa Claus melon, and snake melo'n) .
Pumpkin ( Cucurbita spp.) .. ..............
.........................................
Squash, summer (Cucurbita pepo var. melopepo) (includes crookneck squash, scallop squash, straightnecksquash, vegetable marrow, zucchini) ........ ...................... . ...... .... .. ......... ......................................................
Squash, winter {Cucurbita maxima; C. moschata) {includes butternut squash, calabaza, hubbard squash); {C.
............................. imixta; C. pepo) (includes acorn squash, spaghetti squash)
Watermelon (includes hybrids and/or varieties of Citruflus /anatus)(iii) Table. The following table 2 iden­tifies the crop subgroups for Crop
Group 9, specifies the representative

98
9A
98
9B
9B
98
9A
9B
9B
9B
9A

commodities for each subgroup, andlists all the commodities included ineach subgroup.

TABLE 2-CROP GROUP 9 SUBGROUP LISTING
Representative commodities

Commodities

Crop Subgroup 9A. Melon subgroup
Cantaloupes .
Crop Subgroup 9B. Squash/cucumber
subgroup
One cultivar of summer squash and cucumber.

(14)

Crop Group 10.

Citron melon; muskmelon; watermelon
Chayote (fruit): Chinese waxgourd; cucumber; gherkin; gourd, edible;
Momordica spp.; pumpkin; squash, summer; squash, winter.

Citrus Fruit

Group.

(i) Representative commodities. Sweet
orange; lemon and grapefruit.
(ii) Commodities. The following is alist of all the commodities in Crop
Group 10:
CROP GROUP 10: CITRUS FRUITS (CITRUS SPP.,FORTUNELLA SPP.) GROUP-COMMODITIESCalamondin

mitis)(Citrus mitis x Citrofortunella

Citrus citron (Citrus medica)
Citrushybrids(Citrus spp.)
chironja, tangelo, tangor)
Grapefruit (Citrus paradisi)(includes

Kumquat (Fortunella spp.)
Lemon (Citrus jambhiri, Citrus limon)
Lime (Citrus aurantiifolia)
Mandarin (tangerine) (Citrus reticulata)
Orange, sour (Citrus aurantium)
Orange, sweet (Citrus sinensis)
Pummelo (Citrus grandis, Citrus maxima)

Satsuma mandarin (Citrus unshiu)(15) Crop Group 10-10. Citrus Fruit

Group.
(i) Representative commodities. Orange
or Tangerine/Mandarin, Lemon orLime, and Grapefruit.
(ii) Commodities. The following is alist of all the commodities in Crop
Group 10-10.

TABLE 1-CROP GROUP 10-10: CITRUS FRUIT GROUP
Related crop sub­groupsCommodities.......................................................
. ·······---- - ----1...........
.. ........

Australian desert lime, Eremocitrus g/auca (Lindi.) Swingle ..
Australian finger lime, Microcitrus australasica {F. Muell.) Swingle .
.............................
Australian round lime, Microcitrus australis (A. Cunn. Ex Mudie) Swingle
Brown River finger lime, Microcitrus papuana Winters ...................................................................
Calamondin, Citrofortune/ia microcarpa (Bunge) Wijnands
Citron, Citrus medica L.. ............. ............... ............. ...... .. .. . .. ............. .........,....
Citrus hybrids, Citrus spp. Eremocitrus spp., Fortune/la spp., Microcitrus spp., and Poncirusspp .. .........
Grapefruit, Citrus paradisi Macfad-·-· ..· .......................... ·..-- .....·-···-···.···.···,·--·
499

67

10-10B
10-10910-10B
10-10B
10-10A
10-10A
10-10A
10-10CAppendix 4. 40 CFR 180.41 Crop Group Tables

40 CFR Ch. I (7-1-17 Edition)§180.41

TABLE 1-CROP GROUP 10-10: CITRUS FRUIT GROUP-Continued
Related crop sub­groupsCommodities
Japanese summer grapefruit, Citrus natsudaidal Hayata ..
Kumquat, Fortunel(a spp .
Lemon, Citrus limon {L.) Burm. f ..
Lime, Citrus aurantiifo/ia (Christm.) Swingle
Mediterranean mandarin, Citrus de/iciosa Ten ..
Mount White lime, Microcitrus garrowayae (F.M. Bailey} Swingle
New Guinea wild lime, Mlcrocitrus warburgiana (F.M. Bailey) Tanaka .
Orange, sour, Citrus aurantium L .
........................
Orange, sweet, Cirrus sinensis (L.) Osbec�
Pummelo, Citrus maxima (Burm.) Merr ..................
Russell River lime, Microcitrus inodora (F.M. Balley) Swingle
Satsuma mandarin, Citrus unshiu Marcow . .
Sweet lime, Citrus limetta Risso ................. .
Tachlbana orange, Citrus tachibana (Makino) Tanaka ............ .. ............................................................. ..........
Tahiti lfme, Citrus latifofia (Yu. Tanaka) Tanaka .......................................................................................... I....
Tangelo, Citrus xtange/o J.W. Ingram & H.E. Moore
Tangerine (Mandarin), Citrus reticulata Blanco .
Tangor, Citrus nob/Ifs Lour .
Trifoliate orange, Poncirus trifoliata (L.) Raf ........................... .
Uniq fruit, Citrus aurantium Tangelo group ..... ..
Cultivars, varieties and/or hybrids of these.

(iii) Table. The following Table 2
identifies the crop subgroups for Crop
Group 10--10, specifies the representa-

10-10C
10-10B
10-10B
10-10B
10-10A
10-10B
10-10B
10-10A
10-10A
10-10C
10-10B
10-10A
10-10B
10-"10A
10-10B
10-10A, 10-10C
10-10A
10-10A
10-1DA
10-10C

tive commodities for each subgroupand lists all the commodities includedin each subgroup.

TABLE 2-CROP GROUP 10-10: SUBGROUP LISTING
Commodities

Representative commodities
Crop Subgroup 10-10A. Orange subgroup
Orange or tangerine/mandarin ....

Calamondln; citron; citrus hybrids; mediterranean mandarin; or­ange, sour; orange, sweet; satsuma mandarin; tachibana or­ange; tangerine (mandarin); tangelo; tangor; trifoliate orange;
cultivars, varieties, and/or hybrids of these.

Crop Subgroup 10-108. Lemon/Lime subgroup
Lemon or llme .. . . ............................ . ........................

Australian desert lime; Australian finger lime; Australian round
lime; brown river finger lime; kumquat; lemon; lime; mountwhite lime; New Guinea wild lime; Russell River lime; sweetlime; Tahiti lime; cultivars , varieties, and/or hybrids of these.

Crop Subgroup 10-10C. Grapefruit subgroup
Grapefruit ....

Grapefruit; Japanese summer grapefruit; pummelo; tangelo;
uniq fruit; cultivars, varieties, and/or hybrids of these.

(16) Crop Group 11: Pome Fruits
Group.
(i) Representative commodities. Appleand pear.
(ii) Commodities. The following is alist of all the commodities included in
Crop Group 11:
CROP GROUP 11: POME FRUITS GROUP-COMMODITIESApple (Malus domestica)
Crabapple (Malus spp.)
Loquat (Eriobotrya japonica)
Mayhaw (Crataegus aestivalis, C. opaca, and C.
rufula)Pear (Pyrus communis)
Pear, oriental (Pyrus pyrifolia)

Quince (Cydonia oblonga)(17) Crop group 11-10. Pome Fruit
Group.
(i) Representative commodities. Appleand Pear(ii) Commodities. The following is alist of all the commodities in Crop
Group 11-10.
CROP GROUP 11�10: POME FRUIT GROUPCOMMODITIESApple, Malus domestica Borkh.
Azarole, Crataegus azarolus L.
Crabapple, Malus sylvestris (L.) Mill., M.
prunifolia (Willd.) Borkh.
Loquat. Eriobotrya japonica (Thunb.) Lindl.

500

68

Appendix 4. 40 CFR 180.41 Crop Group Tables

Environmental Protection Agency§180.41

Mayhaw, Crataegus aestivalis (Walter) Torr. &A. Gray, C. opaca
Hook. & Arn., and C. rufula Sarg.
Medlar, ivlespilus germanica L.
Pear, Pyrus communis L.
Pear, Asian, Pyrus pyrifolia (Burm. f.) Nakai
var. culta (Makino) Nakai

Pseudocydonia sinensis (Thouin) C.K. Schneid.
Quince, Cydonia oblonga Mill.
Chaenomeles speciosa
Chinese,Quince,(Sweet) Nakai,
Quince,Japanese,
Chaenomeles(Thunb.) Lindi. ex Spachjaponica

Tejocote, Crataegus mexicana DC.

Cultivars, varieties and/or hybrids of these.

(18) Crop Group 12. Stone Fruits
Group.
(i) Representative commodities. Sweet
cherry or tart cherry; peach; and plumor fresh prune (Prunus domestica,
Prunus spp.)(ii) Commodities. The following is alist of all the commodities included in
Crop Group 12:

CROP GROUP 12: STONE FRUITS GROUPCOMMODITIESApricot (Prunus armeniaca)
Cherry, sweet (Prunus avium),

Cherry, tart (Prunus cerasus)

Nectarine (Prunus ,Persica)
Peach (Prunus persica)
Plum (Prunus domestica, Prunus spp.)
Plum, Chickasaw (Prunus angustifolia)
Plum, Damson (Prunus domestica

spp.

insititia)
Plum, Japanese (Prunus salicina)
Plumcot (Prunus. armeniaca xP. domestica)
Prune (fresh) (Prunus domestica, Prunus spp.)(19) Crop Group 12-12: Slone Fruit
Group.
(i) Representative commodities. Sweet
cherry or Tart cherry; Peach; andPlum or Prune plum.
(ii) Commodities. The following Table
1 is a list of all commodities includedin Crop Group 12--12.

TABLE 1-CROP GROUP 12-12: STONE FRUIT GROUP
Related crop
subgroup

Commodities
Apricot (Prunus armen/aca L.) . ... . ........ ................ .
Apricot, Japanese (Prunus mume Siebold & Zucc.) ......
Capulln (Prunus serotina Ehrh. var. salicifolia (Kunth) Koehne) .
Cherry, black (Prunus serotina Ehrh.) ...
Cherry, Nanking (Prunus tomentosa Thunb.)
Cherry, sweet (Prunus avium {L.) L.) ............
Cherry, tart (Prunus cerasus L.) .....
Jujube, Chinese (Ziziphus jujuba Mill.) .
Nectarine (Prunuspersica (L.) Batsch var. nucipersica (Suckow) C.K. Schneid) .
Peach (Prunus persica (L.) Batsch var. persica) ..........
Plum (Prunus domestica L. subsp. domestic.;t) .
Plum, American (Prunus americana Marshall) ....
Plum, beach (Prunus maritima Marshall)Plum, Canada (Prunus nigra Aiton)
Plum, cherry (Prunus cerasifera Ehrh.) .
Plum, Chickasaw (Prunus angustffolia Marshall) .
Plum, Damson (Prunus domestica L. subsp. insititia (L.) C.K. Schneid.)
Plum, Japanese (Prunus salicina Lindi.; P. sa/icina Lindi. var. sa/icina)
Plum, Klamath (Prunus subcordata Benth.) .
Plum, prune (Prunus domestica L. subsp. domestica)
Plumcot (Prunus hybr.) ...................................
. .. . . ................
Sloe (Prunus spinosa L) .
Cultivars, varieties, and/or hybrids of these.

(iii) Crop subgroups. The following
Table 2 identifies the crop subgroups
for Crop Group 12--12, specifies the rep-

12-12C12-12C12-12A
12-12A
12-12A
12-12A
12-12A
12-12Ci2-12B
12-12B
12-12C12-12C12-12C12-12C12-12C12-12C12-12C12-12C12-12C12-12C12-12C12-12C

resentative commodities for each sub­group, and lists all the commodities in­eluded in each subgroup.

TABLE 2-CROP GROUP 12-12: SUBGROUP LISTING
Commodities

Representative commodities

Crop subgroup 12-12A. Cherry subgroup
Cherry, sweet or Cherry, tart ..

Cap·ulin; Cherry, black; Cherry, Nanklng; Cherry, sweet; Cherry, tart; cultivars, varieties, and/or
hybrids of these.

501

69

Appendix 4. 40 CFR 180.41 Crop Group Tables§180.41

40 CFR Ch. I (7-1-17 Edition)
TABLE 2-CROP GROUP 12-12: SUBGROUP LISTING-Continued

Representative commodities

Commodities
Crop subgroup 12-12B. Peach subgroup

Peach

Peach; Nectarine; cultlvars, varieties, and/or hybrids of these.

Plum or Prune plum .

Apricot; Apricot, Japanese; Jujube, Chinese; Plum; Plum, American; Plum, beach; Plum, Can­ada; Plum, cherry; Plum, Chickasaw; Plum, Damson; Plum, Japanese; Plum, Klamath;
Plumcot; Plum, prune; Sloe; cultivars, varieties, and/or hybrids of these.

Crop subgroup 12-12C. Plum subgroup(20) Crop Group 13. Berries Group.
(i) Representative commodities. Any
one blackberry or any one raspberry;and blueberry.

(ii) Table. The following table 1 lists
all the commodities included in Crop
Group 13 and identifies the related sub­groups.

TABLE 1-CROP GROUP 13: BERRIES GROUP
Related crop
subgroups

Commodities
Blackberry (Rubus eubatus) (including bingleberry, black satin berry, boysenberry, Cherokee blackberry,
Chesterberry, Cheyenne blackberry, coryberry, darrowbeny, dewberry, Dirksen thornless berry,
Himalayaberry, hullberry, Lavacabeny, lowbsrry, Lucretiaberry, mammoth blackberry, marionberry,nectarberry, olallieberry, Oregon evergreen berry, phenomenalberry, rangeberry, ravenberry, rossberry,
Shawnee blackberry, youngberry, and varieties and/or hybrids of these)
Blueberry (Vaccinium spp.) .
Currant (Ribes spp.) ..
Elderberry (Sambucus spp.) ..
Gooseberry (Ribes spp.)
Huckleberry (Gaylussacia spp.) . .
Loganberry (Rubus loganobaccus) . . ...............
Raspberry, black and red (Rubus occidentalis, Rubus strigosus, Rubus idaeus) ...

(iii) Table. The following table 2 iden­tifies the crop subgroups for Crop
Group 13, specifies the representative

13A
13B
13B
13B
13B
13B
13A
13A

commodities for each subgroup, andlists all the commodities included ineach subgroup.

TABLE 2-CROP GROUP 13 SUBGROUPS LISTING
Commodities

Representative commodities
Crop Subgroup 13A. Caneberry (blackberry
and raspberry) subgroup.
Any one blackberry or any one raspberry. .......
Crop Subgroup 13B. Bushberry subgroup ..
Blueberry, highbush.

Blackberry; loganberry; red and black raspberry; cultivars and/or hybrids ofthese.
Blueberry, highbush and lowbush; currant; elderberry; gooseberry; huckleberry.

(21) Crop Group 13-07. Berry and Small
Fruit Crop Group(i) Representative commodities. Any
one blackberry or any one raspberry;highbush blueberry; elderberry or mu!-berry; grape; fuzzy kiwifruit, andstrawberry.
(ii) Table. The following Table 1 Usts
all the commodities listed in Crop
Group 13-07 and identifies the related
crop subgroups.

TABLE 1-CROP GROUP 13-07: BERRY AND SMALL FRUIT CROP GROUP
Commodities

Related crop subgroups

Amur river grape (Vitis amurensis Rupr'j
Aronia berry (Aronia spp.)
Bayberry (Myrlca spp.)

13-07D, 13-0?E, 13-07F
13-07813-07C

502

70

Appendix 4. 40 CFR 180.41 Crop Group Tables

Environmental Protection Agency§ 180.41

TABLE 1-CROP GROUP 13-07: BERRY AND SMALL FRUIT CROP GROUP-Continued
Commodities

Related crop subgroups

Bearberry (Arctostaphy/os uva-urs,)
Bilberry (Vaccinium myHiflus L.) . ......... . ..................................... . ......... ...
. .............
Blackbeny (Rubus spp.) (including Andean blackberry, arctic blackberry, bingleberry, black satin
berry, boysenberry, brombeere, California blackberry, Chesterberry, Cherokee blackberry, Chey­
enne blackberry, common blackberry, coryberry, darrowberry, dewberry, Dirksen thornless berry,
evergreen blackberry, Himalayaberry, hullberry, lavacaberry, loganberry, lowberry, Lucretiaberry,mammoth blackberry, marlonberry, mora, mures deronce, nectarberry, Northern dewberry,olallleberry, Oregon evergreen berry, phenomenalberry, rangeberry, ravenberry, rossberry, Shaw�
nee blackberry, Southern dewberry, tayberry, youngberry, zarzamora, and cultivars, varieties and/or
hybrids of these.) ................ .
Blueberry, highbush (Vaccinium spp.) .
Blueberry, lowbush (Vaccinium angustifolium Aiton) .
Buffalo currant (Ribes aureum Pursh) . .
Buffaloberry (Shepherdia argentea (Pursh) Nutt.)
Che (Cudrania tricuspidata Bur. Ex Lavallee) ..
Chilean guava (Myrtus ugni Mal.)
Chokecherry (Prunus virginlana L.) .... .
Cloudberry (Rubus chamaemorus L.) .......
Cranberry (Vaccinium macrocarpon Aiton) .
Currant, black (Ribes nigrum L.)
Currant, red (Ribes rubrum L.)
Elderberry {Sambucus spp.) ....., ..... ,.. ,.. ,....................................................................................................... .
European barberry (Berberis vwgans L.J .
Gooseberry (Ribes spp.)
Grape (Vitis spp.) . ..
Highbush cranberry (Viburnum opufus L. var. Americanum Aiton) . ......... .
Honeysuckle, edible (Lonicera caerula L. var. emphy!loca/yx Nakai, Lonicera caerula L var . edu/isTurcz. ex herder') .....
Huckleberry (Gayfussacla spp.) .. .
Jostaberry (Ribes x nidigrolaria Rud. Bauer and A. Bauer) .
Juneberry (Saskatoon berry) (Amelanchier spp.) .
Kiwifruit, fuzzy (Actinidia del!ciosa A. Chev.) (C.F. Ljang and A.A. Fergusons, Actinida chlnensis
Planch.) .......
..........
........... . ............ ...........
Kiwifruit,.hardy (Actinidia arguta (Siebold and Zucc.) Planch. ex Miq) ......
Lingonberry (Vaccinium vitis-idaea L.) .
Maypop (Passiflora incamata L.} ........ ...........
Mountain pepper berries (Tasmannia /anceo/ata)(Poir.) A.C.Sm.
Mulberry (Morus spp.)
Muntries (Kunzea pomifera F. Muell.) . ....... .
Native currant (Acrotriche depressa R. BR.)
Partridgeberry (Mitche!la repens L.) ..
Phalsa (Grewia subinaequa/is DC.) .
Pincherry (Prunus pensyfvanica L.f.) . ..
Raspberry, black and red (Rubus spp.) .
Riberry { Syzyglum fuehmannil)
Sala! (Gaulrheria shallon Pursh.) .............................
.
....................................................................................
Schisandra berry (Schisandra chinensis (Turcz.) Baill.) ...
Sea buck.thorn (Hippophae rhamnoides L.) .
Serviceberry (Sorbus spp.) .............................. .
Strawberry (Fragaria x ananassa Duchesne) .
Wild raspberry (Rubus muelleri Lefevre ex P.J. Mull) ..
Cultivars, varieties, and/or hybrids of these.

(iii) Table. The following Table 2
identifies the crop subgroups for Crop
Group 13-07, specifies the representa-

13-07G, 13-071-l
13-07G, 13-07H

13-0?A
13-07B
13-0?B
13-0?B
i3-07C
13-07C
13-07B
13-0?C
13-07G, 13-07H
13-07G, 13-07H
13-0?B
13-07813-07B, i3-07C
13-078
13-0?B, 13-070, 13·
07E, 13-0?F
13-07D, 13-0?F
13-07B
13-07B
13-07B
13-0?B
13-0?B, i3-07C
13-070, 13-0?E
13-07D, 13-07E, i3-07F
13-0?B, 13-0?G 13-07H
13-0?E, i3-D7F
13-0?C
13-07C
13-0?G, 13-0?H
13-07B
13-0?G, 13-07H
13-07C
13-0?G
13-0?A
13-07C
13-07B, 13-07C
13-07D, 13-07E, 13-0?F
13-0?B
13-0?C
13-07G
13-0?A

tive commodities for each subgroupand lists all the commodities includedin each subgroup. ·TABLE 2-CROP GROUP 13-07: SUBGROUP LISTING
Representative commodities

Commodities

Crop Subgroup 13-07A. Caneberry subgroup
Any one blackberry or any one rasp�
berry..
Crop Subgroup 13-07B. Bushberry subgroup.

Blackberry; loganberry; raspberry, red and black; wild raspberry; cultivars, varieties, and/or hybrids of these.

503

71

Appendix 4. 40 CFR 180.41 Crop Group Tables

40 CFR Ch. I (7-1-17 Edition)§ 180.41

TABLE 2-CROP GROUP 13-07: SUBGROUP LISTING-Continued-------------.-----------------------Representative commodities:

Commodities.. IAronia ben-y; blueberry, highbush; blueberry, lowbush; buffalo currant; Chiloan

Blueberry, highbush.

1

guava: currant, black; currant red; elderberry; European, barberry; goose-iberry; cranberry, highb-ush; honeysuckle, edible; huckleberry; jostaberry;
Juneberry; llngonberry: native currant; sata!; sea bucklhorn; cultivars, vari­

I eties: and/or hybrids of these.

Crop Subgroup 13-07C. Large shrub/tree !
berry subgroup.
Elderbert)' or mulberry.

······· I Bai:~~r~~~~0u~;{~;c~~~:~~~~~~;7~t::~;~~1!r::::b:~~;n;a~~ui:rs::

Crop Subgroup 13-070. SmaU

varieties, and/or hybrids of these.

fruit vine :I

c:limbing subgroup.
Grape and 1uzzy kiwifrLJit. . ................

Amur river grape: goosebeny; grape; kiwifruit, fuzzy; kiwifruit, hardy; Maypop;! schisandra berry; cultfvars, vatietles, and /or hybrids of these,
Crop Subgroup 13-07E, Small fruit vine climb- :ing subgroup. excepl. grape.
l
Fuzzy kiwifruit .................,.................. Amur river grape; gooseberry; kiwifruit. fuzzy; kiwifruit, hardy; Maypop;, schisandra berry; cultivars, varieties, and/or hybrids of these.
Crop Subgroup 13..07F. Small fruit vine climbIng subgroup except fuzzy kiwlfrutt.
Grape.
Amur river grape; gooseberry; grape; kiwifruit, hardy; Maypop; schisandraI

berry; cultivars varieties, andror hybrids of these.
Crop Subgroup 13-07G. Low growing berry
subgroup.
Strawberry.

Bearbeny; bilberry; blueberry, lowbush; cloudberry; cranberry; lingonberry;muntries; partridgeberry; strawberry; cultivars, varieties, and/or hybrids ofthese.
Crop Subgroup 13-07H. Low growing berry
subgroup, except strawberry.
Cranberry

Bearberry; bilberry; blueberry, lowbush; doudberry; cranberry; Jingonberry;muntnes; partridgeberry; cultiva.rs, varieties. and/or cultlvars of these.

(22) Crop Group 14. Tree Nuts Group.
(i) Representative commodities. Almondand pecan.
(ii) Commodities. The following is alist of all the commodities included in
Crop Group 14:
CROP GROUP 14: TREE NUT&-COMMODITIESCROP GROUP 11--12: 'l'REE NUT GROUP

African nut-tree (Ricinodendron heu.delotii(Baill.) Hecl,el)Almond {Prunus dttlcis (Mill.) D.A. Webb)
Beechnut (Fag·us grwidifol'la Ehrh.; F.
syWatica, L.)
Bra,zil nut (Bertliolletia excelsa Humb. &Bonpl.)
Brazilianpine(Ara1lcaria
angustifolia

Almond (Prunus dulcis)
Beech nut (Fagus spp.)
Brazil nut (Bertholletia e:rcelsa)(Berto!.) Kuntze)
Bunya (il'raucaria bidwilli'i Hook.)
Bur oak (Quercus macrocarpa Micbx.)

Butt~rnut (Juglans cinerea)

Butternut (Juglans cinerea L.)
Cajou nut (Anacardium gigante-um Hance ex
Engl.)
Candlenut (Ale11.rites moluccanus (L.) Willd.)
Cashew (Anaca.rdium occidentale L.)
Chestnut (Castanea crenata Siebold & Zucc.;
C. dentata (MarshaJ.1) Borkh.; C. mollissima
Blume; C. sativa M!ll.)
Cllinquapin (Castaneapumila (L.) Mill.)
Coconut (Cocos nucifera L.)
Coqui.to nut (Jubaea chilensis (Molina) Baill.)
Dika nut(lrvingia
gabanmisis(Aubry-Cashew (.4nacardtu.m occide-ntale)
Chestnut (Castanea spp.)
Chinqua.pin (Castanea pumila)
Filbert (hazelnut) (Cory/us spp.)
Hickory nut. 1,Carya spp.)
Macadamia nut (01.u;h nut) (]}facadamia spp.)

Pecan (Carya illinoensis)
Walnut. black and English (Persian) (Juglansspp,)(23) Crop Group 14-12. Tree Nut, Group.
(i) Representati1'e commodities. Almondand Peca.n.
(ii) Commodities. The following is alist of all commodities included in Crop
Group 14-12.

Lecomte ex O'Rorke) Baill.)
Ginkgo (Ginkgo biloba L.)
Guiana chestnut (Pachira aquatica Aubl.)
Hazelnut (F1lbert) (Corytus -americana Marshall; C. a-vellana L.; C. californica (A. DC.)

Rose; C. chinensis Franch.)Heartnut (Juglans ailantifolia CarriBre var.
cordijormis (Makino) Rehder)

504

72

Appendix 4. 40 CFR 180.41 Crop Group Tables

Environmental Protection Agency§180.41

Hickory nut (Carya cathayensis Sarg.; C.
glabra (Mill.) Sweet; C. laciniosa (F. Michx.)
W. P. C. Barton; C. myristiciformis (F.
Michx.) Elliott; C. ovata (Mill.) K. Koch; C.
toinentosa (Lam.) Nutt.)Japanese horse-chestnut (Aesculus turbinate
Blume)
Macadamia nut (Macadamia integrifolia Maid­en & Betche; M. tetraphylla L.A.S. John­son)
Mongongo nut (Schinziophyton rautanenii(Schinz) Radcl.-Sm.)
Monkey-pot (Lecythis pisonis Cambess.)
Monkey puzzle nut (Araucaria araucana(Molina) K. Koch)
Okari nut (Terminalia kaernbachii Warb.)
Paohira nut (Pachira insignis (Sw.) Savigny)
Peach palm nut (Bactris gasipaes Kunth var.
gasipaes)
Pecan (Carya illinoinensis (Wangenh.) K.
Koch)Pequi (Caryocar brasiliense Cambess.; C.
villosum (Aubl.) Pers; C. nuciferum L.)
Pili nut (Canarium ovatum Engl.; C. vulgare
Leenh.)
Pine nut (Pinus edulis Engelm.; P. koraiensis
Siebold & Zucc.; P. sibirica Du Tour; P.
pumila (Pall.) Regel; P. gerardiana Wall. ex
D. Don; P. monophylla Torr. & Frem.; P.
quadrifolia Parl. ex Sudw.; P. pinea L.)
Pistachio (Pistacia vera L.)
Sapucaia nut (Lecythis zabucaja Aubl.)
Tropical almond (Terminalia catappa L.)
Walnut, black (Juglans nigra L.; J. hindsii
Jeps. ex R. E. Sm.; J. microcarpa Berland.)
Walnut, English (Juglans regia L.)
Yellowhorn (Xanthoceras sorbifolium Bunge)
Cultivars, varieties, and/or hYbrids of these(24) Crop Group 15. Cereal Grains
Group.
(i) Representative commodities. Corn(fresh sweet corn and dried field corn),rice, sorghum, and wheat.
(ii) Commodities. The following is alist of all the commodities included in
Crop Group 15:
CROP GROUP 15: CEREAL GRAINS�COMMODITIESBarley (Hordeum spp.)
Buckwheat (Fagopyrum esculentum)Corn (Zea mays)
Millet, pearl (Pennisetum glaucum)
Millet, proso (Panicum milliaceum)
Oats (Avena spp.)
Popcorn (Zea mays- var. everta)
Rice (Oryza sativa)
Rye (Secale cereale)
Sorghum (milo) (Sorghum spp.)
Teosinte (Euchlaena mexicana)
Triticale (Triticum-Secale hybrids)
Wheat (Triticum spp.)
Wild rice (Zizania aquatica)(i) Representative commodities. Corn,wheat, and any other cereal grain crop.
(ii) Commodities. The commodities in­cluded in Crop Group 16 are: Forage,
fodder, stover, and straw of all com­modities included in the group cereal
grains group. EPA may establish sepa­
rate group tolerances on forage, fodder,
hay, stover, or straw, if data on the
representative commodities indicate
differences in the levels of residues on
forage, fodder, stover, or straw.
(26) Crop Group 17. Grass Forage, Fod­der, and Hay Group.
(i) Representative commodities. Ber­muda grass; bluegrass; and bfomegrassor fescue.
(ii) Commodities. The commodities in­cluded in Crop Group 17 are: Forage,
fodder, stover, and hay of any grass,
Gramineae/Poaceae family (either green
or cured) except sugarcane and thoseincluded in the cereal grains group,that will be fed to or grazed by live­
stock, all pasture and range grasses
and grasses grown for hay or silage.
EPA may establish separate group tol­erances on forage, fodder, stover, or
hay, if data on the representative com­modities indicate differences in thelevels of residues on forage, fodder, sto­ver, or hay.
(27) Crop Group 18. Nongrass Animal
Feeds (Forage, Fodder, Straw, andHay) Group.
(i) Representative commodities. Alfalfa
and clover (Trifolium spp.)(ii) Commodities. EPA may establish
separate group tolerances on forage,
fodder, straw, or hay, if data on the
representative commodities indicate
differences in the levels of residues on
forage, fodder, straw, or hay. The fol­lowing is a list of all the commodities
included in Crop Group 18:
CROP GROUP 18: NONGRASS ANIMAL FEEDS(FORAGE,FODDER,STRAW,AND
HAY)
GROUP-COMMODITIES(25) Crop Group 16. Forage, Fodder
and Straw of Cereal Grains Group.

Alfalfa (Medicago sativa subsp. sativa)
Bean, velvet (Mucuna pruriens var. utilis)
Clover (Trifolium spp., Melilotus spp.)
Kudzu (Pueraria lobata)
Lespedeza (Lespedeza spp.)
Lupin (LuPinus spp.)
Sainfoin (Onobrychis viciifolia);
Trefoil (Lotus spp.)
Vetch (Vicia spp.)
Vetch, crown (Coronilla varia)
Vetch, milk (Astragalus spp).

505

73

Appendix 4. 40 CFR 180.41 Crop Group Tables§180.4]40 CFR Ch. I (7-1-17 Edltion)I(28) Crop Group 19. Herbs and Spices
Group.
(i) Representative commodities. Ba.sil(fresh and dried); black pepper; chive;
and celery seed or dill seed.
(ii) Table. The following Lable 1 lists
all the commodities included in Crop
Group 19 and i.dentlfies the related sub­groups.
TABLE 1-CROP GROUP 19: HERBS AND SPICES
GROUP
Relatedcrop
sub­Commodities

groups198

Allspice (Pimenta diofca.)
Angelica (Angelica a.rchangefica) .
A'lise (anise seed) (Pimpinef!a anisum) ...
Anise, star (!llicium vernm)

WA

Caper buds (Capparis spinosa) ..... .

198
198
198
19A
19A
19A
19A
19A
198

Caraway {Cmvm cazv1) ..

198

Annatto (seed) .
Balm (lemon batm) (Melissa oftlcinalis') ...
Basil (Ocimum basilicum) ...
Borage (Borago officinalis)
Burnet {SangwSorba mino,j ..

Camomile_ (Anthemis nobifis) .,.............. .
Caraway, black (Nigel/a satlva} ....... .

198
198
19B
198
19A
198
19A
19A
19A
10B
19A
198

Cardamom (Elettaria ca.rdamomum) ........ .
Cassia bark (Cinnamomum aromaticum) .
Cassia buds ( Ginnamomum aromatfcum) .
Catnip (Nepeta ca.tar/a) ....
Celery seed (Ap/cum graveo/ens) ... ,Chervil (dried} (Anthriscus cerefoliUm)

Ch!ve (Allium schoenoprasum) ....
Chive, Chinese (A.fl/um tube1osum)
Cinnamon (G"innamomum verum)
Clary {Salvia scl8rea) .
Clove buds (Eugenia ca.ryophyliata) ....
Coriander {cilantro or

Chineseparsley)(leaf)(Corlandtum sativum) "Coriander (cilantro) (seed) (Coriandrum sativum) ..
Costmary ( Chrysanthemum bafsa.mita) ....
Culantro (leaf) (Eryngium foet/dum} ....
Cu!antro (seed) (Eryngium foetidum)
Cumin ( Cuminum cyminum) .

Curry (leaf) (Murraya koenig/!) .
om {dil!weed) (Anethum graveo!ens) ..
Dill (seed) (Anethum graveofens) ............ .
Fennel (common) (Foenlculum VLJ!gara}

19A
198
19A
19A
198
198
19A
19A
198
198

TABLE 1-CROP GROUP 19: HERBS AND SPICESIGROUP-Continued
Relatedcrop
sub·groupsCommodities

Fonne!,

Florence(seed)(Foeniculum

vulgareAzoricum Group) .
Fenugreek (Trigonel!a foenumgraecum) ...
Grains of paradlse (Aframomum melegueta) ., .
Horehound (MBrmbium vu(cpre)
Hyssop (Hyssopus officinalls) .. _.
Juniper berry {Juniperus communfs) .
Lavender (Lavandufa officinalls) ....
Lemongrass (Cymbopogon citratus) ..
Lavage {leaf) (Levistlcum offlcinale) .
Lavage (seed) {Levisticum officinaie)
Mace (Myristica tragrans) ...... ;..
Marigold ( Calendufa offfcina!is) ......... ......
Marjoram {Origanum spp.) (includes sweet or an·nual rna1joram. wild ma~oram or oregano, andpot marjoram) ...
Mustard (seed) {Brassica juncea, B. hirta, B. nlgra)
Nasturtium (Tropaeolum majLJS)
Nutmeg (Mynst/ca fragrans) .....
Parsley {dried) (Petroselinvm crispum)
Pennyroyal (Menfha puleglum) ...
Pepper, black (Piper nlgrum) ..•.
Pepper, white .....
Poppy (seed) (Pa.paver somniferum/
Rosemary (Rosemarinus ofticinc:Jlis)
Rue (Ruta graveolens)Saffron (Crocus sativus} .....
Sage (Salvia officinalis) ,Savory, summer and winier (Saturoja. spp.) .
Sweet bay (bay leaf) (Laums nobills) .. .
Tansy (Tanacetum vulgare) ... .
Tarragon (Artemisia dracunculus)
Thyme (Thymus spp.}
Vanilla (Vtmi!la p/anifofia)
Wintl;)rgreen ( Gaultheria pmcumbens) . .
Woodruff ( Galium odorata)
Wormwood (Artemisia absinfhium} ....... ,.

19B'
198
198

19A

19A
198

19A
19A
19A
198
198
19A

i 9A
198
19A
198

i9A
19A
19B
19B
198
19A

19A
198

19A
19A
19A
19A
19A
19A

198
19A
19A

"19A

(iii) Table. The following table 2 iden­lifies the crop subgTou.ps for Crop
Group 19, specifies the representative
commodities for each subgroup, andlists all the commodities included ineach subgroup.

TABLE 2-CFtOP GROUP 19 SUBGROUPS- - - - - - - - - - - -~---·-----------------------R-epresentatlve commodities
Crop Subgroup 19A. Herb subgroup,
Basll (fresh and dried) and chive ....

Commodities

Angelica; balm; basil; borage; burnet; camomile; catnip; chervH (dried); chlve;
Chive, Chinese, c!ary; coriander (leaf); costmary; culantro (leaf}; curry (leaf);
dillweed: horehound; hyssop; lavender; lemongrass; lavage (leaf); marigold;
marjoram (Origanum spp.): nasturtium; parsley (dried); pennyroyal; rose­mary; rue; sage; savory, summer and winter; sweet bay; lansy; tarragon;
thyme; wintergreen; woodruff; and wormwood.

Crop Subgroup 198. Sptce subgroup.

Black pepper: and celery seed or dill seed.

Allspice; anise (seed); anise, star; annatto (seed);, caper (buds); caraway; cara­
way, black; cardamom; cassia {buds); celery (seed); cinnamon; clove (buds);
coriander (seed); culantro (seed); cumin; dill (seed); fennel, common; fennel,
Florence (seed); fenugreek; grains of paradise; juniper (berry); fovage (seed);mace; mustard (seed); nutmeg; pepper, black; pepper, white; poppy (seed);saffron; and vanilla.

506

74

Appendix 4. 40 CFR 180.41 Crop Group Tables

Environmental Protection Agency§ 180.41(29) Crop Group 20. Oilseed Group.
(i)
Representative
commodities.
Rapeseed (canola varieties only); sun­flower, seed and cottonseed.

(ii) Table. The following Table 1 lists

all the commodities listed in Crop
Group 20 and identifies the related crop
subgroups and includes cultivars andior
varieties of these commodities.

TABLE 1-CROP GROUP 20: OILSEED GROUP•
Related crop
subgroups

Commodities
Borage, Borago offlcina/is L ....
Calendula, Calendula officinaiis L ...... .
Castor oil plant, Ricinus communis L ...... .
Chinese tal!owtree, Triadica sebif8ra (L.) Small ,.
Cottonseed, Gossypium hirsutum L. Gossypium spp ...
Crambe, Crambe hispanica L.; C. abyssinica Hochst. ex R.E. Fr
Cupl1ea, Cuphea hyssopHolia Kunth ....
Echium. Echium plantagineum L .
Euphorbia, Eophorbia esula L ..
Evening primrose, Oenothera bkmnis L
Plax seed, Unum usitatlssimum L
Gold of pleasure, Camelina sativa (L.) Crantz
Hare's ear mustard, Conringia on·enta.!is (L.) Dumort .
Jojoba, Simmondsia chlnensls (Link) C.K. Schneid ....
Lesquerella, Lesquere!Ja recurvata {Engelm. ex A. Gray) S. Watson .....
Lunaria, Luna.ria annua L ................................. .
Meadowfoam, Limnanthes alba Hartw. ex Benth
Milkweed, Asclepias spp
Mustard seed, Brassic8 hirta Moench, Sin.apis alba L subsp. Alba.. .
Niger" seed, Guizotia abyssinica (L.f.} Cass ...............
Oil radish, Raphanus sativus L. var. oleiformis Pers ......... .
Poppy seed, Papaver somniferum L. subsp. Somniferum .
Rapeseed, Brassies spp.; R napus L
Rose hip, Rosa rubigmosa L

20A
208
20B
208
20C
20A
20A
20A
208 ·
208
20A
20A
20A
208
20A
20A
20A
20A
20A
208
20A
20A
20A
208
208
20A
20B
20B
20A
20B
20B
208

Safflower, Catthamus rlnctorious L .
Se!lame, Sesamum indicum L. S. radiatum Schumacti. & honn
Stokes aster, Stokesia /aevis (Hill) Greene ....
Sunflower, Helianthus annuus L
Sweet rocket, Hesperls matronafis L .
Tallowwood, Ximenia americana L .
Tea oil plant, Camellia. o!eifera C. Abel .....
Vernonia, Vernonia galamens1s (Cass.) Less
Cu!tlvars, var1otles, and/or hybrids of these,(iii) Table. The following Table 2
identifies the crop subgroups for Crop
Group 20, specifies the representative

commodities for each subgToup andlists all the commodities included 1neach subgroup.

t

TABLE 2--CROP GROUP 20: SUBGROUP LISTING---------------Representative commodities.Rapese~d,~:~~:~~:,:~~~~~~

R;peseed ;.,bgro~~---- ..

Commodities

orage; cramba; cuphea; echium; flax seed: gold of pleasure;hare's ear mustard; lesquerel!a: lunaria; meadowfoam; milk­
weed; mustard seed; oi! radish; poppy seed; rapeseed; ses­ame; sweet rocket <.,'Ultivars, varieties, and/or hybrids ofthese.
Crop subgroup 208. Sunflower subgroup

Sunflower, seed

Calendula; castor oil plan!; chinese tallowtree; euphorbla;
evening primrose; jojoba; n!ger seed; rose hip; safflower;
stokes aster; sunflower; ta!lowwood: tea oil plant: vernonia;
cultivars, varietfes, and/or hybrids of these.
Cottonseed; cultivars, varioties, ancVor hybrids of these.

507

75

Appendix 4. 40 CFR 180.41 Crop Group Tables§180.41

40 CFR Ch. I (7-1-17 Edition)(30) Crop Group 21. Edible fungiGroup.
(i) Representative commodities. White
button mushroom and any one oyster
mushroom or any Shiitake mushroom.
(i.i) Table. 'l'he following is a list of all
the commodities in Crop Group 21.
'l'here are no related subgroups.

CROP GROUP 21-EDIBLE FUNGI GROUP­COMMODITIES
Blewitt (Lepista nuda)
Bunashimeji {Hypsizygus ma.rrmoreus)
Chinese mushroom (Volvarieffa volvacea} (Bull.) Singer

Enokl (Flammufjna velutipes) (Curt.) Singer
Hjme-Matsutake (Agaricus blaw1) Murill
Hirmeota tAuricularia aurlculalj
Mattake (Grlfola frondosa)
Morel (Morchelfa spp.)
Nameko (Rho/iota nameko)
Net Bearing (Diatyopho1a)
Oyster mushroom (PJeurotus spp.)
Porn Porn (Hi'mcium erinaceus)
Reishl mushroom (Ganoderma !ucidum (Leyss. Fr.} Karst.)
Rodman's agarious (Agaricus bitorquis) (Quel.) Saccardo
Shlitake mushroom (Lent/nu/a edodes (Berle) Peg!.)
Shimeji (Tricflo/oma conglobalum)*:~:~~:,~r::riaspp.) ~White button mushroom (Agarfcus
Imbach)White Jelly Fung! (Tremelfa fuciformls}

blsporous(Lange)(31) Crop Group 22. Stalk, Stem andLeaf Petiole Vegetable Group.
(i) Representative cornmodtf:ies. Aspar­
agus and celery.
(Ii) Commodities. The following Table
1 lists all commodities included in Crop
Group 22.
TABLE 1-CROP GROUP 22: STALK, STEM AND LEAF PETIOLE VEGETABLE GROUP
Related crop

Commoditi0S

subgroups

Agave (Agave spp.) .............................................................,.............. ,.... ,.................. ,.............." ............................. i~;fi~~iJi:$1f:!:;:;~k:b~;~·~~::••i;i:~~~i:;;i;i:;•ii ;;i~i~:~·:pi:•;i~;;~•;ii:: I
G!gantochloa spp., Nastus elatus; Phylfostachys spp.; Thyrsostachys spp.) .
Cardoon (Cynara cardunculus L)

1................ :I

Celery (Apium graveo/sns var. dulcl3 (MUI.) Pars.) ...........................
..... :.......... lCelery, Chinese {Apium gravEolens L. var. secalfnum (Alef.) Manst.) .
. .................... :Ge!tuce (Lactuca sativa var. angustana LH. Balley)................ .. .................................
i
Fennel, Florence, fresh !eaves and stalk (Foentculum vufgare subsp. vu!gare var. azoricum (Mill.)
Fem, edlbla, fiddlehead ....
.......... .
Fuki (Petasites japonicus (Siebold & Zucc.) Maxim.)
Kale, sea (Grambo maritima L.) .................
!

~~r!t~~:(::::s~;=~· ~~~Th~1"i:)"':..........

I:::::::·:··::::::::

i

no..

gongyl~~~-~:! ..:::::::......

Prickly pear, pads (Opuntia ficus-fndica (L.) Mill.. Opuntia spp.)......
....... 1
Pr~l~~a\i)a~,.. ~~~· .:.~~~.~-~:.!,~~~-~ngelmannli Salm-Dyck ex Engelm. var. llndhelmerl (Engelm.) B.D. Parfitt ~. ,Rhubarb (Rheum x rhabarbarum L.) ...... .
Udo (Amlia cordata Thunb. ) ....................
Zuiki (Co/ocasia gigantea (Blume) Hook. f.)
Cu!tivars, varieties, and hybrids oi these commodities.

1
... !

(iii) Crop subgroups. The following
Ta.ble 2 identifies the crop subgroups
for Crop Group 22, spectfies the rep-

22A
22B
22B
22B
22A
22A
22A
22B
22A
22A
22A
22A
22A
22B
22B
228

resentative commodities for each sub­gToup, a.nd lists all the commodities in­cluded in each subgroup.

508

76

22A
22A
22A

Appendix 4. 40 CFR 180.41 Crop Group Tables

Environmental Protection Agency§180.41

TABLE 2-CROP GROUP 22: SUBGROUP LISTING
Representative commodities

Commodities

Crop Subgroup 22A. Stalk and stem vegetable subgroup
Asparagus ....

Agave; aloe vera; asparagus; bamboo, _shoots; cell.tJce; fennel, flor­
ence, fresh leaves and stalk; fElrn, edible, fiddlehead; kale, sea;kohlrabi; palm hearts; prickly pear, pads; prickly pear, Texas, pads;
cultivars, varieties, and hybrids of these commodities.
Crop Subgroup 228. Leaf petiole vegetable subgroup

Celery ....

Cardoon; celery; celery, Chinese; fukl; rhubarb; udo; zuiki; cultivars,
varieties, and hybrids of these commodities.

(32) Crop Group 23. Tropical and Sub­
tropical Fruit, Edible Peel Group.
(i) Representative commodities. Date,fig, guava, and olive.

(ii) Commodities. The following Tablel lists all commodities includ�d in Crop
Group 23.

TABLE 1-CROP GROUP 23: TROPICAL AND SUBTROPICAL FRUIT, EDIBLE PEEL GROUP
Related crop
subgroups

Commodities
Agai (Euterpe oferacea Mart.)
Acerola (Mafpighia emarginata DC.) ..............................................
Achachairll (Garcinia gardneriana (Planch. & Triana) Zappi)
African plum (Vitex doniana Sweet) .. .
Agritos (Berberis trifo/iolata Morie.) .
Almondette (Buchanan/a Janzan Spreng.) ......
Ambarella (Spondias dufcis Sol. ex Parkinson)
Apak palm (Brahea dulcis (Kunth) Mart.) ..
Appleberry (Billard/era scandens Sm.) .
Araza (Eugenia stipitata McVaugh) .
Arbutus berry (Arbutus unedo L.)..............
Babaco (Vasconcelleax hef!bomii(V.M. Badillo) V.M. Badillo)
Bacaba palm (Oenocarpus bacaba Mart.) ......
Bacaba-de-leque ( Oenocarpus distichus Mart.) . ..
Bayberry, red (Morella rubra Lour.) ...
Bignay (Anfidesma bunius (L.) Spreng.) .
Bilimbi (Aver!hoa billmbi L.) ..........
Boroj6(Borojoa patinoi Cuatrec.) ..
Breadnut (Brosimum a/icastrum Sw.) .... ..
Cabeluda (Plinia glomerata (0. Berg) Amshoff) .
Cajou, fruit (Anacardium giganteum Hance ex Engl.) .
Cambuca (Mar/ierea edu/is Nied.) .
Carandas�plum (Carissa edulis Vahl) .... ..
Carob (Ceratonia silfqua L.) .
Cashew apple (Anacardium occidentale L.) ................
Ceylon iron wood (Manilkara hexandra {Roxb.) Dubard) .
Ceylon olive (Efaeocarpus serratus L.) ..................
Cherry-of-the-Rio-Grande (Eugenia aggregata (Veil.) Kiaersk.) .
Chinese olive, black (Canarium tramdenum C.D. Dai & Yakovlev)
Chinese olive, white (Canar/um album (Lour.) Raeusch.) ...... .
Chirau!i-nut (Buchanan/a /atifo/ia Roxb.) . ............
Ciruela verde (Bunchosia armeniaca (Gav.) DC.)
Cocoplum (Chrysobalanus icaco L.) .
Date (Phoenix dacty/ifera L.) . .
Davidson's plum (Davidsonia pruriens F. Muell.)Desert-date (Balanltes aegyptiacus (L.) Delile) ..
Daum palm coconut (Hyphaene thebaica (L.) Mart.) .
False sandalwood (Ximenia americana L.)
Feijoa (Acea se/lowiana (0. Berg) Burret)
Fig (Ficus carica L.)...............
Fragrant manjack (Cordia dlchotoma G. Forst.) .
Gooseberry, abyssinian (Dovya/is abyssinica (A Rich.) Warb.) . ... .
Gooseberry, Ceylon (Dovyalis hebecarpa (Gardner) Warb.)
Gooseberry, Indian (Phylfanthus emblica L.) ............
Gooseberry, otaheite (Phyllanthus acidus (L.) Ske01s) .
Governor's plum (Flacourtia ind/ca (Bunn. F.) Merr.) .
Grumichama (Eugenia brasfliensis Lam) ..

23C
23A
238
23A
23A
23A
238
23C
23A
238
23A
238
23C
23C
23A
23A
238
23B
23A
23A
238
23B
23A
238
238
23A
23A
23A
23A
23A
23A
238
23A
23C
23B
23A
23C
23A
238
238
23A
23A
23A
238
23A
23A
23A

509

77

Appendix 4. 40 CFR 180.41 Crop Group Tables§180.41

40 CFR Ch. I (7-1-17 Edition)

TABLE 1-CROP GROUP 23: TROPICAL AND SUBTROPICAL FRUIT, EDIBLE PEEL GROUP-Continued
Related crop
subgroups

Commodities
Guabiroba (Campomanesia xanthocarpa O. Berg)
Guava (Psidium guajava L.) ..
Guava berry (Myre/aria f!oribunda (H. West ex Willd.) o. Berg) .
Guava, Brazilian (Psidium guineense Sw.)
Guava, cattley (Psldium cattleyanum Sabine) ....
Guava, Costa Rican (Psidium friedrichstha/ianum (0. Berg) Nied.) .
Guava, Para (Psidium acutangulum DC.) .................................
Guava, purple strawberry (Psidium catt�eyanum Sabine var. catt/eyanum) ...
Guava, strawberry (Psidium catrleyanum Sabine var. /ittorale (Raddi) Fosberg) .
Guava, yellow strawberry (Psidium catt/eyanum Sabine var. cattleyanum forma lucidum 0. Deg.}
Guayabillo (Psidium sarlorianum (0. Berg) Nied.) .
lllawarra plum (Podocarpus elatus R. Br. Ex Endl.)
lmb9 (Garcinia fivingstoneiT. Anderson) . .
lmbu (Spondias tuberosa Arruda ex Kost.) .............
Indian-plum (Flacouttia jangomas (Lour.). basionym)......... ···!···
Jaboticaba (Myrciaria cau/iflora (Mart.) O. Berg) .
Jamaica-cherry (Muntingia calabura L.) ..
Jambolan (Syzygium cumini (L.) Skeels)
Jelly palm (Butia capitata (Mart.) Becc.)
Jujube, Indian (Ziziphus mauritiana Lam.) .
Kaffir-plum (Harpephyllum caffrum Bernh. Ex C. Krauss) .
Kakadu plum (Terminafia /atipes Benth. subsp. psilocarpa Pedley)
Kapundung (Baccaurea racemosa (Relnw.) Mull. Arg.)
Karanda (Carissa carandas L.)
Kwai muk (Attocarpus hypargyreus Hance ex Benth.) .
Lemon aspen (Acronychia acidu/a F. Muell) .........
Mangaba (Hancomia speciosa Gomes)
Marian plum (Bouea macrophy!la Griff.)
Mombin, malayan (Spondias pinnata (J. Koenig ex L. f.) Kurz)
Mombln, purple (Spondias purpurea L.) .
Mombin, yellow (Spondias mombin L.) ....
...... .................
Monkeyfruit (Artocarpus /acucha Buch. Ham.) .............
Monos plum (Pseudanamomis umbel/ulitera (Kunth) Kausel) ..
Mountain cherry (Bunchosia cornifolia Kunth) .
Nance (Byrsonima crassifolia (L.) Kunth) .
Natal plum (Carissa macrocarpa (Eckl.) A. DC.)
Noni (Marinda citrifolia L.)
Olive (Olea europaea L. subsp. europaea) .....
Papaya, mountain ( Vasconce//ea pubescens A. DC.) .... .
PatauS. (Oenoca,pus bataua Mart.) .......... ...................
Peach palm, fruit (Bactris gasipaes Kunth var. gasipaes) .....
Persimmon, black (Diospyros texana Scheele)
Persimmon, Japanese (Diospyros kaki Thunb.) .
Pitomba (Eugenia /uschnathiana Klotzsch ex 0. Berg)
Plum-of-Martinique (Flacourtia inermis Roxb.) ..
Pomerac (Syzygium malaccense (L.) Merr. & L.M. Perry)
Rambai (Baccaurea motleyana (Mull. Arg.) Mull. Arg.)
Rose apple (Syzygium jambos (L.) Alston)
Rukam (Flacourtla rukam Zoll. & Moritizi) .. .
Rumberry (Myrciaria dubia (Kunth) McVaugh Myrtaceae}
Sea grape (Cocco/oba uvifera (L.) L.)
Sentul (Sandoricum koetjape (Burm. F.) Merr.) ........... .
Sete-capotes (Campomanesia guazumifo!ia (Cambess.) 0. Berg) ..
Silver a.span (Acronychia wilcoxian (F. Muell.) T.G. Hartley) .
Siarfruit (Averrhoa carambola L.) ..
Surinam cherry (Eugenia unif!ora L.) .....
Tamarind (Tamarindus indica L.)
Uvalha (Eugenia pyriformis Cambe.s.s )Water apple (Syzygium aqueum (Burm. F.) Alston) .
Water pear (Syzygium guineense (Willd.) DC) ...........
Water berry (Syzygium cordatum Hochst. Ex C. Krauss) ..................
Wax jambu (Syzygium samarangense (Blume) Merr. & L.M. Perry} .....
Cultivars, varieties, and hybrids of these commodities.

(iii) Table. The following Table 2
identifies the crop subgroups for Crop
Group 23, specifies the representative

commodities for each subgroup, andlists all the commodities included ineach subgroup.

510

78

23A
238
23A
23A
238
23A
238
238
238
238
23A
23A
238
238
23A
238
23A
23A
23C
238
23A
23A
23A
23A
238
23A
238
238
23B
238
23A
238
23A
23A
238
238
238
23A
238
23C
23C
23A
238
23A
23A
238
238
238
23A
23A
23A
238
23A
23A
238
238
238
238
23A
23A
23A
23A

Appendix 4. 40 CFR 180.41 Crop Group Tables

Environmental Protection Agency§180.41

TABLE 2-CROP GROUP 23: SUBGROUP LISTING------Representative-------------�---------------commodities
Commodities
Crop Subgroup 23A. Tropical and Subtropical, Small fruit, edlble peel subgroup
Olive . .

-l.

Acerola; Afrfcan plum; agrilos; almondetle; appleb?rry; arbutus berry;bayberry, red; bignay; breadnut;•cabeluda; carandas-plum; Ceylon
Iron wood; Ceylon olive; cherry-of-the-Rio-Grande; Chinese olive,
black; Chinese olive, white; chirauli-nut; cocoplum; desert-date;false sandalwood; fragant man]ack; gooseberry, abyssinian; goose­berry, Ceylon; gooseberry, otaheite; governor's plum; grumichama;guablroba; guava berry; guava, Brazi,lian; guava, Costa Rican;
guayabillo; mawarra plum; Indian-plum; Jamaica-cherry; jambolan;
kaffir-plum; kakadu plum; kapundung; karanda; lemon aspen;
mombin, yellow; monos plum; mountain cherry; olive; persimmon,
black; pitomba; plunH1f,Martinique; rukam; rumberry; sea grape;sete·oapotes; sliver aspen; water apple; water pear; water berry;
wax Jambu; culttvars, varieties, and hybrids of these.commodities.

Crop Subgr�u� 238. Tropical and Subtropical� Medium to large ti-uit, edible peal subgrouJ

Fig and guava .

·aAchachaltU; ambarella; arazfl; babaco; bilimbi; boroj6; cajou, fruit;cambuc:8.; carob; cashew apple; ciruefa verde; davidson's plum;
feijoa; fig; gooseberry, Indian; guava; guava, cattley; guava, Para;
1 guava, purple strawberry; guava, strawberry; guava, yellow slraw,
berry; imb8; imbu; jaboticaba; jujube, Indian; kwai muk; mangaba;
Marian plum; mombln, Malayan; mombin, purple; monkeyfrult;
nance; natal plum; noni; papaya, mountain; persimmon, Japanese;
pomerac; rambai; rose apple; seritul; starfruit; Surinam cherry; tam­arind; walha: outtlvars, varieties, and hybrids of these commodities.

------------- --------------------------

Crop Subgroup 23C. Tropical and Subtropical, Palm fruit, edible peel subgroup

." I

Date

(33) Crop Group 24. Tropical and Sub­
tropical Fruit, Inedible Peel Group.
(i)------

Ayaf; a_pak palm; bacaba palm; bacaba·de·leque; dale; doum palm
, coconut; jelly palm; patauS:; peach palm, fruit; cu!tivars, varieties,
ities.
e commoda
and hybrids ot thesa__a_ _ _a__a_...l____a _

Rep-resentative

commodities.

Atemoya or sugar apple, avocado, ba­nana or pomegranate, dragon fruit,

lychee, passionfruit 1 pineapple, andprickly pear, fruit.
(ii) Commodities. The following 'I'able
I list� all commodities included in Crop
Group 24.

TABLE 1-CROP GROUP 24: TROPICAL AND SUBTROPICAL FRUIT, INEDIBLE PEEL GROUP- - - - - - - - - - - - - - - - - - - - - - - - - - ---Com/1lCldities

Abiu (Pouteriri! caimito (Ruiz & Pav,} Radlk)
Alsen (Boscia senega!ensis (Pers.) Lam.) .
Akee apple (Bfighia sapida K.D. Koenig) ..
Atemoya (Annona cherimo/a Mill. X A squamosa L)
Avocado (Perse-a amedcana MU!.)
Avocado, Guatemalan (Porsaa americana. Mill. var. guatema./ensis) ....................a.a..........a...
Avocado, Mexican (Persea americana Mill. var. drymifo!ia (Schltd!. & Cham.; S,F. 8/ak}
Avocado, West Indian (Persea amer/cans var. americana)
Bacury (Platania insignis Mart.} ................
Bael fruit (Aegfe marmetos (L) CorrOO} ....
Banana (Musa spp.) .
�anana, dwarf {Musa hybrids; Musa acuminaJa Colla)
Blnjai (Manglfera caesia Jack)... ,.... .
Biriba (Annona mucosa Jacq.) . .
Breadfruit (Artocarpus air/Ifs {Parkinson) Fosberg) . . .
Btmnese grape (Baccaurea ramiflora Lour.)Canistel (Pouteria c..,mpechiana (Kuntll) Baelml) ........a................
Cat's-eyes (Dimocarpus fonga.n Lour. subsp. malesianus Leenh.) .
Champedak (Artocarpus integer{Thunb.) Merr.) . .
Cherimoya (AnnDna chertmola Mill.) ...................... _ ..............a....,.....
CupuacU (Theobroma grandlflorum {Wmd, Ex Spreng.) K. Schum.)
Custard applo (Annona reticut.ata L.)......
Dragon fruit (Hyfocereus undatus (Haw.) Britton & Rose) .
Durian (Durio zfbethinus L.)

511

79

Related crop
subgtoups
248
24A
248
24C
248
248
248
24B
248
24A
248
248
24B
24C
24C
24A
24B
24A
24C
24C
24B
24C
24D
24C

Appendix 4. 40 CFR 180.41 Crop Group Tables§ 180.41

40 CFR Ch. I (7-1-17 Edition)

TABLE 1-CROP GROUP 24: TROPICAL AND SUBTROPICAL FRUIT, INEDIBLE PEEL GROUP-Continued- - - - - - ----- - - - - - - - ~--------------------

Related crop
Commod1tias- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - ___s_u_bg_ro__

Elephant-apple (Limonia acidissima L.)
Etambe (Mang!fera zeylanica. (Blume) Hook. F.)
Granadilla (Pass/flora ligularis Juss.) ...
Granadilla, giant (Passiffora quadrangularis L) .
.... ............................. .
llama {Annona macroprophyllata Donn. Sm.)
Inga (fnga vera Wi!ld. subsp. afflnls (DC.) T.D. Penn.) .
Jackfruit (Artocarpus heterophyllus Lam.)
Jatob.i (Hymenaea courbarlf L.)
Karuka (Pandanus Julianettii Martelli) ,u,........
Kei apple {Oovya/is ca.!fra {Hook. F. & Ha1v.) Warb.)
Langsat (Lansium domestfcum Correa) ......
·1
Lanjut (Manglfera /agenlfera Griff.)
Longan (Dimocarpus longan Lour.)Lucuma (Pouteria /ucuma {Ruiz & Pav.} Kuntze) ...
Lychee (Utchi chinensis Senn.)
Mabolo (Dlospyros blancof A. DC.) .
•······ :Madras-thorn (Pithece!lobium du/ce {Aoxb.) Benth.)
Mammy-apple (Mammea amerlca.na L.J...... 1
Manduro (BatanitP-S maughamif Sprague)
Mango (Mangifera ind/ca L) ......................
. ................... !
Mango, horse (Mangifera foetida Lour.) ....
...... ............. ..
Mango, Saipan (Mangifera odorata Griff.)
Mangosteen (GBrcinia mangostana L. )Marang (Artocarpus odoratisslmus Slanco) . .
Marmaladebox {Gan/pa americana L.)
f·.ilatisla (Matisia cordata Humb. & Bonpl.) .
Mesquite (Prosopis juliflora (Sw.) DC.) ............, ................... ..
Mongongo, fruit (Schinziophyton ra.utanenii {Schlnz) Radcl.-Sm)
Monkey-bread-tree {Adansonia dig!tata L) ...
Monstera (Monstera de!iciosa Llebm.) •...
.........
Nicobar-breadfruit (Pandanus leram Jones ex Fontana) .. .
Paho (Mangifera a!tissima Blanco)
Pandanus (Pandanus ut,tis Bory)
Papaya (Carrea papaya L.) ... ., ....
Passionflower, winged-stem (Passfflora alat8 Curtis) .. .
Passlontni!t (Passfflora adu/ls Sims) ..................................... .
Passionfruit, banana (Passif/ora mpartita var. molfissima (Kunth) Holm-N!els. & P. Jorg.)
Passionfruit, purple (Passff/ora edulis Sims forma edulis) . ..
Passlontruit, yellow (Pass/flora edulis Sims torma flavicarpa O. Deg.) ..
Pawpaw, common (Asimina triloba (L.) Dunal) .
Pawpaw, small-flower (Asfmfna palViffora (Mlchx.) Dunal)P�lipisan (Mangifera casturi Kosterm.)
Pequi (Caryocar brasiliense Cambess)
Poquia (Catyoear villosum (Aubl.) Pors.) ..........
Persimmon, American (Dfospyros virginiana. L.)
Pineapple (Ananas comosus (L} Merr.} ....................... ... .........
Pitahaya (Hylocereus polyrhiz[!s (FAC. Weber) Britton & Rose) ........................
Pitaya (f-lylocereus sp. including H. meg.-.Wnthus (H. ocamponis and H. pofych,.zus)
P1taya, amarilla (1-lyfocereus triangu/aris Britton & Rose)
Pitaya, roja (HyJocereus ocamponis {Salm·Dyck) Britton & Rose) ..
Pitaya, yellow {Hylocereus megalanthus (K. Schum. ex Vaupel) Ralf Bauer}
Plantain (Musa paradis1aca L)
Pomegranate (Pun/ca granatum L.) .
Poshte (Annona liebmanniana Bail!.) .
Prickly pear, fruit (Opuntia ficus-indica (L.) Mill.)..........."'"""" ..................................................
Prickly pear, Texas, fruit (Opuntfa cngcJmannii Salm·Dyck ex Engelm. var. Jlndhoimen (Enge!m.) B.D. Parfitt &Plnkav) . .
Pulasan (Nephe/ium ramboutan--ake (Labill.) Leenh.)
Quandong {Santa/um aouminatum {R. Br.) DC.)
Rambutan (M;,phelillm lappaceum L ) . .
Saguaro (Camegiea gigantea (Engelm.) Britton & Rose) ...
SapodiUa (Manilkara zapota (L) P. Royen)
Sapote, black (Diospyros digyna Jaoq.) ..
Sapote, green (Pouteria viridis (PJttier) Cronquist) .......... .
Sapote, mamey (Pouteria sapota {Jacq.) H.E. Moore & Steam)
Sapote, white ( Casimiroa edu!is La LI ave & Lex) .
Sataw (Parkia speciosa Hassk.) .....
Satinleaf (Chrysophy/Jum ollvifonne L.)
Screw-pine (Pandanus tectorius Parkin::ion) ··"···
Sierra Leone-tamarind (Dialium guineenss WiUd.)
Soncoya (Annona purpurea Moc. & Sesse ex Dunal) ..

. ······: ::::----·--r·I
I.i:::.::i

.,

I

512

80

~?.~24C
24B
24E
24E
24C
24A
24C
24B
24C
24B
248
24B
24A
24B
24A
24B
24A
24C
24A
248
24B
24B
24B
24C
24C
24A
24A
24A
24C
24E
24C
24B
24C
24B
24E
24E
24E
24E
24E
24B
24A
24B
24B
24B
24B
24C
24D
24D
24D
24D
24D
24B
24B
24B
24D
240
24C
24B
24C
24D
24C
24B
24B
24C
24B
24B
24A
24B
24A
24C

Appendix 4. 40 CFR 180.41 Crop Group Tables

Environmental Protection Agency§ 180.41

TABLE 1-CROP GROUP 24: TROPICAL AND SUBTROPICAL FRUIT, INEDIBLE PEEL GROUP-Continued-ad crop
Commodities
groups-Soursop (Annona muricata L.) .................
24C
Spanish lime (Meiicoccus bijugatus Jacq.)
24A
Star apple (Chrysophylfum c,,1/nito L.) ...........
24B
Sugar apple (Annona squamosa L.)
24C
Sun sapote \Licanla platypus (Hems!.) Frrr5ch) ............
.....a..............a..a.....a..a.... j.24C
Tamarind-of-the-Indies (Vanguerla mada.gascarlensisJ.F. GmeL)
24B
Velvet tamarind (Dialium lndum L.) ..
...
24A
Wampl (C/ausena lansium (Lour.) Skeels) ...
................. ,24AWhite star apple (Chrysophyllum afbidum G. Don) .
Wild !oquat (Uapaca kirfdana MUii. Arg.) .........
Cultivars, varieties, and hybrids of these commodities.
·············· 1
�::- - - - · - - · · · - - · - · · · · - - · · · - - - · - - ··-·····-··

---

.. I

(111) ?'able. The following Table 2
identifies the crop subgroups for Crop
Group 24, specifies the representative

commodities for each subglloup, andlists all the commodities included ineach subgroup.

TABLE 2-CROP GROUP 24: SUBGROUP LISTING--- - -----------··
����sentative c��amoditres["a -·-- ---·��
--·Crop Subgroup 24A. Tropical and Subtropical, Small fruit, inedibie peel subgroup..a....... : Aisen; baei fruit; Burmese grape: cat's-eyes; ing€l.; !ongan; lychee: ma­: dras-thom; manduro: matisia; mesquite; mongongo, fruit; pawp.'.lw,
small-Jlower; satinleaf; Sit;irra Leone-tamarind; S�anish lime; velvet
tamarind; wampi; white star apple; cultivars, vanetfes, and hybridsof these commodities.

Lychee ..a.

I

Crop Subgroup 24B. Tropical and Subtropical, Medium to large fruit, smooth, inedible peel subgroup·· ·-------------·-------�------------ --··:
akee apple; avocado; avaocado, Guatemalan; avocado, Mexican;
Avocado, plus pomegranate or banana .....a.a.................. Abiu;
avocado, West Indian; bacvry; banana; banana, dwarf; blnjai;
canistel; cupuacU; etambe;.a jatob8.; kei apple; langsat; !anjut;
lucuma; mabolo; mango: mango, home; mango, Saipan;
mangosteen; paho; papaya; pawpaw, common; peUpisan; pequi;la pequia; persimmon, American; plantain; pomegranate; poshte;
quandong; sapote, black; sapote, green; sapote, white; sataw;
screw-pine; star apple; tamarind-oHhe-!ndies; wild loquat; cultivars,
varieties, and hybrids of these comrnod1t1es.
-------��----·---�----· ·- --------------------------- �a .
Crop Subgroup 24C. Tropical and Subtropical, Medium to large fruit, rough or hairy, Inedible peel subgroup. --·--··---------Atemoya; biriba; breadfruit; champedak; cherimoya; custard apple;
Pineapple, plus atemoya or sugar apple ...
durian; elephant-apple; ilama; jackfrult; karuka; mammy- apple;
marang; mannaladebox; monkey-bread tree; nicobar-breadfruit;pandanus; pineapple; pulasan; rambutan; �poqilla; sapote,mamey; soncoya; soursop: sugar apple; sun sapote; cultlvars, vari­eties, and hybrids of these commodltieS.
Crop Subgroup 240. Tropical and Subtropical, Cactus, inedible peel subgroup
Dragon fruit and Prickly pear fruit ..

;ass1onfru1t.

.

Dragon fruit; pitahaya; pitaya; pitaya, amaril!a; pitaya, roJa; pltaya, yel­low; prickly pear, fruit; prickly pear, Texas, fruit; saguaro; cul!ivars,
varieties, and hybrids of these commodlties.

Crop Subgroup 24E. Tropical and Subtropical, Vine, inedible peel subgroup- -.a --.a ..
-. . . · Granadll�:-;-nadrlla, giant, monstera, passionflower, wrngad-stem,
1 passronfrn1t, pass1onfrutt, banana passtonfru1t, purple pass1onfruit,
yellow cult1va.rs va11et1e.s, and J1ybrk!s of these commod1t!es------ --- -----------·-- ---·--

---

513

81

Appendix 5. Physicochemical Properties of Imidacloprid, Clothianidin,
Thiamethoxam, and Dinotefuran

Property

Imidacloprid

Dinotefuran

Clothianidin

Thiamethoxam(E)-1-(2-Chloro-1,3thiazol-5-ylmethyl)3methyl-2nitroguanidine

3-(2-Chlorothiazolyl-5ylmethyl)-5-methyl[1,3,5]oxadiazinan4-ylidene-Nnitroamine

Chemical
Structure:
IUPAC
Namea
N-{1-[(6-Chloro-3pyridyl)methyl]-4,5dihydroimidazol-2-yl}nitramide

Molecular
Formulaa
Molecular
Weight(CAS No.)a
Water
Solubility(WS)a
Octanol:
Water
Coefficient(Kow)aSoil
Adsorption
Coefficient(Koc)aHenry’s
Law
Constant(H)aVapor
Pressure(VP)bTerrestrial
Field
Dissipation
Half-Life(TFD)ba.

b.

N-methyl-N’-nitro-N”[(tetrahydro-3furanyl)methyl)]guanidine

C9H10ClN5O2

C7H14N4O3

C6H8ClN5O2S

C8H10ClN5O3S

255.7 g/mole (13826-41-3)

202.2 g/mol (165252-70-0)

249.7 g/mole(210880-92-5)

291.7 g/mole(153719-23-4)

580 mg/L@ 20 °C

39,830 mg/L@ 20 °C

327 mg/L@ 20 °C

4100 mg/L @ 25 °C

3.7 @ 21 °C

0.283 @ 25 °C

4.4 (EPISuite v4.11)

0.74 @ 25 °C

277 – 411 mL/g

6 – 45 mL/g

84 – 345 mL/g

33 – 177 mL/g

9.9 x 10-13 atm m3 mol-1

8.63 x 10‐14 atm m 3 mol-1

2.9 x 10-16 atm m3
mol-1

4.63 x 10-15 atm m3
mol-1

1 x 10-7 mmHg @ 20 oC

1.28 x 10-8 mmHg @ 30 oC

1 x 10-7 mmHg @20 oC

5 x 10-11 Hg @ 25oC

27 - 146 Days

23 - 77 Days

282 - >982 Days

83-91 Days

Data obtained from the following U.S. EPA preliminary pollinator assessments:•
U.S. EPA, & DPR. (2015). Preliminary pollinator assessment to support the registration review of imidacloprid. Report
Number EPA-HQ-OPP-2008-0844-0140. Washington, D.C.: U.S. EPA.
•
U.S. EPA. (2017a). Draft assessment of the potential effects of dinotefuran on bees. Report Number EPA-HQ-OPP-20110920-0014. Washington, D.C.: Author.
•
U.S. EPA. (2017b). Preliminary bee risk assessment to support the registration review of clothianidin and thiamethoxam.
Report Number EPA-HQ-OPP-2011-0865-0173. Washington, D.C.: Author.
DPR, 2018. DPR Pesticide Chemistry Database. Department of Pesticide Regulation Environmental Monitoring Branch Internal
Website. Accessed: April 30, 2018.

82

Appendix 6. Tier I Toxicity Values for Imidacloprid, Clothianidin, Thiamethoxam, and Dinotefuran

Endpoints Used in Tier I Risk Determinations
Adult Acute Contact Adult Acute Oral
LD50
LD50
Imidacloprid
0.043 µg ai/bee
0.0039 µg ai/bee(96 hours) a(48 hours) a
Clothianidin
0.0275 µg ai/bee
0.00368 µg ai/bee(48 hours) c(48 hours) c
Thiamethoxam
0.024 µg ai/bee
0.0044 µg ai/bee(96 hours) d(48 hours) d
Dinotefuran
0.024 µg ai/bee
0.0076 µg ai/bee(96 hours) e(48 hours) e
Notes:-

Adult Chronic Oral
NOEL / LOEL
0.00016 / 0.00024 μg ai/bee(10 Days) a
0.00036 / 0.00072 μg ai/bee/day(10 Days) c
0.00245 / (unknown) μg ai/bee/day(10 Days) d
0.0015 / 0.0035 μg ai/bee/day(10 Days) e

Larval Acute
LD50> 0.0018 μg ai/larva/day(7 Days) b> 0.0018 μg ai/larva/day(7 Days) c
Unknown> 3.75 μg ai/larva/day(8 Days) f

Larval Chronic
NOEL / LOEL
0.0018 / > 0.0018 µg ai/larva/day(21 Days) a
0.0009 / 0.0018 μg ai/larva/day(22 Days) c
0.0157 / (unknown) μg ai/larva /day
(22 Days) d
3.75 / > 3.75 μg ai/larva/day(22 Days) e

Adult acute contact and oral toxicity tests are single exposure tests followed by observation periods. A 48 hour observation period is standard, but the observation period must
be extended if there is an increase in mortality from 24 to 48 hours (OCSPP 850.3020; OECD 213).
In the adult chronic oral tests bees are fed a diet containing the test chemical continuously for ten days (OECD 245).
The larval acute and larval chronic toxicity endpoints are derived from a single toxicity test in which larvae are exposed to the test chemical on Days 4, 5, and 6 (three days of
exposure), and then observed until emergence (Day 22). Mortality is assessed at 8 days to determine an 8-day LD50. Emergence (the inverse of mortality) is assessed at 22days to determine a 22-day NOEL (OECD 239).

Referencesa
2016, U.S. Environmental Protection Agency, California Department of Pesticide Regulation, Preliminary Pollinator Assessment to Support the Registration Review of Imidacloprid, EPA-HQ-OPP-20080844-0140.
b
2011, Nikolakis, A., Theis, M., and Przygoda, D., Imidacloprid tech.: Effects of Exposure to Spiked Diet on Honeybee Larvae (Apis mellifera carnica) in an In Vitro Laboratory Testing Design. Bayer
CropScience AG, Unpublished Report. No.: E 318 4110-8; September 23, 2011; MRID 49090506.
c
2017, Pest Management Regulatory Agency, Health Canada, Proposed Re-evaluation Decision, Clothianidin and Its Associated End-use Products: Pollinator Re-evaluation, PRVD2017-23.
d2017, Pest Management Regulatory Agency, Health Canada, Proposed Re-evaluation Decision, Thiamethoxam and Its Associated End-use Products: Pollinator Re-evaluation, PRVD2017-24.
e
2017, U.S. Environmental Protection Agency, Draft Assessment of the Potential Effects of Dinotefuran on Bees, EPA-HQ-OPP-2011-0920-0014.
f
2016, Patnaude, M.R., Dinotefuran Technical Grade: 22-Day Survival of Honey Bee Larvae, Apis mellifera L., during an In Vitro Exposure; Unpublished study prepared by Smithers Viscient; Laboratory
Report ID: 10934.6161; MRID: 49860001.
Guidelines Cited
OCSPP 850.3020 – Honey Bee Acute Contact Toxicity Test – Available via <https://www.epa.gov/test-guidelines-pesticides-and-toxic-substances/final-test-guidelines-pesticides-and-toxic>; accessed
June 13, 2018.
OECD 213 – Honeybees, Acute Oral Toxicity Test – Available via <https://www.oecd-ilibrary.org/environment/test-no-213-honeybees-acute-oral-toxicity-test_9789264070165-en>; accessed June 13, 2018.
OECD 245 – Honey Bee (Apis Mellifera L.), Chronic Oral Toxicity Test (10-Day Feeding) – Available via <https://www.oecd-ilibrary.org/environment/test-no-245-honey-bee-apis-mellifera-l-chronic-oraltoxicity-test-10-day-feeding_9789264284081-en>; accessed June 13, 2018.
OECD 239 – Guidance Document on Honey Bee Larval Toxicity Test following Repeated Exposure – Available via <https://one.oecd.org/document/ENV/JM/MONO(2016)34/en/pdf>; accessed
June 13, 2018.

83

Appendix 7. Open Literature References Considered for Use in this Risk
Determination Document
1. Alburaki, M., Boutin, S., Mercier, P.-L., Loublier, Y., Chagnon, M., & Derome, N. (2015).
Neonicotinoid-Coated Zea mays Seeds Indirectly Affect Honeybee Performance and Pathogen
Susceptibility in Field Trials. PLoS ONE, 10(5), e0125790. DOI:10.1371/journal.pone.0125790
2. Alburaki, M., Cheaib, B., Quesnel, L., Mercier, P.-L., Chagnon, M., & Derome, N. (2016).
Performance of honeybee colonies located in neonicotinoid-treated and untreated cornfields inQuebec. J. Appl. Entomol., 141(1-2), 112-121. DOI: 10.1111/jen.12336.
3. Alkassab, A.T., & Kirchner, W.H. (2017). Sublethal exposure to neonicotinoids and related side
effects on insect pollinators: honeybees, bumblebees, and solitary bees. J. Plant Dis. Prot., 124(1), 130. DOI:10.1007/s41348-016-0041-0.
4. Bonmatin, J.M., Marchand, P.A., Cotte, J.F., Aajoud, A., Casabianca, H., & Courtiade, M. (2007).
Bees and systemic insecticides (imidacloprid, fipronil) in pollen: subnano-quantification by
HPLC/MS/MS and GC/MS. Environmental Fate and Ecological Effects of Pesticides, 837-845.
MRID 49719606.
5. Bortolotti, L., Montanari, R., Marcelino, J., Medryzycki, P., Maini, S., & Porrini, C. (2003). Effectsof sub-lethal imidacloprid doses on the homing rate and foraging activity of honey bees. Bulletin of
Insectology, 56(1), 63-67. MRID 47800505.
6. Calatayud-Vernich, P., Calatayud, F., Simó, E., Suarez-Varela, M.M., & Picó, Y. (2015). Influence ofpesticide use in fruit orchards during blooming on honeybee mortality in 4 experimental apiaries.
Science of the Total Environment, 541(2016), 33–41.
7. Campbell, P., Coulson, M., Ruddle, N., Tornier, I., & Pilling, E. (2015). Authors’ response on Hoppeet al. (2015) “Effects of a neonicotinoid pesticide on honey bee colonies: a response to the field study
by Pilling et al. (2013)” Environ. Sci. Eur., (2015), 27–28. Environ. Sci. Eur., (Dec. 2015), 27-31.
DOI:10.1186/s12302-015-0064-3.
8. Cresswell, J.E., & Thompson, H.M. (2012). Comment on "A Common Pesticide Decreases Foraging
Success and Survival in Honey Bees". Science, 337(6101), 1453. DOI:10.1126/science.1224618.
9. Culter, G.C., Scott-Dupree, C.D., Sultan, M., McFarlane, A.D., & Brewer, L. (2014). A large-scale
field study examining effects of exposure to clothianidin seed-treated canola on honey bee colony
health, development, and overwintering success. Peer J, 2, e652. DOI:10.7717/peerj.652.
10. Dively, G.P., & Kamel, A. (2012). Insecticide residues in pollen and nectar of a cucurbit crop andtheir potential exposure to pollinators. J. Agric. Food. Chem., 60(18), 4449‐4456.
DOI:10.1021/jf205393x.
11. Dively, G.P., Embrey, M., & Pettis, J. (2009). Assessment of sublethal effects of imidacloprid on
honey bee and colony health. Department of Entomology, University of Maryland. North American
Pollinator Protection Campaign. MRID 47775502.

84

Appendix 7. Open Literature References Considered for Use in this Risk
Determination Document
12. Eiri, D.M., & Nieh, J.C. (2012). A nicotinic acetylcholine receptor agonist affects honey bee sucrose
responsiveness and decreases waggle dancing. J. Exp. Biol., 215(12), 2022-2029.
DOI:10.1242/jeb.143727. MRID 49719615.
13. Faucon, J.-P., Aurières, C., Drajnudel, P., Mathieu, L., Ribière, M., Martel, A.-C., Zeggane, S.,
Chauzat, M.-P., & Aubert, M.F. (2005). Experimental study on the toxicity of imidacloprid given insyrup to honey bee (Apis mellifera) colonies. Pest Management Science, 61(2), 111-125. DOI:
10.1002/ps.957. MRID 47523406.
14. Fischer, J., Müller, T., Spatz, A.-K., Greggers, U., Grünewald, B., & Menzel, R. (2014).
Neonicotinoids interfere with specific components of navigation in honeybees. PLoS ONE, 9(3),e91364. DOI:10.1371/journal.pone.0091364.
15. Heimbach, F., Russ, A., Schimmer, M., & Boren, K. (2016). Large-scale monitoring of effects ofclothianidin dressed oilseed rape seeds on pollinating insects in Northern Germany: implementationof the monitoring project and its representativeness. Ecotoxicology, 25(9), 1630-1647.
DOI:10.1007/s10646-016-1724-9.
16. Henry M., Béguin, M., Requier, F., Rollin, O., Odoux, J.-F., Aupinel, P., Aptel, J., Tchamitchian, S.,& Decourtye, A. (2012). A common pesticide decreases foraging success and survival in honey bees.
Science, 336(6079), 348-350. DOI: 10.1126/science.1215039.
17. Henry, M., Béguin, M., Requier, F., Rollin, O., Odoux, J.-F., Aupinel, P., Aptel, J., Tchamitchian, S.,& Decourtye, A. (2012). Response to Comment on “A Common Pesticide Decreases Foraging
Success and Survival in Honey Bees.” Science, 337(6101), 1453. DOI:10.1126/science.1224930.
18. Henry, M., Cerrutti, N., Aupinel,P., Decourtye, A., Gayrard, M., Odoux, J.-F., Pissard, A., Rüger, C.,& Bretagnolle, V. (2015). Reconciling laboratory and field assessments of neonicotinoid toxicity tohoneybees. Proc. R. Soc. B., 282(1819). DOI:10.1098/rspb.2015.2110.
19. Hoppe, P.P., Safer, A., Amaral-Rogers, V., Bonmatin, J.-M., Goulson, D., Menzel, R., & Baer, B.
(2015). Effects of a neonicotinoid pesticide on honey bee colonies: a response to the field study by
Pilling et al. (2013). Environ. Sci. Eur., (Nov. 2015), 27- 28. DOI:10.1186/s12302-015-0060-7.
20. Krupke, C.H., Hunt, G.J., Eitzer, B.D., Andino, G., & Given, K. (2012). Multiple routes of pesticide
exposure for honey bees living near agricultural fields. PloS ONE, 7(1), e29268.
DOI:10.1371/journal.pone.0029268.
21. Matsumoto, T. (2013). Reduction in homing flights in the honey bee Apis mellifera after a sublethal
dose of neonicotinoid insecticides. Bulletin of Insectology, 66(1), 1-9.
22. McArt, S.H., Fresch, A., Milano, N.J., Truitt, L.L., & Böröczky, K. (2017). High pesticide risk tohoney bees despite low focal crop pollen collection during pollination of a mass blooming crop.
Science Report, 7(46554). DOI:10.1038/srep46554.

85

Appendix 7. Open Literature References Considered for Use in this Risk
Determination Document
23. Pettis, J.S., Lichtenberg, E.M., Andres, M., Stitzinger, J., Rose, R., & vanEngelsdorp, D. (2013). Crop
pollination exposes honey bees to pesticides which alters their susceptibility to the gut pathogen
Nosema ceranae. PLoS ONE, 8(7), e70182. DOI:10.1371/journal.pone.0070182. MRID 49719624.
24. Pilling, E., Campbell, P., Coulson, M., Ruddle, N., & Tornier, I. (2013). A four-year field program
investigating long-term effects of repeated exposure of honey bee colonies to flowering crops treatedwith thiamethoxam. PLoS ONE, 8(10), e77193. DOI:10.1371/journal.pone.0077193.
25. Pistorius, J., Wehner, A., Kriszan, M., Bargen, H., Knäbe, S., Klein, O., Frommberger, M., Stähler,M., & Heimbach, U. (2015). Application of predefined doses of neonicotinoid containing dusts infield trials and acute effects on honey bees. Bulletin of Insectology, 68(2), 161-172.
26. Pohorecka, K., Skubida, P., Miszczak, A., Semkiw, P., Sikorski, P., Zagibajlo, K., Teper, D.,
Kolowski, Z., Skubida, M., Zdańska, D., & Bober, A. (2012). Residues of Neonicotinoid Insecticides
in Bee Collected Plant Materials From Oilseed Rape Crops and Their Effect on Bee Colonies.
Journal of Apicultural Science, 56(2), 199-208. DOI:10.2478/v10289-012-0029-3.
27. Pohorecka, K., Skubida, P., Semkiw, P., Miszczak, A., Teper, D., Sikorski, P., Zagibajlo, K.,
Skubida, M., Zdanska, D., & Bober, A. (2013). Effects of Exposure of Honey Bee Colonies to
Neonicotinoid Seed-Treated Maize Crops. Journal of Apicultural Science, 57(2), 199-208.
DOI:10.2478/jas-2013-0029. MRID 49719625.
28. Reetz, .JE., Schulz, W., Seitz, W., Spiteller, M., Zühlke, S., Armbruster, W., Wallner, K. (2015).
Uptake of Neonicotinoid Insecticides by Water-Foraging Honey Bees (Hymenoptera: Apidae)
Through Guttation Fluid of Winter Oilseed Rape. Journal of Economic Entomology, 109(1), 31-40.
DOI: 10.1093/jee/tov287.
29. Rogers, R.E.L., & Kemp, J.R. (2003). Imidacloprid, potatoes, and honey bees in Atlantic Canada: isthere a connection?. Bulletin of Insectology, 56(1), 83-88.
30. Rolke, D., Fuchs, S., Grünewald, B., Gao, Z., & Blenau, W. (2016). Large-scale monitoring of effects
of clothianidin-dressed oilseed rape seeds on pollinating insects in North Germany: effects on honeybees (Apis mellifera). Ecotoxicology, 25(9), 1648–1665. DOI:10.1007/s10646-016-1725-8.
31. Rolke, D., Persigehl, M., Peters, B., Sterk, G., & Blenau, W. (2016). Large-scale monitoring ofeffects of clothianidin-dressed oilseed rape seeds on pollinating insects in northern Germany: residuesof clothianidin in pollen, nectar and honey. Ecotoxicology, 25(9), 1691-1701. DOI:10.1007/s10646016-1723-x.
32. Rundlöf, M., Andersson, G.K.S., Bommarco, R., Fries, I., Hederström, V., Herbertsson, L., Jonsson,
O., Klatt, B.K., Pedersen, T.R., Yourstone, J., & Smith, H.G. (2015). Seed coating with aneonicotinoid insecticide negatively affects wild bees. Nature, 521, 77–80.
DOI:10.1038/nature14420.

86

Appendix 7. Open Literature References Considered for Use in this Risk
Determination Document
33. Sandrock, C., Tanadini, M., Tanadini, L.G., Fauser-Misslin, A., Potts, S., & Neumann, P. (2014)
Impact of chronic neonicotinoid exposure on honeybee colony performance and queen supersedure.
PLoS ONE, 9(8), e103592. DOI:10.1371/journal.pone.0103592.
34. Schmuck, R., Schoning, R., Strok, A., Schramel, O. (2001). Risk posed to honeybees (Apis mellifera
L, Hymenoptera) by an imidacloprid seed dressing of sunflowers. Pest Management Science, 57, 225238. MRID 47812303.
35. Schneider, C.W., Tautz, J., Grünewald, B., & Fuchs, S. (2012). RFID Tracking of Sublethal Effectsof Two Neonicotinoid Insecticides on the Foraging Behavior of Apis mellifera. PLoS ONE, 7(1),e30023. DOI:10.1371/journal.pone.0030023. MRID 49719629.
36. Stadler, T., Martinez-Ginés, D., & Buteler, M. (2003). Long-term toxicity assessment of imidacloprid
to evaluate side effects on honey bees exposed to treated sunflower in Argentina. Bulletin of
Insectology, 56(1), 77-81. MRID 47796301.
37. Straub, L., Villamar-Bouza, L., Bruckner, S., Chantawannakul, P., Gauthier, L., Khongphinitbunjong,K., Retschnig, G., Troxler, A., Vidondo, B., Neumann, P., & Williams, G.R. (2016). Neonicotinoid
insecticides can serve as inadvertent insect contraceptives. Proc. R. Roc. B., 283(1835). DOI:
10.1098/rspb.2016.0506.
38. Tan, K., Chen, W., Dong, S., Liu, X., Wang, Y., & Nieh, J.C. (2014). Imidacloprid alters foraging
and decreases bee avoidance of predators. PLoS ONE, 9(7),e102725. MRID 49719631.
39. Thompson, H., Coulson, M., Ruddle, N., Wilkins, S., & Harkin, S. (2015). Thiamethoxam: Assessing
flight activity of honeybees foraging on treated oilseed rape using radio frequency identification
technology. Environmental Toxicology and Chemistry, 35(2), 385–393. DOI: 10.1002/etc.3183.
40. Tremolada, P., Mazzoleni, M., Saliu, F., Colombo, M., & Vighi, M. (2010). Field trial for evaluating
the effects on honeybees of corn sown using Cruiser® and Celest XL® treated seeds. Bull Environ.
Contam. Toxicol., 85(3), 229-234. DOI:10.1007/s00128-010-0066-1.
41. Tsvetkov, N., Samson-Robert, O., Sood, K., Patel, H.S., Malena, D.A., Gajiwala, P.H.,
Maciukiewicz, P., Fournier, V., & Zayed, A. (2017). Chronic exposure to neonicotinoids reduce
honey bee health near corn crops. Science, 356 (6345), 1395-1397. DOI:10.1126/science.aam7470.
42. Williams, G.R., Troxler, A., Retschnig, G., Roth, K., Yañez, O., Shutler, D., Neumann, P., &Gauthier, L. (2015). Neonicotinoid pesticides severely affect honey bee queens. Science Report,5(14621). DOI:10.1038/srep14621.
43. Woodcock, B.A., Bullock, J.M., Shore, R.F., Heard, M.S., Pereira, M.G., Redhead, J., Ridding, L.,
Dean, H., Sleep, D., Henrys, P., Peyton, J., Hulmes, S., Hulmes, L., Sáraspataki, M., Saure, C.,
Edwards, M., Genersch, E., Knäbe, S., & Pywell, R.F. (2017). Country-specific effects ofneonicotinoid pesticides on honeybees and wild bees. Science, 356(6345), 1393-1395.
DOI:10.1126/science.aaa1190.

87

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

Data Evaluation Report
Study Titles:
Bocksch, S. (2014): Honey bee brood and colony level effects
following imidacloprid intake via treated artificial diet in a fieldstudy in North Carolina
Final Report Source:
Eurofins Agroscience Services Inc., unpublished report No: S1303176, 06 Nov 2014
Year of study: 2013-2014
PMRA#: 2474495
PMRA DACO#: 9.2.4.9
MRID: 49510001
Study Type:
Tier II colony feeding study conducted in an open field
Review Date (final): January 28, 2016
PMRA reviewer:
Primary Evaluator: Wayne Hou
Data Statistical Analysis: Keith O'Rourke
EPA reviewer:
Primary Evaluators: Justin Housenger, Biologist
Keith G. Sappington, Senior Science Advisor
Data Statistical Analysis: Christine Hartless, Wildlife Biologist
CDPR reviewer:
Primary Evaluators: Richard Bireley, Sr. Environmental Scientist (Specialist)
Alexander Kolosovich, Environmental Scientist
Brigitte Tafarella, Environmental Scientist
Data Statistical Analysis: John Troiano, Ph.D., Research Scientist III

1
88

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

Table of Contents
Executive Summary....................................................................................................................................... 7
Consideration of Study Strengths, Limitations and Interpretation .......................................................... 9
1.

Study Objective ................................................................................................................................... 12

2.

Study Methods.................................................................................................................................... 12
2.1.

Test crop ..................................................................................................................................... 12

2.2.

Test chemical .............................................................................................................................. 12

2.3.

Test sites ..................................................................................................................................... 12

2.4.

Test organisms ............................................................................................................................ 14

2.5.

Treatments.................................................................................................................................. 15

2.5.1.

Preparation of stock solution.............................................................................................. 16

2.5.2.

Preparation of sugar solution ............................................................................................. 16

2.5.3.

Preparation of feeding solution .......................................................................................... 16

2.5.4.

Artificial Feeding ................................................................................................................. 17

2.6.

Meteorological Data ................................................................................................................... 17

2.7.

Observations ............................................................................................................................... 17

Important activity and dates............................................................................................................... 17
2.7.1.

Colony mortality.................................................................................................................. 19

2.7.2.

Colony Condition Assessments (CCA) ................................................................................. 19

2.7.3.

Evaluation of Disease or Pests in the Hive ......................................................................... 20

2.7.4.

Hive weights........................................................................................................................ 20

2.8.

3.

Residue analysis .......................................................................................................................... 20

2.8.1.

Pollen from outside sources ............................................................................................... 21

2.8.2.

Stored pollen and nectar in test hives ................................................................................ 21

2.8.3.

Feeding solution and stability of test item ......................................................................... 21

Results................................................................................................................................................. 22
3.1.

Land use near test hives.............................................................................................................. 22

3.2.

Pollen sources of test hives......................................................................................................... 23

3.3.

Consumption of spiked sucrose.................................................................................................. 23

3.4.

Examination of pesticides from other sources ........................................................................... 24

3.5.

Confirmation of test concentrations........................................................................................... 25

3.6.

Stability of the test item in feeding solution .............................................................................. 26289

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
3.7.

Residues in hive matrices............................................................................................................ 27

3.7.1.

Background imidacloprid contamination in hives prior to the feeding exposure .............. 27

3.7.2.

Residues in hive matrices during and after feeding exposure............................................ 27

3.8.

Pathogens ................................................................................................................................... 36

3.8.1.
3.9.

Varroa Presence.................................................................................................................. 36

Statistical Analysis....................................................................................................................... 38

3.9.1.

Study Authors Analysis........................................................................................................ 38

3.9.2.

Study Reviewer Analysis ..................................................................................................... 38

3.9.3.

EPA Analysis ........................................................................................................................ 38

3.9.4.

PMRA Analysis..................................................................................................................... 39

3.9.5.

CDPR Analysis...................................................................................................................... 39

3.10.

Hive mortality.......................................................................................................................... 39

3.11.

Colony Condition Assessment Response Variables ................................................................ 41

Life Stages ............................................................................................................................................... 42
3.11.1.

Adults .................................................................................................................................. 42

3.11.2.

Eggs ..................................................................................................................................... 45

3.11.3.

Larvae (Open/Uncapped brood)......................................................................................... 50

3.11.4.

Pupae (Capped brood) ........................................................................................................ 54

3.11.5.

Total individuals in hives ..................................................................................................... 58

Colony Condition Assessments – Food Store Response Variables ......................................................... 65

4.

3.11.6.

Pollen .................................................................................................................................. 65

3.11.7.

Nectar / Honey.................................................................................................................... 68

3.11.8.

Hive Weight......................................................................................................................... 77

Reviewer comments: .......................................................................................................................... 79
General Considerations for Biological Interpretation............................................................................. 79
Control Performance .............................................................................................................................. 80
Consideration of CCA8 ............................................................................................................................ 81
Control overwintering mortality ......................................................................................................... 81
Response Variables from Surviving Hives ........................................................................................... 81
Consideration of Study Strengths, Limitations and Interpretation ........................................................ 82

5.

Conclusions ......................................................................................................................................... 85

390

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

List of Tables
Table 1. Details about the test substance.................................................................................................. 12
Table 2: GPS-coordinates of the test apiary sites ....................................................................................... 13
Table 3. Treatment groups, feeding rates and feeding volume ................................................................. 16
Table 4. Hive assignment to test apiaries .................................................................................................. 16
Table 5. Chronological list of key dates and activities ................................................................................ 18
Table 6. Schedule for colony assessment and beekeeper checks .............................................................. 19
Table 7. LOD for imidacloprid and its metabolites ..................................................................................... 20
Table 8. Sampling schedule for feeding solution and stability of test chemical......................................... 21
Table 9: Percent (%) land use pattern......................................................................................................... 22
Table 10: Cultivated cropping area near each test apiary .......................................................................... 23
Table 11. Residues from outside sources in pollen samples from pollen trap (non-GLP).......................... 25
Table 12. Dosing solution residue data from 2 July 2013 (Week 1), 12 July 2013 (Week 2) and 2 August
2013 (Week 5)............................................................................................................................................. 26
Table 13. The stability of imidacloprid in feeding solution on 3 Jul, 12 Jul, and 2 Aug, 2013. .................. 26
Table 14. Detailed hive pollen residue data, pre-study collection (30 May 2013) .................................... 27
Table 15. Detailed hive nectar residue data, pre-study sample collection (30 May 2013) ....................... 27
Table 16. Imidacloprid concentrations (ppb) in hive pollen sampled three weeks after the start ofartificial feeding on 18 July 2013 (CCA4). ................................................................................................... 29
Table 17. Imidacloprid concentrations (ppb) in uncapped hive nectar sampled three weeks after the startof artificial feeding on 18 Jul, 2013 (CCA4). ................................................................................................ 30
Table 18. Imidacloprid concentrations (ppb) in beebread and uncapped hive nectar sampled one week
after the end of artificial feeding on 14 Aug, 2013 (CCA5)......................................................................... 31
Table 19: Imidacloprid concentrations (ppb) in bee bread and honey sampled after overwintering on
March 24, 2014 (CCA8). .............................................................................................................................. 32
Table 20. Imidacloprid (ppb) detected in test hives before the exposure (CCA2), and in control hives
during the exposure (CCA4) and after the exposure (CCA5 and CCA8)...................................................... 34
2 .................................................................................................................................................................. 34
Table 22. Imidacloprid concentration measured in hive uncapped nectar and hive bee bread................ 36
Table 23a. Hive survival at CCA8 (after overwintering) .............................................................................. 40
Table 23b. Hive mortality after overwintering measure at CCA8.............................................................. 40
Table 24. Estimated percent reduction from control for number of adults............................................... 43
Table 25. Estimated percent reduction from control for number of eggs ................................................. 46
Table 26. Estimated percent reduction from control for number of larvae (open/uncapped brood)....... 50
Table 27. Estimated percent reduction from control for number of pupae .............................................. 54
Table 28. Estimated percent reduction from control for total number of individuals............................... 58
Table 29. Estimated percent reduction from control for pollen stores...................................................... 65
Table 30. Estimated percent reduction from control for nectar/honey stores.......................................... 69
Table 31. Estimated percent reduction from control for hive weights ...................................................... 78

491

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

List of Figures
Figure 1: Location of test apiary sites ......................................................................................................... 13
Figure 2. Layout of test hives in a test site ................................................................................................. 15
Figure 3. Mean total food consumption (mL) per colony during the 6-week exposure period ................ 24
Figure 4. Mean number of Varroa mites per 100 bees ............................................................................. 37
Figure 5. Mean number of Nosema spores per bee ................................................................................... 37
Figure 6. Hive mortality after overwintering. ............................................................................................. 41
Figure 7. Proportion of adults following exposure to varying concentrations of imidacloprid in the dietacross CCA3 – CCA8. ................................................................................................................................... 43
Figure 8. Proportion of adults following exposure to varying concentrations of imidacloprid in the dietacross CCA3 – CCA8 in the control, 12.5, 25, and 50 µg/L groups only...................................................... 44
Figure 9. Proportion of adults following exposure to varying concentrations of imidacloprid in the dietacross CCA3 – CCA8 in the control, 12.5, 25, and 50 µg/L groups.............................................................. 45
Figure 10. Proportion of eggs following exposure to varying concentrations of imidacloprid in the dietacross CCA3 – CCA8. ................................................................................................................................... 47
Figure 11. Proportion of eggs following exposure to varying concentrations of imidacloprid in the dietacross CCA3 – CCA8 in the control, 12.5, 25, and 50 µg/L groups only...................................................... 48
Figure 12. Proportion of eggs following exposure to varying concentrations of imidacloprid in the dietacross CCA3 – CCA8 in the control, 12.5, 25, and 50 µg/L groups.............................................................. 49
Figure 13. Proportion of larval cells following exposure to varying concentrations of imidacloprid in thediet across CCA3 – CCA8. ............................................................................................................................ 51
Figure 14. Proportion of larval cells following exposure to varying concentrations of imidacloprid in thediet across CCA3 – CCA8 in the control, 12.5, 25, and 50 µg/L groups only............................................... 52
Figure 15. Proportion of larval cells following exposure to varying concentrations of imidacloprid in thediet across CCA3 – CCA8 in the control, 12.5, 25, and 50 µg/L groups. ..................................................... 53
Figure 16. Proportion of pupal (capped) cells following exposure to varying concentrations ofimidacloprid in the diet across CCA3 – CCA8.............................................................................................. 55
Figure 17. Proportion of pupal (capped) cells following exposure to varying concentrations ofimidacloprid in the diet across CCA3 – CCA8 in the control, 12.5, 25, and 50 µg/L groups only. .............. 56
Figure 18. Proportion of pupal (capped) cells following exposure to varying concentrations ofimidacloprid in the diet across CCA3 – CCA8 in the control, 12.5, 25, and 50 µg/L groups. ...................... 57
Figure 19. Proportion of total individuals (adult, eggs, larvae, pupae) following exposure to varying
concentrations of imidacloprid in the diet across CCA3 – CCA8. ............................................................... 59
Figure 20. Summary of living organism parameters at the 12.5 µg/L treatment group ............................ 60
Figure 21. Summary of living organism parameters at the 25 µg/L treatment group ............................... 61
Figure 22. Summary of living organism parameters at the 50 µg/L treatment group ............................... 61
Figure 23. Trends of life stages across treatment groups within CCA4 ...................................................... 62
Figure 24. Trends of life stages across treatment groups within CCA5 ...................................................... 63
Figure 25. Trends of life stages across treatment groups within CCA ........................................................ 63
Figure 26. Trends of life stages across treatment groups within CCA7 ...................................................... 64
Figure 27. Trends of life stages across treatment groups within CCA8 ..................................................... 65
592

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Figure 28. Proportion of pollen stores following exposure of honey bees to varying concentrations ofimidacloprid in the diet across CCA3 – CCA8.............................................................................................. 66
Figure 29. Proportion of pollen stores following exposure to varying concentrations of imidacloprid in
the diet across CCA3 – CCA8 in the control, 12.5, 25, and 50 µg/L groups only. ....................................... 67
Figure 30. Proportion of pollen stores following exposure to varying concentrations of imidacloprid in
the diet across CCA3 – CCA8 in the control, 12.5, 25, and 50 µg/L groups. ............................................... 68
Figure 31. Proportion of honey stores following exposure of honey bees to varying concentrations ofimidacloprid in the diet across CCA3 – CCA8.............................................................................................. 70
Figure 32. Proportion of honey stores following exposure to varying concentrations of imidacloprid in
the diet across CCA3 – CCA8 in the control, 12.5, 25, and 50 µg/L groups only. ....................................... 70
Figure 33. Proportion of honey stores following exposure to varying concentrations of imidacloprid in
the diet across CCA3 – CCA8 in the control, 12.5, 25, and 50 µg/L groups. ............................................... 71
Figure 34. Summary of hive food supply parameters at the 12.5 µg/L treatment group .......................... 73
Figure 35. Summary of hive food supply parameters at the 25 µg/L treatment group ............................. 73
Figure 36. Summary of hive food supply parameters at the 50 µg/L treatment group ............................. 74
Figure 37. Trends of food store parameters across treatment groups within CCA4.................................. 75
Figure 38. Trends of food store parameters across treatment groups at CCA5......................................... 75
Figure 39. Trends of food store parameters across treatment groups at CCA6......................................... 76
Figure 40. Trends of food store parameters across treatment groups at CCA7......................................... 76
Figure 41. Trends of food store parameters across treatment groups at CCA8......................................... 77
Figure 42. Proportion of hive weight following exposure of honey bees to varying concentrations ofimidacloprid in the diet across CCA3 – CCA8.............................................................................................. 78
Figure 43. Fate of control hives as compared to number of adults across CCAs....................................... 81
Figure 44. Fate of control hives as compared to the honey stores across CCAs ....................................... 82

Table of Appendices
Appendix A: Details of EPA Statistical Analysis ........................................................................................... 91
Appendix B: Details of PMRA Statistical Analysis..................................................................................... 107
Appendix C: Details of CDPR Statistical Analysis....................................................................................... 146

693

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

Executive Summary
A colony feeding study was conducted with honey bees to assess the potential for long-term effects,
including overwintering survival, resulting from exposure to imidacloprid. The study was
conducted in twelve test areas of low agricultural cultivation (Apiaries A – L) in North Carolina
from June 21, 2013 to March 24, 2014. Eighty-four hives were divided according to hive strength(number of brood frames) with the strongest 7 hives ssigned to Apiary A and the weakest 7 hives
assigned to Apiary L. Within each apiary, the 7 hives were randomly assigned to treatment groups.
At each apiary, five test hives were artificially fed with 50% sugar solution spiked withimidacloprid at 12.5, 25, 50, 100 or 200 µg ai/L for six weeks continuously in the field, with two
hives at each apiary serving as controls. Assuming the density of a 50% sugar solution is 1.2296
g/ml, the reviewer calculated that the test concentrations at 12.5, 25, 50, 100, and 200 µg/L areequivalent to 10.2, 20.3, 40.7, 81.3, and 162.7 ppb (µg/kg), respectively. The average measured
ppb (µg/kg) concentrations in dosing solutions was confirmed to be 11.0, 23.3, 46.7, 96.3, 189.6
ppb (µg/kg), respectively.
Eight Colony Condition Assessments (CCAs) were conducted during the study. Three CCAs
(CCA1 - 3) were conducted prior to feeding to determine hive strength and initial hive conditions.
A CCA was conducted during exposure with another one conducted one week after termination of
exposure (CCA4 and CCA5, respectively) which characterize hive conditions during exposure.
Two more CCAs were conducted at 5 and 10 weeks after exposure (CCA6 and CCA7, respectively)
to assess the chronic effect following exposure to imidacloprid and to characterize preoverwintering hive conditions. A final CCA was conducted after overwintering in March 2014
(CCA8) to assess potential exposure impact on survival and chronic colony level effects. Multiple
parameters, such as hive weight, number of individuals at different life stages in the hive, hive
honey and pollen stores, and hive overwintering survival, were measured during the course of thestudy.
Levels of imidacloprid residues in hives were measured before, during and after the feeding
exposure. Potential contamination of pesticides from other food sources was monitored using
pollen collected in additional hives at each apiary that served as monitoring hives. The results
showed that while there were a few instances of imidacloprid detected in the pollen and nectar of
the control hives, the frequency and magnitude of these detections is not expected to confound theresults of this study. Residues measured in hive matrices demonstrated that higher treatment
exposures corresponded well to higher residues in hive matrices. There were individual hive
variations in measured residues, with some overlap in measured hive concentrations, particularly
at the lower doses. This variability likely originates from the limited spatial and temporal sampling
methodology (i.e. one sample from one side of the comb on one frame to represent a hive, andonly at 3 CCAs) employed for this study. Overall during the exposure period, imidacloprid
concentration in hive nectar averaged 63.7% of the nominal concentration in the feeding solution,
whereas imidacloprid concentration in hive pollen averaged 30.2% of the nominal concentrationin feeding solution. This dilution is expected since bees could forage on outside pollen and nectar
sources, and hive pollen (bee bread) includes only some nectar. See Section 3.7 for more details
regarding the residues of imidacloprid in the dosing solutions and hive matrices.

794

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
During the review of the study, a joint review effort was conducted by the United States
Environmental Protection Agency (EPA), Health Canada’s Pest Management Regulatory Agency(PMRA), and the California Department of Pesticide Regulation (CDPR). As part of that effort,a separate statistical analysis was conducted by each regulatory entity as an independent
verification of the results from the analysis provided by the registrant. These analyses (describedin detail in Appendices A, B, and C for EPA’s, PMRA’s and CDPR’s approaches, respectively)
were distinct in approach but generally yielded similar statistical results. When weighing theseresults as well as biological concerns, particularly as they relate to honey bee biology at the colonylevel, EPA, PMRA, and CDPR arrived at the same conclusions and are therefore harmonized interms of the determination of an overall No Observed Adverse Effect Concentration (NOAEC)
and Lowest Observed Adverse Effect Concentration (LOAEC) for this study.
As will be discussed in Section 3 (Results) the PMRA, EPA, and CDPR analyses determinedstatistically significant imidacloprid dose-related effects in the 50, 100, and 200 µg/L treatmentgroups across multiple CCAs for the majority of response variables. Indeed, for the 100 and 200µg/L treatment groups, significant effects (p<0.05) were determined for every response variable,
persisted across multiple CCAs and eventually resulted in loss of nearly all hives in those treatmentgroups after the overwintering period. The 50 µg/L treatment group also showed significant effects
for multiple response variables across multiple CCAs, and poorer colony condition in surviving
hives after overwintering in comparison to controls.
Conversely, there was not a strong indication from the PMRA, EPA, and CDPR analyses of an
impact at the colony level for the 12.5 and 25 µg/L treatment groups for individual life stages. Thisis evidenced not only by a general lack of statistically significant effects (p>0.1) at these treatment
levels but in cases where significant effects were determined, they either did not show strong doseresponsiveness, did not persist across multiple CCAs, or were considered potential transient effects
which did not persist after overwintering. This latter point was the case for the statisticallysignificant effects noted at CCA6, which included pupal cell and total individual effects for which
the PMRA analyses determined significant effects at all treatment levels, and EPA determinedsignificant effects for pupae at 12.5, 100 and 200 µg/L (but not at 25 or 50 µg a.i/L). As well at
CCA6, PMRA determined significant effects with eggs and larvae at 25 µg/L treatment (but not
at the 50 µg/L). For the two lowest treatment groups (12.5 and 25 µg/L), the colony condition ofsurviving hives at CCA8 following overwintering was similar to controls, indicating the effects
observed at CCA6 were likely transient and the colony was able to compensate for these effects.
When examining the effects on food stores (pollen and nectar), the PMRA, EPA, and CDPR
analyses did not determine any consistent and significant reductions in pollen and nectar stores at
the 12.5 and 25 µg/L treatment groups. This is distinguished from the 50 µg/L group where effects
on nectar in particular were very apparent, when compared alongside the response of the control,
in terms of the level of nectar buildup before hive preparation for overwintering at CCA7. Thisfinding was also confirmed statistically in all three analyses with significant reductions in honeystores at CCAs 6, 7, and 8 (CCA8 data excluded from the EPA analysis for the 100 and 200 µg/Lgroups). Significant reductions in pollen stores were also confirmed at CCAs 4 and 5 (i.e. during
the exposure period) at the 50 µg/L treatment group.
Specifically, when considering the proportion of adults as well as honey and pollen stores response
variables, the differences from control were apparent both visually and statistically, particularly in
895Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
the three highest treatment groups. For the proportion of adults, the onset of a decline in numbers
occurred one CCA earlier in these groups than in the control, 12.5 and 25 µg/L treatment groups.
For honey stores, the buildup that occurred starting at CCA5 in the 50 µg/L treatment group,
reached only half the level reached in the control, 12.5, and 25 µg/L treatment groups by CCA7.
Pollen stores were also reduced at CCA4 and CCA5 compared to controls for the three highest
treatment groups, as well as at CCA6 and CCA7 at the highest treatment group. These effects were
statistically significant (p<0.05) and indicate that the 50 µg/L treatment group was associated withtrends and proportions of abundance for life stages and food stores not observed in the control,
12.5, and 25 µg/L treatment groups.
The study is considered to be informative and will be used as a line of evidence in the pollinatorrisk assessment. While there were uncertainties that were generally related to inherent aspects ofany semi-field or full field study design (described in the section below) this study still provides
information on a number of colony health parameters about the long term (including overwintering)
exposure to imidacloprid at the colony level. When weighing biological significance and thenatural seasonal changes of honey bee colonies, as well as supporting conclusions from thestatistical approaches used by PMRA, EPA, and CDPR, the NOAEC and LOAEC for thisstudy are determined to be 25 and 50 µg/L, respectively.

Consideration of Study Strengths, Limitations and Interpretation
It is important to recognize the inherent strengths and limitations of this study as results areinterpreted and potentially considered in risk assessment.
In the context of available field studies involving honey bees and imidacloprid, this study contains
a number of strengths including:
•
•
•
•
•
•
•

Use of a high degree of replication (n=12) to achieve a reasonable level of statistical power
Demonstration of a generalized concentration-response relationship with respect to theconcentration of imidacloprid in sucrose solution and the magnitude and duration ofadverse effects
Quantification of exposure to parent (imidacloprid) and toxicologically-relevant
metabolites in diet and in hive matrices (uncapped nectar, pollen, honey, bee bread)Use of a 6-week exposure duration to represent a “high end” exposure scenario
Inclusion of multiple colony-level endpoints reflecting hive strength, brood development
and food stores
Detailed QA/QC results regarding quantification of chemical residues in various matrices
Availability of raw data for conducting statistical analysis.

A number of limitations are also noted with this study, including:•Exposure of bees to imidacloprid occurred through nectar (sucrose) alone, whereas bees in
the field are likely exposed through both pollen and nectar routes. Therefore, the designof this study may not reflect a “worst case” exposure scenario in which bees areexperiencing prolonged exposure to both contaminated nectar and pollen. While exclusion
996

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Documentof the pollen route is expected to reduce overall exposure, the impact of this exclusion on
the study results is uncertain and will likely depend on the life stage/caste of bee.
•

It is noted that imidacloprid was found in both hive nectar and hive pollen (beebread), atconcentrations lower than the feeding solutions. Dilution compared to the treatment
feeding solution is expected since bees could also forage on outside nectar and pollen
sources. Additionally, hive pollen contains only some hive nectar, thus would not be
expected to have a concentration equivalent to nectar alone, and it is mixed with pollen
which will come from outside sources. Therefore exposure through both hive pollen andnectar occurred via exposure to the sucrose feeding solution, but how this compares toexposure through contaminated pollen directly is not known. A recent paper by Dively(2015) 1 showed that higher residues throughout the hive resulted from feeding pollen
treatments compared to feeding sucrose solution treatments. It is also noted that while
nectar is considered the dominant exposure route for forager bees; other hive bees andlarvae consume both nectar and pollen. In addition, since bees were forced to forage forpollen in this study, the potential impact of imidacloprid exposure on reducing pollen
foraging efficiency of bees could be incorporated into the overall expression of adverse
effects, as suggested by published literature. Had contaminated pollen been provided tobees, it is not known if the potential impact on pollen foraging efficiency would have beenmasked.

•The quantity of nectar provided to hives (2 L per week per hive) likely did not fulfill thecomplete carbohydrate needs of the colony, as indicated by colony bioenergetics and thelack of remaining sucrose solution upon their renewal. This suggests that bees could be
exposed to a greater dose of imidacloprid in nectar had a greater volume of spiked sucrose
been provided. Although one can infer that the dosing regimen may have underestimatedexposure through sucrose relative to 100% contaminated diet, it is also noted that bees had
to supplement their spiked sucrose by foraging on their own for other sources of nectar.
As with the previous discussion of pollen, it is noted that had 100% of the carbohydrate
needs of the colony been provided via feeders, the potential impact of purported reductions
in nectar foraging efficiency may have been masked to some degree.

•

Overwintering success of controls was impacted (36% hive mortality). This may have
reduced the ability to detect adverse effects related to hive loss following overwintering.
Although comparable to overwintering losses of commercial beekeepers, it is possible thatelements of the study design may have contributed to this loss (e.g., lack of supers to allow
for colony growth, delayed supplemental feeding during fall).

•Hive detections with pesticides from food sources other than the artificial feeding was
detected during the exposure period and post-exposure periods through collection of pollen
from pollen traps. Although the study was deliberately conducted in a low agricultural area
in order to minimize the potential for pesticide contamination from other sources, the bees
still appeared to be foraging on contaminated pollen and possibly nectar. During both

1

Dively GP, Embrey MS, Kamel A, Hawthorne DJ, Pettis JS (2015) Assessment of Chronic Sublethal Effects of
Imidacloprid on Honey Bee Colony Health. PLoS ONE 10(4): e0126043. doi: 10.1371/journal.pone.0126043
1097Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
exposure and post-exposure periods, a high level of multiple pesticides that may cause
concern for bees were detected in most monitoring hives, such as spiromesifen (maximum
at 961 ppb) and piperonyl butoxide (maximum at 591 ppb). Trace amounts of other beetoxic pesticides, such as chlorpyifos (LOD = 1.0 ppb) and malathion (LOD = 4.0 ppb) werealso detected. The test chemical imidacloprid was found at 12.1 ppb in pollen from one(apiary L) of the total of six sites analysed. This level is similar to one of the test
concentrations.
•

Residues of imidacloprid in uncapped nectar and bee bread within the hives at CCAs 4, 5,
and 8 represent a single sample per hive on a single frame rather than a composite sample
from multiple portions of the comb within a hive. This means that residue results may
reflect “hit or miss” scenario with respect to detecting residues in nectar laid down fromcontaminated (fed) vs. outside sources.

•

The exposure, based on residues measured in the hive (hive nectar and hive pollen)
indicated that, overall, higher measured hive residues correlated with higher nominal
residues in feeding solutions. However, individual hive residue values varied, and there
was some overlap in measured values, particularly among the three lowest doses. Given
the limited spatial and temporal sampling methodology (as mentiond above), there is
uncertainy in whether these residues represent actual in hive residues across all portionsof the frame. Specifically, one sample of one area of the comb on one side of the frame
to represent the nectar or pollen residues of an entire hive may not reflect the true natureof the residues across all portions of a given hive.

•

Exposure dilution during the study was evident. Pollen storage was observed consistently
in the control hives and hives exposed to lower test concentrations during the exposure
period, indicating that test bees were foraging on food sources other than the spiked sugar
solution. Remarkably lower residue concentrations detected in bee bread and hive nectarin some test hives compared to the feeding concentrations may also indicate foraging onother food sources. This uncertainty is inherent in any semi-field or full-field study design.

1198Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

1. Study Objective
To determine the potential long term effects on the honey bee (Apis mellifera L.) colony healthduring and after dietary intake of imidacloprid, including the potential effects on overwintering.
The long term exposure allows for the characterization and distinction of short-term versus apersistent nature of effects.

2. Study Methods
2.1.

Test crop

Not applied. The study was conducted in an open field where multiple field flowers were available
and may serve as food sources for the test bees, in addition to the artificial feeding of spiked sugar
solution.

2.2.

Test chemical

The test substance was technical imidacloprid. Further details are provided in Table 1 below.
1Table 1. Details about the test substance
Name
Test item code:
Formulation type:
Active ingredient:
CAS number:
Density (20 °C)
analysed:
Date of analysis:
Stability in solution:

2.3.

Imidacloprid TC
NC-0116
Technical compound
imidacloprid
138261-41-3
Not applicable

Test Item
Batch number:Appearance / colour:
Intended Usage:
Content of a.i. analysed:

17 July 2014
sufficient for the test purpose(at least 1h)

EDE0015669
Solid / beige, light
Insecticide
98.7 %

Risk symbol(s):Not available

Expiry date:
Storage conditions:17 July 2016
ambient

Test sites

The field and sampling phases of this study were conducted by Eurofins Agroscience Services Inc.,
Cedar Grove Research Station Mebane, NC, USA; the analytical phase was conducted by Bayer
CropScience in Durham, NC, USA. The apiary sites were located in the vicinity of the EASI Cedar
Grove Research Station in Orange, Caswell, Person and Alamance counties, North Carolina.
There were 12 apiaries separated by more than 1 mile. Land use surveys in 1- mile radius and 3mile radius were conducted. Pollen species identification and multiple pesticide analysis wereconducted using pollen samples collected from the monitoring hives to characterize outside foodsources of the test bees and contamination. Pollen samples were collected for a period of 24-48
hours using pollen traps at 5 times during the feeding exposure period (Jun 28, July 3, 12, and 19,
and Aug 2, 2013) and 1 time after the exposure (Oct 17, 2013).

1299

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

1Figure 1: Location of test apiary sites
2Table 2: GPS-coordinates of the test apiary sites
ApiaryApiary A
Apiary B
Apiary C
Apiary D
Apiary E
Apiary F
Apiary G
Apiary H
Apiary I
Apiary J
Apiary K
Apiary L

GPS-coordinates
36°12’01.33” N, 79°06’33.76” W
36°13’55.12” N, 79°08’58.15” W
36°15’12.77” N, 79°06’11.58” W
36°13’20.79” N, 79°10’51.85” W
36°14’55.69” N, 79°14’13.95” W
36°9’59.15” N, 79°10’18.26” W
36°11’22.53” N, 79°15’59.81” W
36°15’41.51” N, 79°11’47.16” W
36°16’50.40” N, 79°11’00.11” W
36°13’22.39” N, 79°12’29.23” W
36°13’55.09” N, 79°14’21.00” W
36°11’22.12” N, 79°10’10.64” W

From Table 2, page 19 of the study report.

13100Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
2.4.

Test organisms

The test species was the honey bee (Apis mellifera), Italian race (Apis mellifera ligustica). Hives
were established from package bees bought from the commercial bee supplier (J J’s Honey, 5748
Chancey Road, Patterson, GA 31557, USA), typical of the bee stock used in commercial
beekeeping operations. A new queen was introduced into each colony. All queens were purchasedfrom the package supplier. The colonies were maintained in 10-frame Langstroth boxes with anempty deep super on top as a feeder box. In the test field, hives were raised above ground level.
Eighty-four hives that met the conditions provided below at the third Colony Condition
Assessments (CCA3) were selected for the study. More than 100 inspected hives were screened
based on the outcome of CCA2. Hives were checked for the “appearance” of a healthy colony
with no visible symptoms of Varroa or Nosema, as well as having all stages of brood, a queen, andsome food stores.
••

4-8 brood combs containing eggs, larvae or capped cells (except one colony inthe control which contained only 2 brood combs at test start);
6-10 food combs containing honey and pollen;

Reviewer note: Although the number of adult bees was not considered here as a criteria, it wasestimated to be 7000-8000 bees per hive according to the study report.
Eighty-four hives were blocked into 12 apiary sites by brood strength of the colony, starting with
Apiary A as the strongest group of hives, and Apiary L as the weakest group of hives. Assignmentof apiaries to the geographic locations was done randomly.
Hives were moved on 18/19 Jun 2013 to the Cedar Grove site temporarily from their original
apiary locations. On the night of 19 Jun 2013, hives for Apiary sites A-F were moved to theirstudy locations and had their CCA3 on 21 Jun 2013. Hives for Apiary sites G-L were moved on
21 Jun 2013 and had their CCA3 on 23 Jun 2013. After evaluating the assessments, 6 hives weredeemed unsuitable and were replaced the morning of 26 Jun 2013, just before exposure began.
CCA3 on the 6 replaced hives were conducted. The replaced hives were A1, B8, G1, F2, I4, andJ1.
There were eight hives at each site (7 hives for biological assessments and one as the monitoring
hive for pollen sample collection). Each hive was spatially isolated from other treatment rates by
30 feet (9 m) spacing at each apiary site (Figure 2). Hives were arranged in a semi-circular pattern,facing east to west, with 125 feet (38 m) spacing between the two end hives.

14101Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

2Figure 2. Layout of test hives in a test site
During the study, all hives were treated with one application of Apiguard® (active ingredient:
thymol) following typical apicultural practice for the region. The initial application occurred
immediately after the CCA6 (17 Sep, 2013) to prevent high mite loads. No treatments for any otherhive pests, predators or diseases were administered to any hives.
To minimize the potential for robbing amongst test hives, hives at 100 and 200 ppb treatments
were removed from all test apiaries in week 10 (5-6 Sep, 2013) immediately before CCA6. The
hives were placed at a separate apiary. Information on the separate apiary was not provided. For
over wintering, the surviving colonies were fed with 1 L of 2:1 sugar syrup on 13 Dec 2013, 19
Dec 2013, 13 Jan 2014, 20 Jan 2014, 27 Jan 2014, 07 Feb 2014, 18 Feb 2014, 02 Mar 2014 and
11 Mar 2014.
The monitoring hives were used for outsource pollen sample collection. In addition, test solutions
were sealed and placed in monitoring hives in order to assess imidacloprid stability under field test
conditions. These stability solutions were not available as a food source to the monitoring hives.

2.5.

Treatments

There were:o 6 treatment groups (5 test concentrations and control): 0, 12.5, 25, 50, 100, or 200µg/L. At each site, there were 2 control hives, and one hive for each test concentration.
o 12 replicates per treatment group (apiaries),
The individual treatment groups, the respective feeding rates and the respective feeding volumes
are summarized in3Table 3.

15102Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
3Table 3. Treatment groups, feeding rates and feeding volume
Treatment GroupCode

1 : UTC

Feeding Timing

UTC(T1+T2)
T3
T4
T5
T6
T7

2 : Lowest Rate
3 : Low rate
4 : Moderate rate
5: High rate
6: Highest rate

From Table 3, page 21 of the study report.

Twice a week

Concentration
a.i.
---

Feeding
Volume1000 mL

Twice a week
Twice a week
Twice a week
Twice a week
Twice a week

12.5 ppb
25 ppb
50 ppb100 ppb
200 ppb1000 mL
1000 mL
1000 mL
1000 mL
1000 mLThe assignment of each test hive at 12 apiaries is summarized in Table 4.
4Table 4. Hive assignment to test apiaries
Treatment
group

Apiary
A

B

C

D

E

F

G

H

I

J

K

L

UTC

A1

B2

C7

D4

E1

F4

G6

H5

I5

J1

K3

L7

UTC

A2

B8

C6

D6

E8

F7

G5

H3

I8

J7

K4

L5

12.5 ppb

A4

B3

C3

D8

E5

F5

G4

H7

I7

J3

K2

L6

25 ppb

A6

B4

C1

D1

E4

F3

G8

H6

I6

J4

K6

L2

50 ppb

A8

B7

C5

D5

E7

F2

G1

H2

I3

J2

K5

L3

100 ppb

A5

B6

C2

D7

E2

F8

G3

H8

I4

J5

K1

L4

200 ppb

A7

B1

C8

D3

E6

F1

G7

H1

I2

J6

K8

L8

A3
B5
C4
D2
Monitoring
From Table 4, page 22 of the study report.

E3

F6

G2

H4

I1

J8

K7

L1

2.5.1. Preparation of stock solution
Stock solution was created by combining 0.051 g of Imidacloprid Technical Compound, dissolved
in approx. 20 mL of acetone, in 1000 mL of distilled water. After preparation, the stock solution
was re-stored in a refrigerator until use or replacement. Stock solution was replaced once during
feeding on 16 Jul 2013.

2.5.2. Preparation of sugar solution
Sugar syrup was created by combining 10, 100 mL tap water with 10,100 g of sugar in a 5-gallon(19 L) container to make approximately 17 L of sugar syrup.

2.5.3. Preparation of feeding solution
o 12.5 µg/L: mixing 4.25 mL of stock solution into the 17 L of sugar solution.
o 25 µg/L: mixing 8.5 mL of stock solution into the 17 L of sugar solution
16103Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Documento 50 µg/L: mixing 17 mL of stock solution into the 17 L of sugar solutiono 100 µg/L: mixing 34 mL of stock solution into the 17 L of sugar solution
o 200 µg/L: mixing 68 mL of stock solution into the 17 L of sugar solution.
The test concentrations were reported as “ppb” in the study report. However, the values are in fact
in the unit of µg/L, not ppb (ug/kg). For example, 12.5 µg/L: can be calculated by 4.25 ml * 0.051
g /1020 ml)/17 L.
The test solution density was not provided. Assuming the density of a 50% sugar solution is 1.2296
g/ml 2, the reviewer calculated that the test concentrations at 12.5, 25, 50, 100, and 200 µg/L areequivalent to 10.2, 20.3, 40.7, 81.3, and 162.7 µg/L (ug/kg), respectively.

2.5.4. Artificial Feeding
Each hive had an empty deep super on top, between the lid and the inner cover to allow dark space
to place the feeder inside the hive. This allowed the feeder to be placed on the inner cover so thatthe bees had easy access without allowing the feeder to come into constant contact with light.
The treated sugar syrup was prepared one day in advance for each feeding event and stored
overnight at room temperature. The feeding started on 26 Jun, 2013 and continued for 6 weeks.
All of the hives were artificially fed with 1 liter of 50% sugar solution, two times per week. The
remaining feeding syrup was removed from the feeder and weighed to determine the consumed
amount. The study observation period was 21 Jun, 2013 – 24 Mar, 2014, which includes theoverwintering period.

2.6.

Meteorological Data

Temperature, humidity and rainfall data were obtained from two apiary sites (from the EASI
weather stations located at Apiaries K and J; distance to the other apiaries between 0.1 to 7.5 miles).
A total of 11.93 inches (303 mm) of rainfall accumulated throughout the exposure period –including CCA3 (from 21 Jun 2013 until 08 Aug 2013), with 2.63 inches (67 mm) in June, 7.96
inches (202 mm) in July and 1.34 inches (34 mm) in August. For this period the on-site temperature
minimum was 13 °C (55.4 °F) and the temperature maximum was 34 °C (93.2 ° F). The humidity
ranged from 38-100 %.

2.7.

Observations

Important activity and dates are summarized in Table 5.

Cell Biology Laboratory Manual, http://homepages.gac.edu/~cellab/chpts/chpt3/table3-2.html, accessed on Dec 12,
2014

2

17
104

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
5Table 5. Chronological list of key dates and activities
Week Date-7
12 May 2013

Activity
CCA1

Week
6

Date
07 Aug 2013

-4
-4
-1
-1
-1

30 May 2013
30 May – 13 Jun 2013
21 Jun 2013
21 – 25 Jun 2013
21 – 23 Jun 2013

6
7
7
7
7

08 Aug 201312 Aug 2013
14 Aug 201314/15 Aug 201314/15 Aug 201300

26 Jun 201328 Jun 2013

Hive samples (uncapped nectar, bee bread)
CCA2
Recording of hive weights
CCA3
Hive bee sampling for Varroa and Nosema
assessment
Feeding
Feeding; Measurement of remaining food8
10

21 Aug 201305/06 Sep 2013

0
1
1
1
1
2
2
2
2
3
3
3
3
3
3
4
4
4
5

28 Jun 201301 Jul 201303 Jul 201303 Jul 201303 Jul 201309 Jul 201312 Jul 201312 Jul 201312 Jul 201316 Jul 201317 Jul 201317/18 Jul 201318 Jul 201319 Jul 201319 Jul 201322 Jul 201324 Jul 201326 Jul 201327/28 Jul 2013

Pollen samples from pollen trap
Feeding; Measurement of remaining foodStability samples
Feeding; Measurement of remaining foodPollen samples from pollen trap
Feeding; Measurement of remaining foodFeeding; Measurement of remaining foodStability samples
Pollen samples from pollen trap
New stock solution
Feeding; Measurement of remaining food
CCA4
Hive samples (uncapped nectar, bee bread)
Feeding; Measurement of remaining foodPollen samples from pollen trap
Recording of hive weights
Feeding; Measurement of remaining foodFeeding; Measurement of remaining foodApiary C vandalized11
10/11 Sep 201312
17 Sep 201312
18/20 Sep 201315
08 Oct 201316
16/17 Oct 201316
17 Oct 201316/17 18/23 Oct 2013-13 Dec 2013-19 Dec 2013-13 Jan 2014-20 Jan 2014-27 Jan 2014
-07 Feb 2014-18 Feb 2014
-02 Mar 2014
-11 Mar 2014
After over-wintering-22 Mar 2014
-22 Mar 201455
531 Jul 201302 Aug 201302 Aug 2013

Feeding; Measurement of remaining foodFeeding; Measurement of remaining foodStability samples

----

5

02 Aug 2013

Pollen samples from pollen trap

22 Mar 2014
24 Mar 201415 Apr 2014

Activityst
1 sample shipment
Measurement of remaining food
Recording of hive weights
Hive samples (uncapped nectar, bee bread)
CCA5
Hive bee sampling for Varroa and Nosema
assessment
Varroa counts CCA5
Removal of 100 and 200 ppb hives to separate
apiaryCCA6 (UTC, 12.5 ppb, 25 ppb, 50 ppb)
Recording of hive weights
CCA6 (100 ppb, 200 ppb)
Recording of hive weights
CCA7 (UTC, 12.5 ppb, 25 ppb, 50 ppb)
Pollen samples from pollen trap
CCA7 (100 ppb, 200 ppb)
Feeding 1 L 2:1 sugar syrup per hive
Feeding 1 L 2:1 sugar syrup per hive
Feeding 1 L 2:1 sugar syrup per hive
Feeding 1 L 2:1 sugar syrup per hive
Feeding 1 L 2:1 sugar syrup per hive
Feeding 1 L 2:1 sugar syrup per hive
Feeding 1 L 2:1 sugar syrup per hive
Feeding 1 L 2:1 sugar syrup per hive
Feeding 1 L 2:1 sugar syrup per hive
CCA8
Hive bee sampling for Varroa and Nosema
assessment
Hive samples (capped honey, bee bread)
Recording of hive weightsnd
2 sample shipment

18105Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
2.7.1. Colony mortality
Any colony (hive) that did not show the presence of a queen and had no open brood or eggs, or was
devoid of worker (female) bees was considered “dead”. If a hive was considered “dead” at the time ofassessment, it was no longer used in the analysis of endpoints (e.g., adult bee numbers, hive weight). The
number of individual dead bees was not recorded.

2.7.2. Colony Condition Assessments (CCA)
Observations were blocked by the observer, with the same person always observing the same set of hives
to avoid viewer discrepancies in the data, Apiary A, B, C, G, J, and L were inspected by one inspector andapiaries D, E, F, H, I, and K by another inspector.
Eight CCAs were conducted during the entire study. CCA1 (day -45), and CCA2 (day -13 to -27) wereconducted during the hive establishment. CCA3 (day -1 to -5 days) was conducted 1 week prior to thefeeding exposure which served as initial hive conditions prior to the feeding exposure. CCA4 (17/18 Jul
2013) was conducted 3 weeks after the start of feeding exposure. After the end of feeding exposure (Week
6), the following additional CCAs were conducted: CCA5 (week 7), CCA6 (week 11), CCA7 (week 16)
and after overwintering CCA8 on 22-24 Mar 2014. Each CCA period in 2013 took two or more days tocomplete. For summary statistics, the first day is used to characterize any given CCA.
The time schedule of CCAs is summarized in Table 6 .
6Table 6. Schedule for colony assessment and beekeeper checks
Date

Timing

Evaluation/Activity

12 May 201330, 31 May, and 3, 4, 5, 9,
10, 12 and 13 Jun 2013
21, 23 and 25 Jun 201345 DBE

CCA1 (non-GLP)

27 to 13 DBE

CCA2 (non-GLP)

5 to 1 DBE

17 and 18 Jul 2013

21/22 DAE

CCA3 (before start of feeding; feeding began
26 June)
CCA4 (during exposure, 3 weeks after start of
exposure)
CCA5 (1 week after exposure ended)
CCA6 (5 weeks after exposure ended)*
CCA7 (prior to over-wintering)
CCA8 (after over-wintering)

14 and 15 Aug 201310, 11, 18 and 20 Sep 201316, 17, 18 and 23 Oct 2013
22 Mar 201449/50 DAE
76/77/86 DAE
112 to 119 DAE
269 DAE

DBE: Days before start of exposure; DAE: Days after start of exposure*CCA6 timing allows all bee individuals (eggs, larvae, pupae) present during the exposure period to complete their development cycle
to adults.

During the colony condition assessments, each frame was removed and inspected one at a time, withmeasurements for endpoints taken as percent of total frame area covered by honey / nectar, bee bread /pollen, eggs, open brood (larvae), capped brood (pupae), and adult bees.

19

106

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
The estimation was made by:o Each hive consisted of 20 observed panels (10 frames with two sides of each frame), with an areaof 860 cm2 per side, or a total area of 17,200 cm2 for all 10 frames.
o There were 130 bees per 100 cm2.
o The total number of cells per frame is 3440. Estimated to be 4 cells/cm2.

2.7.3. Evaluation of Disease or Pests in the Hive
Colonies were also checked for visible symptoms of disease or pests, such as Nosema, foulbrood, Varroa
mites or small hive beetle.
To assess the presence of Varroa in the hive, bee samples were taken at the CCA3, CCA5 and CCA8. Bees
were washed in alcohol to remove mites. The number of mites per 100 bees was calculated.

2.7.4. Hive weights
Hive weights were recorded after 10 a.m. once a month from June to October, as well as after over-wintering,on 21 Jun 2013 (week -1), 22 Jul 2013 (week 4), 12 Aug 2013 (week 7), 17 Sep 2013 (week 12), 08 Oct
2013 (week 15) and 24 Mar 2014 (week 39).

2.8.

Residue analysis

All residue and stability samples collected from feeding solution, pollen traps, and test hives were analysed
for imidacloprid, olefin- and 5-hydroxy imidacloprid. Samples from pollen traps in the monitoring hives
were also analysed for residues of multiple pesticides from outside sources at the National Science
Laboratories of USDA in Gastonia (non-GLP). The residue results were reported as ng per g of sample
matrix (ppb), which is different from the test solution that was reported in µg/L,
The LOQ was 5 ppb for imidacloprid, olefin- and 5-hydroxy imidacloprid in feeding solution and hive
nectar samples, and the LOQ was 2 ppb for pollen samples. The LODs are listed in Table 7.
7Table 7. LOD for imidacloprid and its metabolites
Imidacloprid
OlefinDosing/Stability Solutions
2.07 ppb
Hive Collected Nectar
1.38 ppb
Pollen0.74 ppb
Taken from page 175 of the study report
Matrix

Imidacloprid 5hydroxy
2.22 ppb
1.43 ppb
0.18 ppb

Imidacloprid
0.38 ppb
1.43 ppb
0.36 ppb

Olefin- and 5-hydroxy imidacloprid were not detected in any samples except for two samples. The reported
average residue results included only imidacloprid. For the values <LOD, a half of the LOD value was usedin order to calculate the means. Multiple pesticide analysis was conducted in order to monitor pesticidecontamination from outside food sources using pollen collected from pollen traps on the monitoring hives.
20
107Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
All samples for residue analysis were protected from sunlight by using amber vials and transported to freezer
storage after field collection. All samples were placed in frozen storage upon receipt at the test facility.
Samples were maintained frozen (≤ -15° C) (up to -13.9° C for a short period of time) at the test facility
until shipment under frozen conditions to the test site for residue analysis. Daily minimum/maximum
temperatures were recorded for the duration of the storage period at the test facility.

2.8.1. Pollen from outside sources
Pollen samples were collected from pollen traps attached for 24-48 hours to the monitoring hives at eachsite to assess the potential contaminant exposure from outside sources. Pollen amounts collected from eachhive were variable and samples were not available from every site each time. Pollen samples from themonitoring hives were taken at weeks 0 (CCA3), 1, 2, 3 (CCA4), 5, and 16 (17 Oct 2013).

2.8.2. Stored pollen and nectar in test hives
Stored bee bread and bee-collected nectar were collected within the study hives for imidacloprid residue
analysis. Samples weighed at least 500 mg each. Bee bread and uncapped nectar were collected at weeks 3 (CCA4), 4, and 7 (CCA5). Bee bread and capped honey were collected at CCA8 (after overwintering).
All test hives were sampled at CCA4, but only part of them were sampled at the other sampling times.
However, bee bread and honey were not available from every colony each time.

2.8.3. Feeding solution and stability of test item
The monitoring hives were used for dose verification and to evaluate stability of the test item in a hive
environment. Monitoring hives were set up in the same manner as test hives except the colony was denied
access to the spiked or unspiked sucrose. Residue samples comprising approx. 5 g each from the sugar syrup
were taken on week 1 (3 July 2013), week 2 (12 July 2013) and week 5 (2 August 2013).
8Table 8. Sampling schedule for feeding solution and stability of test chemical.
TimingApiary / replicate
UTC12.5 ppb
25 ppb
50 ppb100 ppb
200 ppb
X = samples taken but no sample ID available

Week 1
03 Jul 2013
X
X
X
X
X
X

Week 212 Jul 2013
X
X
X
X
X
X

Week 5
02 Aug 2013
X
X
X
X
X
X

21
108

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

3. Results
3.1.

Land use near test hives

Land use pattern within a 1-mile and 3-mile radius around the 12 apiaries are summarized in Table 9.
The cultivated crop area occupied 0.2-5.5% of the total land within 1 mile radius, and 1.1-2.7% within a 3
mile radius range from the test apiaries. Using the raw data provided, the reviewer calculated the area ofcultivated crops as summarised in Table 10. The mean area of cultivated cropping land was 19 and 168
ha within 1 mile and 3 miles, respectively, of the radius from each apiary.
9Table 9: Percent (%) land use pattern
1 Mile RadiusLand Use Category
Open Water
Developed, Open Space

ApiaryA
0.7
5.7

Developed, Low Intensity
1.1
Developed, Medium Intensity 0.0
Developed, high Intensity
0.0
Barren Land
0.0
(Rock/Sand/Clay)
Deciduous Forest
44.5
Evergreen Forest
5.9
Mixed Forest
2.3
Shrub/Scrub
1.2
Grassland/Herbaceous
4.5
Pasture/Hay
31.6
Cultivated Crops
0.7
Woody Wetlands
1.8
Emergent Herbaceous
0.0
Wetland
Apiary3 Mile RadiusLand Use Category
A
Open Water
0.6
Developed, Open Space
5.6
Developed, Low Intensity
1.0
Developed, Medium Intensity 0.1
Developed, High Intensity
0.0
Barren Land
0.1
(Rock/Sand/Clay)
Deciduous Forest
41.2
Evergreen Forest
6.3
Mixed Forest
2.2
Shrub/Scrub
1.6
Grassland/Herbaceous
3.5
Pasture/Hay
34.9

B
0.8
6.4

C
0.2
2.6

D
1.0
6.9

E
0.7
1.6

F
6.1
5.0

G
0.5
3.5

H
0.6
1.9

I
0.9
1.4

J
0.7
6.5

K
0.8
4.3

L
1.3
7.1

1.5
0.1
0.0
0.1

0.6
0.0
0.0
2.4

1.8
0.1
0.0
0.2

0.8
0.0
0.0
0.0

0.1
0.0
0.0
0.2

1.0
0.1
0.1
0.0

2.9
0.0
0.0
0.0

1.9
0.0
0.0
0.1

2.9
0.2
0.0
0.0

0.8
0.0
0.0
0.0

1.7
0.0
0.0
0.1

38.7
4.2
2.3
1.8
3.2
39.1
1.9
0.1
0.0

52.2
2.5
3.6
2.1
4.7
27.1
1.8
0.3
0.0

33.7
8.5
4.0
0.1
3.4
36.7
2.9
0.7
0.0

49.9
9.6
4.2
1.1
3.6
25.7
1.4
1.3
0.0

48.3
3.3
2.1
3.7
2.0
29.0
0.2
0.0
0.0

39.1
7.7
3.5
2.6
5.1
35.7
0.6
0.4
0.2

46.5
5.8
3.8
0.9
4.0
31.1
2.4
0.1
0.0

35.4
5.5
4.6
2.7
7.3
34.7
5.5
0.0
0.0

32.6
4.4
2.7
0.7
3.0
41.1
5.0
0.0
0.0

40.1
6.8
4.2
2.9
3.2
33.9
2.8
0.2
0.0

28.8
4.7
2.4
0.8
2.1
48.3
2.4
0.0
0.0

B
0.6
4.8
1.6
0.0
0.0
0.4

C
0.5
3.5
1.2
0.0
0.0
0.3

D
0.8
5.6
2.0
0.1
0.0
0.1

E
0.5
3.3
1.6
0.1
0.0
0.0

F
2.3
5.1
0.8
0.0
0.0
0.0

G
0.6
4.9
1.2
0.1
0.0
0.0

H
0.4
2.6
1.7
0.0
0.0
0.1

I
0.6
2.1
1.6
0.0
0.0
0.1

J
0.8
5.2
1.8
0.1
0.0
0.1

K
0.5
4.2
1.4
0.1
0.0
0.0

L
2.5
6.1
1.4
0.1
0.0
0.1

41.2
5.6
2.7
1.6
4.6
34.1

44.4
4.8
2.7
2.0
4.4
33.2

39.4
5.5
2.7
1.4
4.7
34.7

44.0
6.5
3.1
2.6
7.1
28.4

45.8
5.0
2.2
2.2
3.0
31.7

40.2
8.1
2.8
1.9
4.2
33.9

43.4
6.7
3.8
2.6
7.9
27.8

44.0
7.2
3.8
3.1
9.8
24.7

39.2
5.9
3.1
1.5
5.2
33.9

42.6
6.3
3.1
1.8
5.3
31.5

38.3
4.9
2.2
1.5
3.3
36.8

22
109

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
10Table 10: Cultivated cropping area near each test apiary
A

B

C

D

ApiaryE
FG
1 mile radius (813 ha)

H

I

J

K

L

Mean

CultivatedCrops (%)
Area ofcultivatedcrop (ha)

0.7

1.9

1.8

2.9

1.4

0.2

0.6

2.4

5.5

5.0

2.8

2.4

2.3

5.7

15.4

14.6

23.6

11.4

1.6

4.9

19.5

44.7

40.7

22.8

19.5

18.7

CultivatedCrops (%)
Area ofcultivatedcrop (ha)

1.7

2.4

2.5

2.5

2.6

1.1

1.7

2.7

2.7

2.6

2.7

2.0

2.3

124.5

175.8

183.1

183.1

190.4

80.6

124.5

197.7

197.7

190.4

197.7

175.8

168.4

3.2.

3 mile radius (7323 ha)

Pollen sources of test hives

Monitoring hives were used at each test apiary to collect pollen for assessment of the local pollen flora (nonGLP). Pollen trap samples from the monitoring hives were taken at CCA3 (28 Jun 2013; week 0) and CCA4(03 Jul 2013; week 1), as well as on 12 Jul 2013 (week 2), 19 Jul 2013 (week 3) and 02 Aug 2013 (week 5),
and at CCA5 (week 7) and after CCA7 (week 16).
The major pollen was from non-cultivated crops, such as Parthenocissus, Melilotus, Plantago, Rhus, andAsteraceae. Cultivated crops such as Zea mays (maize) and Fagopyrum esculentum (Buckwheat) wereidentified occasionally, and took up the maximum of 13% and 21% of the total pollen particles, respectively.
Full results can be found in Table 49 of the study report (pages 128-129).

3.3.

Consumption of spiked sucrose

Hive consumption rates for the feeding solution (sugar syrup) ranged from 10,290 mL to 12,000 mL of thetotal 12,000 mL per hive provided during a 6-week period (i.e 1 litre per colony 2 times a week for a totalof 12,000 mL per colony during the exposure period). All colonies consumed most or all of the sugar
solution (see 3Figure 3) with a slightly lower consumption in 100 µg/L and 200 µg/L treatment.

23
110Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

3Figure 3. Mean total food consumption (mL) per colony during the 6-week exposure period

3.4.

Examination of pesticides from other sources

Monitoring hives were used to assess the potential contaminant exposure from outside sources (non-GLP)
at each site. Pollen trap samples from the monitoring hives were taken after CCA3 (28 Jun 2013; week 0)
and CCA4 (03 Jul 2013; week 1), as well as on 12 Jul 2013 (week 2), 19 Jul 2013 (week 3) and 02 Aug
2013 (week 5), and at CCA5 (week 7) and after CCA7 (week 16). The amount of pollen collected fromtraps on the monitoring hives varied. A large portion of the pollen samples collected on Aug 2, and Oct 17
were reported as either “No sample” or “no sample sent to USDA”. For these, samples with no pollen
collected were indicated as “No sample”, while those samples without enough to meet the mass requirement
for pesticide analysis were indicated as ‘No sample sent to USDA’. It is noted that out of 16 weeks fromthe beginning of feeding exposure to the last CCA before overwintering, pollen samples were collected 6
times with each collection over a period of 1-2 days and a few of them were not analyzed for residue analysis
due an insufficient amount of pollen for analysis. Pesticide contamination was unknown for those intervals
when pollen samples were not collected.
Dimethenamid (maximum at 87 ppb), Fenamidone (maximum at 345 ppb), Spiromesifen (maximum at 961
ppb) were the major pesticides detected in the pollen samples originating from outside food sources (Table
11). High levels of piperonyl butoxide (maximum at 591 ppb) were detected in the monitoring hive at
Apiary A along with several other pesticides. Out of 6 test sites where the pollen samples were collected
on 02 Aug 2013, imidacloprid was detected in one pollen sample at Apiary L at 12.1 ppb. The detection
frequency by the test sites was 1/6. It was found that the contaminated pollen sample at Apiary L consistedof 92.4% of Rhus, 5.2% plantago, 2.3% of Lagerstroemia indica, none of which are major cultivated crops.

24111Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
11Table 11. Residues from outside sources in pollen samples from pollen trap (non-GLP)
Apiary6/28/13

7/3/13

7/12/13

Sampling Date
7/19/13A

No detects

Chlorpyrifos(trace)No detects

B

No detects

No detects

C

No detects

No detects

No sample sent to
USDA
No detects

D

Hydroprene(trace)No detects

No detects

E

No sample

No sample

F

No detects

No sample
sent to USDANo detects

G

No detects

No detects

No detects

H

No detects

No detects

I

No detects

No detects

Dimethenamid (44.9)
Fenamidone (134)Fenhexamid (trace)J

No detects

No detects

Dimethenamid (26.5)

K

No detects

No detects

L

No detects

No sample

Dimethenamid (10.3)
Spiromesifen (80.1)
MGK-326 (trace)

No sample sent to
USDA

DDD p,p’ (14.4)
Dimethenamid (87.0)
Fenamidone (345)Fenhexamid (trace)
Metalaxyl (trace)
Piperonyl butoxide(591)
Quinoxyfen (474)
Spiromesifen (961)
No sample sent to
USDA
Metalaxyl (6.0)
Methamidophos (62.3)
Dimethenamid (71.7)
Fenamidone (215)
Spiromesifen (584)No sample
Dimethenamid (47.0)
Fenamidone (90.5)
Spiromesifen (362)
Dimethenamid (14.6)
Spiromesifen (148)
No sample sent to
USDA
No detects
No sample sent to
USDA
Trifluralin (trace)
No detects

THPI = tetrahydrophthalimide
Residue values in parentheses are ppb.
From Table 64, page 142 of study report.

3.5.

8/2/13

10/17/13

No detects

No sample sent
to USDATHPI (299)No sample

No sample sent
to USDANo sample

No detects

No detects

No sample

No sample

No sample sent to
USDA

No sample

No detects

No sample sent
to USDANo sample

No sample sent to
USDA
No sample sent to
USDA
No sample sent to
USDA
No detects
Imidacloprid (12.1)
Malathion (trace)

No sample sent
to USDA
Thymol (193)
No sample
No detects

Confirmation of test concentrations

Imidacloprid and its major transformation products were analyzed from feeding solutions sampled after they
were prepared before start of feeding, three times on 2 July (week1), 12 July (week 2) and 2 August 2013
(week 5). The averages of measured concentrations were <LOD, 11.0, 23.3, 46.7, 96.3, and 189.6 ppb forthe nominal concentrations of control, 12.5, 25, 50, 100, and 200 µg/L, respectively. It is noted thatimidacloprid and 5-OH-imidacloprid were detected in one control feeding solution at 0.45 and 4.22 ppb
respectively, sampled at week 2 on 12 July 2013. It is unknown which control hives were fed with thecontaminated feeding solution. The data are tabulated below in Table 12.

25112Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
12Table 12. Dosing solution residue data from 2 July 2013 (Week 1), 12 July 2013 (Week 2) and 2
August 2013 (Week 5)
Average ofmeasuredconcentrations
(ppb)
<LOD†
11.0
23.3
46.7
96.3
189.6

Nominal concentrations(µg/L)(ppb)

0 (Control)
12.5
25
50
100
200

0
10.2
20.3
40.7
81.3
162.7

Measured imidacloprid concentrations(ppb) (n=6)
<LOD
11.3
24.24
44.34
99.15
193.51

<LOD
11.96
23.45
46.71
99.23
195.06

<LOD
10.62
22.91
48.99
91.04
186.9

0.45*
10.2
22.14
43.5
96.1
190.07

<LOD
10.73
23.45
48.66
96.87
189.04

<LOD
11.22
23.75
48.11
95.12
182.94-Regenerated from Table section 5.5, on page 189-190 in the study report†: LOD=0.38 ppb for imidacloprid;*: In the same control sample, 5-OH-imidacloprid was also detected at 4.22 ppb

3.6.

Stability of the test item in feeding solution

Stability of imidacloprid in the sugar solution during the feeding period was examined from diet collectedfrom closed-off feeding solutions placed in the monitoring hives, sampled three times on 3 July 12 July, and2 August 2013. No reduction of test concentrations in the feeding solution was noticed during the feeding
period. The stability of imidacloprid at 200 µg/L in the feeding solution was not provided, but a significant
reduction is not expected based on the reported data for all other concentrations. It is noted that imidacloprid
was detected at 0.56 ppb in one of the control solution for the control hive H4 sampled on 12 July 2014. No
imidacloprid olefin or imidacloprid 5-hyrdoxy was detected in any of the samples (LOD of 2.07 ppb and2.22 ppb, respectively). Average imidacloprid residue data for the stability solution are presented in Table
13.
13Table 13. The stability of imidacloprid in feeding solution on 3 Jul, 12 Jul, and 2 Aug, 2013.
Nominalconcentration(µg/L)

Average of measured
concentrations (ppb)

Number ofsamplesmeasuredControl

<LOD†

20

12.5
25
50
100
200

11.4
23.2
47.4
93.6
N/A*

12
10
10
12
N/A

- Regenerated from Section 5.6, on page 191-193 in the study report†: LOD=0.38 ppb for imidacloprid;*: N/A: data not available.

Measured imidacloprid
concentrations (ppb)
03 Jul,12 Jul,02 Aug,
2013
2013
2013
0.56 ppb in one out of 20 samples.
<LOD in 19 samples;
11.74
11.86
10.65
23.65
23.40
22.89
46.62
46.09
51.78
95.77
92.09
92.98
N/A

26
113

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
3.7.

Residues in hive matrices

It is noted here as it was in the uncertainties section that the residue samples from the different hive matrices
represent a single sample from a single hive. Therefore there is variation in the residues that likely stems
from the sampling procedure employed for this study (single sample, one side of the comb).

3.7.1. Background imidacloprid contamination in hives prior to the feeding exposure
The background imidacloprid contamination in test hives was examined using hive bee bread (hive pollen)
and nectar collected about a month (30 May 2013) prior to the beginning of feeding exposure. Imidacloprid
was detected in two out of a total of six hive pollen samples at 0.43 ppb and 1.19 ppb, respectively (Table14), with a mean of 0.81 ppb and a detection frequency of 33% (2 hives out of total 6 hives). It is noted thatthe limit of detection for imidacloprid in pollen for this study was 0.36 ppb. Imidacloprid was not detectedin any hive nectar samples collected prior to the feeding exposure (Table 15). Residue analysis for otherpesticides was not conducted prior to exposure.
14Table 14. Detailed hive pollen residue data, pre-study collection (30 May 2013)
Location

Imidacloprid Olefin (ppb)

LODs
Pope
Maple
Greenhouse
Corbett Ridge
Cedar Grove
Prospect Hill

0.74
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD

Imidacloprid 5 Hydroxy
(ppb)
0.18
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD

Imidacloprid (ppb)

Residue values were not corrected for recovery or moisture content of the sample.
Residues below are LOD are reported as <LOD.
From page 183 of the study report.

0.36
<LOD
<LOD
0.43
<LOD
<LOD
1.19

15Table 15. Detailed hive nectar residue data, pre-study sample collection (30 May 2013)
Location
LODs
Pope
Maple
Greenhouse
Corbett Ridge
Cedar Grove
Prospect Hill

Imidacloprid Olefin
(ppb)
1.38
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD

Imidacloprid 5 Hydroxy
(ppb)
1.43
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD

Imidacloprid (ppb)
1.43
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD

Brix (%)*
>80
79
78
80
80
80

Taken from page 194 of the study report. *Brix % is the percentage of sugar content in honey by mass, measured by a refractometer.

3.7.2. Residues in hive matrices during and after feeding exposure
Imidacloprid and its two major transformation products in hives (imidacloprid olefin and 5-hydroxyimidacloprid) were examined three times after the feeding started using hive bee bread and hive nectar. All
test hives were sampled at 1st batch of sampling (CCA4, 18 July 2013) during the exposure phase, but only
part of test hives were sampled at the other two sampling times, CCA5 which was one week after the feeding
exposure, and CCA8 after the overwintering).
27
114

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
3.7.2.1. Residues in hive matrices at CCA4 (after 3 weeks of exposure)
The level of imidacloprid in hive bee bread and uncapped nectar after 3 weeks of feeding (CCA4) wassummarized in Table 16 and 17. All test hives were sampled at CCA4 (18 July 2013). A dose-response
correlation was observed between the imidacloprid concentrations in the feeding solution and measured
concentrations in both bee bread and uncapped hive nectar. However, the imidacloprid concentration in
hive uncapped nectar and pollen was 64% and 26% of the mean concentrations in feeding solution,
respectively. It is possible that dilution of stored pollen and nectar from other food sources occurred during
the exposure period since, as indicated in the study, a significant degradation of imidacloprid in test solution
was not detected in the study.
Imidacloprid in bee bread at CCA4: The level of imidacloprid in hive bee bread after 3 weeks of feeding(CCA4) was summarized in the Table 16. Imidacloprid was detected in all measured treatment samples.
No imidacloprid metabolites were detected. It was noted that not all residue information in pollen wasavailable. No residue information for treatment at 200 ug/L in bee bread was provided. Out of 12 hives, four
hives at 100 ug/L and eight hives at 50 ug/L were measured, respectively.
The results showed a dose-response correlation between the average concentrations measured in hive beebread and the concentrations in the feeding solution. However, the concentrations varied within eachtreatment group (see Table 16). The mean of the measured concentrations in bee bread within eachtreatment group of 12.5, 25, 50 and 100 ug/L was 2.86 (range: 0.77-5.34), 5.37 (range: 1.45-9.41), 10.84(range: 4.2-19.41), and 17.89 ppb (range: 2.66-35.1), respectively. By average, the measured concentration
was 25.8% (range 22-28.1%) of the concentration in feeding solution, and 27.8% (range 24.9-31.8 %) of
the measured concentrations in uncapped hive nectar (data not shown in the table). The results showed thatafter 3 weeks of feeding, imidacloprid concentrations in hive bee bread appeared remarkably lower thanthat in the feeding solutions and in hive nectar. The lower concentration in bee bread is expected due to thedilution since bee bread is a mixture of nectar and pollen from various sources.

28
115

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
16Table 16. Imidacloprid concentrations (ppb) in hive pollen sampled three weeks after the start ofartificial feeding on 18 July 2013 (CCA4).
Measured imidacloprid concentrations (ppb) (LOD = 0.38 ppb)*
Nominal concentration (ug/L)
ApiaryControl 1

Control 2

12.5

25

50

100

200

Nominal concentration (ppb)
10.2
20.3
40.7
‡

A
B
C
D
E
F
G
H
I
J
K
L
Number of samples
measured
Averageconcentration

81.3

162.7

<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
1.24
<LOD
<LOD
<LOD
<LOD

<LOD
1.05
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD

3.02
3.33
3.47
0.77
1.59
2.09
1.54
5.34
4.15
3.51
2.4
3.17

4.01
2.34
7.17
2.32
3.03
5.19
6.38
1.45
9.41
7.81
8.05
7.31

7.17
4.2
9.56
5.79
10.1
11.19
19.31
19.41

9.84
23.97
2.66
35.1
-

-

12

12

12

12

8

4

0

2.86

5.37

10.84

17.89

-

0

0

<LOD

% of the feeding
Not applicable
28.1
26.4
26.7
22.0
concentration††% of the average
31.8
27.3
27.4
24.9
detection in hive
Not applicable
Nectar†††* regenerated from the additional residue information (email forwarded by Keith Sappington (EPA) to Tina Singal (PMRA) on March 10,2015) ; “-“ indicates that data are not available‡Nominal concentration in ppb is estimated from the concentration in µg/L by assuming the volume density of the test solution to be
1.2296 g/ml.
†† % Feeding concentration: the average of measured concentration compared with the nominal feeding concentrations in ppb.
††† % of the average detection in hive Nectar: the average of measured concentration in pollen compared with the average measured
concentration in nectar ppb without corrections for sugar.

Imidacloprid in hive uncapped nectar at CCA4: The level of imidacloprid in hive uncapped nectar during
the feeding exposure (CCA4) was summarized in Table 17. All twelve test hives were measured.
Imidacloprid was detected in the majority of the measured treatment samples. Out of 12 hives measured
for each concentration, <LOD was reported in two hives at 12.5 ug/L (Apiary E and H) and two hives at
200 ug/L (Apiary D and F). No imidacloprid metabolites were detected.
The results showed a dose-response correlation between the average concentrations measured in uncapped
hive nectar and the concentrations in the feeding solution. However, the concentrations varied remarkably
within each treatment group (see Table 17). Remarkably lower concentrations were detected in nine test
hives, including all 5 treatment hives at apiary D, E5, E7, F1, and H7. <LOD was reported for treatment at
200 µg/L in hive D3 and F1. After correction with Brix values to 50% sugar concentration, the mean of themeasured concentrations in uncapped hive nectar within each treatment group of 12.5, 25, 50, 100, and 200
ug/L was 6.31 (range: 0.88-9.42), 13.24 (range: 1.19-20.53), 27.66 (range: 2.31-40.59), 46.87 (range: 2.129
116

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
80.15), and 109.14 ppb (range: 0.89-152.94) respectively. By average, the measured concentration in hive
nectar was 64% (range 62.0-68.0%) of the concentration in feeding solution. The results showed that after
3 weeks of feeding, imidacloprid concentrations in hive nectar appeared lower than that in the feeding
solutions, which indicated that the foraging bees also foraged on nectar sources other than the provided
sugar sources which diluted the level of treatment. It is noted that this result is expected, as bees wereallowed to freely forage, and also, under natural conditions bees typically forage on multiple plant pollen
and nectar sources.
17Table 17. Imidacloprid concentrations (ppb) in uncapped hive nectar sampled three weeks after thestart of artificial feeding on 18 Jul, 2013 (CCA4).
Measured imidacloprid concentrations (ppb) (LOD=1.43 ppb)*
ApiaryNominal concentration (ug/L)
Control 1†

Control 2†

12.5

25

50

100

200

Nominal concentration (ppb) ‡
A

0

0

<LOD

<LOD

10.2

20.3

40.7

81.3

162.7

9.42

14.06

40.36

48.85

96.98

B
<LOD
2.98
7.91
20.53
31.12
57.45
124.53
C
<LOD
<LOD
6.29
7.97
38.61
66.88
134.77
D
<LOD
<LOD
3.37
1.19
2.91
22.58
0.89
E
<LOD
<LOD
0.89
7.12
2.31
80.15
90.19
F
<LOD
<LOD
8.04
13.89
34.71
62.15
0.89
G
<LOD
<LOD
7.6
14.82
16.71
58.9
150.79
H
<LOD
<LOD
0.88
10.47
32.32
26.17
137.27
I
<LOD
<LOD
8.02
13.69
18.15
23.55
136.91
J
<LOD
<LOD
7.66
15.51
40.59
47.7
139.32
K
<LOD
<LOD
7.01
19.67
38.83
2.1
144.27
L
<LOD
<LOD
8.57
20
35.25
65.95
152.94
Number of samples
12
12
12
12
12
12
12
Average concentration
<LOD
6.31
13.24
27.66
46.87
109.14% Feeding
62.0
65.1
68.0
57.6
67.1
concentration††* Concentrations in all treatments except for the controls are corrected to 50% sugar using Brix values that are not listed in the table, butwere in the table section 5.9 on page 195-197 of the study report† Concentrations in the controls are measured concentrations in hive uncapped without corrections for sugar concentrations.
‡Nominal concentration in ppb is estimated from the concentration in µg/L by assuming the volume density of the test solution to be
1.2296 g/ml.
†† % Feeding concentration: the average of measured concentration compared with the nominal feeding concentration in ppb.

3.7.2.2. Residues in Hive Matrices at CCA5 (1 week after end of exposure)
The level of imidacloprid in hive bee bread and uncapped nectar one week after the end of feeding exposure(CCA5, 14-15 Aug 2013) was summarized in Table 18. Only three apiaries were sampled (Apiaries A, B,
and L). Again, the level of imidacloprid residues in hive nectar was reported for all treatment concentrations
but not for all the bee bread, especially for 100 and 200 µg/L. In summary, similar to CCA4, a dose-response
correlation was observed between the average concentrations of imidacloprid measured in both bee breadand uncapped hive nectar and the concentrations in the feeding solution. However, the imidacloprid
concentration in hive uncapped nectar and beebread was lower than what was in the feeding solutions,indicating dilution of stored bee bread and nectar from other food sources.
30
117Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Shown in Table 18, a dose-response correlation was shown between the average concentrations ofimidacloprid measured in both bee bread and uncapped hive nectar and the concentrations in the feeding
solution. However, the concentrations varied remarkably within some treatment groups despite the low
number of samples measured. In bee bread, the mean of the measured concentrations for 12.5, 25, 50 and
100 ug/L was 4.22 (range: 3.26-5.25), 5.74 (range: 4.89-6.4), 16.44 (range: 14.37-18.00), and 22.89 ppb (norange, only one measurement), respectively. By average, the measured concentration was 28.1% (range
22.9-33.8%) of the concentration in feeding solution, and 40.3% (range 26.0-51.5 %) of the measured
concentrations in uncapped hive nectar without correction for sugar content (data not shown in the table).
In uncapped hive nectar, after correction with Brix values (amount of sugar dissolved in solution) to 50%
sugar concentration, the mean of the measured concentrations within each treatment group of 12.5, 25, 50,
100, and 200 ug/L was 5.88 (range: 3.36- 7.28), 7.18 (range: 0.89-10.68), 27.46 (range: 22.93-33.39), 54.98(range:5.79-79.79), and 127.93 ppb (range:103.32-144.27) respectively. By average, the measured
concentration in hive nectar was 61.4% (range 35.3-78.7%) of the concentration in feeding solution.
18Table 18. Imidacloprid concentrations (ppb) in beebread and uncapped hive nectar sampled one week
after the end of artificial feeding on 14 Aug, 2013 (CCA5).

MatrixResidues inbee bread

ApiaryA
B
L
Number of samples measured
Average% Feeding Solution (ppb) ††Measured imidacloprid concentrations (ppb) (LOD=0.38 ppb pollen;
1.43 ppb nectar)*
Nominal concentration (ug/L)
Control 1
Control 2
12.5
25
50
100
200
Nominal concentration (ppb) ‡
0

<LOD
<LOD
<LOD
3

0

<LOD
<LOD
0.55
3

<LOD
Not applicable

10.2

20.3

40.7

81.3

162.7

5.25
4.16
3.26
3
4.22
41.5

4.89
5.94
6.4
3
5.74
28.2

14.37
18.00
16.96
3
16.44
40.4

22.89
1
22.89
28.1

0
-

Not applicable
45.6
51.5
38.1
26.0
% Nectar †††Residues in
A
<LOD
<LOD
7.28
0.89
22.93
5.79
136.19uncapped
B
<LOD
<LOD
7.01
10.68
33.39
79.79
144.27nectar after
L
<LOD
<LOD
3.36
9.97
26.06
79.36
103.32
correction to
Number of samples measured
3
3
3
3
3
3
3
50% sugar
Average
<LOD
5.88
7.18
27.46
54.98
127.93
Not applicable
57.8
35.3
67.5
67.6
78.7
% of the Feeding Solution††* Concentration in all treatments except for the controls are corrected to 50% sugar using Brix values. “-“ indicates that data are notavailable‡Nominal concentration in ppb is estimated from the concentration in µg/L by assuming the volume density of the test solution to be
1.2296 g/ml.
†† % Feeding solution: the average of measured concentration compared with the nominal feeding concentrations in ppb.
††† % Nectar: percent of the average of measured concentration in bee bread (hive pollen) compared with the average measured
concentration in nectar (ppb) without corrections for sugar.

3.7.2.3. Residues in hive matrices at CCA8 (after overwintering)
The level of imidacloprid in hive bee bread and capped honey after overwintering (CCA8, 22 March 2014)
was summarized in Table 19. Only surviving hives in four apiaries were sampled (Apiaries E, I, J, and L).
Again, imidacloprid residue was not reported for all hives sampled. In bee bread, imidacloprid was notdetected in treatments of control, 12.5, 25 and 100 ug/L, but was detected at 0.52 ppb (E7), 0.52 ppb (I3),
and 0.40 ppb (J2 in three 50 ug/L treatment hives. No measurement was provided for treatment at 200 ug/L.
31
118

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
In honey, no imidacloprid residues were detected in all measured hives except for one at 13.53 ppb (L4) inthe 100 ug/L treatment group.
The average concentration of imidacloprid in hives after overwintering (CCA8) is considered to be
uncertain, especially for hives at 100 and 200 ug/L. After overwintering, residues were analyzed only fromsurviving hives, not from the dead hives. In the study, a high number of hives was reported dead after
overwintering. At 100 and 200 ug/L treatments, only one and two hives survived, respectively. The
unmeasured level of residues in dead hives presents an uncertainty as to the average of residues that might
represent the level of treatments at CCA8.
19Table 19: Imidacloprid concentrations (ppb) in bee bread and honey sampled after overwintering on
March 24, 2014 (CCA8).

MatrixResidues in bee
bread

Measured imidacloprid concentrations (ppb) (LOD=0.38 ppb pollen; 1.43 ppb
nectar)*Nominal concentration (ug/L)

ApiaryControl 1

Control 2

E

0
<LOD

0
<LOD

I

<LOD

<LOD

<LOD

<LOD

J

<LOD

<LOD

-

L

<LOD

<LOD

Number of samples
measured

4

4

Average% Feeding Solution ††Residues in
uncapped
nectar after
correction to
50% sugar

12.5

25

50

100

200

81.3
-

162.7
-

0.52

-

-

<LOD

0.4

-

-

<LOD

<LOD

<LOD

<LOD

-

3

4

4

1

0

<LOD

<LOD

0.48

<LOD

-

Nominal concentration (ppb) ‡
10.2
20.3
40.7
<LOD
<LOD
0.52

<LOD

Not applicable1.2

Not applicable

E

<LOD

<LOD

<LOD

<LOD

<LOD

-

-

I

<LOD

<LOD

<LOD

<LOD

<LOD

-

-

J

<LOD

<LOD

-

<LOD

<LOD

-

-

L

<LOD

<LOD

<LOD

<LOD

<LOD

13.53

-

Number of samples
4
4
3
4
4
1
0
measured
Average
<LOD
<LOD
<LOD
<LOD
54.98
Not applicable
16.7
Not applicable% Feeding Solution††* “-“ indicates that data are not available‡Nominal concentration in ppb is estimated from the concentration in µg/L by assuming the volume density of the test solution to be
1.2296 g/ml.
†† % Feeding concentration: the average of measured concentration compared with the nominal feeding concentrations in ppb.

3.7.2.4. Detection of imidacloprid in test hives at CCA2, CCA4, CCA5, and CCA832
119

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Imidacloprid was detected during the entire course of the study in test hives before the feeding exposure(CCA2) and in the control hives during the exposure (CCA4) and post exposure (CCA5). However, it was
not detected in the live control hives after overwintering (CCA8). The magnitude and the frequency ofdetection of imidacloprid in control hives are summarized in Table 20. The detection in control hives was
relatively low and was <LOD in the majority of control hives. In a total of 35 bee bread samples fromcontrol hives, imidacloprid was detected in 6 samples with a maximum of 1.24 ppb. In a total of 36 uncapped
nectar samples, imidacloprid was detected in only one control hive at 2.98 ppb. 33% of the hives had been
exposed to the test chemicals before the start of the test (CCA2), and <20% of control hives were
contaminated with the test chemical during (CCA4), and one week after, the exposure (CCA5).
Before the start of the artificial feeding exposure (CCA2), test hives had been contaminated with a low levelof imidacloprid. Out of the total six hives measured, imidacloprid was detected in bee bread of two hives(33% of the measured hives) at 0.43 and 1.19 ppb respectively (LOD= 0.36 ppb). However, it was notdetected in uncapped nectar (LOD= 1.43 ppb) in all six measured hives.
During the exposure period (CCA4), the control hives were contaminated with a low level of imidacloprid.
Out of 23 control hives measured, imidacloprid was detected in hive bee bread in three control hives at 1.05,
1.24, and 0.68 ppb (LOD= 0.36 ppb), respectively. Out of 24 control hives measured, imidacloprid was
detected in uncapped nectar in one control hive at 2.98 ppb (LOD=1.43 ppb). The frequency of detectionsin the control hives was 3/21 in bee bread and 1/24 in nectar.
One week after the end of the feeding exposure (CCA5), a low level of imidacloprid was also detected in
the control hives. Out of five control hives measured, imidacloprid was detected in one control hives in bee
bread at 0.55 ppb (LOD=0.36 ppb). Imidacloprid was not detected in any of the six control hives in nectar(LOD = 1.43 ppb). Out of 8 control hives, no imidacloprid were detected in hive bee bread and honeysamples after overwintering (CCA8). It was noted that the frequency of detection of imidacloprid was lower
in hive nectar than in the bee bread during CCA2, CCA4 and CCA5. This likely resulted from the lesssensitive LOD in nectar as the maximum detection in bee bread was even lower than the LOD for nectar.
In order to consider the potential impact of the detected imidacloprid contamination, the maximum
detections in the control hives were compared with the average residues detected in the treatment hives andexpressed as a percentage in Table 21. The maximum residues in the controls counted for 1.8-41.6% of theaverage detections in treatment hives. The percentage varied by the test concentrations, low in treatmentswith high concentrations but high in treatments with low concentrations. It appeared that the level of
imidacloprid contamination might have a greater impact to the treatments with low concentrations than thehigher ones. Therefore impact of the contamination should not be ignored especially for treatment at low
concentrations. However, due to the low detection frequency in control hives, this impact is likely to be
only on a few individual hives.
Imidacloprid detected in uncapped nectar in the control hives indicated that a slight level of cross foraging
among test hives might have occurred during the exposure period. Overall: (1) imidacloprid (2.98 ppb) was
detected in one control hive nectar during the exposure period at CCA4, but no detection (<LOD =1.43
ppb) in all hives prior to the feeding exposure (Table 20); (2) Although a low level of imidacloprid was
detected in one of the control feeding solutions at 0.45 ppb (Table 14), this level was so low it would unlikely
result in such a high level of the detection in hive nectar (about 6X increase); (3) No other sources ofimidacloprid were detected at CCA4 and earlier. Imidacloprid was not detected (LOD=0.36 ppb) in any
33120

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
pollen samples trapped in the monitoring hives at CCA4 and earlier (Table 21). Considering the doseresponse relationship of the residues detected in the treatment hives, the imidacloprid contamination incontrol hives is expected to have minimal impact to the colony level effect, especially for the treatmentswith high concentrations.
Some individual hives with measured residues had exposure levels more similar to exposure levels measured
in higher or lower doses. While the residue data provides a good indication of exposure, there is someuncertainty regarding the extent of the variability in exposure since residues in all hives were not measured,
residues were measured only at certain timepoints, and as discussed earlier, residue samples were taken only
from one location in the hive. Therefore, there is some uncertainty regarding the true extent of variability inmeasured hive residues and exposure. The residue data does clearly indicate that a dose responserelationship is expected since higher treatment levels had higher mean measured residues in the hive pollen
and nectar.
20Table 20. Imidacloprid (ppb) detected in test hives before the exposure (CCA2), and in control hives
during the exposure (CCA4) and after the exposure (CCA5 and CCA8).

Sampling dates
Sample matrix
LOD (ppb)
Total number ofsamples measured
Number of samples
with quantifiablelevel of residues(Residues in ppb)
% of samples with
detected residue(95% confidence
limit, low-upper)

Pre-exposure(background)
CCA2
30 May, 2013
Uncapped
Bee bread
nectar0.36
1.43

During exposure(26 Jun –8 Aug, 2013)
CCA4
18 Jul, 2013
Uncapped
Bee bread
nectar0.36
1.43

Post exposure(8 Aug 2013 – 22 Mar, 2014)
CCA5
CCA8
14 Aug, 2013
22 Mar, 2014
Bee
Uncapped
Bee bread
Honey
bread
nectar
0.36
1.43
0.36
1.43

6

6

23

24

6

6

8

8

2
(1.19,
0.43)

03(1.05, 1.24,
0.68)

1(2.98)

1(0.55)

0

0

0

33.3
(4.3-77.7)

0.0(0.0-45.9)

13.0(2.8-33.6)

4.2(0.1-21.1)

16.7(0.464.1)

0.0(0.0-45.9)

0.0(0.0-36.9)

0.0(0.0-36.9)

21Table 21. Comparison between the maximum detections of imidacloprid in control hives and theaverage residues detected in the same hive matrices in the treatment hives fed at different concentrationsof imidacloprid for three weeks (CCA4).
Test concentrationin feeding solution

Averageresiduemeasured inbee bread) at
CCA4 (ppb

Average residue
in hive nectar at
CCA4 (ppb)

ug/L

ppb

12.5

10.2

2.86

9

25

20.3

5.37

19.7

% of the maximum detection in control hives in comparison
to the average of measured concentrations in treatment
hives at CCA4
Hive
Bee bread
nectar
PreDuring
Post
Duringexposure
exposure
exposure
exposure(CCA2)(CCA4)(CCA5)(CCA4)(Max=1.19(max=1.25(max=0.55(max=2.98
ppb)
ppb)
ppb)
ppb)
41.6
43.7
19.2
33.1
22.2

23.3

10.2

15.1

34
121

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

Test concentrationin feeding solution

Averageresiduemeasured inbee bread) at
CCA4 (ppb

Average residue
in hive nectar at
CCA4 (ppb)% of the maximum detection in control hives in comparison
to the average of measured concentrations in treatment
hives at CCA4
Hive
Bee bread
nectar
PreDuring
Post
Duringexposure
exposure
exposure
exposure(CCA2)(CCA4)(CCA5)(CCA4)(Max=1.19(max=1.25(max=0.55(max=2.98
ppb)
ppb)
ppb)
ppb)

ug/L

ppb

50

40.7

10.84

39.5

11

11.5

5.1

7.5

100

81.3

17.89

71.8

6.7

7

3.1

4.2

200

162.7

NA

162.4

Not
applicableNot
applicableNot
applicable

1.8-Imidacloprid was not detected in hive nectar (LOD=1.43 ppb) in any control hives in CCA2 and CCA5
-NA: data was not available.
-Measured concentration in hive nectar was not corrected for sugar content.

3.7.2.5. Comparison of concentration in feeding solution and hive matrices
A correlation between the imidacloprid concentrations in the feeding solution and the concentrations
measured in hive beebread and uncapped nectar was observed during the exposure period (CCA4) and one
week after the end of exposure (CCA5). However, imidacloprid measured concentrations in hive matrices
were lower than that in the feeding solution. The average concentrations in hive uncapped nectar and hive
bee bread were 62.7% and 30.2%, respectively, of the concentration in the feeding solution (Table 22).

22Table 22. Imidacloprid concentration measured in hive uncapped nectar and hive bee bread
Nominal concentration in
test feeding solution
Imidacloprid concentrationin hive uncapped nectar in% of the concentration of

µg/L

12.5

25

50

100

200

ppb

10.2

20.3

40.7

81.3

162.7

CCA 4
(n=12)

62.0(6.31)

65.1(13.24)68.0(27.66)57.6(46.87)67.1(109.14)

Average

64.0

35
122

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Nominal concentration in
test feeding solution
nominal feeding solution(average measured
concentration in ppb)¥
Imidacloprid concentrationin hive beebread in % of theconcentration of nominal
feeding solution (averagemeasured concentration in
ppb)

µg/L

12.5

25

50

100

200

ppb

10.2

20.3

40.7

81.3

162.7

CCA 5 (n=3)

57.8(5.88)

35.3(7.18)

67.5(27.46)67.6(54.98)78.7(127.93)

61.4

CCA 4
(n=12)

28.1(2.86)

26.4(5.37)

26.7*(10.84)22.0**(17.89)NA

25.8

CCA 5 (n=3)

41.5(4.22)

28.2(5.74)

40.4(16.44)28.1***(22.89)NA

34.6

Average¥ Measured concentrations in uncapped nectar were corrected for sugar concentration equivalence.
* n=8; **n=4. *** n=1;

The study did not find a significant degradation of imidacloprid in the test solution. No imidacloprid
transformation products (olefin imidacloprid and 5-hydroxy imidacloprid) were detected in almost all thesamples of test solution and hives matrices.
Considering the stability of imidacloprid in the test solution, the reduced concentrations of imidacloprid in
hive matrices likely indicates that test bees were also foraging for pollen and nectar from sources other than
the feeding solution.

3.8.

Pathogens

Besides a standard treatment for Varroa mites, no treatments for any other hive pests, predators or
diseases were administered to any hives.

3.8.1. Varroa Presence
Varroa mite occurrence in the colonies was assessed the week before and after the feeding period, as well
as after over-wintering (CCA3, CCA5 and CCA8). The number of mites per 100 bees was calculated(4Figure4). Hives were treated with one application of Apiguard® (active ingredient: thymol) following
typical apicultural practice for the region immediately after the September CCA’s to prevent high mite loads.
After over-wintering, the colonies of all treatment groups, except the 100 µg/L group, had similar Varroa
infestation levels.
The study showed no correlation between the treatments and the level of Varroa infestation.

36
123Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

4Figure 4. Mean number of Varroa mites per 100 bees

3.8.2. Nosema presence
The number of Nosema spores per bee was determined at three time points at CCA3, CCA5 and CCA8. The
study showed no correlation between the treatments and the level of Nosema infestation.

5Figure 5. Mean number of Nosema spores per bee

37124Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
3.9.

Statistical Analysis

What follows are brief summaries of each of the statistical analyses employed for the review of this study.
It is noted here, and later, when discussing the results that the PMRA, EPA, and CDPR statistical approaches,
and when weighing statistical and biological lines of evidence, that the three Agencies are harmonized in
the determination of the overall NOAEC and LOAEC.

3.9.1. Study Authors Analysis
The study author conducted statistical analysis using SAS (version 9.3). The analysis included colony
strength (as indicated by mean number of adults), brood stages (as indicated by the mean number of eggs,
larval cells, and pupal cells) and food stores (as indicated by the mean number of pollen and nectar/honeycells). For the pre-test data, all tests were done in a two tailed approach, whereas for the data assessed after
application, one tailed (lower) tests were conducted. According to the study author, procedure GLM wasused for ANOVA analysis. Williams’ Trend Test was used to test data that passed statistical tests thatassessed the assumptions of normality, variance homogeneity, and monotonicity. Dunnett’s t-Test was used
to test data that are non-monotonic, but pass tests of normality and variance homogeneity. Dunnett’s T3
Test was used to test data that satisfy the criteria for normality, but fail the criteria for homogeneity ofvariance. For hive mortality, Cochran Armitage Exact Trend Test was used.

3.9.2. Study Reviewer Analysis
During the review of the study, a separate statistical analysis was conducted using the raw data submitted
by the study author. As part of the collaborative review effort of the study by EPA, PMRA, and CDPR, avariety of statistical analyses were conducted for the evaluation of the data. The detailed methods of theseanalyses including statistical model selection and parameterization are presented in Appendices A, B, andC for the EPA, PMRA, and CDPR analyses, respectively. What follows is a brief summarization of eachmethod. It is noted that while each method was distinct in the manner in which the data were analyzed, allthree methods produced similar statistical results, that is, similar findings of significance for a given
response variable at a given treatment level and CCA at a specified alpha level.

3.9.3. EPA Analysis
The general experimental design was a randomized complete block (apiary) with repeated measures (CCA).
Since hives were not randomly assigned and placed in the study apiaries until shortly before CCA3, the data
for the statistical analysis only included CCA3 through CCA8. For the two highest treatment levels (100and 200 µg/L), data obtained from CCA8 was deleted from the analysis as only one and two hives(respectively) were surviving at the CCA8 measurement time. Temporal correlations were evaluated foreach response variable; compound symmetry with heterogeneous variance was selected as the best fitting
covariance structure. PROC MIXED in SAS was used for the data analysis, and the TREATMENT*CCA
interaction was statistically significant for all evaluated response variables. This interaction was exploredby 1) at each CCA, treatment means were compared to the control using a one-sided Dunnett’s test; and 2)
for each treatment level, CCAs 4 through 8 were compared to CCA3 using a two-sided Dunnett’s test.
Further details of the EPA statistical analysis can be found in Appendix A.

38
125

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
The EPA approach controlled for multiplicities by using a Dunnett’s test which holds the family wise error
rate (i.e the probability of make Type I errors or false discoveries of significance) at or below the level of
α.

3.9.4. PMRA Analysis
The differences of each measurement parameter between the treatment and control at the same apiary were
calculated for each apiary site. The means of the differences among 11 sites (Apiary C excluded due tovandalism) were plotted. A formal comparison from the highest to lowest concentration with the control
was carried out using an often used conventional analysis of the block randomised experiments using theraw data with adjustment for baseline measurements: linear modeling (or ANOVA) stratified on Apiary(block) and adjusted for baseline measurements at CCA3 with one-side testing for effect. In the remainderof the document where PMRA analysis results are referred to as “raw data” it is noted that it is actually themodel estimates using the raw data with adjustment for baseline measurements. Taking into consideration
a limited detection power in a typical field level study, alpha levels of both 0.1 and 0.05 were considered asstatistically significant. A list of statistical P values for each measuring parameter is summarized andincluded in this report.
Prior to the data analysis, for the purposes of controlling for multiplicity in the statistical analysis, a primaryparameter for detection of treatment effects was defined as the total individuals in hives (sum of eggs, larvae,
pupa and adults) at CCA6. However, when determining an overall NOAEC all response variables were
examined with equal weight in considering treatment related effects, but were considered against statisticalresults for the primary parameter. Further details of the PMRA statistical approach can be found in
Appendix B.

3.9.5. CDPR Analysis
A multivariate mixed repeated measures model approach was employed and is distinguished from theunivariate approaches above in that all bee life stages or hive food storage variables are simultaneously
analyzed as a single model. Multivariate analyses of variance for fixed effects models are conducted, using
Statistical Analysis System (SAS) software, through implementing the MANOVA option in the PROC
GLM procedure. Recently, multivariate analyses have been extended to mixed models using the PROCMIXED procedure. The MIXED procedure is designed to conduct a mixed model analysis of variance where
fixed and random effects can be specified. Inclusion of random effects in a model provides a broader
application of results. For this study, locations were denoted as apiaries with individual hives as test
subjects. Use of a mixed model with apiaries identified as a random variable provides some assurance thatthe results can be generalized to other locations and hives. Further details of the CDPR analysis can befound in Appendix C.
The CDPR approach controlled for multiplicities by way of a Bonferroni adjustment to fix the family wise
error rate at α.

3.10.

Hive mortality

The study author reported that 72 out of 84 colonies for biological observations were maintained over the
6-week exposure period. Apiary C was vandalized after 8 feedings during the weekend of 27/28th Jul 201339
126

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
during which hives were left open. This allowed all colonies in this apiary access to treated sugar solution
and treated food stores in the hives. Since all hives were compromised, they were no longer used to collectdata for the study and this apiary was thereafter removed from the study and subsequent data analysis.
From the 11 remaining sites with a combined 77 hives, 34 hives were considered dead by 22 March 2014(end of test), as summarized by Table 23 and Figure 6. The author reported that based on an one-sided
Cochran-Armitage exact trend test, mortality in the 100 ppb and 200 µg/L treatments are significantly
different from the control mortality (p = 0.01 and p<0.01 respectively).
After overwintering, only one hive survived (Apiary L) at the treatment 100 µg/L, and two hives survived(Apiary D and F) at the treatment 200 µg/L. The hive mortality showed a U-shaped response to thetreatments (Figure 6). The percent mortality decreased from control (36%) to 25 µg/L (9%), and thenincreased from 25 µg/L to 50 ug/L (36%) to 100 µg/L (91%) and 200 µg/L (82%). It is noted from theresults presented in Table 23a and b and Figure 6 below show that control mortality after overwintering
was higher than it was for the 12.5 and 25 µg/L groups. For this reason, the ability to detect treatment
related decreases in overwintering colony survival may be masked by the magnitude of control colony loss.
23Table 23a. Hive survival at CCA8 (after overwintering)
Treatment
group

Apiary
A

B

C

D

E

F

G

H

I

J

K

L

UTC

A1

-

-

-

E1

-

G6

-

I5

J1

-

L7

UTC

A2

B8

-

-

E8

F7

-

H3

I8

J7

-

L5

12.5 ppb

A4

B3

-

D8

E5

F5

G4

25 ppb

-

B4

-

D1

E4

F3

G8

H7

I7

J3

K2

L6

H6

I6

J4

K6

L2

50 ppb

A8

B7

-

-

E7

-

-

H2

I3

J2

K5

L3

100 ppb

-

-

-

-

-

-

-

-

-

-

-

L4

200 ppb

-

-

-

D3

-

F1

-

-

-

-

-

-

24Table 23b. Hive mortality after overwintering measure at CCA8
Treatment (µg/L)
Number of deceased colonies/total colonies
Colony mortality (%)
Colony survival (%)

Control

12.5

25

50

100

200

8/22

2/11

1/11

4/11

10/11

9/11

36
64

18
82

9
91

36
64

91
9

82
18

40
127

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Hive Mortality (%) After Overwintering
91

100

(

M
o 80
r
t 60
a
l 40
i
t 20
y

36
18

9

0
Control

)

%

36

82

12.5

25

50

100

200

Treatments (µg/l)

6Figure 6. Hive mortality after overwintering.

3.11.

Colony Condition Assessment Response Variables

What follows is a breakdown of each response variable assessed and the significant effects that were
determined at each CCA. A couple of general points are made below when examining the results of the
response variables:
•
•
•

•

•
•

Unless explicitly stated otherwise, all discussion of statistical findings refer to shared determinations
from the PMRA, EPA, and CDPR analyses.
All analyses considered effects at both the 0.05 and 0.1 alpha levels when weighing statisticallysignificant effects with biological considerations.
The tables are the percent differences from control based on raw counts of the data (model
estimations using the raw count data with adjustment for baseline measurements at CCA3) andgenerated by PMRA. The figures with significance “dots” were based off of the proportions offrame coverage for each hive for each response variable (with the exception of hive weight) andwere generated by EPA.
In the EPA analysis, the data in the 100 and 200 µg/L treatment groups were excluded from theanalysis at CCA8. This was done primarily to facilitate the statistical model converging. Excludingthese two treatment groups from the analysis at CCA8 is not expected to have an impact on theinterpretation of results as there was a clear effect at these two treatment groups by the time of CCA8
indicated primarily by hive mortality.
Even though data from CCA8 at the 100 and 200 µg/L groups were included in the PMRA analysis,
the lack of statistical difference from control in these two groups is considered unreliable as there
were only one and two remaining hives in these groups at CCA8, respectively.
The PMRA approach used raw counts of the each response variable while the EPA and CDPR
approaches converted these data into proportion of frame coverage (using methods described inAppendix A) to facilitate convergence of the statistical model. The tables of percent differencesfrom control are based off of the raw counts of the data (model estimates using raw counts adjusted
for baseline measurements) while the figures present the trends of the proportions of life stages andfood stores with significant findings indicated. When differences in statistical findings are
discussed, the findings of PMRA were based off of the raw counts (model estimates using raw counts
41128Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

•
•
•

•

•
•

adjusted for baseline measurements) while the findings of the EPA and CDPR approaches werebased off proportions.
CCA3 was the baseline covariate and therefore is not presented in the tables for each response
variable with percent reductions.
CDPR did not include the “total individuals” endpoint in its analysis so those results will only pertain
to EPA and PMRA findings.
For its simplicity in visualizing the trends and findings of statistical significance simultaneously, theEPA-generated figures are presented below. The figures generated by PMRA can be found in
Appendix B and those generated by CDPR can be found in Appendix C. It is noted here as well as
above that in the discussion of each response variable, the results of all approaches will be discussed
and noted where divergent from each other.
The figures below indicate significance with black and red “dots” denoting a significant finding at
the 0.1 and 0.05 alpha levels, respectively. Although these figures refer to EPA’s analysis, asmentioned previously, the PMRA, EPA, and CDPR statistical results were generally in agreement
and it is noted below where there were differences. As also mentioned previously, although there
were different statistical findings in a few cases depending on the method employed, theinterpretation of the results leads to a shared overall NOAEC/LOAEC of the study.
While the EPA and PMRA analyses looked at each response variable across CCAs, the CDPR is
distinguished from this approach by looking at all life stages or food store variables simultaneously
within a single CCA across treatments.
While it is not depicted in the figures below, it is acknowledged (and addressed in a variety of ways
through the various statistical approaches) that there was considerable variability for some response
variables at certain treatment groups and CCAs. Please refer to Appendix A for summary statistics
tables (i.e. max, min, standard deviation values) of the proportions of each response variable forfurther information.

Life Stages
3.11.1.

AdultsFigure 7 below shows the effects on adult honey bees across CCAs and treatment groups. Compared withthe control, no differences in the number of adults in hives (p>0.1) during the CCA4 exposure period wereapparent in any of the treatments with the exception of the CDPR analysis where a marginally significant(0.05< p< 0.1) reduction was determined at the 200 µg/L group. Additionally, the total number of adults
in the 12.5 and 25 µg/L treatments was not reduced in any of the CCAs. However, the numbers of the adults
in the 100 and 200 µg/L treatments were consistently reduced (p<0.05) at CCA5, CCA6, and CCA7 with
reductions ranging from 24.4 – 59.4% (data from CCA8 excluded from EPA analysis in the 100 and 200µg/L group due to clear effects on hive mortality). An exception is that the EPA and CDPR analyses did
not determine a significant reductions (p>0.05) at the 200 µg/L treatment group at CCA5 while the PMRA
analysis did. However, it is apparent from all analyses that there were impacts to adults at the 200 µg/Lgroup during the course of the study. The number of adults in the 50 µg/L treatment was also reduced with
marginal statistical significance at CCA5 (0.05< p<0.1) but at p<0.05 at CCA6 and CCA8 with percent
reductions from control of 21.7%, 19.8% and 78%, respectively based off raw counts (Table 24). The
PMRA analysis also determined a significant reduction from control at CCA7 while the EPA and CDPR

42
129

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
analyses did not, but like the 100 and 200 µg/L treatment groups, the persistent nature of significant
reductions to adults at the 50 µg/L group is evident.
25Table 24. Estimated percent reduction from control for number of adults
Test
concentration(µg/L)
12.5
25
50
100
200

Estimated reduction from control (%)1
CCA4

CCA5

CCA6

CCA7

CCA82

2.2
2.7
-23.3
5.2
-29.2

-5.4
8.7
21.7*
34**
24.4**

2.3
7.7
19.8*
28.7**
52.8**

-12.6
-4.1
18.6*
51**
59.4**

-11
-4.3
78**
172.5**
7.3

Note: Negative value indicates increased number of adults in comparison to control.
*0.05<p<0.1**p<0.051Percent differences from control are based on the raw counts of adults, not proportions of the adults as Figure 7 below shows.
2At CCA8, comparisons made to the 100 and 200 treatment groups are with uncertainty as 1 and 2 hives survived in these groups,
respectively. These data are included in the PMRA and CDPR analyses but excluded from the EPA analysis.

7Figure 7. Proportion of adults following exposure to varying concentrations of imidacloprid in the dietacross CCA3 – CCA8.
Figure 8 below shows the trends proportions of adults across the CCAs for the control and three lowest
treatment groups only as the impact at the two highest groups was evident, especially when considering
overwintering mortality. Removing the two highest treatment concentrations adjusts the scale of the figures
to see the trends more clearly at the lower treatment groups. There is a clear divergence in the trends at the
43
130Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
50 µg/L treatment group not only in the decline in numbers beginning one CCA earlier (CCA4 as comparedto CCA5 in the control, 12.5, and 25 µg/L groups) but also the average proportion of adults after
overwintering at CCA8 (approximately 20% frame coverage as opposed to 33-35% for control and the lower
groups).

Adults0.6

Proportion of Adults

0.5
0.4
Control

0.3

12.5
25

0.2

50

0.1
0
CCA3

CCA4

CCA5
CCA6
CCA7
Colony Condition Assessment

CCA8

8Figure 8. Proportion of adults following exposure to varying concentrations of imidacloprid in the dietacross CCA3 – CCA8 in the control, 12.5, 25, and 50 µg/L groups only.
In an examination of the trends of adults in the control group, by CCA6, the average number of adults began
to decline to 39% based off the proportions of adults covering the hive frames (Figure 9 below). CCA6
represents the time when the colony as a whole starts to prepare for overwintering and therefore starts tobegin a “shut-down” phase where the numbers of adults and other life stages are clearly decreased by thetime of CCA7. During this pre-overwintering phase, adult proportions decline due to natural die off ofworker bees and reduced rates of replenishment from reduced egg laying by the queen. It is noted that the
average proportion of comb area as adults is similar after overwintering at CCA8 (35%) as compared tobefore exposure at CCA3 (33%) when the hives were initially placed in the test sites.
Also notably, as distinguished from the control and 12.5 and 25 µg/L groups, while the proportions of adults
for those groups generally increased through CCA5 before beginning to decline, the numbers of adults at
the 50 µg/L began to decline as early as CCA4, where these numbers were being built up in the control andlower treatment groups to support the foraging worker bee force for nectar and pollen collection. This again
is evidenced by the average proportion of adults at CCA5 in the 50 µg/L group which was 33% as comparedto 45, 48, and 42% for the control, 12.5 and 25 µg/L groups, respectively (percent reductions ranging from24 – 37% based off the proportions of frame coverage).

44
131Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

0.5

12.5 µg/L
Proportion of Adults

Proportion of Adults

Control
0.4
0.3
0.2
0.1
0

0.5
0.4
0.3
0.2
0.1
0

Colony Condition Assessment

Colony Condition Assessment
AdultsAdults25 µg/L

50 µg/L
0.5
Proportion of Adults

Proportion of Adults

0.5
0.4
0.3
0.2
0.1

0.4
0.3
0.2
0.1

0

0

Colony Condition Assessment

Colony Condition Assessment

AdultsAdults9Figure 9. Proportion of adults following exposure to varying concentrations of imidacloprid in the dietacross CCA3 – CCA8 in the control, 12.5, 25, and 50 µg/L groups.
When weighing statistical and biological significance, the overall NOAEC and LOAEC for adults isdetermined to be 25 and 50 µg/L, respectively.

3.11.2.

Eggs

There were consistently lower numbers of eggs in treatments at 100 and 200 µg/L (p<0.05) during the courseof the CCAs following exposure. There were minor differences in the statistical findings of the three45

132Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
analyses not only at a given CCA and treatment group but also at what alpha level an effect was significant
at, but these differences do not have an impact on the determination that there was not only an early onset
but a persistent nature of a reduction in the numbers of eggs that was not confined to one CCA. At the 100
and 200 µu/L treatment groups, all analyses determined significant reductions (p<0.1) in eggs for the 100
and 200 µg/L treatment groups for at least two CCAs. The sole finding of significance at the 50 µg/L group
was at CCA8 at the 0.05 alpha level.
For the 12.5 µg/L the PMRA and EPA analyses only determined a significant reduction in the number of
eggs at CCA8 (p<0.1). A similar finding was made at CCA4 in the PMRA analysis only. However, there
were no significant reductions (p>0.1) at both CCA4 and CCA8 in the 25 µg/L group, indicating a lack ofdose responsiveness within these time points. The biological significance of this finding at 12.5 µg/L istherefore considered to be low. Similarly, there was a significant reduction in eggs determined at CCA6 in
the 25 µg/L group (PMRA analysis only). However, similar findings of statistical significance at this CCA
was not determined for the 50 µg/L group. Finally, this effect was not observed before CCA6 or in thesubsequent CCAs (CCAs 7 and 8) indicating this effect was isolated to this time point.
At the 50 µg/L treatment level, all analyses determined a significant reduction (p<0.05) at CCA8. While
this effect was isolated to just CCA8 for this treatment group, there is uncertainty as to whether hives could
have compensated for this reduction as could or may not have been shown by an additional CCA.
26Table 25. Estimated percent reduction from control for number of eggs
Test
concentration(µg/L)
12.5
25
50
100
200

Estimated reduction from control (%)1
CCA4

CCA5

CCA6

CCA7

CCA82

22.8*
-1.1
-9.6
37.9**
14.5

11.9
8.3
1.8
39.4**
32.2**

5.9
26.3*
11.7
70.8**
60.1**

0.1
31.3
2.6
46.6**
77.8**

37.7*
5.8
78.2**
138**
153.2**

Note: Negative value indicates increased number of eggs in comparison to control.
*0.05<p<0.1**p<0.05
1Percent differences from control are based on the raw counts of eggs, not proportions of the eggs as Figure 10 below shows.
2At CCA8, comparisons made to the 100 and 200 treatment groups are with uncertainty as 1 and 2 hives survived in these groups, respectively.
These data are included in the PMRA and CDPR analyses analysis but excluded from the EPA analysis.

46133Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

10Figure 10. Proportion of eggs following exposure to varying concentrations of imidacloprid in the dietacross CCA3 – CCA8.
Figure 11 below shows the responses for the control, 12.5, 25, and 50 µg/L treatment groups. Removing
the two highest treatment concentrations adjusts the scale of the figures to see the trends more clearly. It isnoted from this graph the variability present in the groups at CC3 before exposure had started. Particularly,
the variation in egg coverage of the frame at the early CCAs is noted which may have contributed to someof these findings at CCA4.

47
134

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

Eggs
0.09
0.08
Proportion of Eggs

0.07
0.06
0.05

Control

0.04

12.5

0.03

25

0.02

50

0.01
0
CCA3

CCA4

CCA5
CCA6
CCA7
Colony Condition Assessment

CCA8

11Figure 11. Proportion of eggs following exposure to varying concentrations of imidacloprid in the dietacross CCA3 – CCA8 in the control, 12.5, 25, and 50 µg/L groups only.
By CCA8, the average of eggs in the control group increased relative to CCA7, but the average proportion
at CCA8 (4.8%) was approximately half the proportion initially recorded at CCA3 (8.4%). It is noted thatthe 50 µg/L group was the only group of the control and three lowest treatment levels that underwent a
downward trend from CCA7 to CCA8. Additionally, the average proportions of egg cells at CCA8 for the
50 µg/L group are approximately half of the proportion for the control, 12.5, and 25 µg/L groups (Figure
12).

48
135

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

0.1

12.5 µg/L
Proportion of Eggs

Proportion of Eggs

Control
0.08
0.06
0.04
0.02
0

0.1
0.08
0.06
0.04
0.02
0

Colony Condition Assessment

Colony Condition Assessment

Eggs

Eggs

25 µg/L

50 µg/L
0.1
Proportion of Eggs

Proportion of Eggs

0.1
0.08
0.06
0.04
0.02

0.08
0.06
0.04
0.02

0

0

Colony Condition Assessment

Colony Condition Assessment

Eggs

Eggs

12Figure 12. Proportion of eggs following exposure to varying concentrations of imidacloprid in the dietacross CCA3 – CCA8 in the control, 12.5, 25, and 50 µg/L groups.
When weighing statistical and biological significance, the overall NOAEC and LOAEC for eggs isdetermined to be 25 and 50 µg/L, respectively, based on a significant reduction in eggs at CCA8 that
represented a 78.2% reduction based on raw counts as well as being clearly divergent in its responseat CCA8 (based off the proportions) from the control and 12.5 and 25 µg/L groups.

49
136Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
3.11.3.

Larvae (Open/Uncapped brood)

There were consistently and significantly lower numbers of larvae in the 100 and 200 µg/L groups ascompared to control (p<0.05) beginning at CCA4 and persisting throughout the entirety of the study up to
and including after overwintering at CCA8 (data from CCA8 in these groups excluded from EPA analysis).
There were no significant reductions from control at any CCA for the 12.5, 25, and 50 µg/L treatment groups
in the EPA analysis.
In the PMRA analysis at CCA6, a significant reduction from control was determined at 25 µg/L (p<0.1),
but not at 12.5 µg/L and 50 µg/L (p>0.1 for both treatments). Additionally, the effect was not determined
at CCA4, CCA5, and CCA7. Similarly, the CDPR analysis determined a significant difference (p<0.05) at
CCA7 at 12.5 µg/L, but this finding was not determined at the 25 and 50 µg/L treatment groups (p>0.1).
Also in the PMRA analysis only, at CCA8, a statistical reduction was determined at 50 µg/L treatment group(p<0.05). This effect was not determined to be significantly reduced from control from CCA4 to CCA7
(p>0.1). Although this difference was not detected in the EPA and CDPR analyses, the percent reduction inlarval cells at CCA8 for this group was 43% (based on raw counts).
27Table 26. Estimated percent reduction from control for number of larvae (open/uncapped brood)
Test
concentration(µg/L)
12.5
25
50
100
200

Estimated reduction from control (%)1
CCA4

CCA5

CCA6

CCA7

CCA82

12.3
8.1
12.1
37.1**
64**

10.7
16.2
15.3
30.9**
65**

-15.5
23*
-2.1
52.3**
57.2**

-62.6
-25.5
-32.4
64.4**
78.3**

0.8-9.9
42.6**
159.9**
54.1

Note: Negative value indicates increased number of larvae in comparison to control.
*0.05<p<0.1**p<0.05
1Percent differences from control are based on the raw counts of larvae (open) brood, not proportions of the larvae (open) brood as Figure 13
below shows.
2At CCA8, comparisons made to the 100 and 200 treatment groups are with uncertainty as 1 and 2 hives survived in these groups, respectively.
These data are included in the PMRA and CDPR analyses but excluded from the EPA analysis.

Figure 13 below shows the trends of the control and all treatment groups for larval cells across all CCAs
assessed. A clear divergence in the 100 and 200 µg/L groups is evident beginning at CCA4 where thenumbers of larvae in these groups undergo a marked decline while the other treatment groups generally
trend with control.

50137Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

13Figure 13. Proportion of larval cells following exposure to varying concentrations of imidacloprid in
the diet across CCA3 – CCA8.
When examining the trends with the control and three lowest treatment groups alone, differences are lessapparent than when the 100 and 200 µg/L groups depicted alongside with the exception of the separation of
the 50 µg/L treatment group at CCA8 (Figure 14)51138

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

Larvae (Open) Cells
0.12

Proportion of Larvae

0.1
0.08
Control

0.06

12.5

0.04

25
50

0.02
0
CCA3

CCA4

CCA5
CCA6
CCA7
Colony Condition Assessment

CCA8

14Figure 14. Proportion of larval cells following exposure to varying concentrations of imidacloprid in
the diet across CCA3 – CCA8 in the control, 12.5, 25, and 50 µg/L groups only.
When examining the trends in the control group, the average proportion of larval cells increased from CCA7
to CCA8, and at CCA8 had a similar level (8.2%) that was recorded for CCA3 (10%) (Figure 15). Withthe 12.5 µg/L group, the starting average proportions of life stages at CCA3, prior to exposure, were similar
to those in the control group with respect to the proportions of larval cells being approximately 5-8% of the
comb area. The average proportion of larval cells trended down beginning as early as CCA4 (7%) before
experiencing a more marked decline ahead of CCA7 (2%) as with the other life stages, which is anticipatedahead of overwintering. The proportions of larval cells were again relatively stable from the time of CCA3(10%) to CCA6 (10%). This was preceded by a marked decline at CCA7 to an average proportions of 4%.

52
139

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

0.12
0.1
0.08
0.06
0.04
0.02
0

12.5 µg/L
Proportion of Larvae

Proportion of Larvae

Control
0.12
0.1
0.08
0.06
0.04
0.02
0

Colony Condition Assessment

Open

Open

25 µg/L

50 µg/L

0.12

Proportion of Larvae

Proportion of Larvae

Colony Condition Assessment

0.1
0.08
0.06
0.04
0.02
0

0.12
0.1
0.08
0.06
0.04
0.02
0

Colony Condition Assessment

Colony Condition Assessment

Open

Open

15Figure 15. Proportion of larval cells following exposure to varying concentrations of imidacloprid in
the diet across CCA3 – CCA8 in the control, 12.5, 25, and 50 µg/L groups.
It is also noted from the treatment by treatment comparisons below for larval cells across CCAs that
although the trends for the control, 12.5, 25, and 50 µg/L groups are similar, that the 50 µg/L group was the
only group of the 4 that did not end up at approximately 8% coverage of the frame, but instead wasapproximately 6% of frame coverage. The 25 ppb group also look to undergo a more marked decline
beginning at CCA4 as compared to the control, 12.5, and 50 µg/L group but as stated earlier, the difference
was only marginally significant in the PMRA analysis, similar findings were not determined in the 50 µg/Lgroup, and the effect at 25 µg/L was not significant at CCA7 and CCA853
140

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
When weighing statistical and biological significance, the overall NOAEC and LOAEC for larval cells
is determined to be 25 and 50 µg/L, respectively. This is based on a significant reduction at the 50µg/L at CCA8. While this finding is isolated to CCA8, it is an uncertainty what the response would
have been at an additional CCA (i.e., whether the hives could have compensated for this potential
effect). It is also noted, as has been above, that the 50 µg/L group showed a reduced percentage offrame coverage of larval cells at CCA8 as compared to the control and two lower treatment groups.

3.11.4.

Pupae (Capped brood)

In the 50, 100 and 200 µg/L treatment groups, there were significant reductions from control (p<0.05) thatpersisted through most of the study (EPA findings at the 50 µg/L were significant at two CCAs, CDPR at 3
CCAs and the PMRA analysis determined significant reductions from control at 5 CCAs). The percent
reductions from control based on the raw counts of pupal cells in the 100 and 200 µg/L groups ranged from49.7 – 93.5% during CCA4 – CCA7.
At the 12.5 µg/L treatment group, there were significant reductions determined at CCA6 for the EPA and
PMRA analyses. While the findings were not determined at the 25 µg/L treatment group at CCA6 for the
EPA and CDPR analyses, they were for the approach used by PMRA. It is noted that significant reductions
in pupal cells were not determined by any analysis at 12.5 and 25 µg/L in any CCA preceding or subsequentto CCA6, thus the significant effect for the 12.5 and 25 µg/L treatments was isolated to the CCA6 timepoint.
Additionally, although PMRA determined significant reductions at CCA6 for all treatment groups, theeffects did not demonstrate a dose response at the lowest three doses with the percent reductions fromcontrol (based on raw counts of the data) at 22.3, 18.3, 12.5, 49.7 and 75.5% for the 12.5, 25, 50, 100 and
200 µg/L groups, respectively. However, responses at the lower three doses were all similarly reduced
compared to controls (12.5 -22.3%), and some overlap in dose-response might be expected at the lower
doses, given the variability and overlap in exposure among individual hives. It is also noted that theconfidence intervals among the three lowest doses are similar and overlapping (see Bees 8, PMRA analysis
Appendix B). Finally, after overwintering at CCA8, the levels of pupae in the surviving hives at the 12.5
and 25 µg/L treatment groups were actually above the level of control (based on raw counts of the data) by
1.3 and 10.8%, respectively. The percent reduction from control at CCA8 in the 50 µg/L group was 70.6%.
28Table 27. Estimated percent reduction from control for number of pupae
Test
concentration(µg/L)
12.5
25
50
100
200

Estimated reduction from control (%)1
CCA4

CCA5

CCA6

CCA7

CCA82

2.8
17.7
28.1*
51.7**
83.3**-3.7
5.8
34.6**
56.6**
79.5**

22.2**
18.3**
12.5*
49.7**
75.5**

-8.4
18.1
9.7
75.6**
93.5**-1.3-10.8
70.6**
150.9*
42

Note: Negative value indicates increased number of pupae in comparison to control.

*0.05<p<0.1**p<0.05
1Percent differences from control are based on the raw counts of pupae (capped) brood, not proportions of the pupae (capped) brood as

Figure 16 below shows.

At CCA8, comparisons made to the 100 and 200 treatment groups are with uncertainty as 1 and 2 hives survived in these groups,
respectively. These data are included in the PMRA and CDPR analyses but excluded from the EPA analysis.

2

54
141

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

16Figure 16. Proportion of pupal (capped) cells following exposure to varying concentrations ofimidacloprid in the diet across CCA3 – CCA8.
In summarizing the information provided by the different analyses for this response variable, a few points
can be made:••••All analyses find significant differences at the 100 and 200 µg/L levels starting at CCA4 andpersisting until CCA7 (data from CCA8 excluded from EPA analysis at these two treatment groups)
PMRA analysis determined significant difference at the 50 µg/L group (at either 0.1 or 0.05 alphalevel) for all CCAs assessed except CCA7 (for CDPR, same findings except no significant findings
at CCA6 and CCA7)
EPA analysis determined significant differences at 50 µg/L group (at either 0.1 or 0.05 alpha level)at CCA5 and CCA8PMRA determined significant differences at all treatment groups at CCA6 while EPA determinedsignificant effects for the 12.5, 100 and 200 µg/L groups, and CDPR only at 100 and 200 µg/L.

In further exploring this last point, the difference in findings can potentially be explained by the statisticalmodel selections employed for each analyses. The discussion below focuses on additional lines of evidence
to further characterize the findings.
Figure 17 shows the trends in pupal cells over the course of the study in the control and three lowest
treatment groups only. As indicated in both the PMRA and EPA analyses, the impacts to pupal cells occurs
early as there is divergence from the control as early as CCA4.

55
142

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

Capped (Pupal) Cells
0.3
Proportion of Pupae

0.25
0.2
Control

0.15

12.5

0.1

25
50

0.05
0
CCA3

CCA4
CCA5
CCA6
CCA7
Colony Condition Assessment

CCA8

17Figure 17. Proportion of pupal (capped) cells following exposure to varying concentrations ofimidacloprid in the diet across CCA3 – CCA8 in the control, 12.5, 25, and 50 µg/L groups only.
It is noted that the average proportion of comb area as pupal cells in controls is similar after overwintering
at CCA8 (17%) as compared to CCA3 (16%) when the hives were initially placed in the test sites. Also
notably, the average proportion of pupal cells in the 12.5 and 25 µg/L groups (22%) is higher than it wasfor the control group (16%) but this is obviously not a finding related to imidacloprid treatment, given that
exposure had not occurred yet.
In the 12.5 µg/L group, unlike the control group, where the average proportion of pupal cells remained
stable between the time of CCA5 and CCA6 (26%), there was an apparent decrease in the 12.5 µg/L groupfrom CCA5 (26%) to CCA6 (20%) based on average proportions. This decrease continued for the CCA6
to CCA7 interval to an equivalent level as controls (approximately 8%). After the overwintering period in
the 12.5 µg/L group, the proportions of life stages were similar to CCA8 for the control group in that the
proportion of pupal cells 17% at CCA8 (as compared to 17% in the control).
In the 25 µg/L treatment group, there were again no significant differences in the proportions of all life
stages at CCA3 before the start of exposure (p>0.05). As opposed to the steady buildup that was observedin the control and 12.5 µg/L treatment groups from CCA3 to CCA5, the numbers of pupal cells remained
similar from CCA3 to CCA5; they were decreased slightly at CCA4 (21%) as compared to CCA3 (23%)
but at CCA5 (24%) were again to the level of CCA3. As with the other life stages, a decline in numbers
was observed between CCA6 and CCA7 as the hives prepared for overwintering. The average proportionof pupal cells at CCA8 for the 25 µg/L treatment group were similar to the proportions in CCA8 of thecontrol group, that is, 16% and 17% frame coverage at CCA8 for the 25 µg/L group and control group,
respectively.

56
143

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

0.3
0.25
0.2
0.15
0.1
0.05
0

12.5 µg/L
Proportion of Pupae

Proportion of Pupae

Control
0.3
0.25
0.2
0.15
0.1
0.05
0

Colony Condition Assessment

Colony Condition Assessment

CappedCapped

0.3
0.25
0.2
0.15
0.1
0.05
0

50 µg/L
Proportion of Pupae

Proportion of Pupae

25 µg/L
0.3
0.25
0.2
0.15
0.1
0.05
0

Colony Condition Assessment

Colony Condition Assessment

CappedCapped18Figure 18. Proportion of pupal (capped) cells following exposure to varying concentrations ofimidacloprid in the diet across CCA3 – CCA8 in the control, 12.5, 25, and 50 µg/L groups.
As distinguished from the control and 12.5 and 25 µg/L groups, the number of pupal cells at the 50 µg/Lgroup underwent a steady decline beginning at CCA4 and continuing through CCA5 (average proportion at
CCA3 was 19% compared to 16% at CCA5). This is also evidenced by the average proportion of pupal
cells at CCA5 at the 50 µg/L group which was 16% of the comb areas as compared to 26, 26, and 24% at
CCA5 for the control, 12.5, and 25 µg/L groups, respectively. This finding is also statistically significant
for all analyses conducted as indicated above. An examination of the proportions at CCA8 also suggest thepersistent nature of these effects and their lasting impact at this treatment group. The average proportion of
pupal cells at CCA8 for the 50 µg/L group was 9% as compared to 17, 17, and 16% for the control, 12.5,
and 25 µg/L groups, respectively. This finding was significant at the α = 0.1 in the EPA analysis and a α =0.05 for the PMRA analysis (with 70.6 % reduction compared to control).
57
144

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Regarding the statistical analyses, all methods found significant differences at 100 and 200 µg/L that wereapparent at early CCAs and persisted throughout the study. Additionally, effects were noted at multiple
CCAs for 50 µg/L, and the effects continued following overwintering. While all analyses found a significant
effect at the 12.5 µg/L treatment level at CCA6, and the PMRA analysis also found significant effect at the
25 µg/L treatment level, these effects were considered transient. This is because effects at 12.5 and 25µg/L
were isolated to CCA6 with levels returning to those similar to control after overwintering, and at CCA6
the effects lacked a clear dose-response relationship and were similar among all three lower treatment levels(12.5, 25, and 50 µg/L; 22.2, 18.3, and 12.5 % reductions compared to control based on raw data,
respectively). Additionally, the discussion presented above indicates that the average proportions of pupal
cells in the 12.5 and 25 µg/L group at different CCAs resemble the responses found in the control group interms of their level before, during, and after exposure and overwintering. The effects at the 50 µg/L however,appear earlier, persist longer, and have a clear impact, especially after overwintering, when compared to thecontrol.
When weighing statistical and biological significance, the overall NOAEC and LOAEC for pupal cells
is determined to be 25 and 50 µg/L, respectively.

3.11.5.

Total individuals in hives

When evaluating the proportion of frame coverage of total individuals, the pattern of effects has somesimilarity to the proportion of frame coverage of adults and pupae, as these two life stages make up thelargest components of the hive population throughout the course of the study. In the 100 and 200 µg/L
treatment groups, total individuals were significantly reduced from the level of control from CCA4 to CCA7
(p<0.05). The EPA analysis did not find a significant difference (p>0.1) at the 200 µg/L group at CCA4
but the impact at this treatment level is evident at other CCAs. The CDPR analysis did not assess this
response variable.
29Table 28. Estimated percent reduction from control for total number of individuals
Test
concentration(µg/L)
12.5
25
50
100
200

Estimated reduction from control (%)1
CCA4

CCA5

CCA6

CCA7

CCA82

4
10.8
10.3
35.3**
48.6**

1
10.9
25.4*
46**
60.5**

8.7*
17.4**
12.1**
48.2**
65.9**

-16.9
2.2
8.1
60.9**
74.6**-2.6-11.4
49.1**
145.1**
54.1

Note: Negative value indicates increased number of total individuals in comparison to control.
*0.05<p<0.1**p<0.05
1Percent differences from control are based on the raw counts of total individuals, not proportions of the total individuals as Figure 19 below
shows.
2At CCA8, comparisons made to the 100 and 200 treatment groups are with uncertainty as 1 and 2 hives survived in these groups, respectively.
These data are included in the PMRA analysis but excluded from the EPA analysis

58
145Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

19Figure 19. Proportion of total individuals (adult, eggs, larvae, pupae) following exposure to varying
concentrations of imidacloprid in the diet across CCA3 – CCA8.
For the 50 ppb treatment group, reductions relative to the control at CCA5 and CCA6 were significant at
α=0.10 and reductions relative to the control at CCA8 were significant for α=0.05 (CCA6 result wassignificant at 0.05 in the PMRA analysis). For the EPA analysis, there were no further findings of statisticalsignificance which includes all CCAs at the 12.5 and 25 µg/L treatment groups. In the PMRA analysis,
total individuals were significantly reduced at 12.5 (p<0.1) and at the 25 µg/L treatment groups (p<0.05).
It is noted here, as it has been previously for other response variables, that the effects determined at 12.5
and 25 µg/L are isolated to CCA6, with no determinations of significance before and after this CCA,
indicating this may be a transient effect. After overwintering at CCA8, the surviving hives in the 12.5 and25 µg/L groups were actually above the level of control by 2.6 and 11.4% respectively (based on the raw
counts of total individuals) while the 49% reduction from control at CCA8 in the 50 µg/L group was
significantly reduced at a 0.05 alpha level.
When weighing statistical and biological significance, the overall NOAEC and LOAEC for total
individuals is determined to be 25 and 50 µg/L, respectively.
Figures 20-22 below provide another visual representation of the effects across CCAs variables within a
response variable for the various life stages of bees during the course of the study. The bar charts represent
the percent differences from control with negative percent differences from control indicating an increasein a given response variable above the level of control. These figures provide further evidence of the generallack of dose responsiveness in effects at the three lowest treatment groups. Furthermore these charts are
effective in indicating how the percent differences with a given response variable, changed over the courseof the study within a treatment group. The 50 µg/L group chart (Figure 22) in particular shows a progression
59146Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Documentof a continuous impact throughout the study for certain response variables that is still present to a higher
degree after overwintering at CCA8. It is also noted here that the scale for percent difference from control(y-axis) has been standardized across all charts. It is noted here also that negative (“-“) responses refer to apercent increase above the level of control. Charts are not shown for the 100 and 200µg/L groups given theclear impacts on those hives across multiple response variables. Additional charts of the data represented
in a slightly different way, across response variables within a CCA, are provided in Appendix B.
100.0

Difference from control (%)

80.0
60.0

37.7

40.0
20.0

4.0

1.0 8.7

0.0
-20.0
-40.0

-2.6
-16.9

10.7
12.3

22.2
2.2 2.3
-5.4
-11.0
-12.6

2.8
-3.7

-8.4

22.8 11.9
0.8

5.9
0.1

-1.3
-15.5

-60.0
-62.6

-80.0
TotalLife
CCA4

Adult

Pupae

Larvae

Eggs

CCA6

CCA7

CCA8

CCA5

20Figure 20. Summary of living organism parameters at the 12.5 µg/L treatment group60
147Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

Difference from the control (%)

100.0
80.0
60.0
40.0
20.0
0.0

16.2

10.9 17.4
10.8

8.7 7.7
2.7

2.2

-20.0

-4.1

-11.4

17.7 18.3 18.1
8.1
5.8

-4.3

26.3

23.0

31.3

8.3

-9.9

-10.8

5.8

-1.1

-25.5

-40.0
TotalLife

Adult

Pupae

CCA4

CCA5

CCA6

Larvae

Eggs

CCA7

CCA8

21Figure 21. Summary of living organism parameters at the 25 µg/L treatment group

Difference from the control (%)

100.0
78.0

80.0
60.0

78.2

70.6

49.1

40.0

25.4
20.0 10.3 12.1
8.1
0.0

19.8
21.7

18.6

34.6
28.1
12.5
9.7

42.6
15.3
12.1
-2.1

-20.0
-23.3

-40.0
TotalLife
CCA4

Adult

Pupae

CCA5

CCA6

11.7
1.8 2.6-9.6-32.4
Larvae

Eggs

CCA7

CCA8

22Figure 22. Summary of living organism parameters at the 50 µg/L treatment group
61
148

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Figures 23-27 provide an additional visual representation of the effects on life stages during the course of
the study. This representation is distinguished from the figures previously presented for the life stage
response variables in that the trends for each variable are presented within a CCA. It is noted for thesefigures that the scale has been adjusted to match for the y-axis for all CCAs. This helps visualize the trendsof the response variables particularly in the later CCAs as compared to the earlier ones as well as to highlightthe fact that the control hives themselves experience seasonal reductions and increases in certain life stages
through time.
Although the assessment of hive health at CCA4 was taken only 3 weeks after the exposure period began,decreasing numbers of pupal and larval cells are indicated with increase in imidacloprid dose, particularly
at the 100 and 200 µg/L groups (Figure 23). These effects were also confirmed statistically by the three
analyses. Effects on pupal and larval cells numbers were persistent throughout the subsequent CCAs as was
discussed above.

Proportion of Frame Coverage

0.5

CCA4

0.45
0.4
0.35
0.3
0.25

Adult

0.2

Egg

0.15

Larval

0.1

Pupal

0.05
0
Control

12.5

25
50
Treatment Group

100

200

23Figure 23. Trends of life stages across treatment groups within CCA4At CCA5 (Figure 24), the effects become more readily apparent in the 50 µg/L group for adults and pupal
cells especially. In the 100 and 200 µg/L groups, adults, pupal cells, and egg cells continue to be repressedas they were from CCA4. This is also visualized by the sharp dip in the trend lines for adult and pupal cells
in particular at the 50 µg/L groups and above in comparison to the level of control while the responses in
the 12.5 and 25 µg/L groups remain generally at the level of the control.

62
149

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

Proportion of Frame Coverage

0.5

CCA5

0.45
0.4
0.35
0.3
0.25

Adult

0.2

Egg

0.15

Larval

0.1

Pupal

0.05
0
Control

12.5

25
50
Treatment Group

100

200

24Figure 24. Trends of life stages across treatment groups within CCA5

Proportion of Frame Coverage

At CCA6 (Figure 25), reductions that were determined in the three analyses for all life stage response
variables at the two highest treatment groups continue to be repressed from the level of control. At the 50µg/L group, the reductions in adults and pupal cells are also evident. Most noteworthy is the reduction in
pupal cells that was confirmed as statistically significant (p<0.05) by the EPA and PMRA analyses at the12.5 µg/L (the PMRA analysis also determined a significant reduction at the 25 µg/L treatment group). Thisis also evidenced visually by the dip in the proportion of pupal cells of frame coverage at these two groups(12.5 and 25 µg/L) while other response variables in this group (12.5 µg/L) at this CCA are in line with thelevel of the control.
0.5
0.45
0.4
0.35
0.3
0.25
0.2
0.15
0.1
0.05
0

CCA6

Adult
Egg
LarvalPupal
Control

12.5

25
50
Treatment Group

100

200

25Figure 25. Trends of life stages across treatment groups within CCA
63
150

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
As mentioned previously, CCA7 represented the time during the study when hives are in a “shut down”
mode and preparing for overwintering. As a result, the proportions of all life stages are in a natural decline,
independent of imidacloprid exposure. The significant effects for adults, eggs, larvae, and pupal cells at the
100 and 200 µg/L treatment groups that were identified in previous CCAs were again determined by all
three analyses which are also visually evident in Figure 26 with the proportions of life stages in these twogroups clearly being below the level of that in the control. Effects at the 50 µg/L for adults (no othersignificant effects at this group for other life stages) were determined as statistically significant by the
PMRA analysis but the level at CCA7 is reduced from control as well as in previous CCAs.

Proportion of Frame Coverage

0.5

CCA7

0.45
0.4
0.35
0.3
0.25

Adult

0.2

Egg

0.15

Larval

0.1

Pupal

0.05
0
Control

12.5

25
50
Treatment Group

100

200

26Figure 26. Trends of life stages across treatment groups within CCA7
After overwintering at CCA8 (Figure 27a and 27 b), there were a number of hives that were lost due tohive mortality. Despite this, colony health response variables of surviving hives are still consideredinformative in examining the success of certain treatment groups over others (see Section 5 – Reviewer’s
Comments for more details regarding this). As evidenced by the hive mortality data previously discussed
as well as the trends shown below at the 100 and 200 µg/L group, there was a clear impact at these groups.
It is worth noting that significant reductions in these life stage response variables were evident well before
the time of CCA8, with reductions being determined as early as CCA4. What is noteworthy is the levels of
proportions at the 50 µg/L group as compared to the control and 12.5 and 25 µg/L groups. In addition to
the statistically significant findings of all analyses, the levels of proportions in all life stages is shown below
to be reduced from the level of control (EPA and CDPR analyses did not determine significance for larval
cells). This is distinguished from the 12.5 and 25 µg/L groups whose levels are generally in line or above
the level of control.

64151

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

Proportion of Frame Coverage

0.5

CCA8

0.45
0.4
0.35
0.3
0.25

Adult

0.2

Egg

0.15

Larval

0.1

Pupal

0.05
0
Control

12.5

25
50
Treatment Group

100

200

27Figure 27. Trends of life stages across treatment groups within CCA8

Colony Condition Assessments – Food Store Response Variables
3.11.6.

PollenPollen stores were significantly reduced in the 200 µg/L treatment group during from CCA4 to CCA7
(p<0.05), with the CDPR analysis not finding a significant difference at CCA7 only. Pollen stores werereduced at 100 µg/L treatment group at CCA4 andCCA5 (p<0.05). Similarly, pollen stores weresignificantly reduced at the 50 µg/L treatment group at the 0.05 alpha level at CCA4 and CCA5, but not
CCA6 and CCA7 (p>0.1). A marginal reduction at CCA7 (0.05 < p <0.1) for the 50 µg/L was determinedin the PMRA analysis but not in the EPA or CDPR analyses. The reduction of pollen stores was notdetermined in the 12.5 and 25 µg/L treatment groups for any CCA assessed. The reduced pollen store wasmost prevalent during and just after the exposure phase (CCA4 and CCA5) of the treatment levels exhibiting
effects.
30Table 29. Estimated percent reduction from control for pollen stores
Test
concentration(µg/L)
12.5
25
50
100
200

Estimated reduction from control (%)1
CCA4

CCA5

CCA6

CCA7

CCA82

11.8
5.2
56.1**
83.7**
94.5**-6.8
2.1
62.5**
83.6**
90.4**

-53.3
-25.2
15.5
15.3
54.7**

-26.1
-20.7
29.2*
12.9
50.6**-12.2
0.7
63.8**
100.6**
12.3

Note: Negative value indicates increased pollen stores in comparison to control.
*0.05<p<0.1

65152

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document**p<0.05
1Percent differences from control are based on the raw counts of pollen stores, not proportions of the pollen stores as Figure 28 below shows.
2At CCA8, comparisons made to the 100 and 200 treatment groups are with uncertainty as 1 and 2 hives survived in these groups, respectively.
These data are included in the PMRA and CDPR analyses but excluded from the EPA analysis.

At CCA8, in the hives that survived overwintering, the total amount of pollen store was reduced in in the
50 µg/L group (↓64% based off raw counts). The PMRA analysis also determined a significant reductionin the 100 µg/L group (p<0.05) but not in the 200 µg/L group (p>0.1) (this data excluded from EPA’s
analysis). It is noted however, that the lack of statistical difference of pollen stores in the 200 µg/L groupis considered to be uncertain as there were only two hives surviving overwinter and a large confidence
interval.

28Figure 28. Proportion of pollen stores following exposure of honey bees to varying concentrations ofimidacloprid in the diet across CCA3 – CCA8.
Figure 29 below shows the clear divergence of pollen stores in the 50 µg/L group as compared to thecontrol, 12.5, and 25 µg/L groups where stores begin to decline immediately following exposure andcontinue to be repressed throughout the study including after overwintering.

66
153

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

Pollen

Proportion of Polen Stores

0.12
0.1
0.08
Control

0.06

12.5
25

0.04

50
0.02
0
CCA3

CCA4
CCA5
CCA6
CCA7
Colony Condtion Assessment

CCA8

29Figure 29. Proportion of pollen stores following exposure to varying concentrations of imidacloprid in
the diet across CCA3 – CCA8 in the control, 12.5, 25, and 50 µg/L groups only.
In examining the trends of pollen stores in the control (Figure 30 below), there was a buildup that occurredfrom CCA3 (7% of frame coverage area) to CCA5 (10%). This increase supports the queen in her effort tobuild up brood during the late spring and early summer months. Pollen stores experienced a decline in
numbers from CCA5 (10%) to CCA7 (4%) before showing an upward trend from CCA7 to CCA8 (8%).
This downward trend reflects that the fact that up to overwintering, brood production will slow as the hive
prepares for winter and therefore there is a reduced need for pollen within the hive.
As depicted in the Figure 30 below, the trends of the proportions in the 12.5 and 25 µg/L groups tracks very
similarly with the control. Pollen stores at the 50 µg/L group, in contrast to the control, 12.5 and 25 µg/Lgroups, began a decline in stores earlier than the other groups as well as having an average proportion of
approximately 50% of the stores after wintering in CCA8 among the surviving hives.

67154

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

0.12
0.1
0.08
0.06
0.04
0.02
0

12.5 µg/L
Proportion of Pollen Stores

Proportion of pollen stores

Control
0.12
0.1
0.08
0.06
0.04
0.02
0

Colony Condition Assessment

PollenPollen25 µg/L

50 µg/L

0.12

Proportion of Pollen Stores

Proportion of Pollen Stores

Colony Condition Assessment

0.1
0.08
0.06
0.04
0.02
0

0.12
0.1
0.08
0.06
0.04
0.02
0

Colony Condition Assessment

Colony Condition Assessment

PollenPollen30Figure 30. Proportion of pollen stores following exposure to varying concentrations of imidacloprid in
the diet across CCA3 – CCA8 in the control, 12.5, 25, and 50 µg/L groups.
When weighing statistical and biological significance, the overall NOAEC and LOAEC for pollen
stores is determined to be 25 and 50 µg/L, respectively.

3.11.7.

Nectar / Honey

There was consistently and significantly (p<0.1) a lower amount of honey stored in treatment hives at 50,
100 and 200 µg/L than in the control at CCA6 and thereafter (Figure 31 below). One exception is theabsence of a determination of significance at CCA6 for the 100 µg/L group which PMRA determined as
significant but EPA did not. All other findings after CCA6 at the 50, 100 and 200 µg/L (EPA excluded data
68
155

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
at CCA8 for the 100 and 200 µg/L groups) were in general agreement with slight variations of the alphalevel that the effect was significant. No reduction of the honey stores was determined at the 12.5 and 25µg/L during the study, with the exception of the PMRA finding of a reduction at CCA6 at the 12.5 µg/L
treatment group (p<0.1) at CCA6. This statistical difference at 12.5 µg/L was unlikely to be treatment
related, as there were no reductions before or after the CCA6 at the same concentration, nor at the higher
concentration of 25 µg/L at CCA6. While there were no significant findings of impact at CCA8 in the 200µg/L group (EPA excluded this data) it is noted that this lack of finding is considered to be uncertain as
there were only 2 surviving hives at this treatment group.
31Table 30. Estimated percent reduction from control for nectar/honey stores
Test
concentration(µg/L)
12.5
25
50
100
200

Estimated reduction from control (%)
CCA4

CCA5

CCA6

CCA7

CCA8

-7.5
-10.4
-6.2
-8
-21.1

1.3
-15.3
21.6*
7.1
-84.1

15.8*
-2.4
36**
21.8*
70.5**

12.5
10.6
41.2**
52.9**
80**

-10.9
13.3
60.4**
156.6**
5.1

Note: Negative value indicates increased nectar/honey stores in comparison to control.
*0.05<p<0.1**p<0.05
1Percent differences from control are based on the raw counts of nectar/honey stores, not proportions of the nectar/honey stores as Figure 31
below shows.
2At CCA8, comparisons made to the 100 and 200 treatment groups are with uncertainty as 1 and 2 hives survived in these groups, respectively.
These data are included in the PMRA analysis but excluded from the EPA analysis

Figure 32 below for the honey store trends in the control, 12.5, 25, and 50 µg/L groups only show a marked
divergence at the 50 µg/L treatment group beginning at CCA6 and persisting up to and after overwintering
at CCA8.

69
156

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

31Figure 31. Proportion of honey stores following exposure of honey bees to varying concentrations ofimidacloprid in the diet across CCA3 – CCA8.

Proportion of Nectar (honey) stores

Nectar (Honey) Stores
0.35
0.3
0.25
0.2

Control

0.15

12.5

0.1

25

0.05

50

0
CCA3

CCA4
CCA5
CCA6
CCA7
Colony Condition Assessment

CCA8

32Figure 32. Proportion of honey stores following exposure to varying concentrations of imidacloprid in
the diet across CCA3 – CCA8 in the control, 12.5, 25, and 50 µg/L groups only.

70
157

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
For the control group, honey stores underwent an upward trend from CCA3 (16% of the frame coverage) to
CCA4 (20%), before a subsequent decline in average proportion from CCA4 to CCA5 (13%). This wasfollowed by an approximately 140% increase in honey cells from CCA5 to CCA6 (average proportion of30%) that remained stable until CCA7 (30%). This buildup of honey stores took place ahead of CCA7 that
represented the last time point before overwintering. The honey stores declined markedly from CCA7 to
CCA8 (16%) which is expected given lack of foraging and utilization of these reserves during theoverwintering period. It is noted that the proportion of comb cells containing honey stores at CCA3 and at
CCA8 were approximately the same at 16%.

0.35
0.3
0.25
0.2
0.15
0.1
0.05
0

Colony Condition Assessment
Honey

12.5 µg/L
Proportion of nectar/honey stores

Proportion of nectar/honey stores

Control
0.35
0.3
0.25
0.2
0.15
0.1
0.05
0

Colony Condition Assessment
Honey

0.35
0.3
0.25
0.2
0.15
0.1
0.05
0

Colony Condition Assessment

50 µg/L
Proportion of honey/nectar stores

Proportion of honey/nectar stores

25 µg/L
0.35
0.3
0.25
0.2
0.15
0.1
0.05
0

Honey

Colony Condition Assesment
Honey

33Figure 33. Proportion of honey stores following exposure to varying concentrations of imidacloprid in
the diet across CCA3 – CCA8 in the control, 12.5, 25, and 50 µg/L groups.
71
158

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
In the 12.5 µg/L treatment group, honey stores also underwent an initial build up from CCA3 (16%) to
CCA4 (21%) before a subsequent decline from CCA4 to CCA5 (12%), similar to that of the control. Thiswas followed by a large buildup of honey stores from CCA5 to CCA7 (27%). Honey stores declined during
overwintering and represented 15% of the brood area at CCA8, as compared to 16% at CCA3, which asimilar finding to that in the control group. The proportion of honey stores at CCA8 was comparable to thatof the control group (15% for both 12.5 µg/L and control). A similar picture was found for the 25 µg/L
treatment group, in that honey stores underwent an initial build from CCA3 (15%) to CCA4 (21%) before
a decline at CCA5 (14%). The subsequent build up reached 29% at CCA7 before a decline throughoverwintering to CCA8 (14%). CCA8 concentrations of nectar and pollen were comparable to that of thecontrol at CCA8 (honey: 14% and 15%; 25 µg/L and control groups, respectively).
These trends in the control and two lower treatment groups are distinguished from the response at the 50µg/L group. While honey stores underwent an initial buildup and then decline from CCA 3 (17%) to CCA4(21%), the subsequent larger buildup leading up to CCA7 that took place in the control and lower treatmentgroups was much less pronounced with the 50 µg/L group. Specifically, the proportion of honey storesfrom CCA3 to CCA7 roughly double in numbers from 15 to 30% of the brood comb in the control, 12.5
and 25 µg/L treatment groups. This is distinguished from the 50 µg/L group were the buildup that occurredfrom CCA5 to CCA7 reached a marginally higher level that the starting proportion at CCA3 (18% and 17%
respectively). That is to say, that the amount of honey stores at CCA7 (before the overwintering period) inthe 50 µg/L group was approximately half of that in the control, 12.5, and 25 µg/L treatment groups (18%for 50 µg/L as compared to 30, 27, and 29% at CCA7 for the control, 12.5 and 25 µg/L treatment groups,
respectively). Notably, it is also the only group out of these 4 in which the proportion of honey stores at
CCA8 was markedly lower than that of CCA3 (8% at CCA8 and 17% at CCA3).
It is noted that the feeding solutions (sugar solutions) provided during the exposure period might haveaffected natural honey storage patterns; however, effects on honey storage are still able to be considered as
all treatments were compared to control hives (which also received feeding solutions).
When weighing statistical and biological significance, the overall NOAEC and LOAEC for honeystores is determined to be 25 and 50 µg/L, respectively.
Figures 34-36 below show an additional visual representation of the impacts on food stores across the CCAs
for each treatment group (Figures not shown for the 100 and 200 µg/L treatment groups due to the clear
impacts on the hives at that level, primarily on hive mortality). It is noted that the scale for percent difference
from the control (y-axis) was standardized to the level of the 50 µg/L chart. When visualized this way, theimpact of the early on and persistent nature of effects (particularly with pollen) at the 50 µg/L is clearly
divergent from the responses at the 12.5 and 25 µg/L, which generally show a buildup in food stores that
was also observed in the control group. It is noted here that negative (“-“) responses refer to a percent
increase above the level of control.

72
159

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

Difference from the control (%)

20.0

Honey

15.8

11.8

10.0

Pollen

12.5

1.3

0.0
-10.0

-6.8

-7.5

-10.9 -12.2

-20.0
-30.0

-26.1

-40.0
-50.0

-53.3

-60.0

CCA4

CCA5

CCA6

CCA7

CCA8

34Figure 34. Summary of hive food supply parameters at the 12.5 µg/L treatment group
Honey

Difference from control (%)

80.0

Pollen

60.0
40.0
20.0

10.6

5.2

2.1

0.0
-20.0

-10.4

0.7
-2.4

-15.3
-25.2

-40.0
CCA4

13.3

CCA5

CCA6

-20.7
CCA7

CCA8

35Figure 35. Summary of hive food supply parameters at the 25 µg/L treatment group73160

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

Honey

80.0
Difference from control (%)

70.0

62.5

56.1

60.0

60.4 63.8

50.0

41.2

36.0

40.0
30.0

21.6

20.0

Pollen

29.2
15.5

10.0
0.0
-10.0

-6.2

-20.0
CCA4

CCA5

CCA6

CCA7

CCA8

36Figure 36. Summary of hive food supply parameters at the 50 µg/L treatment group
Figures 37-41 provide an additional visual representation of the effects on food store parameters during thecourse of the study. This representation is distinguished from the figures previously presented for the foodstore response variables in that the trends for each variable are presented within a CCA8 across treatmentgroups. It is noted for these figures that the scale has been adjusted to match for the y-axis for all CCAs.
This helps visualize the trends of the response variables particularly when examining the level of control ateach CCA as honey stores for example initially fall then build up across multiple CCAs in preparation for
overwintering heading into CCA7.

74
161

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
At CCA4, 3 weeks into the exposure period, honey stores across all treatment groups remained at the levelof the control, as shown below, and this was also confirmed statistically by all analyses. Pollen storeshowever, began to undergo an immediate reduction at the 50, 100, and 200 µg/L groups while the 12.5 and25 µg/L remain generally at the level of control.

Proportion of Frame Coverage

CCA4
0.35
0.3
0.25
0.2
0.15

Honey

0.1

Pollen0.05
0Control

12.5

25
50
Treatment Group

100

200

37Figure 37. Trends of food store parameters across treatment groups within CCA4
The responses at CCA5 are the same as those at CCA4 in terms of honey responses although the trend at
the 200 µg/L group rises above the level of the control. Pollen stores continue to be suppressed at the 50,
100 and 200 µg/L, while the responses at 12.5 and 25 µg/L are generally at the level of the negative
control.

Proportion of Frame Coverage

0.35

CCA5

0.3
0.25
0.2
0.15

Honey

0.1

Pollen0.05
0Control

12.5

25
50
100
200
Treatment Group
38Figure 38. Trends of food store parameters across treatment groups at CCA575
162

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
At CCA6, significant reductions in honey stores began to become apparent and were confirmed statisticallyat the 50 and 200 µg/L levels. These groups also appear visually reduced from the level of control. Of noteis that the proportions of honey in the control are above that of the level of CCA5 by approximately 2.5
fold, indicating a buildup in stores in the weeks before overwintering.

Proportion of Frame Coverage

0.35

CCA6

0.3
0.25
0.2
0.15

Honey

0.1

Pollen0.05
0Control

12.5

25
50
Treatment Group

100

200

39Figure 39. Trends of food store parameters across treatment groups at CCA6At CCA7, just before the overwintering phase begins, there is a marked decline in honey stores in the 50,
100 and 200 µg/L groups below that of the level of control. This is noteworthy as the reduced stores provide
an indication that these hives will have reduced success after overwintering. In the case of the 100 and 200µg/L groups, these hives largely did not survive overwintering.

Proportion of Frame Coverage

0.35

CCA7

0.3
0.25
0.2
0.15

Honey

0.1

Pollen0.05
0Control

12.5

25
50
Treatment Group

100

200

40Figure 40. Trends of food store parameters across treatment groups at CCA776
163

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Finally, at CCA8 after overwintering, honey stores were markedly decreased in the control group from theirlevel heading into overwintering at CCAs 6 and 7. The levels at the 100 and 200 µg/L groups are clearlysuppressed as these groups had one and two hives surviving, respectively. The response at the 50 µg/L
level was also reduced from control while the response at the 12.5 and 50 groups again are in line with orabove the level of control.

Proportion of Frame Coverage

0.35

CCA8

0.3
0.25
0.2
0.15

Honey

0.1

Pollen0.05
0Control

12.5

25
50
Treatment Group

100

200

41Figure 41. Trends of food store parameters across treatment groups at CCA8

3.11.8.

Hive Weight

There were significant reductions from control observed at the two highest dose levels (100 and 200 µg/L)
beginning at CCA4 and persisting until CCA8 (p<0.05, data from CCA8 excluded from EPA analysis).
Additionally, there were no significant reductions from control in the 12.5 and 25 µg/L treatment groups
determined for all CCAs assessed (p>0.1). For the 50 µg/L group, there were significant reductions at boththe 0.05 and 0.1 alpha level starting as early as CCA4 (CDPR and PMRA analyses). The PMRA analysis
determined significant reductions at the 0.1 alpha level for CCA4 and CCA6 and at the 0.05 alpha level for
CCAs 5, 7, and 8. The EPA analysis determined significant reductions from the control at the 0.1 alphalevel at CCAs 7 and 8 only. Despite these differences in statistical findings, there is an apparent effect onhive weight at the 50 µg/L level that is supported by both analyses indicating significant reductions at
multiple CCAs.
While there was no difference (p>0.1) in the hive weight at the 200 µg/L treatment groups (data not includedin EPA analysis), the lack of statistical difference is considered to be uncertain as there were only two hives
surviving overwintering.

77
164Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
32Table 31. Estimated percent reduction from control for hive weights
Test
concentration(µg/L)
12.5
25
50
100
200

Estimated reduction from control (%)
CCA4

CCA5

CCA6

CCA7

CCA81

-0.5
2.7
4.1*
7.1**
10.1**

2.4
-0.4
11**
14.9**
10.4**

0.2
-1.5
12.3*
15**
30.4**

-1.6
0.6
15**
18.9**
33.3**-1.1
1.2
20.9**
67.7**-25.5

Note: Negative value indicates increased hive weight in comparison to control.
*0.05<p<0.1**p<0.05
2At CCA8, comparisons made to the 100 and 200 treatment groups are with uncertainty as 1 and 2 hives survived in these groups, respectively.
These data are included in the PMRA analysis but excluded from the EPA analysis

42Figure 42. Proportion of hive weight following exposure of honey bees to varying concentrations ofimidacloprid in the diet across CCA3 – CCA8
When weighing statistical and biological significance, the overall NOAEC and LOAEC for hive
weight is determined to be 25 and 50 µg/L, respectively.

78
165

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

4. Reviewer comments:
What follows is brief discussion of some of the elements taken into consideration when evaluating the resultsof this study.

General Considerations for Biological Interpretation
While the hive mortality is considered as the most relevant measurement of survival at the colony level,
sublethal effects at the colony level were estimated by measuring multiple parameters during the course of
study. Each measured parameter is expected to reflect only part of the colony conditions, and allmeasurements have to be integrated for a better understanding of the hive status at the colony level. A
honey bee colony is a super-organism in which live individuals and food supply are the two major
components in maintaining the proper function of the colony. There are interactions between the twocomponents and even within each component.
Bee individuals are present in the colony as eggs, larvae, pupa and adults and they develop from one stage
to another and interact with each other to perform a variety of tasks to maintain the integrity of the colony.
The measurement of each stage of the bees is expected to provide information on the potential treatment
effect on a specific life stage of bees during their development.
Hive food supplies including hive pollen and nectar are collected and processed by adults and are expected
to have a large impact on the development of all stages of bees in hives. However, the amount of hive foodstorage is dependent on not only the power/number of foragers available for food collection, but also thenumber of individuals that consume the food. In addition, the seasonal availability of outside pollen andnectar sources also affects the amount of storage, thus impacting hive development. As well, sucrose
feeding solutions were provided to the hives as a means of treatment and as a supplement for hive
overwintering, which may have affected foraging and food storage during those time periods.
Hive weight was measured during the study. However, it is largely affected by the honey storage and numberof bees that consume the food. A strong colony with a high number of bees likely consumes a high amountof stored honey and may result in a reduced hive weight. In this study, additional sugar solution wasprovided as the means of treatment and a supplement for hive overwintering in the study. Such feeding
likely further confounds the relevance of the hive weight to the treatment effect. In addition, hive weights
were taken after 10 a.m. However, weighing hives at different time periods of the day may result in an
increased variation of the measurement due to the fact that foragers may not be present in the hive when theweight is measured. Hive weights may be artificially lower in hives which contain a high number of forager
bees that may be out collecting food during a different time of the day.
Considerations regarding the measurement time points:••

CCA3 represents the background hive conditions as the first colony assessment after the hives
were placed in the test fields prior to the exposure.
CCA4 and CCA5 represent the hive conditions during the exposure phase. It was noted that the
CCA5 was conducted a week after the end of the 6-week exposure period, but is expected torepresent effects during the exposure period.

79
166Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document•••

CCA6 was measured at 5 weeks after the end of exposure. It allows all bee individuals, including
eggs, larvae and pupa that were exposed to treatment to finish their development cycle and become
adults.
CCA7 represents the hive conditions prior to overwintering. It is considered that hives were
physiologically preparing for overwintering by reducing the production of immature bee
individuals. Treatment effects may be masked by the natural decline of hive individuals.
CCA8 represents hive conditions of surviving hives after overwintering. Additionally, hive
overwintering mortality at CCA8 is expected to be directly relevant to the treatment effect at thecolony level.

Control PerformanceControl mortality:
The level of colony loss after overwintering in controls (36%), though not desirable, is consistent with that
historically experienced by beekeepers on average across the United States 3; the 2014-2015 preliminary
results estimate US overwintering loss on average to be 23.1%. There are, however, differences in bee
management practices associated with the test hives compared to commercial hives, making a directcomparison of expected overwintering success challenging. Commercial hives could experience very
different beekeeping practices than test hives, including being transported for pollination services, being
harvested for honey production, given additional feedings, receiving different pest and disease control
treatments, having different sized hives and different preparation for overwintering. The test hives, whileclosely monitored, may also experience invasive disruptions during the colony condition assessments, havedifferent/less supplemental feeding or mite treatments than commercial hives, have a lower colony size or
be prepared differently going into overwintering as was the case with hives in this study which were notgiven supers to allow for growth as well as having supplemental feeding delayed.
Sublethal effects on life stages and food stores
As described with discussion of the response variables in Section 3 above, the parameters measured indicatethat control hives behaved as would be expected through the seasonal changes that a honey bee colony
undergoes. Although a large variation among apiaries was detected for each parameter in all control hives,
the average of the total number of individuals in hives, as well as eggs, larvae, pupae and adults increasedor remained at similar levels during the exposure period from CCA3 to CCA5. After the end of the exposure,
all these biological parameters also increased or remained at a similar level at CCA5 and CCA6, and thenall decreased sharply from CCA6 to CCA7.
The similarity in the dynamics of all parameters for the individual living organisms at various stages
indicates that control hives were well developed during CCA3 to CCA6, and the hives were preparing foroverwintering at CCA7 as expected in the late fall in October. The increase of hive food supply (pollen andhoney store) and hive weight also confirms that hives were actively developing during the exposure period
from CCA3 to CCA5. The increased level of honey store from CCA5 to CCA6 and CCA7 indicates that
there were plenty of outside nectar sources in the test area after the end of feeding exposure period.
3

http://beeinformed.org/2015/05/colony-loss-2014-2015-preliminary-results/
80
167Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
However, the pollen store reduction observed during CCA5 to CCA7 might have resulted from increased
pollen consumption due to the increase of live individuals and/or limited availability of outside pollen
sources. If the outside pollen source were limited, it may have impacted the further development of control
hives after CCA5. However, the decline of pollen stores in the control hives after CCA5 is likely normal
for the local region of the study, and control hives did not appear to be impacted, therefore, this is notexpected to have biased the study.

Consideration of CCA8Control overwintering mortality

While the level of control mortality after overwintering is generally in line with historical findings from
North American beekeepers as described above, it is noted that the use of data from CCA8 to distinguishtreatment-related effects on colony survival (i.e. dead or alive) is compromised due to the level of control
mortality observed. That is to say, the ability to detect treatment-related decreases in overwintering colony
survival may be masked by the magnitude of control hive loss.
Response Variables from Surviving Hives

While the measure of overwintering success may be compromised by the control hive loss, the evaluationof life stage and food store metrics at CCA8 for surviving hives is considered to be useful and important tointerpreting the study results. Specifically, the data suggest that the weaker control hives were not able tosurvive overwintering because they had disproportionately fewer numbers of adults and honey storescompared to control hives that survived overwintering. (Figure 43).

43Figure 43. Fate of control hives as compared to number of adults across CCAs
Control hives that survived overwintering had 2.5X more honey on average at CCA7 than those that did notsurvive overwintering (Figure 44)81
168

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

44Figure 44. Fate of control hives as compared to the honey stores across CCAs
This, coupled with the experimental design which limited colony development due to lack of supers,
provides a plausible explanation for the loss of control hives. Assuming this hypothesis, the bias introducedby this “culling” of weaker hives would theoretically render the remaining control hives at CCA8 stronger(on average) than the initial population of control hives. This could conceivably improve the ability todetect treatment-related colony condition effects at CCA8, since the actual values for controls would be
weaker (on average) if the data from the dead hives were available for inclusion, and because there weremore non-survived hives among controls than the two lowest treatments.

Consideration of Study Strengths, Limitations and Interpretation
It is important to recognize the inherent strengths and limitations of this study as results are interpreted andpotentially considered in risk assessment.
In the context of available field studies involving honey bees and imidacloprid, this study contains a numberof strengths including:
•
•
•
•
•

Use of a high degree of replication (n=12) to achieve a reasonable level of statistical power
Demonstration of a generalized concentration-response relationship with respect to theconcentration of imidacloprid in sucrose solution and the magnitude and duration of adverse effects
Quantification of exposure to parent (imidacloprid) and toxicologically-relevant metabolites in dietand in hive matrices (uncapped nectar, pollen, honey, bee bread)Use of a 6-week exposure duration to represent a “high end” exposure scenario
Inclusion of multiple colony-level endpoints reflecting hive strength, brood development and foodstores82

169Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document••

Detailed QA/QC results regarding quantification of chemical residues in various matrices
Availability of raw data for conducting statistical analysis.

A number of limitations are also noted with this study, including:••Exposure of bees through nectar (sucrose) alone, whereas bees in the field are likely exposed throughboth pollen and nectar routes. Therefore, the design of this study may not reflect a “worst case”
exposure scenario in which bees are experiencing prolonged exposure to both contaminated nectar
and pollen. While exclusion of the pollen route is expected to reduce overall exposure, the impactof this exclusion on the study results is uncertain and will likely depend on the life stage/caste ofbee.
It is noted that imidacloprid was found in both hive nectar and hive pollen (beebread), atconcentrations lower than the feeding solutions. Dilution compared to the treatment feeding solution
is expected since bees could also forage on outside nectar and pollen sources. As well, hive pollen
contains only some hive nectar, thus would not be expected to have a concentration equivalent tonectar alone, and it is mixed with pollen which will come from outside sources. Therefore exposure
through both hive pollen and nectar occurred via exposure to the sucrose feeding solution, but how
this compares to exposure through contaminated pollen directly is not known. A recent paper by
Dively (2014) showed that higher residues throughout the hive resulted from feeding pollen
treatments compared to feeding sucrose solution treatments. It is also noted that nectar is considered
the dominant exposure route for forager bees; other hive bees and larvae consume both nectar andpollen. In addition, since bees were forced to forage for pollen in this study, the potential impact ofimidacloprid exposure on reducing pollen foraging efficiency of bees could be incorporated into theoverall expression of adverse effects, as suggested by published literature. Had contaminated pollen
been provided to bees, it is not known if the potential impact on pollen foraging efficiency would
have been masked.

•

The quantity of nectar provided to hives (2 L per week per hive) likely did not fulfill the complete
carbohydrate needs of the colony, as indicated by colony bioenergetics and the lack of remaining
sucrose solution upon their renewal. This suggests that bees could be exposed to a greater dose of
imidacloprid in nectar had a greater volume of spiked sucrose been provided. Although one caninfer that the dosing regimen may have underestimated exposure through sucrose relative to 100%
contaminated diet, it is also noted that bees had to supplement their spiked sucrose by foraging on
their own for other sources of nectar. As with the previous discussion of pollen, it is noted that had
100% of the carbohydrate needs of the colony been provided via feeders, the potential impact of
purported reductions in nectar foraging efficiency may have been masked to some degree.

•

Overwintering success of controls was impacted (36% hive mortality). This may have reduced theability to detect adverse effects related to hive loss following overwintering. Although comparable
to overwintering losses of commercial beekeepers, it is possible that elements of the study design
may have contributed to this loss (e.g., lack of supers to allow for colony growth, delayed
supplemental feeding during fall).

•Hive contamination with pesticides from food sources other than the artificial feeding was detectedduring the exposure period and post-exposure periods through collection of pollen from pollen traps.
83
170Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Although the study was deliberately conducted in a low agricultural area in order to minimize thepotential for pesticide contamination from other sources, the bees still appeared to be foraging on
contaminated pollen and possibly nectar. During both exposure and post-exposure periods, highlevels of multiple pesticides that may cause concern for bees were detected in most monitoring hives,
such as spiromesifen (maximum at 961 ppb) and piperonyl butoxide (maximum at 591 ppb). Traceamounts of other bee-toxic pesticides, such as chlorpyifos (LOD = 1.0 ppb) and malathion (LOD =4.0 ppb) were also detected. The test chemical imidacloprid was found at 12.1 ppb in pollen fromone (apiary L) of the total of six sites analysed. This level is similar to one of the test concentrations.
•

Residues of imidacloprid in uncapped nectar and bee bread within the hives at CCAs 4, 5, and 8
represent a single sample per hive on a single frame rather than a composite sample from multiple
portions of the comb within a hive. This means that residue results may reflect “hit or miss” scenario
with respect to detecting residues in nectar laid down from contaminated (fed) vs. outside sources.

•

The exposure, based on residues measured in the hive (hive nectar and hive pollen) indicated that,
overall, higher measured hive residues correlated with higher nominal residues in feeding solutions.
However, individual hive residue values varied, and there was some overlap in measured values,
particularly among the three lowest doses.

•

Exposure dilution during the study was evident. Pollen storage was observed consistently in thecontrol hives and hives exposed to lower test concentrations during the exposure period, indicatingthat test bees were foraging on food sources other than the spiked sugar solution. Remarkably lower
residue concentrations detected in bee bread and hive nectar in some test hives compared to thefeeding concentrations may also indicate foraging on other food sources. This uncertainty is inherent
in any semi-field or full-field study design.

84
171Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

5. Conclusions
The study is considered to be informative and will be used as a line of evidence in the pollinator risk
assessment. While there were uncertainties that were generally related to inherent aspects of any semi-fieldor full field study design (such as dilution of the test chemical through alternative sources of forage,
detection of other chemicals in the monitoring hives), this study still provides information on a number ofcolony health parameters about the long term (including overwintering) exposure to imidacloprid at thecolony level.
As indicated in the results section above, the PMRA, EPA, and CDPR analyses determined significant
effects (at both the 0.05 and 0.1 alpha levels) in the 50, 100, and 200 µg/L groups across multiple CCAs forthe majority of response variables. Specifically, for the 100 and 200 µg/L treatment groups, significant
effects (p<0.05) were determined for every response variable and persisted across at least 2 CCAs, alongwith very high overwintering mortality. While the 50 µg/L group had overwintering mortality similar to thecontrols, colony condition effects were different from controls with an early onset of effects which tended
to persist, and notably poorer colony condition in surviving hives after overwintering in comparison tocontrols.
Conversely, there was not a strong indication from the PMRA, EPA, and CDPR analyses of an impact at
the colony level at the 12.5 and 25 µg/L treatment groups. This is evidenced not only by a general lack ofstatistical findings (p>0.1) at these treatment levels but in cases where significant effects were determined,
they either did not show strong dose-responsiveness, did not persist across multiple CCAs, or wereconsidered potential transient effects (e.g. at CCA6) which did not persist after overwintering. This latter
point was the case for the total life and pupal cell findings in which the PMRA analysis determinedsignificant effects at all treatment levels at CCA6 (EPA also determined a significant reduction in pupal
cells at the lowest treatment group of 12.5 µg/L at CCA6). As well at CCA6, PMRA determined significant
effects with eggs and larvae at 25 µg/L treatment (but not at the 50 µg/L). For these two lowest treatmentgroups (12.5 and 25 µg/L), the colony condition of surviving hives at CCA8 following overwintering was
similar to controls, indicating the effects observed at CCA6 were likely transient and the colony was able
to compensate for these effects.
The figures below present the trends of life stages across all CCAs in the 12.5, 25, and 50 µg/L treatmentgroups compared alongside the response of that in the control.

85172

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

Control
Proportion of Abundance

0.5
0.4
0.3
0.2
0.1
0
CCA3

CCA4

Adults

CCA5
CCA6
Colony Condition Assessment
Eggs

Open

CCA7

CCA8

Capped12.5 µg/L
Proportion of Abundance

0.5
0.4
0.3
0.2
0.1
0
CCA3

CCA4

Adults

CCA5
CCA6
Colony Condition Assessment
Eggs

Open

Significantly reduced from control in the
PMRA, EPA, and CDPR analyses (either
0.1 or 0.05 level depending on theanalysis)

CCA7

CCA8

CappedProportions at CCA7 and CCA8 areapproximately the same as control (8%and 17% of frame
coverage,respectively)

86
173

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

Control
Proportion of Abundance

0.5
0.4
0.3
0.2
0.1
0
CCA3

CCA4

Adults

CCA5
CCA6
Colony Condition Assessment
Eggs

Open

CCA7

CCA8

Capped

25 µg/L
Proportion of Abundance

0.5
0.4
0.3
0.2
0.1
0
CCA3

CCA4

Adults

CCA5
CCA6
Colony Condition Assessment
Eggs

Open

CCA7

CCA8

CappedProportions at CCA7 and CCA8 areapproximately the same as control (7% intreatment vs 8% in control and 16% intreatment vs 18% in control, respectively

Significantly reduced from control in the
PMRA, EPA, and CDPR analyses (either
0.1 or 0.05 level depending on theanalysis)87
174Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

Control
Proportion of Abundance

0.5
0.4
0.3
0.2
0.1
0
CCA3

CCA4

CCA5
CCA6
Colony Condition Assessment

Adults

Eggs

Open

CCA7

CCA8

Capped

50 µg/L
Proportion of Abundance

0.5
0.4
0.3
0.2
0.1
0
CCA3

CCA4

CCA5
CCA6
Colony Condition Assessment

AdultsSignificantly reduced from control in the PMRA,
EPA, and CDPR analyses (either 0.1 or 0.05 level
depending on the analysis). Effects begin at a time
when buildup of adults is still taking place incontrols and two lower treatment groups andrepression continues up to and including after
overwintering.

Eggs

Open

CCA7

CCA8

CappedSignificantly reduced from control in the PMRA and EPA (at 0.05
level). The 50 µg/L is the only group of the three lower treatmentgroups that does not show an upward trend from CCA7 to CCA8.

Significantly reduced from control in EPA, CDPR and PMRA analyses(p<0.05 or p<0.01, depending on the analysis, CCA6 not determined in EPA
analysis). Effects are clearly divergent from control in which a buildup in
pupal cells occurs in the control from CCA3 to CCA6 whereas the 50 µg/Lgroup undergoes an initial decline from CCA4 to CCA588
175

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

Control
0.35
0.3
0.25
0.2
0.15
0.1
0.05
0
CCA3

CCA4

CCA5

CCA6

Pollen

Honey

CCA7

CCA8

CCA7

CCA8

50 ppb
0.35
0.3
0.25
0.2
0.15
0.1
0.05
0
CCA3

CCA4

CCA5

CCA6

PollenHoney

Significantly reduced from control in the PMRA,
EPA, and CDPR analyses (either 0.1 or 0.05 level
depending on the analysis). The level of honeystores at CCA8 in the 50 µg/L group isapproximately half of that of the control at CCA8.

Level of honey stores at their peak incontrol group heading into overwintering
at CCA7.
The control group had
approximately 30% of frame coverage ofhoney stores at CCA7 while the 50µg/Lgroup had 19%89176

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
When examining the effects on food stores (pollen and nectar), the PMRA, EPA, and CDPR analyses did
not determine any consistent and significant reductions in pollen and nectar stores at the 12.5 and 25 µg/L
treatment groups. This is distinguished from the 50 µg/L group where effects on nectar in particular werevery apparent when compared alongside the response of the control in terms of the level of nectar buildup
before the hive preparation for overwintering at CCA7. This finding was also confirmed statistically in all
three analyses with significant reductions in honey stores at CCAs 6, 7, and 8 (CCA8 data excluded fromthe EPA analysis for the 100 and 200 µg/L groups). Significant reductions in pollen stores were also
confirmed at CCAs 4 and 5 at the 50 µg/L treatment during the exposure period.
Specifically, when considering the adult and honey and pollen stores response variables, the differencesfrom control were apparent both visually and statistically, particularly in the three highest treatment groups.
For the proportion of adults, the onset of a decline in numbers occurred one CCA earlier in these groups
than in the control, 12.5 and 25 µg/L treatment groups. For honey stores, the buildup that occurred startingat CCA5 in the 50 µg/L treatment group, reached only half the level reached in the control, 12.5, and 25µg/L treatment groups by CCA7. Pollen stores were also reduced at CCA4 and CCA5 compared to controls
for the three highest treatment groups, as well as at CCA6 and CCA7 at the highest treatment group. These
effects were statistically significant (p<0.05) and indicate that the 50 µg/L treatment group was associatedwith trends and proportions of abundance for life stages and food stores not observed in the control, 12.5,
and 25 µg/L treatment groups.
Therefore, when weighing biological significance and the natural seasonal changes of honey beescolonies, as well as supporting conclusions from the statistical approaches used in PMRA, EPA, and
CDPR, the NOAEC and LOAEC for this study is determined to be 25 and 50 µg/L, respectively.

90
177

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

1.Appendix A: Details of EPA Statistical Analysis
In the statistical analysis of the Tier II colony feeding study for imidacloprid and honey bees, there were
three main questions that were investigated:
1. For given CCA (colony condition assessment) and treatment level, what treatments were having aneffect on the measures of hive health, as determined relative to control?
2. Were the observed treatment effects consistent over time?
3. What was nature of the onset of effects over time for various treatments?
To answer these questions, a variety of statistical approaches were considered and a repeated measure mixed
model analysis was deemed best to address the above questions.
For this analysis the time by treatment interaction was evaluated across all CCAs and treatment groups foreach response variable. In this way, the trends for each response variable (i.e. adult, eggs, etc.) across all
CCAs for a given treatment group could be examined. The details of the parameterization of the repeatedmeasure mixed model along with the statistical results (p-values) are provided below.
Background on data manipulation
•

•
•

•

•

Data utilized for the statistical analysis were provided in an Excel file by the study author. This file
was made available to the Agency on May 15, 2015. Additional QA of the entire data file wascompleted by the study author, as some transcription errors were found in an earlier electronic filesubmitted by the study author.
Data to be included in the data analysis were data that were collected on or after CCA3. CCA3
represents the first time hive parameters were measured after the hives were placed in their treatment
apiaries/locations.
Zeros in the data set represent instances when no frame contained >5% coverage of a given matrix.
Missing values for all matrices during a given timepoint indicate the hive was dead and nomeasurements were taken. Definition of a dead hive is provided in the study report. For these data
analyses, all entered zeros were maintained in the data set. Missing values were kept as ‘missing’.
These parameters were not replaced with zeros.
Time (days) between measurements was roughly even for CCA3 through CCA7, while CCA8 was
measured during the following spring (to evaluate any impacts on hive overwintering). Number ofdays was estimated using the median number of days between CCAs. Assuming that CCA3 was on
day 0, CCA4 through CCA8 occurred on the following days: 25, 53, 84, 119, and 272.
In the initial analyses, there were difficulties with convergence of the PROC MIXED algorithm formany of the more complicated temporal covariance structures. John Troiano (California Departmentof Pesticide Regulation) provided an article from SAS suggesting that by re-scaling the response
variable, the likelihood of convergence would be improved. Given the nature of the response variable(most were count cells of a given matrix within in a hive), a re-scaling was straight-forward. For
eggs, open, capped, pollen, and honey cells, the count (the variable recorded in the excel spreadsheet)
was divided by 68800 (total number of cells in the hive). This resulted in the proportion of cells
occupied by a given matrix (values between zero and one). Number of adult bees, as recorded in the
spreadsheet, was derived from the percent coverage, the density estimate of 1.3 bees/cell, and thedensity estimate of 4 cells/cm2. Therefore, the conversion from the data stored in the spreadsheet
91
178

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document•

was to divide by 68800, divide by 1.3, and multiply by 4. The resulting number is the proportion ofcells occupied by adult bees. Following the re-scaling of the response variables, the model
convergence improved dramatically.
The variables to be analyzed included:o Proportion of hive covered in adults
o proportion of hive covered in eggs
o proportion of hive covered in open cells (larvae)o proportion of hive covered in capped cells (pupae)o Proportion of hive covered in live bees (at any life stage). This was obtained by summing the
proportions of the four life stages (described above). Note that it is possible for the proportionof a hive covered in live bees to be greater than 1.0, as adult bees will be observed on top ofcells containing eggs, larvae, or pupae.
o Proportion of hive covered in pollen cells
o proportion of hive covered in honey cells, ando Hive weight.

Examination of the associations among the variables••

Based on physical hive constructs and the nature of the honey bees, it is generally accepted that thecolony condition assessment (CCA) variables may be correlated over time and may also becorrelated within a time point (sampling time). Given this background, a series of scatterplots,
correlation matrices, and principal component analyses for this bee study was prepared (Further
details are provided in the section entitled “Scatterplot and Principal Component Discussion”
and the full SAS output is included as Attachment 3).
Some of the general summary points are:o Variables tended to have stronger correlations at adjacent time points than at farther time
points (i.e., correlations for CCA3 and CCA4 tended to be stronger than correlations for
CCA3 and CCA7 or for CCA4 and CCA7).
o CCA8 tended to have weaker pairwise correlations with all the other time points than CCA3
through CCA7 had with each other.
o The first principal component for each of the CCAs explained 20-50% of the total
variation. The lowest was capped, and the highest were adults, honey, and pollen.
o The general interpretation of the first principal component was a weighted average over alltime points (weights varied depending on variable).

Analysis Approach and Model Setup
The general experimental design was a randomized complete block with repeated measures. Apiary was theblock effect and the repeated measures were the CCAs. Within each block, the control treatment was
replicated 2x and each treatment occurred one time. Since hives were not placed till shortly before CCA3,
the data for the statistical analysis only included CCA3 through CCA8. Exploring the interaction between
treatment and CCA will address the first two questions above.
Once the design component of the analysis was established, the next part of the analysis was to determine
which correlation structure (across time) was the best fitting for these data. One of the challenges was thatmany hives died before the end of the study (especially the 100 and 200 ppb treatments in CCA8), creating
92
179

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
censored data or an imbalanced design. PROC MIXED in SAS can handle an unbalanced design; however,convergence may not be attained for some correlation structures. In this case the imbalance was due totreatment (the higher treatments had a higher rate of hive death) as well as random mortality (control hive
mortality rate during overwintering was similar to background mortalities of commercial hives). After
exploring several options (which are detailed in the section entitled “Options for Addressing the Data
Imbalance”), the data were analyzed after removing data from the few remaining hives in treatment groups
100 and 200 ppb from CCA8. Data from hives treated with 100 or 200 ppb imidacloprid remain in the data
set and analysis for CCA3 through CCA7 permitting utilization of a majority of the data. These deletions
create a 5x6 factorial design matrix with two of the cells (CCA8, treatment= 100) and (CCA8,
treatment= 200) missing all data. CCA*treatment interaction means can still be calculated. Least squaremeans on the main effects (treatment and CCA) cannot be estimated for Concentration= 100,Concentration=200, and CCA8. Since the expectation was that the interaction would be significant and all
further statistical tests would be exploring the interaction, this was not considered a significant hindrance.
To address the primary research questions above, the “treatment * CCA” interaction was evaluated in two
ways:• At each CCA, was there a reduction in the response relative to the control? This was evaluated using
a one-sided Dunnett’s test.
• At each treatment level, was there a difference in the response relative to the baseline? This wasevaluated using a two-sided Dunnett’s test comparing CCA4 through CCA8 against CCA3.

Scatterplot and Principal Component Discussion
Based on physical hive constructs and the nature of the honey bees, it is generally accepted that the colony
condition assessment (CCA) variables may be correlated over time and may also be correlated within a time
point (sampling time). Given this background, a series of scatterplots, correlation matrices, and principal
component analyses (PCA) for this bee study were prepared. The full printout is included as Attachment
3. Some of the general summary points are:
The first series looked at: for a given response variable, what were the pairwise correlations over time, andhow would a principal components analysis best explain the observed variation. Data were plotted andsubjected to a PCA without accounting for treatment (i.e., all data were included in a single series of plots
and PCAs; separate assessments were not done for each treatment). Some general interpretations are:• Scatterplot and correlation matrices indicated that variables tended to have stronger correlations at
adjacent time points than at farther time points.
• CCA8 tended to have weaker pairwise correlations with all the other time points than CCA3 through
CCA7 had with each other.
• The first principal component for each of the variables explained 26-53% of the total variation. The
lowest was proportion frame coverage of eggs, and the highest were adults, honey, and hive weight.
The general interpretation of the first principal component was a weighted average over all time
points (weights varied depending on variable). For most variables, CCA3 tended to carry the least
weight in the weighted average.
• The second principle component explained an additional 16 to 27 percent of the total variation. For
most endpoints, a general interpretation of the principle component eigenvector was a difference
between measured taken early in the study and measure recorded later in the study (e.g., average of
CCA3 and CCA4 minus the average of CCA7 and CCA8).
93
180

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
These scatterplots, correlation matrices, and principle component analyses were used to inform the choiceof covariance structure used in the repeated measure analysis. Some summary points are:• Variance for a given response variable was not consistent across all CCAs. This may indicate thatthe correlation structures with a constant variance for all CCAs (e.g., CS, SP(pow)(1), and AR(1))
may not fit as well as those that allow for heterogenous variance (e.g., CSH, ToepH(1), andToepH(2)).
• There did not appear to be a consistent decrease in correlation if the paired CCAs were farther apart(i.e., correlation between CCA4 and CCA8 (three time steps apart) was not consistently less thancorrelation between measurements 2 time steps apart, e.g., (CCA4, CCA6), (CCA5, CCA7), and(CCA6, CCA8). This may indicate that the AR(1) and SP(pow)(1) covariance structures may not fit
the data as well as other structures.
The second series of scatterplots and PCAs looked at: for a given CCA, what were the pairwise correlations
across matrices, and how would a principal components analysis best explain the observed variation. Data
were plotted and subjected to a PCA without accounting for treatment (i.e., all data were included in a single
series of plots and PCAs; separate assessments were not done for each treatment). Some of the generalsummary points are:o Honey had the weakest correlations (honey and any of the other measured matrices) amongst
all the pairwise correlations.
o The first principal component explained 36-66% of the total variation. The CCAs with thelowest percent of variation explained were CCA3 and CCA4. The percent variation
explained tended to increase over time. At each time point the first principle component
tended to be interpreted as a weighted average, with honey receiving the least weight.
o The second principal component explained an additional 15 to 24% of the total variation.
The interpretations of the eigenvectors from the second principal component were less clear
and consistent. They tended to be an average of a measure of hive food stores (pollen, honey,
and/or hive weight) or an average of the hive food stores contrasted with a subset of the
population matrices (e.g., eggs, open, capped, adult, and/or total).

Options for Addressing the Data Imbalance
Once the design component of the analysis was established, the next part of the analysis was to determine
which correlation structure (across time) was the best fitting for these data. One of the challenges was thatmany hives died before the end of the study (especially the 100 and 200 ppb treatments in CCA8), creating
censored data or an imbalanced design. PROC MIXED in SAS can handle an unbalanced design; however,convergence may not be attained for some correlation structures. In this case the imbalance was due totreatment (the higher treatments had a higher rate of hive death) as well as random mortality (control hive
mortality rate during overwintering was similar to background mortalities of commercial hives). Several
options were explored for addressing the data imbalance and convergence issues: 4

4These analyses were conducted utilizing an earlier version of the data set that was provided by the study author. It was later
determined that there were some data entry errors. The errors were corrected and the main analyses were re-run. These
exploratory analyses discussed in Appendix B were not re-run utilizing the corrected data set; however, the scope of the data
errors was such that it was unlikely that the analysis choices would have changed.
94

181

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document•••

Analyze the data with treatment groups 100 and 200 deleted for all CCAs. The reasoning behind thispath forward are:o There was consensus that treatments 100 and 200 impacted hive health with no recovery
amongst all evaluators.
o Removing these treatment groups greatly improves the percentage of covariance structuresthat were able to be fitted in PROC MIXED.
o During the study, hives treated with 100 and 200 concentrations of test material were
physically moved away from the initial placements in the field to minimize the potential
impact these hives may have on the other nearby hives. Thus data obtained on these hives
after moving may not be comparable to data obtained before moving.
Analyze the data with CCA8 removed and conduct separate analysis for CCA8. The justificationsare:o The majority of hive deaths occurred between CCA7 and CCA8; therefore, data are betterbalanced from CCA3 to CCA7 and convergence success will be much higher for all
responses.
o Number of days between CCAs was very similar from CCA3 to CCA8; therefore, the AR(1)covariance structure which requires equal spacing between time points could be evaluated.
Analyze the data analyzed after removing data for the few remaining hives in treatment groups 100
and 200 ppb from CCA8. The justifications are:o The vast majority of the data can still be included in the analysis.
o Data on some hives after overwintering can be included in the full analysis.
o Pulled error terms (and standard errors) will utilize the majority of the data set, thusincreasing confidence in the estimates.

After exploring these options, the data were analyzed after removing data from the few remaining hives intreatment groups 100 and 200 ppb from CCA8 (third option above). Data from hives treated with 100 or
200 ppb imidacloprid remain in the data set and analysis for CCA3 through CCA7 permitting utilization ofa majority of the data. These deletions create a 5x6 factorial design matrix with two of the cells (CCA8,
treatment=100) and (CCA8, treatment=200) missing all data. CCA*treatment interaction means can still becalculated. Least square means on the main effects (treatment and CCA) cannot be estimated forConcentration=100, Concentration=200, and CCA8. Since the expectation was that the interaction would
be significant and all further statistical tests would be exploring the interaction, this was not considered asignificant hindrance.

Determining the temporal covariance structure
Before conducting Dunnett’s test, several different correlation structures to best fit the temporal correlation
were evaluated. The structures that were fitted included:• Compound symmetry (CS): assumes equal correlation for all pairwise correlations (regardless of
distance of timepoint).
• Compound symmetry with heterogeneous variance (CSH): Estimates a unique variance at eachtime point, but assumes equal correlation for all pairwise correlations (regardless of distance of time
point).
• Sp(pow)(1): this is a correlation structure that fits an AR(1) but it adjusts for unequal spacing
between time points.

95
182

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
AR(1): autoregressive correlation. Assumes equal correlation between adjacent timepoints. Timepoints further apart have a lesser correlation.
• Heterogeneous Toeplitz TOEPH(1): models a unique variance for each timepoint. Correlation
between timepoints was zero.
• Heterogeneous Toeplitz TOEPH(2): models a unique variance for each timepoint and a common
correlation for adjacent timepoints. Correlation for timepoints not immediately adjacent was
assumed to be zero.
More information about each of the correlation structures can be found here:
http://support.sas.com/documentation/cdl/en/statug/63033/HTML/default/viewer.htm#statug_mixed_sect0
19.htm . The full SAS output is provided in Attachment 4.
•

To compare structure fit, Bayesian Information Criterion (BIC) was utilized. The BIC is similar to the AIC
and both are functions of the log likelihood with a penalty for an increase in the number of covarianceparameters to be fitted. The BIC value for each fitted model for all eight response variables is reported inTable A-1; smaller values of the BIC indicate a better fit (bolded). For many of the endpoints, heterogeneityof variance at different time points was indicated as CSH, ToepH(1), or ToepH(2) were the covariancestructures providing the best fits. This is not surprising as unequal variances were observed in the
exploratory multivariate/principle component analysis. Although the AR(1) model was fit here, it may not
be appropriate as the spacing between measurement times is not consistent (minor deviations as between
CCA3, CCA4, CCA5, CCA6, and CCA7 are acceptable, but CCA8 is clearly not equidistant from CCA7).
For the variables that represent the individual life stages (adults, eggs, open cells, and capped cells),
ToepH(2) is the one covariance structure that provides a good fit for all four matrices of the evaluatedstructures. In addition, the BIC statistics suggests that CSH, AR(1) and ToepH(1) provide adequate fits forat least one of the life stages. For proportion frame coverage of total individuals, ToepH(2) provides thebest fit to the covariance structure of all the evaluated models. For pollen, CSH, ToepH(1) and ToepH(2)provided the best fits to the data. For honey, SP(pow)(1) and AR(1) provided the best fits to the covariancestructure; ToepH(2) did not converge (infinite likelihood). For hive weight, SP(pow)(1), AR(1), andToepH(1) were the best fitting covariance structures. Compound symmetry (CS) was not identified as
quality fit to the data for any of the eight evaluated response variables.
Residual plots were also evaluated for each of the response variables and covariance structures.
Patterns indicative of heterogeneous variance of the residuals were evident for many of the response
variables and models where an assumption of equal variance at each time point was made. It was recognizedthat many of the response variables were proportions, hence the distribution of the response variable andthe residuals may not meet assumptions of normality. Review of the residual plots indicated that estimating
unique variances at each CCA (e.g., CSH, ToepH(1), or ToepH(2) covariance structures) appears to address
the concern of unequal variance.
The varying strength and pattern of correlation seen in the pairwise scatterplots (CCAx vs. CCAy for any
given response variable for x and y equal to 3, 4, 5, 6, 7, and 8), the BIC comparisons, and the residual plot
review indicate that there is not a single covariance structure that is clearly best for all eight response
variables. General conclusions that can be made from the scatter plots and covariance analysis are thatwithin a response variable, variance varies over time and that there is correlation in response variable overtime; however, the pattern of correlation is not particularly strong nor is it consistent. Given these96
183

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
interpretations along with the BIC analysis and residual plots, the CSH covariance was chosen for the mixed
model structure.
Table A-1. BIC values for fitted models. CCA3 - CCA8; concentrations 100 and 200 ppb deleted from
CCA8
Variable →Adults
Eggs
Open
CappedTotal
PollenHoney
Hive
Model ↓
indiv.
weightSP(pow)(1)
-389
-1457
-1268
-735
1.95
-1518
2502
-724
CS
-374
-1459
-1265
-724
33.3
-1518
-679
2588
CSH
-385
-1321
-5.85
-1534
-709
2533
-1549
-816
AR(1)
-1456
-1268@-738@-2.27@-1522-401*@-729
2492@
ToepH(1)
-381
1.99
-711
2547
-1552
-1320
-815
-1532
ToepH(2)Inf. lklhd 2484
-406
-1550
-1319
-824
-35.5
-1535
*Within a response variable, bolded BIC values indicate better covariance model fit. Kass and Raferty (1995) suggested thatdifferences of greater than 10 in BIC values provides very strong evidence that model fits are not equivalent.
@Convergence was attained, but estimated G matrix was not positive definite.

Treatment by time interaction and follow-up contrasts
The text box provides the SAS code for the mixed model that was used for the Dunnett’s comparisons.
Table A-2 provides the results from the Dunnett’s comparisons in which treatment means were tested tosee if significantly less than control at each CCA. For these analyses, the CSH covariance matrix was used
for each of the variables.
title 'concentration 100 and 200 deleted for CCA=8, covariance=csh';proc mixed data=cca3_8 ;title2 "Dunnett's tests - adult";class apiary cca concval replicate;model adult_p = concval|cca /DDFM=SATTERTHWAITE;
random apiary ;repeated cca/ subject=replicate*concval(apiary) type=csh ;lsmeans concval*cca;
slice concval*cca /sliceby=cca diff=controll adjust=dunnett;
slice concval*cca /sliceby=concval diff=control adjust=dunnett;
run;

Treatment effects within a CCA
The table of p-values resulting from the Dunnett’s tests for evaluating whether within a CCA, the treatment
mean are significantly less than control means) are summarized in Table A-2. Figures 1-7 below show theresults for each response variable across all CCAs analyzed (CCA3-CCA8) and all treatment levels. It isnoted as discussed previously that the 100 and 200 ppb treatment groups were excluded from CCA8 due tohigh hive mortality. For all figures presented below, significant reductions from the negative control withp-values below the 0.05 alpha level are denoted by a red dot at a given treatment level and CCA and those
reductions with p-values between 0.05 and 0.1 are denoted by a black dot. Statistical NOAECs and
LOAECs will be determined using an alpha-level of 0.05. Additional comparisons using and alpha-level of
0.10 are included for additional characterization. The tables of p-values resulting from the Dunnett’s test
97
184

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
are summarized in Table A-2. The associated SAS output containing the full results of the Dunnett’s
comparisons can be found in Attachment 5.
Table A-2. Results of one-sided Dunnett’s test (comparing control to each treatment group) with 100 and
200 concentrations deleted from CCA8, correlations modeled using CSH.*
Adults

Eggs

Open

Capped

CCA3
CCA4

NS
NS

NS
100

NS
100
200

NS
100
200

CCA5

50
100

100

100
200

CCA6

50
100
200
100
200

100
200

100
200

200

100
200

50
100
200
12.5
100
200
100
200

CCA7
CCA8

Total
indiv.
NS
100

50
100
200
50
100
200
100
200

Pollen

Honey

Hive weight

NS
50
100
200
50
100
200
200

NS
NS

NS
100
200

NS
50
200

50
100
200
100
200

200

50
100
200
50

50
100
200
50

12.5
NS
50
50
50
50
*Listed concentrations are those that were significantly less than the control following the results of Dunnett’s
test. NS indicates that there were no test concentrations with means significantly less than the control (p>0.10).
Bolded concentration = significantly less than control (p< 0.05)
Italicized concentration = less than control (0.05< P < 0.10)50

Temporal trends within a treatment levelA second component to evaluating the “treatment x CCA” interaction is to look at the temporal changes
within a treatment group. This was accomplished by comparing each CCA (CCA4 through CCA8) to CCA3
by use of a two-sided Dunnett’s test (Table A-3 and Table A-4). This suite of comparisons is not as
informative as the contrasts of control against the treatment group within a CCA for establishing a NOAEC
and LOAEC. However, it may aid in interpretations and further biological understanding of temporal shifts
in the life stages and food components present in the hive. Differences in patterns of temporal shifts between
the control and various treatment groups can provide further understanding of the potential impacts ofimidacloprid on beehive population dynamics.
Table A-3. Results of two-sided Dunnett’s test (comparing CCA3 to each following CCA) with 100 and
200 concentrations deleted from CCA8, correlations modeled using CSH.*
Trt
Group
Control

Adults
CCA5>CCA3

12.5
25

NS
NS

Eggs
CCA7 and CCA8< CCA3NS
CCA7 < CCA3

Response Variable
Open
Capped
CCA7<CCA3
CCA4-6 > CCA3
CCA7 < CCA3NS
CCA7 < CCA3
CCA7<CCA3
CCA7 < CCA3

Total indiv
CCA7 < CCA3
CCA7 < CCA3
CCA7 < CCA3

98
185

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
CCA7 and CCA8
CCA7<CCA3< CCA3
CCA7<CCA3
CCA6 and CCA7
CCA7<CCA3
100*
<CCA3
CCA6 and
CCA6 and CCA7
CCA4-7 < CCA3200*
CCA7<CCA3<CCA3* CCA8 not included for test concentrations 100 and 200
NS – No significant differences from control (p>0.05)
50

NS

CCA7 < CCA3

CCA7-8 < CCA3

CCA5-7 < CCA3

CCA6-7<CCA3

CCA4-7 < CCA3

CCA4-7<CCA3

Table A-4. Results of two-sided Dunnett’s test (comparing CCA3 to each following CCA) with 100 and
200 concentrations deleted from CCA8, correlations modeled using CSH.*
Trt
Group
Control
12.5
25
50

PollenCCA5>CCA3
CCA7 < CCA3
CCA5 and CCA8>CCA3
CCA5>CCA3
CCA4 and CCA7<CCA3

Response Variable
Honey
CCA6-7 > CCA3

CCA6-7 > CCA3
CCA6-7 > CCA3
CCA5 and CCA8< CCA3

CCA4-5<CCA3
NS100*
CCA4-5 and CCA7<CCA3
CCA4-5> CCA3200*
* CCA8 not included for test concentrations 100 and 200
NS – No significant differences from control (p>0.05)

Hive weight
CCA4-7> CCA3
CCA4, 6-7 >CCA3
CCA4-7> CCA3
CCA4>CCA3
CCA8<CCA3
CCA4>CCA3
CCA4>CCA3

Tables A-5 – A-12 tabulate the summary statistics (including mean and standard deviation) of each
response variable for all treatment levels across CCAs 3-8.

99
186

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Table A-5. Summary statistics for adults
TreatmentGroup µg/L
0Parameter

CCA3

CCA4

CCA5

CCA6

CCA7

CCA8

N

21

22

22

21

20

14

0

MIN

0.157513

0.127504

0.077504

0.155009

0.130009

0.047496

0MAX

0.489982

0.632513

0.742487

0.875

0.532513

0.605009

0MEAN

0.328452

0.340230.451138

0.394288

0.297250.364110STD

0.0992380.150640.170406

0.170447

0.117737

0.169285

12

N

11

11

11

11

11

9

12

MIN

0.15

0.130009

0.212522

0.194991

0.130009

0.180009

12

MAX

0.727504

0.680009

0.687522

0.555009

0.485018

0.577504

12MEAN

0.374093

0.36932

0.484327

0.375224

0.342958

0.369728

12STD

0.177567

0.183432

0.157566

0.124439

0.09466

0.13788

25

N

11

11

11

11

11

10

25

MIN

0.155009

0.219991

0.175

0.139982

0.117487

0.077504

25

MAX

0.705009

0.614982

0.672496

0.597496

0.6

0.632513

25

MEAN

0.379094

0.357501

0.422731

0.361364

0.328639

0.346498

25

STD

0.16288

0.141233

0.1731790.169438

0.137110.191584

50

N

11

11

11

10

9

7

50

MIN

0.175

0.175

0.155009

0.147496

0.172496

0.039982

50

MAX

0.535018

0.707513

0.562522

0.407513

0.432513

0.305501

50

MEAN

0.302053

0.396361

0.334323

0.264244

0.255282

0.184718

50

STD

0.098915

0.174624

0.148201

0.086622

0.077175

0.10027

100

N

11

11

11

10

10

1

100

MIN

0.155009

0.117487

0.027504

0.037522

0.042487

0.010018

100

MAX

0.560018

0.664982

0.647496

0.597496

0.397496

0.010018

100

MEAN

0.301374

0.312047

0.281591

0.253001

0.144253

0.010018

100

STD

0.132728

0.157151

0.211170.21868

0.137779

200

N

11

11

11

11

10

2

200

MIN

0.214982

0.202504

0.225

0.112522

0.037522

0.035018

200

MAX

0.589982

0.787522

0.632513

0.302504

0.260018

0.077504

200

MEAN

0.378175

0.479541

0.351374

0.172508

0.143256

0.056261

200

STD

0.120328

0.180595

0.133209

0.059981

0.069188

0.030043

CCA4

CCA5

CCA6

CCA7

CCA8

Table A-6. Summary statistics for eggs
TreatmentGroup µg/L
0Parameter

CCA3

N

21

22

22

21

20

14

0

MIN

0

0.025

0.00750.03

00.0175

0

MAX

0.165

0.15250.12

0.12250.0675

0.08

0

MEAN

0.083929

0.069205

0.070909

0.073690.025

0.04875

0STD

0.052331

0.026574

0.028301

0.027552

0.015749

0.019728

100
187

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
12

N

11

11

11

11

11

9

12

MIN

0

0

0

0.03

0.0125

0

12

MAX

0.15

0.08750.1125

0.175

0.05

0.092512MEAN

0.058636

0.055909

0.065227

0.06750.02750.031389

12

STD

0.041418

0.026369

0.0304850.039655

0.011990.027475

25

N

11

11

11

11

11

10

25

MIN

0

0

0

0

0

0.0275

25

MAX

0.105

0.12

0.11750.1175

0.035

0.1

25

MEAN

0.06750.071364

0.065227

0.055909

0.017727

0.04575

25

STD

0.034605

0.030951

0.034414

0.034229

0.010214

0.021572

50

N

11

11

11

10

9

7

50

MIN

0

0.01250.01250.03750.01250.007550

MAX0.14250.14750.14

0.11250.04750.037550

MEAN

0.080227

0.077045

0.069318

0.068

0.024444

0.017857

50

STD

0.039851

0.037213

0.038342

0.023682

0.01191

0.011586

100

N

11

11

11

10

10

1

100

MIN

0

0

0

0

0

0.0025

100

MAX

0.15

0.105

0.11250.06750.04250.0025100

MEAN

0.066818

0.043864

0.044091

0.02550.01625

0.0025100

STD

0.053445

0.039376

0.046088

0.026557

0.014589

200

N

11

11

11

11

10

2

200

MIN

0

0

0

0

0

0.015

200

MAX

0.12250.1175

0.1

0.065

0.015

0.0175200

MEAN

0.071136

0.060682

0.045682

0.029545

0.007

0.01625200

STD

0.036372

0.037167

0.035019

0.021961

0.006433

0.001768

Table A-7. Summary statistics for larval (open) cells
TreatmentGroup µg/L
0Parameter

CCA3

CCA4

CCA5

CCA6

CCA7

CCA8

N

21

22

22

21

20

14

0

MIN

0

0.015

0

0.0425

0

0

0

MAX

0.215

0.185

0.17250.18

0.06750.14250MEAN0.101905

0.110795

0.100682

0.099048

0.034250.083036

0STD

0.055778

0.037736

0.044207

0.033001

0.017698

0.042508

12

N

11

11

11

11

11

9

12

MIN

0

0.005

0

0.04250.0275

0

12

MAX

0.15

0.15250.14250.2275

0.085

0.15

12

MEAN

0.087045

0.094773

0.09

0.113864

0.053636

0.078333

12STD

0.05073

0.041132

0.040481

0.055389

0.017189

0.049418

25

N

11

11

11

11

11

10

25

MIN

0

0.075

0

0

0

0.012525

MAX

0.17750.16

0.15750.11250.10750.1475101
188

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
25

MEAN

0.11

0.102955

0.084318

0.072727

0.039773

0.07925

25

STD

0.049422

0.025637

0.047593

0.036408

0.027075

0.04026

50

N

11

11

11

10

9

7

50

MIN

0

0.022500.0575

0.02

0.01

50

MAX

0.20750.155

0.17750.14750.0725

0.125

50

MEAN

0.1

0.096591

0.085455

0.09550.041667

0.058929

50

STD

0.054118

0.040316

0.058372

0.029411

0.018028

0.042127

100

N

11

11

11

10

10

1

100

MIN

0

0

0

0

0

0.0025

100

MAX

0.12

0.1325

0.15

0.09

0.045

0.00250.0025100

MEAN

0.071818

0.056591

0.063409

0.04450.01425100

STD

0.041399

0.049816

0.063946

0.03704

0.014484

200

N

11

11

11

11

10

2

200

MIN

0.0425

0

0

0

0

0.005

200

MAX

0.13750.1125

0.15

0.085

0.02250.0275200

MEAN

0.099318

0.039545

0.034318

0.039091

0.007

0.01625200

STD

0.03406

0.042849

0.043288

0.030522

0.00840.01591

Table A-8. Summary statistics for pupal (capped) cells
TreatmentGroup µg/L
0Parameter

CCA3

CCA4

CCA5

CCA6

CCA7

CCA8

N

21

22

22

21

20

14

0

MIN

0

0

0

0.19

0.04

0

0

MAX

0.345

0.36

0.40750.36750.1525

0.31

0

MEAN

0.163810.228523

0.259318

0.260595

0.080875

0.17625

0STD

0.123803

0.093985

0.119229

0.053003

0.031843

0.093627

12

N

11

11

11

11

11

9

12

MIN

0.0625

0.195

0.13

0

0.0325

0

12

MAX

0.285

0.30250.3575

0.315

0.16

0.287512MEAN

0.224545

0.246818

0.257045

0.197955

0.084773

0.178611

12STD

0.068628

0.037518

0.062897

0.104228

0.038705

0.085961

25

N

11

11

11

11

11

10

25

MIN

0.18

0.04

0

0

0.01750.032525

MAX

0.33750.32750.35

0.32750.115

0.272525

MEAN

0.233409

0.214091

0.235227

0.208409

0.065

0.165525

STD

0.042519

0.086373

0.120931

0.084656

0.034893

0.085965

50

N

11

11

11

10

9

7

50

MIN

0

0

0

0.145

0.05

0.02

50

MAX

0.33250.34750.325

0.29750.115

0.162550MEAN

0.189773

0.170909

0.162955

0.21850.077222

0.092143

50

STD

0.108975

0.114511

0.112704

0.051763

0.024253

0.058531

100

N

11

11

11

10

10

1

102
189

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
100

MIN

0

0

0

0

0

0.005

100

MAX

0.355

0.24250.31750.255

0.0825

0.005

100

MEAN

0.211136

0.128864

0.105

0.1215

0.025

0.005

100

STD

0.097477

0.082016

0.128826

0.110001

0.024438

200

N

11

11

11

11

10

2

200

MIN

0.1425

0

0

0

0

0.01

200

MAX

0.33

0.3025

0.195

0.165

0.03

0.045

200

MEAN

0.250682

0.065227

0.041136

0.057955

0.01150.0275200

STD

0.053934

0.095796

0.059091

0.062077

0.012315

0.024749

Table A-9. Summary statistics for total individuals
TreatmentGroup µg/L
0Parameter

CCA3

CCA4

CCA5

CCA6

CCA7

CCA8

N

21

22

22

21

20

14

0

MIN

0.242513

0.210013

0.102504

0.515009

0.212509

0.064996

0MAX

1.012491

1.142513

1.242487

1.47750.664996

1.107509

0MEAN

0.678095

0.748753

0.882047

0.827622

0.437375

0.672146

0STD

0.237041

0.254076

0.312739

0.233682

0.147805

0.30578

12

N

11

11

11

11

11

9

12

MIN

0.21250.450004

0.617522

0.51750.205009

0.180009

12

MAX

1.267504

1.132504

1.160013

0.972487

0.714991

1.067496

12MEAN0.744321

0.76682

0.896599

0.754542

0.508867

0.658061

12

STD

0.264013

0.236201

0.212789

0.132926

0.140911

0.277451

25

N

11

11

11

11

11

10

25

MIN

0.477509

0.479991

0.175

0.15

0.149987

0.152504

25

MAX

1.112509

1.087482

1.184996

1.049996

0.735

1.107513

25

MEAN

0.790003

0.745910.807504

0.698409

0.451139

0.636998

25

STD

0.197306

0.219977

0.332487

0.246998

0.151117

0.325348

50

N

11

11

11

10

9

7

50

MIN

0.47

0.45

0.219996

0.462491

0.270004

0.092482

50

MAX

1.062518

1.150013

1.080022

0.787496

0.580013

0.575018

50

MEAN

0.672053

0.740907

0.65205

0.646244

0.398616

0.353646

50

STD

0.187031

0.2611040.317216

0.122113

0.094003

0.204817

100

N

11

11

11

10

10

1

100

MIN

0.347509

0.124991

0.027504

0.064982

0.059996

0.020018

100

MAX

1.152518

0.860018

1.199996

0.95250.492496

0.020018

100

MEAN

0.651147

0.541365

0.494091

0.444501

0.199753

0.020018

100

STD

0.235144

0.251096

0.4312120.370248

0.179209

200

N

11

11

11

11

10

2

200

MIN

0.519996

0.212504

0.3

0.117487

0.050022

0.065018

200

MAX

1.042522

1.262522

0.952513

0.610004

0.260018

0.167504

200

MEAN

0.799312

0.644996

0.472511

0.299099

0.168756

0.116261

103
190

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
200

STD

0.175532

0.291210.196226

0.155814

0.070029

0.072469

Table A-10. Summary statistics for nectar (honey) cells
TreatmentGroup µg/L
0Parameter

CCA3

CCA4

CCA5

CCA6

CCA7

CCA8

N

21

22

22

21

20

14

0

MIN

0.03750.06250.00750.09750.0325

0.055

0

MAX

0.305

0.345

0.25

0.48750.53250.29250MEAN

0.156667

0.202045

0.125341

0.295476

0.293625

0.168214

0STD

0.069211

0.07982

0.059833

0.12647

0.156801

0.086937

12

N

11

11

11

11

11

9

12

MIN

0.06250.06

0.0125

0.115

0.105

0.032512

MAX

0.4275

0.415

0.285

0.44750.55

0.337512MEAN

0.160909

0.211591

0.121818

0.259773

0.270227

0.168056

12

STD

0.098503

0.110478

0.098190.121431

0.135906

0.080833

25

N

11

11

11

11

11

10

25

MIN

0.01250.012500.1075

0.075

0.065

25

MAX

0.27250.35750.2675

0.55

0.49

0.29

25

MEAN

0.152045

0.213636

0.137045

0.311591

0.288864

0.139

25

STD

0.091240.120781

0.093252

0.138912

0.153913

0.067063

50

N

11

11

11

10

9

7

50

MIN

0.08

0.1

0.02

0.0475

0.06

0

50

MAX

0.2775

0.405

0.195

0.32750.445

0.232550

MEAN

0.168636

0.213409

0.101136

0.18975

0.193889

0.085

50

STD

0.066533

0.08095

0.05968

0.099467

0.122226

0.072414

100

N

11

11

11

10

10

1

100

MIN

0.055

0.085

0.01750.03

0.01750.0325100

MAX0.27750.32250.3

0.46750.355

0.0325100

MEAN

0.173864

0.219773

0.125682

0.22725

0.13975

0.0325100

STD

0.080602

0.084915

0.085089

0.162235

0.112023

200

N

11

11

11

11

10

2

200

MIN

0.035

0.09750.055

0.01750.0025

0.08

200

MAX

0.25

0.33

0.3575

0.17

0.16

0.09

200

MEAN

0.135227

0.22250.207273

0.092727

0.07375

0.085

200

STD

0.06755

0.076722

0.10878

0.053074

0.0482650.007071

104
191

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Table A-11. Summary statistics for pollen cells
TreatmentGroup µg/L
0Parameter

CCA3

CCA4

CCA5

CCA6

CCA7

CCA8

N

21

22

22

21

20

14

0

MIN

0.017500.00250.02

0.00250.0225

0

MAX

0.18

0.1375

0.205

0.1

0.09

0.12

0

MEAN

0.069286

0.066136

0.103182

0.05250.043125

0.076607

0STD

0.043805

0.031156

0.048936

0.022389

0.02484

0.027185

12

N

11

11

11

11

11

9

12

MIN

0.0225

0.005

0.05

0.02250.0275

0.03

12

MAX

0.105

0.10250.205

0.14750.1125

0.13

12

MEAN

0.054091

0.054773

0.104318

0.075455

0.051136

0.0875

12STD

0.024425

0.035151

0.045772

0.039351

0.024555

0.032089

25

N

11

11

11

11

11

10

25

MIN

0.01

0.03250.02750.00750.0125

0.035

25

MAX

0.115

0.095

0.17

0.13250.10250.147525

MEAN

0.045682

0.055455

0.086591

0.059545

0.047273

0.075525

STD

0.026671

0.023044

0.053083

0.039605

0.02425

0.032783

50

N

11

11

11

10

9

7

50

MIN

0.0125000.00750.0025

0.015

50

MAX

0.16

0.06250.11

0.09

0.0825

0.08

50

MEAN

0.067727

0.02750.037045

0.04225

0.03250.039643

50

STD

0.040488

0.025471

0.035686

0.026574

0.026071

0.026826

100

N

11

11

11

10

10

1

100

MIN

0.0275000.0175

0.01

0.015

100

MAX

0.125

0.07

0.07750.07250.0925

0.015

100

MEAN

0.062727

0.008636

0.013409

0.04350.0395

0.015

100

STD

0.024886

0.021429

0.023218

0.018379

0.024743

200

N

11

11

11

11

10

2

200

MIN

0.025

0

0

0

0

0.0125

200

MAX

0.085

0

0.01250.08

0.0875

0.015

200

MEAN

0.04681800.001136

0.020227

0.01650.01375200

STD

0.019464

00.003769

0.027916

0.026358

0.001768

105
192

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Table A-11. Summary statistics for hive weight
TreatmentGroup µg/L
0Parameter

CCA3

CCA4

CCA5

CCA6

CCA7

CCA8

N

21

22

22

21

21

14

0

MIN

32

49

31

30

31.5

25.5

0

MAX

50

66

56.5

68

66.5

53.5

0

MEAN

40.19524

58.06818

47.43182

52.59524

52.14286

41.71429

0STD

4.284913

5.005462

5.910635

10.71286

10.99675

7.392081

12

N

11

11

11

11

11

9

12

MIN

33.5

50

37

39

41

27.5

12

MAX

49

71.5

56.5

68

74

49

12

MEAN

42.13636

58.5

45.86364

52.27273

53

40.11111

12STD

5.822761

6.492303

5.263511

8.866689

9.721111

6.436118

25

N

11

11

11

11

11

10

25

MIN

33.5

48.5

42

37.5

41

34

25

MAX

51

64

54.5

69.5

64.5

48.5

25

MEAN

42.36364

57.31818

47.59091

54.09091

53.18182

42.1

25

STD

5.186959

5.891828

4.597924

9.194613

9.453234

4.629615

50

N

11

11

11

10

10

7

50

MIN

33.5

47.5

36

30

22.5

30

50

MAX

55.5

65

47

55.5

57.5

43

50

MEAN

42.95455

55.31818

41.54545

45.85

44

35

50

STD

6.254816

6.108489

4.1439998.131045

9.436925

4.481443

100

N

11

11

11

10

10

1

100

MIN

35.5

47

32.5

33

32.5

26.5

100

MAX

50

65

50.5

67

62.5

26.5

100

MEAN

39.90909

53.31818

40

44.05

42.15

26.5

100

STD

4.559705

6.569904

6.160357

11.48296

10.78592

200

N

11

11

11

11

11

2

200

MIN

35.5

47

38

34.5

33.5

32

200

MAX

52

60

49.5

43.5

42

32

200

MEAN

41.90909

52.63636

42.54545

38.18182

36.5

32

200

STD

5.337688

4.080998

3.173756

3.356134

3.138471

0

106
193

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

2Appendix B: Details of PMRA Statistical Analysis
During the review of the study, a separate statistical analysis was conducted with the program R (version
3.1.2 ) 5using the raw data submitted by the study author.

Statistical analysis
Analysis Strategy
Hive condition data:
To analyze colony condition data which contains many components over many assessments at different
times, a primary analysis was set out to effectively prevent multiplicities from interfering with theinterpretation of p_values and confidence intervals. These multiplicities arise from having multiple doselevels, multiple outcomes and multiple time points, and are dealt with as follows:••

The multiplicities from having multiple dose levels was dealt with by using step down testing, thehighest dose group’s data was compared directly to the control group’s data, if statisticallysignificant at a chosen alpha level the next lowest dose group’s data was compared to the control
group’s data and this was continued down to the dose where statistical significance was no longer
achieved. A technical reference for this step down testing would be Multiple Comparison
Procedures in Dose Response Studies. Tamhane, Ajit C. and Logan, Brent R., in Dose Finding in
Drug Development edited by Ting, Naitee. Springer New York 2006. This step down procedure(referred to as the SD2PC procedure in the technical reference) was chosen as it provides good powerfor detecting the minimum effective dose (lowest does where effect is present) when monotonic dose
effects are expected while providing stringent control of type one error, regardless of the true patternof dose effects. That is, with minimal assumptions, the procedure strongly controls family wise typeone error rate while maintaining good power for effect patterns that are expected.
The applicant’s choice of multiplicity adjustment procedure, which was William’s trend test
(Williams 1972), presumably chosen to be in accord with OECD. 2003. Draft guidance document
for the statistical analysis of ecotoxicity data. They are both step down procedures but ours differsfrom William’s in that it uses only within dose group data based estimates of means rather thanmaximum likelihood estimates of dose group means using all group’s data simultaneously - undermonotonicity assumption (i.e. order restricted or isotonic means) additionally assuming
homogeneous variances . Although these additional assumptions may not be problematic and arewithin the OECD guidelines, we simply chose not to rely on them (and by doing so, exceed theOECD guidelines.)
The multiplicities from having multiple outcomes, was dealt with by choosing to focus on theassessment of total life in the hive – simply the number of viable life forms at any stage in the hive.
It is considered that the total number of individuals includes all live individuals in hives and is
expected to be a better indicator of the hive status at the colony level than any single stage of bees
alone. This outcome would provide good power when background knowledge is lacking on thestage most likely to be affected (i.e. it cannot be well anticipated) and it is not expected that there

R Core Team (2014). R: A language and environment for statistical computing. R Foundation for StatisticalComputing, Vienna, Austria. URL http://www.R-project.org/.

5

107
194

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document•

will be simultaneous trade-offs effects between the stages. That is, when it is not expected that atoxic effect on one stage would have a beneficial effect for another stage at the same point in time.
The multiplicities from having multiple time points was dealt with by choosing to focus on the time
when the effects were believed most pronounced both in terms of having an impact on total life andhaving a high powered assessment of that. In this case CCA6 was selected for the following reasons.
o CCA6 (five weeks after the end of feeding exposure) was selected as it maximises the time
period for detecting a potential latent effect from exposure and occurs before the start of hive
decline prior to overwintering.
o CCA7 was not chosen simply due to the natural decline of hive size in the late fall that may
mask the effect of treatment.
o CCA8 was not selected because of the higher hive mortality observed in the controls incomparison to lower dose groups, and because data was available only for surviving hives.
The hive mortality in the control was higher than the two lowest test concentrations (12.5
and 25 ug/l) and equal to the 50 ug/l treatment. The uncertainty regarding the cause of deadhives, (i.e. whether mortality resulted from the treatment effects or random background effector both), is considered to reduce the confidence of using CCA8 data as the primary time
point. Additionally, no hive condition data were available on hives that died during theoverwintering, meaning only the condition of hives that survived overwintering(informatively selected on ability to survive) were able to be compared. The lack of data on
dead hives poses difficulties in the statistical analysis for hive condition at CCA8.

While the total individuals at CCA6 is considered as a primary parameter to control multiplicity forstatistical analysis, all parameters including eggs, open brood and capped brood, adults, hive weight, pollen
and nectar store, that were observed during the entire study including CCA4, CCA5, CCA6, CCA7 andCCA8 were also considered in the review. Given that the primary analysis has prevented multiplicities frominterfering with the interpretation of p_values and confidence intervals, if statistical significance has beenachieved (at given dose levels), further analysis with all other outcomes is undertaken “with prejudice” forthe assessment of similar effects as being significant. More formally, re-allowance for multiplicities is not
required and less stringent alpha levels are allowed. Essentially the price has been paid for searching for thepattern in the primary analysis (measures taken to prevent multiplicities) and it need not be re-paid
evaluating the same pattern elsewhere. On the other hand, if statistical significance has not been achieved(at given dose levels), further analysis with all other outcomes is undertaken “with prejudice” for assessmentof other effects as likely being just noise. Here though dramatic effects should not be ignored but carefully
considered and noted.
With the primary focus on CCA6 to discern treatment effects, the later assessment of recovery from detected
effects at CCA7 and CCA8 was subsequently addressed. Here the use of confidence intervals was chosen
to provide assurance that important underlying differences at the later period had been reliably ruled out –that is the upper confidence limit did not include worrisome differences. Given the arguable need to makeimportant bias adjustment for confidence intervals in CCA8 but little to no background information toaccurately make these bias adjustments, the assessment of recovery was limited to CCA7. There was noformal analysis of whether and when treatment effects detected at CCA6 were present before CCA6. The
consideration of recovery at CCA7, while considered, was not formally presented because of concern
regarding relying on parameters at CCA7 which are all decreasing as colonies prepare for overwintering.
Hive mortality data:

108
195

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
The analysis of Mortality at CCA8 had been anticipated as the primary or key assessment for this study
design and the only multiplicities to be dealt with there were from the multiple dose levels (for which thestep down method was used). Unfortunately, the observed hive mortality in the control was higher than the
two lowest test concentrations (12.5 and 25 ug/l) and equal to the 50 ug/l treatment which is at odds with
underlying biological understanding and thus greatly reduced the confidence of using CCA8 mortality as
the primary assessment.
Analysis methods for hive conditions
For all hive conditions total life, eggs, open brood and capped brood, adults, hive weight, pollen and nectar
store at CCA4, CCA5, CCA6, CCA7 and CCA8 a conventional analysis of block randomised experimentswith a baseline measurements was undertaken. In line with the statistical strategy discussed above, the focus
was on total life at CCA6 (with step down adjustment for multiplicities applied) but identical analysis was
carried out (less the step down adjustment) on all other hive conditions assessed at the given assessment
points (with CCA8 considered biased and problematic). This analysis comprised of linear modeling (orANOVA) stratified on Apiary (block) and adjusted for baseline measurements at CCA3 with one-side
testing for harm using only the control group data and the data from a single dose group at a time, startingwith the highest and then through lowest dose groups. It is a series of robust “t.test like” analyses thatconservatively implement the step down testing procedure. Under the assumption of no effect in the single
dose group being tested (relevant to type one error control), the means and variances and covariate effects
should be identical in both the control group and the single dose group being tested. (In an analysis that
includes all dose group data together e.g. William’s procedure, an impact of a treatment effect on thevariance and covariate effects at a higher dose, in addition to an effect on the mean, would invalidate theassumptions needed to control type one error rate in the lower doses.) The results of these analyses arepresented in tables of unadjusted p_values (adjusted p_values can be simply read off as the maximum of all
p_values in higher doses), effect estimates and upper and lower confidence intervals (currently labelled as
Table A-2) as well as plots of the confidence intervals (pdf file Bees8.pdf).
The code snippet to implement these analyses in R was:glm(outcome~Apiary + baseline + exposed, data= x[x$exposed == " control " | x$exposed == dose,])Sensitivity analysis was undertaken on the primary analysis (total life) by taking logs. The resulting p_values
were 0.089, 0.043, 0.042, 0.000 and 0.000 for dose of 13, 25, 50, 100 and 200 the largest difference for theoriginal scale being .013.
Analysis methods for hive mortality
The analysis of hive mortality at CCA8, like all other outcomes was also blocked by Apiary, but unlike
other outcomes, it was not adjusted for a baseline measurement at CCA3. Additionally, given the sparsityof the outcome when blocked, common methods of analysis for binary data like Cochran–Armitage test fortrend, Pearson's chi-square test or logistic regression should not be used as they are well known to be biased.
Instead, conditional logistic regression or exact tests are required. The code snippet to implement conditional
logistic regression in R was:clogit(Mortality ~ Conc + strata(Apiary), data=filter(mm,Conc =="CON" | Conc== dose)).

109
196

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
As with other outcomes, step down testing from highest dose, and then in turn lower doses was used tocontrol for multiplicity.
Transcript/program of analyses carried out
The file RunJune25.2015 contains the transcript of the final run of the R program used to carry out theanalysis and generate the tables and plots.
Supporting graphs
The following graphs were produced as part of the analysis.
Bees1.pdf – Plots of individual hive condition assessments over-time group by Apiary.
Bees2.pdf – Plots of control versus exposed condition assessments over-time group by Apiary.
Bees3a.pdf – Plots of mean and Apiary mean control condition assessments over time.
Bees5.pdf – Plots of model estimated differences and confidence intervals (title revised).
Bees6.pdf – Plots of model estimated difference versus observed mean for total life.
Bees7.pdf – Plots of individual exposed hive versus control condition assessments for all parameters.
Bees8.pdf – Plots of effect estimates and confidence intervals for all parameters.

110
197

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Table B-1: Summary of the differences between treatment and controls on the basis of observations and model estimations, and P values
Standard
error
observed
mean

n

Model
estimatemeandifference
fromcontrol4

Time
(CCA)

Test
conc.
(µg/l)2

Observedmeandifference
fromcontrol3

TotalLife

3

200

-5284

3194

11

TotalLife

3

100

1344

3438

TotalLife

3

50

-63

TotalLife

3

25

TotalLife

3

TotalLife
TotalLife

Standard
error ofestimatedmean

P value forcomparison
with thecontrol

Estimated
reduction
fromcontrol (%)5

Estimated
reductionfrom control(number)

Obeserved means
in control

Ttest
confidence
limit

0

32147

-1.729

11

0

32147

-1.729

4496

11

0

32147

-1.729

-4600

3028

11

0

32147

-1.729

13

-1690

5092

11

0

32147

-1.729

4

200

13608

3399

11

17344.8

3970.078

0

0.678

0.293

0.486

24210

35714

-1.729

4

100

12959

3492

11

12608

3851.589

0.002

0.54

0.167

0.353

19268

35714

-1.729TotalLife

4

50

3147

3246

11

3667.244

3939.317

0.182

0.293

-0.088

0.103

10479

35714

-1.729TotalLife

4

25

998

2684

11

3863.666

3887.011

0.166

0.296

-0.08

0.108

10585

35714

-1.729TotalLife

4

13

108

2566

11

1412.154

3701.529

0.354

0.219

-0.14

0.04

7813

35714

-1.729TotalLife

5

200

23543

3240

11

24026.84838.476

0

0.815

0.394

0.605

32393

39734

-1.729TotalLife

5

100

18818

4838

11

18258.98

5313.62

0.001

0.691

0.228

0.46

27447

39734

-1.729TotalLife

5

50

10399

6533

11

10080.39

6256.939

0.062

0.526

-0.019

0.254

20899

39734

-1.729TotalLife

5

25

3809

6432

11

4333.576

6380.06

0.253

0.387

-0.169

0.109

15366

39734

-1.729TotalLife

5

13

540

3894

11

385.6814958.292

0.469

0.225

-0.206

0.01

8959

39734

-1.729TotalLife

6

200

25992

1870

11

25425.35

2545.607

0

0.774

0.545

0.659

29840

38559

-1.734

TotalLife

6

100

19875

4884

10

18601.92

3977.831

0

0.662

0.303

0.482

25522

38559

-1.74

TotalLife

6

50

6104

2836

10

4674.351

2657.457

0.048

0.241

0.001

0.121

9297

38559

-1.74

TotalLife

6

25

7290

3261

11

6715.559

3326.913

0.029

0.324

0.025

0.174

12485

38559

-1.734

TotalLife

6

13

4072

1865

11

3357.782424.369

0.091

0.196

-0.022

0.087

7562

38559

-1.734

TotalLife

7

200

11178

1781

10

11783.5

1864.738

0

0.952

0.54

0.746

15039

15794

-1.746

TotalLife

7

100

9015

2385

10

9612.191

2061.392

0

0.836

0.381

0.609

13211

15794

-1.746

TotalLife

7

50

1212

779

9

1275.11411.714

0.19

0.237

-0.076

0.081

3750

15794

-1.753

TotalLife

7

25

18

2073

11

353.425

1937.944

0.429

0.236

-0.191

0.022

3725

15794

-1.74

Parameter

190%
confidence
upper limit

90%
confidence
lower limit

111
198

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

n

Model
estimatemeandifference
fromcontrol4

Standard
error ofestimatedmean

P value forcomparison
with thecontrol90%
confidence
upper limit

90%
confidence
lower limit

Estimated
reduction
fromcontrol (%)511-2666.66

1985.142

0.902

0.05

-0.387-0.169

Time
(CCA)

Test
conc.
(µg/l)2

Observedmeandifference
fromcontrol3

TotalLife

7

13

-3289

TotalLife

8

200

14495

NA

1

15094.09

19208.88

0.238

2.009

-0.927

TotalLife

8

100

42178

NA

1

40478.37

16670.41

0.036

2.725

TotalLife

8

50

15852

5456

7

13690.55

6342.992

0.028

TotalLife

8

25

-2246

6876

8

-3173.88

7643.389

TotalLife

8

13

936

2165

8

-724.634

4642.539

Eggs

3

200

1134

976

11

0

Eggs

3

100

1431

1163

11

Eggs

3

50

508

1151

Eggs

3

25

1384

Eggs

3

13

Eggs

4

Eggs

Standard
error
observedmean
2471

Estimated
reductionfrom control(number)

Obeserved means
in control

Ttest
confidence
limit

787

15794

-1.74

0.541

56044

27899

-2.132

0.177

1.451

76017

27899

-2.132

0.903

0.079

0.491

25187

27899

-1.812

0.657

0.378

-0.606-0.114

10553

27899

-1.796

0.561

0.273

-0.325-0.026

7613

27899

-1.7966028-1.72906028-1.729

11

0

6028

-1.729

1043

11

0

6028

-1.729

1994

1280

11

0

6028

-1.729

200

586

907

11

691.939847.57

0.212

0.453

-0.162

0.145

2157

4761

-1.729

4

100

1743

628

11

1805.239

706.582

0.01

0.636

0.123

0.379

3027

4761

-1.729

Eggs

4

50

-539

1014

11

-457.571

898.501

0.692

0.23

-0.422-0.0961096

4761-1.729

Eggs

4

25

-149

737

11

-54.052

763.511

0.528

0.266

-0.289-0.0111266

4761-1.729

Eggs

4

13

915

573

11

1087.188

680.722

0.063

0.476

-0.019

0.228

2264

4761

-1.729

Eggs

5

200

1736

1002

11

1571.261

864.911

0.043

0.629

0.016

0.322

3067

4879

-1.729

Eggs

5

100

1845

1193

11

1920.253

1006.424

0.036

0.75

0.037

0.394

3660

4879

-1.729

Eggs

5

50

109

794

11

88.23

736.676

0.453

0.279

-0.243

0.018

1362

4879

-1.729

Eggs

5

25

391

1094

11

405.731941.939

0.336

0.417

-0.251

0.083

2034

4879

-1.729

Eggs

5

13

391

840

11

578.592788.155

0.236

0.398

-0.161

0.119

1941

4879

-1.729

Eggs

6

200

2916

744

11

2973.088

707.187

0

0.849

0.353

0.601

4199

4949

-1.734

Eggs

6

100

3449

721

10

3505.81669.65

0

0.944

0.473

0.708

4671

4949

-1.74

Eggs

6

50

396

497

10

578.756

531.111

0.146

0.304

-0.07

0.117

1503

4949

-1.74

Eggs

6

25

1102

860

11

1302.118787.695

0.058

0.539

-0.013

0.263

2668

4949

-1.734

Eggs

6

13

305

1045

11

291.007940.826

0.38

0.388

-0.271

0.059

1922

4949

-1.734

Parameter

1

112
199

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

n

Model
estimatemeandifference
fromcontrol4

Standard
error ofestimatedmean

P value forcomparison
with thecontrol90%
confidence
upper limit

90%
confidence
lower limit

Estimated
reduction
fromcontrol (%)5

Time
(CCA)

Test
conc.
(µg/l)2

Observedmeandifference
fromcontrol3

Eggs

7

200

1170

322

10

1259.78371.642

0.002

1.179

0.378

0.778

Eggs

7

100

662

405

10

754.486410.884

0.042

0.909

0.023

Eggs

7

50

-38

242

9

41.97

335.082

0.451

0.389

Eggs

7

25

399

376

11

506.208404.398

0.114

Eggs

7

13

-274

437

11

2.106

429.799

Eggs

8

200

3956

NA

1

4993.274

Eggs

8

100

3612

NA

1

4496.733

Eggs

8

50

2027

387

7

Eggs

8

25

-54

428

Eggs
Open
brood
Open
brood
Open
brood
Open
brood
Open
brood
Open
brood
Open
brood
Open
brood
Open
brood
Open
brood
Open
brood

8

13

1032

3

200

3

Parameter

Standard
error
observedmean

Estimated
reductionfrom control(number)

Obeserved means
in control

Ttest
confidence
limit

1909

1618

-1.746

0.466

1472

1618

-1.746-0.337

0.026

629

1618

-1.7530.747-0.122

0.313

1210

1618

-1.74

0.498

0.463

-0.461

0.001

750

1618

-1.74

916.963

0.003

2.132

0.932

1.532

6948

3258

-2.132793.551

0.002

1.899

0.861

1.38

6188

3258

-2.1322548.007

434.937

0

1.024

0.54

0.782

3336

3258

-1.812

8

189.139542.807

0.367

0.357

-0.241

0.058

1164

3258

-1.796723

81227.919

719.885

0.058

0.774

-0.02

0.377

2521

3258

-1.796

235

1230

11

0

7068

-1.729

100

2127

990

11

0

7068

-1.729

3

50

188

1511

11

0

7068

-1.729

3

25

-500

1105

11

0

7068

-1.729

3

13

1079

1612

11

0

7068

-1.729

4

200

4902

1036

11

4876.767

1033.897

0

0.874

0.405

0.64

6665

7623

-1.729

4

100

3729

1093

11

2825.234

1049.719

0.007

0.609

0.133

0.371

4640

7623

-1.729

4

50

977

1014

11

918.7811063.42

0.199

0.362

-0.121

0.121

2758

7623

-1.729

4

25

539

663

11

618.534

861.531

0.241

0.277

-0.114

0.081

2108

7623

-1.729

4

13

1102

717

11

935.28920.463

0.161

0.331

-0.086

0.123

2527

7623

-1.729

5

200

4566

1188

11

4505.546

1123.44

0

0.931

0.37

0.65

6448

6927

-1.7291

113
200

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

Parameter

Open
brood
Open
brood
Open
brood
Open
brood
Open
brood
Open
brood
Open
brood
Open
brood
Open
brood
Open
brood
Open
brood
Open
brood
Open
brood
Open
brood
Open
brood
Open
brood
Open
brood
Open
brood

n

Model
estimatemeandifference
fromcontrol4

Standard
error ofestimatedmean

P value forcomparison
with thecontrol90%
confidence
upper limit

90%
confidence
lower limit

Estimated
reduction
fromcontrol (%)5

Time
(CCA)

Test
conc.
(µg/l)2

Observedmeandifference
fromcontrol3

5

100

2564

931

11

2137.769

1035.374

0.026

0.567

0.05

0.309

5

50

1048

1563

11

1060.273

1359.971

0.223

0.493

-0.186

5

25

1126

1360

11

1125.489

1237.741

0.187

0.471

5

13

735

991

11

743.7131041.675

0.242

6

200

4042

491

11

3848.744

692.846

6

100

3844

797

10

3519.266

6

50

189

652

10

6

25

1728

989

6

13

-1102

7

200

7

Standard
error
observedmean

Estimated
reductionfrom control(number)

Obeserved means
in control

Ttest
confidence
limit

3928

6927

-1.729

0.153

3412

6927

-1.729-0.146

0.162

3266

6927

-1.7290.367-0.153

0.107

2545

6927

-1.729

0

0.75

0.393

0.572

5050

6731

-1.734851.435

0

0.743

0.303

0.523

5000

6731

-1.74

-144.032

726.133

0.577

0.166

-0.209-0.021

1119

6731

-1.74

11

1548.347

905.214

0.052

0.463

-0.003

0.23

3118

6731

-1.7341420

11-1046.67

1215.331

0.8

0.158

-0.469-0.1551061

6731-1.734

1737

385

10

1738.782

405.588

0

1.102

0.464

0.783

2447

2220

-1.746

100

1462

421

10

1431.022433.195

0.002

0.985

0.304

0.644

2187

2220

-1.746

7

50

-698

370

9

-719.181

396.1110.955-0.011-0.637-0.324-25

2220-1.753

7

25

-516

681

11

-565.735

573.919

0.831

0.195

-0.704-0.255

433

2220

-1.74

7

13

-1470

476

11

-1389.37

456.9410.996-0.268-0.984-0.626

-594

2220

-1.74

8

200

3096

NA

1

2958.927

3590.736

0.228

1.942

-0.859

0.541

10614

5466

-2.132

8

100

8944

NA

1

8738.39

3111.431

0.024

2.812

0.385

1.599

15371

5466

-2.132

8

50

2138

1418

7

2328.118

1194.927

0.04

0.822

0.03

0.426

4494

5466

-1.812

8

25

-677

1406

8

-543.731466.604

0.641

0.382

-0.581-0.0992090

5466-1.7961

114
201

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

Parameter

Open
brood
Cappedbrood
Cappedbrood
Cappedbrood
Cappedbrood
Cappedbrood
Cappedbrood
Cappedbrood
Cappedbrood
Cappedbrood
Cappedbrood
Cappedbrood
Cappedbrood
Cappedbrood
Cappedbrood
Cappedbrood
Cappedbrood
Cappedbrood

Modelestimatemeandifference
fromcontrol4

Standard
error ofestimatedmean

P value forcomparison
with thecontrol90%
confidence
upper limit

90%
confidence
lower limit

Estimated
reduction
fromcontrol (%)5856.197

0.479

0.29

-0.273

0.008

Time
(CCA)

Test
conc.
(µg/l)2

Observedmeandifference
fromcontrol3

8

13

43

585

8

45.576

3

200

-5684

2040

11

3

100

-2963

2161

3

50

-1493

3

25

3

Standard
error
observedmean

Estimated
reductionfrom control(number)

Obeserved means
in control

Ttest
confidence
limit

5466

-1.796011563-1.729

11

0

11563

-1.729

3468

11

0

11563

-1.729

-4495

1327

11

0

11563

-1.729

13

-3886

2448

11

0

11563

-1.729

4

200

11235

1859

11

13101.38

2806.568

0

1.142

0.525

0.833

17954

15722

-1.729

4

100

6857

1666

11

8125.651

2490.725

0.002

0.791

0.243

0.517

12432

15722

-1.729

4

50

3964

2316

11

4411.824

2826.411

0.068

0.591

-0.03

0.281

9299

15722

-1.729

4

25

993

1631

11

2778.589

2653.562

0.154

0.469

-0.115

0.177

7367

15722

-1.729

4

13

-1259

1275

11

433.306

2337.261

0.427

0.285

-0.229

0.028

4475

15722

-1.729

5

200

15011

1722

11

14191.99

2739.248

0

1.061

0.53

0.795

18929

17841

-1.729

5

100

10617

2093

11

10099.86

2683.038

0.001

0.826

0.306

0.566

14739

17841

-1.729

5

50

6630

3684

11

6173.291

3431.445

0.044

0.679

0.013

0.346

12107

17841

-1.729

5

25

1657

3361

11

1028.506

3529.735

0.387

0.4

-0.284

0.058

7132

17841

-1.729

5

13

156

2183

11

-665.442

2729.606

0.595

0.227

-0.302-0.037

4054

17841

-1.729

6

200

14026

1220

11

13595.89

1412.206

0

0.891

0.619

0.755

16045

18013

-1.734

6

100

9477

2550

10

8945.157

2165.524

0

0.706

0.287

0.497

12712

18013

-1.74

1

n

1583

115
202

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

n

Model
estimatemeandifference
fromcontrol4

Standard
error ofestimatedmean

P value forcomparison
with thecontrol90%
confidence
upper limit

90%
confidence
lower limit

Estimated
reduction
fromcontrol (%)5

Time
(CCA)

Test
conc.
(µg/l)2

Observedmeandifference
fromcontrol3

6

50

2761

1515

10

2244.065

1367.303

0.06

0.257

-0.007

0.125

6

25

3675

1774

11

3304.405

1729.37

0.036

0.35

0.017

6

13

4394

2297

11

3994.561

2028.775

0.032

0.417

7

200

4730

610

10

5046.549

808.018

0

7

100

3750

848

10

4078.307875.563

7

50

506

353

9

523.439

7

25

923

755

11

7

13

-438

1038

8

200

4816

8

100

17716

8

50

6917

8

25

8

AdultsStandard
error
observedmean

Estimated
reductionfrom control(number)

Obeserved means
in control

Ttest
confidence
limit

4623

18013

-1.74

0.183

6303

18013

-1.734

0.026

0.222

7513

18013

-1.734

1.197

0.674

0.935

6457

5395

-1.746

0

1.039

0.473

0.756

5607

5395

-1.746697.699

0.232

0.324

-0.13

0.097

1747

5395

-1.753974.261846.077

0.133

0.453

-0.092

0.181

2446

5395

-1.74

11

-454.128917.895

0.686

0.212

-0.38

-0.084

1143

5395

-1.74

NA

1

4816

12138.26

0.356

2.674

-1.835

0.42

30693

11476

-2.132NA

117320.16

10822.64

0.092

3.52

-0.501

1.509

40392

11476

-2.1322328

78097.621

4134.314

0.039

1.359

0.053

0.706

15591

11476

-1.812

-785

2926

8

-1239.48

3942.057

0.62

0.509

-0.725-0.108

5840

11476

-1.796

13

172

1273

8

-148.408

2879.268

0.52

0.438

-0.464-0.013

5022

11476

-1.796

3

200

-968

577

11

0

7488

-1.729Adults

3

100

749

896

11

0

7488

-1.729Adults

3

50

734

638

11

0

7488

-1.729Adults

3

25

-988

904

11

0

7488

-1.729Adults

3

13

-877

926

11

0

7488

-1.729Adults

4

200

-3115

980

11

-2220.6

1189.671

0.961-0.021-0.562-0.292-1647608-1.729Adults

4

100

630

1345

11

391.8891414.562

0.392

0.373

-0.27

0.052

2838

7608

-1.729Parameter

Cappedbrood
Cappedbrood
Cappedbrood
Cappedbrood
Cappedbrood
Cappedbrood
Cappedbrood
Cappedbrood
Cappedbrood
Cappedbrood
Cappedbrood
Cappedbrood
Capped
brood

1

116
203

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

n

Model
estimatemeandifference
fromcontrol4

Standard
error ofestimatedmean

P value forcomparison
with thecontrol90%
confidence
upper limit

90%
confidence
lower limit

Estimated
reduction
fromcontrol (%)5

Time
(CCA)

Test
conc.
(µg/l)2

Observedmeandifference
fromcontrol3

Adults

4

50

-1255

1159

11

-1771.83

1144.327

0.931

0.027

-0.493-0.233

Adults

4

25

-386

741

11

205.3291175.032

0.432

0.294

-0.24

Adults

4

13

-650

1085

11

168.161163.063

0.443

0.286

Adults

5

200

2231

598

11

2466.328

1208.532

0.028

Adults

5

100

3791

1342

11

3425.479

1451.632

Adults

5

50

2612

1430

11

2191.531

Adults

5

25

635

1120

11

874.801

Adults

5

13

-742

1148

11

Adults

6

200

5008

728

Adults

6

100

3105

Adults650Adults6AdultsStandard
error
observedmean

Estimated
reductionfrom control(number)

Obeserved means
in control

Ttest
confidence
limit

207

7608

-1.729

0.027

2237

7608

-1.729-0.242

0.022

2179

7608

-1.729

0.452

0.037

0.244

4556

10087

-1.729

0.015

0.588

0.091

0.34

5936

10087

-1.7291483.044

0.078

0.471

-0.037

0.217

4756

10087

-1.7291378.486

0.267

0.323

-0.15

0.087

3258

10087

-1.729-544.515

1369.016

0.652

0.181

-0.289-0.054

1823

10087

-1.729114677.835

932.194

0

0.71

0.345

0.528

6294

8865

-1.7341397

102544.891

1196.879

0.024

0.522

0.052

0.287

4627

8865

-1.74

2759

1078

10

1754.005

1053.102

0.057

0.404

-0.009

0.198

3586

8865

-1.74

25

785

642

11

686.452

950.173

0.24

0.263

-0.108

0.077

2334

8865

-1.734

6

13

475

953

11

199.536910.841

0.415

0.201

-0.156

0.023

1779

8865

-1.734Adults

7

200

3541

1095

10

3895.534

993.559

0.001

0.858

0.329

0.594

5630

6561

-1.746Adults

7

100

3142

965

10

3347.475

917.098

0.001

0.754

0.266

0.51

4949

6561

-1.746Adults

7

50

1441

650

9

1222.715

799.636

0.074

0.4

-0.027

0.186

2625

6561

-1.753

Adults

7

25

-788

1243

11

-272.138

1019.519

0.604

0.229

-0.312-0.041

1501

6561

-1.74

Adults

7

13

-1108

959

11

-826.715

874.128

0.821

0.106

-0.358-0.126

694

6561

-1.74

Adults

8

200

2627

NA

1

564.155

2791.126

0.425

0.846

-0.7

0.073

6514

7699

-2.132

Adults

8

100

11906

NA

1

13280.14

2402.712

0.003

2.39

1.06

1.725

18402

7699

-2.132

Adults

8

50

4770

1434

7

6008.543

1473.09

0.001

1.127

0.434

0.78

8678

7699

-1.812

Adults

8

25

-730

2337

8

-329.695

2346.527

0.555

0.505

-0.59

-0.0433884

7699-1.796Adults

8

13

-311

695

8

-845.547

1275.142

0.74

0.188

-0.407

-0.11

1444

7699

-1.796

Honey

3

200

1407

1690

11

0

10711

-1.729

Honey

3

100

-1251

1594

11

0

10711

-1.729

Honey

3

50

-891

1300

11

0

10711

-1.729Parameter

1

117
204

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

Standard
error
observed
mean

n

Model
estimatemeandifference
fromcontrol4

Time
(CCA)

Test
conc.
(µg/l)2

Observedmeandifference
fromcontrol3

Honey

3

25

250

1801

11

Honey

3

13

-360

2276

Honey

4

200

-1407

Honey

4

100

Honey

4

Honey
Honey

Standard
error ofestimatedmean

P value forcomparison
with thecontrol90%
confidence
upper limit

Estimated
reduction
fromcontrol (%)5

Estimated
reductionfrom control(number)

Obeserved means
in control

Ttest
confidence
limit

0

10711

-1.729

11

0

10711

-1.7292205

11-2934.5

1555.065

0.963-0.018-0.405-0.211

-246

13901

-1.729

-1220

1573

11

-1111.34

1055.672

0.847

0.051

-0.211

-0.08

714

13901

-1.729

50

-782

2012

11

-864.527

1431.841

0.723

0.116

-0.24

-0.062

1611

13901

-1.729

4

25

-797

1894

11

-1438.89

1134.467

0.89

0.038

-0.245-0.104523

13901-1.729

4

13

-657

1710

11

-1036.17

1160.622

0.808

0.07

-0.219-0.075971

13901-1.729

Honey

5

200

-5637

2476

11

-7251.97

1690.564

1-0.502-1.18-0.841

-4329

8623

-1.729

Honey

5

100

-23

1814

11

609.0481392.03

0.333

0.35

-0.208

0.071

3016

8623

-1.729

Honey

5

50

1665

1294

11

1862.689

1342.59

0.091

0.485

-0.053

0.216

4184

8623

-1.729

Honey

5

25

-805

2119

11

-1323.61

1297.1

0.84

0.107

-0.414-0.153919

8623-1.729

Honey

5

13

242

2130

11

112.2481320.202

0.467

0.278

-0.252

0.013

2395

8623

-1.729

Honey

6

200

14526

2808

11

14734.61

2475.396

0

0.91

0.499

0.705

19027

20906

-1.734

Honey

6

100

4059

3930

10

4547.091

3224.44

0.088

0.486

-0.051

0.218

10156

20906

-1.74

Honey

6

50

7327

3279

10

7524.1252905.267

0.01

0.602

0.118

0.36

12578

20906

-1.74

Honey

6

25

-532

2597

11

-507.008

2427.394

0.582

0.177

-0.226-0.024

3702

20906

-1.734

Honey

6

13

3033

2428

11

3297.32

2397.491

0.093

0.357

-0.041

0.158

7455

20906

-1.734

Honey

7

200

16194

3296

10

16768.37

3080.791

0

1.057

0.543

0.8

22147

20961

-1.746

Honey

7

100

10243

3273

10

11091.16

3101.539

0.001

0.787

0.271

0.529

16506

20961

-1.746

Honey

7

50

8485

4632

9

8636.163

3907.923

0.022

0.739

0.085

0.412

15487

20961

-1.753

Honey

7

25

1087

3820

11

2226.627

3127.457

0.243

0.366

-0.153

0.106

7667

20961

-1.74

Honey

7

13

2369

3644

11

2614.224

3427.576

0.228

0.409

-0.16

0.125

8577

20961

-1.74

Honey

8

200

516

NA

1

559.0447145.141

0.471

1.447

-1.345

0.051

15791

10912

-2.132

Honey

8

100

17114

NA

1

17084.95

6067.538

0.024

2.751

0.38

1.566

30020

10912

-2.132

Honey

8

50

6217

2261

7

6589.059

2225.801

0.007

0.973

0.234

0.604

10623

10912

-1.812

Honey

8

25

1333

3139

8

1450.279

2523.066

0.288

0.548

-0.282

0.133

5981

10912

-1.796

Parameter

190%
confidence
lower limit

118
205

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Modelestimatemeandifference
fromcontrol4

Standard
error ofestimatedmean

P value forcomparison
with thecontrol90%
confidence
upper limit

90%
confidence
lower limit

Estimated
reduction
fromcontrol (%)52059.405

0.712

0.23

-0.448-0.109

Time
(CCA)

Test
conc.
(µg/l)2

Observedmeandifference
fromcontrol3

Honey

8

13

-1161

1972

8

-1185.77

Pollen

3

200

1392

645

11

Pollen

3

100

297

717

Pollen3

50-47Pollen3

25

Pollen3

Pollen4

Pollen4

PollenStandard
error
observedmean

Estimated
reductionfrom control(number)

Obeserved means
in control

Ttest
confidence
limit

10912

-1.79604613-1.729

11

0

4613

-1.729

1057

11

0

4613

-1.729

1470

764

11

0

4613

-1.729

13

891

833

11

0

4613

-1.729

200

4550

478

11

4297.751

690.417

0

1.207

0.682

0.945

5492

4550

-1.729

100

3956

651

11

3806.351

713.408

0

1.108

0.565

0.837

5040

4550

-1.729

4

50

2658

385

11

2551.377

594.965

0

0.787

0.335

0.561

3580

4550

-1.729Pollen

4

25

735

615

11

236.8

695.034

0.369

0.316

-0.212

0.052

1439

4550

-1.729Pollen

4

13

782

541

11

536.343685.609

0.222

0.378

-0.143

0.118

1722

4550

-1.729Pollen

5

200

7021

642

11

6419.711

1061.603

0

1.163

0.646

0.904

8255

7099

-1.729Pollen

5

100

6176

899

11

5935.685

1078.422

0

1.099

0.573

0.836

7800

7099

-1.729Pollen

5

50

4550

704

11

4438.326

1047.955

0

0.88

0.37

0.625

6250

7099

-1.729Pollen

5

25

1141

1327

11

145.606

1282.746

0.455

0.333

-0.292

0.021

2364

7099

-1.729Pollen

5

13

-78

1005

11

-485.925

1177.574

0.658

0.218

-0.355-0.0681550

7099-1.729Pollen

6

200

2119

493

11

1921.621

506.636

0.001

0.798

0.297

0.547

2800

3510

-1.734

Pollen

6

100

731

610

10

537.423519.822

0.158

0.411

-0.105

0.153

1442

3510

-1.74

Pollen

6

50

628

595

10

544.13502.694

0.147

0.404

-0.094

0.155

1419

3510

-1.74

Pollen

6

25

-586

699

11

-885.905

606.289

0.919

0.047

-0.552-0.252165

3510-1.734

Pollen

6

13

-1681

584

11

-1872.63

550.502

0.998-0.262-0.805-0.533-918

3510-1.734

Pollen

7

200

1531

748

10

1400.095

688.615

0.029

0.94

0.071

0.506

2602

2768

-1.746

Pollen

7

100

292

660

10

357.835

605.124

0.281

0.511

-0.252

0.129

1414

2768

-1.746

Pollen

7

50

860

506

9

807.288538.712

0.077

0.633

-0.05

0.292

1752

2768

-1.753Pollen

7

25

-485

568

11

-573.884

549.941

0.844

0.138

-0.553-0.207

383

2768

-1.74

Pollen

7

13

-751

776

11

-723.548

633.978

0.865

0.137

-0.66

-0.261

379

2768

-1.74

Parameter

1

n

2513

119
206

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Modelestimatemeandifference
fromcontrol4

Standard
error ofestimatedmean

P value forcomparison
with thecontrol90%
confidence
upper limit

90%
confidence
lower limit

Estimated
reduction
fromcontrol (%)5

Time
(CCA)

Test
conc.
(µg/l)2

Observedmeandifference
fromcontrol3

Pollen

8

200

688

NA

1

611.9222401.029

0.406

1.153

-0.9070.123

Pollen

8

100

4902

NA

1

4997.097

2082.223

0.037

1.899

0.112

Pollen

8

50

2949

672

7

3170.529

780.622

0.001

0.923

Pollen

8

25

-172

1125

8

34.4731041.741

0.487

Pollen

8

13

-580

787

8

-605.432

809.1640.765

Weight

3

200

-2

2

11

Weight

3

100

0

2

11

Weight

3

50

-3

2

Weight3

25-2

Weight3

13

Weight4

WeightStandard
error
observedmean

Estimated
reductionfrom control(number)

Obeserved means
in control

Ttest
confidence
limit

5731

4969

-2.132

1.006

9436

4969

-2.132

0.353

0.638

4585

4969

-1.812

0.383

-0.37

0.007

1905

4969

-1.7960.171-0.414-0.122848

4969-1.796040-1.729040-1.72911

040-1.729

2

11

0

40

-1.729

-2

2

11

0

40

-1.729

200

5

1

11

5.863

1.472

0

0.145

0.057

0.101

8

58

-1.729

4

100

5

2

11

4.147

1.436

0.005

0.114

0.029

0.071

7

58

-1.729Weight

4

50

3

2

11

2.382

1.532

0.068

0.087

-0.005

0.041

5

58

-1.729Weight

4

25

1

2

11

1.569

1.495

0.154

0.072

-0.017

0.027

4

58

-1.729Weight

4

13

0

2

11

-0.271

1.798

0.559

0.049

-0.058-0.0053

58-1.729Weight

5

200

5

2

11

4.929

1.972

0.011

0.176

0.032

0.104

8

47

-1.729Weight

5

100

7

2

11

7.088

2.034

0.001

0.224

0.075

0.149

11

47

-1.729Weight

5

50

6

2

11

5.241

2.189

0.014

0.19

0.031

0.11

9

47

-1.729Weight

5

25

0

2

11

-0.188

2.015

0.537

0.069

-0.077-0.004347-1.729Weight

5

13

2

2

11

1.146

2.222

0.306

0.105

-0.057

0.024

5

47

-1.729Weight

6

200

15

3

11

16.153

3.421

0

0.415

0.192

0.304

22

53

-1.734Weight

6

100

8

4

10

7.991

3.955

0.03

0.28

0.021

0.15

15

53

-1.74

Weight

6

50

8

3

10

6.564

3.966

0.058

0.253

-0.006

0.123

13

53

-1.74

Weight

6

25

-1

3

11

-0.802

3.677

0.585

0.105

-0.135-0.015653-1.734Weight

6

13

1

3

11

0.088

3.5

0.49

0.116

-0.112

0.002

6

53

-1.734Weight

7

200

16

3

11

17.512

3.434

0

0.446

0.22

0.333

23

53

-1.734

Parameter

1

n

120
207

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

n

Model
estimatemeandifference
fromcontrol4

Standard
error ofestimatedmean

P value forcomparison
with thecontrol90%
confidence
upper limit

90%
confidence
lower limit

Estimated
reduction
fromcontrol (%)5

Time
(CCA)

Test
conc.
(µg/l)2

Observedmeandifference
fromcontrol3

Weight

7

100

9

4

10

9.946

3.841

0.01

0.316

0.062

0.189

Weight

7

50

9

4

10

7.903

4.104

0.036

0.286

0.015

Weight

7

25

-1

4

11

0.306

3.853

0.469

0.133

Weight

7

13

0

3

11

-0.825

3.756

0.586

Weight

8

200

-6

NA

1

-10.3315.62

Weight

8

100

26

NA

1

27.447Weight

8

50

8

2

7

8.475

Weight

8

25

-1

4

8

0.503

Standard
error
observedmean

Estimated
reductionfrom control(number)

Obeserved means
in control

Ttest
confidence
limit

17

53

-1.74

0.15

15

53

-1.74

-0.121

0.006

7

53

-1.7340.108-0.139-0.016653-1.734

0.93

0.041

-0.551-0.2552

41-2.132

3.867

0.001

0.881

0.474

0.677

36

41

-2.132

2.777

0.006

0.333

0.085

0.209

14

41

-1.812

3.511

0.444

0.168

-0.143

0.012

7

41

-1.796Weight813-1
1
8-0.454
1.484
0.617
0.055
-0.077-0.011
2
Notes:
1, Observation dates for hive weight measurement were slight different from the dates when colony assessments were conducted.
2, The test concentration labelled as “13” in the table was originated from the raw data submitted by the study author. The actual concentration is expected to be 12.5 µg/l .
3, Mean of observations in controls minus the observation in the treatment.
4, Difference between the mean of observation in controls and estimated number in treatment after adjustment for covariance for CCA3 to be a 0 baseline.
5, The percentage of the estimated difference between the treatment and control divided by the number in the control.[Value in column must be multiplied by 100 to be a %]41

-1.796

Parameter

1

121
208

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Results
The following tables and graphs present results for individual measurement endpoints (total
individuals, eggs, larvae, pupae, pollen stores, nectar stores, hive weight). The percent reductions
are the means of the differences between each treatment and control at the same apiary, based on
observations and expected values estimated by the statistical model that adjusted baseline
measurement for CCA3, using raw count data.
Table B-2. Estimated percent reduction from control for total number of individuals

Estimated reduction from control (%)(P value)
CCA4
CCA5
CCA6
CCA7
CCA8
4
1
8.7
-16.9
-2.6
12.5
(0.354)(0.469)(0.091)*(0.902)(0.561)
10.8
10.9
17.4
2.2
-11.4
25
(0.166)(0.253)(0.029)**(0.429)(0.657)
10.3
25.4
12.1
8.1
49.1
50
(0.182)(0.062)*(0.048)**(0.19)(0.028)**
35.3
46
48.2
60.9
145.1
100
(0.002)**(0.001)**(<0.001)**(<0.001)**(0.036)**
48.6
60.5
65.9
74.6
54.1
200
(<0.001)**(<0.001)**(<0.001)**(<0.001)**(0.238)
Note: Negative value indicates increased number of individuals in comparison to control.
*0.05<P<0.1**P<0.05
Where two p values are listed, the first is the non-adjusted p value, the second is the p value adjusted forthe step-down approach. The step-down adjustment was shown only if it changed the significance level.
At CCA8, the step-down approach was not applied to the 200 or 100 treatment levels where very few
hives survived.
Test
concentration(µg/L)

122209

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Figure B-1: Estimates and 90% CIs for Total Life with thresholds of statistical (red) significance shown. The green
line shows 10% difference from control.

Figure B-2: Mean of the differences of the total individuals in hives between treatments and control at thesame apiary.

A

B

123
210

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Figure A: Total number of individuals in control hives at each apiary (n=11).
X-axis represents the number of CCA; Y-axis represents the total individuals in control hives.
Apiaries are coded with various colors.
Black dash line represents the mean of all apiaries.
Figure B: Mean of the differences of the total individuals in hives between the treatments and control at the same apiary(n=11) with 90% upper and lower confidence interval.
X-axis represents the number of CCA; Y-axis represents the mean of the differences of the total individuals in hives between
the control and treatment at the same apiary (control minus treatment).
The line of “0” indicates no effect from the control within the same Apiary and it was adjusted for baseline covariate at CCA3
for each CCAs.
Labels at the end of each line are the treatment concentrations (µg/L).
A positive Y value indicates there are more live individuals in hives in the control than in the treatment, and a negative value
indicates reversely.
A dash line indicates the 90% confidence interval estimated by a GLM model for a solid line with the same color.

Table B-3: Estimated percent reduction from control for number of adults

Estimated reduction from control (%)(P value)
CCA4
CCA5
CCA6
CCA7
CCA8
2.2
-5.4
2.3
-12.6
-11
12.5
(0.443)(0.652)(0.415)(0.821)(0.74)
2.7
8.7
7.7
-4.1
-4.3
25
(0.432)(0.267)(0.24)(0.604)(0.555)
-23.3
21.7
19.8
18.6
78**
50
(0.931)(0.078)*(0.057)*(0.074)*(0.001)
5.2
34
28.7
51
172.5
100
(0.392)(0.015)**(0.024)**(0.001)**(0.003)**-29.2
24.4**
52.8**
59.4**
7.3
200(0.961)(0.028)(<0.001)(0.001)(0.425)
Note: Negative value indicates increased number of adults in comparison to control.
*0.05<P<0.1**P<0.05
Where two p values are listed, the first is the non-adjusted p value, the second is the p value adjusted forthe step-down approach. The step-down adjustment was shown only if it changed the significance level.
At CCA8, the step-down approach was not applied to the 200 or 100 treatment levels where very few
hives survived.
Test
concentration(µg/L)

124
211

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Figure B-3: Estimates and 90% CIs for adult bees with thresholds of statistical (red) significance shown. The
green line shows 10% difference from control.

Figure B-4: Mean of the differences of adult bees between treatments and control at the same apiary.

A

B

125
212

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Figure A: Total member of adults in control hives at each apiary (n=11).
X-axis represents the number of CCA; Y-axis represents the number of adults in control hives.
Apiaries are coded with various colors.
Black dash line represents the mean of all apiaries.
Figure B: Mean of the differences of the number of adults in hives between the treatments and control at the same apiary(n=11).
X-axis represents the number of CCA; Y-axis represents the mean of the differences of the number of adults in hives
between the control and treatment at the same apiary (control minus treatment).
The line of “0” indicates no effect from the control within the same Apiary and it was adjusted for baseline covariate at CCA3
for each CCAs.
Labels at the end of each line are the treatment concentrations (µg/L).
A positive Y value indicates there are more adult bee in hives in the control than in the treatment, and a negative value
indicates reversely.
A dash line indicates the 90% confidence interval estimated by a GLM model for a solid line with the same color.

Table B-3: Estimated percent reduction from control for number of eggs

Estimated reduction from control (%)(P value)
CCA4
CCA5
CCA6
CCA7
CCA8
22.8
11.9
5.9
0.1
37.7
12.5
(0.063*/0.69)(0.236)(0.38)(0.498)(0.058*/0.36)
-1.1
8.3
26.3
31.3
5.8
25
(0.528)(0.336)(0.058*/0.14)(0.114)(0.367)
-9.6
1.8
11.7
2.6
78.2
50
(0.692)(0.453)(0.146)(0.451)(<0.001)**
37.9
39.4
70.8
46.6
138
100
(0.01**/0.21)(0.036)**(<0.001)**(0.042)**(0.002)**
14.5
32.2
60.1
77.8
153.2
200
(0.212)(0.043)**(<0.001)**(0.002)**(0.003)**
Note: Negative value indicates increased number of eggs in comparison to control.
*0.05<P<0.1**P<0.05
Where two p values are listed, the first is the non-adjusted p value, the second is the p value adjusted forthe step-down approach. The step-down adjustment was shown only if it changed the significance level.
At CCA8, the step-down approach was not applied to the 200 or 100 treatment levels where very few
hives survived.
Test
concentration(µg/L)

126
213

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Figure B-5: Estimates and 90% CIs for eggs with thresholds of statistical (red) significance shown. The green line
shows 10% difference from control.

Figure B-6: Mean of the differences of number of eggs in hives between treatments and control at the same
apiary.

A

B

127
214

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Figure A: Total number of eggs in control hives at each apiary (n=11).
X-axis represents the number of CCA; Y-axis represents the number of eggs in control hives.
Apiaries are coded with various colors.
Black dash line represents the mean of all apiaries.
Figure B: : Mean of the differences of the number of eggs in hives between the treatments and control at the same apiary(n=11).
X-axis represents the number of CCA; Y-axis represents the mean of the differences of the number of eggs in hives between
the control and treatment at the same apiary (control minus treatment).
The line of “0” indicates no effect from the control within the same Apiary and it was adjusted for baseline covariate at CCA3
for each CCAs.
Labels at the end of each line are the treatment concentrations (µg/L).
A positive Y value indicates there are more eggs in hives in the control than in the treatment, and a negative value indicatesreversely.
A dash line indicates the 90% confidence interval estimated by a GLM model for a solid line with the same color.

Table B-4: Estimated percent reduction from control for number of larvae

Estimated reduction from control (%)(P value)
CCA4
CCA5
CCA6
CCA7
CCA8
12.3
10.7
-15.5
-62.6
0.8
12.5
(0.161)(0.242)(0.8)(0.996)(0.479)
8.1
16.2
23
-25.5
-9.9
25
(0.241)(0.187)(0.052*/0.57)(0.831)(0.641)
12.1
15.3
-2.1
-32.4
42.6
50
(0.199)(0.223)(0.577)(0.955)(0.04)**
37.1
30.9
52.3
64.4
159.9
100
(0.007)**(0.026)**(<0.001)**(0.002)**(0.024)**
64
65
57.2
78.3
54.1
200
(<0.001)**(<0.001)**(<0.001)**(<0.001)**(0.228)
Note: Negative value indicates increased number of larvae in comparison to control.
*0.05<P<0.1**P<0.05
Where two p values are listed, the first is the non-adjusted p value, the second is the p value adjusted forthe step-down approach. The step-down adjustment was shown only if it changed the significance level.
At CCA8, the step-down approach was not applied to the 200 or 100 treatment levels where very few
hives survived.
Test
concentration(µg/L)

128215

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Figure B-7 Estimates and 90% CIs for larvae with thresholds of statistical (red) significance shown. The green
line shows 10% difference from control.

Figure B-8: Mean of the differences of number of larvae in hives between treatments and control at the same
apiary.

A

B

129
216

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Figure A: Total number of larvae in control hives at each apiary (n=11).
X-axis represents the number of CCA; Y-axis represents the number of larvae in control hives.
Apiaries are coded with various colors.
Black dash line represents the mean of all apiaries.
Figure B: : Mean of the differences of the number of larvae in hives between the treatments and control at the same apiary(n=11).
X-axis represents the number of CCA; Y-axis represents the mean of the differences of the number of larvae in hives
between the control and treatment at the same apiary (control minus treatment).
The line of “0” indicates no effect from the control within the same Apiary and it was adjusted for baseline covariate at CCA3
for each CCAs.
Labels at the end of each line are the treatment concentrations (µg/L).
A positive Y value indicates there are more larvae in hives in the control than in the treatment, and a negative value
indicates reversely.
A dash line indicates the 90% confidence interval estimated by a GLM model for a solid line with the same color.

Table B-5: Estimated percent reduction from control for number of pupae

Estimated reduction from control (%)(P value)
CCA4
CCA5
CCA6
CCA7
CCA8
22.2
12.5
2.8
-3.7
(0.032**/-8.4-1.3(0.427)(0.595)0.06*)(0.686)(0.52)
18.3
25
17.7
5.8
(0.036**/
18.1
-10.8
(0.154)(0.387)0.06*)(0.133)(0.62)
28.1
34.6
12.5
9.7
70.6
50
(0.068)*(0.044)**(0.06)*(0.232)(0.039)**
51.7
56.6
49.7
75.6
150.9
100
(0.002)**(0.001)**(<0.001)**(<0.001)**(0.092)*
83.3
79.5
75.5
93.5
42
200
(<0.001)**(<0.001)**(<0.001)**(<0.001)**(0.356)
Note: Negative value indicates increased number of pupae in comparison to control.
*0.05<P<0.1**P<0.05
Where two p values are listed, the first is the non-adjusted p value, the second is the p value adjusted forthe step-down approach. The step-down adjustment was shown only if it changed the significance level.
At CCA8, the step-down approach was not applied to the 200 or 100 treatment levels where very few
hives survived.
Test
concentration(µg/L)

130217

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Figure B-9: Estimates and 90% CIs for pupae with thresholds of statistical (red) significance shown. The green
line shows 10% difference from control.

Figure B-10: Mean of the differences of number of pupae in hives between treatments and control at the same
apiary

A

B

131
218

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Figure A: Total number of capped brood (pupae) in control hives at each apiary (n=11).
X-axis represents the number of CCA; Y-axis represents the number of pupae in control hives.
Apiaries are coded with various colors.
Black dash line represents the mean of all apiaries.
Figure B: Mean of the differences of the number of capped brood (pupae) in hives between the treatments and control at
the same apiary (n=11).
X-axis represents the number of CCA; Y-axis represents the mean of the differences of the number of pupae in hives
between the control and treatment at the same apiary (control minus treatment).
The line of “0” indicates no effect from the control within the same Apiary and it was adjusted for baseline covariate at CCA3
for each CCAs.
Labels at the end of each line are the treatment concentrations (µg/L).
A positive Y value indicates there are more pupae in hives in the control than in the treatment, and a negative value
indicates reversely.
A dash line indicates the 90% confidence interval estimated by a GLM model for a solid line with the same color.

Table B-6: Estimated percent reduction from control for pollen store

Estimated reduction from control (%)(P value)
CCA4
CCA5
CCA6
CCA7
CCA8
11.8
-6.8
-53.3
-26.1
-12.2
12.5
(0.222)(0.658)(0.998)(0.865)(0.765)
5.2
2.1
-25.2
-20.7
0.7
25
(0.369)(0.455)(0.919)(0.844)(0.487)
56.1
62.5
15.5
29.2
63.8
50
(<0.001)**(<0.001)**(0.147)(0.077*/0.28)(0.001)**
83.7
83.6
15.3
12.9
100.6
100
(<0.001)**(<0.001)**(0.158)(0.281)(0.037)**
94.5
90.4
54.7
50.6
12.3
200
(<0.001)**(<0.001)**(0.001)**(0.029)**(0.406)
Note: Negative value indicates increased pollen store in comparison to control.
*0.05<P<0.1**P<0.05
Where two p values are listed, the first is the non-adjusted p value, the second is the p value adjusted forthe step-down approach. The step-down adjustment was shown only if it changed the significance level.
At CCA8, the step-down approach was not applied to the 200 or 100 treatment levels where very few
hives survived.
Test
concentration(µg/L)

132219

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Figure B-11: Estimates and 90% CIs for pollen store with thresholds of statistical (red) significance shown. The
green line shows 10% difference from control.

Figure B-12: Mean of the differences of the amount of pollen store in hives between treatments and control at
the same apiary.

A

B

133
220

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Figure A: Total amount of pollen store in control hives at each apiary (n=11).
X-axis represents the number of CCA; Y-axis represents the amount of pollen store in control hives.
Apiaries are coded with various colors.
Black dash line represents the mean of all apiaries.
Figure B: Mean of the differences of the amount of pollen store in hives between the treatments and control at the same
apiary (n=11).
X-axis represents the number of CCA; Y-axis represents the mean of the differences of the amount of pollen store in hives
between the control and treatment at the same apiary (control minus treatment).
The line of “0” indicates no effect from the control within the same Apiary and it was adjusted for baseline covariate at CCA3
for each CCAs.
Labels at the end of each line are the treatment concentrations (µg/L).
A positive Y value indicates there are more pollen store in hives in the control than in the treatment, and a negative value
indicates reversely.
A dash line indicates the 90% confidence interval estimated by a GLM model for a solid line with the same color.

Table B-7: Estimated percent reduction from control for honey store

Estimated reduction from control (%)(P value)
CCA4
CCA5
CCA6
CCA7
CCA8
-7.5
1.3
15.8
12.5
-10.9
12.5
(0.808)(0.467)(0.093*/0.58)(0.228)(0.712)
-10.4
-15.3
-2.4
10.6
13.3
25
(0.89)(0.84)(0.582)(0.243)(0.288)
-6.2
21.6
36
41.2
60.4
50
(0.723)(0.091*/0.33)(0.01)**(0.022)**(0.007)**
-8
7.1
21.8
52.9
156.6
100
(0.847)(0.333)(0.088)*(0.001)**(0.024)**
-21.1
-84.1
70.5
80
5.1
200
(0.963)(1)(<0.001)**(<0.001)**(0.471)
Note: Negative value indicates increased honey store in comparison to control.
*0.05<P<0.1**P<0.05
Where two p values are listed, the first is the non-adjusted p value, the second is the p value adjusted forthe step-down approach. The step-down adjustment was shown only if it changed the significance level.
At CCA8, the step-down approach was not applied to the 200 or 100 treatment levels where very few
hives survived.
Test
concentration(µg/L)

134
221

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Figure B-13: Estimates and 90% CIs for honey store with thresholds of statistical (red) significance shown. The
green line shows 10% difference from control.

Figure B-14: Mean of the differences of the amount of honey store in hives between the treatments and control
at the same apiary.

135
222

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
A

B

Figure A: Total amount of honey store in control hives at each apiary (n=11).
X-axis represents the number of CCA; Y-axis represents the amount of honey store in control hives.
Apiaries are coded with various colors.
Black dash line represents the mean of all apiaries.
Figure B: : Mean of the differences of the amount of honey store in hives between the treatments and control at the same
apiary (n=11).
X-axis represents the number of CCA; Y-axis represents the mean of the differences of the amount of honey store in hives
between the control and treatment at the same apiary (control minus treatment).
The line of “0” indicates no effect from the control within the same Apiary and it was adjusted for baseline covariate at CCA3
for each CCAs.
Labels at the end of each line are the treatment concentrations (µg/L).
A positive Y value indicates there is more honey store in hives in the control than in the treatment, and a negative value
indicates reversely.
A dash line indicates the 90% confidence interval estimated by a GLM model for a solid line with the same color.

Table B-8: Estimated percent reduction from control for hive weights

Estimated reduction from control (%)(P value)
CCA4
CCA5
CCA6
CCA7
CCA8
-0.5
2.4
0.2
-1.6
-1.1
12.5
(0.559)(0.306)(0.49)(0.586)(0.617)
2.7
-0.4
-1.5
0.6
1.2
25
(0.154)(0.537)(0.585)(0.469)(0.444)
4.1
11
12.3
15
20.9
50
(0.068)*(0.014)**(0.058)*(0.036)**(0.006)**
7.1
14.9
15
18.9
67.7
100
(0.005)**(0.001)**(0.03)**(0.01)**(0.001)**
10.1
10.4
30.4
33.3
-25.5
200
(<0.001)**(0.011)**(<0.001)**(<0.001)**(0.93)
Note: Negative value indicates increased hive weight in comparison to control.
*0.05<P<0.1**P<0.05
Where two p values are listed, the first is the non-adjusted p value, the second is the p value adjusted forthe step-down approach. The step-down adjustment was shown only if it changed the significance level.
At CCA8, the step-down approach was not applied to the 200 or 100 treatment levels where very few
hives survived.
Test
concentration(µg/L)

136
223

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Figure B-15: Estimates and 90% CIs for hive weight with thresholds of statistical (red) significance shown. The
green line shows 10% difference from control.

Figure B-16 Mean of the differences of the hive weight between the treatments and control at thesame apiary.

A

B

137
224

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Figure A: Total hive weight in control hives at each apiary (n=11).
X-axis represents the number of CCA; Y-axis represents the hive weight in control hives.
Apiaries are coded with various colors.
Black dash line represents the mean of all apiaries.
Figure B: Mean of the differences of the hive weight between the treatments and control at the same apiary (n=11).
X-axis represents the number of CCA; Y-axis represents the mean of the differences of the hive weight between thecontrol and treatment at the same apiary (control minus treatment).
The line of “0” indicates no effect from the control within the same Apiary and it was adjusted for baseline covariate
at CCA3 for each CCAs.
Labels at the end of each line are the treatment concentrations (µg/L).
A positive Y value indicates that the hive weight was more in the control than in the treatment, and a negative value
indicates reversely.
A dash line indicates the 90% confidence interval estimated by a GLM model for a solid line with the same color.

Difference from control (%)

Total individuals
200
150
100
50
0
-50
CCA4
12.5

CCA5
25

CCA6
50

CCA7
100

CCA8
200

X: 0.1>P>0.05, : 0.05>P

Figure B-17. Total Individuals (percent difference from control)

Difference from control (%)

Adults
200
150
100
50
0
-50
CCA4
12.5

CCA5
25

CCA6
50

CCA7
100

CCA8
200

Figure B-18. Adults (percent difference from control)
138
225

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

Difference from control (%)

Eggs
200
150
100
50
0
-50
CCA4

CCA5

CCA6

12.5

25

50

CCA7
100

CCA8
200

Figure B-19. Eggs (percent difference from control)

Difference from control (%)

Larvae
200
150
100
50
0
-50
-100
CCA4

CCA5

CCA6

12.5

25

50

CCA7
100

CCA8
200

Figure B-20. Larvae (percent difference from control)

Difference from control (%)

Pupae
200
150
100
50
0
-50
CCA4

CCA5

CCA6

12.5

25

50

CCA7
100

CCA8
200

Figure B-21. Pupae (percent difference from control)
139
226

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

Difference from control (%)

Honey
200
150
100
50
0
-50
-100
CCA4

CCA5

CCA6

12.5

25

50

CCA7

CCA8

100

200

Figure B-22. Honey (percent difference from control)

Difference from control (%)

Pollen
150
100
50
0
-50
-100
CCA4

CCA5

CCA6

12.5

25

50

CCA7
100

CCA8
200

Figure B-23. Pollen (percent difference from control)

Difference from control (%)

Weight
80
60
40
20
0
-20
-40
CCA4

CCA5

CCA6

12.5

25

50

CCA7
100

CCA8
200

Figure B-24. Weight (percent difference from control)
140
227

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
100.0
Difference from the control (%)

80.0
60.0
40.0
20.0
0.0

37.7
22.8
12.3
4.0 2.8
2.2

22.2

1.0

10.7 11.9 8.72.3

5.9-5.4-3.7

-20.0

-15.5

-40.0

0.8

0.1

-2.6 -1.3
-11.0

-8.4
-12.6
-16.9

-60.0
-62.6

-80.0
CCA4

CCA5

TotalLife

CCA6

Adult

Pupae

CCA7

CCA8

LarvaeEggs

Figure B-25 Summary of living organism parameters at 12.5 µg/L

Difference from the control (%)

100.0
80.0
60.0
40.0
20.0

17.7
10.8
8.1
2.7

0.0

16.2
10.98.7
5.8 8.3

23.026.3
17.4 18.3
7.7

-1.1

31.3
18.1
5.8

2.2
-4.1

-20.0

-4.3 -9.9-11.4 -10.8
-25.5

-40.0
CCA4
TotalLife

CCA5

CCA6

Adult

Pupae

CCA7

CCA8

LarvaeEggs

Figure B-26. Summary of living organism parameters at 25 µg/L

141
228

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

Difference from the control (%)

100.0
78.0 78.2
70.6

80.0
60.0

49.1

40.0
20.0

28.1
12.1

10.3

25.4
21.7 15.3
1.8

0.0

-40.0

19.8
12.1 12.5 11.7

18.6
8.1 9.7

2.6

-2.1

-9.6

-20.0

42.6

34.6

-23.3

-32.4

CCA4

CCA5

TotalLife

CCA6

Adult

Pupae

CCA7

CCA8

LarvaeEggs

Figure B-27. Summary of living organism parameters at 50 µg/L
200
172.5
159.9
150.9
145.1
138

180
Difference from the control (%)

160
140
120
100
70.8

80
60
40
20

56.6
51.7
48.2 49.7
46
52.3
39.4
35.3 37.137.9
34
28.7
30.9

75.6
60.9 64.4
51
46.6

5.2

0
CCA4
TotalLife

CCA5

CCA6

Adult

Pupae

CCA7

CCA8

LarvaeEggs

Figure B-28. Summary of living organism parameters at 100 µg/L

142
229

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
4
3
2

1.146

1

0.088

0
-1

-0.271-0.454-0.825

-2
-3
-4
-5
CCA4

CCA5

CCA6

CCA7

CCA8

Figure B-29. Summary of hive weight at 12.5 µg/L
5
4
3
2

1.569

1

0.306

0.503

CCA7

CCA8

0
-0.188-1-2-0.802

-3
-4
-5
CCA4

CCA5

CCA6

Figure B-30. Summary of hive weight at 25 µg/L

143
230

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
14
12
10
8

6.564
5.241

6
4

8.475

7.903

2.382

2
0
CCA4

CCA5

CCA6

CCA7

CCA8

Figure B-31. Summary of hive weight at 50 µg/L
353027.447

25
20
15
10
5

4.147

7.088

7.991

CCA5

CCA6

9.946

0
CCA4

CCA7

CCA8

Figure B-32. Summary of hive weight at 100 µg/L

144
231

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Table B-9. Summary of observed effects at each treatment level (Note: Values reported in thetable are the % reduction compared to control, based on model estimated raw numbers corrected
for baseline measurements).
Treatment
Observations(µg/l)• Decreased total number of individuals in hive at CCA6 (8.7%, P<0.1)
12.5

25

50

100 and200

OVERALL
ENDPOINT

•
•
•
•
•
•
•
•
•
•
•
•
•

•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•

Decreased number of pupae at CCA6 (22.2%, P<0.05)
Decreased honey store at CCA6 (15.8%, P<0.1)
At CCA8, two out of 11 colonies did not survive overwintering (better survival than control)
At CCA8 among surviving hives, hive condition similar to control
The potential colony effects at CCA6 were considered short-term, colony able to compensate
Decreased total number of individuals in hive at CCA6 (17.4%, P<0.05)
Decreased number of pupae at CCA6 (18.3%, P<0.05)
Decreased number of eggs at CCA6 (26.3%, P<0.1)
Decreased number of larvae at CCA6 (23%, P<0.1)
At CCA8, one out of 11 colonies did not survive overwintering (better survival than control)
At CCA8 among surviving hives, hive condition similar to control
The potential colony effects at CCA6 were considered short-term, colony able to compensate
Decreased total number of individuals in hive at CCA5 (25.4%, P<0.1) and CCA6 (12.1%,
P<0.05)Decreased pupae at CCA4, CCA5, CCA6 (28.1, 34.6, 12.5%, P<0.1, <0.05, <0.1)
Decreased number of adults at CCA5 - CCA7 (18.6 - 21.7%, P<0.1)
No effect on eggs or larvae observed
Decreased pollen store at CCA4, CCA5, CCA7
Decreased honey store at CCA5 – CCA7
Decreased hive weight at CCA4 – CCA7At CCA8, four out of 11 colonies did not survive overwintering, comparable to control
At CCA8 among surviving hives, poorer hive condition compared control
Decreased total number of individuals in hive at CCA4-CCA7
Decreased number of adults at CCA5 - CCA7
Decreased number of eggs at CCA4 – CCA7
Decreased number of larvae at CCA4 – CCA7
Decreased number of pupae at CCA4 – CCA7
Decreased pollen store at CCA4 and CCA5
Decreased honey store at CCA6-CCA7
Decreased hive weight at CCA4 – CCA7High overwintering mortality (only 1 survived at 100; only 2 survived at 200)NOAEL: 25 µg/L sucrose solution (nominal 20.3 ppb; measured 23.3 ppb)
LOAEL: 50 µg/L sucrose solution (nominal 40.7 ppb; measured 46.7 ppb)

145
232

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

3.Appendix C: Details of CDPR Statistical Analysis
Statistical Summary
A clear progression of effects on hive health in response to imidacloprid dose was indicated by
the results of the multivariate mixed model analysis.
•

After only 3 weeks into the exposure period pupal and larval numbers decreased in
response to higher dose levels with effects initiated on pupal cells at the 50 ppb treatment.
Pollen food stores also exhibited decreased numbers as a dose response relationship at thehigher treatment levels with effects initiated at the 50 ppb dose level. Overall hive weightwas decreased at 100 and 200 ppb doses.

•

At 7 weeks after initiation of exposure (1 week after end of treatments), numbers of adultand egg cells were then decreased at the higher treatments with effects initiated on adultcells at the 50 ppb dose level. A clear dose response was also observed for hive weight at
the higher doses with effects now measured at the 50 ppb treatment.

•

Later at 11 weeks after initiation of exposure (5 weeks after end of treatments), number ofhoney cells exhibited a dose response relationship at the higher treatment levels witheffects initiated at the 50 ppb dose level.

•

At the final assessment before overwintering at 16 weeks after initiation of exposure (10weeks after end of treatments) decreases in number of honey cells and hive weight were
measured at the 50 ppb dose level. Numbers of adults and pupal and larval cells weredecreased at the 100 and 200 ppb dose levels. Note that some effects previously measured
between control and 50 ppb treatment levels were not apparent at this assessment.

•

Assessment of the hives after overwintering indicated that dose related effects noted at
CCA7 were expressed in CCA8 where extreme loss of hives was observed at the 100 and
200 ppb. Decreased vigor of hives at the 50 ppb dose level was indicated due to decreased
numbers of adults and pupal and larval cells as compared to control hives.

The general conclusion is that the data indicate a NOEC value at 25 ppb and a LOEC value
at 50 ppb.

Background
The multivariate mixed repeated measures model approach is distinguished from the univariate
approach previously in that all bee life stages or hive food storage variables are simultaneously
analyzed as a single model. Multivariate analyses of variance for fixed effects models are
conducted, using Statistical Analysis System (SAS) software, through implementing the
MANOVA option in the PROC GLM procedure. Recently, multivariate analyses have been
extended to mixed models using the PROC MIXED procedure. The MIXED procedure is designed
to conduct a mixed model analysis of variance where fixed and random effects can be specified.
Inclusion of random effects in a model provides a broader application of results. For this study,
146
233

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
locations were denoted as apiaries with individual hives as test subjects. Use of a mixed modelwith apiaries identified as a random variable provides some assurance that the results can begeneralized to other locations and hives.

Model Setup•••

The Multivariate Analysis of Variance in PROC MIXED is conducted by combining the datafor response variables to be analyzed into two columns of data. One column contains the listof variables to be analyzed, denoted as ‘RESPONSE’ in this analysis, and the other variable
contains the measured value for that variable, denoted ‘VALUE’ in this analysis.
Factors on the effect side of the equation for the multivariate response were dose of
imidacloprid and date sampled, which was denoted as day for each CCA assessment. .
Technically, the RESPONSE variable is listed as a class variable in the multivariate analysis
and then included in the effects side of the model.

The SAS code below illustrates the syntax for one of the programs used to determine theappropriate covariate model to use:proc mixed data=a7;
title4 'Overall Multivariate analysis';
title5 'EPA scaling - Apiary random - CV = un@ar(1)';class day dose response hivenum apiary;model epavaluet=response|day|dose/noint;
random apiary;
repeated response day/subject=dose*rep(apiary) type=un@ar(1);run;

Features in this syntax are:
1. Class Statement: The RESPONSE variable is included in the list of effects variables andcontains the variables to be analyzed for the multivariate analysis.
2. Model Statement:
a. VALUE is the respective measure for the RESPONSE variable. For example,
VALUE contains the value for number of adult bees taken for hive number 2
located in Apiary A and taken at CCA3 at the 12.5 ppb treatment. For the statisticalanalysis, the original values were scaled as in the univariate analyses: Raw values
for pupal, larval, and egg cells were dived by 68800 with adult cells divided by
68800/1.3 then times by 4.
b. The RESPONSE variable is tested for interactive effects with day and dose on theeffects side of the model.
3. Random Statement: Apiary is treated as a random variable because effects are to begeneralized to other locations.
4. Repeated Statement:a. Provides for a repeated measures analysis of variance.
b. The subject= indicates the hive where the repeated measures were taken.
c. Type= UN@AR(1) indicates the covariance model used where the symbols
represent a Compound Symmetry model applied to the response variable and a firstorder autoregressive model applied to day. Various covariance models were tested
147
234

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
to determine the model which provided the best fit. The best model was chosen bycomparing values of informational criteria for -2 Res Log Likelihood, a criteria thatprovided an overall estimate of the amount of variance explained by the model, andthe BIC criteria that adjusts the previous criteria based on the number of additionalparameters added to the model for each structured model. .
The sequence of statistical analyses conducted was:
1. Conduct a full model analysis of variance as reflected in syntax above. Owing to the large
number of dead hives at CCA8 for the 100 and 200 ppb treatments, data from CCA3
through CCA7 were used in this overall analysis to test for interactive effects. Threecovariance models relevant to this design were tested. For bee life stages the UN*AR(1)model provided the best fit, whereas, UN@CS provided the best fit for analysis of foodstores .
2. Upon observation of a significant interactive term between dose, day, and response, further
analyses were conducted at each CCA to determine the differential responses among
variables over time. Data for CCA8 were included in this analysis. Seven covariancemodels were tested for each CCA. The autoregressive-first-order model with heterogeneity
model fit best at CCA3, the compound symmetry model with heterogeneity fit best at
CCA4, and the unstructured model fit best for CCA5 through CCA8.
Multivariate Analysis of Variance••

Two sets of analyses were conducted. One focused on the counts for the various life stages ofbees within the hive, contrasting the numbers of adults, pupal, larval, and eggs over time.
A second analysis explored the relationship between nectar and pollen cells. These values
indicate the level of food stores in the hive over. Analysis on hive weight was conducted
separately.

Results
Life Stages
All effects in the multivariate full model for adult, pupal, larval, and eggs cells were highly
significant (Table C-1). Notably, the triple interactive effect for Day*Dose*Response indicatedthat the various bee stages responded differently over time to imidacloprid dosage. Analyses werethen conducted by CCA to determine the sequence of effects over time.
In order to determine the pattern of response for life stages at each CCA, a regression analysis was
first conducted to measure potential linear and curvilinear effects of dose at each CCA (Table C2). Quadratic and cubic dose effects were included to indicate potential curvature in response. The
second analysis provided a LSMEANS test for each pairwise comparison between levels of dosefor each response (Table C-3). These contrasts provide a basis for estimating potential no observed
effect concentration (NOAEC) and lowest observed effect concentration (LOAEC) values.

148
235

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Table C-1. Results for multivariate mixed model analysis of variance for potential interactive
effects of imidacloprid dose over time on counts of bee stages for adult, pupal, larval and egg cells.
Overall Model Effect

Num DF

Den DF

PR>F

Response

3

213<.0001

Day

4

283

<.0001

Day*Response

12

849

<.0001

Dose

5

61

<.0001

Dose*Response

15

213

<.0001

Day*Dose20283

0.0001

Day*Dose*Response

60

849

<.0001Comparison of the pattern of significant regression results between CCAs provided evidence forthe differential responses in bee life stages over time (Table C-2). In the figures for effects, theresponse for each variable over dose and is plotted. In addition, oversized dots and colors indicate
levels of significant difference between the control value and the value at each treatment level as
indicated from Table C-3.
•

CCA3: Prior to dietary administration of imidacloprid at CCA3, baselines for life stages
assigned to each treatment level were essentially not significantly different (p<0.05) fromcontrol, with the exception of pupal cells (Table C-3). For pupal cells, the initial number ofcells tended to be lower for the control group. The reason for this finding is not known but
differences caused by imidacloprid treatments were measured in subsequent analyses. At thistime, adults were in greatest number followed by pupal cells and then larval and egg cells(Figure C-1).

•

CCA4: Although this assessment of hive health was taken only 3 weeks into the exposure
period, significant regression indicated decreasing numbers of pupal and larval cells withincrease in imidacloprid dose (Figure C-2). For pupal cells the effect was first measured at the
50 ppb treatment (p<.1)) and then at progressively increasing probability levels for 100 and
200 ppb treatments. For larval cells the effects were significant for the 100 ppb treatment andhigher. Effects on pupal and larval cells numbers were persistent throughout the subsequent
CCAs (Figures C-2 to C-6).

•

CCA5: Decreases in numbers of adult and egg cells were now indicated at the higher 100 and
200 ppb dose levels (Figure C-3).

•

CCA6: Decreases noted in the previous CCAs at the 100 and 200 pbb levels of dose were
measured for all life stages. For adult cells, decreased numbers of adults were also measured
for the 50 ppb treatment, indicating a dose response to treatments starting at this level (Figure
C-4).

•

CCA7: At CCA7 higher order regression coefficients were significant for counts of adults
and larval cells (Table C-2). The curvilinear nature of the response reflected the extreme
149236

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
effects on reductions in numbers measured for the 100 and 200 ppb levels of imidacloprid dose
(Figure C-5).
•

CCA8: The pattern noted at CCA7 was reflected at the final assessment at CCA8 where
curvature in response measured for all life stages reflected a grouping of treatment levels:
Results were similar for 0, 12.5, and 25 ppb treatments and with 50, 100, and 200 ppb
treatments reflecting detrimental effects due to imidacloprid treatment (Table C-2, Figure C6). Loss of hives at 100 and 200 ppb treatments was an obvious effect resulting in essentiallyloss of all life stages at these treatments. But additional decreases in numbers of adults, pupal,
and egg cells were measured at the 50 ppb treatment compared to the control, indicating lower
vigor of hives at this treatment.

Table C-2. Regression for regression effects conducted within each CCA and for each bee life
stage.
Regression Results for Each CCA
Bee Life Stage
Adult

Pupal

LarvalEgg

Regression Effect

CCA3

CCA4

CCA5

CCA6

CCA7

CCA8

Pr>t

Pr>t

Pr>t

Pr>t

Pr>t

Pr>t

Dose Linear

0.69450.06220.03410.0003<.0001<.0001

Dose Quadratic

0.17630.17610.017

0.2027

0.09380.034

Dose Cubic

0.32790.143

0.59330.87890.0501

0.0417

Dose Linear

0.0923<.0001<.0001<.0001<.0001<.0001

Dose Quadratic

0.9986

0.41650.13660.28410.17580.0437

Dose Cubic

0.22440.69870.91010.70080.11170.0511

Dose Linear

0.626<.00010.0003<.0001<.0001<.0001

Dose Quadratic

0.19920.35510.88780.10370.57890.0968

Dose Cubic

0.2381

0.34240.91010.20080.00550.0275

Dose Linear

0.76450.24510.021<.00010.0003<.0001

Dose Quadratic

0.6786

0.29950.42790.0421

0.96730.0244

Dose Cubic

0.70190.0996

0.40220.18420.95760.1589150237

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Table C-3. Mean comparison for the response between each level of dose at each CCA for eachbee life stage. The difference is the percent frame coverage of dose1 minus dose2 where a positive
value indicates a higher value for the lower dosage and a negative value indicates a lower value
for the lower dosage.
Significant Pairwise Comparisons Testing Differences Between Each Level of Imidicloprid Dose at Each CCA and Each Life Stage
CCA3
CCA4
CCA5
Life
Life
Life
Stage Dose1 Dose2 PR>t Difference Stage Dose1 Dose2 PR>t Difference Stage Dose1 Dose2 PR>t Difference
Pupal

Larval012.5

0.0969

-0.06

0

25

0.0577

-0.07

0

200

0.0185

-0.09

0

100

0.0972

0.03

25

100

0.0709

0.04

Adult

Pupal

LarvalEgg

0

200 0.0225

-0.13

25

200 0.0812

-0.12

Adult

100

200 0.0177

-0.17

0

50 0.0822

0.06

0100 0.0032

0.1

0200 <0.0001

0.16

12.5

50

0.048

0.08

0

50

0.01720.1

12.5

100 0.0026

0.12

0

100

0.00020.15

12.5

200 <0.0001

0.18

0

200

<0.0001

0.02

Pupal

0

50

0.0636

0.12

0

100

0.0079

0.17

12.5

50

0.0397

0.15

12.5

100

0.006

0.21

12.5

200

0.0674

0.13

25

100

0.0525

0.14

25

100

0.027

0.09

12.5

50

0.04280.09

25

200 0.0002

0.15

12.5

100

0.00140.15

50

200 0.0065

0.11

12.5

200

<0.00010.22

100

200 0.0961

0.06

25

100

0.00560.13

0100 0.0003

0.05

25

200

<0.0001

0.19

0200 <0.0001

0.07

50

200

0.0094

0.12

12.5

200

0.024

0.04

0

100

0.045

0.04

25

100 0.0065

0.05

0

200

0.00050.07

25

200 0.0003

0.06

12.5

200

0.01020.06

50

100 0.0182

0.04

25

200

0.0205

0.05

50

200

0.001

0.06

50

200

0.01790.05

0100 0.0367

0.03

0

100

0.0407

0.03

0

200

0.05380.03

25

100 0.0491

0.03

50

100 0.0184

0.03

Larval

Egg

151
238

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Table C-3 Continued. Mean life stage comparisons continued for CCA6 through CCA8:
Significant Pairwise Comparisons Testing Differences Between Each Level of Imidicloprid Dose at Each CCA and Each Life Stage
CCA6
CCA7
CCA8
Life
Life
Life
Stage Dose1 Dose2 PR>t Difference Stage Dose1 Dose2 PR>t Difference Stage Dose1Dose2PR>t
Difference
Adult

Pupal

LarvalEgg

050

0.0246

0.14

0

100

0.016

0.15

Adult

0

100

0.0036

0.14

0

200

0.0034

0.14

Adult

0

50 0.00707

0.11

0

100

0.0004

0.23

0

200

0.001

0.21

12.5

50

0.0142

0.13

0

200

0.0007

0.22

12.5

50

0.0525

0.14

12.5

100

0.0002

0.21

12.5

50

0.0121

0.19

12.5

100

0.0374

0.15

12.5

200

0.00020.21

12.5

100 <0.0001

0.3

12.5

200

0.0042

0.21

25

50

0.0278

0.12

12.5

200

0.0001

0.3

25

50

0.0811

0.12

25

100

0.0004

0.2

25

50

0.0076

0.2

25

100

0.059

0.13

25

200

0.00040.2

25

100 <0.0001

0.31

25

200 <0.0001

0.3

0

50

0.05

25

200

0.0074

0.19

0

100

<0.0001

0.14

Pupal

0

100

0.0001

0.05

0

200

<0.0001

0.06

Pupal

0.09420200<0.0001

0.19

12.5

100

<0.0001

0.06

0

100

0.0007

0.11

12.5

100

0.02

0.09

12.5

200

<0.0001

0.07

0

200

0.0011

0.11

12.5

200

0.0003

0.14

25

100

0.0044

0.04

12.5

50

0.0189

0.09

25

100

0.0096

0.1

25

200

0.0003

0.05

12.5

100

0.0002

0.15

25

200

0.0001

0.15

50

100

0.0063

0.04

12.5

200

0.0002

0.14

50

100

0.0191

0.09

50

200

0.0005

0.05

25

50

0.014

0.09

50

200

0.0003

0.14

0

12.5

0.0023

-0.02

25

100

0.0001

0.15

0

100

0.0005

0.05

0

100

0.0127

0.02

25

200

0.0002

0.15

0

200

0.0003

0.06

0

200

0.0009

0.02

0

100

0.00130.05

LarvalLarval12.5

250.0178

0.04

12.5

25

0.0958

0.01

0

200

0.0022

0.05

12.5

100

<0.0001

0.07

12.5

50

0.02

0.02

12.5

100

0.0008

0.06

12.5

200

<0.0001

0.07

12.5

100

<0.0001

0.04

12.5

200

0.0012

0.06

25

100

0.0609

0.03

12.5

200

<0.0001

0.05

25

50

0.0609

0.03

25

200

0.0511

0.03

25

100

0.0017

0.03

25

100

0.0002

0.07

50

100

0.0079

0.05

25

200

0.0001

0.03

25

200

0.0003

0.07

50

200

0.0063

0.05

50

100

0.0122

0.02

50

100

0.0435

0.04

0

100

<0.0001

0.05

50

200

0.0012

0.03

50

200

0.0609

0.03

Egg

0

200

0.0006

0.04

0

200

0.0014

0.02

12.5

100

0.0012

0.04

12.5

100

0.0284

0.01

12.5

200

0.005

0.04

12.5

200

0.000425

100

0.0148

0.03

25

200

0.049525200

0.0477

0.03

50

200

0.0192

0.01

50

100

0.0043

0.04

50

200

0.0162

0.03

Egg

0

50

0.0158

0.02

0

100

0.0002

0.03

0.02

0

200

0.0007

0.03

0.01

12.5

25

0.0871

-0.02

12.5

100

0.0071

0.03

12.5

200

0.0156

0.02

25

50

0.00160.03

25

100 <0.0001

0.04

25

200 <0.0001

0.04

152
239

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

Proportion of Frame Coverage

Comparisons to control: Large gray dot 0.05<p<0.1; Large red dot p<0.05.
0.4

CCA3

0.35
0.3
0.25
0.2

Adult

0.15

Egg
Larval

0.1

Pupal

0.05
0
Control

12.5

25
50
Treatment Group

100

200

Figure C-1. Response of each life stage measured prior to the initiation of imidacloprid treatments
at CCA3. Except for pupal cells, the baseline for each group was not significantly different (p<0.05)
from control. For pupal cells, significant differences from the control were determined fortreatment locations except for the 50 ppb group. The reason for these differences is not known.

Proportion of Frame Coverage

Comparisons to control: Large gray dot 0.05<p<0.1; Large red dot p<0.05.
0.5

CCA4

0.45
0.4
0.35
0.3
0.25

Adult

0.2

Egg

0.15

Larval

0.1

Pupal

0.05
0
Control

12.5

25
50
100
200
Treatment Group
Figure C-2. At CCA4, significant dose related effects were measured for larval and pupal life
stages. Sporadic effects were noted for egg and adult cells, which were most likely not related todosing level.

153
240

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

Proportion of Frame Coverage

Comparisons to control: Large gray dot 0.05<p<0.1; Large red dot p<0.05.
0.5

CCA5

0.4
0.3

Adult
Egg

0.2

Larval
0.1

Pupal

0
Control

12.5

25
50
100
200
Treatment Group
Figure C-3. Effects on larval and pupal cells measured at CCA4 were sustained in CCA5 withsignificant reductions from control (p<0.05) at the 50, 100, and 200 ppb levels for pupal cells andat the 100 and 200 level for larval cells.

Proportion of Frame Coverage

Comparisons to control: Large gray dot 0.05<p<0.1; Large red dot p<0.05.
0.4

CCA6

0.35
0.3
0.25
0.2

Adult

0.15

Egg

0.1

Larval

0.05

Pupal

0
Control

12.5

25
50
100
200
Treatment Group
Figure C-4. At CCA 6 effects on pupal and larval cells were again sustained with onset of dose
related effects measured for egg and adult cells. Specifically, significant reductions (p<0.05) in thenumber of adults and eggs were determined, along with effects to larval and pupal cells that were
determined in CCAs 4 and 5.

154
241

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

Proportion of Frame Coverage

Comparisons to control: Large gray dot 0.05<p<0.1; Large red dot p<0.05.
0.4

CCA7

0.35
0.3
0.25
0.2

Adult

0.15

Egg

0.1

Larval
Pupal

0.05
0
Control

12.5

25
50
100
200
Treatment Group
Figure C-5. At CCA7, decreases in all life stages from control group were measured for 100 and
200 ppb levels of imidacloprid.
Comparisons to control: Large gray dot 0.05<p<0.1; Large red dot p<0.05.
0.35
Proportion of Frame Coverage

CCA8

0.3
0.25
0.2

Adult

0.15

Egg
Larval

0.1

Pupal

0.05
0
Control

12.5

25
50
100
200
Treatment Group
Figure C-6. Clear effects of hive death were measured at CCA8 at the 100 and 200 ppb
treatments. Decreased numbers of adults, pupal, and egg cells were also measured at the 50 ppb
treatment.

155
242

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Food Stores
A second multivariate analysis was conducted to determine potential effects on honey and pollen
cells. Univariate mixed model analyses were conducted for hive weight. The triple interaction of
Day*Dose*Response was again significant in the combined analysis of pollen and honey cells(Table C-4).
Table C-4. Analysis of Food Stores (Pollen and honey cells, and hive weight
Overall Model Effect

Num DF

Den DFPr > F

Response

1

116<.0001

Day

4

294

<.0001

Day*Response

4306<.0001

Dose

5

118

0.0002

Dose*Response

5116

0.8633

Day*Dose20294<.0001

Day*Dose*Response

20

306

<.0001

In light of the significant three way interaction, the approach used for bee life stages was followed
where further multivariate analyses were conducted to determine the pattern of response at each
CCA. Results from the regression analysis for each CCA indicated that there was a differential
response over time (Table C-5).
•

CCA3: As indicated in the results for bee life stages initial values for pollen and honey cells
and hive weight were essentially similar between all treatment levels. Some slight differences
between the controls and a few treatment levels for pollen cells were measured but theseappeared to be sporadic and not related to dose levels (Table C-6; Figure C-6 and Figure C12).

•

CCA4: The number of pollen cells and hive weight were first affected at CCA4 withdecreases measured in relation to increasing imidacloprid concentration (Figure C-7 andFigure C-13). Dose related effects on pollen cells were evident at the 50 ppb treatment with
progressive decreases in numbers in relation to increases in level of dose. The pattern for hive
weight mimicked that observed for pollen cells but significant effects were measured at the
100 ppb treatment and higher. Again, these decreases are rather significant because thisassessment was made only 3 weeks into the exposure period.

•

CCA5: The pattern in response for pollen cells and hive weight was similar to that observed
at CCA4 (Figure C-8 and Figure C-13). An additional effect was observed for hive weightin that a significant effect was also observed at the 50 ppb level of dose. Together the effects
on honey cells and hive weight indicated two groupings of effects for the levels of dose: One
where the numbers were similar between 0, 12.5, and 25 ppb treatments and a second where
effects of imidacloprid dose were measured for 50, 100, and 200 ppb treatments There was one
156
243Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
significant effect indicated for honey cells but it was an isolated effect where higher numbers
were recorded for the 200 ppb treatment versus the rest of the treatments.
•

CCA6: At CCA6 decreased numbers of pollen cells were only measured at the 200 ppb levelof imidacloprid dose (Figure C-9). The response of honey cells, on the other hand, now
mimicked the initial response for pollen cells in that decreased numbers compared to thecontrol were measured at 50 ppb and higher dose levels. The two grouping pattern noted at
CCA5 was now reflected in the responses for number of honey cells and hive weight (Figure
C-9 and Figure C-14).

•

CCA7: The response at CCA7 was very similar to that measured at CCA6 with the same notedgrouping of effects for honey cells and hive weight (Figure C-10 and Figure C-15). Numbersof pollen cells were not as plentiful as for honey cells throughout the study. By this assessment
all treatment means were low with values either at or below 0.05% frame coverage. The lower
coverage at this CCA is most likely the cause for diminishing effects of imidacloprid treatment
at this CCA and perhaps noted at the previous CCA6. Effects though were still measured atthe 200 ppb level when compared to the control.

•

CCA8: Similar to the effects measured for life stages, the pattern noted at CCA7 was reflected
at the final assessment at CCA8 where curvature in response was measured for all life stages,
reflecting the extreme loss of hives at the 100 and 200 ppb levels of imidacloprid dose (Table
C-5). Lowered counts for honey and pollen cells were also measured for the 50 ppb treatment
when compared to control values, indicating lower hive vigor (Figure C-11 and Figure C16). These results provide evidence for the sustained influence of effects noted at CCA7 on theover wintering heath of hives.

Table C-5. Regression for linear and quadratic effect conducted within each CCA and for pollen
or nectar cells and hive weight.
Regression Results for Each CCA
Food Storage
Variable
Honey Cells

Pollen Cells

Hive Weight

CCA3

CCA4

CCA5

CCA6

CCA7

CCA8

Regression Effect

Pr>t

Pr>t

Pr>t

Pr>t

Pr>t

Pr>t

Dose Linear

0.50370.58420.00560.0001<.0001<.0001

Dose Quadratic

0.28010.79430.08210.92440.25450.0272

Dose Cubic

0.72630.88150.93650.63550.644

0.1049Dose Linear

0.2855<0.0001<0.0001<0.0001

0.002<.0001

Dose Quadratic

0.58490.00070.00060.91760.68430.0279Dose Cubic

0.222

0.81350.78220.46040.89620.0215

Dose Linear

0.99520.00270.00110.00140.0007<.0001

Dose Quadratic

0.91640.23060.00230.24940.14240.0541

Dose Cubic

0.05810.70240.58480.49480.4931

0.002

157
244

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Table C-6. Mean comparisons for the response between each level of dose for number of honeyand pollen cells and hive weight conducted at each CCA. For pollen and honey cells thedifference is the percent frame coverage of dose1 minus dose2 where a positive value indicates ahigher value for the lower dosage and a negative value indicates a lower value for the lower
dosage. For hive cells the difference represents measurements made in pounds (lbs).
Significant Pairwise Comparisons Testing Differences Between Each Level of Imidicloprid Dose at Each CCA for Food Stores
CCA3
EffectPollenHive
WeightDose1 Dose2

CCA4
PR>t Difference Effect

025

0.0582

0.02

0

200

0.071

0.02

Pollen

CCA5
PR>t Difference Effect

Dose1 Dose2050<.0001

0.04

0

100

<.0001

0.06

Dose1 Dose2

PollenPR>t Difference0

50 <0.0001

0.07

0

100 <0.0001

0.09

0

12.5

0.0149-40200<.00010.07

0200 <0.0001

0.1

12.5

100

0.0081

5.2

12.5

50

0.00670.03

12.5

50 0.0001

0.07

12.5

200

0.0261

4.1

12.5

100

<.00010.05

12.5

100 <0.0001

0.09

12.5

200

<.00010.05

12.5

200 <0.0001

0.1

25

50

0.00550.03

25

50 0.0038

0.05

Hive
Weight25

100<.00010.05

25

100 <0.0001

0.07

25

200

<.00010.06

25

200 <0.0001

0.09

50

100

0.0568

0.02

50

200

0.0063

0.03

0

100

0.0208

4.3

0

200

0.0278

4.1

12.5

50

0.0391

4.4

12.5

100

0.0056

6.1

12.5

200

0.0074

5.9

25

100

0.0273

4.8

25

200

0.0348

4.5

50

200

0.033

0.04

0

200

0.009

-0.08

12.5

200 0.0177

-0.08

25

200 0.0498

-0.07

50

200 0.0036

-0.11

100

200 0.0233

-0.08

0

12.5 0.0305

3.6

0

50 <0.0001

7.40100 <0.0001

10.7

Honey

Hive
Weight0

200 0.0015

5.5

12.5

50 0.0464

3.8

12.5

100 0.0005

7.1

25

50 0.0024

6

100 <0.0001

9.3

25
25

200

0.031150

100

0.0843

4.1
3.3

100

200

0.0081

-5.2

158
245

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Table C-6 Continued. Mean food storage comparisons continued for CCA6 through CCA8:
Significant Pairwise Comparisons Testing Differences Between Each Level of Imidicloprid Dose at Each CCA for Food Stores
CCA6
EffectPollenHoney

Hive
WeightDose1 Dose2

CCA7
PR>t Difference EffectPollenDose1 Dose2

PR>t

Difference

CCA8
Effect Dose1 Dose2 PR>t

0

12.5

0.0086

-0.03

0

200

0.0087

0.02

0

12.5

0.0677

-0.02

0

200

0.0022

0.03

12.5

50

0.0203

0.02

0

50

0.0601

0.02

12.5

50

0.0011

0.04

12.5

200

0.0009

0.04

0

100

0.0003

0.05

12.5

100

0.0015

0.04

25

50

0.0492

0.02

0

200

0.0004

0.05

12.5

200

<0.0001

0.06

25

200

0.0027

0.03

12.5

50

0.017

0.05

25

50

0.0545

0.02

100

200

0.04680.02

12.5

100 <0.0001

0.07

25

100

0.0683

0.04

0

50

0.0392-0.00312.5

200 <0.0001

0.07

25

200

0.0006-0.0010100

0.0083

0.11

50

100

0.0975

0.02

50

200

0.0967

0.02

0

200

0.0002

0.14

0

50

0.08

0.05

100

200

0.078

0.02

12.5

50

0.0649

0.11

0

100

0.00080.1

050

0.0255

0.11

12.5

100

0.0188

0.14

0

200

0.003

0.09

0

200

0.0002

0.19

12.5

200

0.0011

0.2

12.5

50

0.018

0.08

12.5

200

0.0036

0.17

25

50

0.032

0.13

12.5

100

0.0002

0.13

25

50

0.0144

0.14

25

100

0.0082

0.16

12.5

200

0.0007

0.12

25

100

0.0623

0.11

25

200

0.0004

0.22

25

50

0.0394

0.07

25

200

0.0002

0.22

0

50

0.0471

9.8

25

100

0.0006

0.12

100

200

0.0436

0.11

0

100

0.0206

11.5

25

200

0.0019

0.11

0

50

0.0818

8.5

0

200

0.0077

13.3

0

25

0.0618

-11.7

0

100

0.0389

10.2

12.5

50

0.0228130100

0.000224

0200

0.0151

12

12.5

100

0.0105

14.7

0

200

0.0013

20.7

12.5

50

0.0615

10.6

12.5

200

0.004216.5

12.5

100 <0.0001

30.4
27

Honey

Hive
WeightPollenDifference

HoineyHive
Weight12.5

1000.0316

12.2

25

50

0.021

13.2

12.5

200

0.000312.5

2000.0137

14.1

25

100

0.0096

14.9

25

50

0.028116

25

50

0.0292

12.4

25

200

0.003916.7

25

100 <0.0001

35.9

25

100

0.014

14

25

200 <0.0001

32.5

25

200

0.0057

15.9

50

100

0.0069

19.9

50

200

0.0241

16.5

159
246

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

Proportion of Frame Coverage

Comparisons to control: Large gray dot 0.05<p<0.1; Large red dot p<0.05.
0.2

CCA3

0.16
0.12
Honey

0.08

Pollen0.04
0Control

12.5

25
50
100
200
Treatment Group
Figure C-6. Response of pollen and honey cells in relation to treatment group at CCA3.
Comparisons to control: Large gray dot 0.05<p<0.1; Large red dot p<0.05.

Proportion of Frame Coverage

CCA4
0.25
0.2
0.15
Honey

0.1

Pollen0.05
0Control

12.5

25
50
Treatment Group

100

200

Figure C-7. At CCA4, onset of dosage related effects were measured for pupal cells and hive
weight (see Figure C-13).

160
247

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Comparisons to control: Large gray dot 0.05<p<0.1; Large red dot p<0.05.
0.25
Proportion of Frame Coverage

CCA5

0.2
0.15
Honey

0.1

Pollen0.05
0Control

12.5

25
50
100
200
Treatment Group
Figure C-8. At CCA5, the effects on pollen cells and hive weight (See Figure C-14) measured
previously at CCA4 were sustained at the 50, 100, and 200 ppb treatment groups (p<0.05) forpollen cells with additional significant effects at the 50 ppb level (p<0.05) measured for hive
weight. An isolated significant effect for honey was noted at the 200 ppb treatment.

Proportion of Frame Coverage

Comparisons to control: Large gray dot 0.05<p<0.1; Large red dot p<0.05.
0.35

CCA6

0.3
0.25
0.2
0.15

Honey

0.1

Pollen0.05
0Control

12.5

25
50
Treatment Group

100

200

Figure C-9. At CCA6, significant treatment related effects were also measured for the number ofhoney cells with decreases at the 50, 100 and 200 ppb dose levels. Effects measured on hive weightat CCA5 were sustained at CCA6 (See Figure C-15).

161
248

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

Proportion of Frame Coverage

Comparisons to control: Large gray dot 0.05<p<0.1; Large red dot p<0.05.
0.35

CCA7

0.3
0.25
0.2
0.15

Honey

0.1

Pollen0.05
0Control

12.5

25
50
100
200
Treatment Group
Figure C-10. At CCA7 the effects on number of honey cells and hive weight (see Figure C-16)
appear to form two groups where numbers are similar between 0, 12.5 and 25 ppb treatments andthen imidacloprid dose-related effects were measured for the 50, 100, and 200 ppb treatments (SeeFigure C-x for hive weight).

Proportion of Frame Coverage

Comparisons to control: Large gray dot 0.05<p<0.1; Large red dot p<0.05.
0.16

CCA8

0.12
0.08
Honey
Pollen0.04
0Control

12.5

25
50
100
200
Treatment Group
Figure C-11. At CCA8 effects measured at the 100 and 200 ppb treatments resulted in loss ofcolonies after overwintering (see Figure C-17for hive weight graph). Lower vigor at 50 ppb
treatment was also indicated by the lower numbers of adults, pupal, and egg cells whencompared to the control hives.

162
249

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Comparisons to control: Large gray dot 0.05<p<0.1; Large red dot p<0.05.
43.5

CCA3

43
42.5

Hive Weight (lbs)

42
41.5
41
40.5
40
39.5
39
38.5
38
Control

12.5

25

50

100

200

Treatment Group

Figure C-12. Response of hive weight in relation to treatment group at CCA3
Comparisons to control: Large gray dot 0.05<p<0.1; Large red dot p<0.05.
59

CCA4

58

Hive Weight (lbs)

57
56
55
54
53
52
51
50
49
Control

12.5

25

50

Treatment Group

100

200

Figure C-13. Response of hive weight in relation to treatment group at CCA4.

163
250

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Comparisons to control: Large gray dot 0.05<p<0.1; Large red dot p<0.05.
50

CCA5

Hive Weight (lbs)

48
46
44
42
40
38
36
Control

12.5

25

50

100

200

Treatment Group

Figure C-14. Response of hive weight in relation to treatment group at CCA5
Comparisons to control: Large gray dot 0.05<p<0.1; Large red dot p<0.05.
60

CCA6

Hive Weight (lbs)

50
40
30
20
10
0
Control

12.5

25

50

Treatment Group

100

200

Figure C-15. Response of hive weight in relation to treatment group at CCA6

164
251

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Comparisons to control: Large gray dot 0.05<p<0.1; Large red dot p<0.05.
60

CCA7

Hive Weight

50
40
30
20
10
0
Control

12.5

25

50

100

200

Treatment Group

Figure C-16. Response of hive weight in relation to treatment group at CCA7
Comparisons to control: Large gray dot 0.05<p<0.1; Large red dot p<0.05.
50

CCA8

Hive Weight

40
30
20
10
0
Control

12.5

25

50

100

200

Treatment Group

Figure C-17. Response of hive weight in relation to treatment group at CCA8

165
252

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
State of California

Department of Pesticide Regulation

EVALUATION REPORT - Imidacloprid Pollen Colony Feeding StudyJohn Troiano, Research Scientist III
Alexander Kolosovich, Senior Environmental Scientist (Specialist)
June, 2018

A review of:- Dively, G.P., Embrey, M.S., Kamel, A., Hawthorne, D.J., & J.S. Pettis. (2015).
Assessment of chronic sublethal effects of imidacloprid on honey bee colony health. PLoS ONE,10(3), e011874. DOI:10.1371/journal.pone.0118748.

Introduction
The objective of the study was to determine sublethal effects on bee colony health as a result of
exposure to beebread fortified at 0, 5, 20, or 100 ug/kg of imidacloprid. The study was replicatedin two years in 2009 and 2010. The study design differed between the two years. In 2009, 2
replicate hives at each treatment level were located in 5 separate apriaries whereas in 2010 there
was only replicate hive in each treatment placed in 7 separate apriaries. Spacing between apiaries
was not specified but within an apiary the hives were spaced 10 meters apart.
Colony health was assessed by measuring the percentage of frame area covered with drawn cells,
adult bees, capped bees, cells with older larvae, cells packed with beebread and honey, thenumber of hives that survived overwintering, and measures of foraging activity. No significant
effect of imidacloprid treatment was measured in either year on coverage of drawn cells,
beebread, capped brood, and adult bees where measurements made at the end of exposure andprior to overwintering of hives. No dose effects indicating decreases in numbers were indicatedat the individual time intervals where measurements were taken to follow development
throughout the summer and fall. No significant consistent effects were indicated formeasurements made on foraging activity. The area of frame coverage for honey was consistently
greater at the 100 ug/kg treatment in both years. Inconsistent effects between years were noted
for measurements made on frequency of queen events, on number of supesedual cells, and inanalyses conducted on cumulative area under curves.

1
253

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

Statistical Analysis
Conclusions on the effect of treatment on overwintering survival of colonies also indicatedinconsistent effects when a statistical analysis for effect of dose was measured within each year.
In 2009 the statistical test used to measure effect of treatment on survival rate indicated asignificant effect of treatment with less hives surviving in imidacloprid treatments, whereas, in2010 survival percentages were similar between all treatment levels and no associated statisticalsignificance was measured. A further manipulation of the data that combined survival dataobtained prior to wintering and then after wintering produced an overall significant effect oftreatment, indicating reduction in survival rate in response to imidacloprid dose. A potential
oddity in the data was the measure of perfect survival of all hives in the control treatment in 2009
where 10 of 10 hives successfully overwintered. A boxplot of all 8 values composed of the ratemeasured at each treatment level in both years indicates that this may be an extreme value(Figure 1). The proportional mean of the 8 values is 0.62 with a standard deviation of 0.21. All
values except for the for the control value in 2009 fall within 1 standard deviation of the mean
where the range in measured values is 0.43 to 0.8 and the range for one standard deviation isfrom 0.41 to 0.83. The measurement of an overall significant effect is most likely driven by thisone value, especially when the months are combined and this value receives even greater weight.
The validity of measuring this extreme event in this study is not at question. But survival of
100% hives from overwintering in actual practice is an event with an extremely low probabilityof occurrence. The problem statement for investigating effects of stressors on rate ofoverwintering of beehives was based on an elevated rate of decreased survival from a normal
occurrence of around 15% to an increased rate of 25 to 30% of hives not surviving
overwintering. The veracity of results obtained from the study is not questioned. The noted
inconsistencies in effects between years, the lack of an imidacloprid effect on bee life stages, andpotential for skewing of results due to the survival rate for the control in 2009 indicate thatverification of effect of exposure by beebread equires more study. Consequently, derivation of a
LOEC or NOEC from this study would have a large uncertainty associated with it.

Conclusion
Investigation of the effect of exposure of beehives from ingestion of imidacloprid from pollen isof critical importance to determining the relevant endpoints for assuring healthy bee colonies.
However, derivation of a LOEC or NOEC from this study would have large uncertainty
associated with it. Even though the authors provide a basis for continued investigation on effectof ingestion of beebread dosed with imidacloprid, causes for uncertainty are the inconsistencies
measured for survival rate between years and lack of effects on bee life stages. Effects on bee life
stages prior to overwintering would be indicators for weakening of hives prior to overwintering.
2
254

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Inspection of the data indicated that complete survival of hives in the control treatment in 2009 isa result that is odd compared to the rest of the treatments. Furthermore, complete survival is not
an expected biological event. Consequently, this one value most likely exerts extraordinary
influence on the significance measured when data were combined between months and subject tofurther statistical analysis. Replication of the experiment is required in order to verify aconsistent effect of ingestion of beebread dosed with imidacloprid on health and survival of beecolonies.

3255

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Figure 1. Boxplot for distributional statistics for proportion of overwintering survival of
hives. Note that except for the control value proportion at 1.0 (all survived) measured forthe control in 2009, the remaining 7 values are captured within the range for a distribution
based on the mean plus/minus 1 standard deviation.

4256

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

Data Evaluation Report
Study Titles:
Louque, J. (2016): Colony feeding study evaluating the chronic
effects of clothianidin-fortified sugar diet on honey bee (Apismellifera) colony health under free foraging conditions.
Final Report Source:
Smithers Viscient, unpublished report No: 13798.4143 Activity ID
EBTIN114, February, 2016
Years of study: 2014-2015
PMRA#: 2610259
PMRA DACO#: 9.2.4.3
MRID: 49836101
Study Type:
Tier II colony feeding study conducted in an open field
Review Date (final): February 7, 2017
EPA reviewer:
Primary Evaluators: Michael Wagman, Biologist
Amy Blankinship, Senior Scientist
Data Statistical Analysis: Christine Hartless, Wildlife Biologist
PMRA reviewer:
Primary Evaluator: Nicole Lauro, Evaluation Officer
Data Statistical Analysis: Keith O'Rourke , Senior Epidemiologist/Bio-statistician
CDPR reviewer:
Primary Evaluators: Richard Bireley, Sr. Environmental Scientist (Specialist)
Alexander Kolosovich, Environmental Scientist
Russel Darling, Environmental Scientist
Brigitte Tafarella, Environmental Scientist
Denise Alder, Sr. Environmental Scientist (Specialist)
Data Statistical Analysis: John Troiano, Ph.D., Research Scientist III

1257

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

Table of Contents

Executive Summary....................................................................................................................................... 6
Consideration of Study Strengths, Limitations and Interpretation .......................................................... 8
1.

Study Objective ................................................................................................................................... 11

2.

Study Methods.................................................................................................................................... 11
2.1.

Test crop ..................................................................................................................................... 11

2.2.

Test chemical .............................................................................................................................. 11

2.3.

Test sites ..................................................................................................................................... 11

2.4.

Test organisms ............................................................................................................................ 13

2.5.

Treatments.................................................................................................................................. 14

2.5.1.

Preparation of stock solution.............................................................................................. 15

2.5.2.

Preparation of sugar solution ............................................................................................. 15

2.5.3.

Preparation of feeding solution .......................................................................................... 15

2.5.4.

Artificial Feeding ................................................................................................................. 16

2.6.

Meteorological Data ................................................................................................................... 16

2.7.

Observations ............................................................................................................................... 17

Important activity and dates................................................................................................................ 17
2.7.1.

Colony mortality.................................................................................................................. 20

2.7.2.

Colony Condition Assessments (CCA) ................................................................................. 20

2.7.3.

Evaluation of Disease or Pests in the Hive ......................................................................... 21

2.7.4.

Hive weights........................................................................................................................ 21

2.8.

3.

Residue analysis .......................................................................................................................... 21

2.8.1.

Pollen from outside sources ............................................................................................... 22

2.8.2.

Stored pollen and nectar in test hives ................................................................................ 22

2.8.3.

Feeding solution and stability of test item ......................................................................... 22

Results................................................................................................................................................. 23
3.1.

Land use near test hives.............................................................................................................. 23

3.2.

Pollen sources of test hives......................................................................................................... 24

3.3.

Consumption of spiked sucrose.................................................................................................. 24

3.4.

Examination of pesticides from other sources ........................................................................... 25

3.5.

Confirmation of test concentrations........................................................................................... 25

3.6.

Stability of the test item in feeding solution .............................................................................. 26

3.7.

Residues in hive matrices............................................................................................................ 26

3.7.1.

Clothianidin residues in hives prior to the feeding exposure ............................................. 26

3.7.2.

Residues in hive matrices during and after feeding exposure............................................ 262258

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
3.8.

Pathogens ................................................................................................................................... 34

3.8.1.
3.9.

Varroa Presence.................................................................................................................. 34

Statistical Analysis....................................................................................................................... 36

3.9.1.

Study Author’s Analysis....................................................................................................... 36

3.9.2.

Study Reviewer’s Statistical Analysis Approach.................................................................. 36

3.9.3 Treatment Effects Within a CCA ................................................................................................ 44
3.9.4 Temporal Trends Within a Treatment Level .............................................................................. 45
3.9.5 Colony Condition Assessment Response Variables ................................................................... 46
3.9.6 Life Stage Results ........................................................................................................................... 47
3.9.7 Colony Condition Assessments – Food Store Response Variables ............................................... 62
3.9.8 Hive Weight................................................................................................................................ 66
3.9.9
4.0

Hive mortality.......................................................................................................................... 67

Reviewer comments ....................................................................................................................... 69

4.1 General Considerations for Biological Interpretation....................................................................... 69
4.2 Control Performance......................................................................................................................... 70
4.3 Consideration of Study Strengths, Limitations and Interpretation.................................................. 70
5.0

Overall Study Conclusions............................................................................................................... 73

List of Tables
Table 1. Details about the test substance................................................................................................... 11
Table 2: GPS-coordinates of the test apiary sites....................................................................................... 12
Table 3. Treatment groups, feeding rates and feeding volume ................................................................. 14
Table 4. Hive assignment to test apiaries.................................................................................................. 15
Table 5. Chronological list of key dates and activities................................................................................ 18
Table 6. LOD for clothianidin ...................................................................................................................... 21
Table 7. Number of samples and sampling schedule for feeding solution and stability of test chemical. 22
Table 8. Average percent (%) land use pattern across the 12 study apiaries (based on 2014 Cropland
Data Layers (CDL))....................................................................................................................................... 23
Table 9. Average percent (%) land use pattern across the 12 study apiaries (based on 2011 National Land
Cover Database (NLCD)).............................................................................................................................. 23
Table 10. Dosing solution residue data from 03 July 2014 (Week 1) and 28 July 2014 (Week.................. 25
Table 11. The stability of clothianidin in feeding solution on 3 Jul and 28 Jul, 2014. ................................ 26
Table 12. Clothianidin concentrations (ppb) in uncapped hive nectar sampled 27 days after the start ofartificial feeding on 23 Jul, 2014 (CCA4). .................................................................................................... 28
Table 13. Clothianidin concentrations (ppb) in bee bread six weeks after the start of artificial feeding(CCA5). ........................................................................................................................................................ 29
Table 14. Clothianidin concentrations (ppb) in uncapped nectar 40-46 days after the start of artificial
feeding (CCA5). ........................................................................................................................................... 30
Table 15. Clothianidin concentrations (ppb) in capped honey 97--103 days after the start of artificial
feeding (CCA7). ........................................................................................................................................... 323259

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Table 16. Clothianidin concentrations (ppb) in capped honey 305 days after the start of artificial feeding(CCA9). ........................................................................................................................................................ 33
Table 17. Clothianidin concentration measured in hive uncapped nectar, capped honey and hive beebread compared to nominal concentrations in the test feeding solutions ................................................ 34
Table 18. Timeline including major milestones of study ............................................................................ 37
Table 19. BIC values for fitted models. CCA3 – CCA7 -clothianidin............................................................ 41
Table 20. Results of one-sided Dunnett’s test (comparing control to each treatment group), correlations
modeled using CSH. Cells include the treatment groups that were significantly lower than control........ 44
Table 21. Results of two-sided Dunnett’s test (comparing CCA3 to each following CCA), correlations
modeled using CSH. .................................................................................................................................... 45
Table 22. Results of two-sided Dunnett’s test (comparing CCA3 to each following CCA), correlations
modeled using CSH. .................................................................................................................................... 45
Table 23. Estimated percent reduction from control for mean number of adults .................................... 47
Table 24. Estimated percent reduction from control for number of eggs. ................................................ 50
Table 25. Estimated percent reduction from control for number of larvae (open/uncapped brood)....... 52
Table 26. Estimated percent reduction from control for number of capped (pupal) cells. ....................... 55
Table 27. Estimated percent reduction from control for total individuals................................................. 57
Table 28. Estimated percent reduction from control for total brood cells ................................................ 58
Table 29. Estimated percent reduction from control for pollen stores ..................................................... 63
Table 30. Estimated percent reduction from control for nectar/honey stores ......................................... 65
Table 31. Estimated percent reduction from control for food (pollen + nectar) storage .......................... 66
Table 32. Proportion of hive weight following exposure of honey bees to varying concentrations ofclothianidin in the diet for six weeks. ......................................................................................................... 68
Table 33. Hive mortality statistics after overwintering measure at CCA8 ................................................. 68

List of Figures
Figure 1: Location of test apiary sites ......................................................................................................... 12
Figure 2. Layout of test hives in a test site ................................................................................................. 14
Figure 3. Average minimum and maximum temperatures across all apiaries ........................................... 17
Figure 4. Mean total food consumption (L) per colony during the 6-week exposure period .................... 24
Figure 5. Varroa infestation levels in control and treatment groups prior to exposure (CCA 3),
immediately following the termination of exposure (CCA5) and after over-wintering (CCA9). ................ 35
Figure 6. Nosema spore loads in control and treatment groups prior to exposure (CCA 3), immediately
following the termination of exposure (CCA5) and after over-wintering (CCA9)....................................... 36
Figure 7. Studentized residual plots for eggs with covariance structures of (left) compound symmetry(CS) and (right) compound symmetry with heterogeneous variance (CSH)............................................... 42
Figure 8. Number of adult honeybees at colony condition assessments (CCA) 3 thru 7 for each treatmentgroup . ......................................................................................................................................................... 48
Figure 9. Number of adult honeybees at colony condition assessments (CCA) 3 thru 7 for the control andthree lowest treatment groups................................................................................................................... 49
Figure 10. Number of eggs (cells) following exposure to varying concentrations of clothianidin in the dietacross CCA3 – CCA7 . .................................................................................................................................. 50
Figure 11. Number of egg cells following exposure to varying concentrations of clothianidin in the dietacross CCA3—CCA7 in the control, 10, 20, and 40 µg/L groups only… ...................................................... 51
Figure 12. Number of open cells (larvae) at colony condition assessments (CCA) 3 thru 7 for eachtreatment group ......................................................................................................................................... 53
4260

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Figure 13. Number of open cells (larvae) at colony condition assessments (CCA) 3 thru 7 for the control
and three lowest treatment groups............................................................................................................ 54
Figure 14. Number of capped cells (pupae) at colony condition assessments (CCA) 3 thru 7 for eachtreatment group…....................................................................................................................................... 55
Figure 15. Number of capped cells (pupae) at colony condition assessments (CCA) 3 thru 7 for thecontrol and three lowest treatment groups… ............................................................................................ 56
Figure 16. Number of live (adult numbers+cells of brood) at colony condition assessments (CCA) 3 thru 7
for each treatment group… ........................................................................................................................ 57
Figure 17. Number of total live (adult+brood) at colony condition assessments (CCA) 3 thru 7 for thecontrol and three lowest treatment groups……… ...................................................................................... 58
Figure 18. Number of brood at colony condition assessments 3--7 for each treatment group.. .............. 59
Figure 19. Number of brood at colony condition assessments (CCA) 3 thru 7 for the control and threelowest treatment groups…. ........................................................................................................................ 60
Figure 20. Summary of living organism parameters at the 10 µg/L treatment group ............................... 61
Figure 21. Summary of living organism parameters at the 20 µg/L treatment group ............................... 61
Figure 22. Summary of living organism parameters at the 40 µg/L treatment group ............................... 61
Figure 23. Summary of living organism parameters at the 80 µg/L treatment group ............................... 62
Figure 24. Summary of living organism parameters at the 160 µg/L treatment group ............................. 62
Figure 25. Number of pollen cells at colony condition assessments 3-7 for each treatment group. ........ 63
Figure 26. Number of pollen cells at colony condition assessments (CCA) 3 thru 7 for the control andthree lowest treatment groups................................................................................................................... 64
Figure 27. Number of honey cells at colony condition assessments 3-7 for each treatment group. ........ 65
Figure 28. Number of food cells (honey+pollen) at colony condition assessments (CCA) 3 thru 7 for eachtreatment group. ........................................................................................................................................ 66
Figure 29. Proportion of hive weight following exposure of honey bees to varying concentrations ofclothianidin in the diet for six weeks. ......................................................................................................... 67
Figure 30. Overall hive survival after overwintering (reproduced from study report, p. 34). ................... 68

Table of Appendices
Appendix A: Details of PMRA Statistical Analysis …………………………………………………………………………………75
Appendix B. Timeline of addition and removal of supers during the clothianidin field trial ................... 117
Appendix C. Summary Statistics for Each Response Variable for All Clothianidin Treatment Levels Across
CCAs 3—7.................................................................................................................................................. 119

5261

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

Executive Summary
A colony feeding study was conducted with honey bees to assess the potential for long-term
effects, including overwintering survival, resulting from exposure to clothianidin in artificial
nectar (i.e. spiked sugar) diet. The study was conducted in twelve test areas of low agricultural
cultivation (Apiaries A – L) in North Carolina from June 17, 2014 (when hives were moved to thestudy apiaries) to April 27, 2015 (final colony condition assessment). Eighty-four hives weredivided according to hive strength (number of brood frames) with the strongest 7 hives ssigned to
Apiary A and the weakest 7 hives assigned to Apiary L. Within each apiary, the 7 hives wererandomly assigned to control and treatment groups.
At each apiary, five test hives were artificially fed with 50% sugar solution spiked with clothianidin
at 10, 20, 40, 80 or 160 µg ai/L for six weeks continuously in the field, with two hives at eachapiary serving as controls. Assuming the density of a 50% sugar solution is 1.2296 g/ml, thereviewer calculated that the test concentrations at 10, 20, 40, 80 or 160 µg/L are equivalent to 8.1,
16.3, 32.5, 65.1, and 130.1 ppb (µg/kg), respectively. Residue analysis of the dosing solutions on
7/3/14 and 7/28/2014 provided mean measured ppb (µg/kg) concentrations of <LOD (0.5 ppb),
9.5, 19.0, 35.6, 71.8 and 140.0 ppb (µg/kg), respectively with stability samples from hive feeders
indicating 93—105% recoveries in the dosing solutions.
Nine Colony Condition Assessments (CCAs) were conducted during the study. Three CCAs
(CCA1 – 3; May 12, June 2 and 18, respectively) were conducted prior to feeding to determine
hive strength and initial hive conditions. A CCA was conducted during exposure (CCA4; July 15)
with another one conducted within one week after termination of exposure (CCA5; August 5)
which characterize hive conditions during exposure. Two more CCAs were conducted at 5 (CCA6;
Sept. 8) and 10 (CCA7; Oct. 14) weeks after exposure (or 11 for hives in the 80 and 160 ppb
treatment groups, only) to assess the chronic effect following exposure to clothianidin and tocharacterize pre-overwintering hive conditions. Two final CCAs were conducted after
overwintering in mid-March 2015 (CCA8; Mar 17-19 for all treatment groups except for the 80
ppb treatment group whose CCA was delayed to April 2) and mid-late April (CCA9; April 22-27)
to assess potential exposure impact on survival and chronic colony level effects. Multiple
parameters, such as hive weight, number of individuals at different life stages in the hive, hive
honey and pollen stores, and hive overwintering survival, were measured during the course of thestudy.
Levels of clothianidin residues were measured before (in pollen and nectar collected from hives’at CCA2), during (uncapped nectar at CCA4), immediately post-exposure (uncapped nectar andbee bread at CCA5), 10 weeks after the feeding exposure (capped honey at CCA7) and following
overwintering (capped honey at CCA9). Potential contamination of colonies by pesticides fromother food sources was monitored using pollen and uncapped nectar collected in additional hives
at each apiary that served as monitoring hives. The results showed that while there were a few
instances of clothianidin detected in the pollen (bee bread) and nectar (uncapped and capped) of
the control hives, the frequency and magnitude of these detections is not expected to confound theresults of this study. The residue samples collected at CCA2 were from four hives, while residue
samples at CCAs 4, 5, 7 and 9 were from all available hives with sufficient material for analysis.
Mean residues measured in hive matrices generally demonstrated that higher treatment exposurescorresponded well to higher residues in hive matrices. There were individual hive variations in
6262

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
measured residues, with some overlap in measured hive concentrations, particularly at the lower
doses. This variability likely originates from the limited spatial and temporal sampling
methodology (i.e. one sample from one side of the comb on one frame to represent a hive, andonly at 4 CCAs) employed for this study. Mean measured residues at CCA5 (end of exposure) in
uncapped nectar were 68% (5.5 ppb), 62% (10.2 ppb), 61% (19.9 ppb), 57% (37.0 ppb) and 51%(65.7 ppb) and in bee bread were 43% (3.5 ppb), 41% (6.7 ppb), 37% (12.2 ppb), and 55% (35.8
ppb) compared to the nominal concentrations from the feeding solutions (10, 20, 40, 80 and 160µg/L or 8.1, 16.3, 32.5, 65.1 and 130.1 µg/kg; insufficient bee bread was available for sampling in
the nominal 160 µg/L treatment). This dilution is expected since bees could forage on outsidepollen and nectar sources, and hive pollen (bee bread) includes nectar (both from the suppliedsucrose solution and untreated foraged) and pollen (untreated). See Section 3.7 for more details
regarding the residues of clothianidin in the dosing solutions and hive matrices.
Study Endpoint Conclusions:
Colony Survival:
Overwintering mortality was 65%, 75%, 33%, 50%, 17% and 100% in the control, 10, 20, 40, 80
and 160 µg/L treatment groups, respectively. As overwintering losses were so high in the controlsthat statistical differences would not be able to be detected, no statistical analysis was conductedon these colonies for the CCAs following overwintering and no NOAEC or LOAEC could
therefore be determined for this endpoint.
Life Stage Endpoints:
Specifically, when considering the number of adults, pupae, total brood and total live bees, thedifferences from control were apparent both visually and statistically, particularly in the threehighest treatment groups. For the number of adults, the onset of a decline in numbers occurred atleast one CCA earlier (CCA5) in the three highest treatment groups than in the control, 10 and 20µg/L treatment groups. Consistent significant effects were observed at multiple CCAs, showing adose-response relationship beginning at the 40 µg/L treatment group for adults, pupae, total brood(pupae, larvae and eggs combined) and total live (all life stages combined).
Food Stores
When examining the effects on food stores (pollen and nectar), the analyses did not determine any
consistent and significant reductions in pollen and nectar stores at the 10 and 20 µg/L treatmentgroups. This is distinguished from the 40 µg/L group where effects on pollen in particular werevery apparent during and immediately after exposure, when compared alongside the response of
the control (though these effects had lessened by the last two CCAs prior to overwintering).
Similarly, significant dose-repsonse decreases in pollen stores were observed in the 80 and 160µg/L treatment groups at all CCAs following exposure. No significant reductions from the control
were observed in the nectar and total food cells, but higher treatments generally had greaternumbers of cells with nectar than the lower treatments and the control.
Overall Study Conclusions
As will be discussed more fully in Section 3.9 (Results) the analyses determined statisticallysignificant clothianidin dose-related effects in the 40, 80, and 160 µg/L treatment groups acrossmultiple CCAs for the majority of response variables. Indeed, for the 80 and 160 µg/L treatmentgroups, significant effects (p<0.05) were determined for every response variable, except for honeyand total food stores and persisted across multiple CCAs. The 40 µg/L treatment group also

7263

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
showed significant effects for multiple response variables (adults, pupae, total live, total brood andpollen storage) across multiple CCAs.
Conversely, there was not a strong indication of an impact at the colony level for the 10 and 20µg/L treatment groups for individual life stages or food storage. This is evidenced not only by ageneral lack of statistically significant effects (p>0.1) at these treatment levels but in cases where
significant effects were determined, they either did not show strong dose-responsiveness and/or
did not persist across multiple CCAs. This was the case for the statistically significant effects notedby EPA in pollen storage at CCA5 at 10 and 20 µg/L (effects did not persist at subsequent CCAs),in the number of eggs at CCA5 at 20 µg/L (but no statistically significant effects at 40 µg/L andthe effect did not persist at subsequent CCAs) and in the number of adults at CCA6 in the 10 µg/L
treatment group (but no statistically significant effects at 20 µg/L and the effect did not persist toCCA7). The PMRA statistical results were slightly different from EPAs for eggs and adults, but
resulted in the same conclusions. PMRA determined significant effects on the number of eggs at
CCA4 and CCA5 at all test concentrations, but not in subsequent CCAs in the 10, 20 and 40 µg/L
treatment groups, suggesting this effect did not persist following exposure. PMRA also determinedsignificant reductions in the number of adults at CCA6 at all test concentrations, but not in
subsequent CCAs in the 10, 20 and 40 µg/L treatment groups, suggesting this effect did not persist.
The study is considered to be informative and will be used as a line of evidence in the pollinatorrisk assessment. While there were uncertainties that were generally related to inherent aspects ofany semi-field or full field study design (described in the section below) this study still provides
information on a number of colony health parameters about the long term (excluding overwintering)
exposure to clothianidin at the colony level. As control survival was only 35% after the
overwintering period, results from the overwintering period are not considered valid for assessing
the potential chronic risks of clothianidin. When weighing biological and statistical significance,
the NOAEC and LOAEC for this study are determined to be 20 and 40 µg/L, respectively
based on effects to number of adults, pupae, total brood, total live bees and pollen storage atthe 40 µg/L treatment group. These effect levels include the understanding that evaluationof overwintering was not possible which limits the ability to fully evaluate potential longterm effects in the two lower treatments groups, and therefore, remains a major source of
uncertainty.

Consideration of Study Strengths, Limitations and Interpretation
It is important to recognize the inherent strengths and limitations of this study as results areinterpreted and potentially considered in risk assessment.
In the context of available field studies involving honey bees and clothianidin, this study contains
a number of strengths including:•••

Use of a high degree of replication (n=12) to achieve a reasonable level of statistical power
Demonstration of a generalized concentration-response relationship with respect to theconcentration of clothianidin in sucrose solution and the magnitude and duration of adverse
effects
Quantification of exposure to clothianidin in diet and in hive matrices (uncapped nectar,
pollen, capped honey, bee bread)
8264

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document••••

Use of a 6-week exposure duration to represent a “high end” exposure scenario
Inclusion of multiple colony-level endpoints reflecting hive strength, brood development
and food stores
Detailed QA/QC results regarding quantification of clothianidin residues in various
matrices
Availability of raw data for conducting statistical analysis.

A number of limitations are also noted with this study, including:
•

•

•

•

Dosing of bees by clothianidin occurred through sucrose (nectar-substitute) alone, whereas
bees in the field are likely exposed through both pollen and nectar routes. Therefore, thedesign of this study may not reflect a “worst case” exposure scenario in which bees areexperiencing prolonged exposure to both contaminated nectar and pollen. While exclusionof the pollen route is expected to reduce overall exposure, the impact of this exclusion on
the study results is uncertain and will likely depend on the life stage/caste of bee. However,
it is notable that in addition to the nectar exposure route and subsequently through honeystorage, bees would also be exposed (albeit in lower doses) in bee bread, as bee breadwould incorporate both supplied and foraged nectar/sucrose and foraged pollen.
Residues in hive matrices were only analyzed for parent clothianidin. Metabolites ofclothianidin were not considered. Clothianidin degradates (e.g. TZNG) have been
demonstrated in laboratory studies to have much less acute toxicity to adult honey bees,
though data is not available for their chronic effects to adult bees or potential effects toother honey bee life stages.
Clothianidin was found in both hive nectar and hive pollen (beebread), at concentrations
lower than the feeding solutions. Dilution compared to the treatment feeding solution is
expected since bees could also forage on outside nectar and pollen sources. As well, hivepollen contains only some hive nectar, thus would not be expected to have a concentrationequivalent to nectar alone, and it is mixed with pollen which will come from outside
sources. Therefore exposure through both hive pollen and nectar occurred via exposure to
the sucrose feeding solution, but how this compares to exposure through contaminatedpollen directly is not known. It is also noted that nectar is considered the dominant exposure
route for forager bees; other hive bees and larvae consume both nectar and pollen. A recent
paper by Sandrock (2014) 1 indicated that consuming contaminated pollen containing low
levels of both clothianidin and thiamethoxam had effects on many hive parameters. In
addition, since bees were forced to forage for pollen in this study, the potential impact ofclothianidin exposure on reducing pollen foraging efficiency of bees could be incorporated
into the overall expression of adverse effects, as suggested by published literature. Had
contaminated pollen been provided to bees, it is not known if the potential impact on pollen
foraging efficiency would have been masked.
The quantity of nectar provided to hives (4 L per week per hive) likely did not fulfill thecomplete carbohydrate needs of the colony, as indicated by colony bioenergetics and thelack of remaining sucrose solution upon their renewal at some of the test concentrations.

1

Sandrock C, Tanadini M, Tanadini LG, Fauser-Misslin A, Potts SG, et al. (2014) Impact of Chronic Neonicotinoid
Exposure on Honeybee Colony Performance and Queen Supersedure. PLoS ONE 9(8): e103592.
doi:10.1371/journal.pone.0103592
9265

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

•

•

•

•

•

•

This suggests that bees could be exposed to a greater dose of clothianidin in nectar had agreater volume of spiked sucrose been provided. Although one can infer that the dosing
regimen may have underestimated exposure through sucrose relative to 100%
contaminated diet, it is also noted that bees had to supplement their spiked sucrose byforaging on their own for other sources of nectar. As with the previous discussion of pollen
it is noted that had 100% of the carbohydrate needs of the colony been provided via feeders,the potential impact of purported reductions in nectar foraging efficiency may have beenmasked to some degree.
Overwintering success of controls was severly impacted (65% hive mortality). This
prevents the ability to detect adverse effects related to hive loss following overwintering.
The lack of control hive overwintering may reflect the study design that prevented earlier
supplemental feeding in the fall (in order to ensure that treatment hives were consuming
their exposed food stores), while typical beekeeping practice would have permitted
additional feeding earlier in the fall.
Pesticides from food sources other than the artificial feeding were also detected during
the exposure period and post-exposure periods through collection of pollen from pollen
traps from monitoring hives. This contributes to exposure uncertainty and can add
confounding effects when interpreting results. However, it is noted that detections
occurred in <10% of samples from monitoring hives and that the only pesticides detected(propiconazole, chlorothalonil and carbaryl) had relatively low toxicity compared toparent clothianidin (ranging from practically non-toxic for chlorothalonil to moderately
toxic for cararyl).
Residues of clothianidin in uncapped nectar and bee bread within the hives at CCAs 4, 5,
7 and 9 represent a single sample per hive on a single frame rather than a composite sample
from multiple portions of the comb within a hive. This means that residue results may
reflect a “hit or miss” scenario with respect to detecting residues in nectar laid down fromcontaminated (fed) vs. outside sources.
The exposure, based on residues measured in the hive (hive nectar and hive pollen)
indicated that, overall, higher measured hive residues correlated with higher nominal
residues in feeding solutions. However, individual hive residue values varied, and there
was some overlap in measured values, particularly among the three lowest doses.
Exposure dilution during the study was evident. Remarkably lower residue concentrations
detected in bee bread and hive nectar in some test hives compared to the feeding
concentrations indicate foraging on other food sources. This uncertainty is inherent in any
semi-field or full-field study design.
Following standard beekeeping practice, supers (additional hive bodies) were added or
removed on a case-by-case basis from each hive to support growth or restrictions in thesize of the bee colony. Since each hive was treated differently, this may have added
variability and uncertainty into the study design. Additionally, because of this difference
between hives, no analysis could be conducted on the proportion of each hive devoted todifferent life stages and/or food storage.

10266Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

1. Study Objective
To determine the potential long term effects on the honey bee (Apis mellifera L.) colony healthduring and after dietary intake of clothianidin, including the potential effects on overwintering.
The long term exposure allows for the characterization and distinction of short-term versus apersistent nature of effects.

2. Study Methods
2.1.

Test crop

Not applied. The study was conducted in an open field where multiple field flowers were available
and may serve as food sources for the test bees, in addition to the artificial feeding of spiked sugar
solution.

2.2.

Test chemical

The test substance was technical clothianidin. Further details are provided in Table 1 below.
Table 1. Details about the test substance
Name
Test item code:
Formulation type:
Active ingredient:
CAS number:
Density (20 °C)
analysed:
Date of analysis:
Stability in solution:

2.3.

Clothianidin
TMC 14-63
Technical compound
Clothianidin
210880-92-5
Not applicable

Test Item
Batch number:Appearance / colour:
Intended Usage:
Content of a.i. analysed:

14 Jan 2015
sufficient for the test purpose(at least 1h)

AE1283742-01-10
White solid
Insecticide
98.6 %

Risk symbol(s):Not available

Expiry date:
Storage conditions:

14 Apr 2016
+10 to +300C

Test sites

The field and sampling phases of this study were conducted by Smithers Viscient, CRC, Carolina
Research Station, Snow Camp, NC, USA; the analytical phase was conducted by Bayer
CropScience in Research Triangle Park, NC, USA. The apiary sites were located in the vicinity of
the Smithers Viscient CRC site in Guilford, Randolf, Alamance, and Chatham counties, North
Carolina.
There were 12 apiaries separated by at least 1 mile. Land use surveys in 1-, 3- and 5-mile radii
were conducted. The land use pattern based on National Land Cover Database (NLCD) coverage(2011 dataset layer) indicates that the surrounding area of the apiaries is dominated by forests andhay/pasture with only ~0.5% cultivated crops, while the more contemporary Cropland Data Layer(CDL) coverage (2014 dataset layer) indicated that corn and soybeans were the predominant crop
types with approximately 8% coverage in the surrounding area of the apiaries. Pollen species
identification and multiple pesticide analysis were conducted using pollen samples collected from11267

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
the monitoring hives to characterize outside food sources of the test bees and contamination.
Pollen samples were collected for a period of 24-48 hours using pollen traps once prior to exposure(June 18-20, 2014), two times during the feeding exposure period (July 1 and 18, 2014), once
immediately after exposure (Aug 13, 2014) and 3 additional post-exposure times (September 5-13,
Sep 26, and Oct 20 2014). The study authors noted that pollen amounts from these hives werevariable and sufficient sample material were not available from every site at each measurement
time point.

1Figure 1: Location of test apiary sites
Table 2: GPS-coordinates of the test apiary sites
ApiaryGPS-coordinates
New Package Apiary
35°51'48.0"N,79°22'24.0"W
Apiary A
35049'50.0"N,79°21’03.0"W
Apiary B
35°53'01.0"N,79°31’20.5"W
Apiary C
35°52'04.0" N, 79°20'02.0" W
Apiary D
35°51’16.0"N,79°35'54.0"W
Apiary E
35°57'55.0"N,79°31’48.0"W
Apiary F
35°58'45.0" N, 79°28'38.0" W
Apiary G
35°54'13.0"N,79°38'25.0"W
Apiary H
35°48'31.0"N,79°31'55.0"W
Apiary I
35°49'19.0"N,79°32'45.0"W
Apiary J
35°50'06.0"N,79°34'10.0"W
12268

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
ApiaryApiary K
Apiary L
High Rate Apiary
Minimal Strength ApiaryGPS-coordinates
35°53'44.0"N,79°36'19.0"W
35°52'52.0" N, 79°36'28.0" W
35°49'06.9"N, 79021'46.5" W
35053'38.4" N, 79°34'03.4" W

From Table 2, page 18 of the study report.

2.4.

Test organisms

The test species was the honey bee (Apis mellifera), Italian race (Apis mellifera ligustica). Hives
were established from package bees bought from the commercial bee supplier The Carolina Honey
Bee Company (10 South Main Street, Travelers Rest, SC 29690, USA) typical of the bee stock
used in commercial beekeeping operations. A new queen was introduced into each colony. Four
breeder queens which were sister queens were used to generate all the queens used in the study.
All queens were purchased from the package supplier. The colonies were maintained in 10-frameLangstroth boxes with an empty deep super on top as a feeder box. In the test field, hives wereraised above ground level.
Eighty-four hives that met the study author’s criteria (presence of all stages of brood, laying queen
and stored pollen and nectar) at the second Colony Condition Assessments (CCA2) were selected
for the study. More than 100 inspected hives were screened based on the outcome of CCA2. Hives
were checked for the “appearance” of a healthy colony with no visible symptoms of Varroa or
Nosema, as well as having all stages of brood, a queen, and some food stores.
Eighty-four hives were blocked into 12 apiary sites (8 hives/apiary) by brood strength of the colony,
starting with Apiary A as the strongest group of hives, and Apiary L as the weakest group of hives.
Assignment of apiaries to the geographic locations was done randomly.
Hives were moved from the new package apiary on 17/18 Jun 2014 to their study apiaries. CCA3
was initiated on 18 Jun 2014. After evaluating CCA3, 7 hives were deemed unsuitable due tomoving stress that caused swarming or loss of queen and were replaced just before exposure
initiation. The replaced hives were A7, B2, H3, H8, J5, J6 and L4.
There were eight hives at each site (7 hives for biological assessments and one as the monitoring
hive for pollen sample collection). Each hive was spatially isolated from other treatment rates by
30 feet (9 m) spacing at each apiary site (Figure 2). Hives were arranged in a semi-circular pattern,facing east to west, with 125 feet (38 m) spacing between the two end hives.

13269

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

2Figure 2. Layout of test hives in a test site

During the study, all hives were treated for Varroa with one application of Apiguard® (active
ingredient: thymol) following typical apicultural practice for the region. The initial application
occurred immediately after CCA6 (8-12 Sep, 2014) to prevent high mite loads. No treatments forany other hive pests, predators or diseases were administered to any hives. To evaluate Varroa
mite infestations, hive bees were sampled to obtain specific mite counts the week before and after
the exposure period, as well as after over-wintering (3rd, 5th and 9th CCAs)
To minimize the potential for robbing amongst test hives, hives at 80 and 160 ppb treatments were
removed from all test apiaries in week 8 (12 Aug, 2014) following CCA5. The hives were placed
at a separate “high treatment” apiary. For over wintering, the surviving colonies were fed with 1
L of 2:1 sugar syrup on 30 Oct 2014, 06 Nov 2014, 17 Nov 2014, 24 Nov 2014, 01 Dec 2014, 29
Jan 2015, 04 Feb 2015, and 11 Feb 2015
The monitoring hives were used for outsource pollen sample collection. In addition, test solutions
were sealed and placed in monitoring hives in order to assess clothianidin stability under field test
conditions. These stability solutions were not available as a food source to the monitoring hives.

2.5.

Treatments

There were:o 6 treatment groups (5 test concentrations and control): 0, 10, 20, 40, 80, and 160 µg/L.
At each site, there were 2 control hives, and one hive for each test concentration.
o 12 replicates per treatment group (apiaries), with 24 replicates in the control group.
The individual treatment groups, the respective feeding rates and the respective feeding volumes
are summarized in Table 3 below.3Table
Table 3. Treatment groups, feeding rates and feeding volume
Treatment Group

Code

Feeding Timing

Concentrationa.i.

Feeding
Volume14
270

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
1 : UTCUTC(C1+C2)
T1
T2
T3
T4
T5

2 : Lowest Rate
3 : Low rate
4 : Moderate rate
5: High rate
6: Effect rate

From Table 3, page 21 of the study report.
UTC = untreated control

Twice a week---

2000 mL

Twice a week
Twice a week
Twice a week
Twice a week
Twice a week

10 ppb20 ppb40 ppb80 ppb160 ppb2000 mL
2000 mL
2000 mL
2000 mL
2000 mLThe assignment of each test hive at 12 apiaries is summarized in Table 4.
Table 4. Hive assignment to test apiaries
Treatment
group

Apiary
A

B

C

D

E

F

G

H

I

J

K

L

UTC

A3

B2

C1

D4

E1

F2

G2

H1

I6

J7

K5

L4

UTC

A5

B3

C3

D8

E4

F5

G8

H6

I7

J8

K7

L6

10 ppb

A7

B8

C5

D3

E2

F6

G6

H2

I1

J4

K4

L1

20 ppb

A1

B5

C4

D6

E7

F7

G3

H9

I4

J5

K3

L2

40 ppb

A4

B1

C2

D7

E8

F3

G5

H7

I8

J6

K2

L5

80 ppb

A2

B6

C8

D5

E6

F1

G4

H4

I5

J3

K1

L7

160 ppb

A8

B4

C7

D1

E5

F8

G1

H3

I2

J2

K6

L3

A6
B7
C6
D2
Monitoring
From Table 4, page 21 of the study report.

E3

F4

G7

H5

I3

J1

K8

L8

2.5.1. Preparation of stock solution
Stock solution was created by combining 0.051 g of clothianidin dissolved in 20 mL of acetone
and added to 1000 mL of distilled water. After preparation, the stock solution was re-stored in arefrigerator until use or replacement. New stock solution was prepared on 09 Jul 2014, 26 July
2014 and 31 July 2014. The study author did not report whether the control sucrose solution
contained any acetone.

2.5.2. Preparation of sugar solution
Sugar syrup was created by combining 3 gallons of water for every 25 pound bag of sugar to
produce approximately 200 gallons (757L) of sugar syrup.

2.5.3. Preparation of feeding solution
o
o
o
o
o

10 µg/L: mixing 3.0 mL of stock solution into the 15 L of sugar solution.
20 µg/L: mixing 6.0 mL of stock solution into the 15 L of sugar solution
40 µg/L: mixing 12.0 mL of stock solution into the 15 L of sugar solution
80 µg/L: mixing 24.0 mL of stock solution into the 15 L of sugar solution
160 µg/L: mixing 48.0 mL of stock solution into the 15 L of sugar solution.

15271

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
The test concentrations were reported as “ppb” in the study report. However, the values are in fact
in the unit of µg/L, not ppb (ug/kg). For example, 10 µg/L: can be calculated by 3.0 ml * (0.051 g/1020 ml)/15,000 ml.
The test solution density was not provided. Assuming the density of a 50% sugar solution is 1.2296
g/ml 2, the reviewer calculated that the nominal test concentrations at 10, 20, 40, 80 and µg/L areequivalent to 8.1, 16.3, 32.5, 65.1, and 130.1 µg/L (ug/kg), respectively.

2.5.4. Artificial Feeding
A hive top feeder was placed on the top box (either original hive box or an added super) andcovered with a telescoping lid. This allowed easy access only to those bees within the hive andminimized light exposure of the test material.
The treated sugar syrup was prepared one day in advance for each feeding event. The feeding
started on 26 Jun, 2014 and continued for 6 weeks until the last feeding on August 4. All of thehives were artificially fed with 2 liters of 50% sugar solution, two times per week. Prior to eachfeeding, any remaining feeding syrup was removed from the feeder and weighed to determine theconsumed amount. The study observation (commencing when hives were moved to their study
apiaries) period was 17 Jun, 2014 – 27 Apr, 2015, which includes the overwintering period.

2.6.

Meteorological Data

Temperature, humidity and precipitation data were monitored at each study apiary. An average
total of 6.32 inches (161 mm) of rainfall accumulated throughout the exposure period (from 26
Jun 2014 until 11 Aug 2014) across the 12 apiaries. However, Apiary I did not record any data on
9 days during the exposure period and only recorded a total of 1.1 mm precipitation throughout
the exposure period. Removal of Apiary I’s rainfall data would result in an average of 6.90 inches(175 mm) across the 11 apiaries during the exposure period. The minimum and maximum weekly
average temperatures are shown in Figure 3.

Cell Biology Laboratory Manual, http://homepages.gac.edu/~cellab/chpts/chpt3/table3-2.html, accessed on Dec 12,
2014

2

16
272

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

Figure 3. Average minimum and maximum temperatures across all apiaries

From Figure 4, page 32 of the study report

2.7.

Observations

Important activity and dates are summarized in Table 5.

17273

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Table 5. Chronological list of key dates and activities
Week Date-6
12-21 May 2014-3
02-13 June 2014
-2

17 – 18 Jun 2014

Activity
CCA1
CCA2; Hive samples (uncapped nectar, beebread)
Hives moved to study apiaries

Week
6
6

Date
05-11 Aug 201405-11 Aug 2014

Activity
Hive samples (uncapped nectar, bee bread)CCA5

605-11 Aug 2014

Hive bee sampling for Varroa and Nosema
assessment
Removal of 80 and 160 ppb hives to hightreatment apiary
Monitoring hive sampling (uncapped nectar,
pollen)Hive C2 was removed from the study-1

18-23 Jun 2014

CCA3

6

12 Aug 2014-1

18-20 Jun 20146

13 Aug 2014-1

18-20 Jun 20148

25 Aug 2014-1

24 Jun 2014

Hive bee sampling for Varroa and Nosema
assessment
Sampling of Monitoring Hives (uncapped
nectar, pollen)
Seven hives replaced based on CCA3 results

10

05 Sep 201400

26 Jun 2014
30 Jun 2014

1st Feeding
2nd Feeding; measurement of remaining food1013

8-12 Sep 2014
23 Sep 2014

0

01-02 Jul 201413

25 Sep 20141

03 Jul 2014

Monitoring hive sampling (uncapped nectar,
pollen)
3rd Feeding; measurement of remaining food15

14-16 Oct 2014

1
1

07 Jul 201307 Jul 2014

Stability samples
4th Feeding; measurement of remaining food16
17

21-22 Oct 2014
28 Oct 2014

1
2
2
2
3
3
3
3

09 Jul 2014
10 Jul 2014
10 Jul 201414 Jul 201418 Jul 201415-18 Jul 201416-18 Jul 201418 Jul 2014

18
18
19
20
21
22
31
31

30 Oct 201404 Nov 201406 Nov 201417 Nov 2014
24 Nov 201401 Dec 201429 Jan 201504 Feb 20153

21 Jul 2014

New stock solution prepared
5th Feeding; measurement of remaining food
Hive I7 removed from study
6th Feeding; measurement of remaining food
7th Feeding; measurement of remaining food
CCA4
Hive sample (uncapped nectar)
Monitoring hive sampling (uncapped nectar,
pollen)8th Feeding; measurement of remaining food

CCA7 (UTC, 10 ppb, 20 ppb, 40 ppb) capped
honey sample
CCA7 (high treatment apiary)
Additional hive moved to minimal strength
apiaryFeeding 1 L 2:1 sugar syrup per hive
All remaining 160 ppb treatment hives destroyed
Feeding 1 L 2:1 sugar syrup per hive
Feeding 1 L 2:1 sugar syrup per hive
Feeding 1 L 2:1 sugar syrup per hive
Feeding 1 L 2:1 sugar syrup per hive
Feeding 1 L 2:1 sugar syrup per hive
Feeding 1 L 2:1 sugar syrup per hive

32

11 Feb 2015

Feeding 1 L 2:1 sugar syrup per hive4
4

24 Jul 2014
26 Jul 2014

9th Feeding: measurement of remaining foodNew stock solution prepared

After over-wintering37
17-19 Mar 2015

Monitoring hive sampling (uncapped nectar,
pollen)CCA6*Monitoring hive sampling (uncapped nectar,
pollen)Weakest hives moved to minimal strength apiary

CCA8 (UTC, 10 ppb, 20 ppb, 40 ppb)
18274

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Week Date
4
28 Jul 20145
31 Jul 2014
5
5

Activity
10th Feeding; measurement of remaining foodNew stock solution prepared

Week
40
43

01 Aug 2014
01 Aug 2014

Date
02 Apr 201522-23 Mar 2015

Activity
CCA8 (80 ppb)
Hive bee sampling for Varroa and Nosema
assessment
CCA9 (UTC, 10 ppb, 20 ppb, 40 ppb)
Hive samples (capped honey)

11th Feeding; measurement of remaining food 43
22-23 Apr 2015
Stability samples, monitoring hive sampling 43
22-23 Apr 2015
(uncapped nectar, pollen)
5
04 Aug 2014
12th (final) Feeding; measurement of remaining 43
27 Apr 2015
CCA9 (80 ppb)
food
6
07 Aug 2014
Measurement of remaining food
*CCA6 timing allows all bee individuals (eggs, larvae, pupae) present during the exposure period to complete their development cycle to adults.

19275

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
2.7.1. Colony mortality
The study author did not report what defined a “dead hive”. However, the reviewer has assumed
that any colony (hive) that did not show the presence of a queen and had no open brood or eggs,
or was devoid of worker (female) bees was considered “dead”. If a hive was considered “dead”
at the time of assessment, it was no longer used in the analysis of endpoints (e.g., adult bee
numbers, hive weight). The number of individual dead bees was not recorded.

2.7.2. Colony Condition Assessments (CCA)
Beginning with CCA 3, observations were blocked by the observer, with the same person always
observing the same set of hives to avoid viewer discrepancies in the data. Hives at apiaries A, B,
E, G, I, and J were inspected by the study author and those at apiaries C, D, F, H, K and L byanother inspector.
Nine CCAs were conducted during the entire study. CCA1 (day -45 to -36), and CCA2 (day -24
to -13) were conducted during the hive establishment. CCA3 (day -10 to -3 days) was conducted
1 week prior to the feeding exposure which served as initial hive conditions prior to the feeding
exposure. CCA4 was conducted 3 weeks (19—22 days) after the start of feeding exposure. Afterthe end of feeding exposure (Week 6), the following additional CCAs were conducted: CCA5
(week 6, 40-47 days post exposure), CCA6 (days 74—78), CCA7 (days 110—118) and after
overwintering CCA8 in Mar 2015 (days 264—280) and CCA9 in April 2015 (300—305 days postexposure). Each CCA period took multiple days to complete. For summary statistics, the average
day is used to characterize any given CCA. The time schedule of CCAs in relation to other study
activities is summarized in Table 5.
During the colony condition assessments, each frame was removed and inspected one at a time(observations recorded for each side), with measurements for endpoints taken as percent of totalframe area covered by honey / nectar, bee bread / pollen, eggs, open brood (larvae), capped brood(pupae), and adult bees.
The estimation was made by:o Each hive consisted of 20 observed panels (10 frames with two sides of each frame), withan area of 929 cm2 per side, or a total area of 18,580 cm2 for all 10 frames.
o A frame with 100% coverage of adult bees was assumed to have an adult bee density of
1.30 bees/cm2.
o Each cell is a regular hexagon with a flat-to-flat distance of 5.2cm and an area of 0.234
cm2
o Each frame was considered to have 3970 cells/frame side (929 cm2/cell area of 0.234
cm2)
For adult bees, therefore, a frame side with 100% coverage of adult bees would contain 1208
bees (929 cm2 * 1.30 bees/cm2). For the number of cells containing honey/nectar, pollen, cappedor open brood or eggs, the following equation was used:

20

276

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Number of cells (for a given hive matrix) = Σ %frame coverage * cells per frame side (3970)
100%

2.7.3. Evaluation of Disease or Pests in the Hive
At each CCA, colonies were also checked for visible symptoms of disease or pests, such as
Nosema, foulbrood, Varroa mites or small hive beetle. To quantify the presence of Varroa in thehive, bee samples were taken at CCA3, CCA5 and CCA9. Bees were washed in alcohol toremove mites. The number of mites per 100 bees was calculated.

2.7.4. Hive weights
Each hive had a dedicated scale beneath it that was placed just before exposure initiation andremained until the end of the study. Each scale was programmed to record the hive weight every
hour.

2.8.

Residue analysis

All residue and stability samples collected from feeding solution, pollen traps, and test hives wereanalysed for clothianidin residues at Bayer CropScience in Durham, NC. Samples from pollen
traps in the monitoring hives were also analysed for residues of multiple pesticides from outside
sources at the National Science Laboratories of USDA in Gastonia, NC (non-GLP). The residue
results were reported as µg per kg of sample matrix (ppb), which is different from the test solutionthat was reported in µg/L. Samples were not analyzed for residues of clothianidin metabolites (e.g.
TZNG).
The LOQ was 1 ppb for clothianidin in feeding solution and hive nectar samples for pollen samples.
The LODs are listed in Table 6.
Table 6. LOD for clothianidin

Clothianidin
LOD
LOQ
Dosing/Stability Solutions
0.5 ppb
1 ppb
Hive Collected Nectar
0.1 ppb
1 ppb
Pollen0.4 ppb
1 ppb
Taken from page 373-374 of the study report
MatrixFor the values <LOD, half of the LOD value was used in order to calculate the means. For values
between the LOD and LOQ, half of the LOQ value was used to calculate means. Multiple pesticide
analysis was conducted in order to monitor pesticide contamination from outside food sources
using pollen collected from pollen traps on the monitoring hives.
All samples for residue analysis were protected from sunlight by using amber vials and transported
to freezer storage after field collection. All samples were placed in frozen storage upon receipt at
the test facility. Samples were maintained frozen (≤ -15° C) at the test facility until shipment underfrozen conditions to the test site for residue analysis. Daily minimum/maximum temperatures wererecorded for the duration of the storage period at the test facility.

21277Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
2.8.1. Pollen from outside sources
Pollen samples were collected from pollen traps attached for 24-48 hours to the monitoring hives
at each site to assess the potential contaminant exposure from outside sources. Pollen traps were
only activated at seven time points during the study and were occasionally left open longer than
48 hours if sufficient pollen sample was not available at the expected date. Pollen amounts
collected from each hive were variable and samples were not available from every site each time.
Pollen samples from the monitoring hives were taken at weeks -1 (CCA3), 0, 3 (CCA 4), 6 (CCA5),
10 (CCA6), 13, and 16 (CCA7).

2.8.2. Stored pollen and nectar in test hives
Stored bee bread and bee-collected nectar were collected within the study hives for clothianidin
residue analysis. Samples weighed at least 500 mg each and were not available from every colonyeach time. Bee bread was collected at week 6 (CCA5). Uncapped nectar was collected at weeks 3
and 6 (CCAs 4 and 5) and capped honey was collected at weeks 15 and 43 (CCAs 7 and 9).

2.8.3. Feeding solution and stability of test item
The monitoring hives were used for dose verification and to evaluate stability of the test item in ahive environment. Monitoring hives were set up in the same manner as test hives except the colony
was denied access to the spiked or unspiked sucrose. Residue samples comprising approx. 5 g eachfrom the sugar syrup were taken on week 1 (7 July 2014), and week 5 (1 August 2014). Stabilityof the test material was evaluated by placing treated feeding solutions in closed-off vials in thefeeding compartments of hives at representative apiaries.
Table 7. Number of samples and sampling schedule for feeding solution and stability of test chemical.
TimingApiary / replicate
UTC
10 ppb20 ppb40 ppb80 ppb160 ppb-- = no samples taken

Week 107 Jul 2014
-4
4
4
4
4

Week 5
01 Aug 2014
8
4
4
4
4
4

22
278

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

3. Results
3.1.

Land use near test hives

Land use pattern within a 1-mile, 3-mile and 5-mile radii around the 12 apiaries are summarizedin Table 8. Generally, the results indicate that the area around the apiaries during the year the
study was conducted was dominated by forest and grassland/pasture/hay and that corn andsoybean were the predominant crop types. The cultivated crop area occupied 6.7% of the total
land within 1 mile radius, 8.3% within a 3 mile radius range, and 7.7% within a 5 mile radius
range from the test apiaries. Data from the 2011 cropland data layer also indicate forests(particulary deciduous) and hay and pasture land were dominant in the study area prior to test
initiation, but that cultivated crop acreage was lower (Table 9).

Table 1. Average percent (%) land use pattern across the 12 study apiaries (based on 2014 Cropland Data
Layers (CDL))
Land Use Category
Average of 12 Study Apiaries
1 mile radius
3 mile radius
5 mile radius
Corn
2.5%
3.1%
2.7%
Soybean
3.3%
4.5%
4.4%
Other Crops
0.9%
0.7%
0.6%
Developed, Open Space
6.0%
5.7%
5.3%
Developed, Low-High Intensity
3.0%
2.7%
2.3%
Forest
44.4%
45.7%
47.8%
Grassland/Pasture/Hay
38.8%
36.1%
35.2%
Water/Barren/Shrub/Wetland
1.6%
1.5%
1.6%
Table 2. Average percent (%) land use pattern across the 12 study apiaries (based on 2011 National Land
Cover Database (NLCD))
Land Use Category
Average of 12 Study Apiaries
1 mile radius
3 mile radius
5 mile radiusOpen Water
0.4%
0.6%
0.7%
Developed, Open Space
5.8%
5.6%
5.3%
Developed, Low Intensity
2.6%
2.2%
1.8%
Developed, Medium Intensity
0.4%
0.5%
0.4%
Developed, High Intensity
0.1%
0.2%
0.1%
Barren Land
0.1%
0.1%
0.1%
Deciduous Forest
32.5%
34.0%
35.5%
Evergreen Forest
5.5%
5.2%
5.6%
Mixed Forest
2.5%
2.2%
2.4%
Shrub/Scrub
2.0%
2.7%
2.9%
Herbaceous
4.0%
4.3%
4.5%
Hay/Pasture
42.9%
41.3%
39.5%
Cultivated Crops
0.5%
0.5%
0.5%
Woody Wetlands
0.6%
0.7%
0.6%

23
279

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
3.2.

Pollen sources of test hives

Monitoring hives were used at each test apiary to collect pollen for assessment of the local pollen
flora (non-GLP). Pollen trap samples from the monitoring hives were taken at seven times: CCA3
(18-20 Jun 2014; week -1), July 1-2 (week 0) and CCA4 (18 Jul 2014; week 3), as well as at CCA
5 (13 Aug 2014; week 6), on September 5-13 (week 10) and 26 (week 13), at prior to overwintering
at CCA7 (20 Oct 2014; week 16).
Major sources (>10%) of pollen at any measured time point were from clovers, crepe myrtle,
plaintain, vitis, corn, virginia creeper, chickory, pigweed, ragweed, goldenrod/tickseed, and grass.
Corn was the only cultivated crop with significant levels of pollen in monitoring hives (average of
18—23% in July). In the fall, pollen in monitoring hives was dominated by asters (goldenrod,
tickseed and ragweed) with average proportions of 55—88% in September and October) andgrasses (mean 37% in late September). Full results can be found in Table 9 (p. 44) and Tables 6470 (p. 319-324) of the study report.

3.3.

Consumption of spiked sucrose

Individual hive consumption rates (determined by the remaining food in the feeder added up
throughout the entire exposure period) for the feeding solution (sugar syrup) ranged from 11,636
mL to 24,000 mL of the total 24,000 mL per hive provided during a 6-week period (i.e. 2 litres percolony 2 times a week for a total of 24,000 mL per colony during the exposure period). All colonies
in the control, 10, 20, 40 and 80 ppb treatment groups consumed most or all of the sugar solution(see Figure 4) with some colonies in the 160 ppb treatment having substantially lower
consumption. Mean total consumption in control hives was 99.5% of provided sugar syrup(minimum control hive consumption of 93%).

Figure 4. Mean total food consumption (L) per colony during the 6-week exposure period

24280

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
3.4.

Examination of pesticides from other sources

Monitoring hives were used to assess the potential contaminant exposure from outside sources(non-GLP) at each site. Pollen trap samples from the monitoring hives were taken at seven times:
CCA3 (18-20 Jun 2014; week -1), July 1-2 (week 0) and CCA4 (18 Jul 2014; week 3), as well as
at CCA 5 (13 Aug 2014; week 6), on September 5 (week 10) and 23 (week 13), at prior to
overwintering at CCA7 (20 Oct 2014; week 16). The study author reported that the amount ofpollen collected from traps on the monitoring hives varied and that not all hives had samples
collected at each measurement time point. However, the hives and measurement times where there
was insufficient material were not identified in the study report and no list of samples that weresent to the USDA National Science Laboratories in Gastonia, NC was provided.
Out of the 42 pollen samples that were analyzed for pesticide residues, only three found any
residues higher than the LOD. The 01 Jul 2014 sample (week 0) from Apiary E has measured
residues of 203 µg/kg chlorothalonil, the 13 Aug 2014 (week 6) sample from Apiary I had
measured residues of 119 µg/kg carbaryl and the 20 Oct 2014 (week 16) sample from Apiary B
had measured residues of 2010 µg/kg propiconazole. No residues of clothianidin, thiamethoxam
or imidacloprid (LODs all 1.0 ppb) were detected in any pollen sample from any monitoring hive.
The LODs for each pesticide in the residue analysis can be found in Appendix I on p. 346 of theoriginal study report.
59 uncapped nectar samples from monitoring hives were analyzed and none had any detectablepesticide residues.
Pesticide contamination is unknown for those intervals when pollen or nectar samples were notcollected.

3.5.

Confirmation of test concentrations

Clothianidin was analyzed from feeding solutions sampled after they were prepared on 03 July
2014 (week 1) and 28 Jul 2014 (week 4). The averages of measured concentrations were <LOD,
9.5, 19.1, 35.7, 71.9, and 140.0 µg/kg for the nominal concentrations of control, 10, 20, 40, 80,
and 160 µg/L, respectively. The data are tabulated below in Table 10.
3Table 10. Dosing solution residue data from 03 July 2014 (Week 1) and 28 July 2014 (Week
4).
Average ofmeasuredconcentrationsµg/L
ppb (µg/kg)(ppb)
0 (Control)
0
<LOD†
10
8.1
9.5
20
16.3
19.1
40
32.5
35.7
80
65.1
71.9
160
130.1
140.0
†LOD = 0.5 ppb in feeding solutions
Nominal concentrations

Measured clothianidin
Concentrations
(n=2)
<LOD
9.13
18.3
34.9
68.5
129

<LOD
9.89
19.8
36.4
75.2
151

25
281

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
3.6.

Stability of the test item in feeding solution

Stability of clothianidin in the sugar solution during the feeding period was examined from dietcollected from closed-off feeding solutions placed in the monitoring hives, placed two times, on 3
July and 28 July, 2014 (n=4 for all treatment hives at each sampling event) and removed four days
after placement. No reduction of test concentrations in the feeding solution was noticed during
these two samplings. No clothianidin residues were detected (LOD of 0.5 ppb) in any of thecontrol samples (n=8) taken on from test materials placed in closed-off hive feeders on 28 July
2014 and removed on 01 Aug 2014. Control samples were not placed in closed-off feeding
solutions on 03 July 2014. Average clothianidin residue data for the stability solution are presented
in Table 11.
4Table 11. The stability of clothianidin in feeding solution on 3 Jul and 28 Jul, 2014.
Nominalconcentration(µg/L)
Control
10
20
40
80
160

Average of measured
concentrations
across sampling
dates (ppb)
<LOD†
9.59
19.98
36.48
70.88
131.0

Number ofsamplesmeasured
8*
8
8
8
8
8

- Regenerated from Section 5.2, on page 382-383 in the study report†: LOD=0.5 ppb for clothianidin;*All control samples were from the samples placed on 28 July 2014.

3.7.

Range of measured clothianidin
concentrations (ppb)
03 Jul, 2014
N/A*
8.31—12.3
18.9—21.7
31.6—39.4
62—75.5
106—128

28 Jul, 2014
<LOD
9.45—9.82
19.5—20.1
36.9—37.8
73.3—75.3
145—149

Residues in hive matrices

It is noted here as it was in the uncertainties section that the residue samples from the different
hive matrices represent a single sample from a single frame. Therefore there is variation in theresidues that likely stems from the sampling procedure employed for this study (single sample,
one side of the comb).

3.7.1. Clothianidin residues in hives prior to the feeding exposure
Potential background clothianidin contamination in test hives was examined using hive bee bread(hive pollen) and nectar collected about three weeks (03 June 2014) prior to the beginning of
feeding exposure from two hives at each of the initial installation apiaries (2 apiaries). Clothianidin
was not detected in any of four hive pollen samples (LOD = 0.4 ppb) or four nectar samples (LOD
= 0.1 ppb). Residue analysis for other pesticides was not conducted prior to exposure beyond thatreported from the monitoring hives in Section 3.4

3.7.2. Residues in hive matrices during and after feeding exposure
Clothianidin residues in hives were examined five times after the feeding started using hive beebread and hive nectar. All test hives were sampled during or immediately following the exposure
phase, for uncapped nectar at at CCA4, 16-18 July 2014 and uncapped nectar and bee bread at26
282

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
CCA5 (5-11 Aug, 2014), and post exposure in capped honey at CCA7 (14-16 Oct, 2014) and at
CCA9 (after overwintering on 22-23 Apr, 2015).

3.7.2.1. Residues in hive matrices at CCA4 (after 3 weeks of exposure)
The level of clothianidin in uncapped nectar after 3 weeks of feeding (CCA4) is summarized inTable 12. All test hives were sampled at CCA4 (16-18 July 2014). Average clothianidin
concentrations were calculated assuming that values below the LOD contained one-half the LOD(0.05 µg/L) and values below the LOQ contained one-half the LOQ (0.5 µg/L). A dose-response
correlation was observed between the clothianidin concentrations in the feeding solution and themean-measured concentrations in uncapped hive nectar. However, the clothianidin concentrationin hive uncapped nectar ranged from 16.6—38.3% of the mean concentrations in feeding solution,
after accounting for brix content of the uncapped nectar compared to the original 50% sugar
solution. It is possible that dilution of nectar from other food sources occurred during the exposure
period since, as indicated in the study, a significant degradation of clothianidin in test solution was
not detected in the study.
Clothianidin in hive uncapped nectar at CCA4: The level of clothianidin in hive uncapped nectar
during the feeding exposure (CCA4) was summarized in Table 12. All but one control hive andone treatment hive were measured (these were removed from the study due to either technicalissues or vandalism of the test hive). Out of 12 hives measured for each concentration, levels
below either the LOD or LOQ were reported in three hives at 10 µg/L, one hive at 20 µg/L, one
hive at 40 µg/L, three hives at 80 µg/L and one hive at 160 µg/L, while the remaining treatment
hives had quantifiable levels of clothianidin. Clothianidin was undetected in the majority (18/23)
of control samples and was detected at levels below the LOQ in four additional samples. Only
one control sample had a level of clothianidin above the LOQ (1.36 ppb)
The results showed a dose-response correlation between the average concentrations measured inuncapped hive nectar and the concentrations in the feeding solution. However, the concentrations
varied remarkably within each treatment group (see Table 12). After correction with Brix values
to 50% sugar concentration, the mean of the measured concentrations in uncapped hive nectar
within each treatment group of 10, 20, 40, 80 and 160 ug/L (8.1, 16.3, 32.5, 65.1, and 130.1 ppb)
was 2.97 (range: <LOQ-8.04), 6.01 (range: <LOD-13.29), 12.44 (range: <LOQ-35.83), 15.67(range: <LOD-45.4), and 21.61 ppb (range: <LOD-84.18), respectively. By average, the measured
concentration in hive nectar was 30.5% (range 16.6-38.3%) of the concentration in feeding
solution. The results showed that after 3 weeks of feeding, clothianidin concentrations in uncapped
hive nectar appeared lower than that in the feeding solutions, which indicated that the foraging
bees also foraged on nectar sources other than the provided sugar sources which diluted the levelof treatment. It is noted that this result is expected, as bees were allowed to freely forage, and also,
under natural conditions bees typically forage on multiple plant pollen and nectar sources.

27283

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Table 5. Clothianidin concentrations (ppb) in uncapped hive nectar sampled 27 days after the start ofartificial feeding on 23 Jul, 2014 (CCA4).
Measured Clothianidin concentrations (ppb) (LOD=0.1 ppb)*
Nominal concentration (ug/L)
ApiaryControl 1†

Control 2

†

10

20

Nominal concentration (ppb)
A

0

0

<LOD

<LOQ

40

80

160

‡

8.1

16.3

32.5

65.1

130.1

1.84

8.42

5.94

4.89

26.69

B
<LOD
<LOD
<LOQ
1.18
7.34
<LOD
4.67
C
<LOD
<LOD
3.08
7.50
16.81
17.94
16.50
D
<LOQ
<LOD
1.42
6.96
17.34
<LOD
<LOD
E
<LOD
<LOQ
<LOQ
5.18
5.69
11.56
25.63
F
<LOD
<LOD
0.96
4.60
***
13.06
1.18
G
<LOD
<LOD
5.57
<LOD
8.23
32.09
19.38
H
<LOD
<LOD
4.69
9.62
10.06
9.69
28.44
**
I
<LOD
<LOQ
2.44
<LOQ
<LOQ
16.63
J
<LOQ
<LOD
4.59
10.63
22.56
36.81
34.94
K
<LOD
1.36
8.04
2.27
6.69
16.19
84.18
L
<LOD
<LOD
4.57
13.29
35.83
45.40
1.08
Number of samples
12
11
12
12
11
12
12
Average
<LOQ
2.97
6.01
12.44
15.67
21.61
concentration% Feeding
Not applicable
36.7
36.9
38.3
24.1
16.6
concentration††* Concentrations in all treatments except for the controls are corrected to 50% sugar using Brix values that are not listed in thetable, but were in the table section 6 on page 384-387 of the study report (brix values reported as >80, were assumed to be
80% for the purpose of this calculation). The brix corrected residue value reported here is determined using the formula thatthe brix corrected concentration = measured concentration * (feeding solution brix {50%}/hive nectar measured brix)** Hive was removed from the study due to a technical error and no sample collected.
***Hive was removed from study after it was found knocked over and no sample collected.
† Concentrations in the controls are measured concentrations in hive uncapped without corrections for sugar concentrations(brix).
‡Nominal concentration in ppb is estimated from the concentration in µg/L by assuming the volume density of the test solution
to be 1.2296 g/ml.
†† % Feeding concentration: the average of measured concentration compared with the nominal feeding concentration in ppb.

3.7.2.2. Residues in Hive Matrices at CCA5 (1 week after end of exposure)
The level of clothianidin in hive bee bread and uncapped nectar one week after the end of feeding
exposure (CCA5, 5-11 Aug 2014) is summarized in Tables 13-14. As with the uncapped nectar
results from CCA4, these measurements indicate a dose-response correlation between the average
concentrations of clothianidin measured in both bee bread and uncapped hive nectar and theconcentrations in the feeding solution. However, the concentrations varied remarkably withinsome treatments.
Clothianidin in bee bread at CCA5: The level of clothianidin in bee bread (hive pollen) following
exposure (6 weeks of feeding) at CCA5 was summarized in the Table 13. Clothianidin was
detected in all measured treatment samples. It was noted that not all residue information in pollen
was available. No residue information for treatment at 160 µg/L in bee bread was provided,
primarily due to insufficient pollen stores. Out of 12 hives, two hives at 80 µg/L, nine hives at 40µg/L, eleven hives at 20 µg/L and ten hives at 10 µg/L were measured, respectively. For the28284Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
remaining hives that did not have bee bread measurements, the vast majority were due toinsufficient pollen stores for sample collection, especially in the 80 and 160 µg/L treatment groups.
The results showed a dose-response correlation between the average concentrations measured inhive bee bread and the concentrations in the feeding solution. However, the concentrations varied
within each treatment group (see Table 13). The mean of the measured concentrations in bee breadwithin each treatment group of 10, 20, 40, and 80 µg/L (8.1, 16.3, 32.5, and 65.1 ppb) was 3.52
(range: 2.32-5.26), 6.68 (range: 3.54-9.41), 12.16 (range: 2.19-19.2), and 35.8 ppb (range: 30.940.6), respectively. By average, the measured concentration was 44.2% (range 37.4-54.9%) of theconcentration in feeding solution, and 45.9% (range 38.9-61.7 %) of the measured concentrationsin uncapped hive nectar (data not shown in the table). The results showed that after 3 weeks offeeding, clothianidin concentrations in hive bee bread appeared remarkably lower than that in thefeeding solutions and in hive nectar. The lower concentration in bee bread is expected due to thedilution since bee bread is a mixture of nectar and pollen from various sources.
Table 6. Clothianidin concentrations (ppb) in hive pollen (bee bread) sampled six weeks after the start ofartificial feeding on 5-11 August 2014 (CCA5).
Measured clothianidin concentrations (ppb) (LOD = 0.4 ppb)

Apiary

A
B
C
D
E
F
G
H
I
J
K
L
Number ofsamplesmeasured
Averageconcentration

Nominal concentration (ug/L)
Control 1

Control 2

0

0

-<LOD
<LOD
<LOQ
<LOQ
<LOD
<LOQ
<LOD
<LOD
<LOD
<LOQ
<LOD

<LOD
<LOD
<LOQ
<LOQ
<LOQ
<LOD
-<LOD
-<LOD
<LOQ
<LOD

11

10
<LOD

10

20

40

80

160

Nominal concentration (ppb) ‡
8.1
16.3
32.5

65.1

130.1

2.91
2.58
2.4
4.58
2.73
5.26
2.32
3.02
4.79
4.56

9.41
6.4
4.65
3.54
9.03
6.93
5.6
3.78
6.67
8.3
9.2

9.79
15.8
6.15
13
12.2
2.19
13.3
19.2
17.8

30.9
40.6
-

-

10

11

9

2

0

3.52

6.68

12.16

35.75

-

% of the feeding
Not applicable
43.5%
41.0%
37.4%
54.9%
concentration††% of the average
detection in hive
Not applicable
41.0%
41.9%
38.9%
61.7%
Nectar†††“-“ indicates that data are not available‡Nominal concentration in ppb is estimated from the concentration in µg/L by assuming the volume density of the test solution to
be 1.2296 g/ml.
†† % Feeding concentration: the average of measured concentration compared with the nominal feeding concentrations in ppb.
††† % of the average detection in hive Nectar: the average of measured concentration in bee bread compared with the averagemeasured concentration in nectar ppb without corrections for sugar (see Section 7 pp 388-391 in the study report).

29285

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Clothianidin in uncapped hive nectar at CCA5: Similar to CCA4, a dose-response correlation wasobserved between the average concentrations of clothianidin measured in uncapped hive nectar
and the concentrations in the feeding solution. However, the clothianidin concentration in hive
uncapped nectar was lower than what was in the feeding solutions, indicating dilution of storednectar from other food sources. The level of clothianidin in hive uncapped nectar following
feeding exposure (CCA5) is summarized in Table 14. All but one control hive and one treatment
hive were measured (these were removed from the study due to either technical issues or vandalismof the test hive). Clothianidin was detected above the LOQ (0.5 ppb) in all of the measured
treatment samples. Clothianidin was undetected in the majority (19/23) of control samples andwas detected at levels below the LOQ in three additional samples. Only one control sample had alevel of clothianidin above the LOQ (1.4 ppb).
The results showed a dose-response correlation between the average concentrations measured inuncapped hive nectar and the concentrations in the feeding solution. Additionally, these measured
concentrations had much less overlap in ranges than the uncapped nectar concentrations indicatedat CCA 4 (Table 12). After correction with Brix values to 50% sugar concentration, the mean of
the measured concentrations in uncapped hive nectar within each treatment group of 10, 20, 40,
80 and 160 ug/L (8.1, 16.3, 32.5, 65.1, and 130.1 ppb) was 5.51 (range: 3.09-7.06), 10.17 (range:
8.45-13.25), 19.94 (range: 12.66-26.86), 36.99 (range: 25-48.97), and 65.65 ppb (range: 31.4107.69), respectively. By average, the measured concentration in hive nectar was 59.8% (range
50.5-68%) of the concentration in feeding solution. The results showed that after 6 weeks offeeding, although clothianidin concentrations in uncapped hive nectar still appeared lower thanthat in the feeding solutions (indicating that the foraging bees also foraged on nectar sources otherthan the provided sugar sources which diluted the level of treatment), they appeared to utilize thefeeding solutions much more than they had after only 3 weeks of feeding. As the level ofclothianidin in the uncapped nectar compared to the feeding solution appeared similar acrosstreatment groups, this may indicate that there was less available alternate forage during the final 3
weeks of exposure compared with the initial 3 weeks represented by the nectar stores at CCA4.
Table 7. Clothianidin concentrations (ppb) in uncapped hive nectar sampled 40-46 days after the start ofartificial feeding on 5-11 Aug, 2014 (CCA5).
Measured Clothianidin concentrations (ppb) (LOD=0.1 ppb)*
Nominal concentration (ug/L)
ApiaryControl 1†

Control 2

†

10

20

Nominal concentration (ppb)
0

0

A

<LOD

<LOQ

B
C
D
E
F
G
H
I
J
K
L

<LOD
<LOD
<LOQ
<LOD
<LOD
<LOD
<LOQ
<LOD
<LOD
<LOD
<LOD

<LOD
<LOD
<LOD
<LOQ
1.4
<LOD
<LOD
**

<LOD
<LOD
<LOD

40

80

160

‡

8.1

16.3

32.5

65.1

130.1

5.72

10.39

23.13

31.88

31.4

5.79
5.98
6.63
3.79
5.06
5.35
4.81
3.09
5.97
7.06
6.82

10.44
9.87
9.94
9.87
12.04
10.06
8.45
8.56
8.94
10.06
13.25

16.17
22.66
21.32
20.45
19.56
12.66
12.97
23.94
19.69
26.86

27.17
38.86
29.55
28.40
32.78
47.53
42.57
25
45
46.20
48.97

61.58
72.60
61.82
69.23
107.69
41.92
40.83
63.90
77.5
68.99
90.38

30
286

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Number of samples
12
11
12
12
11
12
12
Average
<LOQ
5.51
10.17
19.94
36.99
65.65
concentration% Feeding
Not applicable
68.0
62.4
61.4
56.8
50.5
concentration††* Concentrations in all treatments except for the controls are corrected to 50% sugar using Brix values that are not listed in thetable, but were in the table section 7 on page 388-391 of the study report (brix values reported as >80, were assumed to be
80% for the purpose of this calculation). The brix corrected residue value reported here is determined using the formula thatthe brix corrected concentration = measured concentration * (feeding solution brix {50%}/hive nectar measured brix)** Hive was removed from the study due to a technical error and no sample collected.
***Hive was removed from study after it was found knocked over and no sample collected.
† Concentrations in the controls are measured concentrations in hive uncapped without corrections for sugar concentrations.
‡Nominal concentration in ppb is estimated from the concentration in µg/L by assuming the volume density of the test solution
to be 1.2296 g/ml.
†† % Feeding concentration: the average of measured concentration compared with the nominal feeding concentration in ppb.

3.7.2.3. Residues in hive matrices at CCA7 (prior to overwintering)
The level of clothianidin in capped honey at the last CCA prior to overwintering (CCA7, 14-22
Oct, 2014) is summarized in Table 15. As with the uncapped nectar results from CCAs 4 & 5,these measurements generally indicated a dose-response correlation between the average
concentrations of clothianidin measured in the hive matrix (capped honey) and the concentrationsin the feeding solution. However, the concentrations varied remarkably within some treatments
and the mean concentrations in capped honey were very similar between the nominal 20 and 40µg/L concentrations (means of 6.15 and 6.38 ppb, respectively). The concentration of clothianidin
in the capped honey was lower than in the uncapped nectar at CCA5, indicating that either bees
were continuing to consume the clothianidin-exposed food stores, there was continued dilution of
the stores from other nectar sources and/or there was potential degradation of clothianidin in the
capped honey (which seems unlikely given the storage stability data).
At this point in the study there were more hives that did not have measurements taken (comparedwith the measurements at CCAs 4 and 5), often due to insufficient capped honey stores for samples
to be taken. Five control hives and six treatment hives were not measured. Of the treatment hives
with measurements, levels below either the LOD or LOQ were reported in three hives at 10 µg/L,
one hive at 20 µg/L, two hives at 40 µg/L, three hives at 80 µg/L and one hive at 160 µg/L, while
the remaining treatment hives had quantifiable levels of clothianidin. Clothianidin was undetected
in all but one control samples (18/19) and was detected at levels below the LOQ in the remaining
sample. After correction with Brix values to 50% sugar concentration, the mean of the measured
concentrations in capped honey within each treatment group of 10, 20, 40, 80 and 160 ug/L (8.1,
16.3, 32.5, 65.1, and 130.1 ppb) was 2.14 (range: <LOD-6.81), 6.15 (range: <LOQ-10.88), 6.38
(range: <LOD-21.38), 22.32 (range: <LOD-44.31), and 27.23 ppb (range: <LOD-66.25),
respectively. By average, the measured concentration in capped honey was 27.8% (range 19.637.7%) of the concentration in feeding solution. It is notable that the mean residues in uncapped
nectar in the 20 and 40 µg/L treatment groups were highly similar.

31287

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Table 8. Clothianidin concentrations (ppb) in capped honey sampled 97--103 days after the start ofartificial feeding during CCA7 on either 14-16 Oct, 2014 (all but the highest treatment groups) or 21-22
Oct, 2014 (80 and 160 µg/L groups only).
Measured Clothianidin concentrations (ppb) (LOD=0.1 ppb)*
Nominal concentration (µg/L)
ApiaryControl 1†

Control 2

†

10

20

Nominal concentration (ppb)
A

0

0

<LOD

<LOD

40

80

160

‡

8.1

16.3

32.5

65.1

130.1

--

6.63

6.44

<LOD

39.81

B
<LOD
<LOD
10.88
6.56
32.63
24.29
C
<LOD
<LOD
1.29
3.35
-25.19
<LOD
-D
<LOQ
6.81
5.72
<LOD
<LOQ
22.06
E
<LOD
<LOD
<LOD
3.02
2.28
<LOQ
17.97
F
<LOD
<LOD
2.16
3.06
-16
49.5
G
<LOD
<LOD
<LOD
8.19
5.04
30.13
29.88
-H
<LOD
0.78
7.19
3.7
36.5
45.94
-I
<LOD
3.52
8.94
3.49
39.75
14.69
J
<LOD
<LOD
4.59
10.38
21.38
44.31
5.59
-K
<LOD
-<LOQ
<LOD
30.56
66.25
L
<LOD
<LOD
--14.88
12.09
10.75
Number of samples
10
9
9
11
10
12
12
Average
<LOD
2.14
6.15
6.38
22.32
27.23
concentration% Feeding
Not applicable
26.4
37.7
19.6
34.3
20.9
concentration††* Concentrations in all treatments except for the controls are corrected to 50% sugar using Brix values that are not listed in thetable, but were in the table section 8 on page 392-395 of the study report (brix values reported as >80, were assumed to be
80% for the purpose of this calculation). The brix corrected residue value reported here is determined using the formula thatthe brix corrected concentration = measured concentration * (feeding solution brix {50%}/hive nectar measured brix)“-“ indicates that no data are available (either no sample was taken {due to either minimal capped honey stores or the hive hadalready been removed due to technical errors or vandalism} or no sample was received by the analytical lab {only 1 instance})† Concentrations in the controls are measured concentrations in hive uncapped without corrections for sugar concentrations.
‡Nominal concentration in ppb is estimated from the concentration in µg/L by assuming the volume density of the test solution
to be 1.2296 g/ml.
†† % Feeding concentration: the average of measured concentration compared with the nominal feeding concentration in ppb.
--

3.7.2.4. Residues in hive matrices at CCA9 (following overwintering)
The level of clothianidin in capped honey at the final CCA following overwintering (CCA9,22-23 Apr, 2015 for UTC, 10, 20, and 40 ppb; 27 Apr 2015 for 80 ppb treatment group) issummarized in Table 16. As with the uncapped nectar results from CCAs 4 & 5 and capped honeyfrom CCA 7, these measurements generally indicated a dose-response correlation between the
average concentrations of clothianidin measured in capped honey and the concentrations in thefeeding solution. However, the concentrations varied remarkably within some treatments and as
survival in several treatments was very poor (including in controls), and samples were only takenfrom surviving hives, there were a low number of hive samples in all but the 20 and 80 µg/L
treatment groups.
Of the treatment hives with measurements, levels below the LOQ were reported in two (out of
three remaining) hives at 10 µg/L, three hives (out of eight) at 20 µg/L, one hive (out of five) at
40 µg/L, and two hives (out of ten) at 80 µg/L, while the remaining surviving treatment hives had
quantifiable levels of clothianidin. Out of eight surviving control hives, clothianidin was
32288

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
undetected in six samples and was detected at levels below the LOQ in the remaining two samples.
After correction with Brix values to 50% sugar concentration, the mean of the measured
concentrations in capped honey within each treatment group of 10, 20, 40, and 80 ug/L (8.1, 16.3,
32.5, and 65.1 ppb) was 1.17 (range: <LOQ-2.89), 1.82 (range: <LOQ-6.81), 5.24 (range: <LOQ12.88), and 13.41 ppb (range: <LOQ-31.5), respectively. The average measured concentration incapped honey at CCA9 was 15.6% (range 11.2-20.6%) of the concentration in feeding solution.
No measurement was provided for treatment at 160 ug/L as all colonies in this treatment groupwere destroyed after CCA7 (Dec, 2014). The unmeasured level of residues in dead hives presents
an additional uncertainty as to the average residues that might represent the level of treatments atfollowing overwintering.
Table 9. Clothianidin concentrations (ppb) in capped honey sampled 305 days after the start of artificial
feeding on 22-27 Apr (CCA9).
Measured Clothianidin concentrations (ppb) (LOD=0.1 ppb)*
Nominal concentration (ug/L)
ApiaryControl 1†

Control 2

†

10

20

Nominal concentration (ppb)
A

0

0

--

--

40

80

160

‡

8.1

16.3

32.5

65.1

130.1

--

2.43

12.88

5.89

--

B
--<LOQ
-2.49
1.65
-C
<LOD
--<LOQ
-31.5
-D
-<LOD
--<LOQ
<LOQ
-E
-<LOD
-1.91
-<LOQ
-F
--2.89
-***
12.06
-G
-<LOQ
-0.68
-23.63
-H
-<LOD
<LOQ
--26.28
-**
I
<LOD
-1.78
3.69
--J
-<LOD
-<LOQ
6.81
31.5
-K
<LOQ
--<LOQ-0.98-L---6.81---Number of samples
3
5
3
8
5
10
0
Average
<LOQ
1.17
1.82
5.24
13.41
N/A
concentration% Feeding
Not applicable
14.4
11.2
16.1
20.6
N/A
concentration††* Concentrations in all treatments except for the controls are corrected to 50% sugar using Brix values that are not listed in thetable, but were in the table section 9 on page 396-398 of the study report (brix values reported as >80, were assumed to be 80%
for the purpose of this calculation). The brix corrected residue value reported here is determined using the formula that the brix
corrected concentration = measured concentration * (feeding solution brix {50%}/hive nectar measured brix)“-“ indicates that no data are available (either no sample was taken {due to either a dead hive, minimal capped honey stores or thehive had already been removed due to technical errors or vandalism} or no sample was received by the analytical lab {only 1
instance})† Concentrations in the controls are measured concentrations in hive uncapped without corrections for sugar concentrations.
‡Nominal concentration in ppb is estimated from the concentration in µg/L by assuming the volume density of the test solution to
be 1.2296 g/ml.
†† % Feeding concentration: the average of measured concentration compared with the nominal feeding concentration in ppb.

3.7.2.5. Comparison of concentration in feeding solution and hive matrices
A correlation between the clothianidin concentrations in the feeding solution and theconcentrations measured in hive beebread, uncapped nectar and capped honey was observed in the
middle of the exposure period (CCA4), one week after the end of exposure (CCA5) and continued
33
289

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
through succeeding CSAs (CCAs 7 and 9). However, clothianidin measured concentrations in all
hive matrices were lower than that in the feeding solutions (Table 17).
Table 10. Clothianidin concentration measured in hive uncapped nectar, capped honey and hive bee breadcompared to nominal concentrations in the test feeding solutions
Nominal concentration in
test feeding solution

Clothianidin concentrationin hive uncapped nectar(CCAs 4 and 5) or capped
honey (CCAs 7 and 9) in %of the concentration of
nominal feeding solution(average measured
concentration in ppb)¥

Clothianidin concentrationin hive beebread in % of theconcentration of nominal
feeding solution (averagemeasured concentration in
ppb)

µg/L

10

20

40

80

160

ppb

8.1

16.3

32.5

65.1

130.1

CCA 4

36.7
(2.97)

36.9(6.01)

38.3(12.44)24.1(15.67)

16.6(21.61)

30.5

CCA 5

68.0
(5.51)

62.4(10.17)61.4(19.94)56.8(36.99)50.5(65.5)

59.8

CCA 7

26.4
(2.14)

37.7(6.15)

19.6(6.38)

34.3(22.32)

20.9(27.23)

27.8

CCA 9

14.4
(1.17)

11.2(1.82)

16.1(5.24)

20.6(13.41)

N/A

15.6

CCA 5

43.4
(3.52)

41.0(6.68)

37.4(12.16)54.9(35.75)

N/A

44.2

Average

The study did not test for clothianidin degradation products (e.g. TZNG) in the test solution.
Considering the stability of clothianidin in the test solution, the reduced concentrations ofclothianidin in hive matrices likely indicates that test bees were also foraging for pollen and nectar
from sources other than the feeding solution.

3.8.

Pathogens

Besides a standard treatment for Varroa mites, no treatments for any other hive pests, predators
or diseases were administered to any hives.

3.8.1. Varroa Presence
Varroa mite occurrence in the colonies was assessed the week before and after the feeding period,
as well as after over-wintering (CCA3, CCA5 and CCA9). The number of mites per 100 bees wascounted following washing bees in alcohol to remove the mites. Hives were treated with one
application of Apiguard® (active ingredient: thymol) following typical apicultural practice for theregion immediately after the September CCA’s to prevent high mite loads.
Prior to exposure at CCA3, the hives had similar mite loads (mean ranges of 0.28—0.44 mites/100
bees). Immediately following exposure (CCA5), mite loads were more variable (mean ranges of
0.71-2.40 mites/100 bees), but generally appeared to be positively correlated with treatment dose,though the 160 µg/L treatment group had lower infestation levels compared with the 80 µg/L
treatment group (Error! Reference source not found. 5). After over-wintering, Varroa levels were34
290

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
highly variable (mean ranges of 0.67-2.61mites/100 bees) and did not appear to follow a doseresponse relationship, though this may be confounded by the low number of remaining hives withmeasurements in both contols and treatment groups.

Figure 5. Varroa infestation levels in control and treatment groups prior to exposure (CCA 3),
immediately following the termination of exposure (CCA5) and after over-wintering (CCA9).

3.8.2. Nosema presence
The number of Nosema spores per bee was determined at three time points at CCA3, CCA5 andCCA9. At CCA3, there were 2-3 measurements per hive, while at CCA9 there were 2
measurements and at CCA5 there was only 1 sample measurement. It was unclear from the study
report why the number of samples were different between the CCAs or why different numbers ofsamples were taken at some hives during CCA3. There generally appeared to be no trend between
Nosema infestation and treatment dose, though there were generally more Nosema spores in thehigher treatments following overwintering, although the 10 µg/L treatment group had lower levels
than controls at this measurement time (Figure 6) and the 20 µg/L treatment group was only
slightly elevated compared to the controls (2.29 million spores/bee comapred to 2.15 million
spores/bee). As with Varroa, above, the CCA9 numbers may also be confounded by the low
number of remaining surviving hives in both control and treatment groups.

35291

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

Figure 6. Nosema spore loads in control and treatment groups prior to exposure (CCA 3), immediately
following the termination of exposure (CCA5) and after over-wintering (CCA9).

3.9.

Statistical Analysis

What follows are brief summaries of the study author’s and reviewer’s statistical analyses
employed for the review of this study.

3.9.1. Study Author’s Analysis
The study author conducted statistical analysis using SAS (version 9.3). The analysis included
colony strength (as indicated by mean number of adults), brood stages (as indicated by the mean
number of eggs, larval cells, and pupal cells) and food stores (as indicated by the mean number ofpollen and nectar/honey cells). For the pre-test data, all tests were done in a two tailed approach,whereas for the data assessed after exposure, one tailed (lower) tests were conducted. According
to the study author, after Shapiro-Wilks and Levene’s were used to test assumptions of normality
and equal variances, respectively, procedure GLM was used for the ANOVA analysis. Williams’
Trend Test was used to test data that passed the assumptions of normality, variance homogeneity,and monotonicity. Dunnett’s t-Test was used to test data that were non-monotonic, but passed tests
of normality and variance homogeneity. Dunnett’s T3 Test with Rank Transformed (within blocks)
data was used to test data that were normally distributed, but failed the criteria for equal variance.

3.9.2. Study Reviewer’s Statistical Analysis Approach
As part of the collaborative review effort of the study, separate statistical analyses wereconducted by EPA and PMRA using the raw data submitted by the study author. A descriptionof EPA’s statistical methodology is provided here while PMRA’s methodology is presented inAppendix A. However, the discussions below in the Colony Condition Assessment section(Section 3.9.5) presents the results of both analyses. It is noted that while the Agencies utilized
different statistical analysis approaches, interpretations based on the PMRA analysis tended to be
similar to interpretations from the EPA analysis. Although the PRMA analysis resulted in some36292Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
differences in statistically significant endpoints and time periods, these differences do notsignificantly alter the ultimate biological interpretation of the study regarding colony level
effects leading to a clearly defined, highly-confident protective endpoint.
The general experimental design was a randomized complete block (apiary) with repeatedmeasures (CCA) and data will be analyzed in SAS (v9.4) using the PROC MIXED procedure.
Since hives were not assigned and placed in the study apiaries until shortly before CCA3, thedata for the statistical analysis only included data collected from CCA3 and the following CCAs.
Shortly before CCA3, hives were ranked by strength and the ‘strongest’ hives were placed in the
one apiary. The next eight strongest hives were then placed in an empty apiary. This processcontinued until hives were placed in all apiaries. Within each apiary, the control treatment was
replicated two times and each treatment occurred one time (total of 8 hives in each apiary: sevenhives were randomly assigned as control or treatment group and the eighth hive was used for
additional sampling during the study). Given this design, the blocking factor ‘apiary,’ represents
variation due to geographic location and initial hive strength.
As a large percentage of hives did not survive overwintering, data collected the following spring
will not be included in the statistical analyses. Other than the three hives (removed from thestudy and noted in Table 18), no hive mortality occurred prior to overwintering.
Table 11. Timeline including major milestones of study
Date
12 May 2014
2 Jun 201417-18 Jun 201418 Jun 2014

Study action*
Initiate CCA1 (non-GLP)
Initiate CCA2 (non-GLP)
Hives moved to study locations
Initiate CCA3 (non-GLP)

26 Jun 2014

Initiate clothianidin exposure throughsucrose solution.
Initiate CCA4 (GLP)

15 Jul 2014

5 Aug 2014
7 Aug 2014
8 Sep 2014

Initiate CCA5 (GLP)
End clothianidin exposure throughsucrose solution
Initiate CCA6 (GLP)

Comments
Not included in statistical analysis.
Not included in statistical analysis.
noneFirst CCA to be included in thestatistical analyses.
none
Hive I7 (control) removed fromstudy; possible contamination.
Hive F3 (40 ppb) removed fromstudy; found knocked over.
none
none

Hive C2 (40 ppb) removed fromstudy; found knocked over.
14 Oct 2014
Initiate CCA7 (GLP)
Final CCA to be included in thestatistical analyses.
December 2014
All remaining hives in the 160 ppb treatment group were destroyed as colony
strength was low and they were not expected to survive the winter.
17 Mar 2015
Initiate CCA8 (GLP)
Overwintering survival was 35, 27,
67, 50, and 85% for control, 10 ppb,
20 ppb, 40 ppb, and 80 ppb treatment
22 Apr 2015
Initiate CCA9 (GLP)
groups, respectively. Therefore,
CCA8 and CCA9 were not included
in statistical analyses.
*each CCA took three or more days to complete.

37
293

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Variables recorded at each CCA included number of adult bees in the hive and number of cells
containing each of the following life stages or food stores: eggs, larvae (open cells), pupae(closed cells), pollen, and honey. Following standard bee keeping practices, supers were addedor removed from each hive to best support growth or reductions in the size of the bee colony.
Timing for addition and removal of supers is provided in Appendix B. A queen excluder wasplaced between the initial hive box and added super boxes; this limited the summed number of
egg, pupae, and larvae cells to the number of cells in the initial box (3970 cells). All adult bees,
with the exception of the queen, could move to any added supers, and honey and pollen could be
stored in those additional supers as well. The suite of variables that were subjected to data
analysis were:•
Number of adults•
Number of egg cells•
Number of open (larvae) cells•
Number of capped (pupae) cells
Number of pollen cells••
Number of honey cells•
Total number of individuals (adults + eggs + larvae + pupae)•
Total brood (eggs + larvae + pupae), and•Total food (pollen + honey).
To facilitate computation and algorithm convergence in the SAS Procedures, all data was divided
by 1000 prior to any statistical analysis. Since all response variables were divided by the same
constant, there was no effect on any of the test statistics or p-values. No adjustments for addition
or removal of supers were conducted for the statistical analysis.
Total brood and total food are new summary variables; EPA’s Environmental Fate and Effects
Division (EFED) is still evaluating their utility in providing additional information on biologicaleffects beyond the initial set of variables. PMRA did not analyze these summary variables intheir statistical analysis.
Prior to the repeated measures analysis, the data were evaluated for patterns in temporalcorrelation and correlations across hive components within each of the evaluated CCAs. This
analysis was accomplished through a series of pairwise scatterplots and principle components
analyses (PCA).

3.9.2.1 Scatterplot and Principle Component Analysis
Based on physical hive constructs and the nature of honey bees, it is generally accepted that thecolony condition assessment (CCA) variables may be correlated over time and may also becorrelated within a time point (sampling time). Given this background, a series of scatterplots,
correlation matrices, and principle component analyses was prepared; the full SAS output is
included as Attachments 1-3. For these analyses, there was no adjustment for treatment effects,
only correlation over time was evaluated.
For the single hive components, adults, eggs, larvae, pupae, pollen, and honey, some of thegeneral summary points are:38294

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Honey had the strongest and most consistent pairwise correlations across all the time
points.
• CCA3 tended to have the lowest pairwise correlations with the other CCAs for allcomponents.
• For each of the hive components, the first principle component explained 46 to 85% of
the total variation across all CCAs; the lowest percent of explained variation was forlarvae and the highest was for honey.
• For each of the hive components except honey, the general interpretation of the firstprinciple component was a weighted average with CCA4, CCA5, CCA6, and CCA7
carrying approximately equal weights and CCA3 carrying much less weight. Note thatfor pupae, CCA7 carried slightly less weight than CCA4, CCA5, and CCA6. For honey,
the general interpretation of the first principle component was a weighted average with
all CCAs carrying approximately equal weight.
For the three composite hive variables (live, brood, and food), general summary points are:
• For live, all possible pairwise correlations between CCA4, CCA5, CCA6, and CCA7
ranged from 0.61 to 0.87, while the pairwise correlations between CCA3 and thefollowing CCAs ranged from 0.15 to 0.30. For brood, the pairwise correlations between
CCA4, CCA5, and CCA6 were highest; pairwise correlations with CCA7 were lower,
and pairwise correlations with CCA3 were the lowest.
• For live and brood, the first principle component explained 67% and 57%, respectively,of the total variation. As with the individual components, the general interpretation of thefirst principle component was a weighted average over all time points with CCA3
carrying the least weight. For brood, as with pupae (above), CCA7 carried slightly lessweight than CCA4, CCA5, and CCA6.
• For food, all pairwise correlations were strong (ranged from 0.67 to 0.92), and the generalinterpretation of the first principle component was a weighted average with all CCAs
carrying approximately equal weight.
•

In addition to exploring correlations among CCAs for each of the response variables, correlations
among response variables within a CCA were explored. For this exploratory analysis, only theindividual hive components were evaluated. No adjustment was made for treatment effects (i.e.,
all data were included in a single series of plots and PCAs; separate assessments were not donefor each treatment). Some general interpretations are:
• For all of the CCAs, honey had the weakest pairwise correlations (honey with any of theother measured matrices) amongst all the pairwise correlations. For many of the CCAs,
honey was negatively correlated with some of the other variables.
• For each of the CCAs, the percent of the total variation explained by the first principle
component ranged from 40 to 52%. At each time point the first principle component
tended to be interpreted as a weighted average. The weights and interpretations for the
first principle component were not consistent when compared across CCAs.

3.9.2.2 Analysis Approach and Model Setup
As discussed above, the experimental design was a randomized complete block (apiary) withrepeated measures (CCAs). Exploring the interaction between treatment and CCA can addressthese two questions:39295

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document••

At each CCA, was there a reduction in the response relative to the control?
At each treatment level, was there a difference in the response relative to the baseline
time point (CCA3)?

With the experimental design component of the analysis established, the next part of the analysis
was to determine which correlation structure (across time) was the best fitting for these data. The
scatterplots, correlation matrices, and principle component analyses were used to inform thechoice of covariance structure used in the repeated measure analysis. Some summary points fromthe above exploratory analyses are that temporal correlations within a response variable tended
to be stronger than correlations among response variables within a time point; variance for agiven response variable was not homogenous among the CCAs; and that the pairwise
correlations did not consistently decrease as the distance between the temporal pairs increased.
Before conducting any comparisons among treatments or CCAs, several different correlation
structures to best fit the temporal correlation were evaluated. The structures that were fitted
included:• Compound symmetry (CS): assumes equal correlation for all pairwise correlations(regardless of distance of time point).
• Compound symmetry with heterogeneous variance (CSH): Estimates a uniquevariance at each time point, but assumes equal correlation for all pairwise correlations(regardless of distance of time point).
• Autoregressive correlation (AR(1)). Assumes equal correlation between adjacent time
points. Time points further apart have a lesser correlation.
• Heterogeneous Toeplitz (ToepH): models a unique variance for each time point andseparate correlations for equidistant time points (e.g., correlation between CCA3 andCCA5 is the same as the correlation between CCA4 and CCA6).
More information about each of the covariance structures available in the REPEATED statementin SAS can be found here:
https://support.sas.com/documentation/cdl/en/statug/63033/HTML/default/viewer.htm#statug_m
ixed_sect019.htm . The full SAS output is provided in Attachment 1.
To compare covariance structure fits, Bayesian Information Criterion (BIC) was utilized 3. The
BIC is a function of the log likelihood with a penalty for an increase in the number of covarianceparameters to be fitted. The BIC value for each fitted model for all response variables is reported
in Table 19; smaller values of the BIC indicate a better fit (bolded). For many of the endpoints,
heterogeneity of variance at different time points was indicated as compound symmetry withheterogeneous variance (CSH) and heterogeneous Toeplitz (ToepH) were the covariancestructures providing the best fits. This is not surprising as unequal variances were observed in the
exploratory multivariate/principle component analysis.

3

Schwarz, Gideon. Estimating the Dimension of a Model. Ann. Statist. 6 (1978), no. 2, 461--464.
doi:10.1214/aos/1176344136. http://projecteuclid.org/euclid.aos/1176344136.
40296

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Table 12. BIC values for fitted models. CCA3 – CCA7 -clothianidin

Variable →AdultsEggs Larvae
Pupae
Pollen
Honey
Live
Brood
Food
Model ↓
(open) (capped)
CS
2216
1671
2005
2281
2118
2950
2852
2640
3018
CSH
2216
2938
2829
3012
1614
1990
2248
2062
2619
AR(1)
1670
1998@
2277
2118
2905
2833
2632
2189
2974
ToepH
2197
1609
1983@
2247@
2059@
2893@ 2810@
2614@
2972@
*Within a response variable, smaller BIC values (bolded) indicate better covariance model fit. Kass and Raferty(1995) suggested that differences of greater than 10 in BIC values provides very strong evidence that model fits
are not equivalent.
@Convergence was attained, but estimated G matrix was not positive definite and not all covariance parameters
could be estimated.

For all the evaluated response variables, ToepH was identified as one of the ‘best fitting’covariance structures; however, all covariance parameters could not be estimated for majority ofthe endpoints. CSH was identified as one of the best fitting covariance structures for four of thesix single hive components and one of the three composite hive variable. Compound symmetry(CS) was not identified as quality fit to the data for any of the eight evaluated response variables.
AR(1) was identified as a quality fit for two of the evaluated endpoints.
Residual plots were also evaluated for each of the response variables and covariance structures.
Patterns indicative of heterogeneous variance of the residuals were evident for many of the
response variables and models where an assumption of equal variance at each time point was
made. For many of the residual plots when CS or AR(1) covariance structure was modeled, thevertical spread of the residuals around increased as the predicted mean increased (indicatinglarger variances as the mean increased; see Figure 7, for example). These response variables arecounts, hence the distribution of the response variable and the residuals may not meetassumptions of normality and/or equal variance. More specifically, review of the residual plots
indicates that estimating utilizing a covariance structure that estimated unique variances for each
CCA (e.g., CSH, ToepH covariance structures) appears to improve overall model fit.

41297

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

Figure 7. Studentized residual plots for eggs with covariance structures of (left) compound symmetry (CS) and(right) compound symmetry with heterogeneous variance (CSH). Distribution of the residuals indicates a better
fitting model for the CSH covariance structure.

Of the evaluated models, either CSH or ToepH should be selected as the covariance structure forthe repeated measure of CCA as they provided better fitted models for multiple endpoints. The
additional covariance parameters could not always be estimated in the ToepH model suggestingthat the increase in the number of parameters relative to CSH that were to be estimated is an
overparameterization of the model based on the available data. Therefore, the review teamelected to move forward with the heterogeneous compound symmetry (CSH) covariancestructure for the final analyses.

3.9.2.3 Treatment by Time Interaction and Follow-up Contrasts
The text box below provides the SAS code for the mixed model that was used for follow-up
statistical contrasts to address the following questions:• At each CCA, was there a reduction in the response relative to the control?
• At each treatment level, was there a difference in the response relative to the baseline
time point (CCA3)?

42

298

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document•

The contrasts that were utilized for this analysis were Dunnett’s test. Dunnett’s test is aset of pairwise contrasts in which each treatment mean is compared to the control mean;
the tests can be one- or two-sided. For a given set of contrasts, the experiment-wise errorrate is controlled as the specified alpha-level. In this case, a ‘set of contrasts’ is either (1)comparisons of treatment means to the control for a specific endpoint at a specific CCAor (2) comparison of time-points CCA4, CCA5, CCA6, and CCA7 to the baseline CCA3
for a given endpoint. For all analyses, the CSH covariance matrix was used for each of
the variables.

Text Box 1. SAS Code for the mixed model used to run the statistical analysis
title 'Clothianidin - ColonyFeedingStudy(2015) data analysis';proc mixed data=cca3_7 ;title2 "Dunnett's tests - adult_scale";class apiary cca conc hive;model adult_scale = conc|cca /DDFM=SATTERTHWAITE;
random apiary ;repeated cca/ subject=hive*conc(apiary) type=csh ;lsmeans conc*cca/cl;
slice conc*cca /sliceby=cca diff=controll adjust=dunnett;
slice conc*cca /sliceby=conc diff=control adjust=dunnett;run;Williams’ test was also considered for use for one set of the follow-up contrasts - comparisons oftreatment means to the control for a specific endpoint at a specific CCA. Williams’ test has beenshown to be more powerful than Dunnett’s test when the assumption of monotonicity is met.
Williams’ requires the assumption that if there is an effect of the chemical, it follows the classic
dose-response relationship (i.e., assuming there test material has a negative effect on the
response variable, then as the test concentration increases, mean response is equal to or less thanthe mean response of the next lower dose concentration). The test procedure then determines thelowest dose level for which the mean is significantly less than the control mean. This
concentration is identified as the LOAEC and the next lower concentration is identified as the
NOAEC. Williams’ test was not utilized for this analysis for several reasons:• Review of the treatment means identified several instances when the underlying
assumption of monotonicity does not appear to be met. Given the large variation in themeasured responses in general, it could not be determined if the observed deviations
from monotonicity were due to large background variation or to a non-monotone
treatment response.
• For any one response variable, the data are combined across CCAs into one mixed model
analysis. Incorporating data from all CCAs improves the variance/covariance estimatesand increases the degrees of freedom for hypothesis testing. As the degrees of freedom
for hypothesis testing increases, differences in power between Dunnett’s test andWilliams’ test become small.
• It has not been codified in the PROC MIXED procedure in SAS, and the level of effort
to code and QA the test would be significant.

43299Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
An analysis approach where data from each CCA was analyzed separately as a randomized
complete block design was also considered as SAS has options for use of Williams’ test for
simpler experimental designs. This approach was not selected for several reasons:• Equality of variance would still need to be evaluated. If the assumption of homogenous
variances was not met for some CCAs, then transforming the response or non-parametricanalyses would need to be considered. Incorporating the heterogeneous variances into theerror matrices of the general linear model (GLM) would increase the complexity of themodel such that the Williams’ options in SAS could no longer be utilized.
• A statistical analysis approach that does not utilize the strength of the correlations among
time points to improve estimates of error variance would not be as powerful as one that
does incorporate that additional information about the nature of the responses.

3.9.3 Treatment Effects Within a CCA
The table of p-values resulting from the Dunnett’s tests (for evaluating whether within a CCA,
the treatment mean are significantly less than control means) are summarized in Table 20.
Figures 8-17 below show the results for each response variable across all CCAs analyzed(CCA3-CCA7) and all treatment levels. For all the figures presented below, significant
reductions from the negative control with p-values below the 0.05 alpha level are denoted by a
red dot at a given treatment level and CCA and those reductions with p-values between 0.05 and
0.1 are denoted by a black dot. Statistical NOAECs and LOAECs within a CCA will be
determined using an alpha-level of 0.05. Additional comparisons using and alpha-level of 0.10
are included for additional characterization. Error bars represent one standard error from themean calculated from the model residual mean squares estimate. The associated SAS output
containing the full results of the Dunnett’s comparisons can be found in Attachment 1.
Table 13. Results of one-sided Dunnett’s test (comparing control to each treatment group), correlations
modeled using CSH. Cells include the treatment groups that were significantly lower than control.
Adults Eggs
Larvae Pupae
Pollen Honey Live
Brood
Food
(Open) (Capped)
CCA3 NS
NS
NS
NS
NS
NS
NS
NS
NS
CCA4 160
80
40
NS
NS
160
40
40
40
160
80
80
80
80
160
160
160
160
CCA5 40
20
NS
NS
80
40
10
40
40
80
80
160
80
20
80
80
160
160
160
40
160
160
80
160
CCA6 10
80
NS
NS
80
40
80
40
40
40
160
160
80
160
80
80
80
160
160
160
160
CCA7

80
160

80
160

80
160

160

10
80
160

NS

80
160

80
160

NS

44
300

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
AdultsEggs

Larvae Pupae
Pollen Honey Live
Brood
Food
(Open) (Capped)* NS indicates that there were no test concentrations with means significantly less than the control(p>0.10).
Bolded concentration = significantly less than control (p< 0.05)
Italicized concentration = less than control (0.05< p < 0.10)

3.9.4 Temporal Trends Within a Treatment Level
A second component to evaluating the “treatment x CCA” interaction is to look at the temporalchanges within a treatment group. This was accomplished by comparing each CCA (CCA4through CCA7) to CCA3 by use of a two-sided Dunnett’s test (Table 21 and Table 22). This
suite of comparisons is not as informative as the contrasts of control against the treatment groupwithin a CCA for establishing a statistical NOAEC and LOAEC. However, it may aid ininterpretations and further biological understanding of temporal shifts in the life stages and foodcomponents present in the hive. Differences in patterns of temporal shifts between the control
and various treatment groups can provide further understanding of the potential impacts ofclothianidin on beehive population dynamics.
Table 14. Results of two-sided Dunnett’s test (comparing CCA3 to each following CCA), correlations
modeled using CSH.
Trt
Group
Control10

Response Variable
AdultsEggs
CCA5-6>CCA3 CCA4-7<CCA3
CCA7<CCA3
CCA4-7<CCA3Open
CCA7<CCA3
CCA7<CCA3

Capped
CCA7<CCA3
CCA7<CCA32040

CCA5>CCA3
CCA7<CCA3

CCA4-7<CCA3
CCA4-7<CCA3

CCA7<CCA3
CCA7<CCA3

CCA7<CCA3
CCA5-7<CCA3PollenCCA4-5>CCA3
CCA4>CCA3
CCA7<CCA3
CCA4>CCA3
CCA4>CCA380160

CCA5-7<CCA3
CCA4-7<CCA3

CCA4-7<CCA3
CCA4-7<CCA3

CCA4-7<CCA3
CCA4-7<CCA3

CCA4-7<CCA3
CCA4-7<CCA3

CCA5-7<CCA3
CCA4-7<CCA3

Table 15. Results of two-sided Dunnett’s test (comparing CCA3 to each following CCA), correlations
modeled using CSH.
Trt
Group
Control

40

Response Variable
Honey
CCA4-5>CCA3
CCA6-7<CCA3
CCA4-5>CCA3
CCA7<CCA3
CCA4-5>CCA3
CCA7<CCA3
CCA4-5>CCA380160

CCA4-6>CCA3
CCA4-6>CCA3

10
20

Live
CCA7<CCA3Brood
CCA7<CCA3

CCA7<CCA3

CCA4,5,7<CCA3

CCA7<CCA3

CCA7<CCA4

CCA5-7<CCA3

CCA4-7<CCA3Food
CCA4-5>CCA3
CCA6-7<CCA3
CCA4-5>CCA3
CCA7<CCA3
CCA4-5>CCA3
CCA7<CCA3
CCA4-5>CCA3

CCA4-7<CCA3
CCA4-7<CCA3

CCA4-7<CCA3
CCA4-7<CCA3

CCA4-5>CCA3
CCA4-6>CCA3

45
301

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
3.9.5 Colony Condition Assessment Response Variables
What follows is a breakdown of each response variable assessed and the significant effects thatwere determined at each CCA (after set up and prior to overwintering; i.e., CCAs 3-7). A coupleof general points are made below when examining the results data analysis:
•
•

•

•
•

•

•
•

•
•

Unless explicitly stated otherwise, all discussion of statistical findings refer to shareddeterminations from the PMRA and EPA analyses.
All analyses considered effects at both the 0.05 and 0.1 alpha levels when weighing
statistically significant effects with biological considerations. All analyses consideredeffects at both the 0.05 and 0.1 alpha levels when weighing statistically significant effects
with biological considerations.
For simplicity and consistency in visualizing the trends and findings of statisticalsignificance simultaneously, the EPA-generated tables and figures are presented below
while PMRA-generated tables and figures reflect PMRA’s statistical analysis and arepresented in Appendix A (and as such, estimated values and significance in EPA tables
presented below may differ in some instances from the PMRA generated tables in thisappendix).
As noted above, the EPA-generated tables below indicate the percent differences fromcontrol based on raw counts of the data which have been scaled (divided by 1000) for each
response variable to facilitate convergence of the statistical model.
The EPA-generated table values are the percent reductions of the response model-based
mean for a given treatment relative to the control model-based mean. The model-basedmeans are the Least Square means based on the randomized complete block, repeatedmeasures design and model fit using SAS PROC MIXED algorithms. These Least Squaremeans may differ from arithmetic means due to missing values in the raw data (this also
accounts for some of the differences between calculations of mean percent inhibitions
between EPA and PMRA’s analyses).
The figures with colored significance “dots” representing p-values of <0.05 or <0.10 werebased on the results of the mixed model analyses conducted by EPA. off of these counts
for each hive for each response variable (with the exception of hive weight) and weregenerated by EPA. The figures indicate statistical significance (reduction in treatment
mean relative to control within a CCA) with black and red “dots” denoting a significant
reduction at the 0.10- and 0.05-alpha levels, respectively.
CCA3 was the baseline covariate and therefore is not presented in the tables generated by
PMRA (in Appendix A) for each response variable with percent reductions.
Even though data from CCA8 and CCA9 were included in the PMRA analysis andpresented in the tables in Appendix A, the evaluation of effects at these time points is
considered unreliable (by both EPA and PMRA) due to the high hive mortality observedin the controls (65% mortality at these CCAs).
PMRA did not include “total brood in hives” and “total food storage” in its analysis so
those results pertain solely to EPA findings.
It is acknowledged that there was considerable variability for some response variables atcertain treatment groups and CCAs. In order to better understand the variability oftreatment groups and make comparisons with controls, for certain variables the reviewer
has provided additional graphs focusing on the controls and lower treatment groups (10,46302

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
20, and 40 µg/L) that includes error bars around the means. Please refer to Appendix C
for summary statistics tables (i.e. 95% lower and upper confidence intervals, means andstandard error values) of the proportions of each response variable for further information.

3.9.6 Life Stage Results
The tables and figures below present results from CCA3 thru CCA7 across the different life
stages. As discussed previously, CCA3 is the final assessment just prior to placing theclothianidin-treated sucrose solutions (or untreated control) in the hives. CCA4 occurs during
the 6-week treatment period and CCA5 is just after the treatment period. CCA6 represents thetime of year when the colony as a whole starts to prepare for overwintering and therefore starts
to begin a “shut-down” phase where the numbers of adults and other life stages are clearlydecreased which is noted at CCA7. During this pre-overwintering phase, adult proportions
decline due to natural die off of worker bees and reduced rates of replenishment from reduced
egg laying by the queen.

3.9.6.1 Adults
Table 23 and Figure 8 below show the effects on adult honey bees across CCAs and treatmentgroups. Compared with the control, no differences in the number of adults in hives (p>0.1) during
the CCA4 exposure period were apparent in any of the treatments with the exception of asignificant (p<0.05) reduction determined for the 160 µg/L group, which was also reduced in all
subsequent CCAs (percent inhibtitions ranging from 30.3—96.2% in EPA’s analysis and 32.398.4% in PMRA’s analysis). The number of adults in the 80 µg/L treatment group, though notsignificantly reduced at CCA4, was significantly reduced (p<0.05) compared to controls at all
subsequent CCAs (percent inhibitions ranging from 8.5% [non-statistically significant at CCA 4]
to 56.5%) in EPA’s analysis. For PMRA the number of adults in the 80 µg/L treatment group was
significantly reduced (p<0.05) at CCA4 (13.9% estimated reduction) and in all subsequent CCAs
(estimated percent reduction ranging from 41.2-64.1%). The number of adults in the 40 µg/L
treatment group was also significantly reduced (p<0.05) at CCAs 5 and 6 (inhibitions of 23.7%
and 30.4% for EPA and 23.1% and 29.9% for PMRA), but was not significantly reduced at CCA7
(13.3% fewer adults at CCA7, compared with controls). From CCA3 through CCA7, no significant
reductions relative to controls were observed in the 10 and 20 µg/L treatment groups, except for
at CCA6 where a significant reduction was observed in the 10 µg/L group (EPA: 21.9% reduction;
PMRA: 19.6% reduction), but not for the 20 µg/L treatment group (non-statistically significant
16.5% reduction compared to controls). While the findings were not determined to be statisticallysignificant at 20 µg/L treatment group at CCA6 for the EPA analysis, they were for the analysis
used by PMRA (17% reduction compared to control, p<0.05).
Table 16. Estimated percent reduction from control for mean number of adults
Test
Reduction relative to the control mean
concentration
CCA3
CCA4
CCA5
CCA6
(µg/L)
10
4.4
-1.0
9.0
21.9*
20
-0.6
0.4
1.4
16.5
40
-2.0
-2.1
23.7*
30.4*

CCA7
18.9
6.1
13.3
47

303

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Test
concentration(µg/L)
80
160

Reduction relative to the control mean
CCA3

CCA4

CCA5

CCA6

CCA7

-10.0
-4.3

8.5
30.3*

37.1*
68.9*

56.5*
87.7*

47.6*
96.2*

Note: Negative value indicates increased number of adults in comparison to control.
*p<0.05

Figure 8. Number of adult honeybees at colony condition assessments (CCA) 3 thru 7 for eachtreatment group (based on model residual mean squares estimates).

Figure 9 below shows the trends in mean numbers of adults across the CCAs for the control andthree lowest treatment groups only as the impact at the two highest groups was evident, and adds
standard error bars in order to better compare differences in the populations. Removing the twohighest treatment concentrations adjusts the scale of the figures to see the trends more clearly at
the lower treatment groups. There is a clear divergence in the trends at the 40 µg/L treatmentgroup in comparison to the control group at CCAs 5 and 6, though by CCA7 there appears to be
substantial overlap in the error bars (one standard error from the mean). The 10 and 20 µg/L
treatment groups, which appear similar compared to the control group at CCAs 4 and 5, appear tohave low overlap with the control group at CCA6 (when statistically significant reductions wereobserved in the 10 µg/L, but not the 20 µg/L treatment group), but by CCA7, the control and lowest
treatment groups are much closer together with a distinct lack of dose-response. Also notably, as
distinguished from the control and 10 and 20 µg/L groups, while the proportions of adults for thosegroups generally increased or remained stable through CCA5 before beginning to decline, thenumbers of adults at 40 µg/L began to decline as early as CCA4, where these numbers were being

48
304

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
built up or remained constant in the control and lower treatment groups to support the foraging
worker bee force for nectar and pollen collection.

Figure 9. Number of adult honeybees at colony condition assessments (CCA) 3 thru 7 for the control andthree lowest treatment groups. Error bars represent one standard error from the mean calculated from themodel residual mean squares estimate.

Although treatment means were significantly reduced from the control at the lowest treatmentgroup (10 µg/L) at CCA6 in both EPA and PMRA analyses and were also significantly reduced
from control in the 20 µg/L treatment group at CCA6 (PMRA’s analysis only), these effects wereconsidered to be potentially transient, while the early onset and persistence of significant effects
in the highest treatment groups (40, 80 and 160 µg/L) supports the conclusion that the overall
NOAEC and LOAEC for adults is 20 and 40 µg/L, respectively. It is also notable that no
obvious dose-response relationship is observable in the two lowest doses at CCA6, though thismay also be a function of the overlap in exposure among individual hives, based on residue
analysis of hive matrices.

3.9.6.2 Eggs
Table 24 and Figure 10 below show the effects on eggs across CCAs and treatment groups. For
the EPA analysis at CCA4, compared with the controls, significant differences were observed in
the 40 µg/L (p<0.05) and 80 µg/L (0.05<p<0.1) treatment groups, but no dose-response
relationship was observed across any dosage and a lack of significant inhibition was observed
even at the highest treatment dose at this CCA. However, at CCA5 there were clear significant
reductions (p<0.05) at the 80 µg/L and 160 µg/L treatment groups (68.7% and 92.9% reduction of
eggs relative to controls), which persisted at the subsequent CCAs. At CCA5 there were also
marginaly significant reductions (p<0.1) at the 20 µg/L treatment group (37% reduction), but not
at either the 10 and 40 µg/L treatment groups (32 and 34% reductions, respectively). Further, there
49
305

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
were no significant reductions (p>0.1) for the three lowest treatments at any of the subsequent
CCAs.
For the PMRA analysis treatment means were significantly reduced (p>0.05) from the control at
the lowest three doses (10, 20 and 40 µg/L) during and immediately after the exposure period(CCA4 and CCA5) but not in subsequent CCAs after the exposure period and were significantly
reduced (p<0.05) from the control at the two highest treatment groups (80 and 160 µg/L) at all
CCAs. Similar to the EPA analysis, there was no dose response evident at CCA4. A general dose
response was evident starting at CCA5 which became more pronounced over subsequent CCAs
up to CCA7.
Table 17. Estimated percent reduction from control for number of eggs.
Test
Reduction relative to the control mean
concentration
CCA3
CCA4
CCA5
CCA6
(µg/L)
10
-6.6
30.1
32.0
3.1
20
-0.6
26.4
37.0**
15.1
40
-11.8
48.0*
33.9
12.9
80
8.4
38.1**
68.7*
49.4**
160
3.0
31.9
92.9*
83.1*

CCA7
0.9
26.5
25.6
71.2*
95.2*

Note: Negative value indicates increased number of eggs in comparison to control.
*p<0.05**0.05<p<0.1

Figure 10. Number of eggs (cells) following exposure to varying concentrations of clothianidin in the dietacross CCA3 – CCA7 (based on model residual mean squares estimate).

50306Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Figure 11 below shows the responses for the mean number of eggs for the control, 10, 20 and 40µg/L treatment groups. Removing the two highest treatment concentrations adjusts the scale of the
figures to see the trends more clearly. It is noted from this graph that at CCA4, while significant
differences (p<0.05) were observed for the 40 µg/L treatment group, as well as lower (though nonsignificant) numbers at CCA5, where the 20 µg/L treatment group was significantly (p<0.1)
different from controls, at both of these CCAs there were no obvious dose-response trends within
a CCA and across CCAs for these treatment groups (i.e. substantial overlap of these populations
based on their standard errors). Additionally, at subsequent CCAs the mean number of eggs in all
three treatments appears to have reverted close to control means. However, it is noted that themean and standard error values for the lower treatment groups are lower than the control.

Figure 11. Number of egg cells following exposure to varying concentrations of clothianidin in the dietacross CCA3—CCA7 in the control, 10, 20, and 40 µg/L groups only. Error bars represent one standard
error from the mean calculated from the model residual mean square estimate.

When weighing statistical and biological significance, the overall NOAEC and LOAEC for
eggs is determined to be 40 and 80 µg/L, respectively, based on a significant reduction in eggs
consistent at all CCAs following the end of exposure. Although there is some uncertainty
regarding this endpoint as the means of the three lowest treatments were lower than the control
means at CCAs 4 and 5, the lack of dose-response surrounding these doses and the reversion ofthese three treatments back to control means by CCA6 indicates a potential transient effect andsupports the use of 40 µg/L as the NOAEC and 80 µg/L as the LOAEC for effects from clothianidin
on egg production following a six-week exposure period.

3.9.6.3 Larvae (Open/Uncapped brood)
Table 25 and Figure 12 below show the effects on larvae (open/uncapped brood) across CCAs
and treatment groups. In the EPA analysis, compared with the control, no differences in the number
51
307

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Documentof larvae (open/uncapped cells) in hives (p>0.1) during the CCA4 exposure period were apparent
in any of the treatments with the exception of a significant (p<0.05) dramatic reduction determined
for the 160 µg/L group, which was also reduced in all subsequent CCAs (percent inhibtitions
ranging from 86.8—100%). The number of larval cells in the 80 µg/L treatment group, though
not significantly reduced at CCA4, was significantly reduced (p<0.05) compared to controls at
CCAs 5 and 6 (percent inhibitions ranging from 9.1% [non-statistically significant at CCA 4] to
81.7%), but was not statistically significantly inhibited at CCA7, despite a reduction of 46.2%
relative to controls. From CCA3 through CCA7, no significant (p>0.1) reductions were observedin any of the lowest treatment groups.
In the PMRA analysis treatment means were significantly reduced (0.05< p<0.1) from the control
at the lowest three doses (10, 20 and 40 µg/L) during a single CCA during the exposure period(CCA4) but not in subsequent CCAs and were significantly reduced (p<0.05, 0.05< p<0.1) at all
CCAs at the two highest treatment groups (80 and 160 µg/L). A clear dose response (increase in
the reduction from the control as the dose increases) was evident over all CCAs except at CCA4.
Table 18. Estimated percent reduction from control for number of larvae (open/uncapped brood)
Test
Reduction relative to the control mean
concentration
CCA3
CCA4
CCA5
CCA6
CCA7
(µg/L)
10
-0.9
14.8
1.6
-5.0
33.2
20
9.4
12.7
14.5
4.1
36.8
40
5.1
22.4
19.8
24.0
33.0
80
-15.3
9.1
81.7*
48.7*
46.2
160
3.5
86.8*
100*
93.2*
98.6*
Note: Negative value indicates increased number of larvae in comparison to control.
*p<0.0552
308

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

Figure 12. Number of open cells (larvae) at colony condition assessments (CCA) 3 thru 7 for eachtreatment group (based on model residual mean squares estimates).

Figure 13 below shows the responses for the control, 10, 20 and 40 µg/L treatment groups.
Removing the two highest treatment concentrations adjusts the scale of the figures to see the trends
more clearly. It is noted from this graph that although error bars surrounding the control group
generally have some overlap with those surrounding the 10 and 20 µg/L treatment groups, they
just overlap with the 40 µg/L treatment group from CCAs 5 through 6 and a general dose-response
relationship is observed across these doses during CCAs 5 and 6.

53309

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

Figure 13. Number of open cells (larvae) at colony condition assessments (CCA) 3 thru 7 for the control
and three lowest treatment groups. Error bars represent one standard error from the mean calculated fromthe model residual mean squares estimate.

When weighing statistical and biological significance, the overall NOAEC and LOAEC forlarval cells is determined to be 40 and 80 µg/L, respectively. This is based on persistentsignificant effects at the 80 µg/L and 160 µg/L treatment groups and lack of persistent effects
at the three lowest treatment groups (10, 20 and 40 µg/L). However, there is someuncertainty in this endpoint, given the lack of overlap of the 40 µg/L and control group
populations at all CCAs from the beginning of exposure through CCA6 and the appearanceof a dose-response relationship beginning at 40 µg/L.

3.9.6.4 Pupae (Capped Brood)
In the 40, 80 and 160 µg/L treatment groups in the EPA analysis, there were significant reductions
from the control in pupae (capped brood) (p<0.05) that persisted through multiple measurement
points (CCA’s 4-6). The percent reductions from control based on the raw counts of pupal cells
ranged from 16.4—47.1%, 26.1—83.4% and 46.3—100% in the 40, 80 and 160 µg/L treatmentgroups, respectively during CCAs 4-6 (Table 26 and Figure 14, below).
In the PMRA analysis, the number of pupal cells was significantly reduced (p<0.05) compared tothe control at the three highest treatment groups (40, 80 and 160 µg/L) which persisted overmultiple CCAs (16.3—47.0%, 30.9—87.7% and 46.9—99.6% reduction in the 40, 80 and 160µg/L treatment groups, respectively during CCAs 4-6 and 98.2% reduction at CCA7 in the 160µg/L treatment group). No significant reduction (p>0.1) in the number of pupae was observed at
54
310

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
the lowest two treatment groups (10 and 20 µg/L) during any of the CCAs with the exception of
CCA5 where the number of pupae was significantly reduced by 13.1% in the 20 µg/L treatmentgroup (p=0.039) and by 19.9% in the 10 µg/L treatment group (p=0.05). This analysis considers
that the overlap in dose-response at the lower doses is not unexpected since the dose levels are
similar and measured exposures indicate overlap in exposure among individual hives, particularly
at the lower two doses.
Table 19. Estimated percent reduction from control for number of capped (pupal) cells.
Test
Reduction relative to the control mean
concentration
CCA3
CCA4
CCA5
CCA6
(µg/L)
10
-8.3
0.4
17.2
10.3
20
-7.8
3.9
9.7
3.7
40
1.1
16.4*
47.1*
36.7*
80
-7.7
26.1*
83.4*
59.3*
160
-0.9
46.3*
100*
98.1*

CCA7
27.0
4.7
19.6
7.9
97.3*

Note: Negative value indicates increased number of pupae in comparison to control.

*p<0.05

Figure 14. Number of capped cells (pupae) at colony condition assessments (CCA) 3 thru 7 for eachtreatment group ( based on model residual mean squares estimate).

Figure 15 below shows the responses for pupae for the control, 10, 20 and 40 µg/L treatmentgroups. Removing the two highest treatment concentrations adjusts the scale of the figures to see
the trends more clearly. It is noted from this graph that although error bars surrounding the control
group, and the 10 and 20 µg/L treatment groups generally overlap with each other, they do notshow any overlap with the 40 µg/L treatment group from CCAs 4 through 6.
55
311

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
When weighing statistical and biological significance, the overall NOAEC and LOAEC for
pupal cells is determined to be 20 and 40 µg/L, respectively.

Figure 15. Number of capped cells (pupae) at colony condition assessments (CCA) 3 thru 7 for the control
and three lowest treatment groups. Error bars represent one standard error from the mean calculated fromthe model residual mean squares estimate.

3.9.6.5 Total Individuals in Hives
When evaluating the total number of live individuals (total adults + combined number of cells of
eggs, larvae and pupae), significant effects (p<0.05) were observed at the three highest treatments
for CCAs 4-6 and in the two highest treatments at CCA7 (Table 27), generally following thepattern observed earlier for total adults and pupae, the two life stages that made up the largest
components of the hive population throughout the course of the study (Figures 8, 14 and 16). No
significant differences (p>0.1) were observed for the 10 and 20 µg/L treatment groups relative tothe controls in the EPA analysis.
In the PMRA analysis, significant reductions were observed for the 20 µg/L treatment group at
CCA4 and for the 10 and 20 µg/L treatment groups at CCA5 (0.05< p<0.1) A general dose
response (increase in the reduction from the control as the dose increases) at the three highest
treatment groups (40, 80 and 160 µg/L ) was evident over all CCAs. For the two lowest treatmentgroups, the reduction in total individuals in the 10 µg/L treatment group was consistently higher
than or equivalent to the 20 µg/L treatment group over all CCAs (6.1-23.7% and 6.7-11.1%
reduction in the 10 and 20 µg/L treatment groups, respectively) and in particular, CCA7. This
56

312

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
analysis considers that the overlap in dose-response at the lower doses is not unexpected since thedose levels are similar and measured exposures indicate overlap in exposure among individual
hives, particularly at the lower two doses.
Table 20. Estimated percent reduction from control for total individuals
Test
Reduction relative to the control mean
concentration
CCA3
CCA4
CCA5
CCA6
(µg/L)
10
-2.6
5.1
12.4
11.4
20
-1.2
6.3
9.6
9.1
40
-0.8
14.0*
32.5*
30.6*
80
-7.8
18.1*
65.0*
55.8*
160
-0.6
46.6*
88.1*
92.5*

CCA7
21.3
10.4
17.9
40.3*
96.6*

Note: Negative value indicates increased number of total individuals in comparison to control.
*p<0.05

Figure 16. Number of live (adult numbers+cells of brood) at colony condition assessments (CCA) 3 thru 7
for each treatment group (based on model residual mean squares estimate).

Figure 17 below shows the responses for total live for the control, 10, 20 and 40 µg/L treatmentgroups. Removing the two highest treatment concentrations adjusts the scale of the figures to see
the trends more clearly. It is noted from this graph that although error bars surrounding the control
group, and the 10 and 20 µg/L treatment groups generally overlap with each other, they do notshow any overlap with the 40 µg/L treatment group from CCAs 4 through 6. When weighing
statistical and biological significance, the overall NOAEC and LOAEC for total individuals
is determined to be 20 and 40 µg/L, respectively.

57
313

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

Figure 17. Number of total live (adult+brood) at colony condition assessments (CCA) 3 thru 7 forthe control and three lowest treatment groups. Error bars represent one standard error from themean calculated from the model residual mean squares estimate.

3.9.6.6 Total Brood in Hives
When evaluating the total number of brood cells (eggs, open and capped), significant effects(p<0.05) were consistently observed at the three highest treatments for CCAs 4-6 and in the highest
treatment at CCA7 (Table 28 and Figure 18). No significant differences (p>0.1) were observed
for the 10 and 20 µg/L treatment groups relative to the controls.
Table 21. Estimated percent reduction from control for total brood cells
Test
Percent reduction relative to the control mean (%)
concentration
CCA3
CCA4
CCA5
CCA6
(µg/L)
10
-5.8
8.4
14.4
5.4
20
-1.4
9.5
14.4
5.0
40
-0.2
22.5*
37.5*
31.0*
80
-6.8
23.2*
81.2*
55.4*
160
1.1
55.2*
99.2*
95.3*

CCA7
25.5
17.6
25.4
28.3
97.4*

58
314

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

Figure 18. Number of brood at colony condition assessments (CCA) 3 thru 7 for each treatment group(based on model residual mean squares estimate).

Figure 19 below shows the responses for total brood for the control, 10, 20 and 40 µg/L treatmentgroups. Removing the two highest treatment concentrations adjusts the scale of the figures to see
the trends more clearly. It is noted from this graph that although error bars surrounding the control
group, and the 10 and 20 µg/L treatment groups generally show some overlap with each other,
there is clear and consistent divergence of the 40 µg/L treatment group compared to controls andthe lower treatment groups. Therefore, when weighing statistical and biological significance,
the overall NOAEC and LOAEC for total brood is determined to be 20 and 40 µg/L,
respectively.

59
315

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

Figure 19. Number of brood at colony condition assessments (CCA) 3 thru 7 for the control andthree lowest treatment groups. Error bars represent one standard error from the mean calculated
from the model residual mean squares estimate.

Figures 20-24 below provide another visual representation of the effects across CCAs variables
within a response variable for the various life stages of bees during the course of the study for thethree lowest treatments. The bar charts represent the percent differences from control with negative
percent differences from control indicating an increase in a given response variable above the levelof control. Although these figures show what appear to be substantial decreases compared tocontrols for some endpoints at select CCAs (e.g. larvae at CCA7), these figures provide further
evidence of the general lack of dose responsiveness in effects at the lowest treatment groups, while
a clear dose-response relationship is observed between the 40, 80 and 160 µg/L treatment groups.
Furthermore these charts are effective in indicating how the percent differences with a given
response variable, changed over the course of the study within a treatment group. It is also noted
here that the scale for percent difference from control (y-axis) has been standardized across all
charts and that negative (“-“) responses refer to a percent increase above the level of control.

60316Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Figure 20. Summary of living organism parameters at the 10 µg/L treatment group

Figure 21. Summary of living organism parameters at the 20 µg/L treatment group

Figure 22. Summary of living organism parameters at the 40 µg/L treatment group61317

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Figure 23. Summary of living organism parameters at the 80 µg/L treatment group

Figure 24. Summary of living organism parameters at the 160 µg/L treatment group

3.9.7 Colony Condition Assessments – Food Store Response Variables
3.9.7.1 Pollen
Pollen stores were significantly reduced (p<0.05) in the 80 and 160 µg/L treatment groups from
CCA4 to CCA7 (inhibitions of 46.8—98% and 96.7—100%, respectively in the 80 and 160 µg/L
treatment groups in the EPA analysis and 47.5-99.4% and 94.3-100%, respectively in the PMRA
analysis). Pollen stores were also significantly reduced in the 40 µg/L treatment group during
CCAs 4 and 5 (inhibitions of 29.3 and 64.7%, respectively in the EPA anlaysis and 21.9 and 55.3%
in the PMRA analysis), though at CCA4 this difference was more marginal in the EPA analysis(0.05<p<0.1; Figure 25). In the two lower treatment groups, pollen stores were significantly
reduced (p<0.05) at CCA5 and for EPA’s analysis only, at CCA7, though at CCA7 these were notobserved to follow a dose-response trend (Table 29).

62
318

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Table 22. Estimated percent reduction from control for pollen stores
Test
Reduction relative to the control mean
concentration
CCA3
CCA4
CCA5
CCA6
(µg/L)
10
1.2
6.9
32.7*
26.1
20
9.3
16.1
36.2*
3.3
40
11.3
29.3**
64.7*
34.1
80
-2.3
46.8*
98.0*
60.4*
160
2.9
96.7*
100*
99.8*

CCA7
62.2
36.1*
19.0
70.9*
100*

*p<0.05**0.05<p<0.1

Figure 25. Number of pollen cells at colony condition assessments (CCA) 3 through 7 for each treatmentgroup.

Figure 26 below shows the clear divergence of pollen stores in all three lower treatment groups at
CCA5 as compared to the control, but pollen stores than appear to overlap at CCAs 6 through 7,
except for in the 10 µg/L treatment group which does not appear to have any dose-response
relationship with the other doses at CCA7. The data indicate clear and consistent effects during(and immediately after) the feeding exposure on the 40 µg/L group, however are approachinglevels near the control following exposure. There is more uncertainty surrounding the two lower
treatment groups, for which a significant decrease in pollen storage is observed at a single
measurement (CCA5) without any statistical significance or dose-response at CCA6 and 7, withthe 10 µg/L treatment having reduced pollen storage compared to controls while the 20 µg/L
treatment group more closely tracks the control pollen storage.
63319

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

Figure 26. Number of pollen cells at colony condition assessments (CCA) 3 thru 7 for the control and threelowest treatment groups. Error bars represent one standard error from the mean calculated from the model
residual mean squares estimate.

Due to consistent effects in the 40 µg/L treatment group at CCAs 4 and 5, the overall LOAEC
for pollen stores is determined to be 40 µg/L. There is some uncertainty surrounding thisendpoint given the significant reduction at all treatment doses at one measurement point(CCA5), that generally follows a dose-response relationship. However, as this only occurs at
one CCA and thereafter no significant effects or dose-response are observed, the NOAEC is
therefore considered to be 20 µg/L.

3.9.7.2 Nectar / Honey
There were no significant decreases (p>0.1) for honey/nectar storage at any CCA for any treatment
dose (Table 30 and Figure 27). However, there was a general trend of more honey storage inhigher treatments compared to the controls and lower treatment groups. It is noted that honeystorage in the 10 and 20 µg/L treatment groups did not differ appreciably from contols (<20%difference at all CCAs), while in the two highest treatments there was substantially more honeystored (>50%) by CCAs 6 and 7. Given the lack of statistical significance for honey, a second
graph focusing on the control and three lowest treatment groups was not generated for this endpoint.
It is noted that the feeding solutions (sugar solutions) provided during the exposure period might
have affected natural honey storage patterns; however, effects on honey storage are still able to beconsidered as all treatments were compared to control hives (which also received feeding
solutions).

64320Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Table 23. Estimated percent reduction from control for nectar/honey stores
Test
Reduction relative to the control mean
concentration
CCA3
CCA4
CCA5
CCA6
(µg/L)
10
15.7
4.0
1.1
1.8
20
-4.3
-7.2
-13.1
-19.4
40
5.3
-7.6
-17.2
-39.5
80
-4.8
-15.8
-29.7
-75.4
160
-3.4
-20.0
-21.3
-93.6

CCA7
12.6
-6.8
-46.7
-86.5
-114.1

Note: Negative value indicates increased nectar/honey stores in comparison to control.

Figure 27. Number of honey cells at colony condition assessments (CCA) 3 thru 7 for each treatment group.

As no significant effects were observed and there were no evidence of adverse effects onhoney storage, the overall NOAEC and LOAEC for honey stores is determined to be 160 and>160 µg/L, respectively.

3.9.7.3 Total Food Storage
There were no significant decreases (p>0.1) for total food storage at any CCA for any treatment
dose (Table 31 and Figure 28). However, similar to the honey storage above, there was a generaltrend of more food cells in higher treatments compared to the controls and lower treatment groups.
It is noted that food storage in the 10 and 20 µg/L treatment groups did not differ appreciably fromcontols (<20% difference at all CCAs except for a 21.4% decrease at CCA7 in the 10 µg/L
treatment), while in the two highest treatments there was substantially more food stored (>50%)
by CCAs 6 and 7. Given the lack of statistical significance for food storage, a second graphfocusing on the control and three lowest treatment groups was not generated for this endpoint. It
is noted that the feeding solutions (sugar solutions) provided during the exposure period might
have affected natural food storage patterns; however, effects on food cells are still able to be65
321Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
considered as all treatments were compared to control hives (which also received feeding
solutions).
Table 24. Estimated percent reduction from control for food (pollen + nectar) storage
Test
Reduction relative to the control mean
concentration
CCA3
CCA4
CCA5
CCA6
(µg/L)
10
14.0
4.4
5.2
6.0
20
-2.7
-2.9
-6.8
-15.5
40
6.0
-0.7
-6.7
-26.7
80
-4.5
-4.0
-13.2
-51.5
160
-2.7
2.1
-5.6
-59.7

CCA7
21.4
0.9
-35.1
-58.2
-75.6

Figure 28. Number of food cells (honey+pollen) at colony condition assessments (CCA) 3 thru 7 for eachtreatment group.

As no significant effects were observed and there were no evidence of adverse effects on total
food storage, the overall NOAEC and LOAEC for this endpoint is determined to be 160 and>160 µg/L, respectively.

3.9.8 Hive WeightAs supers were added and removed based on the study author’s considerations to best support
growth or reductions in the size of the bee colony and the weights of individual (empty) hive bodies
were not reported in the study report, no statistical analysis was conducted by either EPA or PMRAon the hive weight parameters. Daily hive weight data can be found in Appendix E of the study
report on pages 211—319. The figure below is taken directly from the author’s study report. Hive
weights generally oscillated similarly.

66
322

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

Figure 29. Proportion of hive weight following exposure of honey bees to varying concentrations ofclothianidin in the diet for six weeks.

3.9.9 Hive mortality
Hive survival following overwintering is described below in Tables 32-33 and Figure 29. The
study author reported that 81 out of 84 colonies for biological observations were maintained overthe 6-week exposure period and survived until the last CCA before overwintering (CCA7). Threecolonies were removed due to technical issues between treatment initiation and CCA7. One hivein the control (I7) was removed due to potential contamination of the feeder following a technicalerror during feeding on 10 Jul 2014 and two hives in the 40 ppb treatment were removed after
being knocked over (F3 on 07 Jul 2014 and C2 on 25 Aug 2014). All colonies in the 160 ppb
treatment group had greatly reduced adult bee strength that was determined to be insufficient tosurvive overwintering and were subsequently destroyed following CCA7 in December 2014.
As 65% of control hives did not survive overwintering, the study lacks the capability to reliably
determine differences in treatments compared to controls regarding colony survival.

67323

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Table 25. Proportion of hive weight following exposure of honey bees to varying concentrations ofclothianidin in the diet for six weeks.
Treatment
group

Apiary
A

B

C

D

E

UTC

-

-

C1

-

-

UTC

-

-

-

D8

10 ppb

-B8-

20 ppb

A1-

40 ppb

A4

80 ppb

A2

F

G

H

I

-

-

-

I6

E4

-

G8

H6

1

-

-

F6

-

C4

-

E7

-

G3

B1

1

--

D7

B6

C8

D5

J

K

L

-

K5

-

--

J8

-

-

H2

-

-

-

-

-

I4

J5

K3

L2

-

1

--

-

-

I8

J6

-

-

E6

F1

G4

H4

-

J3

K1

-

160 ppb- = hive dead--1 = hive was removed from study due to technical error or vandalism prior to overwintering.

-

Table 26. Hive mortality statistics after overwintering measure at CCA8
Treatment (µg/L)
Control
1020
40
Number of deceased colonies /total
15/23
9/12
4/12
5/10
colonies
Colony mortality (%)
65%
75%
33%
50%
Colony survival (%)
35%
25%
67%
50%
Treatment (µg/L)
Control
102040
1All colonies in the 160 ppb treatment group were destroyed after CCA7 (Dec 2014)

80

1601

2/12

11/11

17%
83%
80

100%
0%
1601

Figure 30. Overall hive survival after overwintering (reproduced from study report, p. 34).
68
324

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

4.0

Reviewer comments

What follows is brief discussion of some of the elements taken into consideration when evaluating
the results of this study.

4.1 General Considerations for Biological Interpretation
While the hive mortality is considered as the most relevant measurement of survival at the colonylevel, sublethal effects at the colony level were estimated by measuring multiple parameters during
the course of study. Each measured parameter is expected to reflect only part of the colony
conditions, and all measurements have to be integrated for a better understanding of the hive status
at the colony level. A honey bee colony is a super-organism in which live individuals and foodsupply are the two major components in maintaining the proper function of the colony. There areinteractions between the two components and even within each component.
Individual bees are present in the colony as eggs, larvae, pupa and adults and they develop fromone stage to another and interact with each other to perform a variety of tasks to maintain the
integrity of the colony. The measurement of each stage of the bees is expected to provide
information on the potential treatment effect on a specific life stage of bees during theirdevelopment.
Hive food supplies including hive pollen and nectar are collected and processed by adults and are
expected to have a large impact on the development of all stages of bees in hives. However, theamount of hive food storage is dependent on not only the number of foragers available for foodcollection, but also the number of individuals that consume the food. In addition, the seasonal
availability of outside pollen and nectar sources also affects the amount of storage, thus impacting
hive development. As well, sucrose feeding solutions were provided to the hives as a means oftreatment and as a supplement for hive overwintering, which may have affected foraging and foodstorage during those time periods.
Hive weight was measured during the study. However, it is largely affected by the honey storageand number of bees that consume the food. A strong colony with a high number of bees likely
consumes a high amount of stored honey and may result in a reduced hive weight. Weighing hives
at different time periods of the day may result in an increased variation of the measurement due tothe fact that foragers may not be present in the hive when the weight is measured. Hive weights
may be artificially lower in hives which contain a high number of forager bees that may be out
collecting food during a different time of the day.
Considerations regarding the measurement time points:••

CCA3 represents the background hive conditions as the first colony assessment after thehives were placed in the test fields prior to the exposure.
CCA4 and CCA5 represent the hive conditions during the exposure phase. It was notedthat the CCA5 was conducted a week after the end of the 6-week exposure period, but is
expected to represent effects during the exposure period.

69
325

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document•••

CCA6 was measured at 4 weeks after the end of exposure. It allows all bee individuals,
including eggs, larvae and pupa that were exposed to treatment to finish theirdevelopment cycle and become adults.
CCA7 represent the hive conditions immediately prior to overwintering. It is consideredthat hives were physiologically preparing for overwintering by reducing the production of
immature bee individuals. Treatment effects may be masked by the natural decline ofhive individuals.
CCA8 and CCA9 represents hive conditions of surviving hives after overwintering. Highmortality in the control hives excluded these assessments from analysis.

4.2 Control PerformanceControl mortality and sublethal effects on life stages and food stores
The control performance in this study offers some challenges relating to the interpretation of theresults. The level of colony loss after overwintering in controls (65%), adds a great deal of
uncertainty when considering the results of individual measurements. The fact that many of thehives in the lower treatment groups performed/trended similarly to the control hives for thesemeasurements could be indicative of either a lack of treatment effects or potentially that the control
hives were suboptimal to begin the study. Because so few hives survived overwintering andtrended relatively closely to the lower level treatment hives during exposure, the overwintering
component would be extra important to determine if the lack of significant reductions comparedto the control in most treatment groups is biologically significant. Almost every parameter for life
stages decreased after exposure ended (endpoints generally reached their apex at CCA5) which
could have been a factor of either the time of year or of treatment. The fact this also happened in
the control groups suggest a performance issue is possible, or at the very least an uncertainty withrespect to if the exposure measurements were taken too late in the year to be able to reliably discern
treatment effects.
The similarity in the dynamics of all parameters for the individual living organisms at various stageacross the control and lower (10-20 µg/L treatments may indicate that control hives were stressed
prior to overwintering. For most parameters in the lower treatment groups the means converged
to those of the control at CCA 6 through CCA 7 (and through CCA8, for those hives that survived
overwintering). The time of year likely influenced control hive performance as colonies arenormally producing far fewer bees at this time of year, but it is still considered uncertain if thehives were developing normally. There was no apparent spike of honey collection or pollen storesfrom the control hives indicating they may not have been developing and storing enough food tosurvive the winter. Pollen stores were decreasing at the same time other biological parameters wereindicating consumption of resources but not replenishment for the hive.

4.3 Consideration of Study Strengths, Limitations and Interpretation
It is important to recognize the inherent strengths and limitations of this study as results areinterpreted and potentially considered in risk assessment.
In the context of available field studies involving honey bees and clothianidin, this study contains
a number of strengths including:70326Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
•
•
•
•
•
•
•

Use of a high degree of replication (n=12) to achieve a reasonable level of statistical power
Demonstration of a generalized concentration-response relationship with respect to theconcentration of clothianidin in sucrose solution and the magnitude and duration of adverse
effects
Quantification of exposure to clothianidin in diet and in hive matrices (uncapped nectar,
pollen, capped honey, bee bread)Use of a 6-week exposure duration to represent a “high end” exposure scenario
Inclusion of multiple colony-level endpoints reflecting hive strength, brood development
and food stores
Detailed QA/QC results regarding quantification of clothianidin residues in various
matrices
Availability of raw data for conducting statistical analysis.

A number of limitations are also noted with this study, including:•Exposure of bees to clothianidin occurred through nectar (sucrose) alone, whereas bees in
the field are likely exposed through both pollen and nectar routes. Therefore, the designof this study may not reflect a “worst case” exposure scenario in which bees areexperiencing prolonged exposure to both contaminated nectar and pollen. While exclusionof the pollen route is expected to reduce overall exposure, the impact of this exclusion on
the study results is uncertain and will likely depend on the life stage/caste of bee.

•

Residues in hive matrices were only analyzed for parent clothianidin. Metabolites ofclothianidin were not considered. Clothianidin degradates (e.g. TZNG) have been
demonstrated in laboratory studies to have much less acute toxicity to adult honey bees,
though data is not available for their chronic effects to adult bees or potential effects toother honey bee life stages.

•

Clothianidin was found in both hive nectar and hive pollen (beebread), at concentrations
lower than the feeding solutions. Dilution compared to the treatment feeding solution is
expected since bees could also forage on outside nectar and pollen sources. As well, hivepollen contains only some hive nectar, thus would not be expected to have a concentrationequivalent to nectar alone, and it is mixed with pollen which will come from outside
sources. Therefore exposure through both hive pollen and nectar occurred via exposure to
the sucrose feeding solution, but how this compares to exposure through contaminatedpollen directly is not known. It is also noted that nectar is considered the dominant exposure
route for forager bees; other hive bees and larvae consume both nectar and pollen. A recent
paper by Sandrock (2014) 4 indicated that consuming contaminated pollen containing low
levels of both clothianidin and thiamethoxam had effects on many hive parameters. In
addition, since bees were forced to forage for pollen in this study, the potential impact ofclothianidin exposure on reducing pollen foraging efficiency of bees could be incorporated

4

Sandrock C, Tanadini M, Tanadini LG, Fauser-Misslin A, Potts SG, et al. (2014) Impact of Chronic Neonicotinoid
Exposure on Honeybee Colony Performance and Queen Supersedure. PLoS ONE 9(8): e103592.
doi:10.1371/journal.pone.0103592
71
327

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
into the overall expression of adverse effects, as suggested by published literature. Had
contaminated pollen been provided to bees, it is not known if the potential impact on pollen
foraging efficiency would have been masked.
•

The quantity of nectar provided to hives (4 L per week per hive) likely did not fulfill thecomplete carbohydrate needs of the colony, as indicated by colony bioenergetics and thelack of remaining sucrose solution upon their renewal at some of the test concentrations.
This suggests that bees could be exposed to a greater dose of clothianidin in nectar had agreater volume of spiked sucrose been provided. Although one can infer that the dosing
regimen may have underestimated exposure through sucrose relative to 100%
contaminated diet, it is also noted that bees had to supplement their spiked sucrose byforaging on their own for other sources of nectar. As with the previous discussion of pollen
it is noted that had 100% of the carbohydrate needs of the colony been provided via feeders,the potential impact of purported reductions in nectar foraging efficiency may have beenmasked to some degree.

•

Overwintering success of controls was severly impacted (65% hive mortality). This
prevents the ability to detect adverse effects related to hive loss following overwintering.
The lack of control hive overwintering may reflect the study design that prevented earlier
supplemental feeding in the fall (in order to ensure that treatment hives were consuming
their exposed food stores), while typical beekeeping practice would have permitted
additional feeding earlier in the fall.

•

Pesticides from food sources other than the artificial feeding were also detected during
the exposure period and post-exposure periods through collection of pollen from pollen
traps from monitoring hives. This contributes to exposure uncertainty and can add
confounding effects when interpreting results. However, it is noted that detections
occurred in <10% of samples from monitoring hives and that the only pesticides detected(propiconazole, chlorothalonil and carbaryl) had relatively low toxicity compared toparent clothianidin (ranging from practically non-toxic for chlorothalonil to moderately
toxic for cararyl).

•

Residues of clothianidin in uncapped nectar and bee bread within the hives at CCAs 4, 5,
7 and 9 represent a single sample per hive on a single frame rather than a composite sample
from multiple portions of the comb within a hive. This means that residue results may
reflect a “hit or miss” scenario with respect to detecting residues in nectar laid down fromcontaminated (fed) vs. outside sources.

•

The exposure, based on residues measured in the hive (hive nectar and hive pollen)
indicated that, overall, higher measured hive residues correlated with higher nominal
residues in feeding solutions. However, individual hive residue values varied, and there
was some overlap in measured values, particularly among the three lowest doses.

•

Exposure dilution during the study was evident. Remarkably lower residue concentrations
detected in bee bread and hive nectar in some test hives compared to the feeding

72
328

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
concentrations indicate foraging on other food sources. This uncertainty is inherent in any
semi-field or full-field study design.

5.0

Overall Study Conclusions

The study is considered to be informative and will be used as a line of evidence in the pollinatorrisk assessment. While there were uncertainties that were generally related to inherent aspects ofany semi-field or full field study design (such as dilution of the test chemical through alternative
sources of forage, detection of other chemicals in the monitoring hives), this study still provides
information on a number of colony health parameters about the long term (however excluding
overwintering) exposure to clothianidin at the colony level.
An evaluation of the observed effects was conducted considering statistical reductions relative tothe control, trends within each treatment and in comparison to the control, recognition of thenatural trends honey bee colonies follow during the course of the year, and finally, the fact thatsuccessful overwintering in the controls was not observed. With regard to the top two test
treatments (160 and 80 ug/L), statistical reductions relative to the control (p<0.05) were observed
across several different endpoints and at many CCAs within an endpoint. Statistically significant
decreases in the number of adults (30-96% reductions in EPA’s analysis, 14—98% reduction in
PMRA’s analysis) and brood (eggs (38-95% in EPA’s analysis, 31-94% in PMRA’s analysis),larvae (49-100% in EPA’s analyis, 12-99% in PMRA’s analysis), and pupae (26-100% in EPA’s
analysis and 31-100% in PMRA’s analysis) were observed compared to the control, starting at
CCA4 with effects being sustained through CCA7, particularly for number of adults and eggs. At
these top two test concentrations, decreases in pollen storage compared to the control was observedwith significant decreases at CCA4 thru 7. At 40 ug/L, significant decreases in pollen comparedto the control were observed at CCA 4 (0.05<p<0.1 in EPA’s analysis, p<0.05 in PMRA’s analysis)
and CCA5 (p<0.05, both analyses). Also at this concentration, significant decreases (p<0.05, bothanalyses) in the number of adults was observed at CCA5 and 6, and in the number of pupae at
CCA4 thru 6, though these responses were at levels similar to the control for CCA7. In addition,
PMRA determined significant reductions at this concentration in the total number of individuals
at CCA4 thru 6 (p<0.05), number of eggs at CCA4 and 5 (p<0.05) and the number of larvae at
CCA4 (p<0.05).
With regards to the lower two test treatments (10 and 20 ug/L), most endpoint responses were
not significantly different from the control (p>0.1). For the EPA analysis there were two
endpoints for which a statistical reduction for one or both of these treatments was observed.
First, the number of adults was statistically reduced (p<0.05) at CCA6 at the lowest treatment(10 ug/L), but was not significant at 20 ug/L (mean number of adults was slightly greater at 20
ug/L compared to 10 ug/L, both at this CCA and consistently following feeding exposure) andwas not significant at any other CCA. Second, significant decreases (p<0.05) in pollen wereobserved at all test concentrations at CCA5. However, at other CCAs for pollen, only the 160
and 80 ug/L treatment groups were significantly decreased, except for an observed (non-dose
responsive) decrease at the lowest test concentration (10 ug/L) at CCA7.

73
329

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
The PMRA results were slightly different from the EPAs for the lower two test treatments but
resulted in the same conclusion. While most endpoint responses were not significantly different
from the control at various timepoints throughout the study (p<0.1), significant reductions wereobserved at both test concentrations in the number of adults (CCA6), eggs (CCA4 and 5), larvae(CCA4), pupae (CCA5), total number of individuals (CCA5 at 10 ug/L and CCA4 and 5 at 20ug/L) and pollen stores (CCA5). These effects were considered to be potentially transient withnumbers returning to control levels in subsequent CCAs.
Hive mortality is considered the most relevant measurement of survival at the colony level. The
level of colony loss after overwintering experienced by controls in this study (65%), precludes theuse of this endpoint in evaluating chronic exposures of colonies to clothianidin. The lack of control
overwintering success also has significant implications in evaluating effects on other measured
parameters in the study. The potential for observed effects (in the ≥ 40 µg/L treatments) to beameliorated following exposure and subsequent recovery cannot be assessed. Furthermore, there
is potential that additional chronic effects, not observed prior to overwintering, may subsequently
manifest themselves at the lower doses (e.g. 10 and 20 µg/L) which could not be adequately
captured from this study.
Therefore, the overall quantitative NOAEC and LOAEC for this study is 20 and 40 µg/L,
respectively, based on impacts on pollen storage, number of adults, number of pupae andtotal brood and total live bees in the ≥40 µg/L treatment groups that were sustained acrossmultiple CCAs prior to overwintering (effects on larvae, though not significant at 40 µg/L
may also have been suggestive of an impact from this dose, as they consistently did not
track well with the control and lower treatment doses). These effect levels include theunderstanding that evaluation of overwintering was not possible which limits the ability to
fully evaluate potential long-term effects in the two lower treatments groups, and therefore,remains a major source of uncertainty.

74
330

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

Appendix A: Details of PMRA Statistical Analysis
During the review of the study, a separate statistical analysis was conducted with the program R(version 3.1.2) 5 using the raw data submitted by the study author.

Statistical analysis
Analysis Strategy
Hive condition data:
To analyze colony condition data which contains many components over many assessments at
different times, a primary analysis was set out to effectively prevent multiplicities from interferingwith the interpretation of p_values and confidence intervals. These multiplicities arise from having
multiple dose levels, multiple outcomes and multiple time points, and are dealt with as follows:•

The multiplicities from having multiple dose levels was dealt with by using step down
testing, the highest dose group’s data was compared directly to the control group’s data, if
statistically significant at a chosen alpha level the next lowest dose group’s data wascompared to the control group’s data and this was continued down to the dose where
statistical significance was no longer achieved. A technical reference for this step down
testing would be Multiple Comparison Procedures in Dose Response Studies. Tamhane,
Ajit C. and Logan, Brent R., in Dose Finding in Drug Development edited by Ting, Naitee.
Springer New York 2006. This step down procedure (referred to as the SD2PC procedurein the technical reference) was chosen as it provides good power for detecting the minimum
effective dose (lowest does where effect is present) when monotonic dose effects are
expected while providing stringent control of type one error, regardless of the true patternof dose effects. That is, with minimal assumptions, the procedure strongly controls family
wise type one error rate while maintaining good power for effect patterns that are expected.
This step down procedure is implemented by PMRA using only data from the control groupand the dose group being tested in that step which alleviates any concern about
heterogeneity of variance across dose groups. Especially with outcome data that involves
estimates of underlying counts, it is expected that effects at a given dose necessarily
involves both the mean and variance. When this is the case - the use of data from a higher
dose with a putative effect in the comparison of a lower dose would thus be inappropriate
and would invalidate the control of type one error.
The applicant’s choice of multiplicity adjustment procedure, which was William’s trend
test (Williams 1972), was presumably chosen to be in accord with OECD, 2003. Draft
guidance document for the statistical analysis of ecotoxicity data. They are both step down
procedures but ours differs from William’s in that it uses only within dose group data based
estimates of means rather than maximum likelihood estimates of dose group means using

R Core Team (2014). R: A language and environment for statistical computing. R Foundation for StatisticalComputing, Vienna, Austria. URL http://www.R-project.org/.

5

75
331

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
all group’s data simultaneously - under monotonicity assumption (i.e. order restricted or
isotonic means) additionally assuming homogeneous variances. Although these additional
assumptions may not be problematic and are within the OECD guidelines, we simply chose
not to rely on them (and by doing so, exceed the OECD guidelines.)•

The multiplicities from having multiple outcomes, was dealt with by choosing to focus on
the assessment of total life in the hive – simply the number of viable life forms at any stage
in the hive. It is considered that the total number of individuals includes all live individuals
in hives (eggs larvae, pupae and adults) and is expected to be a better indicator of the hive
status at the colony level than any single stage of bees alone. This outcome would provide
good power when background knowledge is lacking on the stage most likely to be affected(i.e. it cannot be well anticipated) and it is not expected that there will be simultaneous
trade-offs effects between the stages. That is, when it is not expected that a toxic effect on
one stage would have a beneficial effect for another stage at the same point in time.

•

The multiplicities from having multiple time points was dealt with by choosing to focus on
the time when the effects were believed to be most pronounced both in terms of having an
impact on total life and having a high powered assessment of that. In this case CCA6 wasselected for the following reasons.
• CCA4 and CCA5 were not selected as they represent the hive conditions during the
6-week exposure phase. It is noted that CCA5 was conducted a week after the endof the 6-week exposure period, but it is expected to represent effects during theexposure period.
• CCA6 (4 weeks after the end of feeding exposure) was selected as it maximises
the time period for detecting a potential latent effect from exposure and occurs
before the start of hive decline prior to overwintering at most apiaries.
• CCA7 (9 weeks after the end of feeding exposure) was not selected simply due tothe natural decline of hive size in the late fall that may mask the effect of treatments.
• CCA8 and CCA9 (after over-wintering period) were not selected because of thehigh hive mortality observed in the controls.

While the total individuals at CCA6 is considered as a primary parameter to control multiplicity
for statistical analysis, all parameters including eggs, open brood and capped brood, adults, pollen
and nectar store, that were observed during the entire study including CCA4, CCA5, CCA6 andCCA7 were also considered in the review. Hive weight was also measured throughout the study
however, given the inherent variability of this parameter it was not further considered in thestatistical review. Given that the primary analysis has prevented multiplicities from interferingwith the interpretation of p_values and confidence intervals, if statistical significance has beenachieved (at given dose levels), further analysis with all other outcomes is undertaken “withprejudice” for the assessment of similar effects as being significant. More formally, re-allowance
for multiplicities is not required and less stringent alpha levels are allowed. Essentially the pricehas been paid for searching for the pattern in the primary analysis (measures taken to prevent
multiplicities) and it need not be re-paid evaluating the same pattern elsewhere. On the other hand,
if statistical significance has not been achieved (at given dose levels), further analysis with allother outcomes is undertaken “with prejudice” for assessment of other effects as likely being justnoise. Here though dramatic effects should not be ignored but carefully considered and noted.
76332

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Analysis methods for hive conditions
For all hive conditions total life, eggs, open brood and capped brood, adults, pollen and nectar
store at CCA4, CCA5, CCA6 and CCA7 a conventional analysis of block randomised experimentswith a baseline measurements was undertaken. In line with the statistical strategy discussed above,
the focus was on total life at CCA6 (with step down adjustment for multiplicities applied) but
identical analysis was carried out on all other hive conditions assessed at the given assessment
points. This analysis comprised of linear modeling (or ANOVA) stratified on Apiary (block) andadjusted for baseline measurements at CCA3 with one-side testing for harm using only the control
group data and the data from a single dose group at a time, starting with the highest and thenthrough lowest dose groups. It is a series of robust “t.test like” analyses that conservatively
implement the step down testing procedure. Under the assumption of no effect in the single dosegroup being tested (relevant to type one error control), the means and variances and covariate
effects should be identical in both the control group and the single dose group being tested. (In an
analysis that includes all dose group data together e.g. William’s procedure, an impact of atreatment effect on the variance and covariate effects at a higher dose, in addition to an effect on
the mean, would invalidate the assumptions needed to control type one error rate in the lower
doses.) The results of all analyses are presented in tables of unadjusted p_values (adjusted p_values
can be simply read off as the maximum of all p_values in any higher dose), effect estimates andupper and lower confidence intervals (in file Clot_summariesF) as well as plots of the confidence
intervals (pdf file Bees8.pdf).
The code snippet to implement these analyses in R was:glm(outcome~Apiary + baseline + exposed, data= x[x$exposed == " control " | x$exposed ==dose,])
Sensitivity analysis was undertaken by extensive graphical analyses sometimes using the squareroot transformation as well as calculating non-parametric randomisation (permutation) tests on the
differences between high dose group and control group average within Apiary. These are given in
the column named PermP_value in Clot_summariesF.
Transcript/program of analyses carried out
The file ClothianidinBees2.R contains the transcript of the final run of the R program used to carryout the analysis and generate the tables and plots.
Supporting graphs
The following graphs were produced as part of the analysis.
Bees1a.pdf – Plots of individual hive condition assessments over-CCAs by Apiary.
Bees1b.pdf – Plots of individual hive condition assessments over-CCAs up to CCA7 by Apiary.
Bees2.pdf – Plots of control versus exposed condition assessments over-time group by Apiary.
Bees3.pdf – Plots of overall mean and Apiary mean control condition assessments over time.

77333Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Bees3S.pdf – Plots of overall mean and Apiary mean of the square root of control conditionassessments over time.
Bees7.pdf – Plots of individual exposed hive versus control condition assessments for “everything”.
Bees7S.pdf – Plots of individual exposed hive versus square root of control condition assessments
for “everything”.
Bees7d.pdf – Plots of individual exposed hive versus control condition assessments for“everything” by dose group.
Bees7dS.pdf – Plots of individual exposed hive versus square root of control condition assessments
for “everything” by dose group.
Bees8.pdf – Plots of effect estimates and confidence intervals for “everything”

78334

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Table A-1. Summary of the differences between treatment and controls on the basis of observations and model estimations, and p values.

Parameter

Time(CCA)1Test
conc.
(µg/l)

Adults3

160

Observedmeandifference
fromcontrol2-644nModel estimate
mean
difference
from control3,4Standard errorof estimatedmean4

p_value forcomparison
with thecontrol4

90%
confidence
upper
limit490%
confidence
lower
limit4

1014

12

0

0

0

0

0

Estimated
reduction
fromcontrol(%)4,50Adults

3

80

-1500

683

12

0

0

0

0

0

Adults

3

40

-307

1331

12

0

0

0

0

Adults

3

20

-96

1422

12

0

0

0

Adults

3

10

664

1746

12

0

0

Adults

4

160

5386

1216

12

5604.866

Adults

4

80

1641

1294

12

Adults

4

40

-63

961

11

Adults

4

20

262

1248

Adults41010Adults5160

Adults5AdultsStandard
error
observedmean

Estimated
reductionfrom control(number)4Observedmeans incontrolT-testconfidence
limit

0

15014

-1.717

0

0

15014

-1.717

0

0

0

15014

-1.717

0

0

0

0

15014

-1.717

0

0

0

0

0

15014

-1.7171164.551

0

0.439

0.208

0.323

7609

17340

-1.7212413.834

1201.969

0.029

0.258

0.02

0.139

4482

17340

-1.721-229.053

1262.953

0.571

0.112

-0.139-0.013

1949

17340

-1.72512108.853

1240.316

0.465

0.129

-0.117

0.006

2243

17340

-1.7211124

12-346.826

1325.985

0.602

0.112

-0.152

-0.02

1935

17340

-1.721

13331

1112

12

13491.43

1567.828

0

0.838

0.559

0.699

16189

19310

-1.721

80

7215

1451

12

7947.582

1683.568

0

0.562

0.262

0.412

10845

19310

-1.721

5

40

4577

1383

11

4463.445

1799.951

0.011

0.392

0.07

0.231

7568

19310

-1.725Adults

5

20

360

1250

12

315.4221643.241

0.425

0.163

-0.13

0.016

3143

19310

-1.721

Adults

5

10

1830

1967

12

1480.459

1949.511

0.228

0.25

-0.097

0.077

4835

19310

-1.721

Adults

6

160

16182

825

12

16521.8

1312.766

0

1.019

0.774

0.897

18781

18427

-1.721

Adults

6

80

10505

1396

12

11810.21

1449.897

0

0.776

0.506

0.641

14305

18427

-1.721

Adults

6

40

5786

2450

10

5501.192254.948

0.012

0.51

0.087

0.299

9400

18427

-1.729Adults

6

20

3210

1561

12

3133.999

1664.1

0.037

0.325

0.015

0.17

5997

18427

-1.721

Adults

6

10

4198

1360

12

3611.868

1537.387

0.014

0.34

0.052

0.196

6257

18427

-1.721

Adults

7

160

12279

656

12

12552.58

1663.736

0

1.208

0.76

0.984

15415

12757

-1.721

Adults

7

80

6158

1215

12

7107.94

1837.448

0

0.805

0.309

0.557

10270

12757

-1.721

Adults

7

40

1984

2082

10

1684.602

2381.384

0.244

0.455

-0.191

0.132

5802

12757

-1.729Adults

7

20

934

1167

12

932.356

1855.67

0.31

0.323

-0.177

0.073

4125

12757

-1.721

Adults

7

10

2544

1531

12

1893.609

1756.561

0.147

0.385

-0.088

0.148

4916

12757

-1.72179335

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

Parameter

Time(CCA)1Test
conc.
(µg/l)

Adults8160

Observedmeandifference
fromcontrol2NAnModel estimate
mean
difference
from control3,4Standard errorof estimatedmean4

p_value forcomparison
with thecontrol4

90%
confidence
upper
limit490%
confidence
lower
limit4

NA

0

NA

NA

NA

NA

NA

Estimated
reduction
fromcontrol(%)4,5NA

Adults

8

80

-2252

3112

7

-2474.47

3577.028

0.74

0.817

-1.672

Adults

8

40

-4047

3416

3

-3178.64

5618.408

0.664

5.577

Adults

8

20

-4077

1453

6

-4081.79

1628.141

0.967

Adults

8

10

3020

NA

1

NA

NA

Adults

9

160

NA

NA

0

NA

Adults

9

80

-915

4716

7

Adults

9

40

-1107

6047

3

Adults

9

20

-4510

3073

Adults

9

10

23798

Honey

3

160

Honey

3

Honey

Standard
error
observedmean

Estimated
reductionfrom control(number)4Observedmeans incontrolT-testconfidence
limit

NA

NA

NA

-0.4274733

5791-2.015-6.675-0.549

32295

5791

-6.314-0.105-1.304-0.705-611

5791-2.132

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

-1218.75

5424.588

0.584

0.661

-0.827-0.083

9712

14685

-2.0152664.342

7809.261

0.395

3.539

-3.176

0.181

51970

14685

-6.3146-4280.88

3056.584

0.883

0.152

-0.735-0.292

2235

14685

-2.132

NA

1

NA

NA

NA

NA

NA

NA

NA

NA

NA

-1530

6052

12

0

0

0

0

0

0

0

45266

-1.717

80

-2175

6139

12

0

0

0

0

0

0

0

45266

-1.717

3

40

2407

7070

12

0

0

0

0

0

0

0

45266

-1.717

Honey

3

20

-1927

8031

12

0

0

0

0

0

0

0

45266

-1.717

Honey

3

10

7088

6550

12

0

0

0

0

0

0

0

45266

-1.717

Honey

4

160

-9511

3598

12

-9672.55

2608.755

0.999-0.095-0.26-0.178

-5184

54439

-1.721

Honey

4

80

-7262

4172

12

-7006.23

2471.091

0.995-0.051-0.207-0.129

-2754

54439

-1.721

Honey

4

40

-2851

5334

11

-5838.4

3036.367

0.966-0.011-0.203-0.107

-602

54439

-1.725

Honey

4

20

-2696

5969

12

-2623.74

2657.341

0.833

0.036

-0.132-0.048

1949

54439

-1.721

Honey

4

10

3275

6129

12

-3203.22

2969.458

0.854

0.035

-0.153-0.059

1906

54439

-1.721

Honey

5

160

-110255173

12-11140.3

2971.193

0.999-0.103-0.279-0.191

-6028

58334

-1.721

Honey

5

80

-158224384

12-15678.4

2659.2071-0.19-0.347-0.269-1110358334-1.721

Honey

5

40

-9068

6034

11

-12035.1

3210.807

0.999-0.111-0.301-0.206

-6497

58334

-1.725

Honey

5

20

-6344

5649

12

-6197.92677.684

0.985-0.027-0.185-0.106

-1590

58334

-1.721

Honey

5

10

1795

5304

12

-3832.55

2535.103

0.927

0.009

-0.14

-0.066530

58334-1.721

Honey

6

160

-293534792

12-29434.8

3572.762

1-0.685-1.046-0.865-2328734018-1.72180
336

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

Parameter

Time(CCA)1Test
conc.
(µg/l)

Honey

6

80

Observedmeandifference
fromcontrol2-23382nModel estimate
mean
difference
from control3,4Standard errorof estimatedmean4

p_value forcomparison
with thecontrol4

90%
confidence
upper
limit490%
confidence
lower
limit45113

12-23289.3

3606.3861-0.502-0.867

Estimated
reduction
fromcontrol(%)4,5-0.685

Honey

6

40

-126156754

10-15217.6

4513.887

0.998-0.218-0.677

Honey

6

20

-5070

6073

12

-5105.39

3524.015

0.919

0.028

Honey

6

10

1861

7227

12

-3921.35

4673.865

0.795

Honey

7

160

-268144512

12-26591.2

3857.038

Honey

7

80

-201154172

12-20117.7

Honey

7

40

-11136

4562

10

Honey

7

20

-794

4928

12

Honey

7

10

3887

6456

Honey

8

160

NA

Honey

8

80

Honey

8

Honey

Standard
error
observedmean

Estimated
reductionfrom control(number)4Observedmeans incontrolT-testconfidence
limit-1708434018-1.721-0.447

-7413

34018

-1.729-0.328

-0.15

959

34018

-1.7210.121-0.352-0.115

4121

34018

-1.721

1-0.796-1.325-1.06-1995425077-1.7213402.1061-0.569-1.036-0.802-1426425077-1.721-12889.2

3628.178

0.999-0.264-0.764-0.514

-6616

25077

-1.729-703.192

3778.982

0.573

0.231

-0.287-0.028

5799

25077

-1.72112-691.527

4370.679

0.562

0.272

-0.327-0.028

6829

25077

-1.721

NA

0

NA

NA

NA

NA

NA

NA

NA

NA

NA

-4537

9702

7

-496.727

8503.231

0.522

0.757

-0.802-0.023

16638

21984

-2.015

40

5029

18842

3

-2400.74

5396.559

0.633

1.441

-1.659-0.109

31672

21984

-6.314820-167734392

6-142405284.67

0.973-0.135-1.16-0.648

-2974

21984

-2.132

Honey

8

10

18064

NA

1

NA

NA

NA

NA

NA

NA

NA

NA

NA

Honey

9

160

NA

NA

0

NA

NA

NA

NA

NA

NA

NA

NA

NA

Honey

9

80

-2184

6379

7

1156.033

4362.47

0.401

0.37

-0.284

0.043

9947

26847

-2.015

Honey

9

40

2580

8835

3

-861.1193209.364

0.583

0.723

-0.787-0.032

19402

26847

-6.314

Honey

9

20

-257729308

6-16502.4

8654.203

0.935

0.073

-1.302-0.615

1947

26847

-2.132

Honey

9

10

-397

NA

1

NA

NA

NA

NA

NA

NA

NA

NA

NA

Pollen

3

160

165

737

12

0

0

0

0

0

0

0

5690

-1.717

Pollen

3

80

-132

816

12

0

0

0

0

0

0

0

5690

-1.717

Pollen

3

40

645

654

12

0

0

0

0

0

0

0

5690

-1.717

Pollen

3

20

529

533

12

0

0

0

0

0

0

0

5690

-1.717

Pollen

3

10

66

754

12

0

0

0

0

0

0

0

5690

-1.717

Pollen

4

160

12423

992

12

12105.42

1546.157

0

1.15

0.736

0.943

14766

12836

-1.721

Pollen

4

80

6170

959

12

6098.508

1421.854

0

0.666

0.284

0.475

8545

12836

-1.72181
337

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

Parameter

Time(CCA)1Test
conc.
(µg/l)

Pollen4

40

Observedmeandifference
fromcontrol23844nModel estimate
mean
difference
from control3,4Standard errorof estimatedmean4

p_value forcomparison
with thecontrol4

90%
confidence
upper
limit490%
confidence
lower
limit41207

112816.395

1541.659

0.041

0.427

0.012

Estimated
reduction
fromcontrol(%)4,50.219

Pollen

4

20

2332

1500

12

1396.809

1612.023

0.198

0.325

-0.107

Pollen

4

10

1174

1708

12

921.971781.651

0.305

0.311

Pollen

5

160

8263

986

12

8269.915

1171.897

0Pollen

5

80

8097

945

12

8216.444

997.742

Pollen

5

40

5341

1846

11

4572.426

Pollen

5

20

2853

1011

12

Pollen

5

10

2556

1173

12

Pollen

6

160

7014

1359

Pollen

6

80

4268

Pollen640

Pollen6

PollenStandard
error
observedmean

Estimated
reductionfrom control(number)4Observedmeans incontrolT-testconfidence
limit

5475

12836

-1.725

0.109

4171

12836

-1.721-0.167

0.072

3988

12836

-1.721

1.245

0.757

1.001

10286

8263

-1.721

0

1.202

0.787

0.994

9933

8263

-1.7211468.524

0.003

0.86

0.247

0.553

7105

8263

-1.7252290.263

1038.79

0.019

0.494

0.061

0.277

4078

8263

-1.7212494.555

1271.055

0.032

0.567

0.037

0.302

4682

8263

-1.721126744.362

1805.483

0.001

1.401

0.517

0.959

9851

7030

-1.7211394

124238.004

1667.556

0.009

1.011

0.195

0.603

7107

7030

-1.721

1449

1340

10

537.4071814.263

0.385

0.523

-0.37

0.076

3675

7030

-1.729

20

281

1176

12

-784.589

1635.662

0.682

0.289

-0.512-0.1122030

7030-1.721

6

10

1869

1882

12

1623.023

1908.998

0.202

0.698

-0.236

0.231

4908

7030

-1.721

Pollen

7

160

5450

1225

12

5214.728

1290.707

0

1.364

0.549

0.957

7436

5450

-1.721

Pollen

7

80

3896

1252

12

3983.153

1176.532

0.001

1.102

0.359

0.731

6008

5450

-1.721

Pollen

7

40

1310

1855

10

590.312

1621.575

0.36

0.623

-0.406

0.108

3394

5450

-1.729Pollen

7

20

2043

1187

12

1277.085

1228.752

0.155

0.622

-0.154

0.234

3391

5450

-1.721

Pollen

7

10

3432

1528

12

3257.764

1434.03

0.017

1.05

0.145

0.598

5725

5450

-1.721

Pollen

8

160

NA

NA

0

NA

NA

NA

NA

NA

NA

NA

NA

NA

Pollen

8

80

-3233

2437

7

-5068.17

2502.893

0.951-0.003-1.327-0.665-257617-2.015Pollen

8

40

-5360

5566

3

-2304.84

3426.556

0.688

2.538

-3.143-0.303

19330

7617

-6.314

Pollen

8

20

-6352

2592

6

-5298.69

3324.068

0.907

0.235

-1.626-0.69617887617-2.132

Pollen810-18858

NA

1

NA

NA

NA

NA

NA

NA

NA

NA

NA

Pollen

9

160

NA

NA

0

NA

NA

NA

NA

NA

NA

NA

NA

NA

Pollen

9

80

-4339

3524

7

-6755.88

3762.322

0.934

0.076

-1.328-0.626825

10793-2.015Pollen

9

40

-2978

2066

3

-2037.76

2064.719

0.748

1.019

-1.397-0.189

10998

10793

-6.31482338

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

Parameter

Time(CCA)1Test
conc.
(µg/l)

Pollen920

Observedmeandifference
fromcontrol2-3011nModel estimate
mean
difference
from control3,4Standard errorof estimatedmean4

p_value forcomparison
with thecontrol4

90%
confidence
upper
limit490%
confidence
lower
limit42453

677.5831658.125

0.482

0.335

-0.32

Estimated
reduction
fromcontrol(%)4,50.007

Pollen9

10-11910

NA

1

NA

NA

NA

NA

NA

Capped

3

160

-157

730

12

0

0

0

0

Capped

3

80

-1332

868

12

0

0

0

Capped

3

40

190

874

12

0

0

Capped

3

20

-1348

904

12

0

Capped

3

10

-1431

978

12

Capped

4

160

9007

1797

12

Capped

4

80

5120

1770

Capped

4

40

3266

Capped4

20

Capped4

CappedStandard
error
observedmean

Estimated
reductionfrom control(number)4Observedmeans incontrolT-testconfidence
limit

3612

10793

-2.132

NA

NA

NA

NA

0

0

0

17294

-1.717

0

0

0

0

17294

-1.717

0

0

0

0

0

17294

-1.717

0

0

0

0

0

0

17294

-1.717

0

0

0

0

0

0

0

17294

-1.7179089.959

1665.078

0

0.617

0.321

0.469

11955

19362

-1.721125980.513

1592.068

0.001

0.45

0.167

0.309

8720

19362

-1.7211263

113161.897

1410.491

0.018

0.289

0.038

0.163

5595

19362

-1.725

852

547

12

1215.668

1112.801

0.144

0.162

-0.036

0.063

3131

19362

-1.721

10

174

849

12

390.583

1260.73

0.38

0.132

-0.092

0.02

2560

19362

-1.721

5

160

19726

886

12

19653.92

1278.542

0

1.108

0.885

0.996

21854

19726

-1.721

Capped

5

80

16484

1496

12

17301.22

1564.406

0

1.014

0.741

0.877

19993

19726

-1.721

Capped

5

40

9429

2607

11

9269.012

2196.511

0

0.662

0.278

0.47

13057

19726

-1.725

Capped

5

20

2076

994

12

2591.968

1395.063

0.039

0.253

0.01

0.131

4993

19726

-1.721

Capped

5

10

3532

2541

12

3930.858

2280.717

0.05

0.398

0

0.199

7855

19726

-1.721

Capped

6

160

19478

1271

12

19351.6

1908.239

0

1.141

0.81

0.975

22635

19842

-1.721

Capped

6

80

11885

1902

12

13436.24

2020.548

0

0.852

0.502

0.677

16913

19842

-1.721

Capped

6

40

6908

3851

10

6126.323

3155.141

0.034

0.584

0.034

0.309

11582

19842

-1.729Capped

6

20

1017

1574

12

2215.388

2014.878

0.142

0.286

-0.063

0.112

5682

19842

-1.721

Capped

6

10

2308

1916

12

3332.882

2306.77

0.082

0.368

-0.032

0.168

7302

19842

-1.721

Capped

7

160

4061

1211

12

4102.683

1106.04

0.001

1.438

0.527

0.982

6006

4177

-1.721

Capped

7

80

273

1481

12

845.1231306.775

0.262

0.741

-0.336

0.202

3094

4177

-1.721

Capped

7

40

1092

1641

10

913.244

1230.771

0.234

0.728

-0.291

0.219

3041

4177

-1.729Capped

7

20

141

1264

12

448.692

1206.418

0.357

0.604

-0.39

0.107

2525

4177

-1.72183
339

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

Parameter

Time(CCA)1Test
conc.
(µg/l)

Capped7

10Observedmeandifference
fromcontrol21083nModel estimate
mean
difference
from control3,4Standard errorof estimatedmean4

p_value forcomparison
with thecontrol4

90%
confidence
upper
limit490%
confidence
lower
limit4840

121554.033

920.202

0.053

0.751

-0.007

Estimated
reduction
fromcontrol(%)4,50.372

Capped

8

160

NA

NA

0

NA

NA

NA

NA

NA

Capped

8

80

-3913

3384

7

-555.205

4282.752

0.549

1.379

Capped

8

40

-7609

6002

3

-3432.66

48.8860.995

Capped

8

20

-6782

2295

6

-5455.82

2497.38

Capped

8

10

-1985

NA

1

NA

Capped

9

160

NA

NA

0

Capped

9

80

-2552

5953

7

Capped

9

40

-7212

3853

Capped

9

20

-5326

Capped

9

10

Open

3

Open

Standard
error
observedmean

Estimated
reductionfrom control(number)4Observedmeans incontrolT-testconfidence
limit

3137

4177

-1.721

NA

NA

NA

NA

-1.569-0.0958075

5856-2.015-0.533-0.639-0.586

-3124

5856

-6.3140.953-0.023-1.841-0.932-132

5856-2.132

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

7620.467

4910.237

0.091

1.211

-0.157

0.527

17515

14466

-2.0153-4539.09

478.3880.967-0.105-0.523-0.314

-1519

14466

-6.3142185

6-5617.14

2729.671

0.946

0.014

-0.791-0.388202

14466-2.132

12108

NA

1

NA

NA

NA

NA

NA

NA

NA

NA

NA

160

339

708

12

0

0

0

0

0

0

0

9801

-1.717

3

80

-1497

1258

12

0

0

0

0

0

0

0

9801

-1.717

Open

3

40

505

602

12

0

0

0

0

0

0

0

9801

-1.717

Open

3

20

918

530

12

0

0

0

0

0

0

0

9801

-1.717

Open

3

10

-91

793

12

0

0

0

0

0

0

0

9801

-1.717

Open

4

160

7642

812

12

7723.364

715.022

0

1.017

0.738

0.878

8954

8800

-1.721

Open

4

80

811

996

12

1084.371

794.465

0.093

0.279

-0.032

0.123

2451

8800

-1.721

Open

4

40

1949

978

11

1882.119823.751

0.017

0.375

0.052

0.214

3303

8800

-1.725

Open

4

20

1125

734

12

1077.189

691.449

0.067

0.258

-0.013

0.122

2267

8800

-1.721

Open

4

10

1307

936

12

1322.625

783.507

0.053

0.303

-0.003

0.15

2671

8800

-1.721

Open

5

160

9900

589

12

9725.764

1006.838

0

1.157

0.807

0.982

11458

9900

-1.721

Open

5

80

8097

923

12

8402.752

1204.924

0

1.058

0.639

0.849

10476

9900

-1.721

Open

5

40

2111

1848

11

1943.583

1622.327

0.122

0.479

-0.086

0.196

4742

9900

-1.725

Open

5

20

1480

1089

12

1063.085

1260.766

0.204

0.327

-0.112

0.107

3233

9900

-1.721

Open

5

10

207

1493

12

280.262

1449.109

0.424

0.28

-0.224

0.028

2774

9900

-1.721

84
340

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

Parameter

Time(CCA)1Test
conc.
(µg/l)

Open

6

160

Observedmeandifference
fromcontrol28122nModel estimate
mean
difference
from control3,4Standard errorof estimatedmean4

p_value forcomparison
with thecontrol4

90%
confidence
upper
limit490%
confidence
lower
limit4572

128294.56

1398.848

0

1.228

0.675

Estimated
reduction
fromcontrol(%)4,5
0.951

Open

6

80

4218

947

12

4177.953

1558.807

0.007

0.787

0.172

Open

6

40

1925

1979

10

1958.857

2088.029

0.18

0.639

Open

6

20

314

680

12

555.968

1473.638

0.355

Open

6

10

-480

952

12

-489.901

1528.829

Open

7

160

2274

513

12

2279.459

Open

7

80

1067

723

12

Open

7

40

983

761

10

Open

7

20

852

655

Open

7

10

769

Open

8

160

Open

8

Open

Standard
error
observedmean

Estimated
reductionfrom control(number)4Observedmeans incontrolT-testconfidence
limit

10702

8717

-1.721

0.479

6860

8717

-1.721-0.189

0.225

5569

8717

-1.7290.355-0.227

0.064

3092

8717

-1.721

0.624

0.246

-0.358-0.0562141

8717-1.721

729.205

0.003

1.532

0.444

0.988

3534

2308

-1.721

1274.387

825.177

0.069

1.168

-0.063

0.552

2694

2308

-1.721

953.73887.858

0.148

1.079

-0.252

0.413

2489

2308

-1.72912937.724801.426

0.128

1.004

-0.191

0.406

2317

2308

-1.721691

12761.399801.139

0.176

0.927

-0.267

0.33

2140

2308

-1.721

NA

NA

0

NA

NA

NA

NA

NA

NA

NA

NA

NA

80

-170

2340

7

2026.873

2522.734

0.229

1.365

-0.587

0.389

7110

5211

-2.015

8

40

-5426

3617

3

-1805.65

3635.901

0.647

4.059

-4.752-0.347

21151

5211

-6.314

Open

8

20

-4102

1098

6

-4157.35

1198.747

0.987-0.307-1.288-0.798

-1602

5211

-2.132

Open

8

10

4367

NA

1

NA

NA

NA

NA

NA

NA

NA

NA

NA

Open

9

160

NA

NA

0

NA

NA

NA

NA

NA

NA

NA

NA

NA

Open

9

80

-2297

3193

7

2146.953

2206.34

0.188

1.03

-0.359

0.335

6593

6402

-2.015

Open

9

40

-265

1610

3

-201.982

2976.105

0.522

2.904

-2.967-0.032

18588

6402

-6.314

Open

9

20

-1621

1662

6

-1802.36

1607.44

0.838

0.254

-0.817-0.2821624

6402-2.132

Open

9

10

13895

NA

1

NA

NA

NA

NA

NA

NA

NA

NA

NA

Eggs

3

160

190

717

12

0

0

0

0

0

0

0

6393

-1.717

Eggs

3

80

538

892

12

0

0

0

0

0

0

0

6393

-1.717

Eggs

3

40

-753

841

12

0

0

0

0

0

0

0

6393

-1.717

Eggs

3

20

-41

602

12

0

0

0

0

0

0

0

6393

-1.717

Eggs

3

10

-422

976

12

0

0

0

0

0

0

0

6393

-1.717

Eggs

4

160

1489

972

12

1429.084

795.466

0.043

0.611

0.013

0.312

2798

4582

-1.721

85
341

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

Parameter

Time(CCA)1Test
conc.
(µg/l)

Eggs

4

80

Observedmeandifference
fromcontrol2
1770nModel estimate
mean
difference
from control3,4Standard errorof estimatedmean4

p_value forcomparison
with thecontrol4

90%
confidence
upper
limit490%
confidence
lower
limit4620

121661.358

573.629

0.004

0.578

0.147

Estimated
reduction
fromcontrol(%)4,5
0.363

Eggs

4

40

2445

525

11

2447.167

556.634

0

0.744

0.325

Eggs

4

20

1241

786

12

1242.122

677.598

0.04

0.526

Eggs

4

10

1406

646

12

1380.358

600.909

0.016

Eggs

5

160

3755

804

12

3674.178

726.848

Eggs

5

80

2796

659

12

2811.043

Eggs

5

40

1660

931

11

Eggs

5

20

1538

550

12

Eggs

5

10

1340

669

Eggs

6

160

2614

Eggs

6

80

Eggs

6

Eggs

Standard
error
observedmean

Estimated
reductionfrom control(number)4Observedmeans incontrolT-testconfidence
limit

2648

4582

-1.721

0.534

3407

4582

-1.725

0.017

0.271

2408

4582

-1.721

0.527

0.076

0.301

2414

4582

-1.721

0

1.22

0.6

0.91

4925

4036

-1.721

662.938

0

0.979

0.414

0.696

3952

4036

-1.721

1681.656

826.62

0.028

0.77

0.063

0.417

3107

4036

-1.7251507.223

592.724

0.009

0.626

0.121

0.373

2527

4036

-1.721121416.205

631.272

0.018

0.62

0.082

0.351

2502

4036

-1.721575

122516.76671.964

0.001

1.175

0.435

0.805

3673

3126

-1.721

1588

469

12

1546.563

668.294

0.015

0.863

0.127

0.495

2697

3126

-1.721

40

635

829

10

932.125903.77

0.158

0.798

-0.202

0.298

2495

3126

-1.729

6

20

546

406

12

488.995

614.952

0.218

0.495

-0.182

0.156

1547

3126

-1.721

Eggs

6

10

182

697

12

202.448771.323

0.398

0.489

-0.36

0.065

1530

3126

-1.721

Eggs

7

160

976

312

12

963.467354.762

0.006

1.535

0.344

0.939

1574

1026

-1.721

Eggs

7

80

728

259

12

729.072336.527

0.021

1.276

0.146

0.711

1308

1026

-1.721

Eggs

7

40

218

299

10

428.311367.232

0.129

1.037

-0.202

0.418

1063

1026

-1.729

Eggs

7

20

265

313

12

261.589355.17

0.235

0.851

-0.341

0.255

873

1026

-1.721

Eggs

7

10

0

353

12

17.235378.245

0.482

0.651

-0.618

0.017

668

1026

-1.721

Eggs

8

160

NA

NA

0

NA

NA

NA

NA

NA

NA

NA

NA

NA

Eggs

8

80

425

988

7

558.0451089.023

0.315

1.233

-0.733

0.25

2752

2233

-2.015

Eggs

8

40

-1456

919

3

-461.442

644.484

0.698

1.616

-2.029-0.2073608

2233-6.314

Eggs

8

20

-1654

509

6

-1729.75

345.134

0.996-0.445-1.104-0.775-994

2233-2.132

Eggs

8

10

0

NA

1

NA

NA

NA

NA

NA

NA

NA

NA

NA

Eggs

9

160

NA

NA

0

NA

NA

NA

NA

NA

NA

NA

NA

NA

Eggs

9

80

284

740

7

316.162

833.592

0.36

1.087

-0.743

0.172

1996

1836

-2.01586342

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

Parameter

Time(CCA)1Test
conc.
(µg/l)

Eggs

9

40

Observedmeandifference
fromcontrol2397nModel estimate
mean
difference
from control3,4Standard errorof estimatedmean4

p_value forcomparison
with thecontrol4

90%
confidence
upper
limit490%
confidence
lower
limit41050

3-482.609

1346.899

0.61

4.369

-4.894

Estimated
reduction
fromcontrol(%)4,5-0.263

Eggs

9

20

959

790

6

1069.694

586.629

0.071

1.264

-0.099

Eggs

9

10

2580

NA

1

NA

NA

NA

NA

TotalLife

3

160

-272

1957

12

0

0

0

TotalLife

3

80

-3791

1920

12

0

0

TotalLife

3

40

-365

2729

12

0

TotalLife

3

20

-567

2619

12

TotalLife

3

10

-1279

3075

12

TotalLife

4

160

23524

3038

TotalLife

4

80

9341

TotalLife

440

TotalLife

4

TotalLife

Standard
error
observedmean

Estimated
reductionfrom control(number)4Observedmeans incontrolT-testconfidence
limit

8021

1836

-6.314

0.583

2320

1836

-2.132

NA

NA

NA

NA

NA

0

0

0

0

48503

-1.717

0

0

0

0

0

48503

-1.717

0

0

0

0

0

0

48503

-1.717

0

0

0

0

0

0

0

48503

-1.717

0

0

0

0

0

0

0

48503

-1.7171223503.64

2987.178

0

0.572

0.367

0.469

28644

50084

-1.7213109

1211199.39

2704.951

0

0.317

0.131

0.224

15854

50084

-1.721

7597

2447

11

7599.152

2884.741

0.008

0.251

0.052

0.152

12575

50084

-1.725

20

3479

1395

12

3345.871

2225.651

0.074

0.143

-0.01

0.067

7176

50084

-1.721

4

10

2897

2634

12

3041.294

2823.896

0.147

0.158

-0.036

0.061

7900

50084

-1.721

TotalLife

5

160

46712

2502

12

46430.78

3249.602

0

0.982

0.771

0.877

52023

52973

-1.721

TotalLife

5

80

34592

2824

12

37199.69

3451.789

0

0.814

0.59

0.702

43139

52973

-1.721

TotalLife

5

40

17777

6001

11

17789.99

5455.377

0.002

0.513

0.158

0.336

27199

52973

-1.725

TotalLife

5

20

5455

2440

12

5324.279

3631.571

0.079

0.218

-0.017

0.101

11573

52973

-1.721

TotalLife

5

10

6908

4323

12

7674.205

4250.837

0.043

0.283

0.007

0.145

14989

52973

-1.721

TotalLife

6

160

46396

2367

12

46099.074247.171

0

1.066

0.774

0.92

53407

50113

-1.721

TotalLife

6

80

28196

3656

12

32832.62

4383.394

0

0.806

0.505

0.655

40375

50113

-1.721

TotalLife

6

40

15255

8144

10

15272.1

7556.661

0.029

0.565

0.044

0.305

28339

50113

-1.729TotalLife

6

20

5088

3185

12

4992.654951.257

0.162

0.27

-0.07

0.1

13512

50113

-1.721

TotalLife

6

10

6208

3272

12

7047.944

4953.287

0.085

0.311

-0.029

0.141

15571

50113

-1.721

TotalLife

7

160

19590

1983

12

19560.29

2938.276

0

1.215

0.716

0.965

24616

20267

-1.721

TotalLife

7

80

8226

2257

12

10750.1

2990.428

0.001

0.784

0.277

0.53

15896

20267

-1.721

TotalLife

7

40

4277

4304

10

4546.944

3947.73

0.132

0.561

-0.112

0.224

11373

20267

-1.72987343

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

Parameter

Time(CCA)1Test
conc.
(µg/l)

TotalLife

720

Observedmeandifference
fromcontrol22191nModel estimate
mean
difference
from control3,4Standard errorof estimatedmean4

p_value forcomparison
with thecontrol4

90%
confidence
upper
limit490%
confidence
lower
limit41961

122241.13020.135

0.233

0.367

-0.146

Estimated
reduction
fromcontrol(%)4,5
0.111

TotalLife

7

10

4397

2452

12

4802.467

3065.589

0.066

0.497

-0.023

TotalLife

8

160

NA

NA

0

NA

NA

NA

NA

TotalLife

8

80

-5910

8862

7

5535.519

11348.39

0.323

TotalLife

840-1853713728

3-21082.6

27248.42

TotalLife

820-166164430

6-16421

TotalLife

8

10

5402

NA

1

TotalLife

9

160

NA

NA

0

TotalLife

9

80

-5480

13625

TotalLife

9

40

-8187

TotalLife920

TotalLife

910Standard
error
observedmean

Estimated
reductionfrom control(number)4Observedmeans incontrolT-testconfidence
limit

7438

20267

-1.721

0.237

10078

20267

-1.721

NA

NA

NA

NA

NA

1.488

-0.908

0.29

28403

19090

-2.015

0.71

7.908

-10.116-1.10415095719090-6.3145116.892

0.984-0.289-1.432

-0.86

-5513

19090

-2.132

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

7

20926.94

12019.68

0.071

1.208

-0.088

0.56

45147

37388

-2.01510550

3-219.332

17872.92

0.504

3.012

-3.024-0.00611262637388-6.314-104985444

6-11621.1

5925.836

0.939

0.027

-0.649-0.311

1012

37388

-2.132

52382

NA

1

NA

NA

NA

NA

NA

NA

NA

NA

NA

Notes:
1. Colony Condition Assessment (CCA) Observation dates.
2. Mean of observations in controls minus the observation in the treatment.
3. Difference between the mean of observation in controls and estimated number in treatment after adjustment for covariance for CCA3 to be a 0 baseline.
4. ‘NA’ indicates there was not enough data to do the test (n = number of Apiaries is small) and ‘0’ means rounded to 0 (except for CCA3 where p_values are constrained to be 0 as the exposure has not occurred.)
5. The percentage of the estimated difference between the treatment and control divided by the number in the control. [Value in column must be multiplied by 100 to be a %]88344

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Results
The following tables and graphs present results for individual measurement endpoints (total
individuals, adults, eggs, larvae, pupae, pollen stores, honey stores). The percent reductions are
the means of the differences between each treatment and control at the same apiary, based on
observations and expected values estimated by the statistical model that adjusted baseline
measurement for CCA3, using raw count data.
Control Trends
For a comparison between the numbers of live bees in the three different neonicotinoid colony
feeding studies (clothianidin, imidacloprid, thiamethoxam), refer to the thiamethoxam statisticalanalysis.
Colony Condition Assessments – Life stages in the hive
Total number of individuals (total life)
Table A-2. and Figure A-1. show the estimated effects on total number of individual bees (totallife) across CCAs and treatment groups. Compared to the control, a significant reduction (p<0.05)
in total life was observed at the three highest treatment groups (40, 80 and 160 µg/L) at CCAs 46 (15.2-33.6%, 22.4-70.2% and 46.9-92.0% reduction at 40, 80 and 160 µg/L, respectively) and in
the two highest treatment groups at CCA7 (53% reduction at 80 µg/L, p= 0.001 and 96.5%
reduction at 160 µg/L, p<<0.05). A significant reduction in total life was also observed at CCA4
for the 20 µg/L treatment group (6.7% reduction, p=0.074) and CCA5 for the 10 µg/L (14.5%
reduction, p=0.043) and 20 µg/L (10.1% reduction, p=0.079) treatment groups. While significant
effects were observed at CCA4 and/or CCA5 at the lowest two treatment groups, no significant
effects (p>0.1) were observed at subsequent CCAs (CCAs 6-7) indicating a potential transient
effect on total life at 10 and 20 µg/L.
Figure A-1. shows a general dose responsiveness (increase in the reduction from the control as
the dose increases) at the three highest treatment groups (40, 80 and 160 µg/L ) over all CCAs. For
the two lowest treatment groups, the reduction in total individuals in the 10 µg/L treatment group
was consistently higher than or equivalent to the 20 µg/L treatment group over all CCAs (6.1-23.7%
and 6.7-11.1% reduction in the 10 and 20 µg/L treatment groups, respectively) and in particular,
CCA7. The overlap in dose-response at the lower doses is not unexpected since the dose levels are
similar and measured exposures indicate overlap in exposure among individual hives, particularly
at the lower two doses. In general, the width of the confidence intervals increased with increasing
CCAs. As seen in Figure A-2. the total number of individuals at CCA6 was reduced from thecontrol at 58% (7/12), 58% (7/12), 60% (6/10), 100% (12/12) and 100% (12/12) of the apiaries in
the 10, 20 , 40, 80 and 160 µg /L dose groups, respectively.
When weighing statistical and biological significance, the overall NOAEC and LOAEC for
total life is determined to be 20 and 40 µg/L, respectively. This is based on significant andpersistent reductions in total life throughout the study from CCA4-CCA7 at the highest threetreatment groups (40, 80 and 160 µg/L) and potential transient effects at ≤ 20 µg/L at CCAs
4-5.
89
345

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Table A-2. Estimated percent reduction from control for total number of individuals.
Estimated reduction from control (%)
Testconcentration (p value)(µg/L)
CCA4
CCA5
CCA6
CCA7
CCA8
6.1
14.5
14.1
23.7
NA
10
(0.147)(0.043**/0.079*) (0.085*/0.162) (0.066*/0.233)
6.7
10.1
10.0
11.1
-86.0
20
(0.074*) (0.079*)(0.162)(0.233)(0.984)
15.2
33.6
30.5
22.4
-110
40
(0.008**) (0.002**)(0.029**)(0.132)(0.71)
22.4
70.2
65.5
53.0
29.0
80
(0**)
(0**)
(0**)
(0.001**)(0.323)
46.9
87.7
92.0
96.5
NA
160
(0**)
(0**)
(0**)
(0**)

CCA9
NA
-31.1
(0.939)-0.6(0.504)56.0(0.071*)NA

Note: Negative value indicates increased number of individuals in comparison to control.
*0.05<p<0.1, **p<0.05
NA indicates there was not enough data to do the test (n = number of Apiaries is small) and ‘0’ means rounded to 0
Where two p values are listed, the first is the non-adjusted p value, the second is the p value adjusted for the step-down approach.
The step-down adjustment was shown only if it changed the significance level.
At CCA8 and CCA9, the step-down approach was not applied to the 160 or 10 treatment levels where very few hives survived.

90346

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

Figure A-1. Estimates and 90% CIs for Total Life with thresholds of statistical (red) significance shown.
The green line shows 10% difference from control.

91347

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

Figure A-2. Difference from control for all treatments and apiaries at CCA6 for total life. Apiaries shown
above the zero line had better control outcomes in comparison to the treatment.

AdultsTable A-3 and Figure A-3 show the effects on adult honey bees across CCAs and treatment groups.
Compared with the control group, a significant reduction (p<0.05) in the number of adults wasobserved at the two highest treatment groups (80 and 160 µg/L) starting at CCA4 and persistingover multiple CCAs from CCAs 4-7 (13.9-55.7% and 32.3-98.4% reduction at 80 and 160 µg/L,
respectively). The number of adults in the 40 µg/L treatment group was also significantly reduced
starting at CCA5 (23.1% reduction, p=0.011) and persisting through to CCA6 (29.9% reduction,p=0.012), but was not significantly reduced (p>0.1) at CCAs 4 and 7 (reductions of -1.3 and 13.2%,
respectively). No significant reduction (p>0.1) in the number of adults was observed in the twolowest treatment groups (10 and 20 µg/L) from CCA4 through to CCA7, except at CCA6 where
92348Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
the number of adults was significantly reduced from the control by 19.6% (p=0.014) in the 10 µg/L
treatment group and 17% (p=0.037) in the 20 µg/L treatment group.
Figure A-3 shows a general dose response (increase in the reduction from the control as the dose
increases) at the three highest treatment groups (40, 80 and 160 µg/L) over all CCAs. For the twolowest treatment groups, the reduction in the number of adults from the control in the 10 µg/L
treatment group was consistently higher than or equivalent to the 20 µg/L treatment group over all
CCAs although generally similar (-2.0-19.6% and 0.6-17.0% reduction in the 10 and 20 µg/L
treatment groups, respectively). The overlap in dose-response at the lower doses is not unexpected
since the dose levels are similar and measured exposures indicate overlap in exposure amongindividual hives, particularly at the lower two doses. In general, the width of the confidence
intervals increased with increasing CCAs. As seen in Figure A-4 the number of adults at CCA6
was reduced from the control in 75% (9/12), 75% (7/12), 60% (6/10), 100% (12/12) and 100%
(12/12) of the apiaries in the 10, 20, 40, 80 and 160 µg /L dose groups, respectively.
Significant reductions in the number of adults were found at the highest two treatment levels (80and 160 µg/L) that were apparent from the beginning of exposure (CCA4) and persisted throughto after exposure (CCA7) when populations were in natural decline. Additionally, persistentsignificant effects were noted at 40 µg/L over multiple CCAs after exposure. While treatment
means were significantly reduced from the control at the lowest two doses (10 and 20 µg/L) at asingle CCA (CCA6), these effects were considered to be isolated and potentially transient. Thisfinding supports the use of 20 µg/L as the NOAEC as determined through the primary analysis for
total life at CCA6. The early onset and persistence of significant effects during and/or after the endof exposure at the highest treatment groups (40, 80 and 160 µg/L) supports the use of 40 µg/L as
the LOAEC.
Table A-3. Estimated percent reduction from control for number of adults.
Estimated reduction from control (%)
Testconcentration (p value)(µg/L)
CCA4
CCA5
CCA6
CCA7
-2.0
7.7
19.6
14.8
10
(0.602)(0.228)(0.014**)(0.147)
0.6
1.6
17.0
7.3
20
(0.465)(0.425)(0.037**)(0.31)
-1.3
23.1
29.9
13.2
40
(0.571)(0.011**)(0.012**)(0.244)
13.9
41.2
64.1
55.7
80
(0.029**)
(0**)
(0**)
(0**)
32.3
69.9
89.7
98.4
160
(0**)
(0**)
(0**)
(0**)

CCA8
NA

CCA9
NA

-70.5
(0.967)-54.9(0.664)-42.7(0.74)
NA-29.2(0.883)18.1(0.395)-8.3(0.584)NA

Note: Negative value indicates increased number of individuals in comparison to control.
*0.05<p<0.1, **p<0.05
NA’ indicates there was not enough data to do the test (n = number of Apiaries is small) and ‘0’ means rounded to 0
Where two p values are listed, the first is the non-adjusted p value, the second is the p value adjusted for the step-down approach.
The step-down adjustment was shown only if it changed the significance level.
At CCA8 and CCA9, the step-down approach was not applied to the 160 or 10 treatment levels where very few hives survived.

93349

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

Figure A-3. Estimates and 90% CIs for adults with thresholds of statistical (red) significance shown. The
green line shows 10% difference from control.

94
350

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

Figure A-4. Difference from control for all treatments and apiaries at CCA6 for adults. Apiaries shown
above the zero line had better control outcomes in comparison to the treatment.

Eggs
Table A-4 and Figure A-5 show the effects on the number of honey bee eggs across CCAs andtreatment groups. A significant reduction (p<0.05) in the number of eggs relative to the control
group was observed for all treatment groups (10, 20, 40, 80 and 160 µg/L) during CCA4 (27.153.4% reduction) and CCA5 (35.1-91.0% reduction). The number of eggs was also significantly
reduced (p<0.05) from the control during subsequent CCAs (6 and 7) at the two highest
treatment groups (49.5-93.9% reduction) but not at the three lowest treatment groups (1.7-41.8%
reduction).
95351

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Figure A-5 shows a general dose response (increase in the reduction from the control as the dose
increases) starting at CCA5 which becomes more pronounced over subsequent CCAs up to
CCA7. There was no clear dose response observed at CCA4. In general, the width of theconfidence intervals increased with increasing CCAs. As seen in Figure A-6, egg cells at CCA6
were reduced from the control in 42% (5/12), 67% (8/12), 60% (6/10), 92% (11/12) and 92%(11/12) apiaries in the 10, 20, 40, 80 and 160 µg /L dose groups, respectively.
Treatment means were significantly reduced from the control at the lowest three doses (10, 20
and 40 µg/L) during and immediately after the exposure period (CCA4 and CCA5) but not in
subsequent CCAs after the exposure period and were significantly reduced from the control at
the two highest treatment groups (80 and 160 µg/L) at all CCAs (CCA4-CCA7). While the widthof the confidence intervals increased with increasing CCAs, the observed reversion back tocontrol levels at the three lowest treatment groups during subsequent CCAs indicates a potential
transient effect on eggs and supports the use of 40 µg/L as the NOAEC and 80 µg/L as the
LOAEC which are less sensitive than the endpoints determined through the primary analysis for
total life at CCA6 (NOAEC of 20 µg/L, LOAEC of 40 µg/L).
Table A-4. Estimated percent reduction from control for number of eggs.
Estimated reduction from control (%)
Testconcentration (P value)(µg/L)
CCA4
CCA5
CCA6
CCA7
30.1
35.1
6.5
1.7
10
(0.016**)(0.018**)(0.398)(0.482)
27.1
37.3
15.6
25.5
20
(0.04**)(0.009**)(0.218)(0.235)
53.4
41.7
29.8
41.8
40
(0**)
(0.028**)(0.158)(0.129)
36.3
69.6
49.5
71.1
80
(0.004**)(0**)(0.015**)(0.021**)
31.2
91.0
80.5
93.9
160
(0.043**)(0**)(0.001**)(0.006**)

CCA8
NA

CCA9
NA

-77.5
(0.996)-20.7(0.698)25.0(0.315)NA

58.3(0.071*)-26.3(0.61)
17.2(0.36)
NA

Note: Negative value indicates increased number of individuals in comparison to control.
*0.05<P<0.1, **P<0.05
NA’ indicates there was not enough data to do the test (n = number of Apiaries is small) and ‘0’ means rounded to 0
Where two p values are listed, the first is the non-adjusted p value, the second is the p value adjusted for the step-down approach.
The step-down adjustment was shown only if it changed the significance level.
At CCA8 and CCA9, the step-down approach was not applied to the 160 or 10 treatment levels where very few hives survived.

96352

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

Figure A-5. Estimates and 90% CIs for eggs with thresholds of statistical (red) significance shown. The
green line shows 10% difference from control.

97353

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

Figure A-6. Difference from control for all treatments and apiaries at CCA6 for eggs. Apiaries shown
above the zero line had better control outcomes in comparison to the treatment.

Larvae (Open/uncapped brood)
Table A-5 and Figure A-7 below show the effects on larvae (open/uncapped brood) across CCAs
and treatment groups. Compared with the control group, a significant reduction (p<0.05) in thenumber of larvae was observed at the highest treatment group (160 µg/L) which persisted overmultiple CCAs from CCA4 to CCA7 (87.8-98.8% reduction). The number of larvae was alsosignificantly reduced from the control in the 80 µg/L treatment group over multiple CCAs
including CCA4 (12.3% reduction, p= 0.093), CCAs 5-6 (47.9-84.9% reduction, p<<0.05) andCCA7 (55.2% reduction, p=0.069). No significant reduction (p>0.1) in the number of larvae wasobserved at the lowest three treatment groups (10, 20 and 40 µg/L) during any of the CCAs withthe exception of CCA4, where the number of larvae was significantly reduced from the control by98354

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
15.0% (p=0.053), 12.2% (p=0.067) and 21.4% (p=0.017) at 10, 20 and 40 µg/L treatment groups,
respectively. The effects were observed during a single CCA indicating a potential transient effectat the lowest three doses for this endpoint.
Figure A-7 shows a clear dose response (increase in the reduction from the control as the dose
increases) over all CCAs except at CCA4. In general, the width of the confidence intervals
increased with increasing CCAs. As seen in Figure A-8, larval cells at CCA6 were reduced fromthe control at 33% (4/12), 75% (7/12), 70% (7/10), 83% (10/12) and 100% (12/12) of apiaries in
the 10, 20, 40, 80 and 160 µg /L dose groups, respectively.
Significant reductions in the number of larvae were found at the highest two treatment levels (80and 160 µg/L) that were apparent from the beginning of exposure (CCA4) and persisted throughto after exposure (CCA7) when populations were in natural decline. Treatment means weresignificantly reduced from the control at the lowest three doses (10, 20 and 40 µg/L) during asingle CCA during the exposure period (CCA4) but not in subsequent CCAs. While the width ofthe confidence intervals increased with increasing CCAs, the observed reversion back to control
levels at the three lowest treatment groups during subsequent CCAs indicates a potential transient
effect on larvae at these doses and supports the use of 40 µg/L as the NOAEC and 80 µg/L as the
LOAEC. These endpoints are less sensitive than the endpoints determined through the primary
analysis for total life at CCA6 (NOAEC of 20 µg/L, LOAEC of 40 µg/L).
Table A-5. Estimated percent reduction from control for number of larvae.
Estimated reduction from control (%)
Testconcentration (P value)(µg/L)
CCA4
CCA5
CCA6
CCA7
15.0
2.80
-5.6
33.0
10
(0.053*)(0.424)(0.624)(0.176)
12.2
10.7
6.4
40.6
20
(0.067*)(0.204)(0.355)(0.128)
21.4
19.6
22.5
41.3
40
(0.017**/0.093*) (0.122)(0.18)(0.148)
12.3
84.9
47.9
55.2
80
(0.093*)(0**)(0.007**)(0.069*)
87.8
98.2
95.1
98.8
160
(0**)
(0**)
(0**)
(0**)

CCA8
NA

CCA9
NA

-79.8
(0.987)-34.7(0.647)38.9(0.229)NA-28.2(0.838)-3.2(0.522)33.5(0.229)NA

Note: Negative value indicates increased number of individuals in comparison to control.
*0.05<P<0.1, **P<0.05
NA’ indicates there was not enough data to do the test (n = number of Apiaries is small) and ‘0’ means rounded to 0
Where two p values are listed, the first is the non-adjusted p value, the second is the p value adjusted for the step-down approach.
The step-down adjustment was shown only if it changed the significance level.
At CCA8 and CCA9, the step-down approach was not applied to the 160 or 10 treatment levels where very few hives survived.

99355

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

Figure A-7. Estimates and 90% CIs for larvae with thresholds of statistical (red) significance shown. The
green line shows 10% difference from control.

100
356

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

Figure A-8. Difference from control for all treatments and apiaries at CCA6 for larvae. Apiaries shown
above the zero line had better control outcomes in comparison to the treatment.

Pupae (Capped brood)
Table A-6 and Figure A-9 below show the effects on pupae (capped brood) across CCAs andtreatment groups. Compared to the control, a significant reduction (p<0.05) in the number of pupal
cells was observed at the three highest treatment groups (40, 80 and 160 µg/L) which persisted
over multiple CCAs (16.3—47.0%, 30.9—87.7% and 46.9—99.6% reduction in the 40, 80 and
160 µg/L treatment groups, respectively during CCAs 4-6 and 98.2% reduction at CCA7 in the
160 µg/L treatment group). No significant reduction (p>0.1) in the number of pupae was observedat the lowest two treatment groups (10 and 20 µg/L) during any of the CCAs with the exception
101

357

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Documentof CCA5 where the number of pupae was significantly reduced by 13.1% in the 20 µg/L treatmentgroup (p=0.039) and by 19.9% in the 10 µg/L treatment group (p=0.05). As the effect was only
observed during a single CCA immediately after the exposure period a potential transient effect at
the lowest two doses is indicated.
Figure A-9 shows a clear dose response (increase in the reduction from the control as the dose
increases), at the first CCA during exposure (CCA4). In CCA5 and CCA6, the 10 and 20 µg/L
treatment groups showed about the same level of effect with the 10 µg/L treatment groupoverlapping the 20 µg/L treatment group (16.8-19.9% and 11.2-13.2% reduction from the control
in the 10 and 20 µg/L treatment groups, respectively). The overlap in dose-response at the lower
doses is not unexpected since the dose levels are similar and measured exposures indicate overlap
in exposure among individual hives, particularly at the lower two doses. No clear dose response
was observed at CCA7 for all doses when all hives were in decline. In general, the width of theconfidence intervals increased with increasing CCAs. As seen in Figure A-10, pupal cells at CCA6
were reduced from the control in 75% (7/12), 42% (5/12), 60% (6/10), 92% (11/12) and 100%
(12/12) of apiaries in the 10, 20, 40, 80 and 160 µg /L dose groups, respectively.
Significant reductions in the number of pupae were found at the highest three treatment levels (40,80 and 160 µg/L) that were apparent from the beginning of exposure (CCA4) and persisted
throughout the study until hives would begin to naturally decline before overwintering (CCA7).
While treatment means were significantly reduced from the control at the lowest two doses (10and 20 µg/L) at a single CCA immediately after exposure (CCA5), these effects were consideredtransient. This is because effects at 10 and 20 µg/L were isolated to CCA5 with levels returning tothose similar to the control in subsequent CCAs. This finding supports the use of 20 µg/L as the
NOAEC as determined through the primary analysis for total life at CCA6. The early onset andpersistence of significant effects at the highest three treatment groups (40, 80 and 160 µg/L)
supports the use of 40 µg/L as the LOAEC.
Table A-6. Estimated percent reduction from control for number of pupae.
Estimated reduction from control (%)
Testconcentration (P value)(µg/L)
CCA4
CCA5
CCA6
CCA7
2.0
19.9
16.8
37.2
10
(0.38)(0.05*)(0.082*/0.142) (0.053*/0.357)
6.3
13.1
11.2
10.7
20
(0.144)(0.039**)(0.142)(0.357)
16.3
47.0
30.9
21.9
40
(0.018**) (0**)(0.034**)(0.234)
30.9
87.7
67.7
20.2
80
(0.001**) (0**)(0**)(0.262)
46.9
99.6
97.5
98.2
160
(0**)
(0**)
(0**)
(0.001**)

CCA8
NA

CCA9
NA

-93.2
(0.953)-58.6(0.995)-9.5(0.549)NA-38.8(0.946)-31.4(0.967)52.7(0.091*)NA

Note: Negative value indicates increased number of individuals in comparison to control.
*0.05<P<0.1, **P<0.05
NA’ indicates there was not enough data to do the test (n = number of Apiaries is small) and ‘0’ means rounded to 0
Where two p values are listed, the first is the non-adjusted p value, the second is the p value adjusted for the step-down approach.
The step-down adjustment was shown only if it changed the significance level.
At CCA8 and CCA9, the step-down approach was not applied to the 160 or 10 treatment levels where very few hives survived.

102
358

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

Figure A-9. Estimates and 90% CIs for pupae with thresholds of statistical (red) significance shown. The
green line shows 10% difference from control.

103
359

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

Figure A-10. Difference from control for all treatments and apiaries at CCA6 for pupae. Apiaries shown
above the zero line had better control outcomes in comparison to the treatment.

104
360

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Colony Condition Assessments - Food Stores
PollenTable A-7 and Figure A-11 below show the effects on pollen storage across CCAs and treatmentgroups. Pollen stores were significantly reduced (p<0.05) from the control at the highest twotreatment groups (80 and 160 µg/L) from CCA4 through to CCA7 (47.5-99.4% and 94.3- 100%
reduction at 80 and 160 µg/L, respectively). Pollen stores were also significantly reduced in the 40µg/L treatment groups during the exposure period at CCA4 (21.9% reduction, p= 0.041) and CCA5
(55.3% reduction, p= 0.003) but not during subsequent CCAs after the exposure period (7.6 and10.8% reduction at CCA6 and CCA7, respectively, p>0.1). No significant reduction in pollen
stores was observed at the two lowest treatment groups (10 and 20 µg/L) from CCA4 through to
CCA7 except at CCA5 where pollen stores were significantly reduced from the control by 30.2%(p=0.032) in the 10 µg/L treatment group and 27.7% (p=0.019) in the 20 µg/L treatment group andat CCA7 where pollen stores were significantly reduced from the control by 59.8% (p=0.017).
Figure A-11 shows a general dose response where the reduction in pollen stores from the control
increases with increasing dose, during the exposure period at CCA4 and CCA5. There was no clear
dose response observed after the exposure period at CCA6 and CCA7 for the three lowest doses(10, 20 and 40 µg/L). For the 10 µg/L treatment group, in particular, there was a significant
reduction in pollen stores at CCA7 but no significant reductions at the 20 and 40 µg/L treatmentgroup for the same time period. Therefore the biological significance of this finding is considered
to be low. In general, the width of the confidence intervals increased with increasing CCAs. As
seen in Figure A-12, pollen stores at CCA6 were reduced from the control in 58% (7/12), 50%(6/12), 70% (7/10), 75% (9/12) and 100% (12/12) of apiaries in the 10, 20, 40, 80 and 160 µg /Ldose groups, respectively.
Treatment means were significantly reduced from the control at the lowest three doses during
and immediately after the exposure period at CCA4 (40 µg/L) and CCA5 (10, 20 and 40 µg/L)
but not in subsequent CCAs after the exposure except at CCA7 (10 µg/L) and were significantly
reduced from the control at the two highest treatment groups (80 and 160 µg/L) at all CCAs.
While the width of the confidence intervals increased with increasing CCAs, the observed
reversion back to control levels at the three lowest treatment groups during subsequent CCAs
indicates a potential transient effect on pollen storage at ≤ 40 µg/L and supports the use of 40µg/L as the NOAEC and 80 µg/L as the LOAEC. These endpoints are less sensitive than theendpoints determined through the primary analysis for total life at CCA6 (NOAEC of 20 µg/L,
LOAEC of 40 µg/L).
Table A-7. Estimated percent reduction from control for pollen store.
Estimated reduction from control (%)
Testconcentration (P value)(µg/L)
CCA4
CCA5
CCA6
CCA7
7.2
30.2
23.1
59.8
10
(0.305)(0.032**)(0.202)(0.017**/0.36)
10.9
27.7
-11.2
23.4
20
(0.198)(0.019**)(0.682)(0.155)
40
21.9
55.3
7.6
10.8

CCA8
NA

CCA9
NA

-69.6
(0.907)-30.3

0.7(0.482)
-18.9
105

361

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Estimated reduction from control (%)
Testconcentration (P value)(µg/L)
CCA4
CCA5
CCA6
(0.041**)(0.003**)(0.385)
47.5
99.4
60.3
80
(0**)
(0**)
(0.009**)
94.3
100
95.9
160
(0**)
(0**)
(0.001**)

CCA7(0.36)
73.1(0.001**)
95.7
(0**)

CCA8
(0.688)-66.5(0.951)NA

CCA9(0.189)-62.6(0.934)NA

Note: Negative value indicates increased pollen stores in comparison to control.
*0.05<P<0.1, **P<0.05
NA’ indicates there was not enough data to do the test (n = number of Apiaries is small) and ‘0’ means rounded to 0
Where two p values are listed, the first is the non-adjusted p value, the second is the p value adjusted for the step-down approach.
The step-down adjustment was shown only if it changed the significance level.
At CCA8 and CCA9, the step-down approach was not applied to the 160 or 10 treatment levels where very few hives survived.

Figure A-11. Estimates and 90% CIs for pollen stores with thresholds of statistical (red) significanceshown. The green line shows 10% difference from control.

106
362

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

Figure A-12. Difference from control for all treatments and apiaries at CCA6 for pollen stores. Apiaries
shown above the zero line had better control outcomes in comparison to the treatment.

107
363

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Honey
Table A-8 and Figure A-13 below show the effects on honey storage across CCAs andtreatment groups. No significant adverse effects were observed on honey storage at any test
concentration. Honey stores increased in all treatment groups over all CCAs compared to control
hives. In general honey stores increased at each successive CCA for all treatment groups except
at CCA7 where honey stores were lower than the proceeding CCAs for the two lowest treatmentgroups (10 and 20 µg/L). Figure A-13 shows a dose response at CCA4 through to CCA7, withhoney stores increasing with increasing dose. In general, the width of the confidence intervals
increased with increasing CCAs. As seen in Figure A-14, honey stores at CCA6 were reduced
from the control in 33% (4/12), 50% (6/12), 60% (6/10), 100% (12/12) and 100% (12/12) ofapiaries in the 10, 20, 40, 80 and 160 µg /L dose groups, respectively.
Table A-8. Estimated percent reduction from control for honey store.
Estimated reduction from control (%)
Testconcentration (P value)(µg/L)
CCA4
CCA5
CCA6
CCA7
-5.9
-6.6
-11.5
-2.8
10
(0.854)(0.927)(0.795)(0.562)
-4.8
-10.6
-15.0
-2.8
20
(0.833)(0.985)(0.919)(0.573)
-10.7
-20.6
-44.7
-51.4
40
(0.966)(0.999)(0.998)(0.999)
-12.9
-26.9
-68.5
-80.2
80
(0.995)
(1.0)
(1.0)
(1.0)
-17.8
-19.1
-86.5
-100
160
(0.999)(0.999)
(1.0)
(1.0)

CCA8
NA

CCA9
NA

-64.8
(0.973)-10.9(0.633)-2.3(0.522)NA-61.5(0.935)-3.2(0.583)-4.3(0.401)NA

Note: Negative value indicates increased honey stores in comparison to control.
*0.05<P<0.1, **P<0.05
NA’ indicates there was not enough data to do the test (n = number of Apiaries is small) and ‘0’ means rounded to 0
Where two p values are listed, the first is the non-adjusted p value, the second is the p value adjusted for the step-down approach.
The step-down adjustment was shown only if it changed the significance level.
At CCA8 and CCA9, the step-down approach was not applied to the 160 or 10 treatment levels where very few hives survived.

108
364

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

Figure A-13. Estimates and 90% CIs for larvae with thresholds of statistical (red) significance shown.
The green line shows 10% difference from control.

109
365

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

Figure A-14. Difference from control for all treatments and apiaries at CCA6 for honey stores. Apiaries
shown above the zero line had better control outcomes in comparison to the treatment.

Table A-9. Summary of statistically significant (at 0.05 and 0.10) observed effects at eachtreatment level (Note: Values reported in the table are the % reduction compared to control, basedon model estimated raw numbers corrected for baseline measurements). Where two p values arelisted, the first is the non-adjusted p value, the second is the p value adjusted for the step-down
approach. The step-down adjustment was shown only if it changed the significance level.
Treatment
Observations(µg/l)• Decreased total number of individuals in hive at CCA5 (14.5%, p=0.043/0.079)
10• Decreased number of adults at CCA6 (19.6%, p=0.014)• Decreased number of eggs at CCA4 (30.1%, p=0.016) and at CCA5 (35.1%, p=0.018)• Decreased number of larvae at CCA4 (15.0%, p=0.053)
110
366

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

20

40

80

160

• Decreased number of pupae at CCA5 (19.9%, p=0.05)• Decreased pollen stores at CCA5 (30.2%, p=0.032)• The potential colony effects were not consistently sustained through to when colonies were innatural decline. Most endpoint responses were not significantly different from the control(p>0.1)• At CCA8 and CCA9, 75% of hives (9 out of 12 colonies) did not survive overwintering
compared to 65% of control hives• Decreased total number of individuals in hive at CCA4 (6.7%, p=0.074) and CCA5 (10.1%(p=0.079)• Decreased number of adults at CCA6 (17%, p=0.037)• Decreased number of eggs at CCA4 (27.1%, p=0.04) and a t CCA5 (37.3%, p=0.009)• Decreased number of larvae at CCA4 (12.2%, p=0.067)• Decreased number of pupae at CCA5 (13.1%, p=0.039)• Decreased pollen store at CCA5 (27.7%, p=0.019)• The potential colony effects were not consistently sustained through to when colonies were innatural decline. Most endpoint responses were not significantly different from the control(p>0.1)• At CCA8 and CCA9, 33% of hives (4 out of 12 colonies) did not survive overwintering
compared to 65% of control hives• Decreased total number of individuals in hive at CCA4 (15.2%, p=0.008), CCA5 (33.6%,
p=0.002) and CCA6 (30.5%, p=0.029)• Decreased number of adults at CCA5 (23.1%, p=0.011) and at CCA6 (29.9%, p=0.012)• Decreased number of eggs at CCA4 (53.4%, p<<0.05) and at CCA5 (41.7%, p=0.028)• Decreased number of larvae at CCA4 (21.4%, p=0.017/0.093)• Decreased number of pupae at CCA4 (16.3%, p=0.018) and a CCA5 (47.0%, p<<0.05) andCCA6 (30.9% p=0.034)• Decreased pollen store at CCA4 (21.9%, p=0.041) and at CCA5 (55.3%, p=0.003)• The potential colony effects were more consistently sustained across multiple CCAs prior towhen colonies were in natural decline (CCA7).
• At CCA8 and CCA9, 50% of hives (5 out of 10 colonies) did not survive overwintering
compared to 65% of control hives• Decreased total number of individuals in hive at CCA4-CCA7 (22.4-70.2%, p<<0.05) andCCA9 (56%, p=0.071)• Decreased number of adults CCA4-CCA7 (13.9-64.1%, p<0.05)• Decreased number of eggs at CCA4-CCA7 (36.3-69.6% p<0.05)• Decreased number of larvae at CCA4 (12.3%, p=0.093), CCA5-CCA6 (47.9-84.9%, p<0.05)
and at CCA7 (55.2%, p=0.069)• Decreased number of pupae at CCA4-CCA6 (30.9-87.7%, p<0.05)• Decreased pollen store at CCA4-CCA7 (47.5-99.4%, p<<0.05)• The potential colony effects were consistently sustained across multiple CCAs through to whencolonies were in natural decline (CCA7).
• At CCA8 and CCA9, 17% of hives (2 out of 12 colonies) did not survive overwintering
compared to 65% of control hives• Decreased total number of individuals in hive at CCA4-CCA7 (46.9-96.5%, p<<0.05)• Decreased number of adults CCA4-CCA7 (32.3-98.4%, p<<0.05)• Decreased number of eggs CCA4-CCA7 (31.2-93.9%, p<0.05)• Decreased number of larvae CCA4-CCA7 (87.8-98.8%, p<<0.05)• Decreased number of pupae CCA4-CCA7 (46.9-99.6%, p<0.05)• Decreased pollen store CCA4-CCA7 (94.3-100%, p<<0.05)• The potential colony effects were consistently sustained across multiple CCAs through to whencolonies were in natural decline (CCA7).
• At CCA8 and CCA9, 100% of hives (11 out of 11 colonies) did not survive overwintering
compared to 65% of control hives
111367

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
OVERALL
Potential colony effects were more consistently sustained across multiple CCAs prior to
ENDPOINT when colonies were in natural decline (CCA7) at ≥40 µg/L. The high overwintering
mortality in the control hives limits the ability to fully assess recovery in the treatment
hives, in particular for the lowest two test doses. In the interim the overall quantitative
NOAEC and LOAEC for this study are considered to be:• NOAEC: 20 µg/L sucrose solution• LOAEC: 40 µg/L sucrose solution

The LOAEC is based on significant effects on the number of adults, pupae and total
number of individuals.

Graphical representation of all parameters at CCA6

112
368

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

113
369

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

114
370

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

115
371

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

116
372

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

Appendix B. Timeline of addition and removal of supers during the clothianidin field trial
Hive

Apiary

Tmt

A3
A5
B2
B3
C1
C3
D4
D8
E1
E4
F2
F5
G2
G8
H1
H6
I6
I7
J7
J8
K5
K7
L4
L6
A7
B8
C5
D3
E2
F6
G6
H2
I1
J4
K4
L1
A1
B5
C4
D6

A
A
B
B
C
C
D
D
E
E
F
F
G
G
H
H
I
I
J
J
K
K
L
L
A
B
C
D
E
F
G
H
I
J
K
L
A
B
C
D

C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
T1
T1
T1
T1
T1
T1
T1
T1
T1
T1
T1
T1
T2
T2
T2
T2

CCA1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
.
1
1
1
1
1
1
1
.
.
1
1
1
1
1
1
1
1

CCA2
2
3
.
2
2
2
2
2
2
2
1
2
2
2
1
2
2
1
1
1
1
1
.
2
.
2
2
2
1
2
1
2
1
1
1
1
2
2
2
2

Number of hive boxes at each CCA measurement
CCA3
CCA4
CCA5
CCA6
CCA7
2
2
2
2
2
3
3
3
3
3
2
2
2
2
2
2
2
3
3
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
3
2
2
2
2
3
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
1
.
.
.
.
2
2
2
2
2
1
2
2
2
2
2
2
2
2
2
1
1
2
2
2
1
2
2
2
2
2
2
2
2
2
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
2

CCA8
.
.
.
.
2
.
.
2
.
2
.
.
.
2
.
2
2
.
.
2
2
.
.
.
.
2
.
.
.
2
.
2
.
.
.
.
2
.
2
.
117

373

CCA9
.
.
.
.
2
.
.
2
.
2
.
.
.
2
.
2
2
.
.
2
2
.
.
.
.
2
.
.
.
2
.
2
.
.
.
.
2
.
2
.

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
E7
F7
G3
H8
I4
J5
K3
L2
A4
B1
C2
D7
E8
F3
G5
H7
I8
J6
K2
L5
A2
B6
C8
D5
E6
F1
G4
H4
I5
J3
K1
L7
A8
B4
C7
D1
E5
F8
G1
H3
I2
J2
K6
L3

E
F
G
H
I
J
K
L
A
B
C
D
E
F
G
H
I
J
K
L
A
B
C
D
E
F
G
H
I
J
K
L
A
B
C
D
E
F
G
H
I
J
K
L

T2
T2
T2
T2
T2
T2
T2
T2
T3
T3
T3
T3
T3
T3
T3
T3
T3
T3
T3
T3
T4
T4
T4
T4
T4
T4
T4
T4
T4
T4
T4
T4
T5
T5
T5
T5
T5
T5
T5
T5
T5
T5
T5
T5

1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
.
1
1
1
1
1
1
1
2
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1

2
2
1
.
2
.
2
2
2
2
1
2
2
2
2
2
2
.
2
1
2
2
2
2
2
2
2
2
1
1
2
2
2
2
2
2
1
1
2
1
2
2
1
1

2
2
1
1
2
1
2
1
2
2
2
2
2
2
2
2
2
1
2
1
2
2
2
3
2
2
2
2
2
2
2
2
2
2
2
2
2
1
2
2
2
2
1
2

2
2
2
2
2
2
2
1
2
2
2
2
2
.
2
2
2
1
2
1
2
2
2
3
2
2
2
2
2
2
2
2
2
2
2
2
2
1
2
2
2
2
1
2

2
2
2
2
2
2
2
1
2
2
2
2
2
.
2
2
2
1
2
1
2
2
2
3
2
2
2
2
2
2
2
2
2
2
2
2
1
2
2
2
2
2
1
2

2
2
2
2
2
2
2
1
2
2
.
2
2
.
2
2
2
1
2
1
2
2
2
3
2
2
2
2
2
2
2
1
2
2
2
2
1
2
2
2
2
2
1
1

2
2
2
2
2
2
2
1
2
2
.
2
2
.
2
2
2
1
1
1
2
2
2
2
2
2
2
2
2
2
2
1
2
2
2
2
1
1
2
2
2
2
1
1

2
.
2
.
2
2
2
1
2
2
.
2
.
.
.
.
2
1
.
.
2
2
2
2
2
2
2
2
.
2
2
.
.
.
.
.
.
.
.
.
.
.
.
.

118
374

2
.
2
.
2
2
2
1
2
2
.
2
.
.
.
.
2
1
.
.
2
2
2
2
2
2
2
2
.
2
2
.
.
.
.
.
.
.
.
.
.
.
.
.

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

Appendix C. Summary Statistics for Each Response Variable for All
Clothianidin Treatment Levels Across CCAs 3—7
Table C-1. Summary Statistics for Adults
CCA
3
4
5
6
7
3
4
5
6
7
3
4
5
6
7
3
4
5
6
7
3
4
5
6
7
3
4
5
6
7

TreatmentGroup µg/L

0
10
20
40
80
160
0
10
20
40
80
160
0
10
20
40
80
160
0
10
20
40
80
160
0
10
20
40
80
160

Mean

SE

DF

Lower Bound

Upper Bound

15.0143
14.3501
15.110315.321616.5143
15.6587
17.1515
17.329717.0782
17.5195
15.6988
11.9543
19.2155
17.480718.9505
14.6602
12.09515.979718.2291
14.229315.2168
12.6790
7.92272.244912.5891
10.2127
11.823110.9166
6.59870.4782

1.03931.45001.45001.45001.45001.45000.80781.0991
1.09911.1361
1.0991
1.09910.98601.35151.3515
1.3972
1.35151.35151.05611.45031.4503
1.54741.45031.45030.99901.36991.36991.4614
1.36991.3699

70.4
72.8
72.8
72.8
72.8
72.8
66.9
79.6
79.6
81.7
79.6
79.6
74.5
80.8
80.8
82.8
80.8
80.8
70.6
78.7
78.7
83
78.7
78.7
68.7
77.7
77.7
82
77.7
77.7

12.9418
11.460212.220412.431713.624412.7688
15.5390
15.1423
14.890715.259413.51149.766817.251114.7916
16.2613
11.88109.40593.290516.1232
11.3423
12.32979.60125.03570
10.59597.48549.09578.00953.8713017.0869
17.2400
18.0001
18.2115
19.4041
18.5485
18.7640
19.5172
19.2656
19.7796
17.8863
14.1417
21.1800
20.1699
21.639717.4393
14.78428.668820.3350
17.1163
18.1038
15.7568
10.8097
5.131914.582212.9401
14.5505
13.8238
9.32613.2056

Table C-2. Summary statistics for eggs
CCA

3
3
3
3
3

TreatmentGroup µg/L

0
10
20
40
80

Mean

SE

DF

Lower Bound

Upper Bound

6.39366.81546.4351
7.14635.85600.62020.85410.85410.85410.8541

83.1
76.5
76.5
76.5
76.5

5.1601
5.11454.73425.4453
4.1551

7.62728.51638.13608.84727.5569119375

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
CCA

3
4
4
4
4
4
4
5
5
5
5
5
5
6
6
6
6
6
6
7
7
7
7
7
7

TreatmentGroup µg/L

160
0
10
20
40
80
160
0
10
20
40
80
160
0
10
20
40
80
160
0
10
20
40
80
160

Mean

SE

DF

Lower Bound

Upper Bound

6.20344.5425
3.1762
3.34172.36412.8123
3.09343.96242.69642.49802.62001.24080.28143.03842.94462.58062.64601.53850.51311.03491.02580.76100.77010.29780.04967

0.85410.50330.67120.67120.69620.67120.6712
0.46610.61720.61720.63990.61720.61720.4526
0.5976
0.5976
0.64230.5976
0.5976
0.26940.32020.3202
0.33830.32020.3202

76.5
80.6
77.8
77.8
78.5
77.8
77.8
85.8
89.6
89.6
90.4
89.6
89.6
81.3
87.2
87.2
89.3
87.2
87.2
17.7
30.7
30.7
34.8
30.7
30.7

4.5025
3.54091.83992.00540.9782
1.47611.75723.03591.4702
1.27181.34890.0146102.13791.75681.39281.36990.350700.46820.37240.10770.08317007.90435.54404.51244.67793.75004.14864.42964.88893.92263.7242
3.89122.46711.5076
3.93894.13243.76843.92212.72631.70091.6016
1.67911.41431.4571
0.95120.7030

Table C-3. Summary statistics for larval (open) cells
CCA

3
3
3
3
3
3
4
4
4
4
4
4
5
5
5

TreatmentGroup µg/L

0
10
20
40
80
160
0
10
20
40
80
160
0
10
20

Mean

SE

DF

Lower Bound

Upper Bound

9.80129.89228.88339.2967
11.2982
9.46218.79097.49377.67556.8202
7.98991.15809.85099.69358.41990.68550.95850.95850.95850.95850.95850.63290.86510.86510.90230.86510.86510.71990.98700.9870

75.8
67.1
67.1
67.1
67.1
67.1
78.2
72
72
71.9
72
72
77.6
79.9
79.9

8.43597.97916.97027.38369.3851
7.54907.53105.76915.95095.02146.265308.41767.72936.4557

11.1666
11.8053
10.7963
11.2098
13.2113
11.3752
10.0507
9.21829.40018.61909.71452.8826
11.2841
11.6577
10.3842120

376

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
CCA

5
5
5
6
6
6
6
6
6
7
7
7
7
7
7

TreatmentGroup µg/L

40
80
160
0
10
20
40
80
160
0
10
20
40
80
160

Mean

SE

DF

Lower Bound

Upper Bound

7.90441.8032
1.94E-14
8.76309.19758.40336.65874.49970.59562.3045
1.53871.45601.54471.24080.033171.0296
0.98700.98700.74591.02351.0235
1.11691.02351.02350.42870.57680.57680.62830.57680.5768

80
79.9
79.9
79.1
79.5
79.5
79.6
79.5
79.5
33.4
47.1
47.1
48.8
47.1
47.1

5.8555007.27837.16056.36634.43582.462701.43270.37830.29570.28180.0804209.95333.7674
1.9642
10.2478
11.2345
10.44038.88166.53672.6326
3.17622.69902.61632.80752.40111.1935

Table C-4. Summary statistics for pupal (capped) cells
CCA

3
3
3
3
3
3
4
4
4
4
4
4
5
5
5
5
5
5
6
6
6
6
6
6
7
7
7

TreatmentGroup µg/L

0
10
20
40
80
160
0
10
20
40
80
160
0
10
20
40
80
160
0
10
20
40
80
160
0
10
20

Mean

SE

DF

Lower Bound

Upper Bound

17.294618.7254
18.642717.1043
18.6263
17.4516
19.267119.1887
18.5104
16.1024
14.2426
10.3553
19.5472
16.194417.650310.33993.2423-955E-16
19.5516
17.534418.8246
12.37677.95680.3642
4.23693.09354.03630.7332
0.97550.97550.97550.97550.97550.88111.17301.17301.21951.17301.17301.15891.56971.56971.63391.56971.56971.25221.70181.70181.84821.70181.70180.7993
1.05451.0545

63.2
72.5
72.5
72.5
72.5
72.5
64.8
78
78
79.2
78
78
76
80.1
80.1
80.6
80.1
80.1
80
81.9
81.9
82.7
81.9
81.9
40.6
52.4
52.4

15.8295
16.7811
16.6983
15.1600
16.681915.5073
17.5072
16.8535
16.1752
13.6750
11.9074
8.0201
17.2392
13.070714.5266
7.08870.1186017.059614.1489
15.43908.70044.571302.62200.97791.920718.7597
20.6697
20.5870
19.0487
20.5706
19.3959
21.0269
21.5239
20.8456
18.5297
16.5778
12.6905
21.8553
19.3181
20.7741
13.59116.3661
3.123722.0435
20.9200
22.2101
16.0530
11.3424
3.74975.8517
5.2091
6.1520121377

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
CCA

7
7
7

TreatmentGroup µg/L

40
80
160

Mean

SE

DF

Lower Bound

Upper Bound

3.40653.90400.11581.14051.05451.0545

55
52.4
52.4

1.12101.788405.69206.01962.2315

Table C-5. Summary statistics for pollen cells
CCA

3
3
3
3
3
3
4
4
4
4
4
4
5
5
5
5
5
5
6
6
6
6
6
6
7
7
7
7
7
7

TreatmentGroup µg/L

0
10
20
40
80
160
0
10
20
40
80
160
0
10
20
40
80
160
0
10
20
40
80
160
0
10
20
40
80
160

Mean

SE

DF

Lower Bound

Upper Bound

5.69065.62435.16125.04555.82295.5253
12.5242
11.662210.50438.8542
6.6666
0.41368.47475.70725.40942.99180.1655
8.35E-15
6.98305.16136.74924.60422.76270.01658
5.33612.01833.40784.3212
1.5551-957E-16

0.47940.67710.67710.67710.67710.6771
1.0534
1.46271.46271.51801.46271.46270.64830.89990.89990.93390.89990.89990.95291.32311.32311.42381.32311.32310.72061.00031.0003
1.07651.00031.0003

69.7
77.7
77.7
77.7
77.7
77.7
77.6
77.7
77.7
78.9
77.7
77.7
74.6
79.6
79.6
81
79.6
79.6
81.8
81.2
81.2
84.2
81.2
81.2
77.2
77.4
77.4
80.3
77.4
77.4

4.73444.27623.8131
3.69734.4747
4.177110.42688.74997.59205.83273.754407.18303.9162
3.61851.1337005.08732.52894.11681.77290.130303.90110.026591.4161
2.1791006.64686.97256.50946.3937
7.1711
6.873414.6217
14.5744
13.4165
11.8758
9.57883.32589.76637.49817.20034.84991.95641.79098.87877.79369.38157.43555.39502.64896.77104.0101
5.39966.4634
3.54681.9917

Table C-6. Summary statistics for nectar (honey) cells
CCA

3
3
3
3
3

TreatmentGroup µg/L

0
10
20
40
80

Mean

SE

DF

Lower Bound

Upper Bound

45.2665
38.178547.193742.859747.44174.6463
6.1934
6.1934
6.1934
6.1934

54.7
77.5
77.5
77.5
77.5

35.9541
25.847134.862330.528335.1104

54.5790
50.509959.5250
55.191059.7731122378

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
3
4
4
4
4
4
4
5
5
5
5
5
5
6
6
6
6
6
6
7
7
7
7
7
7

160
0
10
20
40
80
160
0
10
20
40
80
160
0
10
20
40
80
160
0
10
20
40
80
160

46.7966
53.2940
51.1635
57.1352
57.3690
61.700763.950357.181456.5398
64.6782
67.040174.1566
69.3595
32.7332
32.1572
39.0882
45.6546
57.3998
63.371224.2344
21.1900
25.871235.5606
45.192251.8913

6.1934
3.95805.1172
5.11725.1836
5.1172
5.11724.07225.29115.29115.36035.29115.2911
4.11715.35915.35915.48865.35915.3591
3.76484.82124.82124.93424.82124.8212

77.5
41.4
69.7
69.7
72.4
69.7
69.7
47.9
74.1
74.1
76.9
74.1
74.1
40
65.7
65.7
70.3
65.7
65.7
28.5
50.1
50.1
53.4
50.1
50.1

34.4652
45.3028
40.9567
46.928547.0366
51.4940
53.743648.9933
45.9975
54.1358
56.3661
63.614258.8171
24.412221.4566
28.3876
34.7086
46.699352.6706
16.528111.5069
16.1882
25.665335.509142.2083

59.127961.2851
61.370367.342067.701471.9075
74.157165.3695
67.082275.2205
77.7140
84.6990
79.901941.054342.8577
49.7887
56.6006
68.1004
74.071731.9406
30.873135.554345.4559
54.8752
61.5744

Table C-7. Summary statistics for total live individuals
CCA

3
3
3
3
3
3
4
4
4
4
4
4
5
5
5
5
5
5
6

TreatmentGroup µg/L

0
10
20
40
80
160
0
10
20
40
80
160
0
10
20
40
80
160
0

Mean

SE

DF

Lower Bound

Upper Bound

48.503849.783149.071348.868852.294748.7758
49.7408
47.1883
46.6058
42.7860
40.7437
26.561052.561746.0651
47.5188
35.502118.3814
6.261149.5587

2.34303.26263.26263.26263.26263.2626
1.72872.33552.33552.41792.33552.33552.3162
3.1694
3.16943.28283.1694
3.16942.6826

73.1
68.3
68.3
68.3
68.3
68.3
67.9
78.6
78.6
80.2
78.6
78.6
82.3
85.4
85.4
87.1
85.4
85.4
83

43.834343.2732
42.561442.3590
45.784842.265946.291242.5391
41.9566
37.9745
36.0945
21.911947.954239.7639
41.2176
28.9774
12.0802044.2232

53.1732
56.292955.581155.3787
58.804555.2856
53.1904
51.8374
51.2549
47.5976
45.3928
31.2101
57.1692
52.3663
53.819942.0269
24.6826
12.562354.8942123

379

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
6
6
6
6
6
7
7
7
7
7
7

10
20
40
80
160
0
10
20
40
80
160

43.9058
45.0253
34.4159
21.9177
3.717820.1691
15.8707
18.0764
16.5686
12.04130.67683.68543.6854
3.94983.68543.68541.74972.36562.36562.53162.36562.3656

84
84
87.4
84
84
49.9
67.1
67.1
70.4
67.1
67.1

36.5770
37.6964
26.5658
14.5888016.6546
11.1492
13.3549
11.5200
7.3197051.2347
52.354142.2660
29.2465
11.0466
23.6836
20.592222.7979
21.6171
16.76285.3983

Table C-8. Summary statistics for Total Brood
CCA

3
3
3
3
3
3
4
4
4
4
4
4
5
5
5
5
5
5
6
6
6
6
6
6
7
7
7
7
7
7

TreatmentGroup µg/L

0
10
20
40
80
160
0
10
20
40
80
160
0
10
20
40
80
160
0
10
20
40
80
160
0
10
20
40
80
160

Mean

SE

DF

Lower Bound

Upper Bound

33.489435.433033.961033.5473
35.7804
33.1171
32.610929.8585
29.5276
25.2779
25.0448
14.6067
33.3740
28.5843
28.5682
20.84446.28630.281431.3669
29.6765
29.8085
21.6505
13.9950
1.47287.59225.65796.25335.66465.4426
0.19871.4304
1.92501.92501.92501.92501.92501.35141.77441.7744
1.84271.77441.77441.80642.43082.43082.52842.43082.43081.98322.68242.68242.90872.68242.6824
1.31831.72591.72591.86301.72591.7259

68.5
70.4
70.4
70.4
70.4
70.4
57.3
74.4
74.4
75.9
74.4
74.4
80.1
85.5
85.5
86
85.5
85.5
81.4
84.5
84.5
85.2
84.5
84.5
41.4
55.4
55.4
58.1
55.4
55.4

30.6356
31.5940
30.122029.7083
31.9415
29.278129.9050
26.3232
25.9923
21.6077
21.5095
11.071429.7792
23.751723.7357
15.8180
1.4537027.421324.342724.4747
15.8673
8.661204.9306
2.19972.79511.9356
1.9843036.3433
39.272037.8000
37.3862
39.6194
36.9560
35.3168
33.3938
33.062928.9480
28.580218.142136.9688
33.4169
33.4008
25.870711.1189
5.114035.312435.010335.142327.4336
19.32886.8066
10.2538
9.11629.71169.39378.90083.6569

Table C-9. Summary statistics for Total Food
124
380

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
CCA

3
3
3
3
3
3
4
4
4
4
4
4
5
5
5
5
5
5
6
6
6
6
6
6
7
7
7
7
7
7

TreatmentGroup µg/L

0
10
20
40
80
160
0
10
20
40
80
160
0
10
20
40
80
160
0
10
20
40
80
160
0
10
20
40
80
160

Mean

SE

DF

Lower Bound

Upper Bound

50.957143.802852.354947.9052
53.2647
52.321865.743962.8257
67.6395
66.1805
68.367364.363965.653762.2470
70.0876
70.035974.322169.3595
39.698637.3184
45.837350.2935
60.1625
63.3877
29.5429
23.208329.2791
39.903746.7472
51.89134.87836.52206.52206.52206.52206.52204.43035.80925.80925.88985.80925.80924.23535.51525.51525.59085.51525.51524.40055.76445.76445.91185.76445.76444.0102
5.17295.17295.30155.17295.1729

55.6
77.1
77.1
77.1
77.1
77.1
49.1
73.2
73.2
76
73.2
73.2
46.9
72.5
72.5
75.2
72.5
72.5
43.4
68
68
72.8
68
68
31.5
54.2
54.2
57.9
54.2
54.2

41.1832
30.8162
39.3683
34.918540.2780
39.3352
56.841151.2485
56.062354.450156.790252.7867
57.1330
51.2538
59.094458.899163.3289
58.3663
30.8264
25.8156
34.3346
38.5108
48.6597
51.8850
21.3694
12.8382
18.909029.291136.3771
41.521260.731156.7895
65.341660.8918
66.251365.308574.6466
74.4028
79.2167
77.910979.944575.941174.174473.240281.080781.172885.315280.3527
48.5708
48.821257.3401
62.0763
71.6653
74.890537.7165
33.5785
39.6492
50.516457.1174
62.2615125381

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
State of California

Department of Pesticide Regulation

EVALUATION REPORT - Clothianidin Nectar Colony Feeding Study:
Repeat Study Conducted in 2016-2017
John Troiano, Research Scientist III
June, 2018

A review of: - Louque, J. (2017). Colony feeding study evaluating the chronic effects ofchothianidin-fortified sugar diet on honey bee (Apis mellifera) colony performance under free
foraging conditions. Unpublished study prepared by Smithers Viscient. 609p., Laboratory Report
Number 13798.4162. MRID 49836101.

Introduction
A colony feeding study was conducted to determine the effects of graded levels of clothianidinon the health of honey bee hives where doses mimicked exposure from foraging on nectar.
Clothianidin was dosed directly to hives, supplied in a sugar solution that mimicked a nectar
source for food supply. Hive health was determined by Colony Condition Assessments (CCAs)
where measurements were made over time on the number of individuals in each bee life stage inthe hive, the storage of honey and pollen food supplies in the hives, and the weight of hives. This
study, conducted in 2016-2017, was a repeat of a study conducted in 2014-2015 (Louque, J.,2016). The second study was conducted in the same area as the first study where locations ofapiary sites were distributed throughout a forested area of North Carolina. Not all sites were in
the exact location as in the previous study. The distance between each apiary site wasapproximately 3 miles apart. The majority of land near the apiaries was non-intensively managed
pasture and forest with low potential exposure of bees to pesticides applied for agricultural
purposes.
Measurements made over time were indicated by sequential numbering of the colony conditionassessments (CCAs), which were conducted at approximately monthly intervals. CCAs weremade at similar time intervals for the two studies. The exposure period for both studies was
initiated in early July with the treatment period lasting approximately 6 weeks. The CCAs
included in this analysis are:
 Just prior to initiation of treatments in early July, denoted CCA2 in this analysis
 End of July around 3 weeks into the exposure period, denoted CCA3 in this analysis
 End of August around 6 weeks after initiation of exposure, denoted CCA4 in this analysis
 End of September around 10 weeks after initiation of exposure, denoted CCA5 in thisanalysis1382

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination DocumentEnd of September around 13 weeks after initiation of exposure, denoted CCA6 in thisanalysis

These CCAs were chosen because this was the time period used to determine No Observed
Effects Concentrations (NOECs) and Lowest Observed Effects Concentrations (LOECs) in theprevious neonicotinoid nectar colony feeding studies. The range in levels of dose differed in thissecond repeat study. In the first study dose levels were 0, 10, 20, 40, 80, and 160 ng clothianidin
per g of solution. In the repeat study the highest level was not included where levels of dose were
at 0, 10, 20, 30, 40, and 80 ng/g. Data from the previous study was analyzed jointly by DPR,
U.S. EPA, and Canada’s PMRA staff scientists (U.S. EPA, PMRA, & DPR, 2017).

Statistical Analysis
Evaluation of the data followed the statistical approach used by DPR and EPA scientists toanalyze previously reviewed neonicotinoid colony feeding studies. Since measurements for eachvariable were made in each hive over time, the statistical analysis was conducted as a repeatedmeasures over time. Additionally, a mixed model was used where apiary location was identified
as a random variable and clothianidin levels of dose as a fixed effect. The mixed model waschosen because the results of the analysis were to be applied to the larger population of bee
hives. For data collected in 2016, statistical analysis was conducted on data collected for CCAs
numbered 2-6. As indicated previously, results of statistical analyses collected from the timing ofthese assessments made from July through September were the basis for development of NOEC
and LOEC values derived from data generated in previous neonicotinoid colony feeding studies.
Normality tests were conducted for each CCA within each year as indicated by Shapiro-Wilk andKolmogorov-Smirnov test statistics produced by the PROC CAPABILITY procedure in
Statistical Analysis System (SAS, version 9.4). For comparison, data were also transformed tonatural logarithms to determine if transformation provided better results. The majority of results
indicated that the distributions were normal, with the logarithm transformation indicating many
instances of non-normality. Based on these results the raw data were used in the analyses. The
mixed model approach used to analyze the data included tests to determine the appropriate
covariance model that describes the covariance structure reflected by the data. Inclusion of acovariance model in the analysis accounts for heterogeneity of variances that often are measured
between treatment levels.
The PROC MIXED procedure in the Statistical Analysis System (SAS, version 9.4) was used to
run the repeated measures effects mixed model. Measurements of colony health and hive weight
were conducted approximately 1 month apart so CCAs were treated as equally spaced intervals.
The effects side of the single year model statement included testing differences in the response
between CCAs indicating changes in response over the monthly measurements, between thelevels of clothianidin dose, and the potential interaction for effects of dose over time as indicatedby the CCA factor. SAS Program 1 below reflects the structure of the program used to analyze
the data for single years. Statistical options were included in the ‘Slice’ statement to protect
against falsely discovering a significant multiple comparisons for paired mean values between
the value at the control and each level of dose. The ‘Simulated’ option is a Monte Carlo approachthat computes adjusted p-values from simulated distributions based on distributional statistics
generated during the analysis (Edwards and Berry, 1987). In addition, the ‘Stepdown’ option was
2383

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
invoked because it tends to increase the power of the multi-comparison tests (SAS, version 9.4).
SAS Program 2 indicates the structure of the program used to conduct the analyses for each year.
There were two statements in the mixed model used to analyze the data, where a covariancemodel could be specified. One was in the ‘Random’ statement with apiary indicated as a randomvariable. The second was in the ‘Repeated’ measures statement where each hive was indicated as
the subject for the repeated measure. For the random statement only the Variance Component(VC) model successfully paired with the covariance model specified in the repeated statement:
Specifying more complex covariance models in the random statement resulted in indications ofconverge problems for that model. As observed in the previous colony feeding studies thecorrelation structure indicated greater correlation between samples taken at close time intervals
and, conversely, decreased correlation the further apart the samples were taken in time. Since
this structure is normally represented by autoregressive covariance models, the covariancestructure for the repeated statement was tested using variance component (VC), compound
symmetry (CS), compound symmetry with heterogeneity (CSH), autoregressive first order(AR(1)), autoregressive first order with heterogeneity (ARH(1)), and unstructured (UN) models.
Covariance model selection was based on the statistic generated for the Bayesian Information
Criteria (BIC) where a lower value of the criterion indicated a better fit of the covariance model.
A statistical basis for choosing the appropriate model was determined from Chi-square tests
conducted on the difference of the value of the BIC criteria between the two models tested with
the number of degrees of freedom determined as the difference between the number ofparameters in the model and where the significance level of probability was at 0.01 (Hammer,
2000; Littell et al., 2006). With the VC covariance model specified in the random statement, thebest fit covariance model in the repeated statement for analysis of data collected in 2016 was
ARH(1) for number of adult bees and number of pupal, larval, egg, and pollen cells and AR(1)for number of nectar cells (Table 1). Values for numbers of cells measured for each bee life stage
and food supply were divided by 1000 prior to statistical analysis to minimize potential
convergence problems due to magnitude of values.
SAS Program 1
proc mixed data=a4 order=data;by year;
class apiary dose cca hive rep ;model transvalue =cca dose dose*cca /ddfm=sat htype=1;
random apiary/type=vc;
repeated cca/ subject=hive*rep(dose) type=ar(1);slice dose*cca /sliceby=cca diff=controll stepdown(report) adjust=simulate adjdfe=row;
run;

Results
Data Combined for Years: Means and standard deviation for each response variable measured
at each dose and each CCA are the same as presented in this and the previous report of study
results so they are not reprinted in this analysis (Louque, J., 2016). Results from the datacollected in 2016 indicated numerous effects due to dose of clothianidin (Table 2). The
significant interactive effect of dose with CCA reflects the varying magnitude in the level ofdifferences for significant effects over the sampling interval. For example, there were no3

384

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
differences between the levels of dose for the first CCA2 samples as these were taken prior to
exposure and indicate no bias in treatments at the start of the study (Figures 1 to 6). In later
CCAs, the number of adult bees at the 80 ng/g treatment indicates a downward trend where themagnitude in difference compared to the controls becomes progressively greater at later CCAs
(Figure 1). Results from the pairwise comparisons between values for control and each doselevel indicated numerous instances of significant effects (Table 3 and Figures 1 through 6).
Effects were first indicated for measurements taken at CCA3 where pollens cells were affectedeven at the 30 ng/g level of dose. Initial effects were indicated for numbers of adults which weresignificant throughout the remaining CCAs. With respect to determining the LOEC value, there
were indications of significant effects at the 30 ng/g level of dose whereas the next lowest
treatment at 20 ng/g had only one indication for a potential trend for decreased pollen cells at
CCA3. This pattern of effects indicates that the 20 ng/g dose is the NOEC value and the 30 ng/g
dose is the LOEC value. Actual mean measured values as reported by the authors of the reportwere 28 ng/g for the LOEC value and 19 ng/g for the NOEC value.

Conclusion
Results of this statistical analysis are in agreement with the conclusions drawn by the authors of
the report. The nominal LOEC value was the 30 ng/g level of dose and the NOEC value was the
20 ng/g level of clothianidin dose. The mean measured concentrations of clothianidin in thetreated solutions in the nominal 30 and 20 ng/g treatment groups were 28 and 19 ng/g, which are
the actual LOEC and NOEC values, respectively. This study has been determined to bescientifically sound and can be used quantitatively to assess risks to honey bee colonies.

References
Edwards, D., and Berry, J. J. (1987). “The Efficiency of Simulation-Based Multiple
Comparisons.” Biometrics 43:913–928
Hammer,R.M. 2000. Mixed-Up Mixed Models: Things That Look Like They Should Work But
Don't, and Things That Look Like They Shouldn't Work But Do. Proceedings of the twenty-fifth
annual SAS users group International Conference, April 9-12, 2000, Indianapolis, Indiana.
Available at: http://www2.sas.com/proceedings/sugi25/25/aa/25p020.pdf. (Verified, Feb
12,2018).
Littell, R.C., G.A. Milliken, W.W. Stroup, R.D Wolfinger, and O. Schabenberger. 2006. SAS
System for Mixed Model, Second Edition. SAS Institute Inc., Cary NC.
Louque, J. (2016). Colony feeding study evaluating the chronic effects of clothianidin-fortified
sugar diet on honey bee {Apis mellifera) colony health under free foraging conditions: Final
Report. Unpublished study prepared by Smithers Viscient. 550p., Laboratory Report Number
13798.4143. MRID 49836101 CDPR Study ID TBD.
SAS Institute Inc 2013. SAS/ACCESS® 9.4.. Cary, NC: SAS Institute Inc.
4385

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
U.S. EPA, PMRA, & DPR. (2017). Data evaluation report: Louque, J. (2016) - Colony feeding
study evaluating the chronic effects of clothianidin-fortified sugar diet on honey bee (Apismellifera) colony health under free foraging conditions. Washington, D.C.: U.S. EPA.

5386

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Table 1. Mixed Model Analysis of Variance: BIC goodness-of-fit values generated for eachcovariance model structure tested in the repeated measures analysis of variance program.
Shaded cells indicate the covariance structure used for the analysis. DNC indicates that themodel failed to converge to solution.
CV
Model
Tested
VC
CS
AR(1)
CSH
ARH(1)UN

Model BIC Value for Data Collected in 2016:
Number of
Parameters
2
3
3
7
7
16

Adults2349.52262.12197.52251.42168.92170.4

Pupae

Larvae2048.72043.12032.92020.42006.1
2013.62259.72230.32208.52239.92217.32225.4Eggs
1637
1638.51637.91585.21583.81552.8Nectar3011.62904.32858.82913.32865.42878.6

Pollen2302.92205.72198.82135.42123.62122.2

6387

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Table 2. Combined Years: Results of the repeated measures mixed model testing the
response of each variable to clothianidin dosed surrogate honey.
Mixed Model Results for Repeated Measures Analysis of Variance
All Doses
Variable
EffectDF
Den DFF Value
Pr > F
Adult Bees
AR(1)Pupal Cells

Larval Cells

Egg Cells

Nectar Cells

Pollen Cells

cca
dose

4
5

185
73.1

94.79
2.87

<.00010.0204dose*cca

20

190

1.22

0.2391

cca
dose

4
5

170
85.6

104.015.98<.0001<.0001

dose*cca

20

170

4.37

<.0001

cca
dose

4
5

120
86.3

73.06
2.8

<.0001
0.0217dose*cca

20

122

1.93

0.0160

cca
dose

4
5

135
106

42.43
1.23

<.00010.2999dose*cca

20

142

0.65

0.8654

cca
dose

4
5

269
76

32.72
1.5

<.00010.2009dose*cca

20

271

2.25

0.0020

cca
dose

4
5

166
71.6

57.07
5.44

<.00010.0003dose*cca

20

169

4.56

<.00017388

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Table 3. Data from 2016 Study: Probability value for the contrast of the control to eachclothianidin dose at each CCA and for each variable measured. Dark shaded cells indicate
significance at P<0.01 and lighter shaded cells at 0.1>P>0.05.
Response
and
Dose (ng/g)
Adult Bees

10
20
30
40
80
Pupal Cells
10
20
30
40
80
Larval Cells
10
20
30
40
80
Egg Cells
10
20
30
40
80
Nectar Cells
10
20
30
40
80
Pollen Cells
10
20
30
40
80

Probability Value for Contrast of the Control to
Each Clothianidin Dose at Each CCA
CCA2

CCA3

CCA4

CCA5

CCA6

0.721
0.611
0.721
0.721
0.721
0.709
0.709
0.332
0.709
0.745

0.410
0.410
0.410
0.589
0.063
0.348
0.778
0.444
0.778
0.474

0.519
0.363
0.208
0.519
0.000
0.069
0.263
0.005
0.015
<.0001

0.384
0.551
0.222
0.551
<.0001
0.850
0.850
0.675
0.718
0.005

0.433
0.720
0.325
0.630
0.000
0.362
0.482
0.482
0.379
0.000

0.668
0.758
0.758
0.451
0.949
0.581
0.545
0.573
0.581
0.545
0.930
0.654
0.811
0.930
0.930
0.387
0.491
0.446
0.510
0.841

0.559
0.881
0.881
0.881
0.542
0.865
0.865
0.855
0.702
0.640
0.757
0.637
0.890
0.890
0.999
0.099
0.099
0.031
0.022
<.0001

0.415
0.236
0.052
0.087
<.0001
0.960
0.861
0.555
0.555
0.312
0.902
0.464
0.902
0.902
1.000
0.789
0.789
0.086
0.022
<.0001

0.819
0.819
0.819
0.632
0.013
0.538
0.538
0.537
0.392
0.067
0.547
0.756
0.462
0.678
0.756
0.744
0.744
0.244
0.099
<.0001

0.761
0.761
0.761
0.097
0.005
0.924
0.792
0.924
0.499
0.320
0.168
0.168
0.168
0.121
0.168
0.566
0.900
0.514
0.514
<.00018389

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

Figure 1. Data from 2016 Study: Mean number of adult bee in each treatment groupmeasured at every CCA resulting from nectar feeding at the indicated dose of ofclothianidin.

Number of Adult Bees - Year 2
Dose of Clothianidin (ng/g)
0

10

20

30

40

80

Number of Adult Bees

25000

20000

15000

10000

5000

0

CCA2

CCA3

CCA4

CCA Assessment

CCA5

CCA6

9

390

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Figure 2. Data from 2016 Study: Mean number of pupal cells in each treatment groupmeasured at every CCA resulting from nectar feeding at the indicated dose of ofclothianidin.

Number of Pupal Cells - Year 2
Dose of Clothianidin (ng/g)
0

10

20

30

40

80

25000

Number of Pupal Cells

20000

15000

10000

5000

0

CCA2

CCA3

CCA4

CCA Assessment

CCA5

CCA6

10

391

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Figure 3. Data from 2016 Study: Mean number of larval cells in each treatment groupmeasured at every CCA resulting from nectar feeding at the indicated dose of clothianidin.

Number of Larval Cells - Year 2
Dose of Clothianidin (ng/g)
0

10

20

30

40

80

14000

Number of Larval Cells

12000
10000
8000
6000
4000
2000
0

CCA2

CCA3

CCA4

CCA Assessment

CCA5

CCA6

11

392

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Figure 4. Data from 2016 Study: Mean number of pupal cells in each treatment groupmeasured at every CCA resulting from nectar feeding at the indicated dose of clothianidin.

Number of Pupal Cells - Year 2
Dose of Clothianidin (ng/g)
0

10

20

30

40

80

25000

Number of Pupal Cells

20000

15000

10000

5000

0

CCA2

CCA3

CCA4

CCA Assessment

CCA5

CCA6

12

393

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Figure 5. Data from 2016 Study: Mean number of nectar cells in each treatment groupmeasured at every CCA resulting from nectar feeding at the indicated dose of clothianidin.

Number of Nectar Cells - Year 2
Dose of Clothianidin (ng/g)
0

10

20

30

40

80

Number of Nectar Cells

50000

40000

30000

20000

10000

0

CCA2

CCA3

CCA4

CCA Assessment

CCA5

CCA6

13

394

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Figure 6. Data from 2016 Study: Mean number of pollen cells in each treatment groupmeasured at every CCA resulting from nectar feeding at the indicated dose of clothianidin.

Number of Pollen Cells - Year 20Dose of Clothianidin (ng/g)
10

20

30

40

80

20000

Number of Pollen Cells

18000
16000
14000
12000
10000
8000
6000
4000
2000
0

CCA2

CCA3

CCA4

CCA5

CCA6

CCA Assessment

14

395

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

State of California

Department of Pesticide Regulation

EVALUATION REPORT - Clothianidin Pollen Colony Feeding StudyJohn Troiano, Research Scientist III
June, 2018

A review of: - Bocksch, S., & Werner, S. (2018). Clothianidin Technical - Honey Bee Brood andColony Level Effects Following Clothianidin Intake via Treated Pollen in a Field Study in North
Carolina - USA 2017. Unpublished study prepared by Eurofins Agroscience Services EcoChem
CmbH. 192p., Laboratory Report Number S17-02137. MRID 50478501.
CDPR Study ID 305901.

Introduction
A colony feeding study was conducted to determine the effects of graded levels of clothianidinon the health of honey bee hives where doses mimicked exposure from foraging on pollen. The
dose of clothianidin was supplied in a pollen patty, which was a mixture of pollen powder, sugar
solution, and additives to stimulate feeding. The patties were placed inside the hives andreplenished on a set schedule. The health of hives was determined by Colony Condition
Assessments (CCAs) in which measurements were made over time on the number of individuals
in each bee life stage in the hive, the storage of honey and pollen food supplies in the hives, andthe weight of hives. The design of the study was similar to ones employed in previous feedingstudies on potential effects of nectar feeding of imidacloprid, thiamethoxam, and clothianidin on
bee hive health. Major differences were:

A smaller number of replicate apiaries were used: There were 4 instead of 12 replicate
apiaries.
Two replicates of each treatment were located at each apiary: Previous designs included
replicates for only the control treatment at each apiary.
A smaller number of dose levels were used but covered a greater range: Dose levels were0, 100, 400, and 1600 ng clothianidin/g of patty.

The location of the apiary sites was similar to the sites used for the nectar colony feeding studies
where they were located throughout a forested area of North Carolina. The distance between
each apiary site was approximately 1 mile apart. The majority of land near the apiaries is nonintensively managed pasture and forest with low potential exposure of bees to pesticides applied
for agricultural purposes.

1396Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

Measurements made over time were indicated by sequential numbering of the colony conditionassessments (CCAs). Assessments were made at approximately monthly intervals. For thisanalysis data obtained from CCAs 2 through 5 were included. Observations at CCA2 were taken
one week prior to initiation of clothianidin pollen feeding treatments, which was during the weekof July 3, 2017. Observations at CCA3 were taken during the middle of the six week feeding
period with CCA4 occurring 6 weeks after the initiation of treatments and CCA5 occurring 10
weeks after the initiation of treatments. Observation of bee and colony health taken during thisperiod was the basis for the determination of No Observed Effects Concentrations (NOECs) andLowest Observed Effects Concentrations (LOECs) in previous nectar colony feeding studies.

Statistical Analysis
Evaluation of the data followed the statistical approach used by DPR and EPA scientists toanalyze previously reviewed neonicotinoid colony feeding studies. Since measurements for eachvariable were made in each hive over time, the statistical analysis was conducted as a repeatedmeasures over time. Additionally, a mixed model was used where apiary location was identified
as a random variable and dinotefuran levels of dose as a fixed effect. The mixed model waschosen because the results of the analysis were to be applied to the larger population of bee
hives. Normality tests were conducted for each CCA as indicated by Shapiro-Wilk andKolmogorov-Smirnov test statistics produced by the PROC CAPABILITY procedure in
Statistical Analysis System (SAS, version 9.4). For comparison, data were also transformed tonatural logarithms to determine if transformation provided better results. The majority of results
indicated that the distributions were normal with the logarithm transformation indicating many
instances of non-normality. Based on these results the raw data were used in the analyses. The
mixed model approach used to analyze the data included tests to determine the appropriate
covariance model that describes the covariance structure reflected by the data. Inclusion of acovariance model in the analysis accounts for heterogeneity of variances that often are measured
between treatment levels.
A repeated measures analysis of variance was conducted to determine potential effects ofdinotefuran dose on each measurement of hive health over time. Data collected from colony
condition assessments (CCAs) numbered CCA2 to CCA5 were included because these are thetime intervals (July through September) where effects were observed in the previous
neonicotinoid colony feeding studies. Data obtained from CCA6 was excluded because of
extreme loss of hives at the 1600 ng/g treatment. Also, hive labeled T1a in the report was
excluded from the analysis because the hive was lost and no data was generated after the CCA2
assessment. The PROC MIXED procedure in the Statistical Analysis System (SAS, version 9.4)
was used to run the repeated measures effects mixed model. Since measurements of colony
health and hive weight were conducted approximately 1 month apart, CCAs were treated as
equally spaced intervals. The effects side of the model statement included effects for testing
differences in CCAs over time (CCA), differences in response between the levels of dose (Dose),and the potential interaction for effects of dose over time (SAS Program 1 below). The
regression model was run twice. First, all dose levels were included which were 0, 100, 400, and1600 ng clothianidin/g of patty. Based on these results, a reduced model was run that included
2397

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

the concentrations that appeared to define LOEC and NOEC concentrations. The second run was
intended to remove extraneous variance produced from treatments that were not contributing
information to the model. In order to protect against falsely discovering a significant comparison
between mean values, the ‘Simulated’ option was used to generate comparisons between thecontrol and each dose level (Edwards, D., and Berry, 1987). The ‘Simulated’ option is a Monte
Carlo approach that computes adjusted p-values from simulated distributions based on
distributional statistics generated during the analysis.
There were two statements in the mixed model used to analyze the data, where a covariancemodel could be specified. One was in the ‘Random’ statement with apiary indicated as a randomvariable. The second was in the ‘Repeated’ measures statement where each hive was indicated as
the subject for the repeated measure. For the random statement only the Variance Component(VC) model successfully paired with the covariance model specified in the repeated statement:
Specifying more complex covariance models in the random statement resulted in indications ofconverge problems for that model. As observed in the previous colony feeding studies thecorrelation structure indicated greater correlation between samples taken at close time intervals
and, conversely, decreased correlation the further apart the samples were taken in time. Since
this structure is normally represented by autoregressive covariance models, the covariancestructure for the repeated statement was tested using variance component (VC), compound
symmetry (CS), compound symmetry with heterogeneity (CSH), autoregressive first order(AR(1)), autoregressive first order with heterogeneity (ARH(1)), and unstructured (UN) models.
Covariance model selection was based on the statistic generated for the Bayesian Information
Criteria (BIC) where a lower value of the criterion indicated a better fit of the covariance model.
A statistical basis for choosing the appropriate model was determined from Chi-square tests
conducted on the difference of the value of the BIC criteria between the two models tested with
the number of degrees of freedom determined as the difference between the number ofparameters in the model and where the significance level of probability was at 0.01 (Hammer,
2000; Littell et al., 2006). With the VC covariance model specified in the random statement, thebest fit covariance models in the repeated statement were AR(1) for adult bees, pupae, larvae,
and egg, CS for pollen, ARH(1) for honey and nectar, and UN for hive weight (Table 1). The
greater number of parameters for hive weight was due to more sampling intervals included in theanalysis. Values for numbers of cells measured for each bee life stage and food supply weredivided by 1000 prior to statistical analysis to minimize potential convergence problems due tomagnitude of values.
SAS Program 1
proc mixed data=a3 order=data;class apiary dose cca hive rep;model transvalue =cca dose dose*cca /ddfm=sat htype=1;
random apiary/type=vc;
repeated cca/ subject=hive*rep(dose) type=ar(1);slice dose*cca /sliceby=cca diff=controll adjust=simulate adjdfe=row;
run;

3

398

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

Results
Means and standard deviation for each response variable measured at each dose and each CCA
are presented in Appendix A. Data for CCA2 through CCA5 were included in the repeatedmeasures analysis. For the repeated measures regression model that included all levels of dose
there were numerous indications of effects due to CCA, dose and dose by CCA interactions(Table 2). The Significant CCA effect indicated changes in overall abundance of the numbers ofbee life stages or food supplies over time. Except for egg cells all other variables had anindication of a significant effect due to dose or a dose by CCA interaction. From the patternobserved for the pairwise comparisons, the obvious effect was a sustained and incremental
decrease in all measures due to exposure at the highest level at 1600 ng/g of spiked pollen patty
as compared to the control values (Table 3). The first measurement of statistical significanceoccurred first for adult bees at CCA3, which was taken in the middle of the 6 week exposure(Figures 1 through 8). The early onset of loss in numbers of adult bees appeared to have acascading effect on reduction in the numbers of the other bee life stages and food stores becausesignificant effects for these measures appeared at the later CCAs, starting at CCA4 (Table 3).
These results indicated that the 400 ng/g dose level was potentially an NOEC value and 1600
ng/g an LOEC value. Results from the reduced analyses, using data for 0, 400, and 1600 ng/g
dose levels, confirmed the proposed NOEC and LOEC values (Tables 4). The same timing ofeffects was indicated where reduction in the number of adult bees was observed first at CCA3 at
the 1600 ng/g treatment followed by reductions in numbers for the other measures at later CCAs.
For hive weight, significant reduction was measured at the September 1 sampling interval for the1600 ng/g dose level, though there was indication in a graphical downward trend in the previous
sampling interval (Table 5 and Figure 8). Sampling at September 1 occurred around 2 weeks
after the sampling for CCA4 when most of the measures of bee health indicated decreases at thehighest dose.

Conclusion
Statistical analyses indicated significant effects of clothianidin dosed in pollen patties on nearly
all measures of bee life stages and food stores. The effect was obviously due to the highest
treatment at 1600 ng/g. Adults bees were first affected when sampled at CCA3 which was taken
mid-way through the dosing period of six weeks. The effect on adult bees was sustained
throughout the remainder of the sampling intervals and apparently caused a cascading effectwithin the hives: Reduction in essentially all other measures of bee and hive health occurred insubsequent CCAs. Ultimately 75% of the hives at the 1600 ng/g dose were lost by the lastassessment at CCA6. Reduction in bee life stages and loss of hives provide ample evidence ofthe detrimental effects that feeding pollen laced patties at 1600 ng/g had on health of bees andthe hives. Lack of effects at the next highest dose at 400 ng/g indicated that this is a NOEC value
for this set of treatments. Therefore, nominal concentration for the Lowest Observed EffectConcentration (LOEC) was determined to be 1600 ng/g and for the No Observed EffectConcentration (NOEC) was 400ng/g. The study authors reached the same conclusion, stating that
the NOEC was established at 400 ng/g. The measured value of clothianidin in the pollen patties
in the nominal 400 ng/g treatment group was 372 ng/g, which is the actual NOEC value. This
4399

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

study has been determined to be scientifically sound and can be used quantitatively to assess
risks to honey bee colonies.

References
Edwards, D., and Berry, J. J. (1987). “The Efficiency of Simulation-Based Multiple
Comparisons.” Biometrics 43:913–928
Hammer,R.M. 2000. Mixed-Up Mixed Models: Things That Look Like They Should Work But
Don't, and Things That Look Like They Shouldn't Work But Do. Proceedings of the twenty-fifth
annual SAS users group International Conference, April 9-12, 2000, Indianapolis, Indiana.
Available at: http://www2.sas.com/proceedings/sugi25/25/aa/25p020.pdf. (Verified, Feb
12,2018).
Littell, R.C., G.A. Milliken, W.W. Stroup, R.D Wolfinger, and O. Schabenberger. 2006. SAS
System for Mixed Model, Second Edition. SAS Institute Inc., Cary NC.
SAS Institute Inc 2013. SAS/ACCESS® 9.4.. Cary, NC: SAS Institute Inc.

5400

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

Table 1. Mixed Model Analysis of Variance: BIC goodness-of-fit values generated for each covariance model structure tested
in the repeated measures analysis of variance program. Shaded cells indicate the covariance structure used for the analysis.
Model BIC Value for:CV ModelTested
VC
CS
AR(1)
CSH
ARH(1)
UN

Number of
Parameters
2
3
3
6
6
11

Adults1100.11087.31078.91085.51075.81078.8

Pupae
1114.61111.61101.51113.11104.11096.3

Larvae
988.2
984.1
984.6
985.7
986.3
991

Eggs
941.7
942.2
935.8
945.3
939.4
929.5

Honey
1318.51256.81227.71245.41213
1197.6

Nectar1404.51405.51402.41364.41362.91377.2

Number ofPollen Parameters1012
21006.3
31007.5
3
996.4
8
997.3
8
992.3
22

Hive
Weight1082.21028.9972.4
1017.8
970.4
930.5

6

401

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

Table 2. All Dose Levels: Results of the repeated measures mixed model testing the
response of each variable to clothianidin dosed surrogate honey.

Mixed Model Results for Repeated Measures Analysis of Variance
All Doses of Clothianidin
F
Variable
EffectDF Den DF
Value Pr > F
cca
374
20.65 <.0001
Adult Bees

Pupae

Larvae

Eggs

Honey

NectarPollenTotal Brood

Honey + Nectar

Hive Weight

dose

3

30.1

16.79

<.0001

dose*cca

9

74

8.81

<.0001

cca

3

71.1

36.38

<.0001

dose

3

30.3

5.73

0.0031

dose*cca

9

71.1

2.43

0.0179

cca
dose

3
3

76.9
33.4

18.09
5.85

<.00010.0025dose*cca

9

76.9

1.44

0.19

cca

3

74

6.23

0.0008

dose

3

32.8

1.37

0.27

dose*cca

9

74

1.7

0.10

cca

3

47.5

36.04

<.0001

dose

3

24.6

0.51

0.6759dose*cca

9

47.5

2.51

0.02

cca

3

42.1

6.83

0.0007

dose

3

31.4

6.9

0.0011

dose*cca

9

42.1

3.85

0.0013

cca
dose

3
3

81
24.3

66.42
2.12

<.00010.1244dose*cca

9

81

3.14

0.0027

cca

3

73.6

35.01

<.0001

dose

3

33.1

7.03

0.00

dose*cca

9

73.6

2.43

0.02

cca
dose

3
3

82.8
38.3

3.39
4.36

0.02190.0097dose*cca

9

82.8

2.17

0.03

monthnum

5

27

46.59

<.0001

dose

3

27

1.92

0.15

dose*monthnum

15

27

5.26

<.0001
7402

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

Table 3. All Dose Levels: Probability value for the contrast of the control to eachclothianidin dose at each CCA and for each variable measured.

Probability Value for Contrast of theControl to Each Clothianidin Doseat Each CCA
CCA2
CCA3
CCA4
CCA5

Response
and
Dose (ng/g)
Bees

100
400

0.710
0.733

0.220
0.362

0.477
0.515

0.709
0.906

1,600
100
400
1,600
100
400
1,600
100
400
1,600
100
400
1,600
100
400
1,600
10040
400

0.948
0.830
0.821
0.628
0.790
0.902
0.925
0.746
0.876
0.634
0.870
0.968
0.965
0.935
0.874
0.824
0.755
0.952

<.0001
0.834
0.965
0.668
1.000
0.999
0.828
0.998
0.998
0.995
0.966
0.985
0.998
0.592
0.351
0.251
0.907
0.867

<.00010.707
0.583<.0001
0.996
0.998
0.234
0.705
0.438
0.133
0.915
0.977
0.980
0.381
0.085
0.001
0.839
0.862

<.0001
0.628
0.860
0.001
0.935
0.992
0.071
0.917
0.246
0.016
0.510
0.950
0.301
0.916
0.364
<.0001
1.000
0.942

1,600
Total Brood
100
400
1,600
Honey + Nectar 100
400

0.977
0.855
0.893
0.787
0.888
0.935

0.827
0.993
0.999
0.863
0.785
0.552

0.062
0.896
0.806
0.0002
0.510
0.189

0.083
0.867
0.871
0.0004
0.741
0.796

1,600

0.923

0.555

0.001

0.0002

Pupae

Larvae

Eggs

Honey

NectarPollen8403

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

Table 4. Reduced Dose Levels: Probability value for the contrast of the control to eachclothianidin dose at each CCA and for each variable measured.

Probability Value for Contrast of theControl to Each Clothianidin Doseat Each CCA
CCA2
CCA3
CCA4
CCA5

Response
and
Dose (ng/g)
Bees

400
1,600

0.639
0.905

0.278
<.00010.422<.00010.854<.0001400
1,600
Larvae
400
1,600
Eggs
400
1,600
Honey
400
1,600
Nectar400
1,600
Pollen400
1,600
Total Brood
400
1,600
Honey + Nectar 400
1,600

0.748
0.531
0.850
0.881
0.819
0.540
0.957
0.952
0.815
0.749
0.922
0.962
0.841
0.716
0.893
0.876

0.940
0.577
0.998
0.755
0.996
0.992
0.976
0.996
0.292
0.205
0.800
0.750
0.996
0.792
0.455
0.459

0.490
<.0001
0.996
0.168
0.341
0.087
0.952
0.955
0.013
<.0001
0.800
0.037
0.730
0.001
0.142
0.001

0.794
0.0002
0.983
0.045
0.167
0.009
0.909
0.239
0.317
0.0002
0.904
0.051
0.810
0.00020.719
0.0002

Pupae

9

404

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

Table 5. Reduced Dose Levels-HiveWeight: Probability value for the contrast of the control
to each clothianidin dose at each CCA for hive weight

Responseand
Dose(ng/g)

Probability Value for Contrast of the Control to Each
Clothianidin Dose at Each Weighing Interval
Sep 1

Sep 15

Jul 4

Jul 21

Aug 4

Aug 18

100

0.880

0.937

0.918

0.896

0.29020.2674

400

0.966

0.917

0.805

0.703

0.3928

0.645

1,600

0.974

0.949

0.560

0.196

<.0001<.0001Hive Weight

10

405

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

Figure 1. Mean number of adult bee in each treatment group measured at every CCA.
Treatment levels are 0, 100 , 400, and 1600 ng Clothianidin per g of pollen patty.

Mean Number of Adult Bees
Control

100

400

1600

20000
18000
16000
Mean Number of Adult Bees

14000

12000
10000
8000
6000
4000
2000
0

CCA1

CCA2

CCA3

CCA Assessment

CCA4

CCA5

CCA6

11

406

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

Figure 2. Mean number of pupal cells in each treatment group measured at every CCA.
Treatment levels are 0, 100 , 400, and 1600 ng Clothianidin per g of pollen patty.

Mean Number of Pupal Cells
Control

16000

100

400

1600

Mean Number of Pupal Cells

14000
12000
10000
8000
6000
4000
2000
0

CCA1

CCA2

CCA3

CCA4

CCA Assessment

CCA5

CCA6

12

407

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

Figure 3. Mean number of larval cells in each treatment group measured at every CCA.
Treatment levels are 0, 100 , 400, and 1600 ng Clothianidin per g of pollen patty.

Mean Number of Larval Cells
Control

100

400

1600

8000

Mean Number of Larval Cells

7000
6000
5000
4000
3000

2000
1000
0

CCA1

CCA2

CCA3

CCA Assessment

CCA4

CCA5

CCA6

13

408

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

Figure 4. Mean number of egg cells in each treatment group measured at every CCA.
Treatment levels are 0, 100 , 400, and 1600 ng Clothianidin per g of pollen patty.

Mean Number of Egg Cells
Control

6000

100

400

1600

Mean Number of Egg Cells

5000

4000

3000

2000

1000

0

CCA1

CCA2

CCA3

CCA Assessment

CCA4

CCA5

CCA6

14

409

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

Figure 5. Mean number of honey cells in each treatment group measured at every CCA.
Treatment levels are 0, 100 , 400, and 1600 ng Clothianidin per g of pollen patty.

Mean Number of Honey Cells
Control

100

400

1600

25000

Mean Number of Honey Cells

20000

15000

10000

5000

0

CCA1

CCA2

CCA3

CCA Assessment

CCA4

CCA5

CCA6

15

410

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

Figure 6. Mean number of nectar cells in each treatment group measured at every CCA.
Treatment levels are 0, 100 , 400, and 1600 ng Clothianidin per g of pollen patty.

Mean Number of Nectar Cells
Control

100

400

1600

40000

Mean Number of Nectar Cells

35000
30000
25000
20000
15000
10000
5000
0

CCA1

CCA2

CCA3

CCA Assessment

CCA4

CCA5

CCA6

16

411

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

Figure 7. Mean number of pollen cells in each treatment group measured at every CCA.
Treatment levels are 0, 100 , 400, and 1600 ng Clothianidin per g of pollen patty.

Mean Number of Pollen Cells
Control

100

400

1600

12000

Mean Number of Pollen Cells

10000

8000

6000

4000

2000

0

CCA1

CCA2

CCA3

CCA Assessment

CCA4

CCA5

CCA6

17

412

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

Figure 8. Weight of hives in each treatment group measured at every month. Treatment
levels are 0, 100 , 400, and 1600 ng Clothianidin per g of pollen patty.

Weight of Hives Measured Each MonthControl

100

400

1600

60

Mean Weight of Hives (kg)

50

40

30

20

10

0

Jul 7

Jul 21

Aug 4

Aug 18

Sep 1

Sep 15

Oct 13

Date of Measurement

18

413

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

APPENDIX A
Clothianidin Pollen Feeding Study
Mean Statistics for Response Variables
Measured at Each CCA

19

414

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

Table A-1. Adult Bees: Number of replicate hives (N), mean number of adult bees in hives (Mean), andstandard deviation for the number of cells measured at each CCA (SD) at each treatment level in theclothianidin pollen feeding study.
Clothianidin
Dose
Statistic
0 ng/gN
Mean
SD
100 ng/g
NMean
SD
400 ng/g
N
Mean
SD
1600 ng/g
N
Mean
SD

Counts for Adult Bees Measured at Each CCA
CCA1
CCA2
CCA3
CCA4
CCA5
8
8
8
8
8
15498.25 14708.88 18377.13 14198.75 11550.38
3056.124 3812.937 3184.335 4187.525 5276.044
7
7
7
7
7
14182.71 14454.14 16083.29 13016.71 11275.86
2096.892 2334.947 2548.907 4044.477 4030.799
8
8
8
8
8
15547.25 14610.88 16839.75 13248.25 12465.88
3078.215 1956.657 4615.185 4432.031 3316.341
8
8
8
8
8
15652 16043.5 7113.25
2872 3598.625
2824.109 1982.498 1496.583 1369.262 1706.061

CCA685925.52638.949
73354.143
3660.2985589.875
3165.5888
6011160.37420

415

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

Table A-2. Pupal Cells: Number of replicate hives (N), mean number of cells in each hive with pupae(Mean), and standard deviation for the number of cells measured at each CCA (SD) at each treatment levelin the clothianidin pollen feeding study.

Clothianidin
Dose
Statistic
0 ng/gN
Mean
SD
100 ng/g
NMean
SD
400 ng/g
N
Mean
SD
1600 ng/g
N
Mean
SD

Counts for Pupal Cells Measured at Each CCA
CCA1
CCA2
CCA3
CCA4
CCA5
8
8
8
8
8
8191.5 14426.5
7095
9589
9847
4753.23 3790.068 3784.838 5359.867 4219.888
7
7
7
7
7
6609.714 14865.71 7592.571 9386.286 9263.429
1920.086 3745.833 3837.603 3367.362 4801.058
8
8
8
8
8
7718.514792
8901
8858 10470.5
2695.266 3719.793 1992.635 2818.762 4153.962
8
8
8
8
8
7396 13867.5
6729.51655.53117.54421.628 2519.395 2123.421 1843.205 3297.53

CCA6
85181.52729.545
73857.714
4542.946
8
5289
3402.633
8
0
0

21

416

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

Table A-3. Larval Cells: Number of replicate hives (N), mean number of cells in each hive with lavae(Mean), and standard deviation for the number of cells measured at each CCA (SD) at each treatment levelin the clothianidin pollen feeding study

Clothianidin
Dose
Statistic
0 ng/gN
Mean
SD
100 ng/g
NMean
SD
400 ng/g
N
Mean
SD
1600 ng/g
N
Mean
SD

Counts for Larval Cells Measured at Each CCA
CCA1
CCA2
CCA3
CCA4
CCA5
8
8
8
8
8
4751.5
6536
3655
2666
4708.52719.462 1341.789 1960.563 1710.143 2463.416
7
7
7
7
7
4766.857 6658.857 6216.571 4398.286 5479.429
2540.936 1126.63 2958.248 1328.07 2971.552
8
8
8
8
8
4106.57052
5869.54601
6213.51627.683 2150.246 1743.035 2275.81 2639.814
8
8
8
8
8
4106.57181
3891.51419
2795
1593.571 2445.876 1083.688 936.4584 1055.285

CCA6
81225.5883.9313
71105.714
1263.3798
1096.51019.3818
86243.2447

22

417

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

Table A-4. Egg Cells: Number of replicate hives (N), mean number of cells in each hive with eggs (Mean),and standard deviation for the number of cells measured at each CCA (SD) at each treatment level in theclothianidin pollen feeding study
Clothianidin
Dose
Statistic
0 ng/gN
Mean
SD
100 ng/g
NMean
SD
400 ng/g
N
Mean
SD
1600 ng/g
N
Mean
SD

Counts for Eggs Cells Measured at Each CCA
CCA1
CCA2
CCA3
CCA4
CCA5
8
8
8
8
8
4579.54171
1827.53246.53569
886.3187 2285.077 1479.421 2017.272 2253.416
7
7
7
7
7
4570.286
4128 3317.143 3120.571 4029.714
1118.472 1263.937 2029.586 1972.205 1943.06
8
8
8
8
8
5267.54493.53354
2666
2601.51290.205 1767.115 1019.64 1184.537 1231.547
8
8
8
8
8
4945
3934.53160.5
1978
1548
1239.457 1314.543 985.6558 1488.144 675.6026

CCA6
81268.51140.6877614.2857
665.09498
989
654.95658
193.5
425.6783

23

418

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

Table A-5. Honey Cells: Number of replicate hives (N), mean number of cells in each hive with honey(Mean), and standard deviation for the number of cells measured at each CCA (SD) at each treatment levelin the clothianidin pollen feeding study

Clothianidin
Dose
0 ng/g

100 ng/g

400 ng/g

1600 ng/g

Statistic
N
Mean
SD
N
Mean
SD
N
Mean
SD
N
Mean
SD

Counts for Honey Cells Measured in Hives at Each CCA
CCA1
CCA2
CCA3
CCA4
CCA5
CCA6
8
8
8
8
8
8
15222 16791.5
8062.5
7009
15351
16297
9074.412 11612.93 8879.236 7028.739 11526.66 11892.63
7
7
7
7
7
7
19632.57 17912.57 11720.57 8968.571 11032.57 7985.714
8172.22 8463.862 7816.341 6431.381 8956.356 13603.54
8
8
8
8
8
8
17823.5 21392.5
13287 11674.5
20683
23779
7853.527 10342.88 7605.74 7453.455 14040.25 17802.68
8
8
8
8
8
8
18533 21220.5
15996
11825
8750.52515.510138.52 10908.47 12197.28 10357.51 9078.806 6260.515

24

419

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

Table A-6. Nectar Cells: Number of replicate hives (N), mean number of cells in each hive with honey(Mean), and standard deviation for the number of cells measured at each CCA (SD) at each treatment levelin the clothianidin pollen feeding study.

Clothianidin
Dose
0 ng/g

100 ng/g

400 ng/g

1600 ng/g

Statistic
N
Mean
SD
N
Mean
SD
N
Mean
SD
N
Mean
SD

Counts for Nectar Cells Measured in Hives at Each CCA
CCA1
CCA2
CCA3
CCA4
CCA5
CCA6
8
8
8
8
8
8
21758 12749.5
18662
37754 27025.5 23886.5
10917.79 4640.301 31103.68 14616.1 15436.38 13867.22
7
7
7
7
7
7
17814.29
14448 14742.86 30345.71 30075.43 20345.14
7591.975 6572.861 16093.64 26333.02 8076.939 20059.63
8
8
8
8
8
8
23951 13738.5
9761
22661 22510.5 15329.5
8446.158 5284.978 13910.83 12146.15 9625.169 9003.547
8
8
8
8
8
8
22446 13265.5
7116.57052
3117.5387
10251.79 3587.271 6270.633 4870.104 1902.997 722.4585

25

420

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

Table A-7. Pollen Cells: Number of replicate hives (N), mean number of cells in each hive with pollen(Mean), and standard deviation for the number of cells measured at each CCA (SD) at each treatment levelin the clothianidin pollen feeding study

Clothianidin
Dose
0 ng/g

100 ng/g

400 ng/g

1600 ng/g

Statistic
N
Mean
SD
N
Mean
SD
N
Mean
SD
N
Mean
SD

Counts for Pollen Cells Measured in Hives at Each CCA
CCA1
CCA2
CCA3
CCA4
CCA5
CCA6
8
8
8
8
8
8
6944.57675.52064
2687.52859.51634
1796.762 3352.818 1332.306 1140.687 1366.561 1129.751
7
7
7
7
7
7
9386.286 7690.857 2702.857 2997.714 6241.143 3071.429
3770.76 2372.341 1478.648 2136.443 4035.852 3560.343
8
8
8
8
8
8
10535
8643
2472.53074.53698
2193
4232.888 4032.448 2276.159 2775.606 1129.751 3075.112
8
8
8
8
8
8
10900.58965.5
2322
473
817
43
3511.662 1809.653 1418.348 438.5157 534.1107 121.6224

26

421

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

Table A-8. Hive Weight: Number of replicate hives (N), mean number of cells in each hive with pollen(Mean), and standard deviation for the number of cells measured at each CCA (SD) at each treatment levelin the clothianidin pollen feeding study
Clothianidin
Dose
0 ng/g

100 ng/g

400 ng/g

1600 ng/g

StatisticN
Mean (kg)SD (kg)N
Mean (kg)SD (kg)N
Mean (kg)SD (kg)N
Mean (kg)SD (kg)

Jul 4

8
44.05
7.328808745.85714
6.489699847.95
8.755578
48.4757.660614

Weight of Hives Measured at Each Month
Sep 1
Sep 15
Jul 21
Aug 4
Aug 18
8
8
8
8
8
41.3
39.925
40.35
37.7
41.2
6.55526 3.365264 4.648502 3.732483 5.267691
7
7
7
7
7
44.14286
41.6 41.74286 34.94286 37.94286
6.307365 2.620433 4.11779 4.287523 3.279663
8
8
8
8
8
43.57540.32540.02535.65
40.4758.930486 4.032635 4.396671 4.275846 5.65376
8
8
8
8
8
44.37538.625
36.75
25.6
24.35
7.350364 8.75406 6.989584 6.731589 6.811545

Oct 1
832.275
14.57785
7
24.4
23.54768840.4755.653768
15.55
12.1987127

422

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

Data Evaluation Record
Study Titles:
Bocksch, S. (2015): Thiamethoxam Technical - Honey Bee Brood andColony Level Effects Following Thiamethoxam Intake via Treated Sucrose
Solution in a Field Study in North Carolina
Final Report Source:
Eurofins Agroscience Services, unpublished report No: S14-02633, 30 Oct
2015
Year of study: 2014-2015
PMRA#:PMRA DACO#:
MRID: 49757201
Study Type:
Tier II colony feeding study conducted in an open field
Review Date (final): February 10, 2017
Health Canada Pest Management Regulatory Agency (PMRA) reviewer:
Primary Evaluator: Barbara Martinovic Barrett, Sr. Senior Evaluation Officer
Secondary Evaluator: Connie Hart, Senior Science Advisor
Data Statistical Analysis: Keith O’Rourke, Senior Epidemiologist/Bio-statistician
United States Environmental Protection Agency (EPA) reviewer:
Primary Evaluator: Ryan Mroz, Biologist
Secondary Evaluator: Kristina Garber, Senior Science Advisor
Data Statistical Analysis: Christine Hartless, Wildlife Biologist
CDPR reviewer:
Primary Evaluators: Richard Bireley, Sr. Environmental Scientist (Specialist)
Alexander Kolosovich, Environmental Scientist
Russel Darling, Environmental Scientist
Brigitte Tafarella, Environmental Scientist
Denise Alder, Sr. Environmental Scientist (Specialist)
Data Statistical Analysis: John Troiano, Ph.D., Research Scientist III

1423

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

Table of Contents
Executive Summary....................................................................................................................................... 4
1.

Study Objective ................................................................................................................................... 10

2.

Study Methods.................................................................................................................................... 10
2.1.

Test crop ..................................................................................................................................... 10

2.2.

Test chemical .............................................................................................................................. 10

2.3.

Test sites ..................................................................................................................................... 10

2.4.

Test organisms ............................................................................................................................ 12

2.5.

Treatments.................................................................................................................................. 13

2.5.1.

Preparation of stock solution.............................................................................................. 14

2.5.2.

Preparation of sugar solution ............................................................................................. 14

2.5.3.

Preparation of feeding solution .......................................................................................... 15

2.5.4.

Artificial Feeding ................................................................................................................. 16

2.6.

Meteorological Data ................................................................................................................... 16

2.7.

Observations ............................................................................................................................... 16

2.7.1.

Dates of observation........................................................................................................... 16

2.7.2.

Colony mortality.................................................................................................................. 19

2.7.3.

Colony Condition Assessments (CCA) ................................................................................. 19

2.7.4.

Evaluation of Disease or Pests in the Hive .......................................................................... 20

2.7.5.

Hive weights........................................................................................................................ 20

2.8.

3.

Residue analysis .......................................................................................................................... 20

2.8.1.

Pollen from outside sources ............................................................................................... 21

2.8.2.

Stored pollen and nectar in test apiaries............................................................................ 21

2.8.3.

Feeding solution and stability of test item ......................................................................... 21

Results................................................................................................................................................. 22
3.1.

Land use near test hives.............................................................................................................. 22

3.2.

Sources of pollen from monitoring hives.................................................................................... 23

3.3.

Consumption of spiked sucrose.................................................................................................. 24

3.4.

Examination of pesticides from other sources ........................................................................... 25

3.5.

Confirmation of test concentrations........................................................................................... 25

3.6.

Stability of the test item in feeding solution .............................................................................. 26

3.7.

Residues in hive matrices............................................................................................................ 262424

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
3.7.1.

Thiamethoxam residues in hives prior to the feeding exposure ........................................ 26

3.7.2.

Residues in hive matrices during and after feeding exposure............................................ 27

3.8.

Pathogens ................................................................................................................................... 28
Varroa Presence.................................................................................................................. 28

3.8.1.
3.9.

Hive mortality.............................................................................................................................. 29

3.10.
3.11.

Colony Condition Assessment Response Variables ................................................................ 30

3.11.1.

Statistical Analysis................................................................................................................... 31

3.11.1.1.

Study Authors Analysis.................................................................................................... 31

3.11.1.2.

Study Reviewer Analysis ................................................................................................. 32

3.11.1.2.1.

EPA Analysis ................................................................................................................ 32

3.11.1.2.2.

PMRA Analysis............................................................................................................. 33

3.11.2.

5.

Colony Condition Assessment Variables - Life Stages............................................................. 33

3.11.2.1.

Adults .............................................................................................................................. 33

3.11.2.2.

Eggs ................................................................................................................................. 35

3.11.2.3.

Larvae (Open/Uncapped brood)..................................................................................... 36

3.11.2.4.

Pupae (Capped brood) .................................................................................................... 37

3.11.2.5.

Total individuals in hives ................................................................................................. 39

3.11.3.

4.

Hive Weight......................................................................................................................... 30

Colony Condition Assessments – Food Store Response Variables ......................................... 43

3.11.3.1.

Pollen .............................................................................................................................. 43

3.11.3.2.

Nectar / Honey................................................................................................................ 44

Reviewer comments: .......................................................................................................................... 46
4.1.

General Considerations for Biological Interpretation................................................................. 47

4.2.

Control Performance................................................................................................................... 48

4.3.

Consideration of Study Strengths, Limitations and Interpretation ............................................ 48

Conclusions ......................................................................................................................................... 49
Colony Condition Assessments – Food Stores ..................................................................................... 105

3425

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

Executive Summary
A colony feeding study was conducted with honey bees (Apis mellifera L.) to assess the potential
for long-term effects, including overwintering survival, resulting from exposure to thiamethoxam.
The study was conducted in twelve rural test areas lacking extensive acreages of crops treated withpesticides (Apiaries A – L) in North Carolina from June 27, 2014 (hive installation) to April 28,
2015 (last colony assessment). Colonies were divided into 12 groups of 7 colonies placed at thedifferent apiaries. These 84 hives were used for biological assessments and an additional hive wasplaced in each apiary for residue analysis and pollen source identification (96 total hives). Hives
were divided according to hive strength (total brood coverage) with the strongest 7 hives assigned
to Apiary A and the weakest 7 hives assigned to Apiary L. Within each apiary, the 7 hives wererandomly assigned to treatment groups.
At each apiary, five test hives were artificially fed with 50% sugar solution spiked withthiamethoxam at 12.5, 25, 37.5, 50 or 100 µg a.i/L for six weeks continuously in the field, with
two hives serving as controls and one hive for pesticide residues and pollen source identification(8 hives/Apiary). Assuming the density of a 50% sugar solution is 1.2296 g/ml, the reviewer
calculated that the test concentrations at 12.5, 25, 37.5, 50, and 100 µg/L are equivalent to 10.2,
20.3, 30.5, 40.7, and 81.3 ppb (µg a.i/kg-solution), respectively. Residues of thiamethoxam in thedose verification samples were 9.3 ppb (Tl; 12.5 ppb), 24.1 ppb (T2; 25 ppb), 29.5 ppb (T3; 37.5ppb), 39.7 ppb (T4; 50 ppb) and 73.7 ppb (T5; 100 ppb). Relative to nominal this is equivalent to91.4% (Tl), 118.7 % (T2), 96.8 % (T3), 97.5 % (T4) and 90.7 % (T5). Clothianidin was notdetected in any of the test solutions (Level of Detection = 0.25 ppb).
Ten Colony Condition Assessments (CCAs) were conducted during the study. The two initial
CCAs (CCA1 and CCA2) were conducted prior to exposure to thiamethoxam through feeding.
The objectives of CCAs 1 and 2 were to determine hive strength and initial hive conditions and toselect the 84 hives subject to the treatment and to be used as controls. The third CCA was
conducted just prior (one week) to dosing with thiamethoxam. A CCA was conducted during thefeeding period with another one conducted shortly after the feeding period (CCA4 and CCA5,
respectively) which characterize hive conditions during exposure. Three more CCAs wereconducted at 10, 13, and 16 weeks after exposure (CCA6, CCA7, and CCA8 respectively) to assess
the chronic effect following exposure to thiamethoxam and to characterize pre-overwintering hive
conditions. Two final CCAs were conducted after overwintering in March 2014 (CCA9) and April
2015 (CCA10) to assess potential exposure impact on survival and chronic colony level effects.
Due to substantial overwinter failure in control hives (79%), the biological results from CCA9 and
CCA10 are not considered scientifically valid and are not considered in this DER.
Levels of thiamethoxam and its major metabolite (CGA322704 – clothianidin) residues in hives
were measured before, during and after the feeding exposure. Multiple parameters, such as hive
weight, number of individuals at different life stages in the hive, hive honey and pollen stores, andhive overwintering survival, were measured during the course of the study. Pollen was collectedfrom monitoring hives at each site for assessment of forage availability and the local pollen flora
was also analysed for 174 active ingredients using multi residue methods for assessment ofpotential exposure to pesticides from sources other than the dosing solution. Varroa and Nosema
was also measured throughout the study.
4426

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Potential test colony contamination with pesticides from other food sources was monitored using
pollen collected from the additional hive at each apiary that served as a monitoring hive. The
results showed that while there were a few instances of thiamethoxam detected in the pollen of thecontrol hives, the frequency and magnitude of these detections is not expected to confound theresults of this study. Maximum residues measured at CCA5 (end of exposure) in hive matrices
demonstrated 80, 68, 64, 70, and 43% of nominal concentrations in pollen and in honey 142, 110,75, 124, and 102% of nominal concentrations in the 12.5, 25, 37.5, 50, and 100 µg/L treatmentgroups respectively. Variability was apparent, as many of the hives had minimum measured
residue levels in pollen of 21, 0, 28, 22, and 25%; and in honey of 0, <LOD (0.25 µg a.i./kg), 0,<LOD-, and 0% of nominal concentrations in the 12.5, 25, 37.5, 50, and 100 µg/L respectively.
Dilution is expected since bees forage on outside pollen and nectar sources, and hive pollen (beebread) includes only a small amount of nectar. Clothianidin (CGA322704) residues ranged from
<LOD (0.25 µg a.i./kg) - <LOQ (0.5 µg a.i./kg) in nectar/honey of biological hives, doseverification and stability samples. In one treatment (T3) CGA32274 was detected at the end of
the exposure period with a maximum residue value of 1.362 µg a.i./kg. See Section 3.7 for moredetails regarding the residues of thiamethoxam and CGA32274 in the dosing solutions and hive
matrices.
In the control colonies, the number of adults increased until CCA 5 (august) and then started to
decline. This trend was also observed in open cells and pollen, and was very evident with capped
cells, whereby a dramatic decline was observed following CCA 5. The number of eggs appeared
to drop slightly at CCA 5, but then increased by CCA 6 before a drop in numbers at CCA 7. The
decline in the number of live bees after CCA 5 (August) is likely the result of a later start date forthe thiamethoxam study, which resulted in the 6th colony condition assessment being taken in late
September, which is closer to the period of time in which the colony is preparing for overwintering.
As a result, any effects observed in the thiamethoxam study at CCA 5 were difficult to follow to
CCA 6 (or thereafter) for potential recovery (or reversion) of effects, since the control colony was
declining in numbers at this time, resulting in less sensitivity in the analysis.
In the highest test treatment (100 µg/L), statistical reductions relative to the control (p<0.05) wereobserved across several different endpoints and at several CCAs within an endpoint. Relative tothe control, decreases were observed in the number of adults, brood (eggs, larvae, pupae), and foodstores (pollen). Significant decreases started at CCA4 (mid exposure period) for pollen and pupae
and total number of individuals, and at CCA5 (end of exposure period) for the remaining measured
endpoints. Significant reductions were seen through CCA6 for number of eggs and larvae while
through CCA7, number of individuals, pupae, and pollen. At CCA 8 (late October), most of the
response variables were decreased from CCA7; however, by this time all hives should have been
winding down for winter. Preparation for overwintering could have begun as early as CCA6,
which would comparisons to controls for most endpoints relative to control hives were are also in
decline. Only the number of adults in the 100 µg/µg/L treatment (p<0.05) showed significant
reductions compared to the control past CCA6 for non-food response variables.
Several other significant statistical differences relative to control hives were observed in the next
to highest treatment group (50 µg/L) at CCA5. Pupae, larvae, and total individuals showedstatistically significant reductions (p<0.05), while adults and pollen showed marginal significant
reductions (0.05<p<0.1) at this CCA (the end of the exposure period) compared to the control.
5427Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Since the control colonies were in decline by CCA 6 (possibly in preparation for overwintering),
it is not possible to discern effects from the control at any time point following CCA 5.
With regards to all other treatments (12.5, 25, and 37.5 µg/µg/L), most endpoint responses were
not significantly different from the control (p>0.05). However, there were two endpoints for whicha statistical reduction in these treatments was observed after exposure. The number of pupae werereduced (p<0.05) in the 37.5 µg/µg/L treatment group at CCA5. Also at CCA 4 (0.05<p<0.1), 5
and again at CCA7, the number of pollen cells were reduced (p<0.05) in the 25 µg/µg/L treatmentgroup (no difference at CCA6). Additionally, prior to exposure, hives in the 25 µg/µg/L treatmentgroup were reduced compared to the controls for Eggs (0.05<p<0.1), as well as larvae and pupae(p<0.05). There were also transient reductions of marginal significance (0.05<p<0.1) in the 12.5
treatment group at CCA6 for eggs and CCA5 for honey cells.
Other combination parameters such as total life (adults, eggs, larvae, and pupae), brood, and foodstores (honey and pollen) showed similar patterns of significant reductions i.e. in the highest
treatment group (100 µg/µg/L) relative to the control.
Honey cell counts were not significantly (p<0.05) reduced for any treatment level relative to thecontrols.
In summary, clear decreases in multiple endpoints (relative to controls) and declining trends wereobserved over several CCAs in colonies exposed to 100 µg/L thiamethoxam. In addition, larvae,
pupae, pollen and adults were declining in the 50 µg a.i/L group shortly after exposure. Some
effects were also observed in some endpoints at some CCAs in bees exposed to 25, and 37.5 µg/L
thiamethoxam. At the lowest test level, i.e., 12.5 µg/µg/L, except for eggs at CCA6 and honey at
CCA5 (both 0.05<p<0.1), numbers of bees and food stores were similar in numbers and trends
compared to controls (i.e., no significant differences noted).
It is important to recognize the additional inherent strengths and limitations of this study as results
are interpreted and potentially considered in risk assessment. In the context of available fieldstudies involving honey bees and thiamethoxam, this study contains a number of strengths
including:
•
•
•
•
•
•

Use of a high degree of replication (n=12) to achieve a reasonable level of statistical power
Demonstration of a generalized concentration-response relationship with respect to theconcentration of thiamethoxam in sucrose solution and the magnitude and duration ofadverse effects (at least for the 50 and 100 µg/L treatment groups),
Quantification of exposure to parent (thiamethoxam) and toxicologically-relevant
metabolites (i.e., clothianidin) in diet and in hive matrices (uncapped nectar, pollen, honey,
bee bread)Use of a 6-week exposure duration to represent a “high end” exposure scenario,
Inclusion of multiple colony-level endpoints reflecting hive strength, brood development
and food stores
Detailed QA/QC results regarding quantification of chemical residues in various matrices(screen for 174 pesticides/degradates) to better understand other chemical exposures bees
encounter.
6428

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document•

Availability of raw data for conducting statistical analysis.

A number of limitations are also noted with this study, including:•Exposure of bees to thiamethoxam occurred through nectar (sucrose) alone, whereas beesin the field are likely exposed through both pollen and nectar routes. Therefore, the designof this study may not reflect a “worst case” exposure scenario in which bees areexperiencing prolonged exposure to both contaminated nectar and pollen as may occur
through exposure to a treated crop. While exclusion of the pollen route is expected tounderestimate overall exposure, the impact of this exclusion on the study results is
uncertain and will likely depend on the life stage/caste of bee.

•

Overwintering success of controls was severely impacted (79% hive mortality). In fact, nohives survived overwintering in any significant proportion. This makes interpretation of
results difficult. If control hives had survived, comparing trends or lack thereof with thetreatment hives would be more meaningful.

•

The later date of initiation for the study resulted in CCA 6 (and onward) occurring at thetime of overwintering preparation. Measurements at CCA 5 were taken in August, and
CCA 6 was taken in September. As observed in the control colony, by CCA 6, the numberof total individuals, open cells, capped cells, adults and pollen were all in decline following
CCA 5. This resulted in difficulty assessing potential “reversion of effects” (in comparison
to the control).

•Thiamethoxam was found in both hive nectar and hive pollen (bee bread), at concentrations
ranging from <LOQ to greater than the specific colony feeding solution. Dilution comparedto the treatment feeding solution is possible since bees foraged on outside nectar and pollen
sources. Additionally, bee bread contains only a small quantity of hive nectar, thus would
not be expected to have a concentration equivalent to nectar alone. Therefore, exposure
through both bee bread and nectar occurred via exposure to the sucrose feeding solution.
Since bees were forced to forage for pollen, the potential impact of thiamethoxam exposure
on reducing pollen foraging efficiency of bees could be incorporated into the overall
expression of adverse effects. Had contaminated pollen been provided to bees, it is notknown if the potential impact on pollen foraging efficiency would have been masked.

•

Hive detections of pesticides from food sources other than thiamethoxam and metabolite
was detected during the exposure period and post-exposure periods through analysis ofpollen from pollen traps. Although the study was deliberately conducted in a low
agricultural area in order to minimize the potential for pesticide contamination from othersources, the bees still appeared to be foraging on contaminated pollen and possibly nectar.
During both exposure and post-exposure periods, a high level of multiple pesticides that
may cause concern for bees were detected in most monitoring hives. Acephate (1 sample,
1600 ppb), Carbaryl (1 sample, 214 ppb), Carbendazim (5 samples, traces - 1300 ppb),
Imidacloprid (2 samples, 4.3 and 6.1 ppb), Methamidophos (1 sample, 109 ppb),
Thiamethoxam (2 samples, 8.8 ppb and 11.3 ppb), and Thymol (23 samples, 405 ppb to7429

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
12300 ppb; during period of Varroa treatment) accounted for the highest residue levels
found in pollen samples.
•

Exposure dilution during the study was evident. The exposure, based on residues measured
in the hive (hive nectar and hive pollen) indicated that, overall, higher measured hive
residues correlated with higher nominal residues in feeding solutions. However, individual
hive residue values varied, and there was some overlap in measured values. Pollen storage
was observed consistently in the control hives and hives exposed to lower test
concentrations during the exposure period, indicating that test bees were foraging on foodsources other than the spiked sugar solution. Remarkably lower residue concentrations
detected in bee bread and hive nectar in some test hives compared to the feeding
concentrations may also indicate foraging on other food sources.

This study is considered scientifically valid; however, it is classified Supplemental due to the
key limitations listed below:•••

late timing of exposure that coincides with ramping down trends of colony endpoints,
lower than expected performance of controls, andlack of overwintering success.

Based on the limitations of this study, A NOAEC derived from this study is considered highly
uncertain. Effects were observed to multiple endpoints and multiple CCAs at the highest test level(i.e., 100 µg a.i./L thiamethoxam; 86 µg a.i./L clothianidin-equivalents). Effects to several
endpoints (number of adults, amount of brood) were also observed at CCA5 of the second highest
test level (i.e., 50 µg a.i./L thiamethoxam; 43 µg a.i./L clothianidin-equivalents 1). It is uncertain
whether or not effects at 50 µg a.i./L are meaningful to the colony since these effects were only
observed at CCA5, which is the conclusion of the exposure window, however, potential recovery
could not be determined due to the limitations of CCA6, 7, and 8 (during downward trend ofcolonies) and a lack of overwintering data. Also, the utility of CCAs 6, 7 and 8 in showing
treatment related effects are questionable because of the downward trend in endpoints that is
consistent with preparation of colonies for winter. If effects observed at 50 µg a.i./L arebiologically significant to the colonies, the NOAEC from this study would appear to be 37.5 µg
a.i./L (32 µg a.i./L-clothianidin equivalents). There is uncertainty in whether or not this value isconservative. Since the hives did not perform as expected, and given the late timing of the exposure
window, it is uncertain whether or not effects due to thiamethoxam could be detected.
A comparison can be made between the effects of this study and those in the CFS with clothianidin(MRID 49836101) which was conducted in a similar location and in the same year. In theclothianidin CFS, clear effects were observed at 40 µg a.i./L over multiple endpoints and multiple
CCAs, leading to a NOEC of 20 µg a.i./L (clothianidin). At the NOEC of 20 µg/L, some effects
were observed at CCA5, but these effects did not manifest at later CCAs. This suggests that effects
were observed at lower levels in the clothianidin study compared to the thiamethoxam study.

1

Clothianidin equivalents are used in the preliminary pollinator risk assessment, and concentrations may be
expressed in terms of either thiamethoxam or these clothianidin equivalents (c.e.) in this document.
8430

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Therefore, when considering the uncertainties described above, the apparent NOAEC for
the thiamethoxam CFS is tentatively determined to be 37.5 µg a.i./L (32 µg a.i./L-clothianidin
equivalents), noting that this may value may not be conservative. The apparent NOAEC of
37.5 µg a.i./L (thiamethoxam; 32 µg a.i./L- clothianidin equivalent) along with the effects
levels of 50 and 100 µg a.i./L (thiamethoxam) will be used to characterize the risk ofthiamethoxam to honey bee colonies. Additional information from the clothianidin CFS may
also be used to characterize the risk of thiamethoxam to honey bees.

9431

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

1. Study Objective
To determine the potential long term effects on the honey bee (Apis mellifera L.) colony healthduring and after dietary intake of thiamethoxam, including the potential effects on overwintering.
The long term exposure allows for the characterization and distinction of short-term versus chronicor sub-lethal effects.

2. Study Methods
2.1.

Test cropThis study did not include a test crop. The study was conducted in an open field where flowers
from various wild and cropped areas were available, serving as potential pollen and nectar sources
for the test bees.

2.2.

Test chemical

The test substance was technical thiamethoxam. Further details are provided in Table 1 below.
1Table 1. Details about the test substance
Test Item
Name
Test item code:
Formulation type:
Active ingredient:
CAS number:
Density (20 °C)
analysed:
Certificate ofanalysis:
Stability in solution:

2.3.

Thiamethoxam technicalNC-0421
Technical compound
Thiamethoxam
153719-23-4
Not applicable

Batch number:Appearance / colour:
Intended Usage:
Content of a.i. analysed:
Molecular Weight:
Risk symbol(s):WRS 1239/3;623769
Powder / beige
Insecticide
98.9 %
291.7 g/mol
Xn

19 March 2012

Expiry date:

31 March 2016

sufficient for the test purpose

Storage conditions:ambient

Test sites

The field and sampling phases of this study were conducted by Eurofins Agroscience Services Inc.
(EASI), at Cedar Grove Research Station, located in Mebane, NC, USA. Analysis of samples forresidues of thiamethoxam in nectar and bee bread/pollen collected from hives was performed by
EPL Analytical Laboratory, IL, USA. The apiary sites were located in the vicinity of the EASI
Cedar Grove Research Station in Orange, Caswell, Person and Alamance counties, North Carolina.
There were 12 apiaries separated by at least 1 mile. Land use surveys in 1- mile radius and 3-mileradius were conducted. Pollen species identification and multiple pesticide analysis (174 active
ingredients) were conducted using pollen samples collected from the monitoring hives tocharacterize outside food sources of the test bees and contamination. These pollen samples werecollected for a period of 24-48 hours using pollen traps for 8 sampling events according to theschedule below (Table 2).

10432

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Table 2: Pollen Trap Sample Dates for Residue Analysis and Pollen Source Identification
Pollen sample Event

Date

Timing*S1

7-Jul-2014

CCA3 (1 BE)

S2

16-18 Jul 2014

1 WAE

S3

28-30 Jul 2014

CCA 4 (3 WAE)

S4

15-Aug-2014

5 WAE

S5

20-21, 25 Aug 2015

CCA5(6 WAE)

S6

16-Sep-2014

CCA6 (10 WAE)

S7

7-Oct-2014

CCA7 (13 WAE)

S8

28-Oct-2014

16 WAE*If at a specific CCA that is noted otherwise timing is based in weeks after exposure
WBE = Week before exposure; WAE = Week after exposure

1Figure 1: Locations of test apiary sites (figure taken directly from study report)
2Table 3. GPS-coordinates of the test apiary sites
ApiaryApiary A
Apiary B
Apiary C
Apiary D

GPS-coordinates

36° 12.025'N, 79° 6.560' W
36° 13.921'N, 79° 8.971' W
36° 17.013'N, 79° 13.828'W
36° 13.344'N, 79° 10.861' W

11433

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
ApiaryGPS-coordinates

Apiary E
36° 14.923'N, 79° 14.244'W
Apiary F
36° 9.880'N, 79° 10.286' W
Apiary G
36° 11.376' N, 79° 15.993'W
Apiary H
36° 15.681'N, 79° 11.787'W
Apiary I
36° 14.961'N, 79° 10.517'W
Apiary J
36° 13.372' N, 79° 12.486' W
Apiary K
36° 13.937' N, 79° 14.364' W
Apiary L
36° 11.367'N, 79° 10.161' W
From Table 2, page 19 of the study report.

2.4.

Test organisms

The test species was the honey bee (Apis mellifera), Italian race (A. mellifera ligustica). Hives
were established from package bees bought from the commercial bee supplier (J J’s Honey, 5748
Chancey Road, Patterson, GA 31557, USA), typical of the bee stock used in commercial
beekeeping operations. A new queen was introduced into each colony. All queens were purchasedfrom the package supplier. The colonies were maintained in 10-frame Langstroth boxes with anempty deep super on top as a feeder box. In the test field, hives were placed on a pallet. Morethan 100 inspected hives were screened based on the outcome of the second Colony Condition
Assessment (CCA2). Hives were checked for the “appearance” of a healthy colony with no visible
symptoms of Varroa or Nosema, as well as having all stages of brood, a queen, and some foodstores.
Eighty-four hives that met the following conditions by the third Colony Condition Assessment(CCA3) were used in the biological evaluations in the study:• At least 3 brood combs containing brood (actual: 3-12 brood combs with all brood stages;
exceptions apiary B T2: 0 frames, apiary H T2: 1 frame, apiary D T3: 1 frame, due toprevious swarming)• At least 3 combs containing honey or pollen (actual: 4-16 combs containing food)• Honey bees were free of Nosema and Varroa disease symptoms and other bee diseases.
In addition, a monitoring hive was placed at each apiary for collection of pollen and nectar whichwere analyzed for pesticide residues and pollen source identification (total 96 hives included instudy). At CCA3, the start of the dosing period, each colony consisted of one to two brood boxes
with 10 frames in each box and an mean estimated 8264 (T2) to 11040 (UTC) adult honey bees
per colony.
The eighty-four hives were blocked into 12 apiary sites by colony strength (measured by coverageof brood), starting with Apiary A as the strongest group of hives, and Apiary L as the weakest
group of hives with an additional hive at each site for pollen sample collection. Assignment of
apiaries to the geographic locations was done randomly. Hives were moved from the holding yards
to the study apiary site locations during the period from 27 Jun 2014 to 01 Jul 2014.

12434

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Each hive was spatially isolated from other treatment rates by 30 feet (9 m) spacing at each apiarysite (Figure 2). Hives were arranged in a semi-circular pattern, facing east to west, with 125 feet
(38 m) spacing between the two end hives.

2Figure 2. Layout of test hives in a test site (figure taken directly from study report).
During the study, all hives were treated for Varroa with two applications of Apiguard® (active
ingredient: thymol) following typical apicultural practice for the region. The initial application
occurred immediately after CCA5 (5/6 Sep, 2014) and the second application took place on 22/23
Sep 2014 to attempt to prevent high mite loads. No treatments for any other hive pests, predators
or diseases were administered to any hives.
The monitoring hives were used for pollen sample collection. In addition, one test item dosing
solution in a sealed container was placed in the monitoring hive at each apiary at 2 feeding
events (1 and 5 weeks after exposure initiation), resulting in 2 stability samples for each test item
concentration or controlin order to assess thiamethoxam stability under field test conditions.
These stability solutions were not available as a food source to the monitoring hives.

2.5.

Treatments

There were:• 12 replicates per treatment group (apiaries),• 6 treatment groups (5 test concentrations and control): 0, 12.5, 25, 37.5, 50, or
100 µg/L.
• At each site, there were 2 control hives, one hive for each test concentration, and one
hive for pollen source/contamination monitoring.
• The monitoring hive had different treatment group concentrations (unavailable to bees)
to measure stability.
The individual treatment groups, the respective feeding rates and the respective feeding volumes
are summarized in Table 4. The assignment of each test hive at 12 apiaries is summarized in Table
5.
3
13
435

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Table 4. Treatment groups, feeding rates and feeding volume
Treatment Group

Code

Feeding Timing

1: UTCUTC

2: Lowest test
item rate
3: Low test
item rate
4: Moderate
test item rate
5: High test
item rate
6: Effect test
item rate

T1

Twice a week, overa duration of six
weeks (= 12feeding
events) from 1 week
after CCA3 to
CCA5 (=A1-A12)

T2
T3
T4
T5

NominalFeeding Volume
Concentration(µg a.i./L
solution)--8 X 1000 mL+ 4x
1500mL
12.5
8 X 1000 mL+ 4x
1500mL25
8 X 1000 mL+ 4x
1500mL37.5
8 X 1000 mL+ 4x
1500mL50
8 X 1000 mL+ 4x
1500mL100
8 X 1000 mL+ 4x
1500mL

Total dose
(mg a.i./hive;
nominal)
0
0.6
1.2
1.8
2.4
4.8

From page 23 of the study report.

4Table 5. Hive assignment to test apiaries
Treatment
group

Apiary
A

B

C

D

E

F

G

H

I

J

K

L

UTC

A1

B7

C7

D1

E1

F2

G1

H1

I6

J6

K2

L1

UTC

A4

B4

C2

D7

E7

F4

G8

H6

I8

J7

K8

L7

12.5 ppb

A5

B6

C8

D5

E2

F8

G4

H4

I4

J5

K6

L2

25 ppb

A3

B3

C5

D2

E3

F7

G5

H2

I1

J2

K5

L6

37.5 ppb

A6

B1

C3

D3

E6

F5

G6

H5

I5

J8

K7

L8

50 ppb

A2

B5

C4

D6

E8

F6

G7

H3

I7

J1

K3

L3

100 ppb

A7

B8

C1

D8

E5

F1

G3

H7

I3

J4

K1

L4

A8
B2
C6
D4
E4
F3
G2
H8
I2
J3
K4
L7
Monitoring
From Table 3, page 54 of the study report.
Grey highlighting indicates the treatment level the monitoring hive received in sealed sucrose solution to test
stability. For example, the monitoring hive (position 8) in Apiary A received a 50 ppb (T4) sealed bottle for storage
stability.

2.5.1. Preparation of stock solution
Stock solution was created by combining 0.050 g of technical Thiamethoxam, dissolved in approx.
20 mL of acetone, and diluted to 1000 mL with tap water (2% solvent). After preparation, the stock
solution was stored in a refrigerator until use or replacement. Stock solution was replaced twice
during feeding on 21 Jul 2014 and 01 Aug 2014.

2.5.2. Preparation of sugar solution
For feedings prior to 4 Aug 2014 (1000 mL), sugar solutions were prepared by combining 10.9 kg
tap water with 10.9 kg of sugar in a 5-gallon (19 L) container to make approximately 17 L of sugar
14436

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
solution. For feeding from 4 Aug 2014 onward (1500 mL), sugar solution was created by
combining 5.8 kg tap water with 5.8 kg of sugar to make approximately 9 L of sugar syrup.

2.5.3. Preparation of feeding solution
For the 1000 mL feeding volume 08 Jul 2014 – 03 Aug 2014):• 12.5 µg/L: mixing 4.25 mL of stock solution into the 17 L of sugar solution• 25 µg/L: mixing 8.5 mL of stock solution into the 17 L of sugar solution• 37.5 µg/L: mixing 12.75 mL of stock solution into the 17 L of sugar solution• 50 µg/L: mixing 17 mL of stock solution into the 17 L of sugar solution• 100 µg/L: mixing 34 mL of stock solution into the 17 L of sugar solution
For the 1500 mL feeding volume (04 Aug 2014 onward):• 12.5 µg/L: mixing 2.25 mL of stock solution into the 9 L of sugar solution• 25 µg/L: mixing 4.5 mL of stock solution into the 9 L of sugar solution• 37.5 µg/L: mixing 6.75 mL of stock solution into the 9 L of sugar solution• 50 µg/L: mixing 9 mL of stock solution into the 9 L of sugar solution• 100 µg/L: mixing 18 mL of stock solution into the 9 L of sugar solution
As noted previously, acetone was used in the stock solutions. It appears that no acetone was added
to the control and no attempt was made to equalize the concentration of acetone in the treatmentgroups. Given the small volume of stock solution added to the 17 and 9 L sugar solutions, theconcentration of acetone in the treatment solutions was 0.0005-0.004%. Although having anunequal concentration of acetone in the treatment groups and control represents an additionalvariable in this study, the low concentration of acetone suggests that this aspect of the study design
should not have a substantial impact on the study results.
The test concentrations were reported as “ppb” in the study report. However, the values are in fact
in the unit of µg/L, not ppb (µg/kg). For example, 12.5 µg/L: can be calculated by 4.25 ml * (0.051
g /1020 ml)/17 L.
The test solution density was not provided. Assuming the density of a 50% sugar solution is 1.2296
g/ml, 2 the reviewer calculated that the test concentrations (Table 6) at 12.5, 25, 37.5, 50, and 100µg/L are equivalent to 10.2, 20.3, 30.5, 40.7, and 81.3 (µg/kg), respectively. Residues (mean of
two measurements from week 1 and week 5) of thiamethoxam in the dose verification samples
were 9.327 ppb (Tl; 12.5 µg a.i./L), 24.089 ppb (T2; 25 µg a.i./L), 29.528 ppb (T3; 37.5 µg a.i./L),
39.693 ppb (T4; 50 µg a.i./L) and 73.748 ppb (T5; 100 µg a.i./L). This is equivalent to 91.4% (Tl),
118.7 % (T2), 96.8 % (T3), 97.5 % (T4) and 90.7 % (T5).
Table 6. Nominal and measured test concentrations.
Treatment Group
1: UTC
2: Lowest test item rate

µg a.i./L-solution
Nominal Measured*
UTC--<LOD***
T1
12.5
11.5

Code

Nominal**--10.2µg a.i./kg-solution
Measured
Percent Nominal<LOD***-9.327
91.4

Cell Biology Laboratory Manual, http://homepages.gac.edu/~cellab/chpts/chpt3/table3-2.html, accessed on Dec 12,
2014

2

15
437

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Documentµg a.i./L-solutionµg a.i./kg-solution
Nominal Measured* Nominal**
Measured
Percent Nominal
3: Low test item rate
T2
25.0
29.6
20.3
24.089118.7
4: Moderate test item rate
T3
37.5
36.3
30.5
29.52896.8
5: High test item rate
T4
50.0
48.8
40.7
39.69397.5
6: Effect test item rate
T5
100
90.7
81.3
73.748
90.7*Estimated by multiplying measured test concentration (in µg a.i./kg-solution) by assumed solution density of 1.2296
g/mL**Estimated by dividing nominal test concentration (in µg a.i./L-solution) by assumed solution density of 1.2296
g/mL***Level of Detection (LOD) = 0.25 µg a.i./kg-solution, assumed to be 0.31 µg a.i./L-solution.
Treatment GroupCode

2.5.4. Artificial Feeding
Each hive had an empty deep super on top, between the lid and the inner cover to allow dark
space to place the Boardman feeder inside the hive. This allowed the feeder to be placed on the
inner cover so that the bees had easy access without allowing the feeder to come into constantcontact with light. Control and treatment solutions were sealed inside each hive and were notaccessible outside of the hive.
The treated sugar syrup was prepared one day in advance for each feeding event and stored
overnight at room temperature. The spiked sugar solution was provided to the treated hives twicea week for six weeks, starting on 08 Jul 2014 and ending with last retrieval on 19 Aug 2014. At
each renewal, 1000 or 1500 mL of freshly prepared sugar solution was provided to each colony
and any solution remaining from the previous feeding was removed and measured. Renewal of thesugar solution was separated by at least one day. The study observation period was 20 May, 2014
(CCA1) – 28 Apr, 2015 (CCA10), which includes the overwintering period. In fall and over-winterthe surviving colonies were fed with 1 L of 2:1 sugar solution.

2.6.

Meteorological Data

Temperature, humidity and rainfall data were obtained from two apiary sites (from the EASI
weather stations located at Apiaries K and J; distance to the other apiaries between 0.1 to 7.5
miles). Data from Apiary K were the only data reported as the study authors stated data from theweather station at apiary J (Pope Farm) are nearly identical to the data from the weather station
at Apiary K.
According to weather station at apiary K, a total of 8.3 inches (211 mm) of rainfall accumulated
throughout the exposure period - including CCA3 (from 02 Jul 2014 to 15 Aug 2015), with 4.4
inches (112 mm; 02 - 31 Jul 2014) in July and 3.9 inches in August (99 mm; 01-15 Aug 2014).
The on-site temperature minimum during the exposure period was 14 °C (57.2 °F) and thetemperature maximum was 36 °C (96.8 °F). The humidity ranged from 32-100%

2.7.

Observations

2.7.1. Dates of observation.

16438

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
The important activities and date are below in Table 7.
Table 7. Chronological list of key dates and activities
Activity codea

Study week

Timingb-903 May 2014-7
20-23 May 2014-4
09-13 Jun 2014

9(±1)WBE

Installation of bee packages (before study initiation, non-GLP)7(±l)WBE-2
27 Jun -01 Jul2014-1(±1)
02 -06 Jul 2014

2(±1)WBE

1st visual colony condition assessment (CCA1; before study
initiation, non-GLP)
2nd visual colony condition assessment (CCA2; before study
initiation, non-GLP);
Assignment of hive locations
Move hives to the test locations I apiaries

008 + 10 Jul 201412 07 2014

0WAE

A3 + A4, S2+1
14 + 17 Jul 2014

1WAE

A5 + A6+2
22 + 25 Jul 2014
+3(±1)
28 - 31 Jul 2014

2WAE

+3
29 + 31 Jul 2014+4
04 + 07 Aug
2014
5
11 + 15 Aug
2014

3WAE

4th visual colony condition assessment (CCA4); Assessment ofcolony strength with digital images;
Sampling of nectar and bee bread (pollen) from combs frommonitoring hives;
Sampling of pollen with pollen traps from monitoring hives
Two feedings (A7 and A8)

4WAE

Two feedings (A9 and A10)

5WAE

Two feedings (Al l and Al2);
Sampling of feeding solution before and after feeding frommonitoring
hives;
Sampling of nectar and bee bread (pollen) from combs frommonitoring
hives;
Sampling of pollen with pollen traps from monitoring hives

EA
CCA1
CCA2
EA
CCA3, S1

Al + A2-

CCA4, S3

A7 + A8
A9 + A10
Al l + A12,S44(±1)WBEl(±l)WBE

0WAE

3(±1)WAE

Description

3nd visual colony condition assessment (CCA3); Assessment ofcolony strength with digital images;
Sampling of nectar and bee bread (pollen) from combs from all
biological hives;
Sampling of nectar and bee bread (pollen) from combs frommonitoring hives;
Sampling of pollen with pollen traps from monitoring hives;
Sampling of adult bees for Varroa testing from all biologicalhives; Start of hive weight recording
Start of exposure = 1st application via feeding spiked sugar syrup(two feedings per week = Al and A2)
Installation of hive scales underneath each hive
Two feedings (A3 and A4);
Sampling of feeding solution before and after feeding frommonitoring hives;
Sampling of nectar and bee bread (pollen) from combs frommonitoring hives;
Sampling of pollen with pollen traps from monitoring hives
Two feedings (A5 and A6)17439

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Activity codea
CCA5, S5

Study week

Timingb+6(±1)
20 - 28 Aug 20146(±1)WAE

Description

5th visual colony condition assessment (CCA5); Assessment ofcolony strength with digital images;
Sampling of nectar and bee bread (pollen) from combs from all
biological hives;
Sampling of nectar and bee bread (pollen) from combs frommonitoring hives;
Sampling of pollen with pollen traps from monitoring hives;
Sampling of adult bees for Varroa testing from all biological hives(19-22 Aug 2014)
CCA6, S6+10(±1)10(±1)
6th visual colony condition assessment (CCA6); Assessment of
17 - 23 Sep 2014
WAE
colony strength with digital images;
Sampling of nectar and bee bread (pollen) from combs frommonitoring hives;
Sampling of pollen with pollen traps from monitoring hives
CCA7, S7
+13(±1)13(±1)
7th visual colony condition assessment (CCA7); Assessment of
06 – 10 Oct 2014
WAE
colony strength with digital images;
Sampling of nectar and bee bread (pollen) from combs frommonitoring hives;
Sampling of pollen with pollen traps from monitoring hives
CCA8, S8
+ 16(±1)
16(±1)
8th visual colony condition assessment (CCA8); Assessment of
27 -29 Oct 2014
WAE
colony strength with digital images;
Sampling of nectar and bee bread (pollen) from combs frommonitoring hives;
Sampling of pollen with pollen traps from monitoring hives;
CCA9, S931 Mar 2015
After over- 9th visual colony condition assessment (CCA9); Assessment of
winteringcolony strength with digital images;
Sampling of adult bees for Varroa and Nosema testing from all
biological hives;
Sampling of nectar and pollen I bee bread from combs from all
biological hives;
CCA10
28 Apr 2015
After over- 10th visual colony condition assessment (CCA10); Assessment of
winteringcolony strength with digital images;aActivity code corresponds with EASSM system: A= application, S= sampling, CCA = colony condition assessmentb
WBE I WAE= weeks before I after start of exposure

18
440Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
2.7.2. Colony mortality
Any colony that did not show the presence of a queen and had no open brood or eggs, or was devoid ofworker (female) bees was considered "dead". If a hive was considered "dead" at the time of assessment, it
was no longer used in the analysis of endpoints (e.g., adult bee numbers, hive weight).

2.7.3. Colony Condition Assessments (CCA)
Hive assessments were made by two trained experts. Each expert was assigned a set of apiaries and thisperson made all assessments for the hives at those apiaries, Apiaries A, F, G, J, K and inspected by one
inspector and Apiaries B, C, D, E, H and I by another inspector. Ten CCAs were conducted during the entire
study according to the schedule in Table 8. For summary statistics, the first day is used to characterize any
given CCA.
5Table 8. Schedule for colony assessment and beekeeper checks
Assessment

Timinga

Comments

CCA1

7WBE
First colony assessment for hive selection
20-23 May 2014
CCA2
4WBE
Final assessment for hive selection and assignment of hives to apiaries and
09-13 Jun 2014
treatments (stratification)
CCA3
1WBE
Assessment before start of feeding
02-06 Jul 2014
CCA4
3WAE
Assessment during feeding period
28-31Jul 2014
CCA5
6-7WAE
Assessment shortly after feeding period
20-28 Aug 2014
CCA6
10-11 WAE
17-23 Sep 2014
CCA7
13 WAE
06-10 Oct 2014
CCA8
16 WAE
Last assessment before over-wintering
27-29 Oct 2014
CCA9
After over-wintering First assessment after over-wintering
31 Mar 2015
CCA10
After over-wintering Last assessment of the study
28-29 Apr 2015a
WBE / WAE = weeks before / after start of exposure (feeding); CCA = colony condition assessment

During the colony condition assessments, each frame was removed and inspected, with measurements forendpoints taken as percent of total frame area covered by honey/nectar, bee bread /pollen, capped brood,
larvae, eggs and adult bees. For CCA3 through CCA 10 adult bees was assessed using digital imaging.
The estimation was made by:• Each hive consisted of 20 observed panels (10 frames with two sides of each frame), with an areaof 860 cm2 per side, or a total area of 17,200 cm2 for all 10 frames.
• The observed percentage of each matrix was converted to this area ratio for the estimated area
covered by honey, pollen or brood types.
• Density was assumed to be 130 bees per 100 cm2
19441

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document••

The area in cm2 covered by bees multiplied by 1.30 gives the approximate number of bees in acolonyThe total number of cells per frame is 3440. To calculate total number of cells of brood or foodstores the percentage coverage was multiplied by 3440 for one side of a frame.

The digital estimation of adult bees was made by transferring digital photographs to a computer for
analysis using software Irfanview (version 4.38) and Mousetron (Blacksun Software (2001-2015);
versions 5.0 and 9.1, 10.0). The data were transferred to Report Number: S14-02633 Page 28 of 467 Excel(2010, versions 14.0.6 and 14.0.7) and SAS (version 9.3) for statistical analysis calculations.

2.7.4. Evaluation of Disease or Pests in the Hive
Colonies were also checked for visible symptoms of disease or pests, such as Nosema, foulbrood, Varroa
mites or small hive beetle.
The number of Nosema spores per bee was determined once during the study after over-wintering (CCA9).
To assess the presence of Varroa in the hive, bee samples were taken at the CCA3, CCA5 and CCA9. Bees
were washed in alcohol to remove mites. The number of mites per 100 bees was calculated.

2.7.5. Hive weights
The weight of each hive and temperature (recorded hourly) was monitored continuously using a digitalbalance (B-wareTM Beehive Monitoring System from Solutionbee LLC). The balances were installed
under the hives one week after the third CCA and remained until the final CCA after overwintering.
During the week of CCA3 the hive weights were recorded manually. Several balances failed sporadicallyduring the study due to technical problems so the hive weights were recorded manually (duringmalfunction) with a calibrated balance (note: the study authors provide no additional information on
timing of balance failure or manual recording).

2.8.

Residue analysis

All residue and stability samples collected from feeding solution, pollen traps, and test hives were analysed
for thiamethoxam and its major degradate CGA322704 (clothianidin) at the EPL Bio Analytical Services(Niantic, IL). Samples from pollen traps in the monitoring hives were also analysed for residues of multiple
pesticides from outside sources at USDA Laboratory in Gastonia NC. The residue results were reported asng per g of sample matrix (ppb), which is different from the test solution that was reported in µg/L. The
LOQ and LOD for thiamethoxam and its metabolite CGA322704 in each matrix are listed below:• Bee Bread: LOQ - 1 ppb; LOD - 0.5 ppb• Nectar/Honey: LOQ - 0.5 ppb; LOD - 0.25 ppb• Dose verification/Stability: LOQ - 0.5 ppb; LOD - 0.25 ppb
CGA322704 was only detected above the LOQ in one hive’s bee bread (T3) sample at CCA5. Multiple
pesticide analysis was conducted in order to monitor pesticide contamination from outside food sources
using pollen collected from pollen traps on the monitoring hives.

20442

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
All samples collected for residue analysis and pollen identification were placed in freezer storage as soon
as possible after collection. The samples for residue analysis were shipped from EASI in NC to thelaboratory of EASI in NJ on 10 Sep 2014 and 04 Nov 2014. The samples were then shipped to EPL in IL
on 02 Mar 2015. The samples for pesticide screening were shipped from EPL to the USDA Laboratory in
Gastonia NC on 22 Apr 2015.

2.8.1. Pollen from outside sources
Pollen samples were collected from pollen traps attached for 24-48 hours to the monitoring hives at eachsite to assess the potential contaminant exposure from outside sources. The amount of pollen collected fromeach hive was variable and samples were not available from every site each time. Pollen samples from themonitoring hives were taken at CCA3 (7 Jul 2014), study week +1 (16-18 Jul 2014), CCA4 (28-30 Jul 2014)
study week +5 (15 Aug 2014) CCA5, (20-21, 25 Aug 2014), CCA6 (16 Sep 2014), CCA7 (7 Oct 2014),
and CCA8 (28 Oct 2014).

2.8.2. Stored pollen and nectar in test apiaries
Bee bread (pollen) samples were collected during and after the exposure phase with pollen corers. A
sample consisted of bee bread collected from at least 3 different frames if possible. Samples wereanalyzed for residues of the test item. After start of the exposure phase, bee bread samples were collected
twice from all biological hives. The samples were taken 6 weeks after start of exposure during CCA5 andafter overwintering at CCAl0. After start of the exposure phase, bee bread samples were collected at seventime points from the monitoring hives (1, 3, 5, 6, 10, 13 and 16 weeks after start of exposure).
Honey (nectar) samples were collected during and after exposure with a single use plastic spoon or othersuitable tool. A sample consisted of nectar collected from at least 3 different frames (if available) percolony. After the start of the exposure phase, nectar samples were collected twice from all biologicalhives. Samples were collected at 6 weeks after start of exposure during the CCA5 and after over-wintering
at CCA10. After start of the exposure phase, nectar samples were collected at seven time points from themonitoring hives (1, 3, 5, 6, 10, 13 and 16 weeks after start of exposure).

2.8.3. Feeding solution and stability of test item
Dosing solution concentration and solution stability in hives was evaluated by collection of samples before
and after placement of dosing solution in monitoring hives (Table 9). Monitoring hives were set up in thesame manner as test hives except the colony was denied access to the spiked or un-spiked sucrose. Residue
samples for dose verification were taken on week +1 (14 July 2014), week 2 (12 July 2013) and week +5(11 August 2013).

21
443

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
6Table 9. Sampling schedule dose verification and storage stability of test chemical.
TimingApiary / replicate
UTC12.5 ppb
25 ppb37.5 ppb
50 ppb100 ppb
WAE = Week after exposure

Week +1 (1WAE)
03 Jul 2013
361A
362A
363A
364A
365A
365A

Week 5 (5 WAE)
02 Aug 2013
475A
476A
477A
478A
479A
480A

3. Results
3.1.

Land use near test hives

Land use pattern within a 1-mile and 3-mile radius around the 12 apiaries are summarized in Table 10.
The majority (approximately 70%) of areas near the apiaries is represented by deciduous forest andpasture/hay land covers. The cultivated crop area occupied 0.22-4.76% of the total land within 1-mileradius, and 1.71-2.69% within a 3-mile radius range from the test apiaries. Using the raw data provided,
the reviewer calculated the area of cultivated crops as summarised in Table 11. The mean area ofcultivated cropping land was 15 and 161 ha within 1 mile and 3 miles, respectively, of the radius fromeach apiary.
7Table 10. Percent (%) land use pattern
ApiaryA
0.7
5.3
Developed, Low Intensity
1.5
Developed, Medium
0.0
Intensity
Developed, High Intensity 0.0
Barren Land
0.0
(Rock/Sand/Clay)
Deciduous Forest
43.6
Evergreen Forest
5.6
Mixed Forest
2.3
Shrub/Scrub
2.0
Grassland/Herbaceous
5.0
Pasture/Hay
31.7
Cultivated Crops
0.7
Woody Wetlands
1.8
Emergent Herbaceous
0.0
Wetland
1 Mile RadiusLand Use Category
Open Water
Developed, Open Space

B
0.7
5.9
2.1

C
0.9
0.8
0.8

D
1.0
6.4
2.3

E
0.7
1.5
0.9

F
6.1
4.6
0.4

G
0.5
3.2
1.2

H
0.6
2.3
2.4

I
0.1
3.4
2.2

J
0.7
6.4
3.0

K
0.8
3.9
1.2

L
1.2
11.2
2.2

0.1

0.0

0.1

0.0

0.0

0.1

0.2

0.1

0.2

0.0

0.2

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.1

0.0

0.2

0.0

0.2

0.0

0.0

0.3

0.0

0.0

0.1

38.7
4.5
2.4
2.1
3.0
38.7
1.8
0.8

39.1
3.8
2.5
7.6
13.4
28.0
2.3
1.1

33.4
8.1
3.9
0.7
3.8
36.7
2.9
0.7

47.8
9.0
3.8
3.9
4.0
25.7
1.4
1.3

46.3
3.3
1.9
5.0
2.8
29.1
0.2
0.1

37.5
6.6
3.3
5.3
5.5
35.6
0.6
0.4

45.4
5.5
3.5
2.6
4.2
30.9
2.4
0.1

42.8
7.0
3.6
8.6
6.5
24.7
0.8
0.0

31.6
4.5
2.6
2.8
2.5
40.8
4.8
0.2

39.4
6.5
4.0
4.6
3.1
33.5
2.8
0.2

26.9
4.7
2.2
1.8
3.0
43.9
2.4
0.4

0.0

0.0

0.0

0.0

0.0

0.2

0.0

0.0

0.0

0.0

0.0

22
444

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Apiary3 Mile RadiusLand Use Category
A
Open Water
0.6
Developed, Open Space
5.9
Developed, Low Intensity
1.3
Developed, Medium
0.1
Intensity
Developed, High Intensity 0.0
Barren Land
0.1
(Rock/Sand/Clay)
Deciduous Forest
40.1
Evergreen Forest
6.1
Mixed Forest
2.1
Shrub/Scrub
2.4
Grassland/Herbaceous
4.1
Pasture/Hay
34.3
Cultivated Crops
1.7
Woody Wetlands
1.2
Emergent Herbaceous
0.0
Wetland

B
0.6
5.1
1.8

C
0.4
2.3
1.4

D
0.8
5.9
2.2

E
0.5
3.2
1.6

F
2.4
5.6
1.1

G
0.6
4.6
1.4

H
0.4
2.6
1.7

I
0.5
3.4
1.9

J
0.7
4.9
2.1

K
0.5
4.0
1.6

L
2.5
6.6
1.8

0.1

0.1

0.1

0.1

0.1

0.1

0.1

0.1

0.1

0.1

0.1

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.3

0.1

0.1

0.0

0.0

0.0

0.1

0.1

0.1

0.0

0.1

39.7
5.4
2.6
3.2
4.9
33.5
2.4
0.6

41.9
6.8
2.9
5.5
10.8
24.7
2.1
1.0

38.4
5.3
2.6
3.4
4.2
34.1
2.5
0.6

41.6
5.9
2.8
6.2
7.1
28.1
2.5
0.4

44.3
4.7
2.1
3.3
3.5
31.0
1.1
0.8

37.8
7.3
2.6
4.3
5.3
33.7
1.7
0.6

41.9
6.6
3.5
5.2
7.3
27.7
2.7
0.3

41.8
6.0
3.3
4.5
6.0
29.7
2.5
0.3

37.9
5.5
2.9
4.7
4.1
33.8
2.5
0.7

40.4
5.7
2.9
5.4
5.1
31.3
2.7
0.4

37.2
4.7
2.1
2.7
3.3
36.1
2.0
0.8

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

8Table 11: Cultivated cropping area near each test apiary
ApiaryAB
1-mile radius (813 ha)
Cultivated0.7
1.8
Crops (%)
Area ofcultivated 5.7
14.6
crop (ha)3-mile radius (7323 ha)
Cultivated
1.7
2.4
Crops (%)
Area ofcultivated 125.2 172.8
crop (ha)

3.2.

C

D

E

F

G

H

I

J

K

L

Mean

2.3

2.9

1.4

0.2

0.6

2.4

0.8

4.8

2.8

2.4

1.9

18.7

23.6

11.4

1.6

4.9

19.5

6.5

39.0

22.8

19.5

15.7

2.1

2.5

2.5

1.1

1.7

2.7

2.5

2.5

2.7

2.0

2.2

153.8

181.6

183.8

77.6

125.2

195.5

186.0

183.8

197.0

148.7

161.1

Sources of pollen from monitoring hives

Pollen samples from the monitoring hives were taken at CCA3 (7 Jul 2014), study week +1 (16-18 Jul 2014),
CCA4 (28-30 Jul 2014) study week +5 (15 Aug 2014) CCA5, (20-21, 25 Aug 2014), CCA6 (16 Sep 2014),
CCA7 (7 Oct 2014), and CCA8 (28 Oct 2014).
The majority of the pollen originated from local sources, bees clearly favored five types:
Chenopodium, Plantago, Rhus, Ambrosia, Helianthus and Asteraceae-type. Bees also foraged on plants
such as Parthenocissus, Zea mays and Lagerstroemia. Nearby agricultural fields, included mostly tobacco(Nicotiana) and corn (Zea mays) fields. These were identified occasionally, and took up the maximum of
78.0% and 40.0% of the total pollen particles, respectively. Soybean pollen was also found in highproportions (83.2%) in hives sampled on CCA4. Maximum values found during an assessment are presented

23445

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
below in Table 12. Note these are not necessarily from the same apiary/hive, just the time ofassessment. (Full results can be found in Tables 52-59 of the study report (pages 150-157).
Table 12. Maximum pollen percentages for agricultural crops found in a hive during any assessment.

Soybean-1 WAE

1WAE

--

--

Corn

30
Hive C6

Tobacco7.8
Hive H8

Sunflower

3.3.

40
Hive G2

3WAE
83.2
Hive G2(71.8%
in F3)
22
Hive B2

78
Hive H8

17.8
Hive H85WAE
7Hive F3
1.4
Hive K4

--

6WAE

10WAE

13WAE

16WAE

--

--

--

--

--

--

--

--

--

--

--

4.2
Hive D4

-

77
Hives
D4 andB2(50 and
54% in
A8 andD4)

8.2
Hive J3

66.8
Hive E4(33% in
H8)-

Consumption of spiked sucrose

Hive sugar solution consumption rates ranged from 6910 mL to 14000 mL of the total 14000 mL provided
per hive during 6-week dosing phase. The sugar solution for most hives in all treatments was consumed
completely, excluding the 100 µg a.i/L dose group (see Figure 3). The total food consumption of H7 in the
100 ppb treatment was 6910 mL; while all the other hives in the 100 µg a.i/L consumed between 12320 and
14000 ml of sucrose solution. In the other treatment groups, the least amount or sucrose solution consumed
was 11970 mL (T2 hive J2).

24
446

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

Mean Consumed Sucrose per Hive in mL

14200
14000
13800
13600
13400
13200
13000
12800
12600
UTC

12.5

25

37.5

50

100

Treatment Group

3Figure 3. Mean total food consumption (mL) per colony during the 6-week exposure period

3.4.

Examination of pesticides from other sources

Pollen samples were collected from monitoring hives at each apiary by means of a pollen trap eight times
during the study for subsequent multiple pesticide screening. Acephate (1 sample, 1600 ppb), Carbaryl (1sample, 214 ppb), Carbendazim (5 samples, traces - 1300 ppb), Imidacloprid (2 samples, 4.3 and 6.1 ppb),
Methamidophos (1 sample, 109 ppb), Thiamethoxam (2 samples, 8.8 ppb and 11.3 ppb), and Thymol (23samples, 405 ppb to 12300 ppb; during period of Varroa treatment) accounted for the highest residue
levels found in these pollen samples.

3.5.

Confirmation of test concentrations

Thiamethoxam and CGA 322704 (clothianidin) were analyzed from feeding solutions sampled before andafter placement of dosing solution in monitoring hives. Dosing solution was placed in monitoring hives 1
week and 5 weeks after initiation of the exposure period. Duplicate samples (A for residue analysis and R
for retained or backup sample) of at least 5 g dosing solution were collected at each sampling event. Doseverification samples were collected from just before placement of solutions in hives, one sample per dose =total of 6 samples). The data are tabulated below in Table 13.

25447

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Table 13. Dosing solution residue data from 14-17 July 2014 (Week 1) and 11-15 August 2014 (Week5)

Measured thiamethoxam
Measured CGA322704
Average ofconcentrations(ppb*) (n=2)
concentrations(ppb*) (n=2)
measuredconcentratio(µg/L)(ppb*)
17 Jul, 201415 Aug, 201417 Jul, 201415 Aug, 2014
ns (ppb)^000 (Control)
0
<LOD†
0
0
0
<LOD
12.5
10.2
9.327
10.715
7.939
0
0
25
20.3
24.08926.50321.674
<LOD†
<LOD
37.5
30.5
29.52833.70325.352
<LOD
0
50
40.7
39.69342.02437.362
0
<LOD
100
81.3
73.74871.94175.554†: LOD=0.25 ppb for thiamethoxam; LOQ=0.5ppb^ Equivalent to 91.4 % (Tl), 118.7 % (T2), 96.8 % (T3), 97.5 % (T4) and 90.7 % (T5)*ppb = µg a.i./kg-solution (calculated by dividing nominal µg/L concentration by assumed 50% sugar solution density of 1.2296

Nominalconcentrations

3.6.

Stability of the test item in feeding solution

Stability of thiamethoxam in the sugar solution during the feeding period was examined from diet collectedfrom closed-off feeding solutions placed in the monitoring hives, sampled twice on 17 July 15 August 2014.
One test item dosing solution was placed in the monitoring hive at each apiary at 2 feeding events,
resulting in 2 stability samples for each test item concentration or control (two samples per dosing solution
concentration = total of 12 samples). The solutions were contained and sealed in 15 mL Falcon tubes andwere placed inside the monitoring hives. The samples remained in the hives until the next dosing solutionchange and feeding event. The stability samples were separated from other samples during storage andshipping. Average thiamethoxam residue data for the stability solution are presented in Table 14.
Table 14. The stability of thiamethoxam in feeding solution on 14-17 July 2014 (Week 1) and 11-15
August 2014 (Week 5)
Nominal concentration(µg/L)(µg/L)
Control
12.5
25
37.5
50
100

(ppb*)
0
10.2
20.3
30.5
40.7
81.3

Average of measured
concentrations(ppb)^

Number ofsamples measured

Measured thiamethoxam concentrations(ppb)** †17 Jul, 2014***
15 Aug, 2014***
4
0
0
0
0
4
13.5778.768
10.27910.711
4
22.33416.14222.00621.527
4
28.23840.53738.16831.800446.13542.97538.74444.340495.77771.58581.15280.740**ppb = µg a.i./kg-solution (calculated by dividing nominal µg/L concentration by assumed 50% sugar solution density of 1.2296** All CGA322704 concentrations were reported as 0.0*** Samples were collected 17 Jul 2014 (week 1; placed on 14 Jul
2014 = duration inside hives 3 days) and 15 Aug 2014 (week 5; placed 11 Aug 2014 = duration inside hive 4 days)†: LOD=0.25 ppb for thiamethoxam; LOQ=0.5ppb
010.83420.62734.18643.04982.314^ Equivalent to 106.2 % (Tl), 101.6 % (T2), 112.1 % (T3), 105.8 % (T4) and 101.2 % (T5).

3.7.

Residues in hive matrices

3.7.1. Thiamethoxam residues in hives prior to the feeding exposure

26448

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Bee bread (pollen) and nectar (honey) samples were collected at CCA3 from all hives (biological andmonitoring) before the exposure phase. Samples were collected using pollen corers and spoons, and wereanalyzed for pesticide residues (174 active ingredients or metabolites). A pooled sample was collected frombee bread at least 3 different areas of frames per colony. The limit of detection for thiamethoxam in pollen
for this study was 0.5 ppb, and for honey was 0.25 ppb. Prior to exposure neither thiamethoxam nor
CGA322704 were detected in nectar/honey.

3.7.2. Residues in hive matrices during and after feeding exposure
After start of the exposure phase, nectar/bee bread samples were collected twice from all biological hives.
The samples were taken 6 weeks after start of exposure during CCA5 and after overwintering at CCA9.
After start of the exposure phase, bee bread samples were also collected at seven time points from themonitoring hives (1, 3, 5, 6, 10, 13 and 16 weeks after start of exposure). Ranges in the biological hives
are presented in Table 15 below. Details from all residue analyses is presented in Appendix 4 of the study
report.
Table 15. Residues of thiamethoxam and CGA322704 in Biological Hives1,2
Bee Bread

Honey

Treatment(µg/L)

CCA5

CCA9

CCA3

CCA5

CCA9

UTC

<LOD-1.303<LOD-<LOQ<LOD-<LOQ

<LOD

12.5

2.599-10.020

NS

<LOD
<LOD

<LOD-17.675

NS25<LOD-17.038<LOD-<LOQ<LOD<LOD-27.554<LOD-2.746<LOD-28.060<LOD-5.136<LOD-61.806<LOQ-21.899

37.5

10.368-24.012<LOD-2.42

<LOD

50

10.984-34.960<LOQ-4.084

<LOD
<LOD

100
25.085-43.005
NS<LOD-102.030
NS
Pollen – LOQ = 1ppb, LOD=0.5; Honey - LOQ=0.5 ppb, LOD = 0.25 ppb
NS = None surviving
1– CGA322704 was detected at CCA5 in the 37.5 treatment group in one sample (hive D3) at 1.362 in bee bread
2– CGA322704 was reported as < LOQ or < LOD in all honey samples

There were some detections of thiamethoxam in bee bread of the biological control hives. A CCA5 hives
in Apiary K (hives K2 – 1.269 ppb and K8 – 1.303 ppb) had detects above the LOD. Additionally, twomonitoring hives that thiamethoxam detects above the LOQ (F3 – 0.888 ppb and L7 – 0.752 ppb). These
are considered not to impact the results of this study. At CCA5 (after exposure) Thiamethoxam
concentrations in bee bread ranged from 21-80, 0-68, 28-64, 22-70, and 25-43% of nominal concentrationsin the 12.5, 25, 37.5, 50, and 100 µg/L treatment groups respectively. In honey at CCA5 the thiamethoxam
concentrations ranged from 0-142, <LOD-110, 0-75, <LOD-124, and 0-102% of nominal concentrations in
the 12.5, 25, 37.5, 50, and 100 µg/L treatment groups respectively.
Considering the stability of thiamethoxam in the test solution, the reduced concentrations ofthiamethoxam in hive matrices likely indicates that test bees were also foraging for pollen and nectar fromoutside floral sources. However, the residue concentrations and consumption of the sugar solutions
confirms bees were being exposed to thiamethoxam.

27449

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
3.8.

Pathogens

The colonies were managed as typical for good practice in the region, including applying miticide
treatments Varroa mites and Nosema were quantified in test hives. Besides a standard treatment for
Varroa mites, no treatments for any other hive pests, predators or diseases were administered to any hives.
Applications of Varroa treatments were made based on the assessments of control hives only and were
performed after consultation with the study sponsor. All hives were treated equally when such practices
were employed.

3.8.1. Varroa Presence
Varroa mite occurrence in the colonies was assessed once before start of exposure – CCA3, once after
exposure (before overwintering) – CCA5 and once after overwintering – CCA9. To remove and countmites, bees were washed in alcohol and the number of mites per 100 bees was calculated.
Varroa infestation levels were similar for control, 12.5 µg/L group, lower for 25 µg/L and 37.5 µg/L treatmentgroups, increased at 50 µg/L treatment and was highest at 100 µg/L The highest mite load was found insamples collected at CCA5 (August 2014) ranging from a mean of 1.5 mites per 100 bees (37.5 ppb) to
2.6 mites per 100 bees (100 ppb). After over-wintering, the colonies of all treatment groups were on asimilar infestation level ranging from 0.3 mites per 100 bees (37.5 ppb) to 0.6 mites per 100 bees (25 ppb)

Number of Mites per 100 bees

3
2.5
2
1.5
1
0.5

*

*

0
CCA3

CCA5
UTC

T1

T2

T3

CCA9
T4

T5

* All hives dead

4Figure 4. Mean number of Varroa mites per 100 bee

3.8.2. Nosema presence
The number of Nosema spores per bee was determined once during the study at CCA9. The number ofspores per bee was 1,500,000 (UTC), 750,000 (25 ppb), 3,966,667 (37.5 ppb) and 2,637,500 (T4). Thismeasurement was taken at a time when all the hives in T1 and T5 were dead. There was no correlation
between test item treatment and Nosema infestation level.

28450Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
3.9.

Hive mortality

The study authors reported that 69 out of 84 colonies were considered deceased by 26 April 2015 (Table
16). Colonies were considered dead if they did not show presence of a queen and had no open brood or
eggs or was devoid of worker (female) bees. The study authors maintain that the late start of maintenance(supplemental) feeding after the dosing phase (before winter) led to low bee populations going into winterand in combination with a cold spring, many colonies did not survive the over-wintering period, andconsequently data were not used for interpretation and conclusion related to over-wintering successbetween treatments. Once a hive was declared dead, it was physically removed from the study site andfrom subsequent data analysis. Since successful overwintering in the controls was not achieved, it isdifficult to discern treatment related effects. Control mortality after overwintering was higher (79%) thanmortality in the 25 (67%) and 50 (67%) µg/L groups.
Table 16. Hive survival at CCA9 (after overwintering)
ApiaryDeceased
Colonies(%Mortality)

Treatment
group

A

B

C

D

E

F

G

H

I

J

K

L

UTC

A1

-

-

-

E1

F4

G8

-

-

-

-

L5

UTC
12.5 ppb

-

-

-

-

-

-

-

-

-

-

-

-

12/12 (100%)

25 ppb37.5 ppb

A3
-

-

C5
C3

D2
D3

-

F5

-

-

-

-

-

L6
-

8/12 (67%)
10/12 (83%)

50 ppb

A2

-

-

-

-

F6

G7

-

-

-

-

L3

100 ppb

-

-

-

-

-

-

-

-

-

-

-

-

8/12 (67%)
12/12 (100%)

19/24 (79%)

Hive Mortality (%) After Overwintering
100
90

PErcent Mortaily

80
70
60
50
40
30
20
10
0
UTC

12.5

25

37.5

50

100

Treatment Group

Figure 5. Hive mortality after overwintering.

29451

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
3.10.

Hive Weight

The figure below is taken directly from the author’s study report. Hive weights oscillated similarly with the
exception of the 37.5 (higher) and 100 µg/L (lower) treatment groups in September of 2014.

Figure 6. Proportion of hive weight following exposure of honey bees to varying concentrations ofthiamethoxam in the diet across July through the hives that survived overwintering (taken directly from thestudy report).

3.11.

Colony Condition Assessment Response Variables

What follows is a breakdown of each response variable assessed and the significant effects that were
determined at each CCA (after set up and prior to overwintering; i.e., CCAs 3-8). A couple of general points
are made below when examining the results data analysis:•

Unless explicitly stated otherwise, all discussion of statistical findings refers to the EPA statisticalanalysis. All analyses considered effects at both the 0.05 and 0.1 alpha levels when weighing
statistically significant effects with biological considerations. For simplicity and consistency invisualizing the trends and findings of statistical significance simultaneously, the EPA-generated
figures are presented below.
30452

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
•
•

•

•
•

As noted above, the data counts of individuals (adult bees) or number of occupied cells (immature
life stages and food stores) which have been scaled (divided by 1000) of each response variable tofacilitate convergence of the statistical model.
The table values are the percent reductions of the response model-based mean for a given treatment
relative to the control model-based mean. The model-based means are the Least Square means basedon the randomized complete block, repeated-measures design and model fit using SAS PROCMIXED algorithms. These Least Square means may differ from arithmetic means due to missing
values in the raw data.
The figures with colored significance “dots” representing p-values of <0.05 or <0.10 were based on
the results of the mixed model analyses conducted by EPA. off of these counts for each hive for each
response variable (with the exception of hive weight) and were generated by EPA. The figuresindicate statistical significance (reduction in treatment mean relative to control within a CCA) withblack and red “dots” denoting a significant reduction at the 0.10- and 0.05-alpha levels, respectively.
While it is not depicted in the figures below, it is acknowledged (and addressed in a variety of ways
through the various statistical approaches and discussion) that there was considerable variability forsome response variables at certain treatment groups and CCAs. Please refer to Appendix A or
Appendix B for summary statistics tables (i.e. max, min, standard deviation values) of the
proportions of each response variable for further information.

3.11.1.

Statistical Analysis

What follows are brief summaries of each of the statistical analyses employed for the review of this study.
When discussing the results both statistical and biological lines of evidence are weighted in the final
evaluation.

3.11.1.1. Study Authors Analysis
The study author conducted statistical analysis using SAS (version 9.3) including brood and hive weightdata as well as number of bees. For the exposure data, all tests were done in a two tailed approach, whereas
for the data assessed after application, one tailed (lower) tests were conducted.
Data for the test item treatments (Tl, T2, T3, T4, and T5) and the untreated control UTC were checked fornormality using Shapiro-Wilks Test (p 0.05). Data were analyzed for homoscedasticity using Bartlett Testfor data with well proven normality (p-value according Shapiro-Wilks test above p=0.2) and Levene Testfor data with poor fit to normal distribution. If Box-Cox transformation of the data improved normality or
homoscedasticity in a way that leads to the option to use statistical tests with higher power, Box-Coxtransformed data were used for statistical analysis. For data where normality and homoscedasticity wereproven, Dunnett's t-Test was used to check for possible statistically significant differences of each measured
subject in Tl, T2, T3, T4 and T5 compared to the control. If homoscedasticity was disturbed but normality
was given, Bonferroni-Holms corrected Satterthwaite t-Test (same as Welch-Test) was used for analysis.
In case of disturbed normality, Bonferroni-U Test was conducted. Each of the tests were conducted with p< 0.05 as indicator of statistically significant difference.
An analysis of covariance (ANCOVA) was performed on the Box-Cox transformed data for the data
generated at the colony condition assessments. To eliminate factors with possible impact on the data other31453

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
than effects of treatment, the different apiaries as well as the last values of each observed subject were used
as covariates for ANCOVA models. Three models were tested: ANCOVA with apiaries (blocks) as
covariates, ANCOVA with apiaries and pre-treatment values as covariates, and a one-way ANOVA. The
decision for which parameter was used for ANCOVA or if a simple ANOVA was conducted instead of the
ANCOVA was made based on the significance of the impact of the parameter for the analysis. Impacts ofcovariates on the model were analysed using significance tests (in particular, F-tests) to determine whether
the pre-treatment values or apiary influence the posttreatment values of each parameter. If the covariate wasfound to be significant an analysis of covariance was selected, whereas if the covariate is found to be nonsignificant an analysis of variance was selected. For each assessed subject, the pooled estimate of residual
error variance obtained from the selected form of analysis (ANOVA or ANCOVA) were used to compare
each treatment to the control using a two-sided Dunnett’s t-test at the 5 % significance level.
If an analysis of covariance was selected, the transformed means (and therefore the de-transformed means)
were adjusted for the effect of the covariate. Adjusting the means involves removing all differences between
the treatment groups that can be accounted for by the covariate.

3.11.1.2. Study Reviewer Analysis
During the review of the study, a separate statistical analysis was conducted using the raw data submitted
by the study author. As part of the collaborative review effort of the study EPA, PMRA, and CDPR, will
all be reviewing the study; however, the discussion in the Colony Condition Assessment section is mostly
based on the statistical approach completed by EPA. A detailed description of the methods, including
statistical model selection and parameterization, are presented in Appendix A, what follows is a briefsummarization of the EPA’s method.

3.11.1.2.1.

EPA Analysis

The same statistical analysis approach was used for all the analyzed endpoints. The evaluated endpoints
included:• Number of adults divided by 1000
• Number of egg cells divided by 1000
• Number of open (larvae) cells divided by 1000
• Number of capped (pupae) cells divided by 1000
• Number of pollen cells divided by 1000
• Number of honey cells divided by 1000
• Total number of individuals (adults + eggs + larvae + pupae) divided by 1000
• Total brood (eggs + larvae + pupae) divided by 1000, and• Total food (pollen + honey) divided by 1000.
Total brood and total food are new summary variables; EPA’s Environmental Fate and Effects Division(EFED) is still evaluating their utility in providing additional information on biological effects beyond theinitial set of variables.

32454

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
No adjustments for addition or removal of supers were included in the statistical analysis. The data wereanalyzed as a repeated measures design (multiple CCAs) with a randomized complete block (apiary) andonly data obtained prior to overwintering were included. The covariance structure used for the repeatedmeasures component was compound symmetry with heterogeneous variances at each time point. The
time*treatment interaction was explored two ways:• At each post-treatment CCA, one-sided Dunnett’s test to identify treatments with mean response
significantly less than control.
• For each treatment (including control), two-sided Dunnett’s test to identify change in the responseover time relative to the initial CCA3 measurement.
The interpretations below focus on the one-sided Dunnett’s tests identifying treatment effects at each
CCA. Significant statistical results at two alpha-levels (0.05 and 0.10) were identified.

3.11.1.2.2.

PMRA Analysis

In addition to the statistical approach presented by EPA. PMRA has completed an additional statisticalanalysis. It is noted that while this method utilized a different statistical analysis approach, interpretations
based on the PMRA analysis tended to be similar to interpretations from the EPA analysis. Although the
PRMA analysis resulted in some differences in statistically significant endpoints and time periods, thesedifferences do not significantly alter the ultimate biological interpretation of the study regarding colonylevel effects leading to a clearly defined, highly-confident protective endpoint. Differences from EPA
statistical significance findings are noted (footnotes) in the specific life stages section of the DER and thedetailed method is provided in Appendix B.

3.11.2.

Colony Condition Assessment Variables - Life Stages

3.11.2.1. Adults3
Figure 6 below shows the effects on adult honey bees across CCAs and treatment groups. After the exposure
period at CCA5 the number of adults was marginally significant (0.05<p<0.1) in the 50 µg/L treatmentgroup (24.5% reduction) and significant (p<0.05) at CCA5 through CCA8 in the highest treatment group(44.9-57.2% reduction). By CCA8 adult numbers in all but the highest treatment group had converged tonear the number of adults in the control. The mean number of adults in the control colonies was actually
lower in than the treatment groups, again with the exception of the 100 µg/L treatment group.
It is apparent from all analyses that there were impacts to adults at the 100 µg/L group during the course of
the study. The number of adults in the 50 µg/L treatment was also reduced with marginal statisticalsignificance at CCA5 (0.05< p<0.1) (Table 17). However, the mean number of adults in the subsequent
CCA was not significantly different from the control adding uncertainty to interpreting the effect of thistreatment group on the colony. Qualitatively, after CCA4 the trends of the 37.5 and 50 µg/L group tend totrend closer to the 100 µg/L group (decline), while the 12.5 and 25 µg/L groups tend to trend with the3

PMRA significant difference p<0.05 at CCA4 for 100 µg/L and at CCA 5 for 50 µg/L treatment group.
33455

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
controls (no clear dose trend) as an increase in adults with the decline beginning after CCA 5. From CCA6
onward no significant differences were seen outside of the 100 µg/L group and the mean number of adults
tended to cluster similarly at each assessment. This was a later time in the year in which hives would begin
to wind down for the winter. As the colony as a whole starts to prepare for overwintering the numbers ofadults and other life stages are clearly decreased by the time of CCA6 and CCA7. During this preoverwintering phase, adult proportions decline due to natural die off of worker bees and reduced rates ofreplenishment from reduced egg laying by the queen.
Table 17. Percent reduction from control for mean number of adults
Test
concentration(µg/L)
12.5

CCA3

CCA4

CCA5

CCA6

CCA7

CCA8

18.8

18.5

15.1

15.6

1.2

-4.9

25

25.1

18.4

7.7

3.8

-3.5

-16.9

37.5

4.3

5.6

13.5

14.3

-2.3

-21.1

50

9.4

7.6

24.5*

15.3

11.3

-16.7

100

-5.8

11.0

44.9**

57.2**

56.7**

53.8**

Reduction from control (%)

Note: Negative value indicates increased number of adults in comparison to control.
*0.05<p<0.1**p<0.05
16.00

Compared
to control
0.05<p<0.1
p<0.0514.00

Adults - Counts in 103

12.00
10.00

0
12.5

8.00

25
6.00

37.5
50

4.00

100

2.00
0.00
3
2-6 Jul

428-31 Jul520-28 Aug6
17-23 Sep

7
6-10 Oct827-29 Oct

Colony Condition Assessment (CCA)
Figure 7. Number of adults (103) following exposure to varying concentrations of thiamethoxam in thediet across CCA3 – CCA8. Error bars represent standard error and the shaded box represents the exposure
window.
34456

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
3.11.2.2. Eggs 4
There were similar numbers of eggs in all treatments through CCAs 3 and 4 and again in CCAs 7 and 8.
There was marginal significance (0.05<p<0.1) in the 25 µg/L treatment group at CCA 3 prior to exposure.
Eggs at CCA4 began a sharp decline in the 100 µg/L treatment group and reduction compared to the control
was statistically significant (p <0.05) at CCAs 5 and 6. By CC7 means of egg numbers had converged tosimilar levels of controls in all treatment groups. In general, all treatment groups began a decline in thenumber or eggs after CCA 4 with the exception of the 12.5 ppb treatment group which did not begin its[sharp] decline until after CCA 5 (and appeared variable with number of eggs more similar to the control
by CCA 7). At CCA 6 the 12.5 ppb treatment group was marginally significantly different (0.05<p<0.1)
than the control before all the data converged at CCA7 for all treatment groups. The marginal significance
CCA3 for the 25µg/L group is not considered biologically relevant, as the hives trended similarly with theremaining treatments, including the controls and this was prior to thiamethoxam exposure. By CCAs 7 and8, the average of number eggs converged for all treatments, with the only apparent biological effects at the
100 µg/L treatment level.
Table 18. Percent reduction from control for mean number of eggs
Test
concentration(µg/L)
12.5

CCA3

CCA4

CCA5

CCA6

CCA7

CCA8

11.3

11.0

-16.5

38.6*

5.3

-12.8

25

32.2*

-1.0

6.2

4.9

26.6

-11.9

37.5

20.8

-0.2

-8.1

6.9

-21.0

5.1

50

7.8

-5.7

16.0

13.2

23.2

36.1

100

-7.8

8.6

44.4**

56.0**

37.8

-48.8

Estimated reduction from control (%)

Note: Negative value indicates increased number of eggs in comparison to control.
*0.05<p<0.1**p<0.05

4

PMRA significant difference (p<0.05) at CCA 6 for 12.5 µg/L treatment group35457

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
8

Compared
to control
0.05<p<0.1
p<0.05

7

Eggs - Counts in 103

6
5

0
12.5

4

25
3

37.5
50

2

100

1
0
3
2-6 Jul

428-31 Jul520-28 Aug6
17-23 Sep

7
6-10 Oct827-29 Oct

Colony Condition Assessment (CCA)
Figure 8. Number of eggs (103) following exposure to varying concentrations of thiamethoxam in thediet across CCA3 – CCA8. Error bars represent standard error and the shaded box represents the exposure
window.

3.11.2.3. Larvae (Open/Uncapped brood) 5
There were significantly lower numbers of larvae in the 100 µg/L treatment group as compared to control(p<0.05) beginning at CCA5 and continuing onto CCA6. There was also a significant reduction comparedto the controls in the 50 µg/L treatment group at CCA 5 right after exposure ended. There was also asignificant reduction compared to the controls in the 25 µg/L treatment group before exposure. By CCA6,
excluding the 100 µg/L treatment group, and after a slight increase in the 50 µg/L treatment group, numbersof larva had converged to similar to the control numbers with no significant differences in any treatmentgroup. It is uncertain if the significance after CCA5 is isolated or treatment related. The effects were notsustained at CCA6 indicating some level or recovery; however, there was a decrease in numbers after CCA
4 while the other groups generally sustained numbers or increased.
Table 19. Percent reduction from control for mean number of larvae (open/uncapped brood)

5

PMRA significant difference p<0.05 at CCA7 and marginal significant difference 0.05<p<0.1 at CCA 8 for 25 µg/L treatment
group
36
458

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Test
concentration(µg/L)
12.5

CCA3

CCA4

CCA5

CCA6

CCA7

CCA8

17.2

-3.1

8.7

3.7

-24.3

-5.1

Estimated reduction from control (%)

25

41.9**

3.3

10.0

-1.6

58.4

42.7

37.5

16.5

-4.5

7.2

-2.7

-8.8

-25.2

50

15.7

2.6

35.2**

16.5

11.5

78.8*

100

16.5

32.3

60.3**

59.6**

51.0

33.8

Note: Negative value indicates increased number of larvae in comparison to control.
*0.05<p<0.1**p<0.05

Figure 9 below shows the trends of the control and all treatment groups for larval cells across all CCAs
assessed. A clear divergence in the 100 µg/L groups is evident beginning at CCA4 where the numbers oflarvae in these groups undergo a marked decline while the other treatment groups generally (except 50 µg/L)
trend with control.

Open Cells (larvae - Counts in 103)8

Compared
to control
0.05<p<0.1
p<0.05

7
6
5

0
12.5

4

25
3

37.5
50

2

100

1
0
3
2-6 Jul

428-31 Jul520-28 Aug6
17-23 Sep

Colony Condition Assessment (CCA)

7
6-10 Oct827-29 Oct

Figure 9. Number of larval cells (103) following exposure to varying concentrations of thiamethoxam in
the diet across CCA3 – CCA8. Error bars represent standard error and the shaded box represents the
exposure window.

3.11.2.4. Pupae (Capped brood) 6

6

PMRA significant difference (p<0.05) and marginal significant difference (0.05<p<0.1) at CCA 7 for 50 and 25 µg/L treatmentgroups respectively
37459

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
In the 37.5, 50 and 100 µg/L treatment groups, there were significant reductions from control (p<0.05) at
CCA5. However, the only significant differences beyond CCA5 relative to the control were in the 100 µg/L
treatment group. There was a significant reduction relative to the control in the 25 µg/L treatment groupprior to exposure. The two lowest treatment groups (12.5 and 25 µg/L) trended most similar to the control(peak at CCA5 then sharp drop), while 37.5 and 50 µg/L treatment groups trended similar to each other(slight to no drop CCA3-5 then sharp drop after CCA6) and 100 µg/L trending uniquely (consistent decline
over all CCAs) before CCA8. By CCA6, all treatments, except for 100 µg/L, had converged to numbers
where no significant differences were detected. As with the other variables, there is uncertainty in discerning
treatment related effects beyond CCA5 as the values converge and decline potentially due to preparationfor overwintering. By CCA8 all numbers were similar for all treatment groups. Worth noting, the controls,
12.5 and 25 groups all show an increase in the number of pupae in CCA5 relative to the number of pupae
in CCA4, and that the temporal increase in the number of pupae is not seen in the 37.5, 50 and 100 groups.
This trend is prominent here (Figure 10) and in Figure 12.
Table 20. Percent reduction from control for mean number of pupae
Test
concentration(µg/L)
12.5

CCA3

CCA4

CCA5

CCA6

CCA7

CCA8

13.4

11.5

9.3

-0.6

-39.1

-56.3

25

25.3**

11.9

16.9

0.6

53.8

35.0

37.5

12.1

5.4

26.7**

2.1

-1.8

-32.8

50

9.0

20.8

42.0**

15.9

45.6

-0.7

100

6.6

47.3**

75.5**

58.1**

92.5**

14.3

Estimated reduction from control (%)

Note: Negative value indicates increased number of pupae in comparison to control.

**p<0.05

38
460

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
20

Compared
to control
p<0.05

Capped (pupae - Counts in 103)

18
16
14
12

0

10

12.5
25

8

37.5
50

6

100

4
2
0
3
2-6 Jul

428-31 Jul520-28 Aug6
17-23 Sep

7
6-10 Oct827-29 Oct

Colony Condition Assessment (CCA)
Figure 10. Number of pupal (capped) cells (103) following exposure to varying concentrations ofthiamethoxam in the diet across CCA3 – CCA8. Error bars represent standard error and the shaded box
represents the exposure window.

3.11.2.5. Total individuals in hives
Similar to the individual (adults, larvae, pupae) parameters, the total number of individuals was consistently
significantly reduced compared to the control in the 100 µg/L treatment group. Again, directly after
exposure (CCA5) there was a significant difference in the 50 µg/L treatment in which the numbers begin to
decline after CCA4. There was also a decline in total individuals in the 37.5 µg/L treatment group although
not as dramatic as in the two highest treatment groups. There was a continual decline throughout theremaining CCAs (CCA 6-8). Similar to pupae, the two lowest treatment groups (12.5 and 25 µg/L) trended
most similar to the control (peak at CCA 5 then sharp drop), while 37.5 and 50 µg/L treatment groups
trended similar to each other (slight to no drop CCA3-5 then sharp drop after CCA6) and 100 µg/L trending
uniquely (consistent decline over all CCAs). By CCA6 all treatments (with the exception of 100 µg/L) had
converged to numbers similar to that of the controls and that trend was consistent for the remaining of theassessments. From CCA4-7, numbers of individuals in the 100 µg/µg/L group were significantly lower thanin controls.9
Table 21. Percent reduction from control for mean total number of individuals
Test
concentration(µg/L)
12.5

CCA3

CCA4

CCA5

CCA6

CCA7

CCA8

15.3

11.5

7.8

12.5

-9.2

-12.2

25

29.0**

10.7

11.6

2.3

18.8

-2.1

Estimated reduction from control (%)139461

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Test
concentration(µg/L)
37.5

CCA3

CCA4

CCA5

CCA6

CCA7

CCA8

11.9

3.0

15.5

6.0

-5.6

-19.3

50

10.0

9.3

32.3**

16.3

19.9

-2.6

100

2.1

26.7**

59.8**

58.3**

61.3**

36.6

Estimated reduction from control (%)1

Note: Negative value indicates increased number of total individuals in comparison to control.
*0.05<p<0.1**p<0.05
50

Live (adults + eggs + open + capped - Counts in
103)

Compared
to control
p<0.05

45
40
35
30

0
12.5

25

25

20

37.5
50

15

100

10
5
0
3
2-6 Jul

428-31 Jul520-28 Aug6
17-23 Sep

7
6-10 Oct827-29 Oct

Colony Condition Assessment (CCA)
Figure 11. Number of total (adult, eggs, larvae, pupae) individuals (103) following exposure to varying
concentrations of thiamethoxam in the diet across CCA3 – CCA8. Error bars represent standard error andthe shaded box represents the exposure window.
Similar to pupae after CCA3, there is an upward trend in the total number of individuals in the control,
12.5 and 25 µg/L groups while the trend is downward in the 37.5, 50 and 100 µg/L groups. Without
successful overwintering it is hard to understand the significance (biological if any) of this trend if thecolonies had made it through winter.
Figure 12 below provides another visual representation of the effects across CCAs variables within a
response variable for the various life stages of bees during the course of the study. The graphs are meant toshow the general trends in the life stages for each treatment group.

40462

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
The figure illustrates clear treatment effects for all life stages in the 100 µg/L treatment group. In thatgroup, all life stages begin an immediate decline from CCA3 (which is one week before exposure) to
CCA4 (which is 1 week after exposure) and continue to decline throughout the course of the assessments.
Control, 12, and 25 µg/L treatment groups appeared to have similar trends across life stages. In the threehighest treatment groups the trends begin to diverge at 37.5 and 50 µg/µg/L as the spike in pupae is notobserved and the decline in the number of adults starts sooner. Additionally, the larvae and eggs both
begin to decline together in the highest three treatment groups. The effect is again clearest in the 100µg/µg/L where the sharp decline begins immediately after CCA 3 for pupae and adults, rather thanpeaking around CCA5 for these two life stages. These are meant to qualitatively illustrate the trends.
Variability in the estimates may contribute to the lack of statistical differences which may point to control
performance and uncertainty of the treatment effects.

41463

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

42464

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
The inability to achieve overwintering limits the utility of these results. Additionally, there were also
uncertainties with respect to the health of the hives used particularly the controls and timing of exposure.
Mostly, other than in the highest treatment group, significant reductions compared to the control hives of
biological parameters were only observed in the other treatment groups at CCA5 (the end of exposure).
Other differences occurred transiently prior to exposure concluding but were not consistent enough to besuggestive of colony level effects at other treatment levels. After CCA5 (end of exposure) there was also a
general decline in all parameters in all treatment groups (including controls), suggesting all hives were inpreparation for overwintering and limiting the ability of the study to detect treatment related effects
Healthier control colonies and an earlier exposure window may further refine the effects seen for somemeasurements.

3.11.3.

Colony Condition Assessments – Food Store Response Variables

3.11.3.1. Pollen 7
Pollen stores were significantly reduced in the 100 µg/L treatment group from CCA4 to CCA7 (p<0.05).
Pollen stores were also significantly reduced compared to the control in the 25 µg/L group at CCAs 5 and
7. At CCA4 in the 25 µg/L treatment groups and at CCA5 in the 50 µg/L dose group pollen stores were
marginally significant (0.05<p<0.1). With the exception of a slight increase in the 12.5 µg/L treatmentgroup, all treatment groups had a drop in pollen stores after exposure began (between CCA 3 and CCA 4).
However, to some degree they increased between CCA 4 and 5 with the exception of the 100 µg/L treatment.
There was no significant reduction (relative to controls) of pollen stores in the 12.5 and 37.5 µg/L treatmentgroups for any CCA assessed. The stored pollen numbers converged at the last CCA where no differenceswere detected.
Table 22. Percent reduction from control for mean number of pollen cells
Test
concentration(µg/L)
12.5

CCA3

CCA4

CCA5

CCA6

CCA7

CCA8

17.6

2.0

21.0

6.2

4.8

7.1

25

43.7**

28.3

46.3**

22.7

Estimated reduction from control (%)1

16.4

51.0*

37.5

-9.9

10.0

22.4

11.6

13.9

-14.1

50

21.0

42.1

37.8*

28.3

27.4

9.5

100

-11.1

56.8**

80.7**

74.8**

66.6**

38.6

Note: Negative value indicates increased pollen stores in comparison to control.
*0.05<p<0.1**p<0.05

7

PMRA significant reductions p<0.05 at CCAs 4 and 5 for 50 for µg/L treatment group and CCA 4 for for 25 µg/L treatment
group
43

465

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
6

Compared
to control
0.05<p<0.1
p<0.05Pollen Cells - Counts in 103

5

4
0
12.5

3

25
37.5

2

50
100

1

0
3
2-6 Jul

428-31 Jul520-28 Aug6
17-23 Sep

7
6-10 Oct827-29 Oct

Colony Condition Assessment (CCA)
5Figure 13. Number of pollen cells (103) following exposure of honey bees to varying concentrations ofthiamethoxam in the diet across CCA3 – CCA8. Error bars represent standard error and the shaded box
represents the exposure window.
In examining the trends of pollen stores in the control, there was only a slight buildup between CCA 4 andCCA 5. All treatment groups (excluding 100 µg/L) had the same buildup trend between CCA4 and CCA5
before generally decreasing or leveling off by CCA6 and decreasing thereafter. Pollen stores actually
increased (slightly) in the 12.5 µg/L treatment group until CCA 7. While there were no differences between
these two groups a similar increase would be expected in the control. This increase would support the queen
in her effort to build up brood. Pollen stores experienced (in general) a slight decline in numbers from CCA
5 to CCA 8. This downward trend reflects that the fact that up to overwintering, brood production will slow
as the hive prepares for winter and therefore there is a reduced need for pollen within the hive. The lack ofcontinual increase in the pollen stores of the control add some uncertainty if the control colonies werefunctioning well enough to collect enough food for overwintering.

3.11.3.2. Nectar / Honey 8
There were no significant reductions in honey relative to the control. There was one marginal significance(0.05<p<0.1) for a lower amount of honey stored in treatment hives at 12.5µg/L than in the control at CCA6
and thereafter (Figure 14 below). All hives generally increased in honey stores until CCA 6 with a dropoff between CCA 6 and CCA 7 before increasing again. Trends were similar for all the treatment groups.

8

PMRA marginal statically significant difference 0.05<p<0.1 at CCA 8 for 100 for µg/L treatment group.
44466

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
The 100 actually increased more quickly than the controls which could be an indication of decreased activity
in the hive due to treatment effects. All other treatments trended similar to the control.
10
Table 23. Percent reduction from control for mean nectar/honey stores
Test
concentration(µg/L)
12.5

CCA3

CCA4

CCA5

CCA6

CCA7

CCA8

19.3

24.6

31.6*

14.1

10.1

5.9

25

7.7

8.6

17.5

14.0

-3.0

-5.6

37.5

-14.1

-7.2

-1.8

-11.5

-18.6

-11.8

50

6.3

3.4

4.2

0.8

-15.4

-6.0

100

-3.0

-33.4

-47.5

1.2

-17.2

24.5

Estimated reduction from control (%)1

Note: Negative value indicates increased nectar/honey stores in comparison to control.
*0.05<p<0.1**p<0.05

Figure 14 below for the honey store trends in the control, 12.5, 25, and 50 µg/L groups only show a marked
divergence at the 50 µg/L treatment group beginning at CCA6 and persisting up to and after overwintering
at CCA8.
35

Compared
to control
0.05<p<0.1

Honey Cells - Counts in 103

30

25
0

20

12.5
25

15

37.5
50

10

100
5

0
3
2-6 Jul

428-31 Jul520-28 Aug6
17-23 Sep

7
6-10 Oct827-29 Oct

Colony Condition Assessment (CCA)
6Figure 14. Number of honey cells (103) following exposure of honey bees to varying concentrations ofthiamethoxam in the diet across CCA3 – CCA8. Error bars represent standard error and the shaded box
represents the exposure window.

45467

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
It is noted that the feeding solutions (sugar solutions) provided during the exposure period might haveaffected natural honey storage patterns (because a decline in foraging outside of the hive may not impact
the storage of honey); however, effects on honey storage are still able to be considered as all treatments
were compared to control hives (which also received feeding solutions).
Figure 15 below shows food stores (pollen and honey). The trends are similar to those for honey. With the
only a marginal significant reduction in the 12.5 µg/L treatment group at CCA 5. The higher food stores in
the 100 µg/L could be an indication of decreased activity (higher morality) and increased utilization of thesucrose (rather than outside forage sources and sucrose) in the hive or an uncertainty of how the control
hives were performing. Trends for all the remaining treatment groups were similar to the controls.

35

Compared
to control
0.05<p<0.1

Food Cells- Counts in 103

30

25

0

20

12.5
25

15

37.5
50

10

100

5

0
3
2-6 Jul

428-31 Jul520-28 Aug6
17-23 Sep

7
6-10 Oct827-29 Oct

Colony Condition Assessment (CCA)
Figure 15. Number of food cells (103) following exposure of honey bees to varying concentrations ofthiamethoxam in the diet across CCA3 – CCA8. Error bars represent standard error and the shaded box
represents the exposure window

4. Reviewer comments:
What follows is brief discussion of some of the elements taken into consideration when evaluating the resultsof this study.

46468

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
4.1.

General Considerations for Biological Interpretation

While the hive mortality is considered as the most relevant measurement of survival at the colony level,
sub-lethal effects at the colony level were estimated by measuring multiple parameters during the course of
study. Each measured parameter is expected to reflect only part of the colony conditions, and allmeasurements have to be integrated for a better understanding of the hive status at the colony level. A
honey bee colony is a super-organism in which live individuals and food supply are the two major
components in maintaining the proper function of the colony. There are interactions between the twocomponents and even within each component.
Individual bees are present in the colony as eggs, larvae, pupa and adults and they develop from one stage
to another and interact with each other to perform a variety of tasks to maintain the integrity of the colony.
The measurement of each stage of the bees is expected to provide information on the potential treatment
effect on a specific life stage of bees during their development.
Hive food supplies including hive pollen and nectar are collected and processed by adults and are expected
to have a large impact on the development of all stages of bees in hives. However, the amount of hive foodstorage is dependent on not only the number of foragers available for food collection, but also the numberof individuals that consume the food. In addition, the seasonal availability of outside pollen and nectar
sources also affects the amount of storage, thus impacting hive development. As well, sucrose feeding
solutions were provided to the hives as a means of treatment and as a supplement for hive overwintering,which may have affected foraging and food storage during those time periods.
Hive weight was measured during the study. However, it is largely affected by the honey storage and numberof bees that consume the food. A strong colony with a high number of bees likely consumes a high amountof stored honey and may result in a reduced hive weight. Weighing hives at different time periods of the
day may result in an increased variation of the measurement due to the fact that foragers may not be present
in the hive when the weight is measured. Hive weights may be artificially lower in hives which contain ahigh number of forager bees that may be out collecting food during a different time of the day.
Considerations regarding the measurement time points:
•
•
•

•
•

CCA3 represents the background hive conditions as the first colony assessment after the hives
were placed in the test fields prior to the exposure.
CCA4 and CCA5 represent the hive conditions during the exposure phase. It was noted that the
CCA5 was conducted a week after the end of the 6-week exposure period, but is expected torepresent effects during the exposure period.
CCA6 was measured at 4-5 weeks after the end of exposure. It allows all bee individuals,
including eggs, larvae and pupa that were exposed to treatment to finish their development cycle
and become adults. However, the thiamethoxam study began later in the season, and therefore, by
CCA 6, all colonies, including the controls were in decline.
CCA6, 7 and CCA8 represent the hive conditions prior to overwintering. It is considered thathives were physiologically preparing for overwintering by reducing the production of immature
bee individuals. Treatment effects may be masked by the natural decline of hive individuals.
CCA9 and CCA10 represents hive conditions of surviving hives after overwintering. Mortality inthe control and treatment hives excluded these assessments from analysis.
47469

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
4.2.

Control PerformanceControl mortality and Sub-lethal effects on life stages and food stores
The control performance in this study offers some challenges relating to the interpretation of the results.
The level of colony loss after overwintering in controls (79%) adds a great deal of uncertainty when
considering the results of individual measurements. The fact that many of the treatment hives
performed/trended similarly to the control hives could be indicative of either a lack of treatment effects or
simply the control hives were not optimal to begin the study. Because so few control hives survived
overwintering and trended closely to the treatment hives during exposure, the overwintering component
would be important to determine if the lack of significant reductions compared to the control in mosttreatment groups is biologically significant. Almost every parameter for life stages decreased after exposure
ended which could have been a factor of the time of year or treatment. The fact this also happened in thecontrol groups suggest a performance issue is possible, or at the very least an uncertainty with respect to if
the exposure measurements were taken too late in the year to discern treatment effects.
Additionally, the number of adult bees in the control hives is something to consider. Trends in the adult
bees generally increased during the early CCAs, but no sharp increase was observed. We would expect hives
to build during this time when the packages were first set up in May as there would have been pollen andnectar flows in North Carolina 9. The hive would be expected to ramp up numbers of adults to some degree
to achieve the appropriate number of bees to keep the hive alive throughout the winter. As the high loss inoverwintering colonies in the control suggests, this number of adults (while not necessarily the only factor,
but could be considered a potential contributing factor) was not achieved. In subsequent CCAs after
exposure the control measurements trended similarly to the lowest treatment groups in most cases; although
a comparison was also made difficult by the later initiation date for feeding and all hives were in decline. A
stronger performing group of control hives (potentially with a higher starting number of adults) would
increase the certainty of conclusions based on effects observed relative to these hives.
The similarity in the dynamics of all parameters for the individual living organisms at various stage acrossthe treatments indicates that control hives were may have been stressed. For most parameters and treatmentgroups the means converged to those of the control at CCA 6 through CCA 8. The time of year could haveinfluenced control hive performance, but it is still uncertain if the hives were developing normally. There
was no apparent spike of honey collection or pollen stores from the control hives indicating they may nothave been developing and storing enough food to survive the winter. Pollen stores were decreasing at thesame time other biological parameters were indicating consumption of resources but not replenishment for
the hive.

4.3.

Consideration of Study Strengths, Limitations and Interpretation

9

https://growingsmallfarms.ces.ncsu.edu/wp-content/uploads/2015/02/CALENDAR-FOR-BEEKEEPING-IN-CENTRAL-NORTHCAROLINA.pdf?fwd=no
https://www.ncbeekeepers.org/honey/floral-sourceswhats-blooming
48

470Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
It is important to recognize the inherent strengths and limitations of this study as results are interpreted andpotentially considered in risk assessment.
In the context of available field studies involving honey bees and thiamethoxam, this study contains a
number of strengths. A high degree of replication, demonstration of sustained effects at the highest
treatment level, quantification (and method validation) of both thiamethoxam and CGA322704(clothianidin), high end exposure scenario, multiple measured endpoints in detailed CCAs, and the available
raw data of these endpoints for analysis all add to the strength of the study.
However, there are limitations with this study including: Exposure through nectar (sucrose) alone may notreflect a likely field scenario where bees are probably exposed through both pollen and nectar routes. Thus
the potential effects due to prolonged exposure to both contaminated nectar and/or pollen may beunderrepresented, although, the impact of this exclusion on the study results is uncertain and will likely
depend on the life stage/caste of bee. Additionally, because exposure was channeled through both hivepollen and nectar via exposure to the sucrose feeding solution, there is uncertainty how this compares toexposure through contaminated pollen directly (a potential for foraging bees). Pesticides from food sources
other than the artificial feeding were also detected during the exposure period and post-exposure periods
through collection of pollen from pollen traps. This contributes to exposure uncertainty and can add
confounding effects when interpreting results.
Finally, a key component of this study was achieving overwintering survival. Overwintering success ofcontrols was severely impacted (79% hive mortality). In fact, no hives survived overwintering in any
significant proportion. If control hives had survived, comparing trends or lack thereof with the treatment
hives would be more meaningful. Since no sustained significant effects were seen except in the 100 µg/L
treatment it is uncertain if the control hives were performing normally during the test.

5.

Conclusions

Based on the limitations of this study, a NOAEC derived from this study is considered highly uncertain.
The key limitations include: 1) late timing of exposure that coincides with ramping down trends of colony
endpoints, 2) lower than expected performance of controls, and 3) lack of overwintering success. Effects
were observed to multiple endpoints and multiple CCAs at the highest test level (i.e., 100 µg a.i./Lthiamethoxam; 86 µg a.i./L clothianidin-equivalents). Effects to several endpoints (number of adults,
amount of brood) were also observed at CCA5 of the second highest test level (i.e., 50 µg a.i./Lthiamethoxam; 43 µg a.i./L clothianidin-equivalents). It is uncertain whether or not effects at 50 µg a.i./L
are meaningful to the colony since these effects were only observed at CCA5, which is the conclusion of
the exposure window, however, potential recovery could not be determined due to the limitations of CCA6,
7, and 8 (during downward trend of colonies) and a lack of overwintering data. Also, the utility of CCAs 6,
7 and 8 in showing treatment related effects are questionable because of the downward trend in endpoints
that is consistent with preparation of colonies for winter. If effects observed at 50 µg a.i./L are biologicallysignificant to the colonies, the NOAEC from this study would appear to be 37.5 µg a.i./L (32 µg a.i./Lclothianidin equivalents). There is uncertainty in whether or not this value is conservative. Since the hives
did not perform as expected, and given the late timing of the exposure window, it is uncertain whether or
not effects due to thiamethoxam could be detected.

49471

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
A comparison can be made between the effects of this study and those in the CFS with clothianidin (MRID
49836101; PMRA#), which was conducted in a similar location and in the same year. In the clothianidin
CFS, clear effects were observed at 40 µg a.i./L over multiple endpoints and multiple CCAs, leading to a
NOEC of 20 µg a.i./L (clothianidin). At the NOEC of 20 µg/L, some effects were observed at CCA5, butthese effects did not manifest at later CCAs. This suggests that effects were observed at lower levels in theclothianidin study compared to the thiamethoxam study. Therefore, when considering the uncertainties
described above, the apparent NOAEC for the thiamethoxam CFS is tentatively determined to be 37.5 µg
a.i./L (32 µg a.i./L-clothianidin equivalents), noting that this may value may not be conservative. The
apparent NOAEC of 37.5 µg a.i./L (thiamethoxam; 32 µg a.i./L- clothianidin equivalent) along with theeffects levels of 50 and 100 µg a.i./L (thiamethoxam) will be used to characterize the risk of thiamethoxam
to honey bee colonies. Additional information from the clothianidin CFS may also be used to characterize
the risk of thiamethoxam to honey bees. This study is considered scientifically valid; however, it isclassified supplemental due to the limitations summarized above and throughout this DER.

50
472

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

1.Appendix A: Details of EPA Statistical Analysis
Study Design and Overview of Statistical Analysis Approach
The general experimental design was a randomized complete block (apiary) with repeated measures(CCA) and data will be analyzed in SAS (v9.4) using the PROC MIXED procedure. Since hives were notassigned and placed in the study apiaries until shortly before CCA3, the data for the statistical analysis
only included data collected from CCA3 and the following CCAs. Shortly before CCA3, hives wereranked by strength and the ‘strongest’ hives were placed in the one apiary. The next eight strongest hives
were then placed in an empty apiary. This process continued until hives were placed in all apiaries. Withineach apiary, the control treatment was replicated two times and each treatment occurred one time (total of
8 hives in each apiary: seven hives were randomly assigned as control or treatment group and the eighth
hive was used for additional sampling during the study). Given this design, the blocking factor ‘apiary,’
represents variation due to geographic location and initial hive strength.
As a large percentage of hives did not survive overwintering, data collected the following spring will not
be included in the statistical analyses (Table 23).
Table A.1 Timeline including major milestones of study
Date
20 May 2014
9 Jun 2014
27 Jun-1 Jul 2014
2 Jul 2014

Study action*
Initiate CCA1 (non-GLP)
Initiate CCA2 (non-GLP)
Hives moved to study locations
Initiate CCA3 (GLP)

8 Jul 2014

20 Aug 201417 Sep 2014
6 Oct 2014
28 Oct 2014

Initiate thiamethoxam exposure
through sucrose solution.
Initiate CCA4 (GLP)
End thiamethoxam exposure throughsucrose solution
Initiate CCA5 (GLP)
Initiate CCA6 (GLP)
Initiate CCA7 (GLP)
Initiate CCA8 (GLP)

31 Mar 2015

Initiate CCA9 (GLP)

28 Apr 2015

Initiate CCA10 (GLP)

28 Jul 201419 Aug 2014

*each CCA took two or more days to complete.

Comments
Not included in statistical analysis.
Not included in statistical analysis.
noneFirst CCA to be included in thestatistical analyses.
none
none
none

None
None
None
Final CCA to be included in thestatistical analyses.
Overwintering survival was 21, 0, 33,
17, 33, and 0% in the control, 12.5
ppb, 25 ppb, 37.5 ppb, 50 ppb, and100 ppb treatment groups,
respectively. Therefore, CCA9 and
CCA10 were not included instatistical analyses.

Variables recorded at each CCA included number of adult bees in the hive and number of cells containing
each of the following life stages or food stores: eggs, larvae (open cells), pupae (closed cells), pollen, andhoney. Following standard bee keeping practices, supers were added or removed from each hive to best
support growth or reductions in the size of the bee colony. A queen excluder was placed between theinitial hive box and added super boxes; this limited the summed number of egg, pupae, and larvae cells to
the number of cells in the initial box (68,880 cells: 3440 cells/frame X 2sides/frame X 10 frames/box). All
51
473

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
adult bees, with the exception of the queen, could move to any added supers, and honey and pollen could
be stored in those additional supers as well. The suite of variables that were subjected to data analysis
were:•
Number of adults•
Number of egg cells•
Number of open (larvae) cells•
Number of capped (pupae) cells•
Number of pollen cells•
Number of honey cells•
Total number of individuals (adults + eggs + larvae + pupae)•
Total brood (eggs + larvae + pupae), and•Total food (pollen + honey).
To facilitate computation and algorithm convergence in the SAS Procedures, all data was divided by 1000
prior to any statistical analysis. Since all response variables were divided by the same constant, there wasno effect on any of the test statistics or p-values. No adjustments for addition or removal of supers wereconducted for the statistical analysis.
Prior to the repeated measures analysis, the data were evaluated for patterns in temporal correlation andcorrelations across hive components within each of the evaluated CCAs. This analysis was accomplishedthrough a series of pairwise scatterplots and principle components analyses (PCA).
Scatterplot and Principle Component Analysis
Based on physical hive constructs and the nature of honey bees, it is generally accepted that the colony
condition assessment (CCA) variables may be correlated over time and may also be correlated within atime point (sampling time). Given this background, a series of scatterplots, correlation matrices, andprinciple component analyses was prepared; the full SAS output is included as Attachment 1. For theseanalyses, there was no adjustment for treatment effects, only correlation over time was evaluated.
For the single hive components, adults, eggs, larvae, pupae, pollen, and honey, some of the generalsummary points are:• With the exceptions of adults and honey, CCA8 tended to have the lowest pairwise correlations
with the other CCAs for all components. For adults, there was no CCA that tended to be less (ormore highly) correlated with other CCAs. For honey, CCA3 tended to have the lowest pairwise
correlations with the other CCAs.
• For each of the hive components, the first principle component explained 39 to 65% of the total
variation across all CCAs; the lowest percent of explained variation was for larvae and the highest
was for adults.
• For all the hive components, the first principle component was a weighted average of all theevaluated time points; however, The weights and varied depending on the endpoint. For adults,pollen, and honey, all CCAs carried approximately equal weights (CCA3 and CCA8 had slightly
smaller weights). For eggs and larvae, CCA3, CCA4, CCA5, CCA6, and CCA7 carried
approximately equal weight and CCA8 carried much less weight. For pupae, CCA3, CCA4,
CCA5 andCCA6 had approximately equal weights while CCA7 and CCA8 had smaller weights.
For the three composite hive variables (live, brood, and food), general summary points are:52474

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document•••

For live, the first principle component explained 59% of the total variation and could be described
as a weighted average of all CCAs with CCA3 and CCA8 carrying slightly less weight.
For brood, the first principle component explained 44% of the total variation. The first principle
component could be described as a weighted average with CCA3, CCA4, CCA5 and CCA6
carrying approximately equal weights while CCA7 carried less weight and CCA8 had the least
weight.
For food, the first principle component explained 61% of the total variation, and the generalinterpretation of the first principle component was a weighted average with CCA3, CCA4, CCA5,
CCA6 and CCA7 carrying approximately equal weights while CCA8 carried less weight.

In addition to exploring correlations among CCAs for each of the response variables, correlations among
response variables within a CCA were explored. For this exploratory analysis, only the individual hive
components were evaluated. No adjustment was made for treatment effects (i.e., all data were included ina single series of plots and PCAs; separate assessments were not done for each treatment). For each of the
CCAs, the percent of the total variation explained by the first principle component ranged from 35 to
52%. At each time point the first principle component tended to be interpreted as a weighted average.
Honey had the lowest weight for all of the CCAs was negatively weighted for CCA4 and CCA5.
Analysis Approach and Model Setup
As discussed above, the experimental design was a randomized complete block (apiary) with repeatedmeasures (CCAs). Exploring the interaction between treatment and CCA can address these two questions:• At each CCA, was there a reduction in the response relative to the control?
• At each treatment level, was there a difference in the response relative to the baseline time point(CCA3)?
With the experimental design component of the analysis established, the next part of the analysis was todetermine which correlation structure (across time) was the best fitting for these data. The scatterplots,
correlation matrices, and principle component analyses were used to inform the choice of covariancestructure used in the repeated measure analysis. Some summary points from the above exploratory
analyses are that temporal correlations within a response variable tended to be stronger than correlations
among response variables within a time point; variance for a given response variable was not homogenous
among the CCAs; and that the pairwise correlations did not consistently decrease as the distance between
the temporal pairs increased.
Before conducting any comparisons among treatments or CCAs, several different correlation structures tobest fit the temporal correlation were evaluated. The structures that were fitted included:• Compound symmetry (CS): assumes equal correlation for all pairwise correlations (regardless of
distance of time point).
• Compound symmetry with heterogeneous variance (CSH): Estimates a unique variance at eachtime point, but assumes equal correlation for all pairwise correlations (regardless of distance oftime point).
• Autoregressive correlation (AR(1)). Assumes equal correlation between adjacent time points.
Time points further apart have a lesser correlation.
• Heterogeneous Toeplitz (ToepH): models a unique variance for each time point and separate
correlations for equidistant time points (e.g., correlation between CCA3 and CCA5 is the same as
the correlation between CCA4 and CCA6).
53475

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
More information about each of the covariance structures available in the REPEATED statement in SAS
can be found here:
https://support.sas.com/documentation/cdl/en/statµg/63033/HTML/default/viewer.htm#statug_mixed_sect019.htm . The full SAS output is provided in Attachment 2.
To compare covariance structure fits, Bayesian Information Criterion (BIC) was utilized 10. The BIC is afunction of the log likelihood with a penalty for an increase in the number of covariance parameters to be
fitted. The BIC value for each fitted model for all response variables is reported in Table A.2; smaller
values of the BIC indicate a better fit (bolded). For many of the endpoints, heterogeneity of variance at
different time points was indicated as compound symmetry with heterogeneous variance (CSH) andheterogeneous Toeplitz (ToepH) were the covariance structures providing the best fits. This is not
surprising as unequal variances were observed in the exploratory multivariate/principle component
analysis.
Table A.2. BIC values for fitted models. CCA3 – CCA8 -thiamethoxam

Variable →AdultsEggs
Larvae
Pupae
Pollen
Honey
Live
Brood
Food
Model ↓
(open)(capped)
CS
2281
1910
1959
2598
1849
2982
3152
2980
3044
CSH
2262
1784@
2429
1836
2953
3027
2814
3023
1791
AR(1)
2202@
1891
1949
2593
1821
2913
3093
2959
2964
ToepH
DNC
2172
1767@
2414@
1790
2877
2954
2772@
2932
*Within a response variable, smaller BIC values (bolded) indicate better covariance model fit. Kass and Raferty (1995)
suggested that differences of greater than 10 in BIC values provides very strong evidence that model fits are not equivalent.
@Convergence was attained, but estimated G matrix was not positive definite and not all covariance parameters could be
estimated.
DNC – Model algorithm did not converge.

For all the evaluated response variables except larvae, ToepH was identified as the ‘best fitting’covariance structure; however, all covariance parameters could not be estimated for three of the endpoints.
In addition, convergence was not attained for larvae when fitting the ToepH covariance structure. For
larvae (for which ToepH did not converge), CSH was the best fitting covariance structure. If CS, CSH,
and AR(1) covariance structures are evaluated without considering ToepH, it is noted that CS is never the‘best fitting’ based on BIC comparisons. CSH fits better than AR(1) for five response variables and AR(1)fits better than CSH for four response variables based on BIC alone. Both CSH and AR(1) have one
instance when not all covariance parameters could be estimated.
Residual plots were also evaluated for each of the response variables and covariance structures.
Patterns indicative of heterogeneous variance of the residuals were evident for many of the response
variables and models where an assumption of equal variance at each time point was made. For many of
the residual plots when CS or AR(1) covariance structure was modeled, the vertical spread of the residuals
around increased as the predicted mean increased (indicating larger variances as the mean increased) (seeFigure A.1 for example). These response variables are counts, hence the distribution of the response
variable and the residuals may not meet assumptions of normality and/or equal variance. Morespecifically, review of the residual plots indicates that estimating utilizing a covariance structure that

10

Schwarz, Gideon. Estimating the Dimension of a Model. Ann. Statist. 6 (1978), no. 2, 461--464.
doi:10.1214/aos/1176344136. http://projecteuclid.org/euclid.aos/1176344136.
54
476

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
estimated unique variances for each CCA (e.g., CSH, ToepH covariance structures) appears to improveoverall model fit.
Figure A.1. Studentized residual plots for eggs with covariance structures of (left) compound symmetry (CS)and (right) compound symmetry with heterogeneous variance (CSH). Distribution of the residuals indicates a
better fitting model for the CSH covariance structure.

For this suite of response variables, both the results of the BIC analysis and review of residual plots were
informative in selecting a covariance structure for the mixed model. Comparison of covariance structuresindicated that while ToepH fit better than others for some endpoints, there were also issues of nonestimation of covariance parameters and non-convergence. Comparison of BICs for CS, CSH, and AR(1)did not identify a best covariance structure for a majority of the endpoints. However, a review of theresidual plots did indicate that CSH was providing an advantage as clear evidence of heterogeneity ofvariance was evident in the CS an AR(1) residual plots for a majority of the endpoints. Therefore, thereview team elected to move forward with the heterogeneous compound symmetry (CSH) covariancestructure for the final analyses.
Treatment by Time Interaction and Follow-up Contrasts
The text box below provides the SAS code for the mixed model that was used for follow-up statisticalcontrasts to address the following questions:• At each CCA, was there a reduction in the response relative to the control?
• At each treatment level, was there a difference in the response relative to the baseline time point(CCA3)?

55
477

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
The contrasts that were utilized for this analysis were Dunnett’s test. Dunnett’s test is a set of pairwise
contrasts in which each treatment mean is compared to the control mean; the tests can be one- or twosided. For a given set of contrasts, the experiment-wise error-rate is controlled as the specified alpha-level.
In this case, a ‘set of contrasts’ is either (1) comparisons of treatment means to the control for a specificendpoint at a specific CCA or (2) comparison of time-points CCA4, CCA5, CCA6, CCA7, and CCA8 to
the baseline CCA3 for a given endpoint. For all analyses, the CSH covariance matrix was used for each of
the variables.
Text Box: SAS Code for the mixed model used to run the statistical analysis
title ‘Thiamethoxam - ColonyFeedingStudy(2015) data analysis';proc mixed data=cca3_8 ;title2 "Dunnett's tests - adult_scale";class apiary cca conc hive;model adult_scale = conc|cca /DDFM=SATTERTHWAITE;
random apiary ;repeated cca/ subject=hive*conc(apiary) type=csh ;lsmeans conc*cca/cl;
slice conc*cca /sliceby=cca diff=controll adjust=dunnett;
slice conc*cca /sliceby=conc diff=control adjust=dunnett;run;Williams’ test was also considered for use for one set of the follow-up contrasts - comparisons oftreatment means to the control for a specific endpoint at a specific CCA. Williams’ test has been shown tobe more powerful than Dunnett’s test when the assumption of monotonicity is met. Williams’ requires theassumption that if there is an effect of the chemical, it follows the classic dose-response relationship (i.e.,
assuming the test material has a negative effect on the response variable, then as the test concentration
increases, mean response is equal to or less than the mean response of the next lower dose concentration).
The test procedure then determines the lowest dose level for which the mean is significantly less than thecontrol mean. This concentration is identified as the LOAEC and the next lower concentration is identified
as the NOAEC. Williams’ test was not utilized for this analysis for several reasons:• Review of the treatment means identified several instances when the underlying assumption ofmonotonicity does not appear to be met. Given the large variation in the measured responses ingeneral, it could not be determined if the observed deviations from monotonicity were due to large
background variation or to a non-monotone treatment response.
• For any one response variable, the data are combined across CCAs into one mixed model analysis.
Incorporating data from all CCAs improves the variance/covariance estimates and increases thedegrees of freedom for hypothesis testing. As the degrees of freedom for hypothesis testing
increases, any differences in power between Dunnett’s test and Williams’ test would become verysmall.
• It has not been codified in the PROC MIXED procedure in SAS, and the level of effort to code
and QA the test would be significant.
An analysis approach where data from each CCA was analyzed separately as a randomized complete
block design was also considered as SAS has options for use of Williams’ test for simpler experimental
designs. This approach was not selected for several reasons:56478

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document••

Equality of variance across treatment groups would still need to be evaluated. If the assumption ofhomogenous variances was not met for some CCAs, then transforming the response or nonparametric analyses would need to be considered. Incorporating the heterogeneous variances into
the error matrices of the general linear model (GLM) would increase the complexity of the modelsuch that the Williams’ options in SAS could no longer be utilized.
A statistical analysis approach that does not utilize the strength of the correlations among time
points to improve estimates of error variance would not be as powerful as one that does
incorporate that additional information about the nature of the responses.

Treatment effects within a CCA
The table of p-values resulting from the Dunnett’s tests (for evaluating whether within a CCA, thetreatment means are significantly less than control means) are summarized in Table A.3. Figures 7-15 insection 3 show the results for each response variable across all CCAs analyzed (CCA3-CCA8) and alltreatment levels. For all figures, significant reductions from the negative control with p-values below the0.05 alpha level are denoted by a red dot at a given treatment level and CCA and those reductions with pvalues between 0.05 and 0.1 are denoted by a black dot. Statistical NOAECs and LOAECs will be
determined using an alpha-level of 0.05. Additional comparisons using and alpha-level of 0.10 are
included for additional characterization. These will be integrated into the final biological interpretation.
Error bars represent one standard error from the mean calculated from the model residual mean squares
estimate. The tables of p-values resulting from the Dunnett’s test are summarized in Table A.3. The
associated SAS output containing the full results of the Dunnett’s comparisons can be found in
Attachment 3.
AdultsEggs

Larvae(Open)
25
NS

Pupae(Capped)25
100

Pollen

Honey

Live

Brood

Food

CCA3
CCA4

NS
NS

25
NS

NS
25
100

NS
NS

25
100

25
100

NS
NS

CCA5

50
100

100

50
100

37.5
50
100

25
50
100

12.5

50
100

50
100

NS

CCA6

100

12.5
100
NS

100

100

100

NS

100

100

NS

CCA7

100

NS

100

NS
NS
25
100
100
100
CCA8 100
50
NS
NS
NS
NS
NS
NS
NS
* NS indicates that there were no test concentrations with means significantly less than the control (p>0.10).
Bolded concentration = significantly less than control (p< 0.05)
Italicized concentration = less than control (0.05< P < 0.10)

Temporal trends within a treatment levelA second component to evaluating the “treatment x CCA” interaction is to look at the temporal changes
within a treatment group. This was accomplished by comparing each CCA (CCA4 through CCA8) to57
479

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
CCA3 by use of a two-sided Dunnett’s test (Table A.4 and Table A.5). This suite of comparisons is not as
informative as the contrasts of control against the treatment group within a CCA for establishing a
NOAEC and LOAEC. However, it may aid in interpretations and further biological understanding of
temporal shifts in the life stages and food components present in the hive. Differences in patterns of
temporal shifts between the control and various treatment groups can provide further understanding of thepotential impacts of clothianidin on beehive population dynamics.
Table A.4 Results of two-sided Dunnett’s test (comparing CCA3 to each following CCA), correlations
modeled using CSH.*
Trt
Group

Control

Response Variable

Adults
CCA4** andCCA5**,
CCA7 and
CCA8 <CCA3
CCA8<CCA3

Eggs
CCA7 and
CCA8 <CCA3

Open
CCA7 and
CCA8<CCA3

CCA7 and
CCA7 and
CCA8 <
CCA8<CCA3
CCA3
CCA5, CCA6
CCA4**
CCA6**
25
and CCA8 <CCA7 and
CCA5, CCA7,
andCCA3
CCA8 <CCA3
CCA8<CCA3
CCA8<CCA3
CCA7** and CCA7 and
37.5
CCA8 <
CCA8<CCA3
CCA3
CCA8<CCA3
CCA7 and
CCA7 and50
CCA8 <
CCA8<CCA3CCA3
CCA5CCA5 –CCA5100
CCA8<CCA3
CCA8
CCA8<CCA3<CCA3*Unless otherwise stated significance from CCA3 is (p<0.05)** Significant at 0.05<p<0.1
NS – No significant differences from CCA3 (p>0.05)
12.5

Capped
CCA5,
CCA6**,
CCA7 and
CCA8 < CCA3
CCA7 and
CCA8 < CCA3Live
CCA5, CCA7CCA8<CCA3

Brood
CCA7 and
CCA8<CCA3

CCA5, CCA7CCA8<CCA3

CCA5, CCA7
and CCA8 <CCA3

CCA4-CCA5,CCA7CCA8<CCA3

CCA5**,CCA7CCA8<CCA3
CCA5, CCA7CCA8<CCA3

CCA7** andCCA8 < CCA3

CCA7 and
CCA8<CCA3

CCA7 and
CCA8<CCA3

CCA7 and
CCA8 < CCA3

CCA7 and
CCA8<CCA3

CCA7 and
CCA8<CCA3

CCA5 – CCA8<CCA3

CCA4CCA8<CCA3

CCA4CCA8<CCA3

58
480

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Table A.5. Results of two-sided Dunnett’s test (comparing CCA3 to each following CCA), correlations
modeled using CSH.*
Trt
Group
Control
12.5
25
37.5

Response Variable
PollenCCA8<CCA3NS
CCA4**<CCA3
NS

Honey
CCA6<CCA3
NS
NS
NS

NS
NS
50
CCA4-CCA8<CCA3
CCA4-CCA5 <CCA3
100
Unless otherwise stated significance from CCA3 is (p>0.05)** Significant at 0.05<p<0.1
NS – No significant differences from CCA3 (p>0.05)

Food
CCA5** and CCA6**<CCA3
NS
NS
NS
NS
CCA5<CCA3

Tables A.6 – A.14 tabulate the summary statistics (including mean and standard deviation) of each
response variable for all treatment levels across CCAs 3-8.
Table A.6. Summary statistics for adults
CCA
3
4
5
6
7
8
3
4
5
6
7
8
3
4
5
6
7
8
3
4
5
6
7
8
3
4
5

TreatmentGroup µg/L
0
0
0
0
0
0
12.5
12.5
12.5
12.5
12.5
12.5
25
25
25
25
25
25
37.5
37.5
37.5
37.5
37.5
37.5
50
50
50

Mean

SE

DF

Lower

Upper

11.040212.7383
13.0853
11.5786
9.0371
4.749
8.9627
10.3778
11.1119.77278.92784.98278.263910.3928
12.0837
11.14139.35215.5522
10.569712.0279
11.3155
9.92779.24475.748710.0062
11.77629.88031.0201
0.94931.0846
0.94790.92440.75191.31541.20461.41481.24171.2026
0.89881.31541.20461.41481.2145
1.17670.88371.31541.20461.4426
1.2145
1.17670.88371.31541.20461.4148

42.3
40
56.5
40.5
39.1
19.6
56.6
63
76.6
68.9
67.4
34.8
56.6
63
76.6
65.5
64.3
33
56.6
63
79.7
65.5
64.3
33
56.6
63
76.6

8.982
10.8198
10.9139.6636
7.1675
3.1783
6.32837.97068.2936
7.2955
6.5276
3.15765.62967.98569.26638.71617.00163.75437.93549.62068.44457.5025
6.89413.95087.37189.369
7.062913.0985
14.6568
15.2575
13.4937
10.9067
6.3197
11.597
12.7851
13.9284
12.2498
11.3279
6.807910.8982
12.8001
14.9011
13.5665
11.70277.3501
13.2041
14.4352
14.1865
12.353
11.5953
7.546612.6405
14.1835
12.697759

481

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
CCA
6
7
8
3
4
5
6
7
8

TreatmentGroup µg/L
50
50
50
100
100
100
100
100
100

Mean

SE

DF

Lower

Upper

9.81168.01585.5436
11.6786
11.3337
7.211
4.95393.91552.19471.24171.20260.91571.31541.20461.41481.24171.31270.9643

68.9
67.4
36.8
56.6
63
76.6
68.9
79
42.6

7.33445.6156
3.68789.04438.92654.39362.47671.30270.249512.2888
10.416
7.399414.312913.741
10.0284
7.4311
6.52844.1399

Table A.7. Summary statistics for eggs
CCA
3
4
5
6
7
8
3
4
5
6
7
8
3
4
5
6
7
8
3
4
5
6
7
8
3
4
5
6
7
8
3
4
5
6
7
8

TreatmentGroup µg/L
0
0
0
0
0
0
12.5
12.5
12.5
12.5
12.5
12.5
25
25
25
25
25
25
37.5
37.5
37.5
37.5
37.5
37.5
50
50
50
50
50
50
100
100
100
100
100
100

Mean

SE

DF

Lower

Upper

6.063
6.02
5.289
5.64992.53990.62365.375
5.3607
6.16333.46772.40520.7035
4.1137
6.0773
4.95935.37161.864
0.69794.8017
6.0343
5.715
5.25963.07410.59175.59
6.364
4.44334.9032
1.94990.39836.536
5.504
2.93832.484
1.57970.92780.51550.512
0.4491
0.54280.27680.13820.729
0.72410.63510.81220.41410.20330.729
0.72410.63510.7814
0.39840.19560.729
0.72410.65650.7814
0.39840.19560.729
0.72410.63510.81220.41410.21190.729
0.72410.63510.81220.47940.2353

81.1
82.9
83.4
75
68.8
58.8
81.1
82.9
83.4
76.7
70.4
59.8
81.1
82.9
83.4
75.6
69.3
58.8
81.1
82.9
84.8
75.6
69.3
58.8
81.1
82.9
83.4
76.7
70.4
60.9
81.1
82.9
83.4
76.7
73.5
62.9

5.03735.00154.39584.5686
1.98770.3471
3.92453.92044.90011.8503
1.5794
0.29692.6631
4.637
3.69613.8151
1.06920.30653.3511
4.594
4.40973.70312.27930.2003
4.13954.9237
3.18013.28581.124-0.02539
5.08554.06371.67510.86660.62420.45767.08877.0385
6.1822
6.7313
3.09210.90016.82556.801
7.42655.085
3.23111.11015.56427.5176
6.22256.92812.65881.0892
6.25227.47467.02026.8161
3.86890.983
7.04057.80435.70656.52052.77580.82197.9865
6.94434.20154.10132.5351
1.398160

482

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Table A.8. Summary statistics for larval (open) cells
CCA
3
4
5
6
7
8
3
4
5
6
7
8
3
4
5
6
7
8
3
4
5
6
7
8
3
4
5
6
7
8
3
4
5
6
7
8

TreatmentGroup µg/L
0
0
0
0
0
0
12.5
12.5
12.5
12.5
12.5
12.5
25
25
25
25
25
25
37.5
37.5
37.5
37.5
37.5
37.5
50
50
50
50
50
50
100
100
100
100
100
100

Mean

SE

DF

Upper

Lower

5.9197
6.03436.72235.60751.65130.4781
4.902
6.2207
6.13475.40052.18230.5024
3.44
5.8337
6.04875.69490.68670.2741
4.945
6.3067
6.23955.759
1.79590.59854.988
5.87674.35734.6815
1.46150.1012
4.945
4.085
2.666
2.26440.80910.31630.494
0.60690.52770.65230.32640.11910.695
0.85540.74280.96660.479
0.15740.695
0.85540.74280.9342
0.46320.15290.695
0.85540.76430.9342
0.46320.15290.695
0.85540.74280.96660.479
0.16260.695
0.85540.74280.96660.54530.1771

80.7
82.3
79.3
70.2
60.7
27.3
80.7
82
79.1
72.9
63.5
48.3
80.7
82
79.1
70.9
61.6
45.6
80.7
82
81.5
70.9
61.6
45.6
80.7
82
79.1
72.9
63.5
51.1
80.7
82
79.1
72.9
69.5
57.5

4.93674.827
5.67214.3066
0.99850.23373.51924.519
4.65613.474
1.22520.18592.0572
4.132
4.5701
3.8321-0.2393-0.03376
3.5622
4.605
4.71893.89610.86990.29073.60524.175
2.87882.755
0.5043-0.2253
3.56222.3834
1.18750.3379-0.2786-0.03823

6.90267.24177.77266.90842.30410.72246.28487.9223
7.61327.327
3.13940.81894.82287.5353
7.5272
7.55771.61270.58196.32788.00837.7601
7.62182.722
0.90636.37087.5783
5.83596.608
2.41860.4277
6.32785.7866
4.14454.19091.89670.6709

Table A.9. Summary statistics for pupal (capped) cells
CCA
3
4
5
6
7
8
3

TreatmentGroup µg/L
0
0
0
0
0
0
12.5

Mean

SE

DF

Min

Max

14.0682
13.33
17.3863
10.9905
3.1487
1.166912.1833

0.8515
1.23461.08871.38210.55330.25251.2023

74.9
78.5
80
69.4
57.4
40.5
76.1

12.3719
10.8724
15.21978.23372.04090.65689.788715.7645
15.7876
19.552913.7473
4.25641.677
14.57861483

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
CCA
4
5
6
7
8
3
4
5
6
7
8
3
4
5
6
7
8
3
4
5
6
7
8
3
4
5
6
7
8

TreatmentGroup µg/L
12.5
12.5
12.5
12.5
12.5
25
25
25
25
25
25
37.5
37.5
37.5
37.5
37.5
37.5
50
50
50
50
50
50
100
100
100
100
100
100

Mean

SE

DF

Min

Max

11.7963
15.7667
11.0564.37831.824110.5063
11.73914.44810.9297
1.45470.758112.3697
12.6133
12.7454
10.7653.2046
1.549712.7997
10.5637
10.0763
9.24671.7136
1.175613.1437
7.02334.257
4.60790.23751.0002

1.74471.53822.06480.82390.3646
1.20231.74471.5382
1.98780.79330.35111.20231.74471.58881.98780.79330.35111.20231.74471.53822.06480.82390.37971.20231.74471.53822.06480.9516
0.421

79.2
80.9
72.2
61.2
60.2
76.1
79.2
80.9
70.6
59.7
58.3
76.1
79.2
82.9
70.6
59.7
58.3
76.1
79.2
80.9
72.2
61.2
62
76.1
79.2
80.9
72.2
65.5
65.4

8.323712.7066.94012.73081.09488.11178.266411.3873
6.9658-0.1323
0.055289.975
9.14079.58526.801
1.61760.846910.4057.09117.01575.1307
0.066120.416710.7493.5507
1.19630.4919-1.6628
0.159615.2689
18.8273
15.17196.02582.553512.901
15.211617.5087
14.8937
3.04161.460914.7643
16.0859
15.9055
14.7291
4.7916
2.252515.1943
14.0363
13.13713.3626
3.36111.934615.5383
10.49597.31778.72382.13771.8407

Table A.10. Summary statistics for total individuals
CCA
3
4
5
6
7
8
3
4
5
6
7
8
3
4
5
6
7

TreatmentGroup µg/L
0
0
0
0
0
0
12.5
12.5
12.5
12.5
12.5
12.5
25
25
25
25
25

Mean

SE

DF

Min

Max

37.09138.122642.48333.6219
16.3324
7.092631.42333.7555
39.1757
29.4253
17.8406
7.960426.323934.0428
37.5397
32.8367
13.2651

2.174
2.51692.51862.82081.48490.86413.04143.53093.5333
4.1085
2.1243
1.16353.04143.53093.53334.00812.0737

79.6
82.3
84.7
75.1
64.8
42.1
78.1
80.9
83.2
77.7
68.8
60.9
78.1
80.9
83.2
74.9
66.5

32.764433.11637.4751
28.0028
13.3666
5.349125.3682
26.729932.1483
21.2454
13.6026
5.633820.2691
27.0172
30.512324.85199.125541.417743.129347.4908
39.24119.29818.836237.4778
40.781146.20337.6052
22.0786
10.28732.3787
41.0684
44.56740.821417.404762

484

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
8
3
4
5
6
7
8
3
4
5
6
7
8
3
4
5
6
7
8

25
37.5
37.5
37.5
37.5
37.5
37.5
50
50
50
50
50
50
100
100
100
100
100
100

7.244632.6861
36.982335.9161
31.58817.24788.462333.3838
34.5806
28.7573
28.1544
13.0838
7.273536.3033
27.946117.0723
14.0192
6.3152
4.5

1.13733.04143.53093.60464.00822.07371.13733.04143.53093.5333
4.1085
2.1243
1.19273.04143.53093.5333
4.10852.3386
1.2764

58.8
78.1
80.9
86.1
74.9
66.5
58.8
78.1
80.9
83.2
77.7
68.8
63.2
78.1
80.9
83.2
77.7
77
68.7

4.9686
26.631329.9566
28.7504
23.6032
13.10816.186327.32927.55521.73
19.97458.84574.890130.2485
20.9205
10.0455.83931.6584
1.95359.520538.740944.007943.0818
39.5728
21.3874
10.7383
39.4386
41.606235.7847
36.3343
17.3229.656942.35834.971724.099722.1991
10.972
7.0465

Table A.11. Summary statistics for nectar (honey) cells
CCA
3
4
5
6
7
8
3
4
5
6
7
8
3
4
5
6
7
8
3
4
5
6
7
8
3
4
5
6

TreatmentGroup µg/L
0
0
0
0
0
0
12.5
12.5
12.5
12.5
12.5
12.5
25
25
25
25
25
25
37.5
37.5
37.5
37.5
37.5
37.5
50
50
50
50

Mean

SE

DF

Min

Max

15.3582
16.7557
18.6118
20.1786
13.4115
18.7394
12.3983
12.6277
12.72817.3371
12.0618
17.6326
14.1757
15.3223
15.35117.3619
13.8175
19.7936
17.5297
17.9597
18.9453
22.5023
15.8999
20.9586
14.390716.182317.8307
20.0261

1.3232
1.3322
1.72322.1601
1.57272.05491.8336
1.84662.4081
3.16982.29092.97391.8336
1.84662.40813.07442.22282.8846
1.8336
1.84662.46913.07442.22282.8847
1.8336
1.84662.4081
3.1698

67.1
66.2
73.3
76.7
69.6
61.1
80.1
80.6
78.3
80
79.1
65.4
80.1
80.6
78.3
77.7
76.8
63.7
80.1
80.6
80.7
77.7
76.8
63.7
80.1
80.6
78.3
80

12.7172
14.09615.1778
15.8769
10.2746
14.6305
8.74958.9532
7.934111.0297.501911.6939
10.5268
11.6479
10.5571
11.24099.391214.0304
13.8808
14.2852
14.032216.381211.4736
15.1954
10.7418
12.5079
13.0368
13.71817.9991
19.4153
22.0459
24.4803
16.5485
22.8483
16.0472
16.302117.521923.6452
16.6216
23.571317.8245
18.9968
20.1449
23.4828
18.243925.5568
21.1785
21.634123.8583
28.6233
20.3263
26.721918.0395
19.8568
22.6246
26.3342
63

485

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
7
8
3
4
5
6
7
8

50
50
100
100
100
100
100
100

15.47319.8588
15.82422.3457
27.4483
19.94515.7232
14.1472

2.29093.0726
1.8336
1.84662.4081
3.16982.5766
3.3483

79.1
67.1
80.1
80.6
78.3
80
86.3
70.7

10.9132
13.72612.1751
18.671222.6544
13.6369
10.60137.470420.032925.9915
19.472926.0201
32.242226.2532
20.84520.824

Mean

SE

DF

Min

Max

3.75533.21784.6082
3.76823.77412.43933.096
3.15333.64073.53443.59342.2668
3.139
1.57672.59432.703
2.02561.8856
4.128
2.8953
3.5771
3.3326
3.24852.783
2.967
1.86332.86672.70152.74042.20814.171
1.39030.88870.9494
1.25981.49890.54810.4432
0.48780.41740.44440.36140.75820.60570.67080.59480.63640.50080.75820.60570.67080.57620.61630.48540.75820.60570.68910.57620.61630.48540.75820.60570.67080.59480.63640.51770.75820.60570.67080.59480.72030.5649

75.1
76
72.6
60.6
60
43.3
78.4
78.5
77.2
80.9
75
67.3
78.4
78.5
77.2
78.6
73.3
65.1
78.4
78.5
79.1
78.6
73.3
65.1
78.4
78.5
77.2
80.9
75.1
69.5
78.4
78.5
77.2
80.9
80
74.5

2.66352.3351
3.63592.93342.88521.71071.5867
1.94762.305
2.351
2.32571.2674
1.62970.371
1.25871.556
0.79730.91622.61871.68962.20562.18562.02031.81361.45770.65761.531
1.518
1.4727
1.17552.66170.1846-0.447-0.2341-0.17360.37344.84724.10055.58054.603
4.66293.16794.60534.359
4.97634.71794.8611
3.26634.64832.7824
3.93
3.84993.25392.8551
5.6373
4.101
4.94864.47954.47683.75254.47633.069
4.20233.88494.00823.24075.68032.596
2.22432.13282.69322.6244

Table A.12. Summary statistics for pollen cells
CCA
3
4
5
6
7
8
3
4
5
6
7
8
3
4
5
6
7
8
3
4
5
6
7
8
3
4
5
6
7
8
3
4
5
6
7
8

TreatmentGroup µg/L
0
0
0
0
0
0
12.5
12.5
12.5
12.5
12.5
12.5
25
25
25
25
25
25
37.5
37.5
37.5
37.5
37.5
37.5
50
50
50
50
50
50
100
100
100
100
100
100

64
486

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Table A.13. Summary statistics for Brood
CCA
3
4
5
6
7
8
3
4
5
6
7
8
3
4
5
6
7
8
3
4
5
6
7
8
3
4
5
6
7
8
3
4
5
6
7
8

TreatmentGroup µg/L
0
0
0
0
0
0
12.5
12.5
12.5
12.5
12.5
12.5
25
25
25
25
25
25
37.5
37.5
37.5
37.5
37.5
37.5
50
50
50
50
50
50
100
100
100
100
100
100

Mean

SE

DF

Min

Max

26.0508
25.3843
29.3977
22.1232
7.32222.280222.460323.377728.0647
19.8457
8.93533.007118.06
23.65
25.45621.83423.97281.715522.1163
24.9543
24.683121.67078.05742.741423.3777
22.804318.87718.5525
5.09891.663124.6247
16.61239.86139.17632.61622.3012

1.62711.9894
1.79832.26470.94890.45072.29022.80462.53333.34411.38390.61522.29022.80462.53333.24081.34190.59752.29022.80462.59993.24091.34190.59752.29022.80462.53333.34411.384
0.635
2.29022.80462.53333.34411.56040.6907

76
78.3
79.7
70.7
56.7
30.2
76.5
78.6
80
74.4
61.1
50
76.5
78.6
80
71.9
59.1
47.4
76.5
78.6
82.9
71.9
59.1
47.4
76.5
78.6
80
74.4
61.1
52.6
76.5
78.6
80
74.4
68
58.6

22.8101
21.423925.8187
17.6073
5.42191.36
17.899417.794823.0233
13.1831
6.16811.771413.4991
18.0671
20.4146
15.3735
1.28780.513717.5554
19.3715
19.512
15.21
5.3724
1.539618.8168
17.2215
13.8356
11.88992.33170.389120.0638
11.0295
4.82
2.5137-0.4976
0.919

29.291629.3447
32.9766
26.6391
9.22243.200327.021228.9605
33.10626.5083
11.7025
4.242722.620929.232930.4974
28.29486.65782.917326.6772
30.5372
29.854228.131410.7425
3.943227.938628.3872
23.9184
25.21517.86622.937
29.1856
22.1952
14.9027
15.8389
5.73
3.6833

Mean

SE

DF

Min

Max

19.1135
19.9735
23.22
23.9329
17.1938
21.1434
15.4943

1.56371.41991.853
2.24771.74232.244
2.1798

69.2
65.5
76.4
76.2
67.6
61.9
79.7

15.994217.1382
19.529719.4564
13.7168
16.6576
11.1562

22.232822.8088
26.910328.4093
20.670825.6291
19.8324

Table A.14. Summary statistics for Food
CCA
3
4
5
6
7
8
3

TreatmentGroup µg/L
0
0
0
0
0
0
12.5

65
487

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
CCA
4
5
6
7
8
3
4
5
6
7
8
3
4
5
6
7
8
3
4
5
6
7
8
3
4
5
6
7
8

TreatmentGroup µg/L
12.5
12.5
12.5
12.5
12.5
25
25
25
25
25
25
37.5
37.5
37.5
37.5
37.5
37.5
50
50
50
50
50
50
100
100
100
100
100
100

Mean

SE

DF

Min

Max

15.78116.3687
20.8523
15.5833
19.764717.3147
16.89917.9453
20.03715.8295
21.6347
21.6577
20.85522.520525.7557
19.114323.659717.357718.0457
20.6973
22.625418.1754
21.8537
19.99523.73628.33720.7528
16.839415.1786

1.97312.59393.296
2.5421
3.24942.17981.97312.59393.19962.46843.15442.17981.97312.6577
3.19962.46843.15442.17981.97312.59393.296
2.54213.3543
2.17981.97312.59393.296
2.85183.6481

80.8
78.9
80.6
78.4
66.5
79.7
80.8
78.9
78.3
76.1
64.8
79.7
80.8
81.3
78.3
76.1
64.8
79.7
80.8
78.9
80.6
78.4
68.3
79.7
80.8
78.9
80.6
85.9
72.1

11.8549
11.2055
14.2938
10.5227
13.27812.9766
12.9729
12.782213.6675
10.9134
15.334617.3196
16.928917.2328
19.3861
14.198117.3595
13.0196
14.1196
15.534216.067
13.1148
15.160915.6569
19.809923.1738
14.1943
11.1701
7.906519.7071
21.5318
27.4108
20.6438
26.251421.6528
20.825123.1085
26.4065
20.7456
27.9348
25.9958
24.781127.8082
32.1252
24.0305
29.9599
21.6958
21.971725.8605
29.1839
23.23628.5465
24.333127.662133.500227.311322.5087
22.450766
488

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

Appendix B – PMRA Data Statistical Analysis Report
Analysis Strategy
Hive condition data:
To analyze colony condition data which contains many components over many assessments at
different times, a primary analysis was set out to effectively prevent multiplicities from interfering with
the interpretation of p_values and confidence intervals. These multiplicities arise from having multiple
dose levels, multiple outcomes and multiple time points, and are dealt with as follows:
The multiplicities from having multiple dose levels was dealt with by using step down testing, the highest
dose group’s data was compared directly to the control group’s data, if statistically significant at a chosenalpha level the next lowest dose group’s data was compared to the control group’s data and this wascontinued down to the dose where statistical significance was no longer achieved. A technical reference
for this step down testing would be Multiple Comparison Procedures in Dose Response Studies.
Tamhane, Ajit C. and Logan, Brent R., in Dose Finding in Drug Development edited by Ting, Naitee.
Springer New York 2006. This step down procedure (referred to as the SD2PC procedure in the technicalreference) was chosen as it provides good power for detecting the minimum effective dose (lowest does
where effect is present) when monotonic dose effects are expected while providing stringent control oftype one error, regardless of the true pattern of dose effects. That is, with minimal assumptions, theprocedure strongly controls family wise type one error rate while maintaining good power for effectpatterns that are expected.
This step down procedure is implemented by PMRA using only data from the control group and the dosegroup being tested in that step which alleviates any concern about heterogeneity of variance across dosegroups. Especially with outcome data that involves estimates of underlying counts, it is expected that
effects at a given dose necessarily involves both the mean and variance. When this is the case - the use ofdata from a higher does with a putative effect in the comparison of a lower dose would thus beinappropriate and would invalidate the control of type one error.
The applicant’s choice of multiplicity adjustment procedure, which was William’s trend test (Williams
1972), presumably chosen to be in accord with OECD. 2003. Draft guidance document for the statisticalanalysis of ecotoxicity data. They are both step down procedures but ours differs from William’s in that it
uses only within dose group data based estimates of means rather than maximum likelihood estimates ofdose group means using all group’s data simultaneously - under monotonicity assumption (i.e. order
restricted or isotonic means) additionally assuming homogeneous variances . Although these additional
assumptions may not be problematic and are within the OECD guidelines, we simply chose not to rely onthem (and by doing so, exceed the OECD guidelines.)
The multiplicities from having multiple outcomes, was dealt with by choosing to focus on the assessmentof total life in the hive – simply the number of viable life forms at any stage in the hive. It is consideredthat the total number of individuals includes all live individuals in hives and is expected to be a better
indicator of the hive status at the colony level than any single stage of bees alone. This outcome would
provide good power when background knowledge is lacking on the stage most likely to be affected (i.e. it
cannot be well anticipated) and it is not expected that there will be simultaneous trade-offs effects
67489

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
between the stages. That is, when it is not expected that a toxic effect on one stage would have abeneficial effect for another stage at the same point in time.
The multiplicities from having multiple time points was dealt with by choosing to focus on the time whenthe effects were believed most pronounced both in terms of having an impact on total life and having ahigh powered assessment of that. In this case CCA5 was selected for the following reasons.
1.

2.
3.
4.

CCA5 – assessed effects to the colony after 7 weeks of exposure (which was expected to result in whole
hive exposure), and occurs before the start of hive decline prior to overwintering. In the case of this study,
the initiation of feeding occurred later in the season (in comparison to the clothianidin and imidacloprid
studies), which most likely led to preparation for overwintering at CCA 6.
CCA6 - was not selected as the start of the natural decline of hive size in the fall was clearly apparent andthe width of the confidence intervals started to expand (the precision of estimates declined.).
CCA7 and CCA8 was not selected simply due to the natural decline of hive size in the late fall that may
mask the effect of treatment.
CCA9 and CCA10 were not selected because of the high hive mortality observed in the controls.

While the total individuals at CCA5 is considered as a primary parameter to control multiplicity forstatistical analysis, all parameters including eggs, open brood and capped brood, adults, hive weight,
pollen and nectar store, that were observed during the entire study including CCA4, CCA5, CCA6,CCA7
and CCA8 were also considered in the review. Given that the primary analysis has prevented
multiplicities from interfering with the interpretation of p_values and confidence intervals, if statisticalsignificance has been achieved (at given dose levels), further analysis with all other outcomes isundertaken “with prejudice” for the assessment of similar effects as being significant. More formally, reallowance for multiplicities is not required and less stringent alpha levels are allowed. Essentially theprice has been paid for searching for the pattern in the primary analysis (measures taken to prevent
multiplicities) and it need not be re-paid evaluating the same pattern elsewhere. On the other hand, if
statistical significance has not been achieved (at given dose levels), further analysis with all otheroutcomes is undertaken “with prejudice” for assessment of other effects as likely being just noise. Herethough dramatic effects should not be ignored but carefully considered and noted.
Analysis methods for hive conditions
For all hive conditions total life, eggs, open brood and capped brood, adults, hive weight, pollen andnectar store at CCA4, CCA5, CCA6, CCA7 and CCA8 a conventional analysis of block randomisedexperiments with a baseline measurements was undertaken. In line with the statistical strategy discussed
above, the focus was on total life at CCA5(with step down adjustment for multiplicities applied) but
identical analysis was carried out (less the step down adjustment) on all other hive conditions assessed at
the given assessment points. This analysis comprised of linear modeling (or ANOVA) stratified on
Apiary (block) and adjusted for baseline measurements at CCA3 with one-side testing for harm using
only the control group data and the data from a single dose group at a time, starting with the highest andthen through lowest dose groups. It is a series of robust “t.test like” analyses that conservatively
implement the step down testing procedure. Under the assumption of no effect in the single dose group
being tested (relevant to type one error control), the means and variances and covariate effects should be
identical in both the control group and the single dose group being tested. (In an analysis that includes alldose group data together e.g. William’s procedure, an impact of a treatment effect on the variance andcovariate effects at a higher dose, in addition to an effect on the mean, would invalidate the assumptions
68490

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
needed to control type one error rate in the lower doses.) The results of all analyses are presented in tablesof unadjusted p_values (adjusted p_values can be simply read off as the maximum of all p_values in any
higher dose), effect estimates and upper and lower confidence intervals (in file Thia_summariesF) as well
as plots of the confidence intervals (pdf file Bees8.pdf).
The code snippet to implement these analyses in R was:glm(outcome~Apiary + baseline + exposed, data= x[x$exposed == " control " | x$exposed == dose,])Sensitivity analysis was undertaken by extensive graphical analyses sometimes using the square roottransformation as well as calculating non-parametric randomisation (permutation) tests on the differences
between high dose group and control group average within Apiary.These are in given in the column
named PermP_value in Thia_summariesF.

69491

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

Table B.1. Summary of the differences between treatment and controls on the basis of observations and model estimations, and P values.
Parameter

Time(CCA)1Test
conc.
(µg/l)

mean

SE(standard errorobservedmean)nEstimate(modelestimatemeandifference
fromcontrol)3,4

Std.Error(ofestimated mean)p_value forcompariso
n with thecontrol

Estimated
reductionfrom control(%) 4.5

Estimate(90%
confidenc
e upper
limit) 4)

Estimate(90%
confidence
lower
limit)4,5

Controlt-testMean(observed
means incontrol)

confidence
limit

Adults

3

12.5

2078

983

12

0

0

0

0

0

0

11040

-1.717

Adults

3

25

2776

968

12

0

0

0

0

0

0

11040

-1.717

Adults

3

37.5

470

866

12

0

0

0

0

0

0

11040

-1.717

Adults

3

50

1034

791

12

0

0

0

0

0

0

11040

-1.717

Adults

3

100

-638

992

12

0

0

0

0

0

0

11040

-1.717

Adults

4

12.5

2360

1388

12

465.8541011.542

0.325

0.037

0.173

-0.1

12738

-1.717

Adults

4

25

2345

1626

12

-626.911

1162.225

0.702-0.0490.107-0.20612738-1.717

Adults

4

37.5

710

1194

12

273.457

807.929

0.369

0.021

0.13

-0.08712738-1.717

Adults

4

50

962

1033

12

151.83855.342

0.43

0.012

0.127

-0.10312738-1.717

Adults

4

100

1405

1320

12

1916.704

950.49

0.028

0.15

0.279

0.022

12738

-1.717

Adults

5

12.5

1974

774

12

1332.61

1014.834

0.101

0.102

0.235

-0.03113085-1.717

Adults

5

25

1002

2035

12

-2250.84

1498.899

0.926-0.1720.025-0.36913085-1.71770492

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Parameter

Time(CCA)1Test
conc.
(µg/l)

mean

SE(standard errorobservedmean)nEstimate(modelestimatemeandifference
fromcontrol)3,4

Std.Error(ofestimated mean)p_value forcompariso
n with thecontrol

Estimated
reductionfrom control(%) 4.5

Estimate(90%
confidenc
e upper
limit) 4)

Estimate(90%
confidence
lower
limit)4,5

Controlt-testMean(observed
means incontrol)

confidence
limit

Adults

5

37.5

1481

1535

11

1028.226

1183.261

0.197

0.079

0.234

-0.07713085-1.721

Adults

5

50

3205

1582

12

2400.807

1254.876

0.034

0.183

0.348

0.019

13085

-1.717

Adults

5

100

5874

1782

12

6292.225

1371.698

0

0.481

0.661

0.301

13085

-1.717

Adults

6

12.5

1747

931

10

1652.605

1400.572

0.126

0.141

0.349

-0.06611686-1.729Adults

6

25

183

1727

11

-1443.1

1789.536

0.785-0.1230.141-0.38811686-1.725Adults

6

37.5

1682

1258

11

1490.868

1353.398

0.142

0.128

0.327

-0.07211686-1.725Adults

6

50

2528

1638

11

1954.227

1649.23

0.125

0.167

0.411

-0.07611686-1.725Adults

6

100

6563

1401

10

6890.493

1468.408

0

0.59

0.807

0.372

11686

-1.729Adults

7

12.5

-196

517

10

-526.859

1566.501

0.63-0.0580.24-0.3569079-1.729Adults

7

25

-409

1003

11

-1907.23

1665.135

0.867

-0.21

0.106

-0.5269079-1.725Adults

7

37.5

-28

1053

11

-227.895

1477.933

0.561-0.0250.256-0.3069079-1.725Adults

7

50

1020

1376

10

593.505

1790.472

0.372

0.065

0.406

-0.2769079-1.729Adults

7

100

5594

1491

7

6898.915

2174.979

0.003

0.76

1.178

0.342

9079

-1.746Adults

8

12.5

-324

690

10

-585.345

958.9410.725-0.1190.219-0.4584912-1.734Adults

8

25

-823

964

11

-1746.76

1110.662

0.934-0.3560.035-0.7474912-1.72971493

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Parameter

Time(CCA)1Test
conc.
(µg/l)

mean

SE(standard errorobservedmean)nEstimate(modelestimatemeandifference
fromcontrol)3,4

Std.Error(ofestimated mean)p_value forcompariso
n with thecontrol

Estimated
reductionfrom control(%) 4.5

Estimate(90%
confidenc
e upper
limit) 4)

Estimate(90%
confidence
lower
limit)4,5

Controlt-testMean(observed
means incontrol)

confidence
limit

Adults

8

37.5

-844

683

11

-813.68

871.911

0.819-0.1660.141-0.4734912-1.729Adults

8

50

-668

1000

9

-968.17

1073.057

0.81-0.1970.183-0.577

4912

-1.74

Adults

8

100

2703

992

7

3470.592

1214.753

0.006

0.707

1.14

0.273

4912

-1.753

Adults

9

12.5

NA

NA

0

NA

NA

NA

NA

NA

NA

NA

NA

Adults

9

25

-3968

336

2

-4005.85

NANA-0.966

NA

NA

4148

NA

Adults

9

37.5

-1062

NA

1

NA

NA

NA

NA

NA

NA

NA

NA

Adults

9

50

-3745

2855

4

-2553.83

3178.295

0.747-0.6161.622-2.853

4148

-2.92

Adults

9

100

NA

NA

0

NA

NA

NA

NA

NA

NA

NA

NA

Honey

3

12.5

2960

3129

12

0

0

0

0

0

0

15358

-1.717

Honey

3

25

1182

2275

12

0

0

0

0

0

0

15358

-1.717

Honey

3

37.5

-2172

2115

12

0

0

0

0

0

0

15358

-1.717

Honey

3

50

968

2062

12

0

0

0

0

0

0

15358

-1.717

Honey

3

100

-466

2225

12

0

0

0

0

0

0

15358

-1.717

Honey

4

12.5

4128

3118

12

1413.722

1143.924

0.115

0.084

0.202

-0.03316756-1.717

Honey

4

25

1433

2723

12

389.4841536.613

0.401

0.023

0.181

-0.13416756-1.71772494

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Parameter

Time(CCA)1Test
conc.
(µg/l)

mean

SE(standard errorobservedmean)nEstimate(modelestimatemeandifference
fromcontrol)3,4

Std.Error(ofestimated mean)p_value forcompariso
n with thecontrol

Estimated
reductionfrom control(%) 4.5

Estimate(90%
confidenc
e upper
limit) 4)

Estimate(90%
confidence
lower
limit)4,5

Controlt-testMean(observed
means incontrol)

confidence
limit

Honey

4

37.5

-1204

1886

12

496.2451225.842

0.345

0.03

0.155

-0.09616756-1.717

Honey

4

50

573

2014

12

-192.61306.776

0.558-0.0110.122-0.14516756-1.717

Honey

4

100

-5590

2040

12

-5284.74

1573.762

0.999-0.315-0.154-0.47716756-1.717

Honey

5

12.5

5884

3549

12

3372.694

2650.901

0.108

0.181

0.426

-0.06318612-1.717

Honey

5

25

3261

3410

12

2251.552

2810.035

0.216

0.121

0.38

-0.13818612-1.717

Honey

5

37.5

-399

2505

11

1023.878

2769.842

0.358

0.055

0.311

-0.20118612-1.721

Honey

5

50

781

2703

12

35.8362575.857

0.495

0.002

0.24

-0.23618612-1.717

Honey

5

100

-8836

2497

12

-8492.71

2464.922

0.999-0.456-0.229-0.68418612-1.717

Honey

6

12.5

-1367

2710

10

-1292.68

2532.99

0.692-0.0650.156-0.28619809-1.729

Honey

6

25

1454

4380

11

2013.298

3078.98

0.26

0.102

0.37

-0.16619809-1.725

Honey

6

37.5

-3377

4057

11

-500.93096.905

0.563-0.0250.244-0.29519809-1.725

Honey

6

50

2603

3845

11

1985.883

3009.392

0.258

0.1

0.362

-0.16219809-1.725

Honey

6

100

-585

4332

10

349.3372861.585

0.452

0.018

0.267

-0.23219809-1.729

Honey

7

12.5

-1428

2175

10

-1464.58

2194.118

0.744-0.111

0.177

-0.4

13158

-1.729

Honey

7

25

-1298

2720

11

-954.1172224.217

0.664-0.0730.219-0.36413158-1.72573495

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Parameter

Time(CCA)1Test
conc.
(µg/l)

mean

SE(standard errorobservedmean)nEstimate(modelestimatemeandifference
fromcontrol)3,4

Std.Error(ofestimated mean)p_value forcompariso
n with thecontrol

Estimated
reductionfrom control(%) 4.5

Estimate(90%
confidenc
e upper
limit) 4)

Estimate(90%
confidence
lower
limit)4,5

Controlt-testMean(observed
means incontrol)

confidence
limit

Honey

7

37.5

-3159

3573

11

-491.248

2722.557

0.571-0.0370.32-0.39413158-1.725

Honey

7

50

-2571

2574

10

-2671.61

2224.197

0.878-0.2030.089-0.49513158-1.729

Honey

7

100

-3243

4024

7

-1602.15

3195.269

0.689-0.1220.302-0.54613158-1.746

Honey

8

12.5

-2150

3339

10

-1971.07

4067.710.683-0.1090.282-0.50118017-1.734

Honey

8

25

-2377

2212

11

-2087

3421.135

0.725-0.1160.212-0.44418017-1.729

Honey

8

37.5

-3252

3597

11

-555.28

3891.151

0.556-0.031

0.343-0.40418017-1.729

Honey

8

50

-96

2712

9

-350.486

4035.232

0.534-0.0190.37-0.409

18017

-1.74

Honey

8

100

6155

4291

7

7209.036

5059.696

0.087

0.4

0.892

-0.09218017-1.753

Honey

9

12.5

NA

NA

0

NA

NA

NA

NA

NA

NA

NA

NA

Honey

9

25

-5160

7052

2

34801.33

NA

NA

2.555

NA

NA

13622

NA

Honey

9

37.5

2408

NA

1

NA

NA

NA

NA

NA

NA

NA

NA

Honey

9

50

2881

2159

4

2539.491

2986.889

0.242

0.186

0.827

-0.454

13622

-2.92

Honey

9

100

NA

NA

0

NA

NA

NA

NA

NA

NA

NA

NA

Pollen

3

12.5

659

688

12

0

0

0

0

0

0

3755

-1.717

Pollen

3

25

616

705

12

0

0

0

0

0

0

3755

-1.71774496

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Parameter

Time(CCA)1Test
conc.
(µg/l)

mean

SE(standard errorobservedmean)nEstimate(modelestimatemeandifference
fromcontrol)3,4

Std.Error(ofestimated mean)p_value forcompariso
n with thecontrol

Estimated
reductionfrom control(%) 4.5

Estimate(90%
confidenc
e upper
limit) 4)

Estimate(90%
confidence
lower
limit)4,5

Controlt-testMean(observed
means incontrol)

confidence
limit

Pollen

3

37.5

-373

782

12

0

0

0

0

0

0

3755

-1.717

Pollen

3

50

788

514

12

0

0

0

0

0

0

3755

-1.717

Pollen

3

100

-416

1010

12

0

0

0

0

0

0

3755

-1.717

Pollen

4

12.5

64

575

12

-274.182

529.486

0.695-0.0850.197-0.3683218-1.717

Pollen

4

25

1641

472

12

1338.308

489.69

0.006

0.416

0.677

0.155

3218

-1.717

Pollen

4

37.5

322

747

12

524.949586.77

0.19

0.163

0.476

-0.15

3218

-1.717

Pollen

4

50

1354

440

12

995.281531.877

0.037

0.309

0.593

0.025

3218

-1.717

Pollen

4

100

1828

451

12

1976.704

533.579

0.001

0.614

0.899

0.33

3218

-1.717

Pollen

5

12.5

968

831

12

626.829845.907

0.233

0.136

0.451

-0.1794608-1.717

Pollen

5

25

2014

698

12

1773.621

837.4

0.023

0.385

0.697

0.073

4608

-1.717

Pollen

5

37.5

954

772

11

1039.421

943.221

0.141

0.226

0.578

-0.1274608-1.721

Pollen

5

50

1742

621

12

1388.486

803.124

0.049

0.301

0.601

0.002

4608

-1.717

Pollen

5

100

3720

624

12

3805.417

848.688

0

0.826

1.142

0.51

4608

-1.717

Pollen

6

12.5

146

804

10

72.159802.08

0.465

0.019

0.39

-0.3513741-1.729Pollen

6

25

751

622

11

526.567

707.645

0.233

0.141

0.467

-0.1853741-1.72575497

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Parameter

Time(CCA)1Test
conc.
(µg/l)

mean

SE(standard errorobservedmean)nEstimate(modelestimatemeandifference
fromcontrol)3,4

Std.Error(ofestimated mean)p_value forcompariso
n with thecontrol

Estimated
reductionfrom control(%) 4.5

Estimate(90%
confidenc
e upper
limit) 4)

Estimate(90%
confidence
lower
limit)4,5

Controlt-testMean(observed
means incontrol)

confidence
limit

Pollen

6

37.5

109

558

11

326.383

796.257

0.343

0.087

0.454

-0.28

3741

-1.725Pollen

6

50

1157

701

11

669.109711.586

0.179

0.179

0.507

-0.1493741-1.725Pollen

6

100

2778

851

10

3005.044

922.899

0.002

0.803

1.23

0.377

3741

-1.729Pollen

7

12.5

112

917

10

41.086966.295

0.483

0.011

0.46

-0.4383720-1.729Pollen

7

25

1345

475

11

1121.167

758.894

0.078

0.301

0.653

-0.05

3720

-1.725Pollen

7

37.5

109

468

11

385.35834.496

0.325

0.104

0.491

-0.2833720-1.725Pollen

7

50

731

601

10

236.918

833.164

0.39

0.064

0.451

-0.3243720-1.729Pollen

7

100

1831

884

7

2298.613

1247.892

0.042

0.618

1.204

0.032

3720

-1.746

Pollen

8

12.5

9

666

10

-36.682660.370.522-0.0150.462-0.4922401-1.734

Pollen

8

25

281

456

11

123.306

514.202

0.407

0.051

0.422

-0.3192401-1.729Pollen

8

37.5

-625

433

11

-282.961

527.598

0.701-0.1180.262-0.4982401-1.729Pollen

8

50

134

542

9

-91.816585.371

0.561-0.0380.386-0.462

2401

-1.74

Pollen

8

100

614

914

7

1000.501

906.927

0.144

0.417

1.079

-0.2452401-1.753Pollen

9

12.5

NA

NA

0

NA

NA

NA

NA

NA

NA

NA

NA

Pollen

9

25

-1290

1462

2

-2752

NA

NA

-1.404

NA

NA

1961

NA

76
498

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Parameter

Time(CCA)1Test
conc.
(µg/l)

mean

SE(standard errorobservedmean)nEstimate(modelestimatemeandifference
fromcontrol)3,4

Std.Error(ofestimated mean)p_value forcompariso
n with thecontrol

Estimated
reductionfrom control(%) 4.5

Estimate(90%
confidenc
e upper
limit) 4)

Estimate(90%
confidence
lower
limit)4,5

Controlt-testMean(observed
means incontrol)

confidence
limit

Pollen

9

37.5

172

NA

1

NA

NA

NA

NA

NA

NA

NA

NA

Pollen

9

50

-1935

1004

4

-1239.68

873.4480.854-0.6320.668-1.933

1961

-2.92

Pollen

9

100

NA

NA

0

NA

NA

NA

NA

NA

NA

NA

NA

Capped

3

12.5

1885

1302

12

0

0

0

0

0

0

14068

-1.717

Capped

3

25

3562

1981

12

0

0

0

0

0

0

14068

-1.717

Capped

3

37.5

1698

1597

12

0

0

0

0

0

0

14068

-1.717

Capped

3

50

1268

1033

12

0

0

0

0

0

0

14068

-1.717

Capped

3

100

924

952

12

0

0

0

0

0

0

14068

-1.717

Capped

4

12.5

1534

1940

12

868.4172287.868

0.354

0.065

0.36

-0.23

13330

-1.717

Capped

4

25

1591

1688

12

-203.405

2154.713

0.537-0.0150.262-0.29313330-1.717

Capped

4

37.5

717

1915

12

-485.599

2050.363

0.593-0.0360.228-0.30113330-1.717

Capped

4

50

2766

1328

12

2134.483

1958.734

0.144

0.16

0.412

-0.09213330-1.717

Capped

4

100

6307

1777

12

5894.047

2125.093

0.006

0.442

0.716

0.168

13330

-1.717

Capped

5

12.5

1620

909

12

835.0911238.42

0.254

0.048

0.17

-0.07417386-1.717

Capped

5

25

2938

1835

12

492.2171465.202

0.37

0.028

0.173

-0.11617386-1.71777499

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Parameter

Time(CCA)1Test
conc.
(µg/l)

mean

SE(standard errorobservedmean)nEstimate(modelestimatemeandifference
fromcontrol)3,4

Std.Error(ofestimated mean)p_value forcompariso
n with thecontrol

Estimated
reductionfrom control(%) 4.5

Estimate(90%
confidenc
e upper
limit) 4)

Estimate(90%
confidence
lower
limit)4,5

Controlt-testMean(observed
means incontrol)

confidence
limit

Capped

5

37.5

4605

2016

11

3446.378

1658.596

0.025

0.198

0.362

0.034

17386

-1.721

Capped

5

50

7310

1885

12

7062.109

1769.461

0

0.406

0.581

0.231

17386

-1.717

Capped

5

100

13129

1567

12

12668.53

1508.311

0

0.729

0.878

0.58

17386

-1.717

Capped

6

12.5

-103

1516

10

280.291

1953.853

0.444

0.025

0.33

-0.27911087-1.729Capped

6

25

203

1803

11

-693.872024.965

0.632-0.0630.252-0.37811087-1.725

Capped

6

37.5

876

2190

11

-20.494

2101.09

0.504-0.0020.325-0.32911087-1.725

Capped

6

50

2807

2447

11

2439.957

2383.828

0.159

0.22

0.591

-0.15111087-1.725

Capped

6

100

6312

1736

10

6579.909

2035.95

0.002

0.593

0.911

0.276

11087

-1.729Capped

7

12.5

-1041

942

10

-629.7938.6110.745-0.1890.298-0.6763332-1.729Capped

7

25

1384

977

11

1568.674

1009.999

0.068

0.471

0.993

-0.0523332-1.725

Capped

7

37.5

-164

1206

11

-62.6131133.697

0.522-0.0190.568-0.6063332-1.725

Capped

7

50

1462

600

10

1638.468

845.043

0.034

0.492

0.93

0.053

3332

-1.729Capped

7

100

1867

980

7

1867.429

1045.354

0.046

0.56

1.108

0.013

3332

-1.746

Capped

8

12.5

-654

563

10

-519.544

542.4620.825-0.4070.33-1.1451276-1.734

Capped

8

25

336

209

11

211.226

378.348

0.292

0.166

0.678

-0.3471276-1.72978500

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Parameter

Time(CCA)1Test
conc.
(µg/l)

mean

SE(standard errorobservedmean)nEstimate(modelestimatemeandifference
fromcontrol)3,4

Std.Error(ofestimated mean)p_value forcompariso
n with thecontrol

Estimated
reductionfrom control(%) 4.5

Estimate(90%
confidenc
e upper
limit) 4)

Estimate(90%
confidence
lower
limit)4,5

Controlt-testMean(observed
means incontrol)

confidence
limit

Capped

8

37.5

-367

451

11

-456.96484.771

0.821-0.3580.299-1.0151276-1.729Capped

8

50

-143

297

9

-214.838

464.816

0.675-0.1680.465-0.802

1276

-1.74

Capped

8

100

-467

438

7

-466.857

538.258

0.8-0.3660.374-1.1061276-1.753

Capped

9

12.5

NA

NA

0

NA

NA

NA

NA

NA

NA

NA

NA

Capped

9

25

-5504

2408

2

-5426.32

NA

NA

-0.73

NA

NA

7430

NA

Capped

9

37.5

-1892

NA

1

NA

NA

NA

NA

NA

NA

NA

NA

Capped

9

50

-5031

5148

4

-7475.75

2913.255

0.938-1.0060.139-2.151

7430

-2.92

Capped

9

100

NA

NA

0

NA

NA

NA

NA

NA

NA

NA

NA

Open

3

12.5

1018

831

12

0

0

0

0

0

0

5920

-1.717

Open

3

25

2480

942

12

0

0

0

0

0

0

5920

-1.717

Open

3

37.5

975

824

12

0

0

0

0

0

0

5920

-1.717

Open

3

50

932

653

12

0

0

0

0

0

0

5920

-1.717

Open

3

100

975

690

12

0

0

0

0

0

0

5920

-1.717

Open

4

12.5

-186

1032

12

-858.507

1033.015

0.793-0.1420.152-0.4366034-1.717

Open

4

25

201

876

12

-916.841

1159.785

0.781-0.1520.178-0.4826034-1.71779501

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Parameter

Time(CCA)1Test
conc.
(µg/l)

mean

SE(standard errorobservedmean)nEstimate(modelestimatemeandifference
fromcontrol)3,4

Std.Error(ofestimated mean)p_value forcompariso
n with thecontrol

Estimated
reductionfrom control(%) 4.5

Estimate(90%
confidenc
e upper
limit) 4)

Estimate(90%
confidence
lower
limit)4,5

Controlt-testMean(observed
means incontrol)

confidence
limit

Open

4

37.5

-272

611

12

-931.856

824.6440.865-0.1540.08-0.3896034-1.717

Open

4

50

158

817

12

-678.321

854.933

0.782-0.1120.131-0.3566034-1.717

Open

4

100

1949

903

12

1149.816

929.037

0.114

0.191

0.455

-0.0746034-1.717

Open

5

12.5

588

798

12

65.307804.633

0.468

0.01

0.215

-0.1966722-1.717

Open

5

25

674

750

12

-116.926

953.8280.548-0.0170.226-0.2616722-1.717

Open

5

37.5

555

899

11

293.801

945.175

0.379

0.044

0.286

-0.1986722-1.721

Open

5

50

2365

873

12

2011.076

904.088

0.018

0.299

0.53

0.068

6722

-1.717

Open

5

100

4056

663

12

3523.042

746.233

0

0.524

0.715

0.333

6722

-1.717

Open

6

12.5

335

570

10

39.984

1013.403

0.484

0.007

0.302

-0.2895934-1.729

Open

6

25

235

1166

11

-1328.15

1267.95

0.846-0.2240.145-0.5925934-1.725

Open

6

37.5

360

948

11

3.834

1078.98

0.499

0.001

0.314

-0.3135934-1.725

Open

6

50

1564

1184

11

1240.532

1229.488

0.163

0.209

0.566

-0.1485934-1.725

Open

6

100

3870

940

10

2997.233

1065.815

0.006

0.505

0.816

0.195

5934

-1.729

Open

7

12.5

-206

773

10

-390.251701.5880.708-0.2120.447-0.8711842-1.729

Open

7

25

1048

445

11

1042.041

583.558

0.045

0.566

1.112

0.019

1842

-1.72580502

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Parameter

Time(CCA)1Test
conc.
(µg/l)

mean

SE(standard errorobservedmean)nEstimate(modelestimatemeandifference
fromcontrol)3,4

Std.Error(ofestimated mean)p_value forcompariso
n with thecontrol

Estimated
reductionfrom control(%) 4.5

Estimate(90%
confidenc
e upper
limit) 4)

Estimate(90%
confidence
lower
limit)4,5

Controlt-testMean(observed
means incontrol)

confidence
limit

Open

7

37.5

31

493

11

-117.567

530.0540.587-0.064

0.433

-0.56

1842

-1.725

Open

7

50

318

707

10

280.351661.722

0.338

0.152

0.773

-0.4691842-1.729

Open

7

100

356

725

7

331.341693.158

0.32

0.18

0.837

-0.4771842-1.746

Open

8

12.5

0

186

10

-33.062198.726

0.565-0.0650.612-0.742509-1.734

Open

8

25

164

122

11

38.619180.761

0.417

0.076

0.69

-0.538509-1.729

Open

8

37.5

-133

110

11

-133.331

154.5270.801-0.2620.263-0.787509-1.729

Open

8

50

363

182

9

269.007193.836

0.092

0.529

1.191

-0.134

509

-1.74

Open

8

100

-12

264

7

-40.823241.085

0.566

-0.08

0.75

-0.911509-1.753

Open

9

12.5

NA

NA

0

NA

NA

NA

NA

NA

NA

NA

NA

Open

9

25

-4300

1032

2

-2335.87

NANA-0.606

NA

NA

3853

NA

Open

9

37.5

-2408

NA

1

NA

NA

NA

NA

NA

NA

NA

NA

Open

9

50

-2451

2421

4

-2700.72

3149.925

0.759-0.701

1.686-3.088

3853

-2.92

Open

9

100

NA

NA

0

NA

NA

NA

NA

NA

NA

NA

NA

Eggs

3

12.5

688

781

12

0

0

0

0

0

0

6063

-1.717

Eggs

3

25

1949

1063

12

0

0

0

0

0

0

6063

-1.71781503

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Parameter

Time(CCA)1Test
conc.
(µg/l)

mean

SE(standard errorobservedmean)nEstimate(modelestimatemeandifference
fromcontrol)3,4

Std.Error(ofestimated mean)p_value forcompariso
n with thecontrol

Estimated
reductionfrom control(%) 4.5

Estimate(90%
confidenc
e upper
limit) 4)

Estimate(90%
confidence
lower
limit)4,5

Controlt-testMean(observed
means incontrol)

confidence
limit

Eggs

3

37.5

1261

1056

12

0

0

0

0

0

0

6063

-1.717

Eggs

3

50

473

680

12

0

0

0

0

0

0

6063

-1.717

Eggs

3

100

-473

596

12

0

0

0

0

0

0

6063

-1.717

Eggs

4

12.5

659

671

12

512.178679.245

0.229

0.085

0.279

-0.1096020-1.717

Eggs

4

25

-57

917

12

-539.733

840.224

0.736

-0.09

0.15

-0.3296020-1.717

Eggs

4

37.5

-14

684

12

-383.721

652.1810.719-0.064

0.122

-0.25

6020

-1.717

Eggs

4

50

-344

873

12

-494.63754.6140.741-0.0820.133-0.2976020-1.717

Eggs

4

100

516

775

12

620.721

732.1

0.203

0.103

0.312

-0.1066020-1.717

Eggs

5

12.5

-874

681

12

-1052.4

847.2290.886-0.1990.076-0.4745289-1.717

Eggs

5

25

330

492

12

-146.206

816.494

0.57-0.0280.237-0.2935289-1.717

Eggs

5

37.5

-461

779

11

-1021.58

840.179

0.881-0.1930.08-0.4665289-1.721

Eggs

5

50

874

1117

12

681.408

1012.774

0.254

0.129

0.458

-0.2

5289

-1.717

Eggs

5

100

2351

481

12

2517.267

745.13

0.001

0.476

0.718

0.234

5289

-1.717

Eggs

6

12.5

2348

733

10

1840.078

1025.765

0.044

0.323

0.633

0.012

5705

-1.729

Eggs

6

25

305

922

11

-1022.46

1026.986

0.834-0.179

0.131

-0.49

5705

-1.72582504

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Parameter

Time(CCA)1Test
conc.
(µg/l)

mean

SE(standard errorobservedmean)nEstimate(modelestimatemeandifference
fromcontrol)3,4

Std.Error(ofestimated mean)p_value forcompariso
n with thecontrol

Estimated
reductionfrom control(%) 4.5

Estimate(90%
confidenc
e upper
limit) 4)

Estimate(90%
confidence
lower
limit)4,5

Controlt-testMean(observed
means incontrol)

confidence
limit

Eggs

6

37.5

367

973

11

-441.412

1000.926

0.668-0.077

0.225

-0.38

5705

-1.725

Eggs

6

50

1235

1311

11

776.626

1180.261

0.259

0.136

0.493

-0.2215705-1.725

Eggs

6

100

3466

676

10

3525.367

1042.032

0.002

0.618

0.934

0.302

5705

-1.729

Eggs

7

12.5

112

357

10

76.195674.86

0.456

0.03

0.486

-0.4262558-1.729

Eggs

7

25

610

335

11

396.183

654.163

0.276

0.155

0.596

-0.2862558-1.725

Eggs

7

37.5

-625

468

11

-746.558

674.229

0.859-0.2920.163-0.7462558-1.725

Eggs

7

50

559

403

10

485.487

679.011

0.242

0.19

0.649

-0.2692558-1.729

Eggs

7

100

725

369

7

788.178823.767

0.176

0.308

0.87

-0.2542558-1.746

Eggs

8

12.5

-86

257

10

-74.885240.469

0.62-0.1160.53-0.763645-1.734

Eggs

8

25

-78

231

11

-167.544

222.122

0.77

-0.26

0.336

-0.855645-1.729

Eggs

8

37.5

16

196

11

34.87

209.763

0.435

0.054

0.616

-0.508645-1.729

Eggs

8

50

220

158

9

191.919198.451

0.174

0.298

0.833

-0.238

645

-1.74

Eggs

8

100

-393

516

7

-379.17388.547

0.828-0.5880.468-1.644645-1.753

Eggs

9

12.5

NA

NA

0

NA

NA

NA

NA

NA

NA

NA

NA

Eggs

9

25

-1118

2666

2

3947.4

NA

NA

0.948

NA

NA

4162

NA

83
505

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Parameter

Time(CCA)1Test
conc.
(µg/l)

mean

SE(standard errorobservedmean)nEstimate(modelestimatemeandifference
fromcontrol)3,4

Std.Error(ofestimated mean)p_value forcompariso
n with thecontrol

Estimated
reductionfrom control(%) 4.5

Estimate(90%
confidenc
e upper
limit) 4)

Estimate(90%
confidence
lower
limit)4,5

Controlt-testMean(observed
means incontrol)

confidence
limit

Eggs

9

37.5

-688

NA

1

NA

NA

NA

NA

NA

NA

NA

NA

Eggs

9

50

-1290

2689

4

347.554894.384

0.368

0.083

0.711

-0.544

4162

-2.92

Eggs

9

100

NA

NA

0

NA

NA

NA

NA

NA

NA

NA

NA

TotalLife

3

12.5

5668

3513

12

0

0

0

0

0

0

37091

-1.717

TotalLife

3

25

10767

4383

12

0

0

0

0

0

0

37091

-1.717

TotalLife

3

37.5

4405

3798

12

0

0

0

0

0

0

37091

-1.717

TotalLife

3

50

3707

2515

12

0

0

0

0

0

0

37091

-1.717

TotalLife

3

100

788

2489

12

0

0

0

0

0

0

37091

-1.717

TotalLife

4

12.5

4367

3999

12

942.4313798.456

0.403

0.025

0.196

-0.14638123-1.717

TotalLife

4

25

4080

4212

12

-3289.24

3853.76

0.799-0.086

0.087

-0.26

38123

-1.717

TotalLife

4

37.5

1140

3831

12

-2022.78

3258.133

0.729-0.053

0.094

-0.2

38123

-1.717

TotalLife

4

50

3542

3482

12

733.753323.912

0.414

0.019

0.169

-0.13

38123

-1.717

TotalLife

4

100

10177

3555

12

9634.56

3370.598

0.005

0.253

0.405

0.101

38123

-1.717

TotalLife

5

12.5

3307

2444

12

694.4562864.883

0.405

0.016

0.132

-0.09942483-1.717

TotalLife

5

25

4943

4192

12

-2840.58

3277.988

0.802-0.0670.066-0.19942483-1.71784506

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Parameter

Time(CCA)1Test
conc.
(µg/l)

mean

SE(standard errorobservedmean)nEstimate(modelestimatemeandifference
fromcontrol)3,4

Std.Error(ofestimated mean)p_value forcompariso
n with thecontrol

Estimated
reductionfrom control(%) 4.5

Estimate(90%
confidenc
e upper
limit) 4)

Estimate(90%
confidence
lower
limit)4,5

Controlt-testMean(observed
means incontrol)

confidence
limit

TotalLife

5

37.5

6180

4311

11

3162.085

3439.368

0.184

0.074

0.214

-0.06542483-1.721

TotalLife

5

50

13754

4869

12

11624.68

4131.08

0.005

0.274

0.441

0.107

42483

-1.717

TotalLife

5

100

25411

3685

12

24887.04

3151.779

0

0.586

0.713

0.458

42483

-1.717

TotalLife

6

12.5

4327

2285

10

2865.989

4443.573

0.263

0.083

0.307

-0.14

34411

-1.729TotalLife

6

25

926

4828

11

-6504.93

4896.464

0.901-0.1890.056-0.43434411-1.725

TotalLife

6

37.5

3285

4448

11

135.5294447.651

0.488

0.004

0.227

-0.21934411-1.725

TotalLife

6

50

8134

5407

11

5352.874

5452.809

0.169

0.156

0.429

-0.11834411-1.725

TotalLife

6

100

20211

3425

10

19401.15

4514.48

0

0.564

0.791

0.337

34411

-1.729TotalLife

7

12.5

-1331

1564

10

-1491.87

3041.223

0.685-0.0890.224-0.40216812-1.729TotalLife

7

25

2632

2114

11

1071.462

3265.271

0.373

0.064

0.399

-0.27116812-1.725

TotalLife

7

37.5

-786

2446

11

-1184.94

3148.394

0.645

-0.07

0.253

-0.39316812-1.725

TotalLife

7

50

3359

2432

10

2877.337

3316.56

0.198

0.171

0.512

-0.17

16812

-1.729TotalLife

7

100

8542

2793

7

8845.245

3844.574

0.018

0.526

0.925

0.127

16812

-1.746

TotalLife

8

12.5

-1064

1242

10

-1157.8

1526.336

0.771-0.1580.203-0.5187342-1.734

TotalLife

8

25

-401

1308

11

-1899.2

1466.454

0.895-0.2590.087-0.6047342-1.72985507

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Parameter

Time(CCA)1Test
conc.
(µg/l)

mean

SE(standard errorobservedmean)nEstimate(modelestimatemeandifference
fromcontrol)3,4

Std.Error(ofestimated mean)p_value forcompariso
n with thecontrol

Estimated
reductionfrom control(%) 4.5

Estimate(90%
confidenc
e upper
limit) 4)

Estimate(90%
confidence
lower
limit)4,5

Controlt-testMean(observed
means incontrol)

confidence
limit

TotalLife

8

37.5

-1328

946

11

-1163.46

1322.765

0.805-0.158

0.153

-0.47

7342

-1.729TotalLife

8

50

-228

1263

9

-949.986

1512.1520.731-0.1290.229-0.488

7342

-1.74

TotalLife

8

100

1831

1703

7

2103.263

1790.473

0.129

0.286

0.714

-0.1417342-1.753

TotalLife

9

12.5

NA

NA

0

NA

NA

NA

NA

NA

NA

NA

NA

TotalLife925-148906441

28870.319

NA

NA

0.453

NA

NA

19594

NA

TotalLife

9

37.5

-6050

NA

1

NA

NA

NA

NA

NA

NA

NA

NA

TotalLife

950-1251712936

4-2269.79

1581.988

0.856-0.1160.12-0.352

19594

-2.92

TotalLife

9

100

NA

NA

0

NA

NA

NA

NA

NA

NA

NA

NA

86
508

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

Analysis and discussion of results
Control trends
As shown in Figure B.1, the number of adults increased until CCA 5 (August) and then started to decline. This
trend was also observed in open cells and pollen, and was very evident with capped cells, whereby a dramatic
decline was observed following CCA 5. The number of eggs appeared to drop slightly at CCA 5, but then increased
by CCA 6 before a drop in numbers at CCA 7. Figure B.2 shows a comparison between the numbers of live beesin the three different neonicotinoid colony feeding studies for the control. Live bee counts in the imidacloprid andclothianidin studies both appear to be in decline beginning after CCA 6 (September) in the control colonies,
whereas the decline in live bees started to decline in the thiamethoxam study after CCA 5 (August). This is likely
the result of a later start date for the thiamethoxam study, which resulted in the 6th colony condition assessment
being taken in late September, which is closer to the period of time in which the colony is preparing foroverwintering. As a result, any effects observed in the thiamethoxam study at CCA 5 were difficult to follow to
CCA 6 (or thereafter) for potential recovery of effects, since the control colony was declining in numbers at thistime, resulting in less sensitivity in the analysis. Figure B.3 shows the variability in the mean of the controls for
total life and the beginning of the “die off” of bees at CCA 5.

22
20

Thia Controls

18
16

Adults

14

Eggs

12

Open Cells

106

CappedCells
Pollen

4

Honey

8

2
0
5-30

6-19

7-9

7-29

8-18

9-7

9-27

10-17

11-6

CCA Date
Figure B.1. Control data for all endpoints for CCA 3 to CCA 8.

87509

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

60.0
55.0
50.0

Live counts in 103

45.0

ClothiControl

40.0

Thia
Control

35.0
30.0

Imi
Control

25.0
20.0
15.0
10.0
5.0
0.0
30-May19-Jun09-Jul29-Jul18-Aug

CCA Date

07-Sep27-Sep17-Oct06-Nov

Figure B.2. Comparison between the numbers of live bees in the three different neonicotinoid colony feedingstudies for the controls.

88
510

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

Figure B.3. Mean of two controls within each apiary for CCAs for total life.
Colony Condition Assessments: Life stages in the hive
Total number of individuals (total life)
Figures B.4 and Table B.2 show the effects on total life (total number of individual bees) across CCAs andtreatment groups. CCA 5 is of particular interest in this study since it provides an observation period which has
allowed for the whole colony to be exposed to thiamethoxam for up to 7 weeks following exposure. Subsequent
CCAs (for example CCA 6 and onward) are difficult to use for comparison to the control since all the hives are
beginning to prepare for overwintering. This was the likely consequence of a latter study initiation which resulted in
CCA 6 occurring at the end of September, and CCA 7/8 in October. Compared with the control, no differences in
the number of TOTAL LIFE adults in hives (P>0.1) was observed for any CCA in the 12.5, 25 or 37.5 µg a.i/L dosegroups. In the 50 µg a.i/L group, there was a significant reduction from the control at CCA 5 (27% reduction,p=0.005). There was no other significant reduction in the following CCAs (CCA 6, and 7), although percent
reduction from the control were 15 and 17%. Variability in the data and the preparation of hives for overwinteringresulted in difficulty comparing the treatment and control groups following CCA 5. In the 100 µg a.i/L group, there
was a significant reduction from the control at all CCAs (except CCA 8). Total number of individuals was reduced
by 25% at CCA 4 (p=0.005), 58% at CCA 5 (p<<0.05p<.001), 56% at CCA 6 (p<<0.05 p<.001) and 52% at CCA 7(p=0.018). It is noted that the largest decrease in total life was at CCA 5. Figure B.4 and B.5a shows a generalincrease in the reduction from the control as the dose increases. In the 37.5 µg a.i/L dose group 6/11 apiaries are89511

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
reduced compared to the control, in the 50 µg a.i/L dose group 9/12 apiaries are reduced compared to the control,and in the 100 µg a.i/L all apiaries are reduced. In general, the standard error bars were observed mostly to increase
with increasing CCAs and dose. Figure X5b shows the number of hives which were performing more poorly (fortotal number of individuals) than the control at CCA 3 which is prior to the beginning of exposure. In particular, it
is noted that 10/12 hives in the 25 µg a.i/L had reductions from the control for total life.
Table B.2. Estimated percent reduction from control for total number of individuals (total life).
Estimated reduction from control (%)(P value)
Test
CCA9
concentration
CCA4
CCA5
CCA6
CCA7
CCA8
(After over(µg/L)(3 WAE)(6-7 WAE)(10-11 WAE)
(13 WAE)(16 WAE)
winter)July 28-31
20-28 Aug
17-23 Sep
6-10 Oct27-29 Oct
31 Mar
2.5
1.6
8.3
-8.9
-15.8
NA
12.5
(0.403)(0.405)(0.263)(0.685)(0.771)
-8.6
-6.7
-18.9
6.4
-25.9
45.3
25
(0.799)(0.802)(0.901)(0.373)(0.895)
(NA)
-5.3
7.4
0.4
-7
-15.8
NA
37.5
(0.729)(0.184)(0.488)(0.645)(0.805)
1.9
27
15.6
17
-12.9
-11.6
50
(0.414)(0.005**)(0.169)(0.198)(0.731)(0.856)
25.3
58.6
56.4
52.6
28.6
NA
100
(0.005**)
(0**)
(0**)
(0.018**)(0.129)Note: Negative value indicates increased number of individuals in comparison to control.
*0.05<P<0.1**P<0.05
NA – not applicable because the sample sizes were too low.
NOTE: WAE = weeks after exposure. CCA 1 was 7 weeks before exposure, CCA 2 was 4 weeks before exposure, and CCA 3 was 1 week before exposure.
Tables present post exposure CCA data only.

90
512Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

Figure B.4. Difference from control for the means for total life for CCA 4 to 9.

91
513

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

Figure B.5a. Difference from control for all treatments and apiaries at CCA3 (before exposure) for total
life.

92
514

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

Figure B.5b. Difference from control for all treatments and apiaries at CCA5 for total life.
AdultsFigure B.6 and Table B.3 shows the effects on adult honey bees across CCAs and treatment groups.
Compared with the control, no differences in the number of adults in hives (P>0.1) was observed for any CCA in
the 12.5, 25 or 37.5 µg a.i/L dose groups. In the 50 µg a.i/L group, there was an 18% reduction from the control at
CCA 5 (p=0.034). By the next CCA (6), there was no statistical significance from the control, however, thereduction was very similar at 16.7%. Since the thiamethoxam study started later in the season, by CCA 6 andonward, the colonies appeared to be starting to prepare for overwintering, and therefore, the consistent decline inmost colonies made a comparison difficult. In the 100 µg a.i/L group, there was a consistent trend of significant
93515

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
reduction from the control at all CCAs with the reduction of the number of adults generally increasing with eachsubsequent CCA (CCA 4, 15% reduction from control; CCA 5, 48% reduction; CCA6, 59% reduction; CCA 7, 76%
reduction; and CCA 8, 70% reduction). In the 50 µg a.i/L the decline in adults may have been a large contributing
factor to the decline in total life. Figure B.6 and B.7 shows a general increase in the reduction from the control as
the dose increases. In the 37.5 µg a.i/L dose group 6/10 apiaries are reduced compared to the control, in the 50 µg
a.i/L dose group 8/12 apiaries are reduced compared to the control, and in the 100 µg a.i/L 11/12 apiaries are
reduced. In general, the observed standard error bars mostly increase with increasing CCAs and dose.

Table B.3. Estimated percent reduction from control for adults.
Estimated reduction from control (%)(P value)
Test
concentration
CCA4
CCA5
CCA6
CCA7
CCA8
(µg/L)(3 WAE)(6-7 WAE)(10-11 WAE)
(13 WAE)(16 WAE)
July 28-31
20-28 Aug
17-23 Sep
6-10 Oct27-29 Oct
12.5
25
37.5
50
100

3.7
(0.325)-4.9(0.702)2.1(0.369)1.2(0.43)
15(0.028**)10(0.101)-17(0.926)7.9(0.197)18(0.034**)
48
(0**)

14
(0.126)-12(0.785)12.8(0.142)16.7(0.125)
59
(0**)

-5.8
(0.63)-21(0.867)-2.5(0.561)
6.5(0.372)76(0.003**)-11.9(0.725)-35.6(0.934)-16.6(0.819)-19.7(0.81)
70.7(0.006**)CCA9(After over
winter)
31 Mar
NA
-96.6
(NA)
NA
-61.6
(0.747)NA

Note: Negative value indicates increased number of individuals in comparison to control.
*0.05<P<0.1**P<0.05
NA – not applicable because the sample sizes were too low.
NOTE: WAE = weeks after exposure. CCA 1 was 7 weeks before exposure, CCA 2 was 4 weeks before exposure, and CCA 3 was 1 week before exposure.
Tables present post exposure CCA data only.

94516

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

Figure B.6. Difference from control for the means for number of adults for CCA 4 to 9.

95
517

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

Figure B.7. Difference from control for all treatments and apiaries at CCA5 for adults.

96518

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Eggs
Figure B.8 and Table B.4 shows the effects on eggs across CCAs and treatment groups. Compared with thecontrol, no differences in the number of eggs in hives (P>0.1) was observed for any CCA in the 25, 37.5 or 50 µg
a.i/L dose groups. In the 12.5 µg a.i/L group, there was a significant reduction from control at CCA 6 only (32%reduction, p=0.04). However, in the previous CCAs at 12.5 µg a.i/L, and the higher dose groups (25, 37.5 and 50 µg
a.i/L ) at CCA 6 (and all other CCAs) there were no significant reductions from the control, and many of thetreatment groups actually had more eggs compared to the control over the course of the study. However, in the 100µg a.i/L group there was a significant reduction from the control at CCA 5 (- you to be consistent in the number ofdecimal places you give and how you round or truncate 47.6% reduction, p=0.001) and CCA6 (62% reduction,p=0.002). As previously discussed, despite a lack of statistically observed effects at CCA 7 (30% reduction), it islikely that the colonies preparation for overwintering resulted in a less ability to distinguish effects compared to thecontrol, since all colonies were in decline. Figure B8 and B.9 shows a general increase in the reduction from thecontrol as the dose increases. In the 37.5 µg a.i/L dose group 5/11 apiaries are reduced compared to the control, inthe 50 µg a.i/L dose group 7/12 apiaries are reduced compared to the control, and in the 100 µg a.i/L 10/12 apiaries
are reduced. In general, the observed standard error bars mostly increase with increasing CCAs and dose.

Table B.4. Estimated percent reduction from control for eggs.
Estimated reduction from control (%)(P value)
Test
concentration
CCA4
CCA5
CCA6
CCA7
CCA8
(µg/L)(3 WAE)(6-7 WAE)(10-11 WAE)
(13 WAE)(16 WAE)
July 28-31
20-28 Aug
17-23 Sep
6-10 Oct27-29 Oct
12.5
25
37.5
50
100

8.5
(0.229)-9(0.736)-6.4(0.719)-8.2(0.741)10(0.203)-20(0.886)-2.8(0.57)-19.3(0.881)
12.9(0.254)47.6(0.001**)32(0.04**/0.834)-17.9(0.834)-7.7(0.668)13.6(0.259)62.8(0.002**)3(0.456)15(0.276)-29(0.859)19(0.242)30.8(0.176)-11.6(0.62)-26(0.77)
5.4(0.435)29.8(0.174)-58.8(0.828)CCA9(After over
winter)
31 Mar
NA
94.8
(NA)
NA
8.3
(0.368)NA

Note: Negative value indicates increased number of individuals in comparison to control.
*0.05<P<0.1**P<0.05
NOTE: When two p values are presented, it is the result of a step down approach in the statistical analysis. We gave this rationale for not having to do that
other than total life at CCA5 – but you can if you wish)
NA – not applicable because the sample sizes were too low.
NOTE: WAE = weeks after exposure. CCA 1 was 7 weeks before exposure, CCA 2 was 4 weeks before exposure, and CCA 3 was 1 week before exposure.
Tables present post exposure CCA data only.

97519

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

Figure B.8. Difference from control for the means for number of eggs for CCA 4 to 9.

98
520

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

Figure B.9. Difference from control for all treatments and apiaries at CCA5 for eggs.
Larvae (Open/uncapped brood)
Figure B.10 and Table B.5 shows the effects on open brood (larvae) across CCAs and treatment groups.
Compared with the control, no differences in the open brood (larvae) in hives (P>0.1) was observed for any CCA in
the 12.5, or 37.5 µg a.i/L dose groups. In the 25 µg a.i/L group, there was a significant reduction from control at
CCA 7 only (56.6% reduction, p=0.045). However, in the previous CCAs at 25 µg a.i/L, and the higher dose groups(37.5, 50 and 100 µg a.i/L ) at CCA 7 there were no significant reductions from the control. However, in the 50 and
100 µg a.i/L group there was a significant reduction from the control at CCA 5 (29% reduction, p=0.018 for 50 µg
a.i/L, and 52% reduction, p<<0.05 for 100 µg a.i/L). At 50 µg a.i/L, there was no significant reduction from the99521

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
control at CCA 6 and 7 (with reductions of 20 and 15%, respectively), however, by CCA 8 there was a significant
reduction (52%, p=0.092). In the 100 µg a.i/L group, there was also a reduction at CCA 6 (50%, p=0.006), however,there were no further reductions from the control at any subsequent CCAs. Once again, this could be the result of all
hives (including the control) preparing for overwintering. The reduction in larvae may have contributed to theoverall decline in total individuals. Figure B.10 and B.11 shows a general increase in the reduction from thecontrol as the dose increases. In the 37.5 µg a.i/L dose group 6/10 apiaries are reduced compared to the control, inthe 50 µg a.i/L dose group 8/12 apiaries are reduced compared to the control, and in the 100 µg a.i/L 11/12 apiaries
are reduced. In general, the observed standard error bars mostly increase with increasing CCAs and dose.

Table B.5 Estimated percent reduction from control for larvae (open).
Estimated reduction from control (%)(P value)
Test
concentration
CCA4
CCA5
CCA6
CCA7
CCA8
(µg/L)(3 WAE)(6-7 WAE)(10-11 WAE)
(13 WAE)(16 WAE)
July 28-31
20-28 Aug
17-23 Sep
6-10 Oct27-29 Oct
12.5
25
37.5
50
100

-14
(0.793)1(0.468)7(0.484)-15(0.781)-15(0.865)-12(0.78)
19(0.114)-1.7(0.548)4.4(0.379)29.9(0.018**)
52.4
(0**)

-22.4
(0.846))1(0.499)20.9(0.163)50.5(0.006**)-21(0.708)56.6(0.045**/0.587)-6.4(0.587)15(0.338)18(0.32)-6.5(0.565)
7.6(0.417)-26(0.801)52.9(0.092*)-8(0.566)CCA9(After over
winter)
31 Mar
NA
-60.6
(NA)
NA
-70
(0.759)NA

Note: Negative value indicates increased number of individuals in comparison to control.
*0.05<P<0.1**P<0.05
NA – not applicable because the sample sizes were too low.
NOTE: When two p values are presented, it is the result of a step down approach in the statistical analysis.
NOTE: WAE = weeks after exposure. CCA 1 was 7 weeks before exposure, CCA 2 was 4 weeks before exposure, and CCA 3 was 1 week before exposure.
Tables present post exposure CCA data only.

100
522

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

Figure B.10. Difference from control for the means for open cells (larvae) for CCA 4 to 9.

101
523

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

Figure B.11. Difference from control for all treatments and apiaries at CCA5 for open cells (larvae).

Pupae (Capped brood)
Figure B.12 and Table B.6 shows the effects on capped brood (pupae) across CCAs and treatment groups.
Compared with the control, no differences in the capped brood (pupae) in hives (P>0.1) was observed for any CCA
in the 12.5 µg a.i/L dose group. In the 25 µg a.i/L group, there was a significant reduction from the control at CCA
7 only (47% reduction, p=0.068) which was not observed at any other CCA. In previous CCAs (4, 5 and 6), thenumber of pupae were either close to the control, or higher. In the 37.5 µg a.i/L group, there was a significant
reduction from the control at CCA 5 only (19.8%, p=0.025). In the 50 µg a.i/L group, there was a significant
102524

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
reduction from the control at CCA 5 (40%, p<<0.05p<0.001) and CCA 7 (49%, p=0.034). At CCA 6 there was
22 % reduction, which was not statistically significant. It is interesting to note that the reduction in pupae appeared
dose related at CCA 5, with percent reduction increasing from 19.8% at 37.5 µg a.i/L, to 40% at 50 µg a.i/L, up to
72.9% at 100 µg a.i/L. The reduction in pupae may have contributed to the overall reduced live individuals. Figure
B.12 and B.13 shows a general increase in the reduction from the control as the dose increases. In the 37.5 µg a.i/Ldose group 9/11 apiaries are reduced compared to the control, in the 50 µg a.i/L dose group 10/12 apiaries are
reduced compared to the control, and in the 100 µg a.i/L 12/12 apiaries are reduced. In general, the standard error
bars increase with increasing CCAs and dose.

Table B.6. Estimated percent reduction from control for pupae (capped).
Estimated reduction from control (%)(P value)
Test
concentration
CCA4
CCA5
CCA6
CCA7
CCA8
(µg/L)(3 WAE)(6-7 WAE)(10-11 WAE)
(13 WAE)(16 WAE)
July 28-31
20-28 Aug
17-23 Sep
6-10 Oct27-29 Oct
12.5
25
37.5
50
100

6.5
(0.354)4.8(0.254)2.5(0.444)-1.5(0.537)-3.6(0.593)16(0.144)44(0.006**)2.8(0.37)
19.8(0.025**)
40.6
(0**)
72.9
(0**)

-6.3
(0.632)-2(0.504)22(0.159)59.3(0.002**)-18.9(0.745)47(0.068*/0.522)-1.9(0.522)49.2(0.034**)56(0.046**)-40.7(0.825)
16.6(0.292)-35.8(0.821)-16.8(0.675)
-36.6
(0.8)

CCA9
(After over
winter)
31 Mar
NA
-73
(NA)
NA
-100
(0.938)NA

Note: Negative value indicates increased number of individuals in comparison to control.
*0.05<P<0.1**P<0.05
NA – not applicable because the sample sizes were too low.
NOTE: When two p values are presented, it is the result of a step down approach in the statistical analysis.
NOTE: WAE = weeks after exposure. CCA 1 was 7 weeks before exposure, CCA 2 was 4 weeks before exposure, and CCA 3 was 1 week before exposure.
Tables present post exposure CCA data only.

103
525

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

Figure B.12. Difference from control for the means for capped cells (pupae) for CCA 4 to 9.

104
526

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

Figure B.13. Difference from control for all treatments and apiaries at CCA for capped cells (pupae).
Colony Condition Assessments – Food Stores
PollenFigure B.14 and Table B.7 shows the effects on pollen across CCAs and treatment groups. Compared withthe control, no differences in the pollen stores (P>0.1) was observed for any CCA in the 12.5 and 37.5 µg a.i/L dosegroups. This trend was similar to the larval effects. In the 25 and 50 µg a.i/L groups, there were significant
reductions from the control at CCA 4 and 5 (with lower pollen stores in the lower test concentration). The percent
105
527

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
reduction in the 25 µg a.i/L group was 41.6 % (p=0.006) and 38.5% (p=0.023) at CCA 4 and 5, respectively. In the
50 µg a.i/L group, reduction was 30% at both CCA 4 and 5 (p=0.037 and 0.049, respectively). In the 100 µg a.i/Lgroup, the effects were observed across all CCAs at relatively consistent reductions. Percent reduction from control
ranged from 61 to 82.6% from CCA 4 to 7. By CCA 8, the reduction was not statistically significant, but was still at
41.7%. It is interesting to note that this food store response variable, compared to life forms in the hive, appeared toreflect earlier onset of effects. Figure B.14 and B.15 shows a general increase in the reduction from the control as
the dose increases. In the 25 µg a.i/L dose group 10/12 apiaries are reduced compared to the control, in the 37.5 µg
a.i/L dose group 7/10 apiaries are reduced compared to the control, in the 50 µg a.i/L dose group 10/12 apiaries are
reduced compared to the control, and in the 100 µg a.i/L 11/12 apiaries are reduced. In general, the standard error
bars increase with increasing CCAs.

Table B.7. Estimated percent reduction from control for pollen stores
Estimated reduction from control (%)(P value)
Test
concentratio
CCA4
CCA5
CCA6
CCA7
CCA8
n (µg/L)(3 WAE)(6-7 WAE) (10-11 WAE)
(13 WAE)(16 WAE)
July 28-31
20-28 Aug
17-23 Sep
6-10 Oct27-29 Oct
12.5
25
37.5
50
100

-8.5
(0.695)41.6(0.006**/
0.19)
16.3
(0.19)
30.9(0.037**)61.4(0.001**)

13.6(0.233)38.5(0.023**/0.141)
22.6(0.141)30(0.049**)
82.6
(0**)

1.9
(0.465)1.1(0.483)-1.5(0.522)14(0.233)8.7(0.343)17.9(0.179)80.3(0.002**)30(0.078*)10(0.325)
6.4(0.39)
61.8(0.042**)5.1(0.407)-11.8(0.701)-3.8(0.561)41.7(0.144)CCA9(After over
winter)
31 Mar
NA
-140
(NA)
NA
-63
(0.854)NA

Note: Negative value indicates increased number of individuals in comparison to control.
*0.05<P<0.1**P<0.05
NA – not applicable because the sample sizes were too low.
NOTE: When two p values are presented, it is the result of a step down approach in the statistical analysis.
NOTE: WAE = weeks after exposure. CCA 1 was 7 weeks before exposure, CCA 2 was 4 weeks before exposure, and CCA 3 was 1 week before exposure.
Tables present post exposure CCA data only.

106
528

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

Figure B.14. Difference from control for the means for pollen for CCA 4 to 9.

107
529

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

Figure B.15. Difference from control for all treatments and apiaries at CCA5 for pollen.

Honey
Figure B.16 and Table B.8 shows the effects on honey stores across CCAs and treatment groups. Comparedwith the control, no differences in the honey stores (P>0.1) was observed for any CCA in the 12.5, 25, 37.5, and 50µg a.i/L dose groups. Note, in CCA4 and CCA5 at the 100 µg a.i/L dose the confidence interval is below zero but as
testing is one sided for harm it is not statistically significant. There was only one CCA (8) in the 100 µg a.i/L groupwhich was marginally reduced from the control (40% reduction, p=0.087). It is noted that the feeding solutions (sugar
solutions) provided during the exposure period might have affected natural honey storage patterns; however, effects
on honey storage are still able to be considered as all treatments were compared to control hives (which also received

108
530

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
feeding solutions). Figure B.16 and B.17 shows a general decrease in the reduction from the control as the dose
increases. This may also be the effect of a lower number of individuals available to consume the honey.
Table B.8. Estimated percent reduction from control for honey stores.
Estimated reduction from control (%)(P value)
Test
concentration
CCA4
CCA5
CCA6
CCA7
CCA8
(µg/L)(3 WAE)(6-7 WAE)(10-11 WAE)
(13 WAE)(16 WAE)
July 28-31
20-28 Aug
17-23 Sep
6-10 Oct27-29 Oct
12.5
25
37.5
50
100

8.4
(0.115)2.3(0.401)
3(0.345)-1.1(0.558)-31.5(0.999)18.1(0.108)12(0.216)5.5(0.358)0.2(0.495)-45.6(0.999)-6.5(0.692)10(0.26)-2.5(0.563)10(0.258)1.8(0.452)-11(0.744)-7.3(0.664)-3.7(0.571)-20(0.878)-12(0.689)-10.9(0.683)-11.6(0.725)-3.1(0.556)-1.9(0.534)40(0.087*)CCA9(After over
winter)
31 Mar
NA
255
(NA)
NA
18.6
(0.242)NA

Note: Negative value indicates increased number of individuals in comparison to control.
*0.05<P<0.1**P<0.05
NA – not applicable because the sample sizes were too low.
NOTE: WAE = weeks after exposure. CCA 1 was 7 weeks before exposure, CCA 2 was 4 weeks before exposure, and CCA 3 was 1 week before exposure.
Tables present post exposure CCA data only.

109
531

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

Figure B.16. Difference from control for the means for honey for CCA 4 to 9.

110
532

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

Figure B.17. Difference from control for all treatments and apiaries at CCA5 for honey.

111
533

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Table B.9. Summary of observed effects at each treatment level (Note: Values reported in the table
are the % reduction compared to control, based on model estimated raw numbers corrected forbaseline measurements).
Treatment(µg/l)
12.5

25

37.5

50

100

Observations (excluding overwintering data)
No significant reduction from control for Significant reduction from control for thethe following endpoints
following endpoints
Total number of individuals (total life),
Eggs at CCA 6 only (32% reduction, p=0.04).
Larvae (open),
Pupae (capped),
Adults,Pollen stores,
Honey stores.
Total number of individuals (total life),
Eggs,
Honey stores,
Adults.
NOTE: In the 25 µg a.i/L dose, at CCA 3(before exposure began), 10/12 hives were
performing more poorly than control hives(for example, total number of individuals).
Total number of individuals (total life),
Eggs,
Larvae (open),
Pollen stores,
Honey stores,
Adults.
Eggs,
Honey

Larvae (open) at CCA 7 only (56%reduction, p=0.045),
Pupae (capped) at CCA 7 only (47%
reduction, p=0.068),
Pollen stores at CCA 4 (41% reduction,p=0.006), CCA 5 (38% reduction, p=0.023)
and CCA 7 (30% reduction, p=0.078).
Pupae (capped) at CCA 5 only (19.8%
reduction, p=0.025).

Total number of individuals at CCA 5 only
(27% reduction, p=0.005),
Larvae at CCA 5 (29.9% reduction,p=0.018) and CCA 8 (52.9% reduction,p=0.092),
Pupae at CCA 5 (40% reduction, p<<0.05)
and CCA 7 (49% reduction, p=0.034),
Pollen at CCA 4 (30.9% reduction, p=0.037)
and CCA 5 (30% reduction, p=0.049),
Adults at CCA 5 (18% reduction, p=0.034).
Total number of individuals at CCA 4 (25%reduction, p=0.005), CCA 5 (58.6%
reduction, p<<0.05), CCA 6 (56% reduction,p<<0.05) and CCA 7 (52% reduction,p=0.018).
Eggs at CCA 5 (47% reduction, p=0.001) and
CCA 6 (62.8% reduction, p=0.002),
Larvae at CCA 5 (52% reduction, p<<0.05),
CCA 6 (50% reduction, p=0.006),
Pupae at CCA 4 (44% reduction, p=0.006),
CCA 5 (72.9% reduction, p<<0.05), CCA 6(59% reduction, p=0.002), CCA 7 (56%reduction, p=0.046),
Pollen at CCA 4 (61% reduction, p=0.001),
CCA 5 (82.6% reduction, p<<0.05), CCA 6(80% reduction, p=0.002) and CCA 7 (61.8%
reduction, p=0.042),112
534

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

OVERALL
ENDPOINT

Honey stores at CCA 8 only (40% reduction,p=0.087)
Adults at CCA 4 (15% reduction, p=0.028),
CCA 5 (48% reduction, p<<0.05), CCA 6(59% reduction, p<<0.05), CCA 7 (76%reduction, p=0.003) and CCA 8 (70.7%
reduction, p=0.006).

The thiamethoxam study began later in the season, and thus by CCA 6 the colonies
were likely starting to prepare for overwinter, making a comparison between control
and treatment groups difficult since all colonies were declining in strength. Highoverwinter mortality in the control, resulted in an inability to assess overwintersuccess. Data was highly variable which also led to amibiguity in some of the datainterpretation. Overall, there appears to be effects at the 50 µg/L dose group.
Another study is being conducted, and will be taken into consideration whencompleted and reviewed. In the interim, the quantitative NOEC will be 37.5
µg/L. The quantitative LOEC will be 50 µg/L.

Consideration of combined doses response modelling
Figure B.18 shows a dose modeling of toxicity (the reduction of control life), with vertical lines showing
the best estimate of the dose which causes 10% reduction from control for total life at CCA 5 for thiamethoxam(T), imidacloprid (I) and clothianidin (C), and all data combined (A). Here, the analysis is being specificallyfocussed on a 10% reduction from control as well as contrasting and utilising data from all three neonicotinoids(which are believed to have the same biological mode of action). This may assist in the biological interpretation of
the data for thiamethoxam which had large standard errors for total life at CCA5 (and others) that resulted indifficulties of interpretation. It should be noted that the 25 µg a.i/L dose group was excluded in this analysis due thelarge statistically significant imbalance at baseline (pre-treatment condition of the hives). In the analysis that
accompanies this graph, in addition to the BMD (benchmark dose) (which is the dose expected to result in 10%
reduction of total life from control) the lower confidence for the BMD (the BMDL (benchmark dose lower
confidence limit) was also calculated, which is the dose that with 95% confidence (one sided) can be expected toresult in less than 10% effect (from the control). This provides a “safe” estimate of the dose that would result in lessthan a 10% reduction.
For thiamethoxam alone (considering only the data in that submission), the BMD was 29.19 µg a.i/L and
the BMDL was 14.36 µg a.i/L. Considering all data together under the assumption that all three neonicotinoids havethe exactly same biological mode of action, the BMD was 15.91 µg a.i/L and the BMDL was 9.69 µg a.i/L. To
allow biological mode of action to differ in some aspects according to the data, testing methods suggested in
Hydrogen Sulfide: Integrative Analysis of Acute Toxicity Data for Estimating Human Health Risk. J Stanek, J Gift,
G Woodall, and G Foureman, US EPA/ORD/NCEA, Research Triangle Park, NC, USA were followed. This
compromise method resulted in a BMD of 31.69 µg a.i/L and BMDL 18.07 µg a.i/L (higher in part due to increased
sample size). As a comparison, the compromise BMDs were 20.02 µg a.i/L for imidacloprid, and 13.14 µg a.i/L for
clothianidin with BMDLs 12.53 µg a.i/L and 7.2 µg a.i/L, respectively. On the other hand, if all threeneonicotinoids truly have exactly the same biological mode of action, the BMD and BMDL arguable should betaken as 15.91 and 9.69. [The statement “BMD and BMDL arguable should be taken as 15.91 µg a.i/L and 9.69 µg
a.i/L” represents some real uncertainty given the limitation of the studies even if the data suggested otherwise – it is
not definitive.] Therefore, the observed deviation of thiamethoxam from the other two neonicotinoids introduces
113535

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
some additional uncertainty in the assessment, and provides further support for considering effects at the 50 µg a.i/L
for the LOEC.

Figure B.18. Best estimate of the dose which causes 10% reduction from control for total life at CCA 5 (forT=thiamethoxam, I=imidacloprid, C=clothianidin and A = “all” or combined). The grey lines represent theindividual analysis and the coloured lines represent the BMD of the combined analysis.

Graphical representation of all parameters at CCA 5

114
536

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

115
537

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

116
538

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

117
539

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

118
540

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

119
541

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

120

542

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
State of California

Department of Pesticide Regulation

EVALUATION REPORT - Thiamethoxam Nectar Colony Feeding Study:
Repeat Study Conducted in 2016-2017
John Troiano, Research Scientist III
June, 2018
A review of: - Bocksch, S. (2017). Thiamethoxam Technical – Honey Bee Brood and Colony
Level Effects Following Thiamethoxam Intake via Treated Sucrose Solution in a Field Study inNorth Carolina – USA 2016: Final Report. Unpublished study prepared by Eurofins Agroscience
Service EcoChem Gmbh, & Eurofins Agroscience Services Ecotox GmbH. 481p., Laboratory
Report Number S16-02808. MRID 50432101. CDPR Study ID 304522.

Introduction
A colony feeding study was conducted to determine the effects of graded levels of thiamethoxam
on the health of honey bee hives where doses mimicked exposure from foraging on nectar.
Thiamethoxam was dosed directly to hives, supplied in a sugar solution that mimicked a nectar
source for food supply. Hive health was determined by Colony Condition Assessments (CCAs)
where measurements were made over time on the number of individuals in each bee life stage inthe hive, the storage of honey and pollen food supplies in the hives, and the weight of hives. This
study, conducted in 2016-2017, was a repeat of a study conducted in 2014-2015 (Bocksch, S.,2015). The second study was conducted in the same area as the first study where locations ofapiary sites were distributed throughout a forested area of North Carolina. Not all sites were in
the exact location as in the previous study. The distance between each apiary site wasapproximately 3 miles apart. The majority of land near the apiaries was non-intensively managed
pasture and forest with low potential exposure of bees to pesticides applied for agricultural
purposes.
Measurements made over time were indicated by sequential numbering of the colony conditionassessments (CCAs), which were conducted at approximately monthly intervals. Timing ofassessments were made at similar time intervals for the two studies. The exposure period for bothstudies was initiated in early July with the treatment period lasting 6 weeks. The CCAs includedin this analysis are:
 Just prior to initiation of treatments, denoted CCA2 in this analysis
 3 weeks into the exposure period, denoted CCA3 in this analysis
 6 weeks after initiation of exposure, denoted CCA4 in this analysis
 10 weeks after initiation of exposure, denoted CCA5 in this analysis
 13 weeks after initiation of exposure, denoted CCA6 in this analysis
 16 weeks after initiation of exposure, denoted CCA7 in this analysis
1543

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
These CCAs were chosen because this was the time period used to determine No Observed
Effects Concentrations (NOECs) and Lowest Observed Effects Concentrations (LOECs) in theprevious neonicotinoid nectar colony feeding studies.
The similarity of the study design between the two studies facilitated an analysis of the data
combined between years. Addition of variance between years in the statistical analysis provides
confidence that detection of significant effects are biologically significant and that they are notlimited to the year in which the study was conducted. Data from the previous study was analyzed
jointly by DPR, U.S. EPA, and Canada’s PMRA staff scientists (U.S. EPA, PMRA, & DPR,
2017).

Statistical Analysis
Evaluation of the data followed the statistical approach used by DPR and EPA scientists toanalyze previously reviewed neonicotinoid colony feeding studies. Since measurements for eachvariable were made in each hive over time, the statistical analysis was conducted as a repeatedmeasures over time (McIntosh, 1982). Additionally, a mixed model was used where apiary
location was identified as a random variable and thiamethoxam levels of dose as a fixed effect.
The mixed model was chosen because the results of the analysis were to be applied to the larger
population of bee hives. The analysis was conducted on the data combined from both years. In
the first year, data collected for CCAs number 3-8 corresponded to CCAs in the second year
numbered 2-7. As indicated previously, data collected from the timing of these assessments
conducted from July through September were the basis for development of NOEC and LOEC
values on previous neonicotinoid colony feeding studies. Normality tests were conducted foreach CCA within each year as indicated by Shapiro-Wilk and Kolmogorov-Smirnov test
statistics produced by the PROC CAPABILITY procedure in Statistical Analysis System (SAS,version 9.4). For comparison, data were also transformed to natural logarithms to determine if
transformation provided better results. The majority of results indicated that the distributions of
the raw data were normal with many of the logarithm transformed data indicating many instancesof non-normality. Based on these results the raw data were used in the analyses. The mixed
model approach used to analyze the data included tests to determine the appropriate covariancemodel that describes the covariance structure reflected by the data. Inclusion of a covariancemodel in the analysis accounts for heterogeneity of variances that often are measured between
treatment levels.
The PROC MIXED procedure in the Statistical Analysis System (SAS, version 9.4) was used to
run the repeated measures effects mixed model. Measurements of colony health and hive weight
were conducted approximately 1 month apart so CCAs were treated as equally spaced intervals.
The effects side of the model statement included testing differences in the response between
years, between CCAs indicating changes in response over the monthly measurements, between
the levels of thiamethoxam dose, and the potential interaction for effects of dose over time with
CCA and year factors. SAS Program 1 below reflects the structure of the program used toanalyze the combined data from both years. Statistical options were included in the ‘Slice’statement to protect against falsely discovering significant multiple comparisons for paired meanvalues between the value at the control and each level of dose. The ‘Simulated’ option is a Monte
Carlo approach that computes adjusted p-values from simulated distributions based on
2544

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
distributional statistics generated during the analysis (Edwards, D., and Berry, 1987). In addition,the ‘Stepdown’ option was invoked because it tends to increase the power of the multicomparison tests (SAS, version 9.4). SAS Program 2 indicates the structure of the program used
to conduct the analyses for each year.
There were two statements in the mixed model used to analyze the data, where a covariancemodel could be specified. One was in the ‘Random’ statement with apiary indicated as a randomvariable. The second was in the ‘Repeated’ measures statement where each hive was indicated as
the subject for the repeated measure. For the random statement only the Variance Component(VC) model successfully paired with the covariance model specified in the repeated statement:
Specifying more complex covariance models in the random statement resulted in indications ofconverge problems for that model. As observed in the previous colony feeding studies thecorrelation structure indicated greater correlation between samples taken at close time intervals
and, conversely, decreased correlation the further apart the samples were taken in time. Since
this structure is normally represented by autoregressive covariance models, the covariancestructure for the repeated statement was tested using variance component (VC), compound
symmetry (CS), compound symmetry with heterogeneity (CSH), autoregressive first order(AR(1)), autoregressive first order with heterogeneity (ARH(1)), and unstructured (UN) models.
Covariance model selection was based on the statistic generated for the Bayesian Information
Criteria (BIC) where a lower value of the criterion indicated a better fit of the covariance model.
A statistical basis for choosing the appropriate model was determined from Chi-square tests
conducted on the difference of the value of the BIC criteria between the two models tested with
the number of degrees of freedom determined as the difference between the number ofparameters in the model and where the significance level of probability was at 0.01 (Hammer,
2000; Littell et al., 2006). With the VC covariance model specified in the random statement, thebest fit covariance model in the repeated statement for the combined years analysis was AR(1)for adult bees and ARH(1) for pupae, larvae, eggs, nectar, and pollen cells (Table 1). Values fornumbers of cells measured for each bee life stage and food supply were divided by 1000 prior to
statistical analysis to minimize potential convergence problems due to magnitude of values.
SAS Program 1
proc mixed data=a3 order=data;class apiary dose cca hive rep year;
model transvalue =year cca dose dose*cca year*cca year*dose year*dose*cca/ddfm=sat
htype=1;
random apiary(year)/type=vc;
repeated cca/ subject=hive*rep(dose) type=arh(1);slice dose*cca /sliceby=cca diff=controll stepdown(report) adjust=simulate adjdfe=row;
run;

Results
Data Combined for Years: Means and standard deviation for each response variable measured
at each dose and each CCA are the same as presented in this and the previous report of study
results so they are not reprinted in this analysis (Bocksch, S., 2015). Results from the combined
years repeated measures model indicated numerous effects due to dose of thiamethoxam and its
3545

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
interactions with primarily CCA assessment for all bee life stages and food storage variables(Table 2). There was no interactive effect between the three factors for analysis of number ofadult bees, or pupal, larval, and nectar cells indicating similar responses between for the effectsof treatments where significance was indicated. The interactive effect of dose with CCA reflects
the varying magnitude in the level of differences for significant effects over the samplinginterval. For example, there were no differences between the levels of dose for the first CCA2
samples as these were taken prior to the study and indicate no bias in treatments at the start of thestudy (Figures 1 to 6). In later CCAs, the number of adult bees at the 100 ng/g treatment
indicates a downward trend where the magnitude in difference compared to the controls becomes
progressively greater at CCA4 and 5, but then lessens as hive activity normally decreases toward
the end of the season at the last CCA (Figure 1). Results from the pairwise comparisons between
values for control and each dose level indicate a specific pattern (Table 3). Except for nectar
cells, effects were first indicated on the number of pupal, larval, and pollen cells at CCA3 at thehighest dose of 100 ng/g when measurements were made midway through the exposure period(Figures 2, 3, and 6). These effects were sustained until CCA7 where, as previously indicated, allmeasurement of hive health decreased due to the normal yearly pattern of growth. At the next
CCA (CCA4) the number of adult bees and egg cells were then affected at the 100 ng/g dose andthese effects were also sustained throughout the entire season. Significant (P<0.05) effects werealso measured at the next lowest dose at 50 ng/g for number of adult bees, pupal cells, and pollen
cells that also appear to be sustained for a number of consecutive sampling intervals (Figures 1,
2, and 6). A trend was indicted (P<0.1) at CCA4 for number of larval cells. Sporadic indicationsof effects were noted at the next lowest dose at 37.5 ng/g for number of pupal and pollen cells
but they were not sustained for consecutive sampling intervals, most likely indicating spurious
effects. Effects on egg and nectar cells were minimal (Figures 4 and 5). This pattern of effects
indicates that the 50 ng/g dose is the LOEC value and the 37.5 g/g dose is the NOEC value.
Actual values measured for these concentrations in the dosing solutions as reported in Table 58of the report were 50 ng/g for the LOEC value and 34 ng/g for the NOEC value.
For completeness, graphs for each year are presented in Figures 7-12. Overlap for treatments
below 50 ng/g is obvious, clouding the consistency for effects between years at the 50 ng/g levelof dose. Figures 13 through 18 compare the effects on each variable for only the 0, 50 and 100
ng/g. Graphs for number of adult bees and pupal, larval and pollen cells clearly show theconsistent effect between year for decreased numbers at the 50 ng/g treatment.

Conclusion
In both replicate studies, the authors of the reports concluded that the 100 ng/g treatment was anominal LOEC value and that the 50 ng/g treatment was the NOEC value due to inconsistenteffects at that concentration. Statistical analyses conducted independently by the three agencies
on data generated from the first study disagreed with that conclusion (U.S. EPA, PMRA, & DPR,
2017). This analysis of the combined data between years strongly supports the previous
conclusion that the 50 ng/g level of dose was the LOEC value and the 37.5 ng/g treatment was
the appropriate NOEC value: Effects at the 50 ng/g level were evident and sustained across
CCAs for number of adult bees and pupal, larval, and pollen cells (Tables 2 and 3). The
measured value of thiamethoxam in the sucrose patties in the nominal 37.5 ng/g treatment group4546

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
was 34 ng/g, which is the actual NOEC value based on mean measured concentrations ofthiamethoxam. This study has been determined to be scientifically sound and can be used
quantitatively to assess risks to honey bee colonies.

References
Bocksch, S. (2015). Thiamethoxam Technical - Honey Bee Brood and Colony Level Effects
Following Thiamethoxam Intake via Treated Sucrose Solution in a Field Study in North
Carolina: Final Report. Unpublished study prepared by Eurofins Agroscience Services EcoChem
Gmbh. 468p., Laboratory Report Number S14-02633. MRID 49757201. CDPR Study ID
288917.
Edwards, D., and Berry, J. J. (1987). “The Efficiency of Simulation-Based Multiple
Comparisons.” Biometrics 43:913–928
Hammer, R.M. 2000. Mixed-Up Mixed Models: Things That Look Like They Should Work But
Don't, and Things That Look Like They Shouldn't Work But Do. Proceedings of the twenty-fifth
annual SAS users group International Conference, April 9-12, 2000, Indianapolis, Indiana.
Available at: http://www2.sas.com/proceedings/sugi25/25/aa/25p020.pdf. (Verified, Feb
12,2018).
Littell, R.C., G.A. Milliken, W.W. Stroup, R.D Wolfinger, and O. Schabenberger. 2006. SAS
System for Mixed Model, Second Edition. SAS Institute Inc., Cary NC.
Mcintosh, M. (1983). Analysis of combined experiments. Agronomy Journal (75) 153-155.
SAS Institute Inc 2013. SAS/ACCESS® 9.4.. Cary, NC: SAS Institute Inc.
U.S. EPA, PMRA, & DPR. (2017). Data evaluation report: Bocksch, S. (2015) -Thiamethoxam
Technical - Honey Bee Brood and Colony Level Effects Following Thiamethoxam Intake via
Treated Sucrose Solution in a Field Study in North Carolina. Washington, D.C.: U.S. EPA.

5

547

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Table 1. Mixed Model Analysis of Variance: BIC goodness-of-fit values generated for eachcovariance model structure tested in the repeated measures analysis of variance program.
Shaded cells indicate the covariance structure used for the analysis. DNC indicates that themodel failed to converge to solution.

CV
Model
Tested
VC
CS
AR(1)
CSH
ARH(1)

Combined Years Model BIC Value for:Number of
Parameters
2
3
3
8
8

Adults
DNC
5296.55080.35235.7
DNC

Pupae
DNC
5228.75213.75049.75026.2

Larvae4280.64158.34155.24037.14030.3Eggs
DNC
3836.43826.13722.93713.6

Nectar7262.76930.8DNC
6899.36784.9Pollen4829.94670.94627.84609.34536.1

6548

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Table 2. Combined Years: Results of the repeated measures mixed model testing the
response of each variable to clothianidin dosed surrogate honey.
Mixed Model Results for Repeated Measures Analysis of Variance:
Combined Data from Both Years
Variable
EffectDF Den DF
F Value
Pr > F
Adult Bees Year
1
22.1
3.49
0.0752cca
dose
dose*cca
cca*Yeardose*Yeardose*cca*Year
Pupal Cells Yearcca
dosedose*cca
cca*Yeardose*Yeardose*cca*Year
Larval Cells Yearcca
dosedose*cca
cca*Yeardose*Yeardose*cca*Year
Egg Cells
Year
cca
dose
dose*cca
cca*Yeardose*Yeardose*cca*Year
Nectar Cells Yearcca
dosedose*cca
cca*Yeardose*Yeardose*cca*YearPollen Cells Yearcca
dosedose*cca
cca*Yeardose*Yeardose*cca*Year

5
5
25
5
5
25
1
5
5
25
5
5
25
1
5
5
25
5
5
25
1
5
5
25
5
5
25
1
5
5
25
5
5
25
1
5
5
25
5
5
25

628
164
632
628
165
631
214
419
214
438
417
215
449
59.4
403
196
404
402
197
402
38.9
368
220
378
369
221
388
24.7
396
141
398
395
142
396
31.8
365
156
370
366
156
371

64.99
2.91
1.79
26.94
0.48
0.76
31.79
465.24
9.52
3.43
44.4
0.72
1.02
65.34
261.04
5.83
2.1
15.47
0.16
1.17
1.38
220.65
2.15
1.2
5.66
0.32
1.67
667.71
68.29
0.85
2.41
63.06
0.56
1.26
106.36
65.62
9.13
3.78
54.13
3.95
1.85

<.00010.01520.0110<.0001
0.79250.7973<.0001<.0001<.0001<.0001<.00010.61060.4420<.0001<.0001<.00010.0017<.00010.97780.25960.2464<.0001
0.0611
0.2365<.00010.90010.0236<.0001<.00010.51760.0002<.00010.72740.1817<.0001<.0001<.0001<.0001<.00010.00210.00847549

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Table 3. Combined Years: Probability value for the contrast of the control to eachclothianidin dose at each CCA and for each variable measured. Dark shaded cells indicate
significance at P<0.01 and lighter shaded cells at 0.1>P>0.05.
Responseand
Dose(ng/g)Bees

Pupae

LarvaeEggs

Nectar

12.5
25
37.5
50
100
12.5
25
37.5
50
100
12.5
25
37.5
50
100
12.5
25
37.5
50
100
12.5
25
37.5
50
100
Pollen
12.5
25
37.5
50
100

Probability Value for Contrast of the Control to Each
Dinotefuran Dose at Each CCA
CCA2
CCA3
CCA4
CCA5
CCA6
CCA7
0.774
0.671
0.801
0.801
0.870
0.274
0.187
0.274
0.274
0.309
0.430
0.190
0.430
0.430
0.663
0.742
0.172
0.430
0.778
0.778
0.976
0.982
0.982
0.958
0.961

0.493
0.493
0.493
0.297
0.422
0.467
0.467
0.467
0.053
<.0001
0.626
0.592
0.626
0.488
0.005
0.352
0.762
0.803
0.803
0.339
0.976
0.982
0.982
0.958
0.961

0.212
0.677
0.170
0.031
<.0001
0.090
0.090
0.016
0.000
<.0001
0.166
0.166
0.166
0.078
<.0001
0.795
0.329
0.795
0.337
0.014
0.464
0.717
0.744
0.615
0.958

0.155
0.502
0.176
0.095
<.0001
0.477
0.477
0.454
0.092
0.001
0.296
0.296
0.435
0.117
<.0001
0.281
0.639
0.639
0.435
0.002
0.203
0.520
0.520
0.137
0.203

0.548
0.649
0.548
0.270
0.001
0.477
0.111
0.302
0.184
0.001
0.711
0.225
0.564
0.564
0.049
0.654
0.654
0.654
0.465
0.034
0.187
0.482
0.482
0.080
0.122

0.691
0.691
0.691
0.691
0.040
0.888
0.642
0.690
0.642
0.532
0.819
0.240
0.240
0.240
0.240
0.435
0.494
0.494
0.494
0.411
0.155
0.439
0.439
0.142
0.010

0.942
0.942
0.942
0.931
0.942

0.913
0.827
0.312
0.030
<.0001

0.792
0.748
0.046
0.002
<.0001

0.787
0.550
0.365
0.026
0.000

0.364
0.071
0.071
0.017
<.0001

0.296
0.296
0.296
0.170
0.002

8

550

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Figure 1. Combined Years: Mean number of adult bee in each treatment group measured
at every CCA resulting from nectar feeding at the indicated dose of thiamethoxam.

Mean Number of Adult Bees
0

12.5

25

37.5

50

100

18000

Number of Adult Bees

16000
14000

12000
10000
8000
6000
4000
2000
0
0

1

2

3

4

5

6

7

CCA Assessment

9551

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Figure 2. Combined Years: Mean number of pupal cells in each treatment group measured
at every CCA resulting from nectar feeding at the indicated dose of thiamethoxam.

Mean Number of Pupal Cells
0

12.5

1

2

25

37.5

50

100

18000

Number of Pupal Cells

16000
14000
12000
10000
8000
6000
4000
2000
0
0

3

4

5

6

7

CCA Assessment

10

552

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Figure 3. Combined Years: Mean number of larval cells in each treatment group measured
at every CCA resulting from nectar feeding at the indicated dose of thiamethoxam.

Mean Number of Larval Cells
0

12.5

25

37.5

50

100

9000

Number of Larval Cells

8000
7000
6000
5000
4000
3000
2000
1000
0
0

1

2

3

4

5

6

7

CCA Assessment

11

553

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Figure 4. Combined Years: Mean number of larval cells in each treatment group measured
at every CCA resulting from nectar feeding at the indicated dose of thiamethoxam.

Mean Number of Egg Cells
0

12.5

25

37.5

50

100

7000

Number of Egg Cells

6000
5000
4000
3000
2000
1000
0
0

1

2

3

4

5

6

7

CCA Assessment

12

554

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Figure 5. Combined Years: Mean number of nectar cells in each treatment groupmeasured at every CCA resulting from nectar feeding at the indicated dose ofthiamethoxam.

Mean Number of Nectar Cells
0

12.5

1

2

25

37.5

50

100

70000

Number of Nectar Cells

60000

50000
40000
30000
20000
10000
0

0

3

4

5

6

7

CCA Assessment

13

555

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Figure 6. Combined Years: Mean number of pollen cells in each treatment group measured
at every CCA resulting from nectar feeding at the indicated dose of thiamethoxam.

Mean Number of Pollen Cells
Dose of Thiamethoxam (ng/g)

Number of Pollen Cells

12000

0

12.5

25

CCA2

CCA3

CCA4

37.5

50

100

10000
8000
6000
4000
2000
0

CC56

CCA Assessment

CCA6

CCA7

14

556

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Figure 7. Separate Years: Mean number of adult bees in each treatment group measured atevery CCA resulting from nectar feeding at the indicated dose of thiamethoxam.

A. Year 1 (2014-2015)

Number of Adult Bees
0

Dose of Thiamethoxam (ng/g)
12.5

25

37.5

50

100

14000

Number of Adult Bees

12000
10000
8000

6000
4000
2000
0

CCA2

CCA3

CCA4

CCA5

CCA Assessment

CCA6

CCA7

B. Year 2 (2016-2017)

Number of Adult Bees
Dose of Thiamethoxam (ng/g)
0

25000

12.5

25

37.5

50

CCA3

CCA4

CCA5

CCA6

100

Number of Adult Bees

20000

15000

10000

5000

0

CCA2

CCA Assessment

CCA7

15

557

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Figure 8. Separate Years: Mean number of pupal cells in each treatment group measured
at every CCA resulting from nectar feeding at the indicated dose of thiamethoxam.

A. Year 1 (2014-2015)

Number of Pupal Cells
Dose of Thiamethoxam (ng/g)
0

12.5

25

37.5

50

CCA4

CCA5

100

20000
18000

Number of Pupal Cells

16000
14000
12000
10000
8000
6000
4000
2000
0

CCA2

CCA3

CCA Assessment

CCA6

CCA7

B. Year 2 (2016-2017)

Number of Pupal Cells
Dose of Thiamethoxam (ng/g)
0

20000

12.5

25

37.5

50

100

CCA4
CCA5
CCA Assessment

CCA6

CCA7

Number of Pupal Cells

18000
16000
14000
12000
10000
8000

6000
4000
2000
0

CCA2

CCA3

16

558

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Figure 9. Separate Years: Mean number of larval cells in each treatment group measured
at every CCA resulting from nectar feeding at the indicated dose of thiamethoxam.

A. Year 1 (2014-2015)

Number of Larval Cells
0

Dose of Thiamethoxam (ng/g)
12.5

25

37.5

50

100

8000

Number of Larval Cells

7000
6000
5000
4000
3000
2000
1000

0

CCA2

CCA3

CA4

CCA5

CCA6

CCA7

CCA Assessment

B. Year 2 (2016-2017)

Number of Larval Cells
Dose of Thiamethoxam (ng/g)
0

12.5

25

37.5

50

100

10000

Number of Pupal Cells

9000
8000
7000
6000
5000
4000
3000

2000
1000
0

CCA2

CA3

CCA4
CCA5
CCA Assessment

CCA6

CCA7

17

559

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Figure 10. Separate Years: Mean number of egg cells in each treatment group measured atevery CCA resulting from nectar feeding at the indicated dose of thiamethoxam.

A. Year 1 (2014-2015)

Number of Egg Cells
Dose of Thiamethoxam (ng/g)
7000

0

12.5

CCA2

CCA3

25

37.5

50

100

6000

Number of Egg Cells

5000
4000
3000
2000
1000
0

CCA4

CCA5

CCA Assessment

CCA6

CCA7

B. Year 2 (2016-2017)

Number of Egg Cells
Dose of Thiamethoxam (ng/g)
0

7000

12.5

25

37.5

50

100

CCA6

CCA7

Number of Pupal Cells

6000
5000
4000
3000
2000
1000
0

CCA2

CCA3

CCA4
CCA5
CCA Assessment

18

560

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Figure 11. Separate Years: Mean number of nectar cells in each treatment group measured
at every CCA resulting from nectar feeding at the indicated dose of thiamethoxam.

A. Year 1 (2014-2015)

Number of Nectar Cells
Dose of Thiamethoxam (ng/g)
30000

0

12.5

25

37.5

50

100

Number of Nectar Cells

25000
20000
15000
10000
5000
0

CCA2

CCA3

CCA4

CCA5

CCA Assessment

CCA6

CCA7

B. Year 2 (2016-2017)

Number of Nectar Cells
Dose of Thiamethoxam (ng/g)
0100000

12.5

25

37.5

50

CCA4

CCA5

CCA6

100

90000
80000

Number of Pupal Cells

70000
60000
50000
40000
30000

20000
10000
0

CCA2

CCA3

CCA7

CCA Assessment

19

561

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Figure 12. Separate Years: Mean number of pollen cells in each treatment group measured
at every CCA resulting from nectar feeding at the indicated dose of thiamethoxam.

A. Year 1 (2014-2015)

Number of Pollen Cells
0

Dose of Thiamethoxam (ng/g)
12.5

25

37.5

CCA4

CCA5

50

100

5000
4500

Number of Pollen Cells

4000
3500
3000
2500
2000
1500
1000
500
0

CCA2

CCA3

CCA6

CCA7

CCA Assessment

B. Year 2 (2016-2017)

Number of Pollen Cells
Dose of Thiamethoxam (ng/g)
0

20000

12.5

25

37.5

50

100

Number of Pupal Cells

18000
16000
14000
12000
10000
8000

6000
4000
2000
0

CCA2

CCA3

CCA4
CCA5
CCA Assessment

CCA6

CCA7

20

562

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Figure 13. Separate Years-0, 50 100 ng/g Comparison: Mean number of adult bees in eachtreatment group measured at every CCA resulting from nectar feeding at the indicateddose of thiamethoxam.

A. Year 1 (2014-2015)

Number of Adult Bees
Dose of Thiamethoxam (ng/g)
0

14000

50

100

Number of Adult Bees

12000
10000
8000
6000
4000
2000
0

CCA2

CCA3

CCA4

CCA5

CCA6

CCA7

CCA Assessment

B. Year 2 (2016-2017)

Number of Adult Bees
Dose of Thiamethoxam (ng/g)
0

20000

50

100

Number of Adult Bees

18000
16000
14000

12000
10000
8000
6000
4000
2000
0

CCA2

CCA3

CA4

CCA5

CCA Assessment

CCA6

CCA7

21

563

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Figure 14. Separate Years-0, 50 100 ng/g Comparison: Mean number of pupal cells in eachtreatment group measured at every CCA resulting from nectar feeding at the indicateddose of thiamethoxam.

A. Year 1 (2014-2015)

Number of Pupal Cells
Dose 0of Thiamethoxam(ng/g)
50
100
20000
18000
Number of Pupal Cells

16000

14000
12000
10000
8000
6000
4000
2000
0

CCA2

CCA3

CCA4

CCA5

CCA Assessment

CCA6

CCA7

B. Year 2 (2016-2017)

Number of Pupal Cells
Dose of Thiamethoxam (ng/g)
0

20000

50

100

CCA4
CCA5
CCA Assessment

CCA6

Number of Pupal Cells

18000
16000
14000
12000
10000
8000
6000
4000
2000
0

CCA2

CCA3

CCA7

22

564

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Figure 15. Separate Years-0, 50 100 ng/g Comparison: Mean number of larval cells in eachtreatment group measured at every CCA resulting from nectar feeding at the indicateddose of thiamethoxam.

A. Year 1 (2014-2015)

Number of Larval Cells
Doseof Thiamethoxam(ng/g)
0
50
100
8000

Number of Larval Cells

7000
6000
5000
4000
3000
2000
1000

0

CCA2

CCA3

CCA4

CCA5

CCA Assessment

CCA6

CCA7

B. Year 2 (2016-2017)

Number of Larval Cells
Dose of Thiamethoxam (ng/g)
0

50

100

10000

Number of Pupal Cells

9000
8000
7000
6000
5000
4000
3000
2000
1000
0

CCA2

CCA3

CCA4
CCA5
CCA Assessment

CCA6

CCA7

23

565

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Figure 16. Separate Years-0, 50 100 ng/g Comparison: Mean number of egg cells in eachtreatment group measured at every CCA resulting from nectar feeding at the indicateddose of thiamethoxam.

A. Year 1 (2014-2015)

Number of Egg Cells
Dose of Thiamethoxam (ng/g)
0

50

100

7000
6000

Number of Egg Cells

5000
4000
3000
2000
1000

0

CCA2

CCA3

CCA4

CCA5

CCA Assessment

CCA6

CCA7

B. Year 2 (2016-2017)

Number of Egg Cells
Dose of Thiamethoxam (ng/g)
0

50

100

CCA4
CCA5
CCA Assessment

CCA6

Number of Pupal Cells

7000
6000
5000
4000
3000
2000
1000
0

CCA2

CCA3

CCA7

24

566

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Figure 17. Separate Years-0, 50 100 ng/g Comparison: Mean number of nectar cells in eachtreatment group measured at every CCA resulting from nectar feeding at the indicateddose of thiamethoxam.

A. Year 1 (2014-2015)

Number of Nectar Cells
Dose of Thiamethoxam (ng/g)
0

50

100

30000

Number of Nectar Cells

25000
20000
15000
10000
5000
0

CCA2

CCA3

CCA4

CCA5

CCA6

CCA Assessment

CCA7

B. Year 2 (2016-2017)

Number of Nectar Cells
Dose of Thiamethoxam (ng/g)
0100000

50

100

90000
80000

Number of Pupal Cells

70000
60000
50000
40000
30000
20000
10000
0

CCA2

CCA3

CCA4

CCA5

CCA Assessment

CCA6

CCA7

25

567

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Figure 18. Separate Years-0, 50 100 ng/g Comparison: Mean number of pollen cells in eachtreatment group measured at every CCA resulting from nectar feeding at the indicateddose of thiamethoxam.

A. Year 1 (2014-2015)

Number of Pollen Cells
Dose of Thiamethoxam (ng/g)
0

5000

50

100

Number of Pollen Cells

4500
4000
3500
3000
2500
2000
1500
1000
500
0

CCA2

CCA3

CCA4

CCA5

CCA6

CCA Assessment

CCA7

B. Year 2 (2016-2017)

Number of Pollen Cells
Dose of Thiamethoxam (ng/g)
0

18000

50

100

Number of Pupal Cells

16000
14000
12000
10000
8000

6000
4000
2000
0

CCA2

CCA3

CCA4
CCA5
CCA Assessment

CCA6

CCA7

26

568

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
State of California

Department of Pesticide Regulation

EVALUATION REPORT - Dinotefuran Nectar Colony Feeding StudyJohn Troiano, Research Scientist III
May, 2018

A review of: - Bocksch, S. (2016). Honey Bee Brood and Colony Level Effects Following
Dinotefuran Intake via Treated Sucrose Solution in a Field Study in North Carolina: Final
Report. Unpublished study prepared by Eurofins Agroscience Services EcoChem GmbH, &Eurofins Agroscience Services Ecotox GmbH. 523p., Laboratory Report Number S15-00102.
MRID 50147001. CDPR Study ID 296826.

Introduction
A colony feeding study was conducted to determine the effects of dinotefuran on the health ofhoney bees and their hives. Dinotefuran was dosed directly to hives, supplied in a sugar solutionthat mimicked a nectar source for food supply. Health of the bee colonies was determined bymeasuring the number of individuals in each bee life stage in the hive over time, the storage ofhoney and pollen food supplies in the hives, and the weight of hives over time. The statisticaldesign of the study was the same as that used in previous colony feeding studies on potential
effects of imidacloprid, thiamethoxam, and clothianidin on honey bee colony health. Dinotefuran
was mixed into sugar feeding solutions (i.e. nectar surrogate) at nominal concentrations of 10,
20, 40, 80, and 160 ng/g (ppb; ng of active ingredient per g of sugar solution). Colonies were
exposed to the sugar feeding solutions for six weeks. Each treatment was replicated at 12
different apiary sites that were located throughout forested areas of North Carolina. Assessmentsof land use conducted in a three mile radius around each apiary site indicated that cultivatedcrops comprised a maximum of 3% of the surrounding land, so the potential for exposure toother agricultural pesticides was low. The untreated control group was replicated twice at eachapiary, resulting in 24 replicates at the 0 (control) dose and 12 replicates at each of the treatmentgroup levels of dinotefuran. The distance between each apiary site was sufficient to minimize
potential for bees to cross-contaminate the apiary sites. Measurements made over time wereindicated by numbering of the colony condition assessments (CCA). For this analysis CCAs 3
through 8 were included. Observations at CCA3 were taken prior to initiation of dinotefuran
feeding treatments. Observations at CCA 4 were taken during the middle of the six week feeding(exposure) period with CCA 5 through 8 assessments taken after the exposure. Observations
were taken at approximately 1 month intervals. Additional observations taken after
overwintering were indicated as CCA9 and CCA10. Since determination of No Observed Effects
Concentrations (NOECs) and Lowest Observed Effects Concentrations (LOECS) in previous
1569

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
studies had been based on effects noted during the growing season, data were analyzed for CCA3
to CCA8 in this study, which corresponded to the same time interval in previous studies.
Observations made at CCA9 and CCA10 were not included in this statistical analysis of the data
because of complications which arose in previously evaluated colony feeding studies.

Statistical Analysis
Evaluation of the data followed the statistical approach used by DPR and EPA scientists toanalyze data from previously reviewed neonicotinoid colony feeding studies. Since
measurements for each parameter were made in each hive over time, the statistical analysis was
conducted as a repeated measures over time. Additionally, a mixed model was used where apiary
location was identified as a random variable and dinotefuran levels of dose a fixed effect. The
mixed model was chosen because the results of the analysis were to be applied to the larger
population of bee hives. Normality tests were conducted for each CCA as indicated by ShapiroWilk and Kolmogorov-Smirnov test statistics produced by the PROC CAPABILITY procedurein Statistical Analysis System (SAS, version 9.4). For comparison, data were also transformed tonatural logarithms to determine if transformation provided better results. Results were mixed
where in some cases the probability levels did not agree between the two test statistics or thetransformation gave more instances of non-normality, such as for pupal and honey cell counts.
Based on these results the raw data were used in the analyses. The mixed model approach
included tests to determine the appropriate model that describes the covariance structurereflected by the data. Inclusion of a covariance model in the analysis accounts for heterogeneityof variances that often are measured between treatment levels.
The repeated measures analysis of variance was conducted to determine potential effects ofdinotefuran dose on each measurement of hive health over time. Data collected from colony
condition assessments (CCAs) numbered CCA3 to CCA8 were included because these are thetime intervals where effects were observed in the previous neonicotinoid feeding studies. The
PROC MIXED procedure in the Statistical Analysis System (SAS, version 9.4) was used to run
the repeated measures effects mixed model. Since measurements of colony health wereconducted and hive weight reported approximately 1 month apart, they were treated as equally
spaced intervals in the analysis. A regression model was used to determine the effects of dose oneach response variable (SAS Program 1 below). The regression model was run twice. First, alldose levels were included where the levels of dose were expressed as orthogonal polynomial
contrasts that reflected the uneven spacing between the levels – 0, 10, 20, 40, 80, and 160 ng/g.
Based on these results, a reduced regression model was run that included the concentrations thatappeared to define LOEC and the NOEC concentrations. The second run was intended to remove
extraneous variance produced from treatments that were not contributing information to themodel. The linear and quadratic coefficients for the effect of dose concentration and theirinteraction with CCA assessments were tested in the models. The interaction term provided
information on changes in the relative effect of dinotefuran concentration over the duration of thestudy. For each regression model, an additional analysis was run that compared the probability
values between the control and each level of dinotefuran treatment at each CCA (SAS Program 2
below). This analysis provided guidance on the potential concentration of dinotefuran that2570

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
defined the NOEC and LOEC values. In order to protect against falsely discovering a significant
comparison between mean values, the ‘Simulated’ option was used to generate comparisons
between the control and each dose level (Edwards, D., and Berry, 1987). The ‘Simulated’ option
computes adjusted p-values from simulated distributions that are based on distributional statistics
generated during the analysis.
In the regression mixed model used to analyze the data, there are two places where a covariancemodel could be specified. One is in the ‘Random’ statement where apiary was identified as therandom variable. The second is in the ‘Repeated’ measures statement where each hive was
identified as the subject for the repeated measure. For the random statement only the Variance
Component (VC) model successfully paired with the covariance model specified in the repeatedstatement: Specifying more complex covariance models in the random statement resulted inindications of converge problems for that model. For the repeated measures statement thecorrelation structure generally indicated greater correlation between samples taken at close timeintervals and, conversely, decreased correlation the further apart the samples were taken in time
(Appendix I). Since this structure is normally represented by autoregressive covariance models,the covariance structure for the repeated statement was tested using variance component (VC),
compound symmetry (CS), compound symmetry with heterogeneity (CSH), autoregressive firstorder (AR(1)), autoregressive first order with heterogeneity (ARH(1)), and unstructured (UN)models. Covariance model selection was based on the statistic generated for the BayesianInformation Criteria (BIC) where a lower value of the criterion indicated a better fit of thecovariance model. A statistical basis for choosing the appropriate model was determined from
Chi-square tests conducted on the difference of the value of the BIC criteria between the twomodels tested with the number of degrees of freedom determined as the difference between thenumber of parameters in the model and where the significance level was at 0.01 (Hammer, 2000;
Littell et al., 2006). With the VC covariance model specified in the random statement, the best
fits covariance models in the repeated statement were AR(1) for adult bees and honey, ARH(1)for larvae, eggs, pollen and hive weight, and UN for pupae (Table 1).
SAS Program 1
proc mixed data=b6 order=data;class apiary dose cca hive;model transvalue =cca lin quad lin*cca quad*cca /ddfm=sat htype=1 solution;
random apiary/type=vc;
repeated cca/ subject=hive*dose type=ar(1);run;
SAS Program 2
proc mixed data=b6 order=data;class apiary dose cca hive;model transvalue =cca dose dose*cca /ddfm=sat htype=1;
random apiary/type=vc;
repeated cca/ subject=hive*dose type=ar(1);slice dose*cca /sliceby=cca diff=controll adjust=simulate adjdfe=row;
run;
3

571

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

Results
Means and standard deviation for each response variable measured at each dose and each CCA
are presented in Appendix B. Data for CCA3 through CCA8 were included in the repeatedmeasures analysis. Hive C2 was excluded from the analysis because data were lacking for
CCA6, CCA7, and CCA8. For the repeated measures regression model that included all levels ofdose (i.e. exposure concentrations) there were a few indications of trends in the data (Probability
level between 0.05 and 0.1): Linear effect of dose for number of adult bee cells, a linear
interaction over time (linear*CCA) for pupal cells, and a quadratic interaction over time forlarval cells (Table 2). There was an indication of a significant linear interaction term for honeycells (Probability level <0.05). For the pairwise comparisons of each dose level to the control
value, only trends, indicating decreases in numbers, were observed at the highest dose level (160ng/g) for adult bee and pupal cells (Table 3). These results indicated that 160 ng/g is the LOEC
value, which makes the 80 ng/g concentration the NOEC value.
Results from reduced analyses, using data for 0, 80, and 160 ng/g dose levels, confirmed the
NOEC and LOEC values. The linear effect for dose for adult bees was significant (Probability
level < 0.05). Comparison to the control value indicated significant decreases at CCA6 andtrends indicated at CCA5 and CCA7 (Tables 4 and 5). The graphical comparison of the effects
measured at each CCA indicates that upon exposure after CCA3 the number of adult bee cells
were consistently lower at the 160 ng/g treatment over the rest of the CCAs (Figure 1). The
graph comparing only the 3 treatment levels (0, 80, and 160 ng/g) clearly indicates that thedecrease was maintained over successive colony assessments. Although there was no indicationof significance for pupal cells in the regression table, the individual comparisons reflected thetrend observed in the overall analysis where the effect at CCA6 was significant for thecomparison of the effect at 160 ng/g to the control value (Table 5). The graph comparing thenumber of pupal cells reflects this effect with a clear reduction at the 160 ng/g treatment shownin the graph containing the 3 treatment levels (Figure 2). Although there was an indication of asignificant linear interaction for honeys cells in the reduced analysis, there were no indication of
dose-related effects in the individual comparisons (Figures 3). Potential effects noted in the fullanalysis for larval cells were also non-significant in the reduced analysis.
Potential dose-related effects were indicated for honey and pollen food stores where a significant
linear interaction over time was indicated in the full and reduced analyses (Tables 2 and 4). For
honey, none of the specific dose comparisons were significant, indicating no sustained effects at
any level of dose (Tables 3 and 5 and Figure 5). There were no dose-related effects for pollen
indicated in pairwise comparisons made for the full analyses, but results in the reduced analysis
indicated a trend for a decreases in the number of cells at the 160 ng/g dose at CCA4 and CCA5
(Tables 2 and4). The graph comparing responses between the three treatment levels reflects a
reduction in the mean number of pollen cells at the 160 ng/g dose at those CCAs (Figure 6).
One additional objective of colony feeding studies is to determine potential effects of treatmentson overwintering of hives. The number of surviving hives measured at the last CCA after
4572

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
overwintering (CCA10 on April 27, 2016) indicated a high survival rate for all treatments withno dose-related effects: Rates were 88%, 83%, 67% , 92%, 83%, and 83% for 0, 10, 20, 40, 80,
and 160 ng/g treatments, respectively. Analyses for hive weights also indicated no dose-related
effects (Tables 2 through 5 and Figure 8).

Conclusion
Results of the statistical analyses indicated effects of the highest dinotefuran dose on various bee
life stages and food stores. The most sustained effect was a decrease in the number of adult bees
at 160 ng/g indicated from CCA5 through CCA7. The graph comparing the projected NOEC and
LOEC values to the control values (0 80, and 160 ng/g) show that the number of adult bee cells
between the treatments were clearly similar at CCA 3 prior to initiation of the treatments,
followed by a steady decline at the highest 160 ng/g treatment after imposition of the dinotefuran
treatments (Figure 1). Other effects measured at the 160 ng/g treatment were decreased numberof pupal cells and pollen food stores (Figures 2 and 6). The number of adult bees, often referred
to as colony strength, is one of the primary indicators of colony health. Therefore, the Lowest
Observed Effect Concentration (LOEC) was determined to be 160 ng/g and the No Observed
Effect Concentration (NOEC) was established at a nominal concentration of 80 ng/g. The actual
measured value of clothianidin in the nectar feeding solution was 71 ng/g, which is the actual
NOEC value. The study authors reached the same conclusion, stating that the NOEC wasestablished at 80 ng/g as based on a significant reduction in bee bread storage at CCA4 and on asignificant reduction in the number of cells containing pupae at CCA6. Overall, this study has
been determined to be scientifically sound and can be used quantitatively to assess risks to honeybee colonies.

References
Edwards, D., and Berry, J. J. (1987). “The Efficiency of Simulation-Based Multiple
Comparisons.” Biometrics 43:913–928
Hammer,R.M. 2000. Mixed-Up Mixed Models: Things That Look Like They Should Work But
Don't, and Things That Look Like They Shouldn't Work But Do. Proceedings of the twenty-fifth
annual SAS users group International Conference, April 9-12, 2000, Indianapolis, Indiana.
Available at: http://www2.sas.com/proceedings/sugi25/25/aa/25p020.pdf. (Verified, Feb
12,2018).
Littell, R.C., G.A. Milliken, W.W. Stroup, R.D Wolfinger, and O. Schabenberger. 2006. SAS
System for Mixed Model, Second Edition. SAS Institute Inc., Cary NC.
SAS Institute Inc 2013. SAS/ACCESS® 9.4.. Cary, NC: SAS Institute Inc.

5573

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Table 1. Repeated Measures Analysis of Variance: BIC goodness-of-fit values generated for each covariance model structure
tested in the repeated measures analysis of variance program. Shaded cells indicate the covariance structure used for the
analysis. Number of parameters in parenthesis refers to hive weight analysis

CV ModelTested
VC/VC
VC/CS
VC/AR(1)
VC/CSH
VC/ARH(1)
VC/UN

Model BIC Value for:Number of
Parameters
2
3
3
8
8
22

Adults2735.72690.62601.12686.62601.82587.4

Pupae
2766.32676.62636.82591.82560.72545.8

Larvae2256.02207.32204.42092.72089.22094.8Eggs
2156.32139.52118.3
1980.6
1963.71977.7

Honey
3960.13635.63542.93646.63548.93543.6

Number ofPollen Parameters2753.1
22618.832582.332609.472566.572566.416

Hive
Weight2491.32397.82354.22320.32250.82192.46574

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Table 2. All Dose Levels: Results of the repeated measures mixed model testing the
response of each variable to dinotefuran dosed surrogate honey.

Mixed Model Results for Repeated Measures Analysis of Variance
All Doses
Variable
EffectDF Den DF F Value
Pr > F
Adult Bees

CCA
Lnear
Quadratic
Linear*CCA
Quadratic*CCA

5
1
1
5
5

325
86.9
86.9
325
325

50.29
3.45
0.13
0.21
0.52

<.0001
0.07
0.72
0.96
0.76

Pupae

CCA
LinearQuadratic
Linear*CCA
Quadratic*CCA

5
1
1
5
5

80
79
79
80
80

174.52
0.96
0.63
2.07
1.14

<.0001
0.33
0.43
0.08
0.35

LarvaeCCA
LinearQuadratic
Linear*CCA
Quadratic*CCA

5
1
1
5
5

172
113
113
172
172

183.56
0.02
2.19
0.4
2.06

<.0001
0.88
0.14
0.85
0.07

Eggs

CCA
LinearQuadratic
Linear*CCA
Quadratic*CCA
CCA
LinearQuadratic
Linear*CCA
Quadratic*CCA
CCA
LinearQuadratic
Linear*CCA
Quadratic*CCA

5
1
1
5
5
5
1
1
5
5
5
1
1
5
5

161
119
119
161
161
375
80.5
80.5
375
375
234
75.6
75.7
234
234

166.89
1
0.01
0.69
0.5
40.04
0.29
0.06
2.46
1.29
24.03
1.46
0.48
3.22
0.36

<.0001
0.32
0.93
0.63
0.77
<.0001
0.59
0.81
0.03
0.27
<.0001
0.23
0.49
0.01
0.88

Month
LinearQuadratic
Linear*Month
Quadratic*Month

4
1
1
4
4

73
62.3
62.7
73
73

29.32
0.01
1.46
1.06
0.7

<.0001
0.91
0.23
0.38
0.59

Honey

PollenHive Weight7575

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Table 3. All Dose Levels: Probability value for the contrast of the control to eachdinotefuran dose at each CCA and for each variable measured.

Response
and
Dose (ng/g)

Probability Value for Contrast of the Control to Each
Dinotefuran Dose at Each CCA
CCA3
CCA4
CCA5
CCA6
CCA7
CCA8

Bees

10
20
40
80
160

0.391
0.613
0.691
0.336
0.588

0.137
0.826
0.882
0.257
0.231

0.200
0.328
0.677
0.508
0.131

0.273
0.450
0.901
0.795
0.058

0.805
0.493
0.932
0.848
0.167

0.532
0.520
0.903
0.782
0.279

Pupae

10
20
40
80
160

0.678
0.519
0.799
0.841
0.854

0.668
0.930
0.988
0.985
0.621

0.826
0.663
0.960
0.916
0.550

0.595
0.892
0.524
0.630
0.088

0.940
0.961
0.973
0.887
0.258

0.516
0.487
0.817
0.825
0.781

Larvae

10
20
40
80
160

0.573
0.416
0.935
0.531
0.682

0.760
0.541
1.000
0.997
0.766

0.925
0.674
0.884
0.950
0.773

0.905
0.996
0.986
0.999
0.977

0.840
0.911
0.998
0.995
0.921

0.681
0.973
0.391
0.617
0.478

Eggs

10
20
40
80
160
Honey 10
20
40
80
160

0.090
0.662
0.931
0.871
0.641
0.940
0.163
0.613
0.817
0.739

0.797
0.916
0.964
0.846
0.979
0.997
0.557
0.580
0.901
0.999

0.892
0.954
0.970
0.542
0.954
0.994
0.556
0.906
0.942
0.999

0.583
0.815
0.882
0.998
0.973
0.961
0.349
0.915
0.937
0.934

0.700
0.968
0.863
0.596
0.941
0.998
0.929
0.992
0.995
0.980

0.631
0.631
0.172
0.972
1.000
0.991
0.761
0.930
0.960
0.649

Pollen

0.802
0.921
0.948
0.985
0.978
June
1.000
0.826
0.825
0.740
0.866

0.690
0.982
0.628
0.937
0.113
July
1.000
0.931
1.000
1.000
0.651

0.719
0.671
0.846
0.850
0.811
0.770
0.816
0.954
0.977
0.767
0.881
0.785
0.110
0.434
0.929
August September October
1.000
1.000
0.903
0.511
0.098
0.832
0.997
0.939
0.644
1.000
0.991
0.768
0.840
0.879
0.991

0.594
0.578
0.809
0.520
0.349

10
20
40
80
160
Hive Weight
10
20
40
80
160

8

576

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Table 4 Reduced Dose Levels: Results of the repeated measures mixed model testing the
response of each variable to dinotefuran dosed surrogate honey. Dose levels tested were 0,80, and 160 ng/g because the full analysis indicated 80 ng/g as a LOEC value and 160 ng/g
as a NOEC value.

Mixed Model Results for Repeated Measures Analysis of Variance
Data for 0, 80, and 160 ug/g Analyzed
Variable EffectDF
Den DFF Value
Pr > F
Adult Bees CCA
Lnear
Quadratic
Linear*CCA
Quadratic*CCA

5
1
1
5
5

184
39.6
39.5
184
184

29.96
4.6
0.01
0.26
0.53

<.0001
0.04
0.94
0.93
0.76

Pupae

CCA
LinearQuadratic
Linear*CCA
Quadratic*CCA

5
1
1
5
5

44
40.7
40.6
44
44

106.58
1.44
0.57
1.57
0.66

<.0001
0.24
0.45
0.19
0.65

LarvaeCCA
LinearQuadratic
Linear*CCA
Quadratic*CCA

5
1
1
5
5

99.8
60.4
60.3
99.8
99.8

95.46
0.01
1.19
0.38
1.09

<.0001
0.94
0.28
0.86
0.37

Eggs

CCA
LinearQuadratic
Linear*CCA
Quadratic*CCA
CCA
LinearQuadratic
Linear*CCA
Quadratic*CCA
CCA
LinearQuadratic
Linear*CCA
Quadratic*CCA

5
1
1
5
5
5
1
1
5
5
5
1
1
5
5

67.5
38.9
38.8
67.5
67.5
202
37.7
37.6
202
202
139
44.4
44.3
139
139

88.46
0.17
0.02
0.33
0.66
18.94
0.36
0.02
3.18
0.65
10.66
1.55
0.28
2.81
0.57

<.0001
0.68
0.88
0.89
0.65
<.0001
0.55
0.90
0.01
0.66
<.0001
0.22
0.60
0.02
0.73

Hive Weight Month
LinearQuadratic
Linear*Month
Quadratic*Month

4
1
1
4
4

73
62.5
62.1
73
73

29.24
0.11
1.24
1
0.44

<.0001
0.74
0.27
0.42
0.78

Honey

Pollen9577

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Table 5. Reduced Dose Levels: Probability value for the contrast of the control to eachdinotefuran dose at each CCA and for each variable measured.

Response
and
Dose (ng/g)

Probability Value for Contrast of the Control to Each
Dinotefuran Dose at Each CCA
CCA3

CCA4

CCA5

CCA6

CCA7

CCA8

Bees

80
160

0.179
0.368

0.143
0.126

0.304
0.067

0.578
0.034

0.638
0.087

0.558
0.153

Pupae

80
160

0.640
0.661

0.915
0.398

0.775
0.309

0.397
0.030

0.704
0.144

0.626
0.574

Larvae

80
160

0.327
0.457

0.952
0.566

0.822
0.525

0.990
0.899

0.909
0.736

0.388
0.276

Eggs

80
160

0.685
0.446

0.646
0.888

0.330
0.826

0.956
0.854

0.367
0.801

0.841
0.970

Honey

80
160

0.623
0.529

0.743
0.984

0.801
0.981

0.797
0.790

0.956
0.901

0.844
0.435

80
160
Hive Weight
80
160

0.908
0.883
June
0.475
0.615

0.769
0.067
July
0.997
0.448

0.551
0.673
0.555
0.063
0.239
0.774
August September October
0.972
0.844
0.524
0.574
0.626
0.894

0.316
0.203

Pollen10578

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Figure 1. Mean number of adult bee cells in each treatment group measured at every CCA.
A) All Levels of Dose (ng/g)

Response of Adult Bee Counts
25000

Control

10

20

40

CCA4

CCA5
CCA6
CCA Assessment

80

160

Counts

20000
15000
10000
5000
0
CCA3

CCA7

CCA8

CCA7

CCA8

B) 0, 80 , and 160 ng/g Dose Levels

Response of Adult Bee Counts
Control

25000

80

160

Counts

20000

15000
10000

5000
0
CCA3

CCA4

CCA5
CCA6
CCA Assessment

11

579

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Figure 2. Mean number of pupal cells in each treatment group measured at every CCA.
A) All Levels of Dose (ng/g)

Response of Pupal Counts

Control

10

20

40

80

160

25000

Counts

20000
15000
10000
5000
0
CCA3

CCA4

CCA5
CCA6
CCA Assessment

CCA7

CCA8

B) 0, 80 , and 160 ng/g Dose Levels

12

580

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

Response of Pupal Counts
Control

80

160

25000

Counts

20000

15000

10000

5000

0
CCA3

CCA4

CCA5
CCA6
CCA Assessment

CCA7

CCA8

13

581

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Figure 3. Mean number of larval cells in each treatment group measured at every CCA.
A) All Levels of Dose (ng/g)

Response of Larval Counts

Control

10

20

40

80

160

12000

10000

Counts

8000
6000
4000
2000
0
CCA3

CCA4

CCA5
CCA6
CCA Assessment

CCA7

CCA8

CCA7

CCA8

B) 0, 80 , and 160 ng/g Dose Levels

Response of Larval Counts
Control

80

160

10000

Counts

8000
6000
4000
2000
0
CCA3

CCA4

CCA5
CCA6
CCA Assessment

14

582

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Figure 4. Mean number of egg cells in each treatment group measured at every CCA.
A) All Levels of Dose (ng/g)

Response of Egg Counts

Control

10

20

40

80

160

8000
7000
6000
Counts

5000
4000
3000
2000

1000
0
CCA3

CCA4

CCA5
CCA6
CCA Assessment

CCA7

CCA8

CCA7

CCA8

B) 0, 80 , and 160 ng/g Dose Levels

Responseof Egg 80
CountsControl

160

8000
7000

Counts

6000
5000
4000
3000

2000
1000
0
CCA3

CCA4

CCA5
CCA6
CCA Assessment

15

583

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Figure 5. Mean number of honey cells in each treatment group measured at every CCA.
A) All Levels of Dose (ng/g)
Control

Response of Honey Stores
10

20

40

80

160

100000Counts

80000
60000
40000
20000
0
CCA3

CCA4

CCA5

CCA6
CCA7
CCA8
CCA Assessment

CCA9

CCA10

CCA9

CCA10

B) 0, 80 , and 160 ug/g Dose Levels

Response of Honey Stores
Control

10000080160Counts

80000
60000
40000
20000
0
CCA3

CCA4

CCA5

CCA6
CCA7
CCA8
CCA Assessment

16

584

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Figure 6. Mean number of pollen cells in each treatment group measured at every CCA.
A) All Levels of Dose (ng/g)

Response of Pollen Stores

Control

10

20

40

80

160

14000
12000

Counts

10000
8000
6000
4000
2000
0
CCA3

CCA4

CCA5
CCA6
CCA Assessment

CCA7

CCA8

CCA7

CCA8

B) 0, 80 , and 160 ng/g Dose Levels

Response of Pollen Stores
Control

14000

80

160

12000

Counts

10000
8000
6000
4000
2000
0
CCA3

CCA4

CCA5
CCA6
CCA Assessment

17

585

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Figure 7. Weight of hives in each treatment group measured at every month.
A) All Levels of Dose (ng/g)

Hive Weight
20
40

Control

10

July

August

80

160

70
60

Weight (kg)

50
40
30
20
10
0
June

September

October

80

160

Month

March

B) 0, 80 , and 160 ug/g Dose Levels

Hive WeightControl
70
60

Weight (kg)

50
40
30
20

10
0
June

July

AugustMonth

September

October

March
18

586

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

APPENDIX A
Dinotefuran Feed Study
Correlation Statistics for Measurements Taken at each CCA for
Each Variable

19

587

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Table I-1. Pearson Correlation Coefficients for correlation of cell counts or hive weightmeasured between the CCAs or months.
A) Adult Bee Cells
Adult Bee Cells: Pearson Correlation Coefficients for
Number Measured at Each CCA (N = 83)
CCA
CCA3 CCA4
CCA5
CCA6
CCA7
CCA8
CCA3
1.000
0.689
0.555
0.410
0.384
0.125
<.0001 <.0001
0.000
0.000
0.262
CCA4
1.000
0.801
0.691
0.656
0.361
<.0001 <.0001 <.0001
0.001
CCA5
1.000
0.755
0.656
0.426
<.0001 <.0001 <.0001
CCA6
1.000
0.901
0.746
<.0001 <.0001
CCA7
1.000
0.770
<.0001CCA8
1.000B) Pupal Cells

CCA
CCA3
CCA4
CCA5
CCA6
CCA7
CCA8

Pupal Cells: Pearson Correlation Coefficients for
Number Measured at Each CCA (N = 83)
CCA3
CCA4
CCA5
CCA6 CCA7
CCA8
1.000
0.600
0.475
0.446
0.161
0.055
<.0001 <.0001 <.0001
0.147
0.622
1.000
0.734
0.628
0.373
0.104
<.0001 <.0001
0.001
0.352
1.000
0.583
0.299
0.279
<.0001
0.006
0.011
1.000
0.262
0.100
0.017
0.369
1.000
0.462
<.0001
1.000

20

588

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Table 2. Continued.

C) Larval Cells
Larval Cells: Pearson Correlation Coefficients for
Number Measured at Each CCA (N = 83)
CCA
CCA3 CCA4 CCA5 CCA6 CCA7 CCA8
CCA3
1.000
0.429
0.375
0.380
0.257
0.006
<.0001
0.001
0.000
0.019
0.960
CCA4
1.000
0.548
0.397
0.202
0.108
<.0001
0.000
0.067
0.331
CCA5
1.000
0.389
0.254
0.045
0.000
0.021
0.683
CCA6
1.000
0.271
0.012
0.013
0.914
CCA7
1.000
0.121
0.275
CCA8
1.000

D) Egg Cells

CCA
CCA3
CCA4
CCA5
CCA6
CCA7

Egg Cells: Pearson Correlation Coefficients for
Number Measured at Each CCA (N = 83)
CCA3
CCA4
CCA5 CCA6 CCA7
1.000
0.542
0.384
0.320
0.072
<.0001
0.000
0.003
0.518
1.000
0.513
0.339
0.187
<.0001
0.002
0.090
1.000
0.365
0.308
0.001
0.005
1.000
0.306
0.005
1.000

CCA8

CCA8
0.136
0.222
0.252
0.021
0.148
0.182
0.119
0.285
0.063
0.571
1.000
21

589

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Table 2 Continued.

E) Honey Cells

CCA
CCA3
CCA4
CCA5
CCA6
CCA7
CCA8

Honey Cells: Pearson Correlation Coefficients for
Number Measured at Each CCA (N = 83)
CCA3 CCA4
CCA5
CCA6
CCA7
CCA8
1.000
0.829
0.736
0.701
0.597
0.568
<.0001 <.0001 <.0001 <.0001 <.0001
1.000
0.806
0.744
0.709
0.681
<.0001 <.0001 <.0001 <.0001
1.000
0.837
0.757
0.677
<.0001 <.0001 <.0001
1.000
0.857
0.817
<.0001 <.0001
1.000
0.934
<.00011.000F) Pollen Cells

CCA
CCA3
CCA4
CCA5
CCA6
CCA7
CCA8

Pollen Cells: Pearson Correlation Coefficients for
Number Measured at Each CCA (N = 83)
CCA3 CCA4
CCA5
CCA6
CCA7
CCA8
1.000
0.554
0.337
0.436
0.371
0.253
<.00010.002 <.0001
0.001
0.021
1.000
0.718
0.661
0.593
0.366
<.0001 <.0001 <.0001
0.001
1.000
0.631
0.485
0.357
<.0001 <.0001
0.001
1.000
0.685
0.575
<.0001 <.0001
1.000
0.747
<.0001
1.000
22

590

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document
Table 2 Continued.
G) Hive Weight

Hive Weight: Pearson Correlation Coefficients for
Number Measured at Each CCA (N = 84)
Month
June
July August September October March
June
1.000 0.305
0.210
0.109
0.125 0.192
0.005
0.055
0.323
0.257 0.081
July
1.000
0.535
0.460
0.364 0.136<.0001<.00010.001 0.216
August1.000
0.846
0.764 0.483<.0001 <.0001 <.0001
September
1.000
0.931 0.578<.0001 <.0001
October
1.000 0.635<.0001
March
1.000

23

591

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

APPENDIX B
Dinotefuran Feed Study
Mean Statistics for Response Variables
Measured at Each CCA

24

592

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

Table B-1. Adult Bees: Number of replicate hives (N), mean number of cells in each hive with adult bees
(Mean), and standard deviation for the number of cells measured at each CCA (SD) at each treatment levelin the dinotefuran feeding study.
Dinotefuran
Dose
Statistic
0 ng/gN
Mean
SD
10 ng/gN
Mean
SD
20 ng/gNMean
SD
40 ng/gNMean
SD
80 ng/gNMean
SD
160 ng/g
N
Mean
SD

CCA1
24
5128.831293.4
12
5566.83
1227.37115153.18
1657.19125347.83
1000.95
125235.92
1218.93125804.42
1783.31CCA2
2412377.6
1947.211212475.2
2009.031212489.1
2010.081212465.8
2070.981212530.82157.9712
12600.8
2388.2

Number of Adult Bee Cells Measured at Each CCA
CCA3 CCA4 CCA5 CCA6 CCA7 CCA8
24
24
24
23
23
23
19996
23117
20541
17504 17456.5 14312.9
3140.25 4198.16 4790.43 5598.87 6519.65 5458.08
12
12
12
12
12
12
18130.3 20496.6 18260.8
15242 16900.5 12866.3
3178.42 4431.82 4603.56 3767.35 6360.42 4582.8
12
12
12
12
12
12
18810.5 22905.2 18740.7 15792.7
15871
12830
5629.12 8118.46 7129.21 7102.8 7524.05 5384.53
12
12
12
12
12
12
19052.8 23189.3 19821.3 17473.5
17604 14282.4
4912.33 6847.72 5280.9 5880.49 7535.97 4819.93
12
12
12
12
12
12
17948.7 21032.3 19299.5 16928.4 17096.1 13690.9
3225.53 3932.31 4684.47 5413.23 6969.97 4974.02
12
12
12
12
12
12
18735.9 20934.5 17915.9 14068.3 14724.9 12069.8
4479.31 6477.22 5413.95 5428.6 7225.7 5646.75

CCA9
2112487.1
5629.431013561.3
6937.39911509.1
6413.38
1211734.4
7323.721212265.4
7523.05
11
10778.6
6090.96

CCA10
2115931.5
7560.5510
19291.3
9216.138
18768.4
9396.521114457.7
8483.291012834.7
5611.611014539.6
6676.9625

593

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

Table B-2. Pupae: Number of replicate hives (N), mean number of cells in each hive with pupae (Mean), andstandard deviation for the number of cells measured at each CCA (SD) at each treatment level in the
dinotefuran feeding study.
Dinotefuran
Dose
Statistic
0 ng/gN
Mean
SD
10 ng/gN
Mean
SD
20 ng/gNMean
SD
40 ng/gNMean
SD
80 ng/gNMean
SD
160 ng/g
N
Mean
SD

CCA1
24
9675
2158.58129316.67
1856.61118264.732363.361210205.3
1866
129832.672284
129187.672261.07

CCA2
24
19543.5
2494.48
12
19034.73933.39
12
19235.3
2967.221218203.3
5529.76
12
19894.7
3592.46
12
18834
3701

Number of Pupal Cells Measured at Each CCA
CCA3 CCA4 CCA5 CCA6 CCA7 CCA8
24
24
24
23
23
23
18024.2 17393.5 19572.2 10723.8 6498.61 4920.7
4921.93 5030.83 5333.08 3276.88 2910.15 1805.25
12
12
12
12
12
12
17185.7 16368.7
19178
9546 6779.67
4300
4748.77 4783.97 4295.62 3192.23 3066.72 1557.53
12
12
12
12
12
12
16583.7 17787.7 18346.7 10807.3 6951.67
4257
5842.23 5805.75 9527.66 5809.16 2755.38 1974.94
12
12
12
12
12
12
17716 18905.7 20482.3 9302.33 7080.67
4773
6297.52 4698.1 4875.09 5770.43 3040.63 1685.45
12
12
12
12
12
12
17931 18733.7
19909 9660.67 6507.33 4787.33
4079.59 5077.22 5164.23 6106.2 3691.78 2310.92
12
12
12
12
12
12
18017 16182.3 17859.3
7353
4945 4701.33
3829.31 3990.49 4237.2 3396.83 2470.98 2362.14

CCA9
21
15611
5931.611015136
7067.6914027.6
6719.631213186.7
8836.41215107.3
8521.4111
13072
8138.34CCA10
2110721.3
6854.221012968.86384.478
11266
5688.27119647.647308.871011885.2
8692.181010578
8507.4826

594

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

Table B-3. Lavae: Number of replicate hives (N), mean number of cells in each hive with lavae (Mean), andstandard deviation for the number of cells measured at each CCA (SD) at each treatment level in the
dinotefuran feeding study
Dinotefuran
Dose
Statistic
0 ng/gN
Mean
SD
10 ng/gN
Mean
SD
20 ng/gNMean
SD
40 ng/gNMean
SD
80 ng/gNMean
SD
160 ng/g
N
Mean
SD

CCA1
24
4765.83
1253.18124773
1207.62114581.451445.7712
5805
1405.25125733.33
2153.7124672.67
1601.51CCA2
247324.33
1782.37127582.33
2128.73126794
2179.97126980.33
3092.92126822.672226.16126478.67
1880.36

Number of Larval Cells Measured at Each CCA
CCA3 CCA4 CCA5 CCA6 CCA7 CCA8
24
24
24
23
23
23
6471.5 8019.5 9138.5 5204.87 3155.83 1435.83
2640.27 2929.85 3703.3 2760.4 1723.6 873.357
12
12
12
12
12
12
5676 7668.33
9331
5289
3053 1261.33
2524.68 2258.69 2577.52 2804.4 1645.07 794.434
12
12
12
12
12
12
5375
7267
8471
6493 3239.33 1605.33
2500.19 2501.27 4257.02 3390.49 1840.6 606.266
12
12
12
12
12
12
6665 9732.33
9116 6048.67 4070.67
1075
2588.98 1786.66 2354.92 3909.04 2113.36 668.672
12
12
12
12
12
12
5590 9044.33 9517.33 6779.67
3913 1218.33
2783.58 2181.15 3395.41 3716.92 2405.9 899.612
12
12
12
12
12
12
5891 7682.67
8729 5876.67
3268 1132.33
3093.5 2257.35 1935.04 2500.95 2071.8 929.027

CCA921
8968.57
3322.89108101.23554.1698332.443839.41127123.673897.76127252.674116.151179124601.76CCA10
217838.295952.191010130.84039.018
9804
4701.43114941.095395.48105228.85282.8910
7671.26486.1227595

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

Table A-4. Eggs: Number of replicate hives (N), mean number of cells in each hive with eggs (Mean), andstandard deviation for the number of cells measured at each CCA (SD) at each treatment level in the
dinotefuran feeding study
Dinotefuran
Dose
Statistic
0 ng/gN
Mean
SD
10 ng/gN
Mean
SD
20 ng/gNMean
SD
40 ng/gNMean
SD
80 ng/gNMean
SD
160 ng/g
N
Mean
SD

CCA1
24
4722.831346.69124027.67
1181.36114597.09982.332124902
1697.18124013.33
1203.63124515
2027.35

CCA2
24
7009
2448.3912
6292.33
2039.7127338.672235.8127023.332046.77127467.673807.71127396
1710.59

Number of Egg Cells Measured at Each CCA
CCA3 CCA4 CCA5 CCA6 CCA7 CCA8
24
24
24
23
23
23
6736.67 6528.83 6027.17 5429.22 2153.74 785.217
2727.43 2507.68 2680.25 2555.82 958.876 870.608
12
12
12
12
12
12
4701.33 6206.33 6091.67 4629.67 1920.67 630.667
2182.23 2296.47 1774.58 2071.1 1280.5 504.581
12
12
12
12
12
12
6206.33
6579 6392.67 5188.67 2379.33 630.667
3274.54 3433.84 3206.8 2324.85 1664.1 410.535
12
12
12
12
12
12
7095 6865.67 6507.33 5432.33 2121.33
387
1973.58 2296.47 2818.47 2113.52 794.434 452.848
12
12
12
12
12
12
6808.33 6335.33 5303.33
6751 1820.33 917.333
3856.13 3001.46 2719.39 5354.96 1103.68 871.905
12
12
12
12
12
12
6163.33 7023.33 6392.67
6020
2279 1175.33
3890.42 2752.81 2140.55 2516.14 1426.15 842.092

CCA9
21
4824.192237.6410
5366.42732.5895140.892740.48125246
3540.56124945
2995.45114237.45
2960.47CCA10
214938.86
3707.84105504
2193.785547
3139.04113502.55
4262.18102855.23751.02104506.44454.1828

596

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

Table B-5. Honey: Number of replicate hives (N), mean number of cells in each hive with honey (Mean), andstandard deviation for the number of cells measured at each CCA (SD) at each treatment level in the
dinotefuran feeding study
Dinotefuran
Dose
Statistic
0 ng/gN
Mean
SD
10 ng/gN
Mean
SD
20 ng/gNMean
SD
40 ng/gNMean
SD
80 ng/gNMean
SD
160 ng/g
N
Mean
SD

CCA1
24
11538.3
4080.34
1212719.7
3993.521110929.84881.51
12
11051
3590.81128571.33
2124.151212154.73943.63

Number of Cells Containing Honey Measured in Hives at Each CCA
CCA2 CCA3 CCA4 CCA5 CCA6 CCA7 CCA8 CCA9
24
24
24
24
23
23
23
21
32386.2 52961.7
53234 52237.8 57784.5 61628.3 74206.8
38143
14475.1 19613.8 21175.6 18004.7 19435.1 21043.2 20641.8 15393.8
12
12
12
12
12
12
12
10
32995.3 54452.3 59368.7 57820.7 59755.7
68241
78561 44651.2
12054.7 17139.9 18484.7 17495.8
18457 14130.5 16307.6 16195.3
12
12
12
12
12
12
12
9
31863 42985.7 48260.3 47371.7 50238.3
62221 71566.3 37916.4
11043.9 11430.5 13659.5 18287.6 18983.8 17228.3 15359.4 12115.1
12
12
12
12
12
12
12
12
31605
48762 48532.7
52460 57992.7 66334.7 74848.7
39990
5936.15 17575.5 13165.7 12092.2 15014.7 16914.3 14820.7 14498.3
12
12
12
12
12
12
12
12
31605 51485.3 53291.3
53578 58666.3
66994 75995.3
42484
12436.5 16061.2 16622.7 12317.7 19425.6 19161.7
22149 15720.5
12
12
12
12
12
12
12
11
32766 50295.7
60974 59798.7 58537.3 64786.7 70104.3 35463.3
11300.8 15397.9 17658.8 15406.7 16563.6 18071.1 18756.6 10548.5

CCA10
21
54671.420204.71066650
32580.6
855212
26407.5
1154852.4
27499.81056416
30542.6
1058738
24326.829597

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

Table B-6. Pollen: Number of replicate hives (N), mean number of cells in each hive with pollen (Mean), andstandard deviation for the number of cells measured at each CCA (SD) at each treatment level in the
dinotefuran feeding study
Dinotefuran
Dose
Statistic
0 ng/gN
Mean
SD
10 ng/gN
Mean
SD
20 ng/gNMean
SD
40 ng/gNMean
SD
80 ng/gNMean
SD
160 ng/g
N
Mean
SD

CCA1
24
2142.83784.079122594.33
1712.88112517.45997.816123526
1304.7712
2623
981.579122752
1348.36

Number of Cells Containing Pollen Measured in Hives at Each CCA
CCA2 CCA3 CCA4 CCA5 CCA6 CCA7 CCA8 CCA9
24
24
24
24
23
23
23
21
4973.67 7840.33
12083 12885.7 9407.65 8131.65 7253.91 17724.2
3012.15 3641.94 4280.14 5302.59 4412.19 3690.99 3925.51 11117.3
12
12
12
12
12
12
12
10
6765.33 7467.67 11065.3
11954 8472.33
7912
6192 15462.8
3270.33 3898.45 4088.94 4962.05 4152.93 3572.7 2978.22 7421.71
12
12
12
12
12
12
12
9
5618.678084
13072 12670.7 8972.67
7611
6149 12938.2
2821.8 4004.31 5108.67 6830.31 5015.6 5296.38 3853.81 6983.38
12
12
12
12
12
12
12
12
5575.67
8299 10850.3 12455.7 9904.33 8915.33 6865.67 14390.7
1812.07 3407.89 3263.85 4586.28 4351.58 4667.88 4708.96 8735.39
12
12
12
12
12
12
12
12
6134.67 8929.67 12326.7 12197.7 9316.67 7668.33 5991.33 15580.3
4537.87 4681.47 5406.55 6680.26 5144.78 3746.47 4514.99 11788.6
12
12
12
12
12
12
12
11
4687 8743.33 8872.33
8944 7711.33
8385 5518.33 14776.4
2690.85 3421.84 4906.43 5731.4 3844.52 5285.71 4257.86 9885.27

CCA10
21
20541.79024.441022618
9283.49820769
7532.03
11
17309.5
9007.71020622.810407.1
10
19797.2
8598.2630

598

Appendix 8. Data Evaluations for the Colony Feeding Studies that were
Included in this Risk Determination Document

Table B-7. Hive Weight: Number of replicate hives (N), mean number of cells in each hive with pollen(Mean), and standard deviation for the number of cells measured at each CCA (SD) at each treatment levelin the dinotefuran feeding study
DinotefuranDose
0 ng/g

10 ng/g

20 ng/g

40 ng/g

80 ng/g

160 ng/g

StatisticN
Mean (kg)SD (kg)N
Mean (kg)SD (kg)N
Mean (kg)SD (kg)N
Mean (kg)SD (kg)N
Mean (kg)SD (kg)N
Mean (kg)SD (kg)June
2249.54649.2950112
48.2321
10.486711
52.6477
8.19024950.6675
12.874712
53.080211.701412
52.2784
13.5021Weight of Hives Measured at Each Month
July
August September October March
23
24
23
23
23
54.9398 53.1717
53.95815 58.1913
47.2969
7.9595 7.12342
8.656816 10.3452
8.98094
12
12
12
12
10
53.732 53.0841
53.48075 59.9183
48.2816
7.62297 7.48246
7.493645 6.75777
7.70813
12
12
12
12
11
52.2401 49.9195
49.32529 55.4768
44.7787
4.37797 4.9698
8.233907
7.75818.72248
12
11
11
11
10
53.3843 53.3459
54.43734 59.5305
46.7136
5.09707 4.55789
6.000668 8.33407
7.42551
12
12
12
12
12
53.7356 53.3899
54.86597 60.6274
47.39626.19684 6.45758
7.542952
9.111
9.20402
12
12
12
12
11
51.0845 50.954
51.62743 56.2386
43.06228.55456 7.1154
6.600616 7.18878
4.5969831

599

Appendix 9. Foliar and Soil Residue Studies Considered for Use in this Risk Determination Document
The following tables present the foliar and soil residue studies that were considered for use in this Risk Determination Document. For
reviews of the studies that were found to be acceptable, refer to Appendix 10.
Imidacloprid Soil or Foliar Application Residue Studies
Crop(Lab Study ID #)Application Type and Rate

Notes/Acceptability

8 – Fruiting
Vegetables

Tomato(EBNTN012)

1 soil application at 0.376 lb ai/A
and 2 foliar applications at 0.06 lb
ai/A/each.
Total app. rate = 0.5 lbs ai/A.

Acceptable

8 – Fruiting
Vegetables

Tomato(EBNTL056-05)

2 soil applications at 0.13 lb ai/Aeach OR 1 soil application at 0.18 lb
ai/A/each.

Not acceptable for use in this risk determination
document. Study was not conducted at the
maximum annual application rate.

Melon(EBNTL056-02)

Soil application at 0.29-0.36 lb ai/A
in 2011 (year prior to sampling) andin previous years.

Not acceptable for use in this risk determination
document. Bee-collected matrices (tented)
including pollen from pollen traps and hive
deposited nectar. In addition, applications could
have been made a lot closer to bloom according
to label.

Citrus(EBNTL056-7)

5 different soil treatment trials
testing a variety of citrus crops. Soil
applications were made at 1x and 2x
the maximum annual application
rate. Trials with applications made at
the maximum annual application rate
were used in this risk determination
document. Data was not separated bycrop due to poor replication for
statistical analysis.

Crop Group

9 – Cucurbit
Vegetables

10 – Citrus

600

Acceptable
Only data from applications made at the
maximum annual application rate were includedin this risk determination document. Data was
not separated by crop due to poor replication for
statistical analysis.

Appendix 9. Foliar and Soil Residue Studies Considered for Use in this Risk Determination Document
Imidacloprid Soil or Foliar Application Residue Studies
Crop Group

Crop(Lab Study ID #)Application Type and Rate

Notes/Acceptability

10 – Citrus

Orange(EBNTY007)

2 foliar applications at 0.25 lb ai/A
each.
Total app. rate = 0.5 lbs ai/A.

Acceptable for 2/3 sites which were conducted at
the max annual foliar application rate.

11 – Pome Fruits

Apple(EBNTN014)

1 soil at 0.38 lb ai/A + 2 foliar
applications at 0.06 lb ai/A/each.
Total app. rate = 0.5 lbs ai/A.

Acceptable

12 – Stone Fruits

Cherry(EBNTY008)

5 x 0.1 lbs ai/A foliar applications.
Total app. rate = 0.5 lbs ai/A.

Acceptable

12 – Stone Fruits

Stone fruit(EBNTN013)

One soil (0.38 lb ai/A) and two foliar
applications (0.06 lb ai/A/each)
Total app. rate = 0.5 lbs ai/A.
Multiple stone fruit crops tested.
However, data was not be separated
by crop due to poor replication for
statistical analysis.

13 – Berries

Blueberry(EBNTY006)

1 soil application at 0.5 lb ai/A.

Acceptable

13 – Berries

Strawberry(EBNTL056-04)

1 soil application at 0.5 lb ai/A.

Acceptable

601

Acceptable
However, data was not be separated by crop due
to poor replication for statistical analysis.

Appendix 9. Foliar and Soil Residue Studies Considered for Use in this Risk Determination Document
Imidacloprid Soil or Foliar Application Residue Studies
Crop Group

Crop(Lab Study ID #)Application Type and Rate

Notes/Acceptability

20 – Oilseed

Cotton(EBNTY010)

Seed treatment (0.375 mg ai/seed or
0.047 lb ai/A) and 5 x 0.06 lb ai/A
foliar applications.

Acceptable

20 – Oilseed

Cotton(EBNTN011)

1 soil application (0.34 lb ai/A) and
3 foliar applications (0.058 lb
ai/A/each). The total seasonal
application rate was 0.5 lbs ai/A.

Acceptable

20 – Oilseed

Cotton(EBNTL056-01)

1 foliar app at 0.063 lb ai/A madeduring flowering.

Not acceptable for use in this risk determination
document. Study was not conducted at the
maximum annual application rate.

602

Appendix 9. Foliar and Soil Residue Studies Considered for Use in this Risk Determination Document
Clothianidin Foliar and Soil Application Residue Studies
Crop Group

Crop(Lab Study ID #)Application Type and Rate

1 – Root and Tuber Potato
Vegetables(VP-38985)

Trial 1: 1 soil application at 0.2 lb
ai/A.
Trial 2: foliar application at 0.05 lb
ai/A.

9 – Cucurbit
Vegetables

Cucurbit (VP38938)

1 soil application at 0.20 lb ai/A.

9 – Cucurbit
Vegetables

Pumpkin(VP-38263)

1 soil application at 0.2 lb/ai/A.
Second soil application was made at
3/9 sites a month later (this data is
not included because 2nd application
is not permitted by the label).

9 – Cucurbit
Vegetables

Pumpkin(VP-38313)

2 pre-bloom foliar applications at
0.0935 lb ai/A/application (0.18 lb
ai/A/season).

9 – Cucurbit
Vegetables

Pumpkin(VP-38971)

9 – Cucurbit
Vegetables

Cantaloupe(VP-39242)

TRT-2: 1 pre-emergent soil
application at 0.2 lb ai/A.
TRT-3: 1 post-emergent soil
application at 0.2 lb ai/A.
TRT-4: 1 foliar application at 0.1 lb
ai/A.
1 soil application at 0.2 lb ai/A. Beecollected matrices (pollen traps andbee stomachs) in 3 plots, flower
collected matrices in 1 plot.

603

Notes/Acceptability
Acceptable
Trial 2 is excluded from analysis as it was notconducted at the maximum annual application
rate.
Acceptable
Data was broken up by crop (pumpkin,cucumber, squash and melon) for crop-specificanalysis.
Acceptable
Data for 2nd soil application not included as 2nd
application is not permitted by the label.

Acceptable

Acceptable
Only TRT-3 included for analysis in the risk
determination document as this treatment
represented a worst-case application.

Not acceptable for use in this risk determination.

Appendix 9. Foliar and Soil Residue Studies Considered for Use in this Risk Determination Document
Clothianidin Foliar and Soil Application Residue Studies
Crop Group

Crop(Lab Study ID #)Application Type and Rate
1 soil application at 0.6 g ai/tree.
Applications were made at different
times in relation to bloom for eachplot (anywhere from 21 days – 6months prior to sample collection.
TRT-2, TRT-3, and TRT-4 had soil
applications at 0.59 g
ai/tree/application at 6, 3, and 1
month before bloom, respectively.
TRT-5 had soil applications at both
6 and 1 month before bloom.
Clothianidin applied in 2 soil
applications at
0.6 g ai/tree/application. Multiple
neonicotinoids were applied in the
9 months prior to test initiation.

Notes/Acceptability
Not acceptable for use in this risk determination.
Clothianidin is not registered on Citrus in
California.

10 – Citrus Fruit

Citrus(VP-38685)

10 – Citrus Fruit

Citrus(VP-38980)

10 – Citrus Fruit

Citrus(VP-39259)

10 – Pome Fruits

Apple(VP-38552)

1 post-bloom foliar application at
0.1874 lb ai/A.

Acceptable

12 – Stone Fruits

Peach(VP-38563)

2 post-bloom foliar applications at
0.1 lb ai/A/application.

Acceptable604

Not acceptable for use in this risk determination.
Clothianidin is not registered on Citrus in
California.

Not acceptable for use in risk determination.
Clothianidin is not registered on Citrus in
California.

Appendix 9. Foliar and Soil Residue Studies Considered for Use in this Risk Determination Document
Clothianidin Foliar and Soil Application Residue Studies
Crop(Lab Study ID #)Application Type and Rate

13 – Berries

Grape(VP-38992)

3 trials.
TRT-2: post-bloom foliar application
at 0.1 lb ai/A.
TRT-3: Pre-bloom soil application at
0.2 lb ai/A.
TRT-4: pre-bloom foliar application
at 0.1 lb ai/A

14 – Tree Nuts

Almond(VP-38473)

2 post-bloom foliar applications at
0.1 lb. ai/A/application.

Acceptable

15 – Cereal Grains

Corn(VP-39240)

1 Soil Application at 0.2 lb ai/A.

Not acceptable for use in risk determination.
Clothianidin is not registered for soil
applications to corn in California.

15 – Cereal Grains

Corn(VP-39071)

1 Soil Application at 0.2 lb ai/A.

Not acceptable for use in risk determination.
Clothianidin is not registered for soil
applications to corn in California.

15 – Cereal Grains

Corn(VP-39234)

1 soil application at planting (0.16 lb
ai/A).

Not acceptable for use in risk determination.
Clothianidin is not registered for soil
applications to corn in California.

15 – Cereal Grains

Corn(VP-39422)

1 soil application at planting (0.20 lb
ai/A).

Not acceptable for use in risk determination.
Clothianidin is not registered for soil
applications to corn in California.

Crop Group

605

Notes/Acceptability
Acceptable
TRT-2 and TRT-4 are excluded for analysis as
they were not conducted at the maximum annual
application rate.

Appendix 9. Foliar and Soil Residue Studies Considered for Use in this Risk Determination Document
Clothianidin Foliar and Soil Application Residue Studies
Crop Group

Crop(Lab Study ID #)Application Type and Rate

Notes/Acceptability

20 – Oilseed

Cotton(VP-38259)

2 foliar applications at 0.1 lb.
ai/A/application.

Acceptable

1 pre-bloom foliar application at
0.085 lb. ai/A.

Not acceptable for use in risk determination.
Study was not conducted as maximum annual
application rate.

20 – Oilseed

Cotton(EBNIN115)

606

Appendix 9. Foliar and Soil Residue Studies Considered for Use in this Risk Determination Document
Thiamethoxam Foliar and Soil Application Residue Studies
Crop Group

Crop(Lab Study ID #)Application Type and Rate

6 – Legume
Vegetables(Succulent orDried)

Soybean(TK0250070)

Two foliar applications at 0.063 lbs
ai/A for a total annual rate of 0.126 lbs Acceptable
ai/A.

8 – Fruiting
Vegetables

Pepper(TK0236306)

One soil application at a rate of
0.172 lbs ai/A.

Acceptable

8 – Fruiting
Vegetables

Tomato(TK0025811)

One soil application at a rate of 0.078
lbs ai/A or 0.172 lbs ai/A.

Not acceptable for use in this risk determination.
Samples of whole flowers were taken but not
samples of pollen.

Tomato(TK0222531)

Two foliar applications at 0.086 lbs
ai/A for a total annual rate of 0.172 lbs Acceptable
ai/A.

8 – Fruiting
Vegetables

Tomato(TK0242072)

One soil application at a rate of
0.125 lbs ai/A or 0.172 lbs ai/A.

Acceptable. Only data from the maximum rate
allowed by the label were included in statisticalanalysis.

9 – Cucurbit
Vegetables

Cucumber(TK0024668)

One soil application at 0.172 lbs ai/A.

Acceptable

9 – Cucurbit
Vegetables

Cucumber(TK0222532)

Two foliar applications at 0.086 lbs
ai/A for total annual rate of 0.172 lbs
ai/A.

Acceptable

8 – Fruiting
Vegetables

607

Notes/Acceptability

Appendix 9. Foliar and Soil Residue Studies Considered for Use in this Risk Determination Document
Thiamethoxam Foliar and Soil Application Residue Studies
Crop Group

Crop(Lab Study ID #)Application Type and Rate

Notes/Acceptability
Acceptable

9 – Cucurbit
Vegetables

Cucurbits(Pumpkin)(TK0222530)

One soil treatment at a rate of
0.125 lbs ai/A or 0.172 lbs ai/A.

9 – Cucurbit
Vegetables

Pumpkin(TK0242074)

Two foliar applications at a rate of
0.023 lbs ai/A or 0.086 lbs ai/A. Totalannual rates were 0.046 lbs ai/A or
0.172 lbs ai/A.

9 – Cucurbit
Vegetables

Cucurbits(Muskmelon)(TK0222530)

One soil treatment at a rate of
0.0858 lbs ai/A or 0.172 lbs ai/A.

9 – Cucurbit
Vegetables

Cucurbits(Summer Squash)(TK0222530)

One soil treatment at a rate of
0.172 lbs ai/A.

10 – Citrus Fruit

Orange(TK0124743)

One soil application at a rate of 0.086,
0.172, or 0.558 lbs ai/A for the firstyear. For the second year, one soil
application was made at a rate of
0.256 lbs ai/A.

10 – Citrus Fruit

Citrus(TK0124745)

One soil application at a rate of 0.086,
0.129, 0.172, 0.257, or 0.556 lbs ai/A.

608

From a study with multiple cucurbit crops. Only
data from the maximum rate allowed by the label
were included in statistical analysis.
Acceptable
Only data from the maximum rate allowed by thelabel were included in statistical analysis.
Acceptable
From a study with multiple cucurbit crops. Only
data from the maximum rate allowed by the label
were included in statistical analysis.
Acceptable
From a study with multiple cucurbit crops.
Not acceptable for use in this risk determination.
The maximum label rate was only investigatedafter the first yearin navel oranges, which do not
produce pollen. After the first year, only one rate
was used, which was significantly higher than the
maximum application rate allowed.
Not acceptable for use in this risk determination.
Classified as supplemental per study DER due toonly one geographical location used. Not
included in the risk determination as otheracceptable studies were available that also
assessed soil applications to citrus.

Appendix 9. Foliar and Soil Residue Studies Considered for Use in this Risk Determination Document
Thiamethoxam Foliar and Soil Application Residue Studies
Crop Group
10 – Citrus Fruit

Crop(Lab Study ID #)Citrus(TK0177221)

Application Type and Rate

Notes/Acceptability

One soil application at 0.172 lbs ai/A.

Acceptable
Acceptable

10 – Citrus Fruit

Sweet Orange(TK0250069)

One or two foliar applications at
0.086 lbs ai/A. Total annual rates were
0.086 lbs ai/A or 0.172 lbs ai/A.

11 – Pome Fruits

Apple(TK0250071)

One foliar application at 0.086 lbs
ai/A.

12 – Stone Fruits

Stone Fruit(peach, plum, andsweet cherry)(TK0177222)

Two foliar applications at 0.086 lb
ai/A for a total annual rate of 0.172 lbs Acceptable
ai/A.

13 – Berries

Strawberry(TK0177224)

Three foliar applications at 0.063 lbs
ai/A for a total annual rate of 0.189 lbs Acceptable
ai/A.

Only data from the maximum rate allowed by thelabel were included in statistical analysis.
Acceptable with limitations.
The application rate is lower than the maximum
rate permitted by the label, but residue
concentrations still exceed the NOEC by a wide
margin. In addition, lower than acceptable
recoveries in nectar indicate that actual values
may be even higher than reported.

Acceptable
13 – Berries

Strawberry(TK0250068)

One soil application at either 0.129 lbs
ai/A or at 0.188 lbs ai/A.

609

Only data from the maximum rate allowed by thelabel were included in statistical analysis.

Appendix 9. Foliar and Soil Residue Studies Considered for Use in this Risk Determination Document
Thiamethoxam Foliar and Soil Application Residue Studies
Crop Group

Crop(Lab Study ID #)Application Type and Rate

Notes/Acceptability

13 – Berries

Cranberry(TK0236307)

Three foliar applications at a rate of
0.0626 lbs ai/A for a total annual rateof 0.188 lbs ai/A.

Acceptable

13 – Berries

Blueberry(TK0250072)

One or three foliar applications at a
rate of 0.063 lbs ai/A. Total annual
rates were either 0.063 lbs ai/A or
0.188 lbs ai/A.

15 – Cereal Grains

Corn(TK0258214)

Seed treatment (1.25 mg ai/seed) andtwo foliar applications at a rate of
0.043 lbs ai/A or 0.063 lbs ai/A. Totalannual foliar rates were either 0.086
lbs ai/A or 0.126 lbs ai/A.

20 – Oilseed

Cotton(TK0177223)

Two foliar applications at a rate of
0.063 lbs ai/A for two years.

610

Acceptable
Only data from the maximum rate allowed by thelabel were included in statistical analysis.
Acceptable
Foliar applications were made to corn grownfrom treated seeds. Only data from the maximum
rate allowed by the label were included instatistical analysis.
AcceptableAppendix 9. Foliar and Soil Residue Studies Considered for Use in this Risk Determination Document
Dinotefuran Soil or Foliar Application Residue Studies

1 – Root and Tuber
Vegetables

Crop
Application Type and Rate
(Lab Study ID #)PotatoOne soil application at 0.38 lbs ai/A.
(10934.4100)

8 – Fruiting
Vegetables

Tomato(10934.4103)

Two soil applications at 0.206 &0.330 lbs ai/A or two foliar
applications at 0.089 & 0.179 lbs ai/A.

Acceptable

8 – Fruiting
Vegetables

Bell Pepper(S16-01167)

Two soil applications at
0.206 & 0.330 lbs ai/A.

Acceptable

9 – Cucurbit
Vegetables

Butternut Squash,
Yellow
Crookneck
Squash,Cucumber,
Pumpkin(S16-02009)

Two soil applications at
0.206 & 0.330 lbs ai/A.

A four cucurbit study. Not acceptable for use inthis risk determination document. Nectar samples
collected from within the hives.

9 – Cucurbit
Vegetables

Cantaloupe(S16-01165)

Two soil applications at
0.206 & 0.330 lbs ai/A.

9 – Cucurbit
Vegetables

Cucumber(10934.4102)

Two pre-bloom soil or foliar
applications.

9 – Cucurbit
Vegetables

Pumpkin(10934.4104)

Two soil applications at
0.206 & 0.330 lbs ai/A.

Acceptable

9 – Cucurbit
Vegetables

Pumpkin(S16-02008)

Two foliar applications at a rate of
0.089 & 0.179 lb ai/A.

Not acceptable for use in this risk determination
document. Nectar collected by bees and sampledfrom within the hives.

Crop Group

611

Notes/Acceptability
Acceptable

Not acceptable for use in this risk determination
document. Pollen and nectar samples collectedfrom within the hives.
Not acceptable for use in this risk determination
document. Pollen and nectar collected by bees
and samples collected from within the hives.

Appendix 9. Foliar and Soil Residue Studies Considered for Use in this Risk Determination Document
Dinotefuran Soil or Foliar Application Residue Studies
Crop Group

9 – Stone Fruits

Crop
Application Type and Rate
(Lab Study ID #)Two foliar applications at 0.232 &Cherry0.304 lbs ai/A or one trunk injection(10934.4105)
application of 2 g product per inch ofdiameter at breast height.

Notes/Acceptability

Acceptable

13 – Berries

Cranberry(10934.4101)

Two foliar applications at a rate of
0.18 lbs ai/A.

Acceptable

13 – Berries

Blueberry(10934.4107)

Two foliar applications at a rate of
0.18 lbs ai/A.

Acceptable

20 – Oilseed

Cotton(43411B104)

Two foliar applications at a rate 0.129
to 0.136 lbs ai/A.

Acceptable612

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

Data evaluations for the residue studies that were found to be acceptable and included in thisrisk determination document are presented below. These data evaluations are a collection ofDPR Data Evaluation Reports (DERs), Study Summary Tables (for those residue studies that
did not have full DERs available at the time this document was finalized), and citations for
DERs conducted by and available through U.S. EPA. Measured residue concentrations reported
in the following data evaluations are those reported by the study author and thus may not matchthe residue concentrations used to make risk determinations. DPR independently calculated
descriptive statistics for each of the acceptable studies. These descriptive statistics are presented
in Appendix 11.

Imidacloprid Data Evaluations (begin on next page)613

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49665201

CDPR IMI Soil and Foliar Tomato Study

Reference
Gould, T., and Jerkins, E. (2015) Determination of the Residues of Imidacloprid, 5-Hydroxy
Imidacloprid, and Imidacloprid Olefin in Bee Relevant Matrices Collected from Tomatoes Following
Application of Imidacloprid Over Two Successive Years: Final Report. Project Number: EBNTN012.
Unpublished study prepared by Bayer CropScience 466pg. MRID 49665201, CDPR study ID 285680,
Data Volume 51950-0899, Tracking ID#270950

1. STUDY INFORMATION
Chemical:Test Material:
Study Type:

Sponsor:Sponsor Study
ID:
Study
Completion
Date:
Date of
Amendment:
GLP Status:

Imidacloprid
PC Code129099Admire Pro Systemic
Purity:43.50% a.i. w.w.
Protectant (SC)
Non-Guideline field residue study on tomatoes to establish imidacloprid andmetabolite levels in pollen, nectar, and leaves after at plant soil drip/drench
application followed by two foliar spray applications in each of two successive
years and three different soil types (fine, medium, and coarse).
Bayer CropScience
2T.W. Alexander Drive
Experiment Start andJuly 2, 2013 Research Triangle Park, NC
End Date:
April 9, 2015
USA 27709
EBNTN012

9 Field Trials in thecities of:

Study Locations:

June 29, 2015
April 15, 2016

Fresno, Guadalupe,
Kerman, Porterville,
Sanger, San Joaquin, San
Luis Obispo, California

GLP-compliant; protocol reviewed by EPA, PMRA, CDPR.
[CDPR study ID 254887, Data Volume 51950-0757, Tracking ID# 241047.]

2. REVIEWER INFORMATION

Primary Reviewers:
California
Department of
Pesticide Regulation

John Troiano, Ph.D., Research Scientist III, Environmental Monitoring
BranchRichard Bireley, Senior Environmental Scientist (Specialist), Ecotoxicology
Group, Pesticide Registration Branch
Denise Alder, Senior Environmental Scientist (Specialist), Lead Reevaluation
Coordinator, Pesticide Registration Branch
Russell Darling, Environmental Scientist, Reevaluation Coordinator,
Pesticide Registration BranchSecondary Reviewer:

TBD

3. EXECUTIVE SUMMARY
A total of nine field trials were conducted to measure the magnitude of imidacloprid residues intransplanted tomato pollen and in/on transplanted tomato leaves following three applications ofAdmire Pro Systemic Protectant, EPA Reg. No. 264-827 in each of two successive years. Admire Pro
Systemic Protectant is a suspension concentrate formulation containing 550 g/L imidacloprid.
1614

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49665201

CDPR IMI Soil and Foliar Tomato Study

Across all reported trials and years, treated plots received one soil (in-furrow) drip/drench application of
Admire Pro five to seven days after tomato transplantation followed by two equivalent Admire Pro foliar
spray applications per planting season. Individual soil application rates ranged from 0.37 to 0.38 lb
imidacloprid/acre per application (0.42 to 0.43 kg/ha). The interval between the soil and first foliar
applications was 48 to 78 days. Individual foliar application rates ranged from 0.058 to 0.062 lb
imidacloprid/A/application (0.065 to 0.070 kg/ha). All foliar applications were made to flowering tomato
plants, after the first two sampling events were complete. The interval between foliar applications wasfour to five days. The foliar spray volumes ranged from 50 to 101 gal/A, with the exception of thesecond foliar spray in 2013 to trial NT018-13ZA (48 gal/A). Total seasonal application rates were 0.49 to
0.50 lb imidacloprid/A (0.55 to 0.56 kg/ha).
All applications were made using ground-based equipment. The adjuvant Dyne-Amic (0.25 or 0.5 % v/v)
was used in all foliar applications, with the exception of the first foliar application in trial NT017-13ZB
and both foliar applications in trial NT039-13ZA.
Each trial year, one bee tunnel was erected on an untreated plot (UTC), and two bee tunnels wereerected on a treated plot (TRTD), except in trials NT013-13ZA, NT040-13ZA, and NT041-13ZA, when only
one TRTD tunnel was erected. Bumble bee (Bombus impatiens) colonies (1 to 3 per tunnel) were placedin each tunnel for the collection of pollen. One sample was collected per bee tunnel, yielding two TRTD
samples and one UTC sample at each sampling interval, except in trials NT013-13ZA, NT040-13ZA, andNT041-13ZA, when two replicate samples were collected from the single erected TRTD tunnel.
Additionally, in trial NT042-13ZA, the first pollen sampling of 2015 was made by hand-collecting pollen
directly from the flowers in the field due to a bee shortage.
Tomato leaf and pollen samples were collected at four sampling intervals each year: two samples werecollected after the soil application, approximately 14 days apart (31 to 68 and 45 to 77 days after the soil
application, respectively), and two samples were collected after the last foliar application,
approximately 14 days apart (2 to 8 and 16 to 22 days after the last foliar application, respectively). At
each interval, fresh bumble bee colonies were placed in each bee tunnel, and the bumble bees wereallowed to forage from the tomato flowers for several days. Then, bumble bees carrying pollen werecollected from the tunnels and the pollen was removed from them. To ensure a large enough pollen
sample for analysis was collected, some trials collected bees over multiple days (up to seven) per
sampling event. Multi-day pollen samples from the same sampling interval and bee tent werecomposited together into one sample vial.
During the described sampling intervals, composite samples of tomato leaves were collected fromwithin the tunnels of the treated plots. Composite samples of tomato pollen and leaves were collectedfrom the control plot tunnel of each trial during the same sampling intervals and using the methods as
samples collected from the treated plots.
The residues of Admire Pro Systemic Protectant (imidacloprid, 5-hydroxy imidacloprid, and imidacloprid
olefin) were quantitated by high performance liquid chromatography/triple stage quadrupole mass
spectrometry (LC/MS/MS) and LC/high resolution mass spectrometry (LC/HRMS) using stable
isotopically labeled internal standards. The individual analyte residues were summed to give a total
imidacloprid residue.

2615

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49665201

4. STUDY VALIDITY

Guideline Followed:
Guideline Deviations:
Other Deviations:Classification:
Rationale:
Reparability:

CDPR IMI Soil and Foliar Tomato Study

Non-guideline study (protocol was reviewed by U.S. EPA/PMRA/CDPR)
N/A
N/A
Acceptable For Quantitative Use
The data from the study will provide a basis for developing aquantitative assessment of exposure levels to bees that can be used in arisk assessment scenario.
N/A

5. MATERIALS AND METHODS
Test Material Characterization
Test item:
Description:
Lot No./Batch No.
Material Source:
Material Receipt
Date:
Expiration Date:

Storage of Test
Material:

Admire Pro Systemic Protectant(Imidacloprid) 550 g a.i./L SC
Suspension concentrate (SC)
Batch No. NK41CX0578
Bayer CropScience
Not Reported
12/03/2014
Ambient (35-86ºF)
except trials NT010-13ZA andNT016-13ZA when thetemperature briefly reached
95ºF; trial NT017-13ZB when
the temperature briefly
reached 90ºF; and, trial NT01813ZA when the temperaturereached as low as 32ºF and as
high as 90ºF.

CAS #:
Purity:Density:
Cert. #
Analysis
Date:
Solubility:

138261-41-3

SampleStorage:-27ºC to -7ºC
-16ºF to 19ºF

43.50% w/w
1.41 – 1.54 g/mL
213CJ2446
12/03/2012
0.51 to 0.61 g/L

5A. STUDY DESIGNThis study requirement was part of the imidacloprid special review at the California Department of
Pesticide Regulation (CDPR). The study design and protocol were approved by CDPR prior to study
initiation. This study was conducted using GLP standards and following an approved protocol. The study
initiation date was June 21, 2013. The experimental start date was July 02, 2013 (first application), andthe experimental end date was December 5, 2016 (last analysis).
Nine trials in California were conducted for this study, representing all three soil texture categories(fine, medium, and coarse). Each trial includes one treated plot to be planted and treated for two
consecutive years and one untreated plot.
Indeterminate (continuously flowering and fruiting) tomato varieties representing those commonlygrown in the area of the trials and agronomic practices typical for commercial production of tomato
were used at all trial locations. Once bloom began, the tomato plots were sampled at four intervals. The
plots were sampled twice at early bloom approximately 14 days apart, prior to any foliar sprays, to3616

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49665201

CDPR IMI Soil and Foliar Tomato Study

assess the residues in bee-relevant matrices resulting from the at-plant soil application. The flowering
tomatoes were then given two foliar applications of imidacloprid. The tomatoes were sampled twice
more after the last foliar application, approximately 14 days apart, to assess the residues resulting fromthe soil application at planting plus two additional at-bloom foliar applications.
Homogenization and analysis of the samples from this study were performed by Bayer CropScience in
Research Triangle Park (RTP), NC. Final report preparation was performed by Critical Path Services, LLC,
located in RTP, NC.
All raw data associated with this study are retained along with the protocol, protocol amendments, andfinal report under Notebook Number EBNTN012 at Bayer CropScience, 2 T. W. Alexander Drive, RTP, NC
27709.
5B. APPLICATION TIMING AND RATESThe full study report provides (1) Chronological listing of significant study dates (Appendix 1); (2) Field
report summaries for each trial detailing the actual amount of test substance applied, plot sizes, dates oftreatment, dates of sample collection, maintenance chemicals, climatic data, and irrigation data
(Appendix 2); and (3) Quality assurance statements for each trial (Appendix 3). Information on
application timing is provided in Table 1. Soil and meteorological characteristics of the study sites areprovided in Table 2 and Table 3. Table 4 provides the sampling dates and tomato developmental stages.
BBCH or Biologische Bundesanstalt, Bundessortenamt und CHemische Industrie, identifies the specificphenological development stages of tomato. Plot TRTD received one soil (in-furrow) drip/drench
application of Admire Pro five to seven days after tomato transplantation followed by two equivalent
Admire Pro foliar applications per planting season. Individual soil application rates ranged from 0.37 to
0.38 lb imidacloprid/A per application (0.42 to 0.43 kg/ha). The interval between the soil and first foliar
applications was 48 to 78 days. Individual foliar application rates ranged from 0.058 to 0.062 lb
imidacloprid/A/application (0.065 to 0.070 kg/ha). All foliar applications were made to flowering tomato
plants, after the first two sampling events were complete. The interval between foliar applications wasfour to five days. The foliar spray volumes ranged from 50 to 101 gal/A, with the exception of thesecond foliar spray in 2013 to trial NT018-13ZA (48 gal/A). Total seasonal application rates were 0.49 to
0.50 lb imidacloprid/A (0.55 to 0.56 kg/ha).
Temperature and precipitation data were recorded for each trial and are summarized in Appendix 2 ofthe study report EBNTN012. Temperatures recorded during the field phase of the study were similar to
average historical records except in trial NT013-13ZA, which had a slightly warmer spring 2014.
Recorded rainfall was slightly lower than historical records in trials NT010-13ZA, NT016-13ZA, andNT018-13ZA. However, there were no significantly unusual weather conditions that would affect theconclusions of the study.
CDPR requested that the trial sites be distributed as three coarse, three medium, and three finetextured soils [per USDA’s Soil Survey Geographic database (SSURGO) mapping units]. There are nine
trial sites in this tomato study design: three in fine texture soils, one in medium, and five in coarse; threesandy loams (as determined by SSURGO and shown in the general texture description of wereconsidered coarse textured. Two years of data for each site are presented in this report.

4617

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49665201

CDPR IMI Soil and Foliar Tomato Study

NT01313ZA

Fresno, CA
Region 10

Admire Pro
Systemic
Protectant

TRTD

2013

2014

13

218
(2037)

0.376(0.422)

NA

Foliar Spray

71

57
(530)

0.061
(0.068)57

DyneAmic
0.5% v/v

Foliar Spray

71

57
(535)

0.061
(0.069)4

DyneAmic
0.5% v/v

Soil
Drench/Drip

17

218(2037)

0.376(0.422)

279

Foliar Spray

65

57
(533)

0.061
(0.069)75

DyneAmic
0.5% v/v

Foliar Spray

65

57
(532)

0.061
(0.069)5

DyneAmic
0.5% v/v

Soil
Drench/Drip

19

24
(220)

0.377
(0.422)

NA

Foliar Spray

68

60
(558)

0.060
(0.068)78

DyneAmic
0.25%
v/v

Foliar Spray

69

62
(576)

0.062
(0.070)

5

DyneAmic
0.25%
v/v

Soil
Drench/Drip

19

26
(247)

0.374
(0.419)262

Foliar Spray

71

61

0.061

48

5
618

Total Rateb

0.50
(0.56)

0.50(0.56)

0.50(0.56)

0.50(0.56)

Adjuvant

Soil
Drench/Drip

Retreatment Interval (Days)

Rate, lb a.i./A (kg a.i./ha)

a

2014

Actual Spray Volume, GPA (L/ha)

2013

Timing/Growth Stage (BBCH)

TRTD

MethodFormulation
Admire Pro
Systemic
Protectant

Year

Porterville,
CA Region
10

Plot Name

NT01013ZA

Location (City, State, NAFTA Region)

Trial Identification

Table 1. Summary of soil and foliar application rates and timing*.

NA

NA

NA

NA
Dyne-

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

NT01613ZA

Porterville,
CA
Region 10

Admire Pro
Systemic
Protectant

TRTD

2013

2014

NT01713ZB

Guadalupe,
CA Region
10

Admire Pro
Systemic
Protectant

TRTD

2014

Adjuvant(0.069)Total Rate

Rate, lb a.i./A (kg a.i./ha)(568)

Retreatment Interval (Days)

Actual Spray Volume, GPA (L/ha)

Timing/Growth Stage (BBCH)

MethodYear

Plot Namea

CDPR IMI Soil and Foliar Tomato Study

Formulation

Location (City, State, NAFTA Region)

Trial Identification

MRID 49665201

Amic
0.25%
v/v

Foliar Spray

71

61
(568)

0.062
(0.069)4

Soil
Drench/Drip

12218(2037)

0.376(0.422)

NA

Foliar Spray

71

57
(530)

0.061
(0.068)61

DyneAmic
0.5% v/v

Foliar Spray

68

57
(353)

0.061
(0.069)4

DyneAmic
0.5% v/v

Soil
Drench/Drip

13

218(2037)

0.376(0.422)

280

Foliar Spray

65

57
(532)

0.061
(0.069)75

DyneAmic
0.5% v/v

Foliar Spray

65

57
(532)

0.061
(0.069)5

DyneAmic
0.5% v/v

Soil
Drench/Drip

19

8530
(79781)0.377(0.422)

NA

Foliar Spray

75

56
(520)

0.061
(0.069)61

NA

Foliar Spray

75

55
(516)

0.061
(0.068)

5

DyneAmic
0.25%

6
619

DyneAmic
0.25%
v/v
0.50
(0.56)

0.50(0.56)

0.50(0.56)NA

NA

NA

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

Adjuvant

Total Rate

Retreatment Interval (Days)

Rate, lb a.i./A (kg a.i./ha)Actual Spray Volume, GPA (L/ha)

Timing/Growth Stage (BBCH)

MethodYear

Plot Namea

CDPR IMI Soil and Foliar Tomato Study

Formulation

Location (City, State, NAFTA Region)

Trial Identification

MRID 49665201

v/v
2015

NT01813ZA

Sanger, CA
Region 10

Admire Pro
Systemic
Protectant

TRTD

2013

2014

NT03913ZA

San LuisObispo, CA
Region 10

Admire Pro
Systemic
Protectant

TRTD

2014

Soil
Drench/Drip

16

10353
(96829)0.377(0.422)296

Foliar Spray

79

53
(495)

0.058
(0.065)75

DyneAmic
0.25%
v/v

Foliar Spray

81

54
(507)

0.060
(0.067)

4

DyneAmic
0.25%
v/v

Soil
Drench/Drip

19

6787
(63477)0.380(0.426)NA

Foliar Spray

83

51
(477)

0.062
(0.069)72

DyneAmic
0.5% v/v

Foliar Spray

NR

48
(451)

0.058
(0.066)

5

DyneAmic
0.5% v/v

Soil
Drench/Drip

15

6787
(63477)0.380(0.426)210

Foliar Spray

71

50
(467)

0.060
(0.068)72

DyneAmic
0.5% v/v

Foliar Spray

71

50
(466)

0.060
(0.068)

5

DyneAmic
0.5% v/v

Soil
Drench/Drip

NR

7
(63)

0.378
(0.423)

NA

Foliar Spray

72

51

0.062

64

7
620

c

0.49
(0.55)

0.50(0.56)

0.50(0.56)

0.50(0.56)

NA

NA

NA

NA
NA

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

2015

NT04013ZASan
Joaquin,
CA Region
10

Admire Pro
Systemic
Protectant

TRTD

2014

2015

Adjuvant(0.069)Total Rate

Rate, lb a.i./A (kg a.i./ha)(473)

Retreatment Interval (Days)

Actual Spray Volume, GPA (L/ha)

Timing/Growth Stage (BBCH)

MethodYear

Plot Namea

CDPR IMI Soil and Foliar Tomato Study

Formulation

Location (City, State, NAFTA Region)

Trial Identification

MRID 49665201

Foliar Spray

73

51
(474)

0.062
(0.069)5

Soil
Drench/Drip

NR

7
(63)

0.378
(0.423)256

Foliar Spray

75

50
(468)

0.061
(0.068)

77

DyneAmic
0.5% v/v

Foliar Spray

75

50
(469)

0.061
(0.068)

5

DyneAmic
0.5% v/v

Soil
Drench/Drip

19

26
(247)

0.374
(0.419)NA

Foliar Spray

69

100
(933)

0.061
(0.068)52

DyneAmic
0.25%
v/v

Foliar Spray

71

100
(935)

0.061
(0.069)5

DyneAmic
0.25%
v/v

Soil
Drench/Drip

16

26
(247)

0.374
(0.419)195

Foliar Spray

75

100
(933)

0.061
(0.069)60

DyneAmic
0.25%
v/v

Foliar Spray

76

99
(930)

0.061
(0.068)

5

DyneAmic
0.25%

8
621

NA
0.50
(0.56)

0.50(0.56)

0.50(0.56)NA

NA

NA

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

Adjuvant

Total Rate

Retreatment Interval (Days)

Rate, lb a.i./A (kg a.i./ha)Actual Spray Volume, GPA (L/ha)

Timing/Growth Stage (BBCH)

MethodYear

Plot Namea

CDPR IMI Soil and Foliar Tomato Study

Formulation

Location (City, State, NAFTA Region)

Trial Identification

MRID 49665201

v/v
NT04113ZA

Kerman,CA Region
10

Admire Pro
Systemic
Protectant

TRTD

2014

2015

NT04213ZA

Sanger, CA
Region 10

Admire Pro
Systemic
Protectant

TRTD

2014

Soil
Drench/Drip

19

26
(247)

0.374
(0.419)NA

Foliar Spray

65

101
(942)

0.062
(0.069)50

DyneAmic
0.25%
v/v

Foliar Spray

81

100
(931)

0.061
(0.068)

5

DyneAmic
0.25%
v/v

Soil
Drench/Drip

19

26
(247)

0.374
(0.419)245

Foliar Spray

69

100
(936)

0.061
(0.069)51

DyneAmic
0.25%
v/v

Foliar Spray

71

100
(937)

0.061
(0.069)5

DyneAmic
0.25%
v/v

Soil
Drench/Drip

11

9044
(84585)0.380(0.426)NA

Foliar Spray

72

65
(612)

0.061
(0.068)52

DyneAmic
0.25%
v/v

Foliar Spray

72

55
(514)

0.062
(0.069)5

DyneAmic
0.25%

9
622

0.50
(0.56)

0.50(0.56)

0.50(0.56)NA

NA

NA

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

Adjuvant

Total Rate

Retreatment Interval (Days)

Rate, lb a.i./A (kg a.i./ha)Actual Spray Volume, GPA (L/ha)

Timing/Growth Stage (BBCH)

MethodYear

Plot Namea

CDPR IMI Soil and Foliar Tomato Study

Formulation

Trial Identification

Location (City, State, NAFTA Region)

MRID 49665201

v/v
2015

Soil
Drench/Drip

14

9973
(93277)0.377(0.422)

217

Foliar Spray

85

67
(929)

0.061
(0.068)655

DyneAmic
0.25%
v/v

Foliar Spray

87

68
(632)

0.062
(0.069)5

DyneAmic
0.25%
v/v

a

In trials NT010-13ZA, NT013-13ZA, NT039-13ZA, NT040-13ZA, NT041-13ZA and NT042-13ZA,
additional irrigation (0.2 to 0.75 inches) occurred as part of the drench/drip applications that is not
captured in the listed spray volumes. See Appendix 2 of the study report for details.
b
NA= Not applicable.
c
NR= Not Reported; the BBCH at this application was not reported by the PFI.
*Table 4 of the study report.

10
623

0.50
(0.56)

NA

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49665201

CDPR IMI Soil and Foliar Tomato Study

5C. STUDY SITE LOCATION AND CHARACTERISTICS
Table 2. Soil and meteorological characteristics of the study sites*.

Trial
IDa

Trial Location (City,Country/State, Year, GPS
Coordinatesb)

Soil Characteristicsc
Meteorological Datad
CEC
(meq/
Total
%
%
%
100 g
Rainfall Temp. Range
OM (%) pH soil) Sand Silt Clay Type (in)(°F)
Variety

NT010- Porterville, CA, 2013-2014
13ZA(36.005, -119.0721)

0.97

7.1

9.6

84

13

NT01313ZA

Fresno, CA, 2013-2014(36.7362, -119.87476)

0.93

7.2 12.96922NT016- Porterville, CA, 2013-2014
13ZA(36.0348, -118.9964)

2.2

7.7 31.640

27NT017Guadalupe, CA, 2014
13ZB (N34.96917, W120.60196)

0.81

8.0 14.1

84

9NT018- Sanger, CA, 2013-2014
13ZA (36.739659, -119.576766)

0.25

8.2

6.5

76

18

2.7

7.6 27.77

50

19

Loamy
Sand 4.56
Sandy
9 Loam 3.62
Clay
33 Loam 4.62
Loamy
7
Sand 5.87
Loamy
6
Sand 4.34
Sandy
Clay
31 Loam 12.15

1.8

7.9 46.8

15

20

65

Clay

0.38

6.1

89

8

3

Sand

NT039- San Luis Obispo, CA, 2014
13ZA (35.306478, -120.677548)
NT040- San Joaquin, CA, 2014
13ZA
(36.59885, -120.20671)
NT041Kerman, CA, 2014
13ZA
(36.79380, -120.05320)

3.9

3GardenDelight

29

97

30
29

102 Big Beef
Garden97 Delight

43

81

28

Sungold
Cherry100 Tomato

45

81

Naomi

5.58

37

97

Naomi

6.46

53

98

Naomi
CherryTomato(GoldenGem)

Loamy
0.25 6.6 5.1
75 20 5
Sand 6.27
35
97aSite conditions listed are for the TRTD plot. For UTC plot conditions, see Appendix 2 of the study
report.
b
GPS coordinates are in the form (latitude, longitude).
c
These soil characteristics are based on analyses of soil samples collected from within approximately
500 ft of the treated plot. Abbreviations used: %OM = percent organic matter; CEC = cation exchange
capacity.
dData is for the interval of the month of first application through the month of last sampling.
Meteorological data were obtained from nearby government weather stations.
* Combined table from Table 3A and Table 8 of the study report.
NT042Sanger, CA, 2014
13ZA (36.70034, -119.461982)

11624

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49665201

CDPR IMI Soil and Foliar Tomato Study

Table 3. SSURGO soil characteristics of the study sites*.
General Texture
CDPR
Description
Trial
Trial
Particle
Component Texture Drainage Hydrologic
ID
Location(SSURGO)
Category
Class
Group
Runoff Size%a
Tujunga loamy
Somewhat
NT010- Porterville,
sand,excessively
b
13ZA
CA
85
Coarse drainedA
Negligible
NA
0-2% slopes
NT013-13ZA Fresno, CA

Ramona loam

NT016 Porterville,
-13ZA
CA

Centerville clay,
2-9% slopes
Mocho sandy
NT017 Guadalupe,
loam,
-13ZB
CA
0-3% slopes

80

MediumWelldrained

C

Low

Fine
Loamy

80

Fine

Well
drained

D

NA

Fine

85

CoarseWelldrainedA

Fine
Negligible Loamy

A

Very Low

Somewhat
NT018
Delhi loamy sand,excessively
85
Coarse drained-13ZA Sanger, CA
0-3% slopes
NT039 San Luis
Salinas silty clay
Well
85
Fine
-13ZA Obispo, CA loam, 0-2% slope
Drained
NT040
San
Merced clay,
Very Poorly
85
Fine-13ZA Joaquin, CA
slightly saline
Drained
NT041
Hanford coarse
Well85
Coarse drained-13ZA Kerman, CA
sandy loam
Hanford fine sandy
NT042
Well
loam, gravelly-13ZA Sanger, CA
85
CoarseDrainedsubstratumaMajor component of the soil as a percentage of total soil.
b
NA = Not applicable.

C

NA
Fine
Medium loamy

CMediumA

Fine
CoarseVery Low loamy

A

Very Low

Coarseloamy

5D. SAMPLE COLLECTION, HANDLING, PROCESSING
Tomato Plant Matrices.
Composite samples (one per each bee tunnel) of tomato leaves and pollen were collected from plots
UTC and TRTD in years one and two of the study, except in trials NT017-13ZB and NT039-13ZA throughNT042-13ZA, which were started/restarted in 2014 and have only completed one year of the study. All
tomato leaf and pollen samples were collected from within the erected bee tunnels on both plots atfour sampling intervals. The first and second samples were collected at early bloom approximately
14 days apart, prior to any foliar sprays (31 to 68 and 45 to 77 days after the soil application,
respectively). The third and fourth samples were collected after the last foliar application, approximately
14 days apart (2 to 8 and 16 to 22 days after the last foliar application, respectively). UTC samples werecollected at the same sampling intervals and via the same methods as the TRTD samples.
All samples were protected from sunlight and placed in field coolers containing ice substitute. Upon
arrival at the site facility, all composite leaf samples were placed directly into frozen storage. All

12625

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49665201

CDPR IMI Soil and Foliar Tomato Study

composite pollen samples were placed directly into frozen storage after their removal from the bumble
bees (used as a sampling device).
Composite samples were placed into labeled (study number and sample number) containers forshipment. All samples were frozen within four hours of their collection. Samples were shipped andremained frozen until receipt at Bayer CropScience (RTP, NC).
During the described sampling intervals, duplicate composite samples (one per bee tunnel) of tomato
leaves were collected from the treated plots (one day of sampling only).
All composite leaf samples were collected by hand from different areas of the tomato plants (top andmiddle, left and right). Each composite sample of leaves contained a target of 150 g collected from any
number of healthy-appearing plants located inside the bee tunnel.
Tomato Pollen Samples and Bee Handling.
Details on the pollinator portions of the study are located in Appendix 5. One bee tunnel was erected on
untreated plot UTC, and two bee tunnels were erected on treated plot TRTD, except in trials NT01313ZA, NT040-13ZA, and NT041-13ZA, when only one TRTD tunnel was erected. Bumble bee (Bombus
impatiens) colonies (1 to 3 per tunnel) were placed in each tunnel for the collection of pollen. One
sample was collected per bee tunnel, yielding two TRTD samples and one UTC sample at each samplinginterval, except in trials NT013-13ZA, NT040-13ZA, and NT041-13ZA, when two replicate samples werecollected from the single erected TRTD tunnel.
At each testing location, the control and treated plots were divided into sampling subplots for theconstruction of bee-tight, ventilated mesh-covered tents (tunnels). When the tomato bloom was
sufficient to support pollen sampling after the first (soil) application, the tunnels were erected. The
tunnels were 100 to 210 feet long and 20 to 40 feet wide, and each tunnel enclosed four to eight rowsof tomato plants. In some trials, the tunnel was removed after sampling interval two to allow for thefoliar applications and re-erected prior to sampling interval three.
Normally developed, apparently healthy bumble bees (Bombus impatiens) were used for pollen
collection. One to three bumble bee colonies were placed in each bee tunnel. Bumble bee colonies were
contained in Class A research hives, Class C garden hives, or boxed colonies. In all trials, the bumble beecolonies were allowed access to sugar water feeders or nectar/syrup bags.
Fresh bumble bee colonies were placed in each bee tunnel prior to each sampling event; the bumble
bees were allowed to forage from the tomato flowers for several days. Bumble bees were observed forvisible pollen collected in the pollen basket on their legs. Bees with pollen were collected using nets or
vacuums and placed in containers with dry ice at the field. The bees were transported to the site facility
for pollen removal. All available pollen was collected from the bees using an appropriate tool such as
tweezers or forceps, and the pollen samples were placed in 40-mL amber glass containers.
To ensure a large enough pollen sample for analysis, some trials collected bees over multiple days (up toseven) per sampling event (see Appendix 2). Multi-day pollen samples from the same sampling intervaland bee tent were composited together into one sample vial. Bumble bee colonies were removed fromthe tents after the end of the sampling event.
Bumble bee colonies were removed from the tents after the end of the sampling event
13626

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49665201

CDPR IMI Soil and Foliar Tomato Study

Table 4. Sampling dates and tomato developmental stages for plant residue analysis*

†

BBCH

Year 1
‡
Dates

†

BBCH

Pollen and Leaf SamplingYear 2
Year 1
†
‡
‡
Dates
Dates
BBCH

Fresno = NT013-13ZA
2

XX

XX
XX
XX

62-68 DASA,(-16 to -10
DA1FA)
75-77 DASA,(-3 to -1
DA1FA)
2-8 DA2FA
17-20 DA2FA

31-33 DASA,(-17 to -15 DA1FA)

XX

45-47 DASA,(-3 to -1 DA1FA)XX
XX

4 DA2FA
19-21 DA2FA

1

71

83
85
85

Guadalupe = NT017-13ZB
46 DASA,(-15 DA1FA)

58 DASA(-17 DA1FA)

XX

72

59 DASA,(-2 DA1FA)

72 DASA(-3 DA1FA)

XX

74
79

8 DA2FA
19-20 DA2FA

4-5 DA2FA
18 DA2FAXX
XX

Kerman = NT041-13ZA

XX
XX
XX

XX
XX
XX

44-45 DASA,(-13 to -12
DA1FA)
54-56 DASA,(-3 to -1
DA1FA)
1
4 DA2FA
20-21 DA2FA

34-36 DASA(-15 to -17 DA1FA)
48-50 DASA(-3 to -1 DA1FA)
1
6 DA2FA
1
21 DA2FA

XX

XX
XX
XX
XX

XX

61 DASA,(-14 DA1FA)

XX

72-73 DASA,(-3 to -2 DA1FA)

XX

XX
XX

4-6 DA2FA
17-19 DA2FA

XX
XX

XX

XX
XX

52-54 DASA,(-20 to -18
DA1FA)

71

69 DASA,(-3 DA1FA)

72
86

7 DA2FA
20 DA2FA

36-38 DASA,(-16 to -14
DA1FA)
49-51 DASA,(-3 to -1
DA1FA)
5-6 DA2FA
16-18 DA2FA

58 DASA(-7 DA1FA)
62-64 DASA(-3 to -1 DA1FA)
5-7 DA2FA
1-18 DA2FA

35-37 DASA,-17 to -15
DA1FA
50 DASA,
1-2 DA1FA
4-6 DA2FA
19-22 DA2FA

43-44 DASA(-16 to -17
DA1FA)
57-58 DASA(-3 to -2 DA1FA)
4-6 DA2FA
17-19 DA2FA

San Luis Obispo = NT039-13ZA

Porterville 2 = NT016-13ZA
48-50 DASA,(-13 to -11
DA1FA)
59 DASA,1(-2 DA1FA )70-71 DASA,(-2 to -1
DA1FA)
6 DA2FA
20 DA2FA61

San Joaquin = NT040-13ZA

Porterville 1 = NT010-13ZAXX

55 DASA,(-17 DA1FA)

Sanger 2 = NT042-13ZA

69

XX

BBCH

Year 2‡
Dates

Sanger 1 = NT018-13ZA

XX

33-37 DASA,(-17 to -13
DA1FA)
49 DASA,1(-1 DA1FA )4-7 DA2FA
19-21 DA2FA†

59-61 DASA,(-16 to -14
DA1FA)
72-73 DASA,(-3 to -2 DA1FA)
14
627

XX

50 DASA,1(-14 DA1FA )61-63 DASA,(-3 to -1
DA1FA)
5-6 DA2FA
17-18 DA2FA

61-63 DASA(-14 to -16
DA1FA)
74-76 DASA(-3 to -1 DA1FA)
4-6 DA2FA
14-16 DA2FA

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49665201

†

BBCH

CDPR IMI Soil and Foliar Tomato Study

Year 1
‡
Dates

†

BBCH

Pollen and Leaf SamplingYear 2
Year 1
†
‡
‡
Dates
Dates
BBCH

†

BBCH

Year 2
‡
Dates

1

XX
5 DA2FAXX
4-6 DA2FA
XX
20 DA2FAXX
17-19 DA2FA†
BBCH = Biologische Bundesanstalt, Bundessortenamt und CHemische Industrie growth stage scale for tomato.
‡
DASA = Days after the soil (in-furrow) application; DA1FA = days after the first foliar application;
DA2FA = days after the second foliar (and last) application. A negative number designates days prior to theindicated application. Ranges indicate that samples were collected over several days and composited together tocreate a large enough volume for analysis.
1
Leaf sampling ONLY. No pollen data, insufficient sample weight.
2
BBCH XX indicates no growth stage reported in the field data summary.
*Combination of Appendix 1 and Appendix 5 of the study report.

Sample Storage.
Composite samples were placed into labeled (study number and sample number) containers forshipment. All samples were frozen within four hours of their collection. Samples were shipped andremained frozen until receipt at Bayer CropScience (RTP, NC).
Storage stability studies indicate that the imidacloprid residues would have been stable during frozen
storage for at least 1,080 days (36 months) in tomato leaves prior to analysis. Transit stability samples
showed that imidacloprid residues were stable in pollen for the duration of the study. The maximum
storage period of frozen samples in this study for Admire Pro Systemic Protectant was
561 days for tomato leaves and 560 days for tomato pollen.
Stability studies have indicated that imidacloprid residues are stable (<30% decomposition) for 24months (728 to 769 days) of freezer storage in the following representative crops: an oilseed(tomatoseed), a non-oily grain (wheat), a leafy vegetable (lettuce), a root crop (potato), a tree fruit(apple), and a fruiting vegetable (tomato).4-10 An additional stability study has indicated thatimidacloprid residues are stable (<30% decomposition) for 36 months of freezer storage in wheat(grain), orange (fruit), tomato (fruit), bean (seed), and rape (seed)16. Demonstrated freezer stability inall of the above crops is representative of the freezer stability of imidacloprid, 5-hydroxy imidacloprid,and imidacloprid olefin residues to be expected for tomato leaves from this study.
Based on the available storage stability data,4-11 the imidacloprid residues in tomato leaves would be
representative of the residues to be expected after the use of Admire Pro Systemic Protectant on
tomato.

15628Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49665201

CDPR IMI Soil and Foliar Tomato Study

5E. ANALYTICAL METHODS
The analytical methods1-2 used in this study measured the residues of imidacloprid, 5-hydroxyimidacloprid, and imidacloprid olefin in tomato leaves and pollen. These data are reported inAppendix 4 of the full study report titled, “Analytical Report for EBNTN012 Determination of the
Residues of Imidacloprid, 5-Hydroxy Imidacloprid, and Imidacloprid Olefin in Bee Relevant Matrices
Collected from Tomato Plants Following Soil and Foliar Applications of Admire Pro Over Two Successive
Years.”All neat analytical reference standards were stored frozen prior to dilution. All reference standard
solutions were prepared in parent equivalents and corrected for purity during initial preparation. The
reference standard solutions were stored refrigerated or frozen and have been shown to be stable forthe length of storage time required for this study.
For the tomato leaves1, a 2.5 g sample was weighed into a 50-mL polypropylene conical centrifuge tube,
and 10 mL HPLC-grade water was added. The tube was mixed manually for 1 minute, followed by adding
20 mL of acetonitrile and mechanical shaking (HS501 digital, IKA-Werke, Wilmington, NC) for an
additional 1 minute. Then, 3 g of MgSO4 and 1.5 g of NaCl were added. The sample was amended with amixed internal standard solution, capped, and shaken for 1 minute. For leaf samples which were foundto contain high residues of imidacloprid (>2 ppm), the sample was amended with a 10X mixed internal
standard solution before the salts were added. The sample was centrifuged. For low imidacloprid
residue samples, 20 mL of organic supernatant was transferred into a separate 50-mL polypropylene
conical centrifuge tube containing 0.3 g of Bondesil-PSA and 1.8 g of MgSO4. For high imidacloprid
residue samples, 2.0 mL of organic supernatant and 18.0 mL of acetonitrile were transferred into aseparate 50-mL polypropylene conical centrifuge tube containing 0.3 g of Bondesil-PSA and 1.8 g of
MgSO4, which was capped and shaken for 1 minute. The sample extract was centrifuged, and a 1.25 mL
aliquot of supernatant was transferred into a clean culture tube. The sample aliquot was evaporated tonear dryness on the Turbo-Vap (Biotage, Charlotte, NC). The solid was reconstituted with 1.25 mL of
9:1 H2O:MeOH and transferred into a 2-mL sample vial for LC/MS/MS analysis.
For pollen2, a 0.1 g sample was weighed into a 2-mL centrifuge tube containing 2.8 mm steel balls. If theavailable pollen amount was less than 0.1 g but greater than 0.025 g for a sample, the sample wasconsidered sufficient for analysis. A 1 mL portion of methanol/water (3:1 v/v) was added, and the
mixture was homogenized with a bead mixer at 5000 beats/minute for 1 minute on a Precellys
homogenizer (Bertin Technologies, Rockville, MD). The isotopically labeled internal standards wereadded and mixed, and the mixture was centrifuged at 12,000 rpm for 2 minutes. The supernatant wastransferred into a clean culture tube containing 2.5 mL of water and was evaporated to an aqueous
remainder, then applied to a 3-mL ChemElut SPE cartridge. After 10 to 15 minutes, the cartridge waswashed with 4 mL of hexane/ethyl acetate (1:1 v/v) three times into a clean culture tube. The combined
eluates were evaporated to dryness. The analytes were dissolved from the tube with 0.5 mL of
MeOH/H2O (1:4 v/v). The solution was transferred into a 2 mL sample vial for analysis by LC/highresolution mass spectrometry (LC/HRMS).
Quantitation of each analyte was based on the daughter ion transitions of the analyte and therespective internal standard analog. The responses of the LC/MS/MS and LC/HRMS systems to eachanalyte and its internal standard were measured in samples and in standards, and a relative response
was calculated (as the ratio of the analyte and the stable isotopically labeled internal standard

16629

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49665201

CDPR IMI Soil and Foliar Tomato Study

responses). The relative response of the analyte in each sample was compared to the relative responseof the analyte in the standards.
The relative responses of imidacloprid and its analytes were measured over the range of 0.00012 to 2.0
ppm. The analyte relative responses were fit to a linearity curve calculated using linear regression
analysis with 1/x weighting (Thermo Finnigan XCalibur 2.7.0.20 or 2.2 SP1.48). Correlation coefficientswere calculated with the same software.
All data are reported in parent equivalents, and the individual measured residues of imidacloprid,
5-hydroxy imidacloprid, and imidacloprid olefin are summed to give a total imidacloprid residue.
The methods for determining imidacloprid, 5-hydroxy imidacloprid, and imidacloprid olefin residues
in/on tomato leaves and pollen were validated by measuring the recovery of these analytes from control
matrices fortified at their respective LOQs. Additional recoveries at higher fortification levels validated
the method for the highest residues observed in individual matrices. Concurrent recoveries ofimidacloprid, 5-hydroxy imidacloprid, and imidacloprid olefin from fortified samples were measured
with each set of samples to verify method performance.
5F. QUALITY ASSURANCE RESULTS
The responses of the LC/HRMS and LC/MS/MS systems to imidacloprid, 5-hydroxy imidacloprid, andimidacloprid olefin were linear in solvent over the range of 0.00012 to 2.0 ppm. The coefficients ofdetermination were >0.99. The response data are located in Appendix 4 in the study report.
Control interferences for tomato matrices are discussed in this paragraph; no total imidacloprid residue
was calculated for the UTC samples, so the levels of imidacloprid as an individual analyte are described.
Imidacloprid (parent) residues in UTC tomato pollen ranged from below the analyte LOD to 0.060 ppm(trial NT018-13ZA). Imidacloprid residues in UTC tomato leaves ranged from below the analyte LOD to
0.031 ppm (trial NT017-13ZB).
All recoveries were corrected for any interferences in corresponding controls. The overall means of therecoveries for each matrix at each fortification level were within the acceptable range of 70 to 120%,and the standard deviation values were below 20%. The analytical data summaries are located inAppendix 4 in the study report.
The limit of quantitation (LOQ) is defined as the lowest fortification level of an analyte at which
acceptable recovery has been achieved. The LOQ for a total residue is the highest of the LOQ values
assigned to the individual analytes for a particular matrix.
The limit of detection (LOD) is defined as the lowest concentration of an analyte that can be determined
to be statistically different from a blank. The LODs were determined from method validation dataobtained from control samples fortified at the respective analyte LOQs. The LODs were calculated bymultiplying the standard deviation of recovery measurements at the LOQ by t0.99 [where t0.99 is the onetailed t-statistic at the 99% confidence level for the number of replicates (n)].4 The LOD for the total
imidacloprid residue in each matrix is the highest LOD value of any one individual analyte for thatparticular matrix.

17630Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49665201

CDPR IMI Soil and Foliar Tomato Study

Table 5. Limits of quantification and detection for imidacloprid and metabolites*.
MatrixTomato Leaves

PollenAnalyteImidacloprid
5-Hydroxy Imidacloprid
Imidacloprid Olefin
Total Imidacloprid
Imidacloprid
5-Hydroxy Imidacloprid
Imidacloprid Olefin
Total Imidacloprid

LOQ (ppb)
5.0
5.0
5.0
5.0
1.0
1.0
1.0
1.0

*From page 22 of the study report.

LOD (ppb)
2.2
0.7
1.0
2.2
0.4
0.5
0.3
0.5

6. RESULTS:
6.A. COMPARISON OF CONCENTRATIONS MEASURED FOR PARENT AND DEGRADATESComparison of the distribution of concentrations measured for parent imidacloprid and degradation
products in pollen and leaves are presented in Table 6-1 and Table 6-2. The LOD of values for pollen
were very low ranging from 0.3 ppb to 0.5 ppb. Reporting concentration in ppb in this report indicates aweight to weight basis. The LOD for leaves ranged from 0.7 to 2.2 ppb. Data reported as below the LOD
were assigned one-half the LOD value. Comparison of the contribution of each chemical to the total
residue indicates that parent imidacloprid comprised essentially 90% or greater of the total residue
measured in each plant part. Since a small portion of the degradation products (considered to be as
toxic as parent Imidacloprid) were present in the measured residue, the following discussions will focus
on total imidacloprid residue measured in plant tissues. Statistical procedures used in the Statistical
Analysis System (SAS) software to provide distribution statistics or statistical tests were PROCCAPABILITY, PROC T-TEST, PROC SHEWHART, and PROC NPAR1WAY.
6.B. MAGNITUDE OF RESIDUES IN BEE-RELEVANT MATRICES
Figure 6-1 explains the statistical aspects relayed in the Box-and-Whisker plots used to compare the
distribution of total imidacloprid concentrations calculated for each sampling interval. For each data set
analyzed, the box graphic presents values for the mean, median, minimum, maximum, and 25th and75th percentiles. Replicate samples were taken at each interval. All data have been used to determine
the expected distributional properties for concentration in pollen and leaves because each analysis isrepresentative of the potential distribution encountered in field sampling.
Pollen. Comparison of overall statistics for total imidacloprid residue indicated much greater
concentrations at the third sampling interval than at the other three sampling intervals (Figure 6-2). The
soil application occurred at planting and two samplings were conducted after the soil application but
before the foliar applications. The first sampling, noted as “Interval 1”, occurred at a mean of 49 days
after the soil application with a range of values from 31 to 63 days. The second sampling interval, noted
as “Interval 2”, was also made prior to foliar applications at a mean of 62 days after the soil application
with a range of 45 to 77 days. Median total imidacloprid residues in pollen, which is relevant to potential
bee exposure, were 41 and 30 ppb with maximum values of 679 and 138 ppb for sampling intervals 1
and 2, respectively (Table 6-1).
After the second sampling interval, plants received two additional foliar applications at approximately 5
day intervals. The third sampling interval, noted as “Interval 3”, occurred approximately 6 days after the
18631

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49665201

CDPR IMI Soil and Foliar Tomato Study

second foliar spray. Median concentrations measured at the third interval increased nearly tenfold to
442 ppb with a maximum value of 1763 ppb. The fourth and final sampling interval, noted as “Interval4”, occurred around 19 days after the second foliar application where the median total imidacloprid
residue concentration dropped to 66 ppb and the maximum concentration was also lower at 354 ppb.
With respect to timing after soil application, the means were 74 and 87 days after application forintervals 3 and 4, respectively.
6.C. MAGNITUDE OF RESIDUES IN LEAVESFor leaves, the same pattern was measured as for pollen where median total imidacloprid values were
125, 100, 726, and 96 ppb for sampling intervals 1, 2, 3, and 4, respectively (Table 6-2). The sharp
decrease in concentration between intervals 3 and 4 does not appear to be due to wash-off from rainfall
or irrigation because drench/drip irrigation was indicated at each site. Also, there was essentially norecorded rainfall during the interval between foliar applications and sampling of plant tissues.
6.D. SITE SPECIFIC TRENDS
Temporal patterns in residue concentration for each site are depicted for pollen in Figures 6-3A and 63B and for leaves in Figures 6-4A and 6-4B. For pollen data, five of the individual studies lacked data forsampling interval 3. Based on the pattern noted in section 6.B, interval 3 is obviously an importantsampling event where concentrations were noted to spike upwards. Graphs are not presented for sites
missing data for interval 3, which were Fresno in 2014, Kerman in 2015, Porterville sites 1 and 2 in 2013,
and San Joaquin in 2015. In addition, data were not available for sampling interval 2 at the San Joaquin
and Kerman site in 2014 and for interval 1 at the San Luis Obispo site in 2014, but the curves arepresented for these sites because they conformed to the noted trend for sharp increases at interval 3.
Except for the San Joaquin site in Figure 6-3B, the general pattern of mean concentrations rising nearly
tenfold at the third sampling interval was observed at all other sites (Figures 6-3A and 6-3B). A sharp
decline in concentration between intervals 3 and 4 was also predominant except for the San Luis Obispo
site where there was only a slight decrease at interval 4. The concentrations measured in interval 4 arenearly equal to those measured before the foliar application. An additional treatment that measured
longevity of residues due to the soil application would have provided data to determine if the residues
measured at interval 4 were due to the soil application or simply the dissipation curve for residues
deposited from the foliar application.
For leaves, data were available for all sites and all years. Except for a few sites, a steep rise at the thirdsampling interval was observed with many reflecting an order of magnitude increase (Figures 6-4A and
6-4B). Curves not reflective of the predominant pattern were indicated at the Sanger 1 site in 2014, theFresno site in 2013, and the Kerman site for both years. In contrast, pollen data for these sites reflect asignificant rise in concentration at the third sampling interval. The lack of a similar pattern between
leaves and pollen for these two sites indicate a larger variability in distribution of concentration in leaf
tissue, which might be due to variability in application or sampling methodology.
6.E. Potential Carry-over of Residues
Paired T-tests were conducted at each interval to determine potential carry-over of residues between
years. Two replicates were taken at each interval within each year. These replicates, however, have no
relationship with respect to sampling between years so means were calculated. Tests for normality
indicated that that base 10 logarithm transformations produced distributions that approximatednormality. Paired T-tests were conducted for each interval to reduce potential problems with variancecaused by increased concentrations measured at interval 3. Within each site, the study was conducted
for two years with some sites spanning years 2013 to 2014 and others spanning years 2014 to 2015. T19
632

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49665201

CDPR IMI Soil and Foliar Tomato Study

tests were conducted on the difference between year 1 and year 2 for the logarithmic values of the total
imidacloprid residue concentration. Results generally indicated no difference in values between years
for most of the tests (Table 6-5). There were two instances where significance was indicated but thevalues for year 1 were greater than year 2. These results indicate no potential carry-over between years
for imidacloprid residues under the conditions of this study.
6.F. LEAF AND NECTAR CONCENTRATION IN RELATION TO SOIL TYPE
Originally, the proposed study design suggested sampling sites located in coarse, medium, and finetextured soils with three replicates assigned to each soil type. There is an inherent difficulty in fulfilling
the proposed design when the study is conducted after fields have been planted and then attempting toprocure cooperating growers. The distribution of sites with respect to soil texture category was 5 sites incoarse-textured soil, 1 site in medium-textured soil, and 3 sites in fine-textured soil. Using all replicate
data pooled over all years to represent potential distribution of residues within a soil type, the total
number of samples within each soil type was 20, 4, and 12 for coarse, medium, and fine-textured soil,
respectively. The replication was too low in medium-textured soil so comparisons were limited to coarse
and fine-textured categories. Non-parametric statistical tests were used to test for differences in
distributions between specified comparisons. Non-parametric tests do not require tests for normality as
they are robust to differences in distribution and they are also robust for experimental designs with low
replicates. The PROC NPAR1WAY procedure in the Statistical Analysis System (SAS) statistical package
was used to conduct Wilcoxon-Mann –Whitney (Wilcoxon) and Median non-parametric tests. A
significant result from the Wilcoxon test indicates differences in the shape of distributions and asignificant result from the Median test indicates differences in the location of the medians between
distributions. The Exact option for each statistic was implemented as it provides permutation testing, astatistical method that minimizes the effect of sample size and distributional differences. Under the
Exact option, the Monte Carlo procedure was also implemented which provided 10,000 separate runs
for each statistic to produce the permutation distributions.
Statistics for the distributions of total imidacloprid residue by each soil category for pollen and leaves
are in Tables 6-4 and 6-5, respectively. Significant differences in distributions between soil category
were indicated for pollen and leaves for sampling intervals 1 and 2, which potentially represent
differences in uptake due to soil application (Table 6-6). No significant differences were measured atinterval 3 when residues were greatly increased by the foliar applications. Foliar applications are notexpected to be influenced by soil type because residues are deposited directly onto the plant. By the last
sampling at interval 4, concentrations in leaves appear to retain differences noted prior to foliar sprays.
No difference in pollen concentration was measured at the last sampling interval. Even though theuneven replication amongst the soil categories provides some uncertainty in the statistical analysis, theresults appear to indicate that prior to the foliar sprays, concentrations in leaves and pollen weregreater in the coarse soil category.

20
633

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49665201

CDPR IMI Soil and Foliar Tomato Study

Table 6-1. Cumulative distributional statistics for concentration of imidacloprid and related metabolites in tomato pollen. Acronyms in thetable are: IMI= IMIDACLOPRID; OLEFIN = IMIDACLOPRID OLEFIN; 5-OH = 5 HYDROXY IMIDACLOPRID; TOTAL = SUM OF IMI, OLEFIN, AND 5-OH (IN BOLD);
N=NUMBER OF OBSERVATIONS; SD=STANDARD DEVIATION; CV = COEFFICIENT OF VARIATION. NUMBERED INTERVAL DENOTES TIMING OF SAMPLING WHERE INTERVALS
1 AND 2 WERE APPROXIMATELY AT 49 AND 62 DAYS AFTER THE FIRST SOIL APPLICATION AND INTERVALS 3 AND 4 WERE AT 6 AND 19 DAYS AFTER A SECOND FOLIARSPRAY COINCIDING WITH 74 AND 87 DAYS AFTER THE SOIL APPLICATION.
POLLENSAMPLING
STATISTICS

5-OH

INTERVAL 1
OLEFIN IMINMEAN (PPB)
SD (PPB)
CV (%)MIN (PPB)MEDIAN (PPB)
75TH (PPB)
90TH (PPB)
95TH (PPB)
MAX (PPB)% OF TOTAL

32
5.2
8.5
163.4
0.3
1.6
6.2
11.7
30.3
37.2
4.9

32
2.1
2.6
125.0
0.2
0.8
3.1
6.6
8.1
9.2
2.0

32
99.6
140.6
141.1
6.6
39.3
121.9
225.7
490.0
632.8
93.2

TOTAL

5-OH

INTERVAL 2
OLEFIN IMI

32
106.9
151.1
141.4
7.0
41.0
130.8
242.3
526.9
679.2

27
1.7
1.8
104.9
0.3
1.0
2.3
4.6
5.3
6.4
4.2

27
0.7
1.0
138.1
0.2
0.2
0.7
2.8
3.2
3.3
1.7

TOTAL

5OH
27
27
22
38.0 40.4 25.3
33.1 35.4 14.7
87.1 87.7 58.1
1.4
1.8
9.7
28.7 29.9 19.7
48.6 51.9 30.3
94.8 102.6 43.9
103.3 106.7 58.7
128.6 138.3 63.5
94.1
4.3

21
634

INTERVAL 3
OLEFIN
IMI
22
7.9
4.8
61.2
2.6
7.1
9.6
12.7
15.9
24.1
1.3

22
560.0
381.1
68.1
232.9
420.5
560.4
975.7
1448.81679.7
94.4

TOTAL

5-OH

INTERVAL 4
OLEFIN IMI

22
593.2
397.2
66.9
249.2
442.4
602.9
1015.91520.81762.5

32
5.9
7.3
125.2
0.8
4.3
6.1
9.6
21.8
39.4
7.4

32
2.1
1.7
85.2
0.2
1.3
3.9
4.4
4.6
6.0
2.6

32
71.1
56.6
79.6
15.5
58.8
84.5
141.0
148.7
312.2
90.0

TOTAL
32.0
79.0
64.1
81.2
16.5
66.3
90.7
154.1
174.5
354.0

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49665201

CDPR IMI Soil and Foliar Tomato Study

Table 6-2. Cumulative distributional statistics for concentration of imidacloprid and related metabolites in tomato leaves. Acronyms in thetable are: IMI= IMIDACLOPRID; OLEFIN = IMIDACLOPRID OLEFIN; 5-OH = 5 HYDROXY IMIDACLOPRID; TOTAL = SUM OF IMI, OLEFIN, AND 5-OH (IN BOLD);
N=NUMBER OF OBSERVATIONS; SD=STANDARD DEVIATION; CV = COEFFICIENT OF VARIATION. NUMBERED INTERVAL DENOTES TIMING OF SAMPLING WHERE INTERVALS
1 AND 2 WERE APPROXIMATELY AT 49 AND 62 DAYS AFTER THE FIRST SOIL APPLICATION AND INTERVALS 3 AND 4 WERE AT 6 AND 19 DAYS AFTER A SECOND FOLIARSPRAY COINCIDING WITH 74 AND 87 DAYS AFTER THE SOIL APPLICATION.
Leaf
Sampling
Statistics

Interval 1

Interval 2Interval 3

5-OH

Olefin

IMI

Total

5-OH

Olefin

IMI

Total

5-OH

Olefin

IMI

N

36.0

36.0

36.0

36.0

36.0

36.0

36.0

36.0

36.0

36.0

Mean (ppb)

8.9

7.7

151.0

167.6

5.7

6.2

134.0

145.9

57.5

SD (ppb)

11.8

7.5

148.2

163.5

6.1

5.6

128.3

139.4

CV (%)

132.2

97.4

98.2

97.6

107.7

91.6

95.7

Min (ppb)

0.4

0.5

1.1

2.0

0.4

0.5

Median (ppb)

4.3

4.9

103.7

125.0

3.3

5.0

75th (ppb)

9.8

11.6

182.9

798.3

9.4

90th (ppb)

27.2

18.0

362.6

397.0

95th (ppb)

43.5

24.2

492.9

Max (ppb)

43.5

25.7

541.4

% of Total

5.3

4.6

90.1

Interval 4
Total

5OH

Olefin

IMI

Total

36.0

36.0

36.0

36.0

36.0

36.0

31.8

1139.81229.2

8.7

7.4

130.5

146.5

51.6

22.5

1272.81341.7

6.6

5.0

107.6

117.7

95.6

89.7

70.6

111.7

109.2

76.7

68.5

82.5

80.3

4.6

5.5

1.7

1.5

41.3

44.5

0.1

1.5

12.7

14.3

89.5

100.1

41.8

26.3

660.3

726.2

7.8

6.4

84.8

96.4

11.0

231.7

252.0

93.3

39.9

1133.91271.1

12.0

9.7

143.8

170.0

15.2

14.0

330.5

361.5

111.8

70.9

3222.03427.9

18.5

15.8

320.6

356.1

544.0

18.4

16.8

381.2

417.5

136.6

79.9

3339.83539.8

21.1

19.1

370.8

423.1

592.3

23.2

19.0

459.0

496.4

261.3

81.1

5907.96249.1

31.2

21.1

402.8

434.9

3.9

4.2

91.9

4.7

2.6

92.7

5.9

5.0

89.1

22
635

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49665201

CDPR IMI Soil and Foliar Tomato Study

Table 6-3. Probability levels for paired T-tests for the effects of year on concentration of total
imidacloprid residues.

Paired T-test for Total Imidaclprid Residue
SourcePollenLeavesInterval

N

Pr>t-value

1
2
3

16
12
7

0.88
0.64
0.47

4
1
2
3

16
18
18
18

0.004
0.42
0.03
0.11

4

18

0.55

Comment

Year 1 Greater
Year 1 Greater

23636Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49665201

CDPR IMI Soil and Foliar Tomato Study

Table 6-4. Cumulative distributional statistics for total imidacloprid concentration measured in tomato pollen for categorized texture of soil atthe site plants were grown. Acronyms in the table are; N=NUMBER OF OBSERVATIONS; SD=STANDARD DEVIATION; CV = COEFFICIENT OF VARIATION.
NUMBERED INTERVAL DENOTES TIMING OF SAMPLING WHERE INTERVALS 1 AND 2 WERE APPROXIMATELY AT 49 AND 62 DAYS AFTER THE FIRST SOIL APPLICATION AND
INTERVALS 3 AND 4 WERE AT 6 AND 19 DAYS AFTER A SECOND FOLIAR SPRAY COINCIDING WITH 74 AND 87 DAYS AFTER THE SOIL APPLICATION.
Pollen: Distribution of Total Imidacloprid Residues by Site Soil Type Classification
Statistic

Interval 1
CoarseMediumN
Mean (ppb)
SD (ppb)
CV (%)Min (ppb)
Median (ppb)
75th (ppb)
90th (ppb)
95th (ppb)
Max (ppb)

20
242
182
75
13
176
394
517
568
592

4
93
24
25
71
89
111
124
124
124

Fine

Interval 2
CoarseMedium

Fine

12
69
70
102
2
49
101
197
199
199

20
187
137
73
13
154
282
373
441
496

12
75
109
145
5
35
51
291
322
322

4
150
180
120
29
77
254
418
418
418

24
637

CoarseInterval 3
Medium

20
1174
1520
130
45
529
952
3502
4894
6249

4
541
377
70
68
594
840
906
906
906

Fine

Interval 4
CoarseMedium

Fine

12
1550
1185
76
226
1131
2894
3120
3428
3428

20
205
131
64
14
161
309
390
429
435

12
81
30
37
30
82
96
124
135
135

4
58
17
29
41
58
72
74
74
74

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49665201

CDPR IMI Soil and Foliar Tomato Study

Table 6-5. Cumulative distributional statistics for total imidacloprid concentration measured in tomato leaves for categorized texture of soilat the site plants were grown. Acronyms in the table are; N=NUMBER OF OBSERVATIONS; SD=STANDARD DEVIATION; CV = COEFFICIENT OF VARIATION.
NUMBERED INTERVAL DENOTES TIMING OF SAMPLING WHERE INTERVALS 1 AND 2 WERE APPROXIMATELY AT 49 AND 62 DAYS AFTER THE FIRST SOIL APPLICATION AND
INTERVALS 3 AND 4 WERE AT 6 AND 19 DAYS AFTER A SECOND FOLIAR SPRAY COINCIDING WITH 74 AND 87 DAYS AFTER THE SOIL APPLICATION.

StatisticN
Mean (ppb)
SD (ppb)
CV (%)Min (ppb)
Median (ppb)
75th (ppb)
90th (ppb)
95th (ppb)
Max (ppb)

Leaves: Distribution of Total Imidacloprid Residues by Site Soil Type Classification
Interval 1
Interval 2Interval 3
CoarseMediumFine Coarse
MediumFine Coarse
Medium
Fine
19
4
9
16
3
8
14
2
6
144
82
40
50
55
15
557
1268
453
185
34
53
37
45
12
387
357
195
129
42
131
73
82
76
70
28
43
18
47
7
10
25
2
265
1016
249
41
82
13
44
33
15
442
1268
375
232
111
41
69
107
25
561
1521
603
527
118
170
103
107
30
972
1521
769
679
118
170
138
107
30
1763
1521
769
679
118
170
138
107
30
1763
1521
769

25
638

Interval 4
CoarseMedium
18
4
77
66
35
51
46
78
24
20
70
60
87
109
154
124
155
124
155
124

Fine
10
88
118
16
49
80
264
354
354
354

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49665201

CDPR IMI Soil a

Table 6-6. Results of non-parametric Wilcoxon and Median tests comparing distributions of total
imidacloprid residue concentration in pollen and leaves with coarse and fine-textured soil
categories.

Exact Probability Levels for Wilcoxon and Median Non-Parametric Tests Comparing
Total Imidacloprid Residue Distributions in Coarse to Fine-Textured soilInterval 1
Interval 2Interval 3Interval 4
SourcePollenLeavesWilcoxon
0.028
0.005

Median0.42
0.01

Wilcoxon
0.005
0.024

Median0.026
0.009

Wilcoxon
0.66
0.17

26
639

Median0.64
0.28

Wilcoxon
0.21
0.003

Median0.24
0.01

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49665201

CDPR IMI Soil and Foliar Tomato Study

7. STUDY STRENGTHS, LIMITATIONS AND CONCLUSIONS
The data from this study provide an expected distribution for the concentration of imidacloprid residues
that bees are exposed to in pollen in tomato flowers grown under actual agronomic practices in
California. Relating concentrations measured in flower parts to bee health is possible by comparing theconcentrations measured in bee relevant plant parts to target values that define acute or chronic
exposure scenarios. Although the detected levels of imidacloprid residues indicate substantial presenceof residues in pollen samples, results do not reflect potential maximum exposure concentrations tofoliar applications because samples were taken approximately 6 days after a second foliar application.
Therefore, there is some uncertainty if the sampling schedule reflected a maximal exposure scenario.
In the context of documenting the magnitude of imidacloprid residues in bee-related matrices oftomato, the following strengths are observed in this study.
1. Data provide quantitative values of total imidacloprid residues expected in pollen and leaves oftomato plants grown under California conditions.
2. Measurements were taken at four time intervals in an attempt to quantify levels expected inplant tissues over time and from different methods of application: The first and second intervalreflected concentrations following a period of time after a soil application at planting, and thirdand fourth interval reflected concentrations after two additional foliar applications were madeto the crop.
3. Each site was replicated over two years so potential carry-over effects could be measured.
Limitations noted in this study include:
1. The values most likely do not reflect expected maximum concentrations in pollen because
sampling did not occur directly after foliar application. Sampling occurred on average 6 days
after a second foliar application. Substantial decreases at nearly an order of magnitude werenoted in residues from plants sampled between the 3rd and 4th foliar application interval. The
average sampling between the 3rd and the 4th foliar application was 13 days. Since there was nopotential for redistribution of residues due to water movement from either irrigation or rainfall,
the steep dissipation indicates that concentrations would most likely have been highest if
samples were taken directly after the foliar applications.
2. Data were missing for pollen sampling at interval 3 at three sites. Highest concentrations were
measured at this sampling interval so characterization of the distribution for the third samplinginterval across all sites is incomplete.
3. Since data from coarse soils were disproportionately represented as compared to the medium
and fine soil types, observed statistical effects provide only preliminary evidence for differences
measured in residue uptake in tomato plants due to soil type.
Overall, considering the strengths and limitations of this study, the following conclusions can be drawn:
1. Imidacloprid residues were measured in pollen from soil application: The distribution of total
imidacloprid residues resulting from the soil application (the mean sampling time occurred at
approximately 48 days after application of 0.38 lb ai/A) produced a median concentration of 41
ppb, a maximum value measured at 679 ppb and a 90th percentile value at 242 ppb. These
values represented 32 samples taken over two years from 9 sites sampled.
27
640

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49665201

CDPR IMI Soil and Foliar Tomato Study

2. Imidacloprid residues in pollen increased from additional foliar sprays: The distribution of total
imidacloprid residues from 2 additional foliar applications (approximately 0.068 lb ai/A), whichwere sampled approximately six days after the second application, resulted in a median of 442
ppb, a maximum value of 1763 ppb and a 90th percentile value of 1016 ppb. These values
represented 22 samples taken over two years from 9 sites.
3. Patterns noted in concentration over time were similar between leaf and pollen samples: The
pattern in leaves was similar to that observed for bee-relevant pollen samples. Leaf
concentrations that were measured approximately 48 days after the soil application resulted ina median value of 125 ppb, a maximum of 592 ppb and a 90th percentile value of 347 ppb. These
values were increased for samples taken after the second foliar application where the medianvalue was increased to 726 ppb with a maximum value at 6249 ppb and a 90th percentile valueof 3428 ppb. These values represented 36 samples taken over 2 years from 9 sites.
4. Imidacloprid concentration indicated dissipation between the third and fourth samplingintervals: Residues were observed to drop at a second sampling date taken approximately 19
days after the second foliar application. Median and maximum values for pollen were measured
at 66 and 354 ppb, respectively, and 46 and 435 ppb for leaves, respectively. For the medianvalues, this was an 86% decrease for pollen and a 94% decrease for leaves over theapproximately 11 day period between the two sampling intervals taken after the second foliar
application of imidacloprid.
5. No carry-over effect between years: There was no increase in concentrations over years
measured in either pollen or leaf samples.
6. Concentrations in leaf and pollen tissue was greater in coarse-textured soil due to soil
application: Although the replication was uneven between the soil texture categories, thedistribution of total imidacloprid residues was greater in the coarse-texture soil compared to thefine-textured soil in the sampling intervals conducted after the soil application but prior to foliar
application. Greater uptake in coarse-textured soil has been measured in previous studies.

8. STUDY VALIDITY/CLASSIFICATION

The data from this study provide an expected distribution of the concentration of imidacloprid residues
that bees are exposed to in pollen of tomato plants grown under actual agronomic practices in
California. These data, however, do not represent the maximum expected concentrations in pollen due
to samples not being taken directly after foliar applications. They were taken on average 6 days after asecond foliar application. Therefore, there is uncertainty if these values reflect maximum exposure
scenarios. The study is considered scientifically sound and useful for risk assessment purposes. The
concentrations reported for pollen are high enough to require comparison to target values that define
acute or chronic exposure scenarios when they are calculated. The study is classified as ACCEPTABLE for
quantitative use in risk assessment

28641Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49665201

CDPR IMI Soil and Foliar Tomato Study

9. REFERENCES

1. Brungardt, J. 2010. An Analytical Method for the Determination of Residues of lmidacloprid andits Metabolites lmidacloprid Olefin and 5-Hydroxy lmidacloprid in Bee Relevant Matrices Using
LC/MS/MS. Bayer Method No. NT-005-P10-01.
2. Miller, A. 2014. An Analytical Method for the Determination of Residues of lmidacloprid and its
Metabolites lmidacloprid Olefin and 5-Hydroxy lmidacloprid in Bee Relevant Matrices Using
LC/MS/MS. Bayer CropScience Report Number: NT -006-A 13-01.
3. Office of Pesticide Programs, US EPA. 2000. Assigning values to nondetected/nonquantified
pesticide residues in human health food exposure assessments. EPA Docket #OPP-00570A.

4. Noland, P.A. 1992. lmidacloprid and metabolites- freezer storage stability study in crops. Bayer
CropScience Report No. 103237. MRID 42556135.
5. Noland, P.A. 1993. lmidacloprid and metabolites- freezer storage stability study in crops. Bayer
CropScience Report No. 103237-1. MRID 42810311.
6. Noland, P .A. 1994. lmidacloprid and metabolites- freezer storage stability study in crops. Bayer
CropScience Report No. 103237-2. MRID 43197203.
7. Noland, P .A. 1994. lmidacloprid and metabolites- freezer storage stability study in crops. Bayer
CropScience Report No. 103237-3. MRID 43487302.
8. Lenz, C.A. 1993. Addendum 1. lmidacloprid and metabolites- freezer storage stability study incrops {wheat matrices, tomatoseed, tomato, cauliflower, and lettuce). Bayer CropScience
Report No. 103949-1. MRID 42810313.
9. Lenz, C.A. 1993. Addendum 2. lmidacloprid and metabolites- freezer storage stability study incrops (wheat matrices, tomatoseed, tomato, cauliflower, and lettuce). Bayer CropScience
Report No. 103949-2. MRID 43197201.
10. Lenz, C.A. 1993. Addendum 3. lmidacloprid and metabolites- freezer storage stability study incrops (wheat matrices, tomatoseed, tomato, cauliflower, and lettuce). Bayer CropScience
Report No. 103949-3. MRID 43487301.
11. Schoning, R. 2014. Storage stability of imidacloprid and its 5-Hydroxy and olefin metabolite
in/on plant matrices for 36 Months. Bayer CropScience Report No. P642094733 Amendment No.
1.

29
642

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49665201

CDPR IMI Soil and Foliar Tomato Study

Figure 6-1. Explanation of statistical meaning of the Box-and-Whisker plots.

Maximum Observation

75% Quartile
Mean ValueMedian Value (50%)
25% Quartile
Minimum Observation

30
643

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49665201

CDPR IMI Soil and Foliar Tomato Study

Figure 6-2. Relative concentration of total imidacloprid residues measured in tomato (A) pollen and (B) leaves compared between samplingintervals. Data were averaged over all sites. NUMBERED INTERVAL DENOTES TIMING OF SAMPLING WHERE INTERVALS 1 AND 2 WERE APPROXIMATELY AT 49
AND 62 DAYS AFTER THE FIRST SOIL APPLICATION AND INTERVALS 3 AND 4 WERE AT 6 AND 19 DAYS AFTER A SECOND FOLIAR SPRAY COINCIDING WITH 74 AND 87 DAYSAFTER THE SOIL APPLICATION.

A. Pollen

Interval 1

B. LeavesInterval 2Interval 3Interval 4

Page 31 of 35
644

Interval 1

Interval 2Interval 3Interval 4

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49665201

CDPR IMI Soil and Foliar Tomato Study

Figure 6-3A. Trend in total imidacloprid residue measured in pollen at each site. Within each panel markers, marker color and line styledenote the year, i.e. year 2013 is a solid black circle and a small-dashed black line; year 2014 is a hollow square and a green solid line; year
2015 isa hollow triangle and a large-dashed blue line.
1500

Sanger1 - 2013
Sanger1 - 2014

1200

Total Imidacloprid Residue (ppb)

1500
1200

900

900

600

600

300

300

0

20

30

1500

40

50

60

70

80

90

0

100 110

900

600

600

300

300

20

30

40

50

60

20

30

70

80

90

100 110

0

40

50

60

70

80

90

100

110

70

80

90

100

110

Porterville1 - 2014
Porterville2 - 2014

1200

900

0

Hand-collected

1500

Guadalupe 2014

1200

Sanger2 - 2014
Sanger2 - 2015

20

30

40

50

Days After Imidacloprid Soil Application

60

Specific Site Information:
Sanger1=NT018-13ZA; Sanger2=NT042-13ZA; Guadalupe=NT017-13ZB; Porterville1=NT010-13ZA; Porterville2=NT016-13ZA
Page 32 of 35645

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49665201

CDPR IMI Soil and Foliar Tomato Study

Figure 6-3B. Trend in total imidacloprid residue measured in pollen at each site. Within each panel markers, marker color and line styledenote the year, i.e. year 2013 is a solid black circle and a small-dashed black line; year 2014 is a hollow square and a green solid line; year
2015 is a hollow triangle and a large-dashed blue line.
1500
1200

Total Imidacloprid Residue (ppb)

1500

Fresno 2013

1200

900

900

600

600

300

300
0

0

20
1500

30

40

50

San Luis Obispo - 2014
San Luis Obispo - 2015

60

70

80

90

100

110

20

30

1500
1200

900

900

600

600

300

300

0

20

30

40

50

60

70

80

90

100

110

0

20

30

40

Days After Imidacloprid Soil Application
Specific Site Information:
Fresno=NT013-13ZA; ; San Luis Obispo=NT039-13ZA; San Joaquin=NT040-13ZA; Kerman=NT041-13ZA
Page 33 of 35
646

50

60

70

80

90

100 110

70

80

90

100 110

Kerman - 2014

San Joaquin - 2014
1200

40

50

60

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49665201

CDPR IMI Soil and Foliar Tomato Study

Figure6-4 A. Trend in total imidacloprid residue measured in leaf tissue at each site. Within each panel markers, marker color and line styledenote the year, i.e. year 2013 is a solid black circle and a small-dashed black line; year 2014 is a hollow square and a green solid line; year
2015 is a hollow triangle and a large-dashed blue line. Note both Sanger both sites are plotted on the same graph with site 2 year 2014 a
green dashed line.
4000

4000

Porterville1 - 2013

Sanger1 - 2013
Sanger1 - 2014

Total Imidacloprid Residue (ppb)

3000

Sanger2 - 2014
Sanger2 - 2015

2000

2000

1000

0

Porterville1 - 2014

3000

1000

20

30

4000

40

50

60

70

80

90

0

100 110

3000

3000

2000

2000

1000

1000

0

0
30

40

50

60

70

30

40

50

60

70

80

90

100 110

70

80

90

100 110

4000

Guadalupe - 2014
Guadalupe - 2015

20

20

80

90

100 110

Porterville2 - 2013
Porterville2 - 2014

20

30

40

50

60

Days After Imidacloprid Soil Application
Specific Site Information:
Sanger1=NT018-13ZA; Sanger2=NT042-13ZA; Guadalupe=NT017-13ZB;
Porterville2=NT016-13ZA
Page 34Porterville1=NT010-13ZA;
of 35
647

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49665201

CDPR IMI Soil and Foliar Tomato Study

Figure 6-4B. Trend in total imidacloprid residue measured in leaf tissue at each site. Within each panel markers, marker color and line styledenote the year, i.e. year 2013 is a solid black circle and a small-dashed black line; year 2014 is a hollow square anda green solid line; year
2015 is a hollow triangle and a large-dashed blue line
4000

Fresno - 2013
Fresno - 2014

3000

Total Imidacloprid Residue (ppb)

4000

2000

1000

1000

0

0
30

4000

40

50

60

San Luis Obispo -2015

3000

2000

20

San Luis Obispo - 2014

70

80

90

100

110

20

30

4000

San Joaquin - 2014

3000

2000

2000

1000

1000

0

20

30

40

50

60

70

80

90

100

110

0

50

20

30

40

Days After Imidacloprid Soil Application

Specific Site Information:
Fresno=NT013-13ZA; ; San Luis Obispo=NT039-13ZA; San Joaquin=NT040-13ZA; Kerman=NT041-13ZA
Page 35 of 35
648

60

70

80

90

100 110

Kerman - 2014
Kerman - 2015

San Joaqin - 2015
3000

40

50

60

70

80

90

100 110

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 49090504

CDPR IMI Citrus (2011)

Reference
Byrne, F.; Morse, J.; Visscher, P.; Grafton-Cardwell, E.; Leimkuehler, W. (2011) Determination of exposurelevels of honey bees foraging on flowers of citrus trees previously treated with imidacloprid. Project
Number: EBNTL056/7, M/408424/012. Unpublished study prepared by University of California, Riverside.
70p. MRID 49090504, CDPR Study ID 259131, Data Volume 51950-0787, Tracking ID# 246252

1.

STUDY INFORMATION

Study Reviewed By:

Sponsor:Sponsor Study ID:
GLP Status:
Study Type:

Study Completion Date:
March 30, 2011
Study Location:
1) Hemet, California
2) Lindcove Research and Extension Center
3) Ventura County, California
4) Temecula, California
5) Tulare County, California
6) University of California, Riverside
EBNTL056-7
PC Code:129099
Non-GLP; Final protocol was submitted to CDPR for review.
[CDPR study ID 253951, Data Volume 51950-0756, Tracking ID# 240317]
Non-Guideline field residue study on Southern California Citrus Groves that had beenpreviously treated with Imidacloprid to evaluate bee exposure.
CDPR, U.S. EPA and PMRA
Bayer CropScience
2T.W. Alexander Drive
Research Triangle Park,
NC USA 27709

2. REVIEWER INFORMATION
Primary Reviewers:
John Troiano, Ph.D., Research Scientist III, Environmental Monitoring
California Department Branchof Pesticide
Richard Bireley, Senior Environmental Scientist (Specialist), Ecotoxicology
Regulation
Group, Pesticide Registration Branch
Denise Alder, Senior Environmental Scientist (Specialist), Lead Reevaluation
Coordinator, Pesticide Registration Branch
Russell Darling, Environmental Scientist, Reevaluation Coordinator,
Pesticide Registration BranchSecondary Reviewer:

TBD

3. TEST MATERIAL CHARACTERIZATION
Test Material:
1) Admire Pro
2) Unknown Formulations
Description:
Material Source:
CAS #:

1) Suspension concentrate (SC)2) Unknown
Bayer Corporation
138261-41-3

4. EXECUTIVE SUMMARY

1649

Percent Active
Ingredient:

1) 42.8% A.I.
2) Unknown

Density:

1.41-1.54 g/mL

Solubility:
pH (24°C):

0.51 to 0.61 g/L
7.8

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 49090504

CDPR IMI Citrus (2011)

A series of field investigations were conducted between 2008 and 2011 to determine to what extent
honey bees foraging on citrus blossoms may be exposed to imidacloprid when citrus trees are treatedwith systemic applications (soil treatments) of this insecticide.
The approach that was taken was to compare the imidacloprid residues in floral nectar with nectar
extracted from the crops, foraging honey bees and from stored nectar within their hives. Twoexperimental systems were established. First, honey bee colonies were confined within large tunnel
cage enclosures where foraging access to treated trees was limited. Second, honey bee colonies weresituated in an open-field system, in which hives were placed in a large acreage of commercial citrus that
had been treated with imidacloprid. In this second system, the foraging activity of the honey bees was
not limited to any specific trees or to citrus.
In addition to the honey bee studies, the study quantifies the levels of imidacloprid in nectar from treesthat had been treated under a number of different scenarios. The intent was to determine residues intrees treated in successive years to test for potential imidacloprid accumulation and persistence. For
this, groves that were growing in different soil types were included because of the impact that soil hason the availability of imidacloprid for uptake. The effect of differing application rates were also
evaluated on imidacloprid residues in nectar.
The subject study report consists of five different trials that will make up the composition of this Data
Evaluation Report. Detailed findings, study limitations, study validity and results from each trial arefound below.
5. STUDY VALIDITY
Guideline Followed:
Guideline Deviations:
Other Deviations:Non-guideline study (final protocol submitted by CDPR)
N/A
N/A

Classification:
Rationale:
Reparability:

Supplemental
N/A
N/A

6. SAMPLE COLLECTION, HANDLING, PROCESSING
Nectar Collections by Hand
Nectar was sampled from individual flowers using a micro-capillary tube inserted into a bulb dispenser.
After insertion of the tip of the tube into the nectar at the base of the flower, the nectar was drawn into
the tube by capillary action. The nectar was then transferred to an autosampler vial. The target volume
for each sample was 150 µl. During field trips to collect the nectar, the samples were kept in an ice chest
containing dry ice until they were stored in a -20°C freezer.
Nectar collections by Honey Bees
Nectar was collected from the crops of honey bees returning to their hives from foraging trips todetermine the concentrations of imidacloprid and metabolites. Bees were intercepted at the entrance
to the hives using a small net and were immediately transferred to a cooler containing dry ice toanesthetize them. After 30 seconds in the cooler, individual bees were forced to regurgitate thecontents of their crops by pressing gently on the lateral sides of their abdomen region (the gaster) with2650

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 49090504

CDPR IMI Citrus (2011)

paddle forceps. The nectar formed a droplet at the top of their mandibles where it was collected using a
micro-capillary tube. The nectar was transferred to an autosampler vial and then placed in a cooler withdry ice. For each sample it was necessary to composite nectar from several bees in order to meet theminimum target volume of 75 µl. At least 5 bees were used to prepare 1 sample and a maximum of 20
samples from each hive were collected.
Stored Nectar
In addition to the stomach nectar, nectar was also collected from deposits made to new comb withineach hive. A sample consisted of pooled nectar from comb cells near each other. Sufficient nectar wascollected for residue analysis and to measure sugar concentration using a refractometer to provide
information on possible changes in imidacloprid concentration during conversion from nectar to honey.
The target sample number was 10 samples from each hive.
7. ANALYTICAL METHODS
NectarNectar collected from citrus trees (orange, mandarins and grapefruit) was analyzed by dilution withmobile phase containing the stable isotope internal standards. 50 µL of nectar was pipetted into an
autosampler vial containing a 300 µL conical insert. 50 µL of mobile phase (9:1 water/methanol
containing 10 mM NH4HCO3) containing 10 ng/mL of the stable isotope internal standards was added
and the sample mixed a couple times by pulling up and down with the Gilson Microman pipette. The
final concentration in the diluted nectar of the internal standard was 5 ng/mL. The samples wereanalyzed by LC/MS/MS. Response ratios (NA/IS) were directly compared to a calibration curve having
concentrations of 5 ng/mL for the internal standards and native analyte concentrations ranging from 0.1
to 100 ng/mL and adjusted for dilution, i.e., 50 µL of nectar diluted to 100 µL (2X dilution). Recoveries(QC) samples were prepared by diluting control nectar with the QC dilution solution as described.
PollenA 0.05 to 0.5 g sample of pollen was weighed into a 13 mL polypropylene conical screw cap centrifuge
tube. 1.5 mL of HPLC grade water (pesticide residue grade) was added and the pollen dissolved. 20 ng of
the stable isotope internal standard mixture of imidacloprid, imidacloprid olefin and 5-hydroxyimidacloprid was added followed by 4 mL of ACN. The tube was then shaken vigorously for one minute.
Then 1 g of MgSO4 (anhydrous) and 0.5 g NaCl was added and the tube again shaken and vortexed
vigorously for 1 minute. The tubes were then centrifuged at 4000 RPM for 5 minutes using an Eppendorf
5810 R centrifuge.
The supernatant, nominally 4 mL, was decanted into another 13 mL centrifuge tube containing, 0.040 g
BONDESIL-PSA and 170 g Mg SO4 (anhydrous). The tube was shaken and vortexed vigorously for 1
minute. The tubes were centrifuged as before. A 1.0 mL portion of the supernatant (ACN) wastransferred to a LC autosampler vial and the ACN was avaporated to dryness using a N-Evap or Tubovap.
The residue was re-dissolved in 1.0 mL of HPLC mobile phase (9:1 water/MeOH containing 10 mM
NH4HCO3) in preparation for analysis by LC/MS/MS.
Recoveries (QC) samples (5 ppb) were prepared by fortifying control pollen (500 mg) with mixed native
stds (25 µL of the 0.1 µg/mL mixed standard).
3651

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 49090504

CDPR IMI Citrus (2011)

Imidacloprid, imidacloprid olefin and 5-hydroxy imidacloprid were measured in nectar and pollen.
Nectar was measured directly by dilution with HPLC mobile phase and the pollen was extracted using
QuEChERS methodology. The final extracts were analyzed by LC/MS/MS employing stable isotope
internal standards. The Limit of Detection (LOD) and Limit of Quantitation (LOQ) were calculated basedon 3X and 10X, respectively, of the standard deviation of 4 QC recovery samples from stomach nectar at
1 ppb. From this evaluation, the LOD and LOQ were calculated to be 0.018, 0.36 and 0.30 ppb (LOD) and
0.6, 1.2 and 1 ppb (LOQ) for imidacloprid, 5-hydroxy imidacloprid, and imidacloprid olefin, respectively.

8. TRIALS
7A. TRIAL INFORMATION
Title:
Tunnel Cage Trial
Application Date: September 3, 2009

Sampling Date:

Section of Study:

Trial Location:

Section 2

April 24, 2010
Lindcove Research andExtension Center (LREC)TRIAL SUMMARY
The objective of this component of the study was to examine citrus groves that were treated with a soil
application of imidacloprid systemic insecticide, to understand the levels of imidacloprid that occurredin (a) nectar extracted by hand from citrus flowers, (b) nectar collected by forager honey bees andtransported back to the hive, and (c) nectar or "uncapped honey" deposited by bees in cells of theBrood comb.
Honey bee colonies were confined within tunnel cage enclosures, each containing 3 flowering citrus
trees that had been treated with soil applications of systemic imidacloprid (Admire Pro®). Nectar wascollected by hand from each tree within each tunnel cage. In addition, nectar was extracted from thestomachs of honey bees foraging within the tunnels so that a comparison could be made between handcollected and honey bee-collected nectar. At the end of the confinement period within the tunnels,
stored nectar was sampled from new comb that had been placed within each hive at the start of the
exposure period. Four tunnels were constructed, providing three imidacloprid-treated and one
untreated control data set.
METHODAPPLICATION TIMING AND RATES
Admire Pro® was applied at the maximum label rate of 14 fl oz per acre (0.104 fl oz per tree) on Sept. 3,
2009. Trees were pre-irrigated for 2 hours prior to applications. The insecticide was applied to each treeusing a watering can, taking care to apply the solution evenly within the irrigated area defined by the
sprinklers. Watering cans were used to administer the insecticide to the trees in order to ensure that thecorrect amount of active ingredient, as defined by the insecticide label, was delivered to each tree.
Trees were irrigated for another 4 hours following completion of the applications, and then subjected to4652

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 49090504

CDPR IMI Citrus (2011)

the standard 8 hour, once-per-week irrigation regime during the first 4 weeks post-treatment.
Thereafter, trees were irrigated according to tensiometer measurements.
STUDY SITE LOCATION AND CHARACTERISTICS

This trial was conducted at the Lindcove Research and Extension Center (LREC) in Exeter, California. The
trees were Washington Parent Navels on C35 rootstock, which were planted in June 1992 (17 years oldat the time of the study). The soil type at LREC is a loam soil consisting of 15-25% clay and 00.5-1%
organic matter. There were 134 trees per acre with a tree spacing of 18’ x 18’ in the 3.9 acre block. Eachtree was irrigated by 2 sprinklers located on opposite sides of the trunk with 10.3 gph output on a
weekly schedule.
The enclosures were constructed from transparent netting with a maximum light exclusion of 13%. The
netting was supported on a frame constructed from 1" PVC tubing to provide a semi cylindrical tunnel
that fully enclosed 3 citrus trees. There was sufficient clearance to allow movement of personnel
between the citrus trees and the netting so that all sections of the trees could be used for nectar
sampling. Tunnel dimensions were 26' x 96' x 16'.
The Italian honey bee, Apis mellifera ligustica, was used in the subject trial. When the trees were in fullbloom, one small colony of bees (a nuclear hive consisting of 5 frames of comb, a queen, developing
brood, and workers to cover about 4 frames) was introduced into each tunnel. Hives were initiallyplaced in the tunnels at 8.00 a.m. on April 20, 2010. Due to heavy rainfall, bees were confined within thehives until the morning of April 22, 2010 when the bees were released to begin foraging.
SAMPLING METHOD
Nectar was sampled directly from the flower by hand using a micro-capillary tube. The target volume foreach sample was 150 µl, with nine composite nectar samples collected, three from each tree. Samples
were also collected using honey bees that were contained over the three trees by a tunnel constructedfrom transparent netting (Combined Clear Net 13%) with 1” PVC tube frames. The bees wereintercepted at the hive entrance and chilled with dry ice for 30 seconds. A composite sample from atleast 5 bees was collected. In addition, stored nectar was collected from new combs within each hive. A
pooled sample was collected from cells near each other with a target of ten samples per hive. Nectar
sugar analysis was measured by refractometry.
RESULTS:
The mean residues of total imidacloprid in hand or bee collected citrus nectar ranged from 13.97-21.19
ppb, while mean residues in hive stored nectar ranged from 44.65-72.81 ppb. The highest residues were
found in hive stored nectar, corresponding with the highest sugar concentration.

5653

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 49090504

CDPR IMI Citrus (2011)

Table 1. Summary of imidacloprid residues in nectar collected from orange trees within four tunnel
enclosures that had been treated the previous two years as a soil drench at the maximum labelled rate.
Maximum application rate applied to individual trees on 9/3/2009.

TunnelControl
12
3

Mean forTunnels 13

Lindcove Research and Extension Center (LREC)
Spring 2010Loam Soil [15-25% clay, 0.5 – 1% organic matter]
Mean Residue Concentration (ppb)(minimum – maximum)
Hand Collected Nectar
Bee Collected Nectar
Uncapped Stored Nectar
N1 = 9N1 = 20
N1 = varies[3 samples from each tree, 3[one hive per tunnel, mean of 20[one hive per tunnel, mean of up totrees per tunnel]
samples]
10 samples]
Sampling occurred
Sampling occurred 4/22/2010
Sampling occurred 4/24/2010
4/22/2010
IMI
Total IMI2
IMI
Total IMI2
IMI
Total IMI2
<1.0
<1.0
<1.0
<1.0
<1.0
<1.0
8.3
13.96
10.61
17.55
25.49
44.65
(2.86 –
(5.30 –
(5.6 – 21.22)(9.17 – 37.12)(19.71 – 36.06)(35.40 – 62.75)
13.64)
22.73)
7.64
14.01
8.97
15.08
27.33
49.65
(4.38 –
(8.06 –
(3.02 – 16.17)(4.92 – 29.75)(24.81 – 30.83)(42.52 – 57.58)
12.38)
22.84)
12.81
21.19
8.00
13.38
40.12
72.81
(8.72 –
(9.18 –
(1.66 – 18.99)(2.81 – 34.15)(27.14 – 54.14)(48.75 – 95.18)
21.91)
34.64)
9.58
16.39
9.23
15.40
30.98
55.70
(7.64 –
(13.96 –(8.00 – 10.61)(13.38 – 17.55)(25.49 – 40.12)(44.65 – 72.81)
12.81)
21.19)

1“N” is the total number of samples collected
2 “Total IMI” combines magnitude of residues of imidacloprid plus degradants IMI-olefin and IMI-5-OH

CONCLUSION TO TUNNEL CAGE
In this first trial, oranges (Washington Parent Navels on C35 rootstock) in a 3.9 acre block were treatedin the fall of 2009 with 14 fl oz per acre (the maximum current U.S. label rate). The trial was located at
the Lindcove Research and Extension Center (LREC) in Exeter, CA. The soil type at LREC is a loam soil
consisting of 15-25% clay and 0.5-1% organic matter. Honey bee colonies were confined within tunnel
cage enclosures, each containing 3 flowering citrus trees.
Nectar was collected by hand from each tree within each tunnel cage. In addition, nectar was extracted
from the stomachs of honey bees foraging within the tunnels so that a comparison could be madebetween hand-collected and honey bee-collected nectar. At the end of the confinement period within
the tunnels, stored nectar was sampled from new comb that had been placed within each hive at thestart of the exposure period.
The mean residues of total imidacloprid in hand or bee collected citrus nectar ranged from 13.97-21.19
ppb, while mean residues in hive stored nectar ranged from 44.65-72.81 ppb. The highest residues were
found in hive stored nectar, corresponding with the highest sugar concentration.

6654

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 49090504

CDPR IMI Citrus (2011)

7B. TRIAL INFORMATION
Title:
Open Field Trial
Application Date: Fall 2009

Sampling Date:

Section of Study:

Trial Location:

Section 3

April 25, 2010
Lindcove Research andExtension Center (LREC)TRIAL SUMMARY
Honey bee colonies were situated within a large area of treated commercial citrus. Within the area,
there were multiple citrus varieties, some weeds, but no other commercial crops. The bees wereallowed to forage freely. Nectar was collected by hand from trees within several different blocks within
the perceived foraging area. In addition, nectar was extracted from the stomachs of honey bees foraging
within the citrus region so that a comparison could be made between hand-collected and honey beecollected nectar. Whereas in the tunnel study, hand-collected and honey bee-collected sources were
controlled, the foraging range of the honey bees was not controlled. At the end of the 3-day foraging
period, nectar was sampled from new comb that had been placed within each hive at the start of thetrial. Pollen traps were also included in the hives and these were operated for a 24-hour period prior tothe completion of the experiment.
The Italian honey bee Apis mellifera ligustica was also used in this study. Five large colonies consisting of
10 frames were situated in the citrus area on the evening of April 22. The bees were confined within thehives until the morning of April 23 when the bees were released to begin foraging. The larger hives used
for the open field study were equipped with pollen traps that were set into operation for 24 hours
beginning on April 24, 2010 and concluding on April 25, 2010.
Hand-collect nectar, bee-collected nectar, and stored nectar were collected in the same way as in thetunnel trial. Pollen samples were also collected from pollen traps within a 24-hour collection periodprior to the completion of the experiment. Pollen samples from only two hives were available forresidue analysis. It was found that the pollen load in each trap was very low given the 24-hour collection
period.
Pollen sources were not distinguished for residue analysis. Upon visual examination, there were clearlyseveral pollen sources within the traps indicating that the bees were not foraging exclusively on citrus
flowers.
METHODAPPLICATION TIMING AND RATESAll citrus groves in a radius of 1-2 miles on and around the LREC were treated for aphids withimidacloprid in the fall of 2009. A very large contiguous area of citrus had been treated in 2009 withvarious imidacloprid commercial formulations at half the maximum field rate (0.25 lb. a.i. per acre
regardless of the commercial formulations used). A large block of citrus was selected in the center ofsuch treatments for this honey bee study with the knowledge that bees can forage very long distancesfrom their hives. Although there were other blooming plants in the vicinity of this block that honey bees

7655

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 49090504

CDPR IMI Citrus (2011)

could have foraged from, there were no nearby agricultural crops other than citrus. All nearby citrus
within 1 mile had been treated with the half rate of imidacloprid the previous fall.
STUDY SITE LOCATION AND CHARACTERISTICS

The Lindcove Research and Extension Center (LREC) contains much of the collection of importantrootstock and scions for the Citrus Clonal Protection Program (CCPP) and research projects for the Stateof California. In 2009, a higher than normal number of trees in the region became infected with Citrus
tristeza virus (CTV). CTV is a pathogen vectored by several species of aphids. The LREC has soils
characterized as loam soils (15-25% clay, 0.5-1% organic matter).
RESULTS:In this trial, the mean residues of total imidacloprid in hand collected citrus nectar ranged from 1.819.42 ppb while the mean residues in nectar extracted from foraging honey bees ranged from 1.11-7.59
ppb. Also, the mean residues of total imidacloprid in hive stored nectar ranged from 6.95-11.63 ppb.
Moreover, the maximum mean residues for hive pollen were found to be 10.2 ppb while the average
residue measurement was 9.39 ppb.
There appears to be large variations in the level of residues among citrus varieties. Due to the limited
samples and information provided, it is unknown if this variation was due to citrus varieties or otherfactors, including age of the trees or level or irrigation.
Table 2. Summary of imidacloprid residues in hand collected nectar randomly selected within six blocks
of citrus surrounding the location of five honey bee hives. Commercial citrus trees were treated in theFall of 2009 at half (½X) the labelled rate.

1
2

Lindcove Research and Extension Center (LREC)
Spring 2010Loam Soil [15-25% clay, 0.5 – 1% organic matter]
Mean Residue Concentration (ppb)(minimum – maximum)
Hand Collected Nectar
Citrus Variety
N1
IMI
5-OH
Olefin
10
<1.0
<1.0
<1.0
Valencia Oranges – South East(<1.0 – 1.18)
5
2.73
<1.0
<1.0
Valencia Oranges – South West(1.53 – 3.55)(<1.0 – 2.22)
4
1.79
<1.0
<1.0
Navel Oranges – North East(<1.0 – 2.48)(1.02 – 1.95)
4
3.51
<1.0
<1.0
Navel Oranges – South East
(2.03 – 6.15)(<1.0 – 1.73)
10
6.82
<1.0
1.99
Tangerines(2.84 – 13.26)(<1.0 – 3.67)
5
2.76
<1.0
<1.0
Young Oranges(1.34 – 4.21)(<1.0 – 1.11)
3.29
Means
<1.0
1.19
(<1.0 – 6.82)“N” is the total number of samples collected“Total IMI” combines magnitude of residues of imidacloprid plus degradates IMI-olefin and IMI-5-OH

8656

Total IMI2
1.81
(1.15 – 2.48)
5.18(3.17 – 6.21)
3.51(2.11 – 4.42)
4.87(2.64 – 8.42)
9.42(4.03 – 18.28)
3.73(1.76 – 5.88)
4.75(1.81 – 9.42)

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 49090504

CDPR IMI Citrus (2011)

Table 3. Summary of imidacloprid residues in nectar collected from free-ranging honey bees incommercial citrus trees treated in the fall of 2009 at half the labelled rate.
Lindcove Research and Extension Center (LREC)
Spring 2010Loam Soil [15-25% clay, 0.5 – 1% organic matter]
Mean Residue Concentration (ppb)(minimum – maximum)

Hive

1
2
3
4
5

1
2

Mean for
Hive 1-5

Bee Collected Nectar
N1 = varies[mean of up to
20 samples]
Sampling occurred 4/23/2010

IMI
1.05
(<1.0 – 2.43)
3.77(<1.0 – 9.31)
1.94(<1.0 – 7.56)<1.0(<1.0 – 2.69)
1.94(<1.0 – 4.2)
1.88(<1.0 – 3.77)

Total IMI2
2.11(<1.0 – 5.07)
7.59(1.16 – 16.02)
3.59(<1.0 – 12.16)
1.11(<1.0 – 3.71)
3.23(<1.0 – 7.29)
3.53(1.11 – 7.59)

Uncapped Stored Nectar
N1 = varies[mean of up to
10 samples]
Sampling occurred 4/25/2010

Total IMI2
11.63
(9.31 – 15.53)

IMI
6.25
(4.73 – 8.67)

3.23(2.47 – 4.98)
5.98(3.81 – 8.18)
5.15(3.23 – 6.25)

Hive Pollen[Each value represents asingle measurement for the
analysis of the entire pollen
retrieved from traps within
individual hives]
Sampling occurred
4/25/2010
IMI
Total IMI2
N/A

N/A

N/A

N/A

N/A
6.96
(4.47 – 9.25)
10.60(8.13 – 13.98)
9.72(6.96 – 11.63)

6.58

8.57

5.84

10.2

6.21

9.39

“N” is the total number of samples collected“Total IMI” combines magnitude of residues of imidacloprid plus degradates IMI-olefin and IMI-5-OH

CONCLUSION TO OPEN FIELD TRIAL
The second trial, also at the LREC, consisted of an open field area with multiple citrus varieties. A verylarge contiguous area of citrus had been treated in 2009 with various imidacloprid commercial
formulations at 0.25 lbs. a.i./acre (half the maximum label rate).
Five large 10-frame colonies were situated in the citrus orchard area. Pollen was successfully collectedfrom only two of five hives via pollen traps that were operated for 24 hours. Nectar was either handcollected from randomly selected citrus trees, extracted from honey stomachs of honey bees allowed toforage in the treated orchard and intercepted at the hive, and from the hive as uncapped honey. Nectar
sugar analysis was measured by refractometry.
In this trial, the mean residues of total imidacloprid in hand collected citrus nectar ranged from 1.819.42 ppb while the mean residues in nectar extracted from foraging honey bees ranged from 1.11-7.59
ppb. Also, the mean residues of total imidacloprid in hive stored nectar ranged from 6.95-11.63 ppb.
Moreover, the maximum mean residues for hive pollen were found to be 10.2 ppb while the average
residue measurement was 9.39 ppb.
9657

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 49090504

CDPR IMI Citrus (2011)

7C. TRIAL INFORMATION
Title:One Year Nectar Collections
Application Date: September 3 and 9, 2009

Sampling Date:

Section of Study:

Trial Location:

Section 4

Spring 2010
Lindcove Research andExtension Center (LREC)
and Bakersfield, CA

TRIAL SUMMARYAs research into the potential uses of imidacloprid for the management of Asian Citrus Psylliid(Diaphorina citri), different imidacloprid rates at several locations within the citrus producing areas ofsouthern and central California were evaluated. Nectar from trees treated with either the maximum
label rate (1X) or double the maximum label rate (2X) of imidacloprid (Admire Pro) were evaluated todetermine whether there was a relationship between application rates and residue levels. For thiscomponent of the trial, all data were derived from analysis of hand-collected nectar.
METHODAPPLICATION TIMING AND RATESThe treatments were applied by water can on September 3, 2009 and September 8, 2009 at the LREC
and Bakersfield sites, respectively. Citrus trees were treated with Admire Pro at the maximum label rate
(1X=14 fl oz/ acre) and double the maximum label rate (2X= 28 fl oz/acre) in the Fall of 2009. Residues ofimidacloprid, 5-hydroxy imidacloprid, and imidacloprid olefin were then analyzed from hand-collected
nectar samples.
STUDY SITE LOCATION AND CHARACTERISTICS
For this trial, Lindcove Research and Extension Center (LREC) and a commercial citrus farm in Bakersfield
were used to collect data from. The LREC trees were in the same block of navel oranges that were used
for the honey bee trial. At the commercial citrus farm in Bakersfield, a 3.9 acre block of Valencia oranges
that were planted in 1985 were provided. There were 100 trees per acre compared to the 134 trees
were acre at the LREC site. The LREC has soils classified as loam soil (15-25% clay, 0.5-1% organic
matter). Soil characteristics were not provided in the study for Bakersfield.
RESULTS:The 1X label rate treatments resulted in an average residue measurement of 19.30 ppb in nectar at the
Bakersfield grove and 16.30 ppb at the LREC. While the 2X label rate treatments resulted in an average
residue measurement of 47.36 ppb at the Bakersfield grove and 35.83 ppb at the LREC.
Residues of imidacloprid, 5-hydroxy Imidacloprid and imidacloprid olefin were higher in nectar sampledfrom the trees treated with the 2X label rate compared with the 1X label rate, with the ca. twofold
difference in residue levels reflecting the twofold difference in application rates. The imidacloprid and10658

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 49090504

CDPR IMI Citrus (2011)

total residues were lower at the LREC site. The most likely reason for this difference is the higher tree
density at the LREC site. Since the insecticide was applied on a per acre basis (1X=14 fl oz./acrea and
2X=28 fl oz./acre), the amount of active ingredient per tree would have been higher at the Bakersfieldsite and this could, therefore, account for the higher residues in the nectar.
Table 4. Summary of imidacloprid residues in hand collected nectar resulting from a soil treatment at
maximum label rate (1X) and twice maximum label rate (2X). Application made by watering can toindividual trees in Bakersfield on 9/8/2009 and LREC on 9/3/09.

Location
Citrus Variety
Bakersfield
Valencia Oranges(soil type unknown)
LREC
Navel Oranges[Loam Soil]
1
2

Rate
1X
2X
1X
2X

Spring 2010
Mean Residue Concentration (ppb)(minimum – maximum)
Hand Collected Nectar (N1 = 10)
IMI
5-OH
Olefin
12.13
2.53
4.64
(3.62 – 18.82)(1.10 – 3.61)(1.58 – 6.93)
31.10
5.67
10.60
(9.70 – 92.05)(1.88 – 15.95)(3.12 – 31.15)
9.51
3.09
3.70
(3.31 – 15.49)(1.18 – 5.39)(1.56 – 6.04)
22.23
6.70
6.89
(5.98 – 43.94)(2.25 – 11.74)(1.70 – 13.06)

Total IMI2
19.30
(6.30 – 29.10)
47.36(14.70 – 139.15)
16.30(6.81 – 26.45)
35.82(9.93 – 68.30)“N” is the total number of samples collected“Total IMI” combines magnitude of residues of imidacloprid plus degradates IMI-olefin and IMI-5-OH.

CONCLUSION TO ONE YEAR NECTAR COLLECTIONS
The third trial was conducted at the LREC and a commercial citrus farm in Bakersfield, CA. Navel oranges(LREC) and Valencia oranges (Bakersfield) were treated with either 1X (the maximum label rate) or 2X
the label rate in Fall 2009. Hand collected nectar was quantified in Spring 2010.
Residues of imidacloprid, 5-hydroxy Imidacloprid and imidacloprid olefin were higher in nectar sampledfrom the trees treated with the 2X label rate compared with the 1X label rate, with the ca. twofold
difference in residue levels reflecting the twofold difference in application rates

11659

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 49090504

CDPR IMI Citrus (2011)

7D. TRIAL INFORMATION
Title:
Citrus Nectar Collected from Field Sites Treated in Successive Years
Sampling
Application Years:
2008 & 2009
Spring 2010
Date:1) Hemet, California
2) Temecula, California
Section of Study:
Section 5
Trial Location:3) Lindcove Research andExtension Center (LREC)TRIAL SUMMARY
The objective of this trial was to determine if imidacloprid residues in nectar could persist and/or
accumulate in situations where the insecticide was used on the same trees in successive years. Residues
in nectar sampled from citrus trees that had been treated in 2 successive years were quantified. Sites
were chosen with different soil types for the sampling program. Imidacloprid uptake into trees can beaffected by soil type and this could potentially affect the residues in nectar. All analyses were conducted
on nectar that was extracted by hand.
METHODAPPLICATION TIMING AND RATES
Hemet. The first treatments with Admire Pro were applied in Fall 2008 and the second treatments wereapplied in Fall 2009. The intent was to evaluate both the 1X label rate and the 2X label rate in all
possible application sequences. In order to do this, the block was subdivided into four 2.5 acre plots sothat the following sequences of treatments could be established: 1X (Fall 2008) followed by 1X (Fall2009), 1X (Fall 20.08) followed by 2X (Fall 2009), 2X (Fall 2008) followed by 1X (Fall 2009), and 2X (Fall2008) followed by 2X (Fall 2009). Nectar Samples were taken from trees chosen at random from withineach plot.
Temecula. Six commercial orchards in Temecula, California that had been treated both years of 2008
and 2009 in an area-wide control program were used in this study. The sites consisted of five Grapefruitand one Valencia orange orchard, which were first treated between June 7 and June 16, 2008 and thentreated a second time between April 7 and May 27, 2009. The maximum label rate (1X) for Admire Pro
was used in both treatment years.
Lindcove Research and Extension Center. Five citrus blocks at the LREC had been treated withimidacloprid for two successive years (2008 and 2009). The sites consisted of four navel oranges and one
Valencia orange orchard, which were first treated on September 17 and September 18, 2008 and thentreated a second time between September 10 and September 16, 2009. The maximum label rate (1X) for
Admire Pro was used in both treatment years.
STUDY SITE LOCATION AND CHARACTERISTICS
Hemet. The trial was conducted in a 10 acre block of 50 year old Ruby Red Grapefruit trees on Troyer
Rootstock. Soil type was sandy loam and irrigation was scheduled weekly according to tensiometer
12660

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 49090504

CDPR IMI Citrus (2011)

measurements. Nectar samples were collected from treated trees at random within the commercial
grapefruit orchard.

Temecula. A total of 68.25 acres of Star Ruby Grapefruit and 1.65 acres of Valencia oranges were treatedwith imidacloprid for two successive years. The soil type at all six site locations is considered to be Sandy
Loam. At each site, two trees were randomly selected for hand-collected nectar sampling.
Lindcove Research and Extension Center. Five citrus blocks with 2 successive years of imidacloprid
applications were chosen from the LREC. These blocks were independent from the block used for thetunnel study. The soil type at all five site locations is considered to be Loam Soil. A total of 15.44 acres ofnavel oranges and 2.5 acres of Valencia oranges were treated with imidacloprid. Within each block, 2
trees were randomly selected for hand-collected nectar sampling.
RESULTS:
Imidacloprid nectar residues in Hemet were lowest in the 1X – 1X label rate treatment with an average
residue measurement of 23.84 ppb and highest in the 2X – 2X label rate treatment with an average
residue measurement of 58.66 ppb. Following the 1X – 1X label rate treatments in two successive years
at Temecula and LREC, an average residue measurement of the nectar residues were 5.15 ppb and 11.16
ppb, respectively. The maximum mean residues of 21.65 ppb were measured at LREC.
The imidacloprid and total residues measured in the nectar sampled from trees treated with the 1X-2X
label rate sequence were approximately 2-fold higher than those measured for the 1X- 1X label rate
treatments, suggesting that the residue levels reflected the rate of imidacloprid used in the most recent
application. Thus, residues for the 1X- 1X and 2X-1X rate sequences were not significantly different, anddata for the 1X-2X and 2X-2X rate sequences were not significantly different. While the means for the 2X
treatments exceeded the 1X measurements by more than twofold, the statistical analysis shows thatimidacloprid and its metabolites did not accumulate significantly from one year to the next.
Data from six commercial groves in Temecula that were treated in two successive years (2008 and 2009)
with the maximum label rate of imidacloprid (1X-1X) were completed by June each year. The highest
total residues (9.56 ppb and 15.53 ppb for the two trees sampled) were measured at Site 6, where theyoungest trees (seven years of age) used in the sampling program was located. The overall means for
the Temecula samples were lower than those measured for the Hemet site where the 1X rate wasapplied for two successive years.
Data from five blocks at the LREC that were treated in two successive years (2008 and 2009) with thelabel rate of imidacloprid (1X-1X) had higher residues than those measured at the Temecula site,
possibly reflecting the later timing of treatments. Despite the higher values at the LREC site, the residues
were still lower than those measured for trees at Hemet. The major difference between the LREC and
Hemet sites is the soil type and it seems that the lighter and sandier soil at Hemet allowed better uptakeof imidacloprid into the trees resulting in higher residues in nectar the following spring.

13661

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 49090504

CDPR IMI Citrus (2011)

Table 5. Summary of imidacloprid residues in hand collected nectar of Ruby Red grapefruit trees
resulting from two successive year soil treatments at maximum label rate (1X) and twice maximum labelrate (2X) in Fall 2008/Fall 2009.

Rate
1X – 1X
1X – 2X
2X – 1X
2X – 2X
1
2

Hemet Site
Spring 2010
[Sandy Loam]
Mean Residue Concentration (ppb)(minimum – maximum)
Hand Collected Nectar
IMI
5-OH
Olefin
16.07
5.05
2.72
(9.88 – 25.67)(3.62 – 6.43)(1.57 – 4.00)
35.13
9.70
6.04
(13.28 – 63.60)(6.70 – 12.33)(3.77 – 9.31)
14.50
4.78
2.44
(11.77 – 22.66)(3.63 – 7.49)(1.73 – 3.66)
41.41
10.78
6.47
(28.62 – 62.67)(7.14 – 13.56)(4.38 – 10.48)

N
10
9
7
7

Total IMI2
23.84
(15.57 – 35.47)
50.87(27.54 – 85.03)
21.73(17.62 – 33.81)
58.67(40.14 – 86.71)“N” is the total number of samples collected“Total IMI” combines magnitude of residues of imidacloprid plus degradates IMI-olefin and IMI-5-OH.

Table 6. Summary of imidacloprid residues in hand collected nectar sampled in Spring 2010 from citrus
trees receiving a soil treatment at maximum label rate (1X) in successive years 2008 and 2009.

1
2

Location

Rate

N1

Temecula

1X – 1X

11

LREC

1X – 1X

9

Spring 2010
Mean Residue Concentration (ppb)(minimum – maximum)
Sampling occurred 4/2010
Hand Collected Nectar
IMI
5-OH
Olefin
3.17
<1.0
1.48
(<1.0 – 10.60)(<1.0 – 1.18)(<1.0 – 3.76)
6.51
1.80
2.86
(<1.0 – 13.65)(<1.0 – 5.40)(<1.0 – 8.42)“N” is the total number of samples collected“Total IMI” combines magnitude of residues of imidacloprid plus degradates IMI-5-OH and IMI-olefin

Total IMI2
5.15(<1.0 – 15.54)
11.17(1.53 – 21.64)

CONCLUSION FOR FIELD SITES TREATED IN SUCCESSIVE YEARSThe fourth trial was conducted to determine if imidacloprid residues in nectar could accumulate fromyear to year following successive year applications at three different locations (Hemet, Temecula, andLREC). Hand collected nectar samples were obtained with either 1X or 2X soil applications were made in
two successive years (2008, 2009) prior to sampling during bloom 2010.
Imidacloprid residues at the Hemet site appear to be a function of the rate applied at the most recent
application only, with no evidence of carryover from previous years. The overall means for the Temecula
samples were lower than those measured for the Hemet site where the 1X rate was applied for two
successive years. Data from five blocks at the LREC that were treated in two successive years (2008 and2009) with the label rate of imidacloprid (1X-1X) had higher residues than those measured at theTemecula site, possibly reflecting the later timing of treatments. Despite the higher values at the LREC
site, the residues were still lower than those measured for trees at Hemet. The major difference
14662

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 49090504

CDPR IMI Citrus (2011)

between the LREC and Hemet sites is the soil type and it seems that the lighter and sandier soil at
Hemet allowed better uptake of imidacloprid into the trees resulting in higher residues in nectar thefollowing spring.
7E. TRIAL INFORMATION
Title:
Citrus Nectar Collection from Field Sites Treated in Successive Years
Sampling
Application Years:
2008, 2009 and 2010
Spring 2011
Date:1) Tulare County, California
2) Temecula, California
3) Lindcove Research andTrial
Appendix B
Extension Center (LREC)
Section of Study:
Location:(Supplemental)
4) Ventura County, California
5) University of California,
Riverside6) Hemet, California
TRIAL SUMMARY

The objective of this trial was to determine if residues of imidacloprid and its important metabolites
could persist and/or accumulate in nectar in situations where the insecticide was used on the same
trees in successive years. Also, because imidacloprid uptake into trees can be affected by soil type, sites
were chosen to reflect the variety of soil types where citrus is gown in California. In section 3 of thisData Evaluation Report, data and information was provided for several sites where citrus was grown insoils that ranged from sandy loam to loam. To expand on the previous trial, this study will include dataand information on heavier clay soils and lighter soils. Nectar was extracted from flowers by hand at allsites during bloom and imidacloprid, imidacloprid olefin and 5-hydroxy imidacloprid were quantified by
LC/MS/MS.
METHODAPPLICATION TIMING AND RATES
Tulare County. Five of the citrus sites had been treated with the full label rate of imidacloprid for at least
3 previous years. The sixth citrus site was treated with the full label rate of imidacloprid for 2 successive
years. Two composite nectar samples were collected from each grove and all nectar samples were handcollected.
Temecula Valley. Nectar samples were hand-collected from six groves where the trees have beentreated for three successive years with the full label rate of imidacloprid.
Lindcove Research and Extension Center. Nectar samples were hand-collected from five citrus blocks at
the LREC that had been treated with the full label rate of imidacloprid for three successive years(September 2008, 2009, and 2010).
Ventura County. Nectar samples were hand-collected from a lemon grove where the trees had beentreated with the full label rate of imidacloprid at different timings during the season. The treatment
15663

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 49090504

CDPR IMI Citrus (2011)

timings were in May, July and September of 2010. The subject grove had not been treated in 2009. Two
composite nectar samples were collected from trees treated at application timing 1 and 3, while one
composite nectar sample was collected from trees treated at application 2.
University of California, Riverside. In October of 2010, nectar samples were collected from a citrus block
where the trees had been treated with the full label rate of imidacloprid. Sixteen composite samples
were collected from the trees.
Hemet. In the previous trial titled “Citrus Nectar Collected from Field Sites Treated in Successive Years”(Section 5 of study report), data was provided for a grapefruit grove that had been treated in successive
years (Fall 2008 nad Fall 2009) with different combinations of application rates. The initial trial beganwith either a 1X label rate of imidacloprid (Admire Pro at 14 fl oz/ acre) or a 2X label rate (Admire Pro at
28 fl oz/ acrea). In 2009, the same trees were treateed to give different treatment rate scenarios. To
expand on the subject trial, an additional year of treatment was provided to the trees in 2010.
STUDY SITE LOCATION AND CHARACTERISTICS
Table 7. Site location with information regarding soil type and treated commodity.
SITE AND COMMODITY INFORMATIONTEMELCULA
SOIL TYPECOMMODITY1FALLBROCK ROCKY SANDY LOAM
STAR RUBY GRAPEFRUIT
2FALLBROCK ROCKY SANDY LOAM
STAR RUBY GRAPEFRUIT
3
FALLBROCK ROCKY SANDY LOAM
VALENCIA ORANGE4FALLBROCK ROCKY SANDY LOAM
STAR RUBY GRAPEFRUIT
5
FALLBROCK ROCKY SANDY LOAM
STAR RUBY GRAPEFRUIT
6
GREENFIELD SANDY LOAM
STAR RUBY GRAPEFRUITTULARE COUNTY1PORTERVILLE CLAYTANGELOS2
CENTERVILLE CLAY
NAVEL ORANGES3
PORTERVILLE CLAY
NAVEL ORANGES
4PORTERVILLE CLAY
NAVEL ORANGES
5
PORTERVILLE CLAY
NAVEL ORANGES6PORTERVILLE CLAY
VALENCIA ORANGESOTHER SITES
LINDCOVE RESEARCH AND EXTENSIONSAN JOAQUIN LOAM
NAVEL AND VALENCIA ORANGESCENTER
UNIVERSITY OF CALIFORNIA, RIVERSIDEARLINGTON LOAM
VALENCIA ORANGESVENTURA COUNTYMOCHO LOAMLEMONRESULTS:
Total mean residues in nectar of imidacloprid from trees treated three years in a row at the Temeculasite ranged from 1.02 to 5.91 ppb with an average residue measurement of 2.57 ppb. Mean nectar
residues from Tulare County ranged from 0.29 to 4.21 ppb with an average residue measurement of
1.54 ppb. Total mean nectar residues from trees at the LREC with a loam soil ranged from 0.52 to 23.95
ppb with an average residue measurement of 4.55 ppb. Total mean nectar residues from the UCR site
16664

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 49090504

CDPR IMI Citrus (2011)

with a loam soil ranged from 0.83 to 13.88 ppb with an average residue measurement of 3.68 ppb.
Imidacloprid nectar residues from Ventura County sites treated at various times during 2010 were allless than 1 ppb regardless of the application timing. Following three years of treatments at the Hemetsite at various application rates, the average residue measurement in nectar for the 1X-1X-1X treatment
regimen was 24.40 ppb. The average residue measurement following the third year of treatment forsites treated once per year with 2X the label rate (2X-2X-2X) was 44.81 ppb. For the 1X-2X-0X and 2X-1X0X treatments the average residue measurement was 31.67 and 32.97 ppb. The residues following three
years of applications at 2X the maximum label rate were slightly lower than residues after just two
years.
Table 8. Summary of imidacloprid residues in hand collected nectar sampled in Spring 2011 from citrus
trees receiving a soil treatment at maximum label rate (1X) in successive years 2008, 2009, and 2010.

1
2

Location

Rate

Temecula

1X – 1X – 1X

LREC

1X – 1X – 1X

Spring 2011
Mean Residue Concentration (ppb)(minimum – maximum)
Hand Collected Nectar
1
N
IMI
5-OH
Olefin
1.68
<1.0
12
<1.0
(<1.0 – 3.48)(<1.0 – 1.62)
3.50
<1.0
<1.0
10
(<1.0 – 16.87)(<1.0 – 3.88)(<1.0 – 3.20)

Total IMI2
2.58
(1.01 – 5.91)
4.95(<1.0 – 23.95)“N” is the total number of samples collected“Total IMI” combines magnitude of residues of imidacloprid plus degradates IMI-olefin and IMI-5-OH

Table 9. Supplement to Table E-10. Summary of imidacloprid residues in hand collected nectar
resulting from successive year soil treatments at maximum label rate (1X), twice maximum label rate(2X), and no application (0X) in Fall 2008/Fall 2009/Fall 2010.
Hemet Site
Spring 2011
[Sandy Loam]
Mean Residue Concentration (ppb)(minimum – maximum)Rate
1X – 1X – 1X

IMI
19.68

Total IMI2
24.40

(2.02 – 4.31)(<1.0 – 4.96)(17.56 – 39.86)(23.05 – 30.00)(3.35 – 4.37)(1.44 – 1.92)(28.97 – 36.27)(20.01 – 33.45)(2.28 – 5.43)(1.51 – 3.11)(25.12 – 41.99)(26.67 – 41.92)(3.86 – 5.76)(2.27 – 3.16)
1“N” is the total number of samples collected2“Total IMI” combines magnitude of residues of imidacloprid plus degradates IMI-olefin and IMI-5-OH(32.80 – 50.35)

1X – 2X – 0X
2X – 1X – 0X
2X – 2X – 2X

(12.26 – 33.82)

Hand Collected Nectar (N1 = 10)
5-OH
Olefin
3.13
1.59

26.01
27.02
36.86

3.93
3.87
5.18

17
665

1.73
2.08
2.77

31.67
32.97
44.81

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 49090504

CDPR IMI Citrus (2011)

Table 10. Information on trees used for hand collected nectar collected from six commercial orchards in
Tulare County during bloom in Spring 2011 after three successive years of imidacloprid application forcontrol of glassy-winged sharpshooter 2008, 2009, 2010.
Tulare County[Porterville Clay – 40% clay]
Spring 2011
2008
2009
2010
Treatment
Treatment
Treatment
Site
Citrus Variety
Date
Date
Date
1
TangelosNot treated
6/16/2009
5/21/20102
Navel Oranges
5/22/2008
6/29/2009
6/18/20103
Navel Oranges
7/8/2008
6/18/2009
6/9/2010
4
Navel Oranges
5/17/2008
6/18/2009
6/6/2010
5
Navel Oranges
5/21/2008
6/29/2009
6/15/2010
6
Valencia Oranges
7/3/2008
6/26/2009
6/16/2010
Table 11. Information on trees used for hand collected nectar collections during bloom in Spring 2011
after three successive years of imidacloprid application at the label rate 2008, 2009, 2010.

Site
1
2
3
4
5
6

Site
1
2
3
4
5

Citrus Variety
Star Ruby Grapefruit
Star Ruby Grapefruit
Valencia Orange
Star Ruby Grapefruit
Star Ruby Grapefruit
Star Ruby Grapefruit

Citrus Variety
Atwood Navel
Atwood Navel
Caracara Navel
Parent NavelRed Valencia

Temecula[Sandy Loam]
Spring 20112008
TreatmentDate
6/7/2008
6/9/2008
6/6/2008
6/9/2008
6/7/2008
6/16/2008
LREC[Loam Soil]
Spring 20112008
Treatment
Date
9/18/2008
9/17/2008
9/18/2008
9/18/2008
9/17/2008

18
666

2009
Treatment
Date
5/27/2009
4/13/2009
4/7/2009
4/21/2009
4/10/2009
4/17/20092010
Treatment
Date
5/22/10
6/11/10
6/12/10
5/17/10
6/2/105/14/102009
Treatment
Date
9/10/2009
9/10/2009
9/16/2009
9/16/2009
9/14/20092010
Treatment
Date
9/13/2010
9/10/2010
9/09/2010
9/09/2010
9/13/2010

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 49090504

CDPR IMI Citrus (2011)

Table 12. Summary of imidacloprid residues in hand collected nectar sampled in Spring 2011 from citrus
trees receiving a soil treatment at maximum labelled rate as part of the area side control program forglassy-winged sharpshooter in successive years 2008, 2009 and 2010.
Spring 2011
Mean Residue Concentration (ppb)(minimum – maximum)

Location

N1

IMI
1.29

1X3 – 1X – 1X

12(<1.0 – 3.31)“N” is the total number of samples collected2“Total IMI” combines magnitude of residues of imidacloprid plus IMI-olefin and IMI-5-OH
3
One site did not receive an application in 2008.
1

Tulare County

Rate

Hand Collected Nectar
4/5-OH
Olefin<1.0<1.0

Total IMI2
1.70(<1.0 – 4.21)

CONCLUSION
In 2011, the researchers followed up with a third year of treatments at the Temecula, LREC, and Hemet
sampling sites. Nectar was again collected from six groves previously treated in Temecula where the soil
type is sandy loam and five citrus blocks at the LREC with a loam soil (20% clay).
In addition, sites were added at University of California, Riverside (UCR), Ventura County and Tulare
County to address different soil types not previously represented. Nectar was collected from six citrus
groves in Tulare County grown in Porterville clay (clay content 40%). Of these six sites, five had beentreated with 1X imidacloprid for the past three years and the remaining site was treated similarly thepast two years. A new site in Ventura County with a soil consisting of 23% clay/35% sand was sampled
following applications at the full label rate of imidacloprid at different times during the season. The
treatment timings for this site were May, July and September 2010 and untreated in 2009. Also, a citrus
block from the farm at UCR, where the soil type is loam, was treated in October 2010 and sampled in
2011.
In Temecula and Tulare County, the 2010 treatments at the 1X label rate were made in mid-May to midJune. The LREC and UCR 2010 treatments at 1X label rate were made in September and October.
Treatments at the four Hemet sites were 1X-1X-1X, 1X-2X-0X, 2X-1X-0X, or 2X-2X-2X representing years
2008, 2009 and 2010, where 1X-1X-1X represents a single application at the maximum label rate per
year for three consecutive years.
9.

STUDY VALIDITY/CLASSIFICATION AND STUDY LIMITATIONSClassification/Utility for Bee Risk Assessment. This study is classified as supplemental for use in risk
assessment due to no pollen data available and a potential underestimation of a worst case scenario
due to current labels not restricting pre bloom and during bloom applications whereas this study done
post bloom. The study results characterize expected imidacloprid residues in citrus nectar fromapplications in various soils ranging from fine to coarse and following different application rates andapplication timing in California. These results may not extrapolate directly to expected results in otherregions of the U.S.

19
667

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 49090504

CDPR IMI Citrus (2011)

Concentrations in nectar extracted from the stomachs of free-ranging bees (open field study) were
somewhat lower than samples collected directly from flowers of nearby trees. This may reflect a"dilution effect" from bees foraging on other (untreated) flower types. The few pollen samples obtained
during the open field study had imidacloprid concentrations roughly equal to the nectar sampled fromthe same hives. Concentrations in flower nectar samples appear to be linearly related to the application
rate, based on ca. twofold increases in residue levels with doubling the application rate in the Hemet
trials.
Temporal Variability in Residues. Nectar samples were obtained from two locations (citrus blocks in the
Temecula region and at LREC) where the 1X soil application rate of imidacloprid had been made in two
successive years (2008, 2009) prior to sampling in April 2010. Residue levels at these 11 sites averaged
8 ppb and ranged from 1 to 18 ppb. The application timing (May, July, September, October) appears to
be an important factor in determining residue levels in flower nectar the following year particularly forsites planted to coarse soils which consistently yielded the higher imidacloprid residues. Fall (Sept)applications resulted in about twofold higher residue concentrations than spring (April-June)applications.
Spatial Variability in Residues. The six locations for the citrus trials were in relatively close proximity.
Soil types reflect sandy loam, loam or clay compositions (20-40% clay) and low organic carbon content(0.35-1.9%). Weather conditions (temperature and precipitation) were similar across the three trials. As
a result of the close proximity of trial sites, this study provides very limited information on how
differences in environmental conditions across different areas of the U.S. may affect accumulation of
total imidacloprid in pollen and nectar.
Pesticide Carryover. The authors speculated that imidacloprid residues at the Hemet site appear to be afunction of the rate applied at the most recent application only, with no evidence of carryover fromprevious years. However, following the third year of application at the Hemet site, residues were higher
at the two sites receiving no treatment in 2010 than at the site treated all three years with 1X. This
indicates some degree of carryover from previous application years, at least for sites treated with the 2X
rate during one of the two years prior to the no treatment year. This was the only site where samples
were collected following a year without treatment.

20
668

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 49662101

CDPR IMI Apple DER

Reference
Miller, A., and Jerkins, E. (2016) Determination of the Residues of Imidacloprid and its Metabolites 5Hydroxy Imidacloprid and Imidacloprid Olefin in Bee Relevant Matrices Collected from Apple Trees
following Soil and Foliar Applications of Imidacloprid over Two Successive Years: Final Report. Project
Number: EBNTN014. Unpublished study prepared by Bayer Cropscience LP. 406. MRID 49662101,
CDPR Study ID 289057, Data Volume 51950-0901, Tracking ID# 273842
1. STUDY INFORMATION
Chemical:Imidacloprid
PC Code129099Admire Pro Systemic
Percent Active
42.8%
Test Material:
Protectant
Ingredient:
Field residue study on apple orchards to measure the residues of imidacloprid andStudy Type:metabolite levels in nectar, pollen and on leaves following one soil and two foliar
applications.
Bayer CropScience
2T.W. Alexander Drive
Experiment Start andAugust 1, 2013 – July
Sponsor:Research Triangle Park, NC
End Date:
22, 2015
USA 27709
Sponsor StudyEBNTN014
ID:
Nine Apple Orchard
Study Locations:field trials located in
Study
California.
Completion
January 13, 2016
Date:
GLP; protocol reviewed by CDPR.
GLP Status:[CDPR Study ID 289057, Data Volume 51950-0901, Tracking ID# 273842]
2. REVIEWER INFORMATION
Study Reviewed by:Richard Bireley, Sr. Environmental Scientist (Specialist)
California Department
John Troiano, Ph.D., Research Scientist III
of Pesticide Regulation
Alexander Kolosovich, Environmental Scientist
Brigitte Tafarella, Environmental Scientist
Denise Alder, Sr. Environmental Scientist (Specialist)
Russell Darling, Environmental Scientist

3. EXECUTIVE SUMMARY
A total of nine field trials were conducted to measure the magnitude of imidacloprid residues in apple
nectar, pollen and leaves following one soil and two foliar applications of Admire Pro® SystemicProtectant in each of two successive years. Admire Pro Systemic Protectant is a suspension concentrate
formulation containing 550 g/L imidacloprid. Applications were made in the fall of 2013 and 2014, postbloom.

669

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 49662101

CDPR IMI Apple DER

Across both years, individual soil application rates ranged from 0.38 to 0.39 lb. imidacloprid/acre. Theinterval between the soil application and first foliar application was 3 to 5 days. For all foliar
applications, individual rates ranged from 0.059 to 0.064 lb. imidacloprid/acre. The interval between
first and second foliar application was 8 to 10 days. Application volumes ranged from 13,000 to 15,200
gal/acre (GPA) for the soil applications and from 55 to 75 GPA for the foliar applications. Total seasonal
application rates ranged from 0.50 to 0.52 lb. imidacloprid/acre.
In 2013, trials NT031-13ZA and NT036-13ZA made applications prior to apple harvest, while the other
trials made all applications post-harvest. Soil applications were made at BBCH growth stages 79 to 99,
while the two foliar applications were made at BBCH growth stages 81 to 99 and 85 to 99, respectively.
In 2014, all applications were made prior to apple harvest. Soil applications were targeted for 21 days
prior to apple harvest and made at BBCH growth stages 75 to 89. The two foliar applications weretargeted such that the last would occur 7 days prior to harvest, with sprays made at BBCH growth stages
65 to 85 and 67 to 89, respectively.
All applications were made using ground-based equipment. The adjuvant Dyne-Amic (0.25 % v/v) wasused in all foliar applications.
Apple flower (also called blossom) and leaf samples were collected once in the spring of 2014, following
the fall 2013 applications, and once in the spring of 2015, following the fall 2014 applications. At eachsampling interval, two composite samples of apple flowers (to be hand-processed to obtain apple nectar
and pollen) and apple leaves were collected by hand when the apple trees were at bloom.
Single composite samples of apple flowers and leaves were collected from the control plot of each trial
on the same days that samples were collected from the treated plots.
After their collection, apple flowers were hand-processed at the field site to obtain the bee relevant
matrices of apple nectar and pollen. The processed flowers were discarded.
The residues of Admire Pro Systemic Protectant (imidacloprid, 5-hydroxy imidacloprid, and imidacloprid
olefin) were quantitated by high performance liquid chromatography/triple stage quadrupole mass
spectrometry (LC/MS/MS) and LC/high resolution mass spectrometry (LC/HRMS) using stable
isotopically labeled internal standards. The individual analyte residues were summed to give a total
imidacloprid residue.
4. STUDY VALIDITY
Guideline Followed:
Guideline Deviations:
Other Deviations:Classification:
Rationale:
Reparability:

See Section 7; Protocol was reviewed and accepted by CDPR
N/A
N/A
Acceptable
N/A
N/A

670

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49662101

CDPR IMI Apple DER

5. MATERIALS

Test item:
pH (20°):CAS #:

Test Material Characterization
Admire Pro Systemic
Percent A.I.:
Protectant
7.8
Density (20°C):
138261-41-3
Solubility:

42.8% A.I.
1.41 to 1.54 g/mL
0.51 to 0.61 g/L

5A. STUDY DESIGNThis study requirement was part of the Neonicotinoid Reevaluation at the California Department of
Pesticide Regulation (CDPR). The study design and protocol were approved by the CDPR prior to study
initiation. This study was conducted using GLP standards and following an approved protocol. The study
initiation date was August 01, 2013. The experimental start date was August 30, 2013 (first application),and the experimental end date was July 22, 2015 (last analysis).
Nine trials were conducted for this study, representing each of the three soil texture categories (fine,medium, and coarse) throughout multiple locations in California. Each trial includes one untreatedcontrol plot and one TRTD plot to be treated in two consecutive years. Apple varieties in this study
represented those commonly grown in the area and agronomic practices typical for commercial
production of apples were used at all trial locations.
Homogenization and analysis of the leaf, nectar, and pollen samples from this study were performed byBayer CropScience in Research Triangle Park (RTP), NC. Final report preparation was performed byCritical Path Services, LLC, located in RTP, NC.
5B. APPLICATION TIMING AND RATESApplications were made in the fall of 2013 and 2014, post-bloom. Across both years, individual soil
application rates ranged from 0.38 to 0.39 lb. imidacloprid/acre. The interval between the soil
application and first foliar application was 3 to 5 days. For all foliar applications, individual rates ranged
from 0.059 to 0.064 lb. imidacloprid/acre. The interval between first and second foliar applications was
8 to 10 days. Application volumes ranged from 13,000 to 15,200 gal/acre (GPA) for the soil applications
and from 55 to 75 GPA for the foliar applications. Total seasonal application rates ranged from 0.50 to
0.52 lb. imidacloprid/acre.
In 2013, trials NT031-13ZA and NT036-13ZA made applications prior to apple harvest, while the other
trials made all applications post-harvest. Soil applications were made at BBCH growth stages 79 to 99(BBCH 79: fruit about 90% final size; BBCH 99: harvested product), while the two foliar applications weremade at BBCH growth stages 81 to 99 (BBCH 81: beginning of ripening, first appearance of cultivarspecific color) and 85 to 99 (BBCH 85: advanced ripening, increase in intensity of cultivar-specific color),
respectively. In 2014, all applications were made prior to apple harvest. Soil applications were targetedfor 21 days prior to apple harvest and made at BBCH growth stages 75 to 89 (BBCH 75: fruit about half
final size; BBCH 89: fruit ripe for consumption, fruit have typical taste and firmness). The two foliar
applications were targeted such that the last would occur 7 days prior to harvest, with sprays made at
BBCH growth stages 65 to 85 (BBCH 65: full flowering, at least 50% of flowers open, first petals falling)and 67 to 89 (BBCH 67: flowers fading, majority of petals fallen), respectively.
671

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49662101

CDPR IMI Apple DER

All applications were made using ground-based equipment. The adjuvant Dyne-Amic (0.25 % v/v) wasused in all foliar applications.
Table 1. Application and Location Information
Location(City,
State,NAFTA
Application
Trial
Region)Year
Identification

2013
NT028-13ZA

Clarksburg,
California
Region 10
2014

2013
NT029-13ZA

Stockton,
California
Region 10
2014

2013
NT030-13ZA

Linden,California
Region 10
2014

MethodTiming
BBCHa

Rate, lb.
a.i./Acre

Drip
Application
Airblast
Application
Airblast
Application
Drip
Application
Airblast
Application
Airblast
Application
Drip
Application
Airblast
Application
Airblast
Application
Drip
Application
Airblast
Application
Airblast
Application
Drip
Application
Airblast
Application
Airblast
Application
Drip
Application
Airblast
Application

99

0.381

99

0.059

99

0.060

76

0.381

81

0.060

81

0.060

99

0.381

99

0.060

99

0.060

NRb

0.379

81

0.061

81

0.060

99

0.380

99

0.060

99

0.060

77

0.379

81

0.061

672

Total Rate,lb. a.i./Acre

0.50

0.50

0.50

0.50

0.50

0.50

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49662101

Trial
Identification

CDPR IMI Apple DER
Location(City,
State,
NAFTA
Region)Application
Year

2013
NT031-13ZA

Clarksburg,
California
Region 10
2014

2013
NT032-13ZA

Linden,California
Region 10
2014

2013
NT033-13ZA

Linden,California
Region 10
2014

MethodTiming
BBCHa

Rate, lb.
a.i./Acre

Airblast
Application

81

0.060

Drip
Application
Airblast
Application
Airblast
Application
Drip
Application
Airblast
Application
Airblast
Application
Drip
Application
Airblast
Application
Airblast
Application
Drip
Application
Airblast
Application
Airblast
Application
Drip
Application
Airblast
Application
Airblast
Application
Drip
Application
Airblast
Application
Airblast
Application

79

0.381

81

0.060

85

0.060

78

0.381

81

0.060

81

0.060

99

0.381

99

0.060

99

0.060

75

0.381

81

0.060

81

0.060

99

0.380

99

0.060

99

0.060

76

0.381

81

0.061

81

0.060

673

Total Rate,lb. a.i./Acre

0.50

0.50

0.50

0.50

0.50

0.50

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49662101

Trial
Identification

CDPR IMI Apple DER
Location(City,
State,
NAFTA
Region)Application
Year

Method2013

Timing
BBCHa

Rate, lb.
a.i./Acre

Total Rate,lb. a.i./Acre

Drip
99
0.391
Application
Airblast
99
0.063
0.52
Application
Airblast
99
0.063
NT034-13ZA
Madera,Application
California
Drip
NR
0.391
Region 10
Application
2014
0.52
Airblast
65
0.062
Application
Airblast
67
0.063
Application
Drip
99
0.377
Application
NT035-13ZAc
Madera,2013
0.50
Airblast
99
0.063
California
Application
Region 10
Airblast
99
0.063
Application
Drip
87
0.380
Application
2013
0.50
Airblast
91
0.061
Application
Airblast
91
0.059
NT036-13ZA
Sanger,
Application
California
Drip
89
0.380
Region 10
Application
2014
0.50
Airblast
85
0.059
Application
Airblast
89
0.059
ApplicationaTypical commercial apple harvest generally occurs between BBCH 87 and 89. In 2013, trials NT03113ZA and NT036-13ZA made applications prior to apple harvest, and all other trials applied post-harvest(BBCH 99). In 2014, all applications were made prior to apple harvest.
b
NR = Not reported; the BBCH growth stage at this application was not reported by the PFI.
c
Trial NT035-13ZA was not able to complete the second year of sampling, so only one year of test
substance application data are reported.
5C. STUDY SITE LOCATION AND CHARACTERISTICS

674

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49662101

CDPR IMI Apple DER

A variety of soil types were included in the study design. Bayer CropScience conducted the study inthree soil texture types, fine, medium, and coarse, based on Soil Survey Geographic (SSURGO) Databasemapping units. There are nine trial sites in this apple study design: three in fine texture soils, one inmedium, and five in coarse. Two years of data for each site are presented in this report with the
exception of trial NT035-13ZA (coarse soil), which could not complete year 2 because the apple trees
were removed from the plot, so only first year data are reported from this trial.
Table 2. Trial Site Conditions for Apple Orchard
Trial (Field)
Trial
OM pH CEC%%
Identification
Location(%)(meq/100g Sand Silt
(County,soil)
State)
NT028-13ZA
Clarksburg, 6.1 6.5
26.9
16
30
California
NT029-13ZA
Stockton,
2.8 7.4
28.2
22
38
California
NT030-13ZA
Linden,2.5 7.1
22.8
48
24
California
NT031-13ZA
NT032-13ZA
NT033-13ZA
NT034-13ZA
NT035-13ZA
NT036-13ZA

Clarksburg, 4.7 6.7
California
Linden,5.0 7.0
California
Linden,1.6 6.9
California
Madera,1.3 7.0
California
Madera,0.88 7.1
California
Sanger,0.83 6.1
California%
Clay

Soil Types

Rainfall Temperature(in)Range (°F)

54

Clay (Fine)

23.0

28 - 89

40

Clay Loam(Fine)
Sandy Clay
Loam
(Coarse)Clay (Fine)

17.6

29 - 92

17.6

29 - 92

23.0

28 - 89

Clay Loam(Medium)
Sandy Loam
(Coarse)Sandy Loam
(Coarse)Sandy Loam
(Coarse)Sandy Loam
(Coarse)17.6

29 - 92

17.6

29 - 92

10.3

31 - 97

3.6

28 - 77

10.3

31 - 97

28

26.2

12

30

58

21.6

26

40

34

11.6

68

20

12

8.0

68

22

10

8.4

68

20

12

6.1

67

26

7

5D. SAMPLE COLLECTION, HANDLING, PROCESSINGApple flower (also called blossom) and leaf samples were collected once in the spring of 2014, following
the fall 2013 applications, and once in the spring of 2015, following the fall 2014 applications. The
exception is trial NT035-13ZA, in which the year 2 (2015) sample collection was cancelled because theapple trees were removed from the trial field. Each TRTD plot was divided into two subplots. At eachsampling interval, two composite samples (one from each subplot) of apple flowers (to be handprocessed to obtain apple nectar and pollen) and apple leaves were collected by hand when the apple
trees were at bloom, BBCH 65 to 69 (BBCH 69: end of flowering, all petals fallen). Exceptions are the leaf
samples collected in 2014 from trials NT034-13ZA and NT035-13ZA and in 2015 from trial NT036-13ZA,
which were collected at BBCH 71 (BBCH 71: fruit size up to 10 mm, fruit fall after flowering). In 2014,
apple flower samples were collected at 138 to 193 days after the last application (DAA), and apple leaf
samples were collected at 151 to 214 DAA. In 2015, apple flower samples were collected at 131 to 287
DAA, and apple leaf samples were collected at 147 to 293 DAA.

675

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49662101

CDPR IMI Apple DER

Single composite samples of apple flowers and leaves were collected from the control plot of each trial
on the same days that samples were collected from the treated plots.
Apple flowers and leaves were collected by hand into Ziplock bags from all four quadrants (high, low,
inside, and outside) of the trees in the subplot (UTC plot: 6 to 24 trees; TRTD subplots: 8 to 15 trees).
Each composite flower sample contained a minimum of 125 g, and each composite leaf sample
contained a minimum of 100 g.
After their collection, apple flowers were hand-processed at the field site to obtain the bee relevant
matrices of apple nectar and pollen. Nectar processing began the same day as flower collection. Nectar
from the floral nectary was removed by a micropipette and placed into a pre-weighed glass collection
vial. The blossoms were then allowed to dry overnight at room temperature to desiccate the pollen. The
next day, pollen was removed from the apple blossoms either by vacuum aspiration with collection in
filter tips or by tapping the pollen from the blossoms onto wax paper and collection of the accumulated
pollen into a vial. All resulting nectar and pollen samples were labeled and placed in the freezer
immediately after they were generated. After processing was completed, the flowers were discarded.
Composite samples of apple nectar, pollen, and leaves were placed into labeled (study number andsample number) containers for shipment. All leaf, nectar, and pollen samples were placed in frozen
storage within 6 hours and 10 minutes of collection.
Sample Storage.
Upon arrival at Bayer CropScience, all leaf, nectar, and pollen samples were immediately transferred tofrozen storage. The leaf samples were homogenized with dry ice using a Robot Coupe chopper andwere returned to frozen storage immediately following homogenization. Pollen and nectar were usedwithout further processing. All samples remained frozen at all times except during subsampling foranalysis.
Stability studies have indicated that imidacloprid residues are stable (<30% decomposition) for 24months (728 to 769 days) of freezer storage in the following representative crops: an oilseed(tomatoseed), a non-oily grain (wheat), a leafy vegetable (lettuce), a root crop (potato), a tree fruit(apple), and a fruiting vegetable (tomato)4-10. An additional stability study has indicated thatimidacloprid residues are stable (<30% decomposition) for 36 months of freezer storage in wheat(grain), orange (fruit), tomato (fruit), bean (seed), and rape (seed)11. Demonstrated freezer stability in allof the above crops is representative of the freezer stability of imidacloprid, 5-hydroxy imidacloprid, andimidacloprid olefin residues to be expected for apple leaves from this study. The apple leaves analyzed
in this study were held in frozen storage for a maximum of 561 days (18 months) prior to extraction.
To demonstrate that imidacloprid, 5-hydroxy imidacloprid, and imidacloprid olefin were stable in theapple nectar and pollen, samples of nectar surrogate and commercial pollen were fortified with amixture of imidacloprid, 5-hydroxy imidacloprid, and imidacloprid olefin, each at a level of 100 or 200
ppb. These transit stability samples were shipped to the field site at the start of each study year andwere subsequently stored with the study samples. The transit stability samples were analyzed after all
sample analyses were complete. The transit stability analyses indicate that the imidacloprid residues arestable (<30% decomposition) during concurrent freezer storage with the nectar and pollen samples fromthis study report.

676

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49662101

CDPR IMI Apple DER

5E. ANALYTICAL METHODS
The analytical methods1-2 used in this study measured the residues of imidacloprid, 5-hydroxyimidacloprid, and imidacloprid olefin in apple nectar, pollen, and leaves. All neat analytical reference
standards were stored frozen prior to dilution. All reference standard solutions were prepared in parent
equivalents and corrected for purity during initial preparation.
For the apple leaves1, a 2.5 g sample was weighed into a 50-mL polypropylene conical centrifuge tube,
and 10 mL of HPLC-grade water was added. The tube was mixed manually for 1 minute, followed byadding 20 mL of acetonitrile and manual shaking for an additional 1 min. Then, 3 g of MgSO4 and 1.5 gof NaCl were added. The sample was amended with a mixed internal standard solution and manually
mixed for 1 minute. For leaf samples found to contain high imidacloprid residues (>2 ppm), as
determined by an initial run in which the response exceeded the calibration curve, the sample wasamended with a 10X mixed internal standard solution before the salts were added. The sample wascentrifuged, and 20 mL of the organic supernatant was transferred into a separate 50-mL polypropylene
conical centrifuge tube containing 0.3 g of Bondesil-PSA and 1.8 g of MgSO4. For the high imidacloprid
residue samples, 2.0 mL of the organic supernatant and 18.0 mL of acetonitrile were instead transferred.
The sample was again manually mixed for 1 minute. The sample extract was centrifuged and a 1.25 mL
aliquot of supernatant was transferred into a clean culture tube. The sample aliquot was evaporated tonear dryness on a Turbo-Vap. The extract was reconstituted with 1.25 mL of 9:1 water/methanol andtransferred into a 2-mL sample vial for high performance liquid chromatography/triple stage quadrupole
mass spectrometry (LC/MS/MS) analysis.
For nectar2, a 0.1-mL sample was weighed into a 20 x 150 mm culture tube and dissolved in 4 mL ofwater. If the total sample volume was less than 0.1 mL, the entire sample was weighed and recorded.
The mixture was amended with isotopically labeled internal standards, and the resultant solution was
mixed well and applied to an Agilent BondElut Solid Phase Extraction (SPE) cartridge (50 mg resin;previously conditioned with methanol then water). The cartridge was washed with 1 mL ofwater/methanol (19:1 v/v), and the combined eluates were discarded. The analytes were extracted fromthe cartridge with 0.5 mL of water/methanol (4:1 v/v). The eluate was collected into a 2 mL sample vial
for analysis by LC/MS/MS.
For pollen2, a 0.1-g sample was weighed into a small Precellys vial containing 2.8 mm steel balls. If theavailable pollen sample amount was not sufficient for an analysis, samples of the same interval werecomposited and analyzed. The composite sample was weighed and recorded. A 1-mL portion ofwater/methanol (3:1 v/v) was added, and the mixture was homogenized with a bead mixer at 5000
beats/minute for 1 minute on a Precellys homogenizer. The isotopically labeled internal standards wereadded and mixed, and the mixture was centrifuged at 12,000 rpm for 2 minutes. The supernatant wastransferred into a clean culture tube containing 2.5 mL of water and was evaporated to an aqueous
remainder, then applied to a 3-mL ChemElut SPE cartridge. After 10 to 15 minutes, the cartridge waswashed with 4 mL of hexane/ethyl acetate (1:1 v/v) three times into a clean culture tube. The combined
eluates were evaporated to dryness. The analytes were dissolved in 0.5 mL of water/methanol (4:1 v/v).
The solution was transferred into a 2 mL sample vial for analysis by LC/high resolution mass
spectrometry (LC/HRMS).

677

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49662101

CDPR IMI Apple DER

Quantitation of each analyte was based on the daughter ion transitions of the analyte and therespective internal standard analog. The responses of the LC/MS/MS and LC/HRMS systems to eachanalyte and its internal standard were measured in samples and in standards, and a relative response
was calculated (as the ratio of the analyte and the stable isotopically labeled internal standard
responses). The relative response of the analyte in each sample was compared to the relative responseof the analyte in the standards.
The relative responses of imidacloprid and its analytes were measured over the range of 0.12 to 2000
ppb. The analyte relative responses were fit to a linearity curve calculated using linear regression
analysis with 1/x weighting (AB Sciex Analyst 1.6.1, 1.6.2 or Thermo Finnigan XCalibur 2.7.0.20, 2.2
SP1.48; Appendix 4 of the study report). Correlation coefficients were calculated with the same
software.
All data are reported in parent equivalents, and the individual measured residues of imidacloprid, 5hydroxy imidacloprid, and imidacloprid olefin are summed to give a total imidacloprid residue.
5F. QUALITY ASSURANCE RESULTS
The responses of the LC/HRMS and LC/MS/MS systems to imidacloprid, 5-hydroxy imidacloprid, andimidacloprid olefin were linear in solvent over the range of .12 to 2000 ppb. The coefficients ofdetermination were >0.99.
No total imidacloprid residue was calculated for the UTC samples, so the levels of imidacloprid as anindividual analyte are described. Imidacloprid (parent) residues in UTC apple nectar ranged from below
the analyte LOD to below the analyte LOQ. Imidacloprid (parent) residues in UTC apple pollen ranged
from below the analyte LOD to 23 ppb (trial NT034-13ZA). Imidacloprid residues in UTC apple leaves
were all below the analyte LOD with the exception of the year 2 samples from trial NT029-13ZA, which
was likely, contaminated and had an imidacloprid residue of 2200 ppb.
The limit of quantitation (LOQ) is defined as the lowest fortification level of an analyte at which
acceptable recovery has been achieved. The LOQ for a total residue is the highest of the LOQ values
assigned to the individual analytes for a particular matrix.
The limit of detection (LOD) is defined as the lowest concentration of an analyte that can be determined
to be statistically different from a blank. The LODs were determined from method validation dataobtained from control samples fortified at the respective analyte LOQs. The LODs were calculated bymultiplying the standard deviation of recovery measurements at the LOQ by t0.99 [where t0.99 is the onetailed t-statistic at the 99% confidence level for the number of replicates (n)]3. The LOD for the total
imidacloprid residue in each matrix is the highest LOD value of any one individual analyte for thatparticular matrix.
The LOQs and LODs are summarized in the table below.
Summary of LOQs and LODs
MatrixAnalyteLOQ (ppb, parent equivalents)
Apple Nectar
Imidacloprid
1.05-hydroxy Imidacloprid
1.0
Imidacloprid olefin
1.0678

LOD (ppb, parent equivalents)
0.3
0.7
0.6

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49662101
MatrixApple Pollen

Apple Leaves

CDPR IMI Apple DER
AnalyteTotal Imidacloprid
Imidacloprid
5-hydroxy Imidacloprid
Imidacloprid olefin
Total Imidacloprid
Imidacloprid
5-hydroxy Imidacloprid
Imidacloprid olefin
Total Imidacloprid

LOQ (ppb, parent equivalents)
1.0
1.0
1.0
1.0
1.0
5.0
5.0
5.0
5.0

LOD (ppb, parent equivalents)
0.7
0.4
0.5
0.3
0.5
0.9
0.5
0.8
0.9

6. RESULTS:
The imidacloprid residue data for apple nectar, pollen, and leaves are provided in Table 3. Only residue
values above the respective analyte LODs are reported. Any residue value that was below the LOD isreported as less than the LOD (<LOD). The total imidacloprid residue is a sum of the analyte residue
values that are greater than the respective analyte LODs. If the analyte value was less than the LOD, adefault value equal to half of the analyte LOD (half-LOD) was added into the sum. For samples with areported total residue of less than LOD, the sum of the analyte half-LOD values for the respective matrix
was used to calculate the average residue values.

Imidacloprid Olefin Residues(ppb)

5-Hydroxy Imidacloprid
Residue (ppb)

Imidacloprid Residue (ppb)

Total Imidacloprid Residue(ppb)

DAA (Days After the Last
Application)

0.7
26.0

0.3
8.9

0.7
36.3
Avg.: 36.3

Total Rate, lb. a.i./acre

0.6
1.4

Soil TypeApple Nectar
NT028Clarksburg,
13ZA
California
Region 10
2014NT028Clarksburg,
13ZA
California
Region 10
2015NT029Stockton,
13ZA
California
Region 10

GrannySmith

Fine

LODs (ppb):
0.50
193

Granny
Smith

Fine

0.50

279

0.7
<LOD

<LOD
<LOD

<LOD
<LOD

1.1
0.8
Avg.: 1.0

York
Imperial

Fine

0.50

177

<LOD
<LOD

0.9
<LOD

1.1
0.4

2.3
1.0
Avg.: 2.0

Crop Variety

Location (City, State, NAFTA
Region, Sampling Year)

Trial Identification

Table 3. Results of Imidacloprid and Imidacloprid Metabolite Residue from Apple679

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

NT03313ZA

NT03313ZA

York
Imperial

Fine

0.50

278

0.7
<LOD

<LOD
<LOD

<LOD
<LOD

1.2
0.8
Avg.: 1.0

GrannySmith

Coarse

0.50

186

<LOD
<LOD

<LOD
<LOD

<LOD
1.2

0.8
1.8
Avg.: 2.0

GrannySmith

Coarse

0.50

287

<LOD
1.0

<LOD
<LOD

1.0
0.7

1.6
2.0
Avg.: 2.0

Fuji

Fine

0.50

193

<LOD
<LOD

<LOD
<LOD

1.2
2.8

1.9
3.4
Avg.: 3.0

Fuji

Fine

0.50

273

<LOD
<LOD

<LOD
<LOD

<LOD
<LOD

0.8
0.8
Avg.: 1.0

Shirely
Ranch

Medium

0.50

177

<LOD
<LOD

0.8
<LOD

0.9
0.4

2.0
1.0
Avg. 2.0

Shirley
Ranch

Medium

0.50

278

<LOD
<LOD

<LOD
<LOD

<LOD
<LOD

0.8
0.8
Avg.: 1.0

GrannySmith

Coarse

0.50

186

0.7
<LOD

<LOD
<LOD

0.4
<LOD

1.4
0.8
Avg.: 1.0

GrannySmith

Coarse

0.50

287

1.3
0.9

<LOD
<LOD

0.7
0.7

2.3
2.0

Crop Variety

Total Imidacloprid Residue(ppb)

NT03213ZA

Imidacloprid Residue (ppb)NT03213ZA

5-Hydroxy Imidacloprid
Residue (ppb)NT03113ZA

Imidacloprid Olefin Residues(ppb)

NT03113ZADAA (Days After the Last
Application)

NT03013ZA

Total Rate, lb. a.i./acre

NT03013ZA

2014
Stockton,
California
Region 10
2015
Linden,California
Region 10
2014
Linden,California
Region 102015
Clarksburg,
California
Region 102014
Clarksburg,
California
Region 10
2015
Linden,California
Region 10
2014
Linden,California
Region 10
2015
Linden,California
Region 10
2014
Linden,California

CDPR IMI Apple DER

Soil TypeNT02913ZA

Location (City, State, NAFTA
Region, Sampling Year)

Trial Identification

MRID 49662101

680

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

Region 10
2015NT034Madera,
13ZA
California
Region 10
2014NT034Madera,
13ZA
California
Region 10
2015NT035Madera,
13ZA
California
Region 10
2014
NT036Sanger,
13ZA
California
Region 10
2014
NT036Sanger,
13ZA
California
Region 10
2015
Apple Pollen
NT028Clarksburg,
13ZA
California
Region 10
2014NT028Clarksburg,
13ZA
California
Region 10
2015NT029Stockton,
13ZA
California
Region 10
2014

Total Imidacloprid Residue(ppb)

Imidacloprid Residue (ppb)5-Hydroxy Imidacloprid
Residue (ppb)

Imidacloprid Olefin Residues(ppb)

DAA (Days After the Last
Application)

Total Rate, lb. a.i./acre

Soil TypeCDPR IMI Apple DER

Crop Variety

Location (City, State, NAFTA
Region, Sampling Year)

Trial Identification

MRID 49662101

Avg.: 2.0
Fuji

Coarse

0.52

145

<LOD
<LOD

<LOD
<LOD

<LOD
<LOD

0.8
0.8
Avg.: 1.0

Fuji

Coarse

0.52

265

<LOD
0.8

<LOD
<LOD

<LOD
<LOD

0.8
1.3
Avg.: 1.0

Gala

Coarse

0.50

138

<LOD
<LOD

<LOD
<LOD

0.6
0.5

1.2
1.2
Avg.: 1.0

Pink LadyApple

Coarse

0.50

151

1.6
1.8

<LOD
<LOD

0.9
0.7

2.8
2.8
Avg.: 3.0

Pink LadyApple

Coarse

0.50

131

2.0
2.5

0.7
<LOD

1.3
1.2

4.0
4.0
Avg.: 4.0

Granny
Smith

Fine

0.50

LODs (ppb):
193

0.3
<LOD
<LOD

0.5
0.8
<LOD

0.4
6.6
8.0

0.7
7.7
8.4
Avg.:8.0

Granny
Smith

Fine

0.50

279

0.5
3.4

<LOD
0.8

4.7
2.8

5.4
7.1
Avg.: 6.0

York
Imperial

Fine

0.50

177

0.9
<LOD

1.1
0.5

28.4
18.8

30.4
18.8
Avg.: 25.0681

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

NT03313ZA

NT03313ZA

York
Imperial

Fine

0.50

278

7.9
1.3

2.0
<LOD

4.8
5.6

14.6
7.1
Avg.: 11.0

GrannySmith

Coarse

0.50

186

<LOD
<LOD

<LOD
0.5

20.5
20.2

20.9
20.8
Avg.: 21.0

GrannySmith

Coarse

0.50

287

14.7
9.0

3.6
2.4

34.2
91.3

52.4
102.7
Avg.: 78

Fuji

Fine

0.50

193

<LOD
<LOD

0.7
1.1

19.2
45.9

20.1
47.2
Avg.: 34.0

Fuji

Fine

0.50

273

52.1
14.2

13.9
3.0

10.2
6.6

76.2
23.8
Avg.: 50.0

Shirely
Ranch

Medium

0.50

177

0.3
0.4

<LOD
0.5

29.5
20.5

30.1
21.4
Avg.: 26.0

Shirley
Ranch

Medium

0.50

278

<LOD
4.2

<LOD
1.0

5.0
2.0

5.4
7.2
Avg.: 6.0

GrannySmith

Coarse

0.50

186

<LOD
<LOD

<LOD
<LOD

14.2
0.7

14.6
1.1
Avg.: 8.0

GrannySmith

Coarse

0.50

287

1.8
8.0

<LOD
1.8

13.6
28.7

15.6
38.5

Crop Variety

Total Imidacloprid Residue(ppb)

NT03213ZA

Imidacloprid Residue (ppb)NT03213ZA

5-Hydroxy Imidacloprid
Residue (ppb)NT03113ZA

Imidacloprid Olefin Residues(ppb)

NT03113ZADAA (Days After the Last
Application)

NT03013ZA

Total Rate, lb. a.i./acre

NT03013ZA

Stockton,
California
Region 10
2015
Linden,California
Region 10
2014
Linden,California
Region 102015
Clarksburg,
California
Region 102014
Clarksburg,
California
Region 10
2015
Linden,California
Region 10
2014
Linden,California
Region 10
2015
Linden,California
Region 10
2014
Linden,California
Region 10

CDPR IMI Apple DER

Soil TypeNT02913ZA

Location (City, State, NAFTA
Region, Sampling Year)

Trial Identification

MRID 49662101

682

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

Soil Type

Total Rate, lb. a.i./acre

DAA (Days After the Last
Application)

Imidacloprid Olefin Residues(ppb)

5-Hydroxy Imidacloprid
Residue (ppb)

Imidacloprid Residue (ppb)

Total Imidacloprid Residue
(ppb)

2015
NT034Madera,
13ZA
California
Region 10
2014NT034Madera,
13ZA
California
Region 10
2015NT035Madera,
13ZA
California
Region 10
2014
NT036Sanger,
13ZA
California
Region 10
2014
NT036Sanger,
13ZA
California
Region 10
2015
Apple Leaves
NT028Clarksburg,
13ZA
California
Region 10
2014NT028Clarksburg,
13ZA
California
Region 10
2015NT029Stockton,
13ZA
California
Region 10
2014NT029Stockton,

CDPR IMI Apple DER

Crop Variety

Location (City, State, NAFTA
Region, Sampling Year)

Trial Identification

MRID 49662101

Fuji

Coarse

0.52

145

<LOD
<LOD

<LOD
<LOD

13.7
8.7

14.1
9.1
Avg.: 12.0

Fuji

Coarse

0.52

265

<LOD
0.8

<LOD
<LOD

7.5
1.3

7.9
2.3
Avg.: 5.0

Gala

Coarse

0.50

138

<LOD
<LOD

<LOD
<LOD

3.8
1.4

4.2
1.8
Avg.: 3.0

Pink LadyApple

Coarse

0.50

151

1.0
1.9

<LOD
0.6

5.1
13.2

6.3
15.8
Avg.: 11.0

Pink LadyApple

Coarse

0.50

131

9.4
9.5

1.2
1.2

92.8
47.8

103.4
58.5
Avg.: 81.0

GrannySmith

Fine

LODs (ppb):
0.50
214

0.8
<LOD
<LOD

0.5
<LOD
<LOD

0.9
<LOD
<LOD

0.9
1.1
1.1
Avg.: 1.0

Granny
Smith

Fine

0.50

293

1.9
1.4

<LOD
<LOD

1.1
<LOD

3.2
2.1
Avg.: 3.0

York
Imperial

Fine

0.50

198

2.1
4.0

<LOD
<LOD

2.4
3.6

4.7
7.8
Avg.: 6.0

York

Fine

0.50

293

11.7

30.0

3533.23575.0683

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

NT03013ZA

NT03113ZA

NT03113ZA

NT03213ZA

NT03213ZA

NT03313ZA

NT03313ZA

Imidacloprid Residue (ppb)

Total Imidacloprid Residue(ppb)

DAA (Days After the Last
Application)

Total Rate, lb. a.i./acre

Soil Type

Crop Variety
Imperial

5-Hydroxy Imidacloprid
Residue (ppb)NT03013ZA

California
Region 10
2015
Linden,California
Region 10
2014
Linden,California
Region 102015
Clarksburg,
California
Region 102014
Clarksburg,
California
Region 10
2015
Linden,California
Region 10
2014
Linden,California
Region 10
2015
Linden,California
Region 10
2014
Linden,California
Region 10
2015

CDPR IMI Apple DER

Imidacloprid Olefin Residues
(ppb)

13ZA

Location (City, State, NAFTA
Region, Sampling Year)

Trial Identification

MRID 49662101

13.2

25.3

3164.03202.5Avg.:
3400.015.3
16.9
Avg.: 16.0

GrannySmith

Coarse

0.50

204

6.0
6.2

1.2
1.5

8.1
8.9

GrannySmith

Coarse

0.50

293

4.5
4.9

0.8
0.6

8.0
5.9

13.3
11.4
Avg.: 12.0

Fuji

Fine

0.50

214

0.8
1.0

<LOD
<LOD

<LOD
<LOD

1.5
1.7
Avg.: 2.0

Fuji

Fine

0.50

293

1.9
3.1

<LOD
<LOD

<LOD
<LOD

2.6
3.8
Avg.: 3.0

Shirely
Ranch

Medium

0.50

198

1.5
1.8

<LOD
<LOD

1.0
3.4

2.8
5.4
Avg.: 4.0

Shirley
Ranch

Medium

0.50

293

3.9
4.9

<LOD
0.6

3.4
4.9

7.6
10.4
Avg.: 9.0

GrannySmith

Coarse

0.50

204

9.4
5.5

1.5
1.4

8.5
7.5

19.3
14.4
Avg.: 17.0

GrannySmith

Coarse

0.50

293

8.6
10.0

1.1
1.5

7.8
10.3

17.5
21.8
Avg.: 20.0684

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

Imidacloprid Olefin Residues(ppb)

5-Hydroxy Imidacloprid
Residue (ppb)

Imidacloprid Residue (ppb)

Total Imidacloprid Residue(ppb)

NT03613ZADAA (Days After the Last
Application)

NT03613ZA

Total Rate, lb. a.i./acreNT03513ZA

Soil TypeNT03413ZA

Madera,California
Region 10
2014
Madera,California
Region 10
2015
Madera,California
Region 10
2014
Sanger,California
Region 10
2014
Sanger,California
Region 10
2015

CDPR IMI Apple DER

Crop Variety

NT03413ZA

Location (City, State, NAFTA
Region, Sampling Year)

Trial Identification

MRID 49662101

Fuji

Coarse

0.52

175

6.4
3.7

1.4
0.9

2.9
1.8

10.7
6.3
Avg.: 9.0

Fuji

Coarse

0.52

265

12.9
14.0

1.3
1.1

6.7
5.8

20.9
20.9
Avg.: 9.0

Gala

Coarse

0.50

175

8.6
7.5

1.7
1.9

7.1
8.0

17.4
17.4
Avg.: 17.0

Pink LadyApple

Coarse

0.50

151

28.6
30.6

4.7
4.3

27.0
21.7

60.3
56.7
Avg.: 59.0

Pink LadyApple

Coarse

0.50

147

68.7
81.1

14.2
17.2

54.1
70.4

137.0
168.6
Avg.:
150.0

7. STUDY VALIDITY/CLASSIFICATION AND STUDY LIMITATIONSClassification/Utility for Bee Risk Assessment. This study is classified as ACCEPTABLE with limitations.
This study provides a snapshot of imidacloprid, 5-hydroxy imidacloprid, and imidacloprid olefin residues
in apple leaves, pollen, and nectar during bloom. The residue values presented should be considered tobe reliable. However, it is important to note that it is unclear if concentrations were increasing or
decreasing at the time the samples were collected. It is also important to note that there are many
concerns regarding the timing of applications in this study. In 2013, applications to 7 of the 9 trial sites
were made at BBCH 99. At this growth stage, apples have been harvested and all of the trees’ leaves
have fallen. This may be an inappropriate application time for a residue study as leaves play animportant part in uptake of residues by the plant, especially for foliar applications. In addition, the label
for Admire Pro® Systemic Protectant states that applications may not be made during pre-bloom or
bloom periods. However, in 2014, applications at 1 of the 9 sites were made during BBCH 65-67, which iswhen the plant is in full flowering.
685

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49662101

CDPR IMI Apple DER

Temporal Variability in Residues. This study was not designed for temporal analysis of declining
concentrations, but rather, to provide a snapshot of residue concentrations during flowering.
Spatial Variability in Residues. All nine sites in this study were located in central California andexperienced similar climatic conditions throughout the study duration. Three of the sites had fine soil,
one had medium soil, and five sites had course soil. Statistical comparison for potential differences insoil type was conducted in Section 8 comparing the coarse soil to fine-texture soil data. The mediumtextured soil did not contain an adequate number of replicates for comparison. The findings indicatedthat there were significantly higher concentrations in leaves of plants grown in coarse soils that in thosegrown in fine soils. This pattern was not reflected in nectar or pollen samples.
Pesticide Carryover. The extent to which prior year applications of imidacloprid contributed to year-toyear carryover was not analyzed by the study authors. In Section 8, analyses were conducted todetermine if there were significant differences in distribution of imidacloprid residues between years.
The findings indicated that, generally, there were no significant differences between the two years.
Thus, it is not expected that residues that resulted from the first year of applications contributed to theresidue levels that resulted from the second year of applications.
Magnitude of Residues. A summary of imidacloprid residue data for apple nectar, pollen, and leaves areprovided in Table 4 of the study (copied below). Only residue values above the respective analyte’s LOD
are reported. Any residue value that was below the LOD is reported as less than the LOD (<LOD). The
total imidacloprid residue is a sum of the analyte residue values that are greater than the respective
analyte LODs. If the analyte value was less than the LOD, a default value equal to half of the analyte LOD(half-LOD) was added into the sum. For samples with a reported total residue of less than LOD, the sumof the analyte’s half-LOD value for the respective matrix was used to calculate the average residue
values.
The maximum total imidicloprid residues were 36, 100, and 3600 ppb in nectar, pollen, and leaves,respectively. The mean total imidicloprid residues were 3, 24, and 220 ppb in nectar, pollen, and leaves,respectively.

138-1930.50-0.52

17

1.0

36

3.0

1.0

4.0

8.0

2015

131-287

0.50-0.52

16

1.0

4.0

3.0

1.0

2.0

1.0

2014, 2015 131-287
2014 138-193

0.50-0.52
0.50-0.52

33
18

<LOD
1.0

36
47

3.0
30

1.0
15

3.0
16

6.0
12

131-287

0.50-0.52

16

2.0

100

89

15

33

35

2014, 2015 131-287

0.50-0.52

34

1.0

100

57

15

24

27

2015

686

Mean

Standard
Deviation

90th
percentile

2014

MedianMax

Seasonal
Application
Rates(lb a.i./A)

DAAa

Min

Pollen TRTD

Total Imidacloprid Residue Levels (ppb)bnc

Nectar TRTD

Years

Plot

Matrix

Table 4. Summary of Residue Data for Imidacloprid in/on Apple, All Trials.*

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49662101

Leaves TRTD

CDPR IMI Apple DER
2014

151-2140.50-0.52

18

1.0

60

31

9.0

14

17

2015

147-2930.50-0.52

16

2.0

3600d

1700

15

450

1100

2014, 2015 147-293

0.50-0.52

34

1.0

3600

110

12

220

810

a DAA = Days after the last application.

b For the purpose of calculating the total imidacloprid residues, any individual analyte value reported as<LOD was summed into the total at a default value equal to ½ the LOD.
c n = Number of individual treated samples analyzed.
d The next highest residue from 2015 leaf samples in trial NT029-13ZA was 4.0 ppb.

* Values rounded to the nearest whole number

8. STATISTICAL ANALYSIS
1. There were a number of values below the limit of detection (LOD). These values were substituted
with ½ LOD values for the represented year and plant tissue. The total was a simple addition of all
analytes.
2. Analyses were conducted to determine if there were significant differences in distribution of
imidacloprid residue concentrations between years in the plant samples. Tables S-1 through S-3 containa comparison of the distributions for each analyte between years for leaves, nectar, and pollen,
respectively. Figures S-1 through S-3 provide a graphical view of the distributional differences between
years for parent imidacloprid and total residues in leaves, nectar, and pollen, respectively. The data arepresented as distributions of the natural logarithms because they provide a clearer separation of thedata. As shown in the graph for leaves, there were 2 rather large values of 3,575 and 3,202.5 ppb
measured from samples taken at the Stockton site in 2015 (Figure S-1). Generally, there were no
significant differences between the two years as indicated by non-parametric tests for Wilcoxon ranksum, which tests for a general difference in distributions, and Median tests, which tests for differencesin the location of the median value between the distributions (Table S-4).
3. Tables S-5 through S-7 contain the distributions combined from both years for data in each soil typethat was measured for leaves, nectar and pollen, respectively. The highest nectar value at 36.3 ppb wasmeasured in the fine-textured soil with the next highest value measured at 4 ppb in the coarse-textured
soil (Table S-6). For pollen, the highest values were measured in coarse soil at 103.4 and 102.7 ppb,
respectively (Table s-7). Comparison of the median values between nectar and pollen indicate that thepollen distribution tended to contain higher values. The medians for the 3 soils typed for nectar ranged
from 0.9 to 1.5 ppb compared to 14.3 to 16.7 ppb for pollen.
4. Statistical comparison for potential differences in soil type were conducted comparing the coarse soil
to fine-texture soil data: the medium-textured soil did not contain an adequate number of values forcomparison to the other two categories. Although there were a few rather large values measured forthe fine-textured soils for imidacloprid in leaves, the non-parametric tests indicated a significant
difference in the distribution. The findings indicated that leaves in coarse-textured soils tended to have agreater number of higher values than in the fine-textured soils (Table S-8). The patterns in the graphic
for leaves in Figure S-4 illustrate that although there were a few large values in the fine-texture soil, thebulk of the concentration were at the lower tail compared to the coarse-texture soils distribution. No
differences were noted for nectar and pollen (Figures S-5 and S-6).

687

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 49662101

CDPR IMI Apple DER

Conclusion: The median pollen concentration was generally 10x greater than nectar thus requiring acomparison between these values and benchmark values to determine biological significance.
Difference in concentration due to soil type became evident as higher concentrations were measured inleaves of plants grown in coarse soils. This pattern was not reflected in nectar or pollen samples.

688

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 49662101

CDPR IMI Apple DER

Table S-1. Leaves: Comparison of statistics for the distribution between years 2014 and 2015 forconcentrations of imidacloprid, its degradation products and their total in leaves of apples. Residues arereported in parts per billion (ppb).

OlefinStatisticN
MeanSD
CV (%)MinMedian
75th
90th
Max
% of
Total

Hydroxy

Imidacloprid

2014
18
6.9
8.7
126.4
0.4
4.8
7.5
28.6
30.6

2015
16
15.4
23.7
153.9
1.4
6.8
13.1
68.7
81.1

2014
18
1.3
1.3
106.5
0.3
1.1
1.5
4.3
4.7

2015
16
5.9
9.9
167.5
0.3
1.0
7.9
25.3
30.0

2014
18
6.3
7.3
116.0
0.5
3.5
8.1
21.7
27.0

2015
16
429.8
1141.6
265.6
0.5
6.3
32.2
3164.03533.0

47.6

3.4

8.6

1.3

43.6

95.3

689

Total Residue
2014
18
14.5
17.3
119.1
1.1
9.3
17.4
56.7
60.3

2015
16
451.2
1149.8
254.8
2.1
15.4
79.4
3202.53575.0Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 49662101

CDPR IMI Apple DER

Table S-2. Nectar: Comparison of statistics for the distribution between years 2014 and 2015 forconcentration of imidacloprid, its degradation products and their total in nectar of apples. Residues arereported in parts per billion (ppb).
OlefinStatisticN
MeanSD
CV (%)MinMedian
75th
90th
Max
% of
Total

Hydroxy

Imidacloprid

2014
17
0.6
0.5
92.9
0.3
0.3
0.3
1.6
1.8

2015
16
0.8
0.7
86.5
0.3
0.5
1.0
2.0
2.5

2014
17
1.9
6.2
323.7
0.4
0.4
0.4
0.9
26.0

2015
16
0.4
0.1
23.5
0.4
0.4
0.4
0.4
0.7

2014
17
1.2
2.1
172.0
0.2
0.6
1.1
2.8
8.9

2015
16
0.4
0.4
95.2
0.2
0.15
1.2
1.3
1.3

15.0

49.0

52.5

23.6

33.1

28.0

690

Total Residue
2014
17
3.7
8.5
230.6
0.8
1.4
2.3
3.4
36.3

2015
16
1.6
1.1
68.5
0.8
1.2
2.0
4.0
4.0

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 49662101

CDPR IMI Apple DER

Table S-3. Pollen: Comparison of statistics for the distribution between years 2014 and 2015 forconcentration of imidacloprid, its degradation products and their total in pollen of apples. Residues arereported in parts per billion (ppb).

OlefinStatisticN
MeanSD
CV (%)MinMedian
75th
90th
Max
% of
Total

Hydroxy

Imidacloprid

2014
18
0.4
0.5
129.3
0.2
0.2
0.3
1.0
1.9

2015
16
8.6
12.6
147.0
0.2
6.1
9.5
14.7
52.1

2014
18
0.5
0.3
63.5
0.3
0.3
0.6
1.1
1.1

2015
16
2.0
3.3
165.0
0.3
1.1
2.2
3.6
13.9

2014
18
15.5
11.5
74.2
0.7
14.0
20.5
29.5
45.9

2015
16
22.4
30.2
134.6
1.3
7.1
31.5
91.3
92.8

2.2

26.0

2.8

6.2

95.1

68.0

691

Total Residue
2014
18
16.3
11.7
71.8
1.1
15.2
20.9
30.4
47.2

2015
16
33.0
35.2
105.6
2.3
15.1
55.5
102.7
103.4

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 49662101

CDPR IMI Apple DER

Table S-4. Exact probability levels for non-parametric tests between years 2014 and 2015 for
distribution of concentrations of imidacloprid and its degradation products in leaves, nectar, and pollenof apples.
Probability Level for Non-Parametric Test Between Years
Plant
TissueLeavesNectarPollenOlefinWilcoxon
0.13
0.2<0.0001Median1
0.2
0.0003

Hydroxy
Wilcoxon
0.66
0.3
0.02

Imdacloprid

Median
1
0.6
0.19

692

Wilcoxon
0.34
0.1
0.63

Median
0.72
0.3
0.3

Total Residue
Wilcoxon
0.12
0.4
0.49

Median0.73
0.7
1Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 49662101

CDPR IMI Apple DER

Table S-5. Leaves: Distribution statistics for imidacloprid and degradates for applications made in three soil texture categories and with data
combined over 2014 and 2015. Residues are reported in parts per billion (ppb).

StatisticN
MeanSD
CV (%)MinMedian
75th
90th
Max
% of Total

Coarse Textured Soil
5-OH Olefin
IMI
Total
18
18
18
18
3.2
17.6
15.0
35.9
4.7
22.3
18.4
45.2
144.8
126.3
122.5
125.8
0.6
3.7
1.8
6.3
1.5
8.6
8.0
17.5
1.9
14.0
10.3
21.8
14.2
68.7
54.1
137.0
17.2
81.1
70.4
168.6
9.0
49.1
41.9

5-OH
4
0.3
0.2
51.9
0.3
0.3
0.4
0.6
0.6
5.2

Medium Textured Soil
Olefin
IMI
Total
4
4
4
3.0
3.2
6.6
1.6
1.6
3.2
54.3
50.8
49.3
1.5
1.0
2.8
2.9
3.4
6.5
4.4
4.2
9.0
4.9
4.9
10.4
4.9
4.9
10.4
46.3
48.5

693

5-OH
12
4.8
10.7
222.4
0.3
0.3
0.3
25.3
30.0
0.8

Fine Textured Soil
Olefin
IMI
12
12
3.5
559.0
4.3
1305.4
123.9
233.6
0.4
0.5
1.9
0.5
3.6
3.0
11.7
3.6
13.2
3533.2
0.6
98.5

Total
12
567.3
1320.3
232.8
1.1
2.9
6.3
3202.53575.0Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 49662101

CDPR IMI Apple DER

Table S-6. Nectar: Distribution statistics for imidacloprid and degradates for applications made in three soil texture categories and with data
combined over 2014 and 2015. Residues are reported in parts per billion (ppb).

StatisticN
MeanSD
CV (%)MinMedian
75th
90th
Max
% of Total

5-OH
18
0.4
0.1
22.3
0.4
0.4
0.4
0.4
0.7
20.6

Coarse Textured Soil
Olefin
IMI
Total
18
18
18
0.9
0.6
1.8
0.7
0.4
1.0
83.1
67.2
57.5
0.3
0.2
0.8
0.5
0.7
1.5
1.3
0.9
2.3
2.0
1.2
4.0
2.5
1.3
4.0
47.2
33.3

5-OH
4
0.5
0.2
48.7
0.4
0.4
0.6
0.8
0.8
40.0

Medium Textured Soil
Olefin
IMI
Total
4
4
4
0.3
0.4
1.2
0.0
0.4
0.6
0.0
88.4
50.0
0.3
0.2
0.8
0.3
0.3
0.9
0.3
0.7
1.5
0.3
0.9
2.0
0.3
0.9
2.0
26.1
34.8

694

5-OH
11
2.7
7.7
282.6
0.4
0.4
0.4
0.9
26.0
59.6

Fine Textured Soil
Olefin
IMI
11
11
0.5
1.4
0.4
2.6
73.3
188.5
0.3
0.2
0.3
0.2
0.7
1.2
0.7
2.8
1.4
8.9
10.3
30.3

Total
11
4.6
10.6
230.3
0.8
1.1
2.3
3.4
36.3

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 49662101

CDPR IMI Apple DER

Table S-7. Pollen: Distribution statistics for imidacloprid and degradates for applications made in three soil texture categories and with data
combined over 2014 and 2015. Residues are reported in parts per billion (ppb).

StatisticN
MeanSD
CV (%)MinMedian
75th
90th
Max
% of Total

5-OH
18
0.8
0.9
121.0
0.3
0.3
1.2
2.4
3.6
2.9

Coarse Textured Soil
Olefin
IMI
Total
18
18
18
3.2
23.7
27.2
4.6
27.9
32.1
145.2
20.0
118.0
0.2
0.7
1.1
0.5
13.7
15.1
8.0
28.7
38.5
9.5
91.3
102.7
14.7
92.8
103.4
11.7
86.9

5-OH
4
0.5
0.4
70.7
0.3
0.4
0.8
1.0
1.0
3.1

Medium Textured Soil
Olefin
IMI
Total
4
4
4
1.3
14.3
16.0
2.0
13.0
11.8
155.3
91.3
74.6
0.2
2.0
5.4
0.4
12.8
14.3
2.3
25.0
25.8
4.2
29.5
30.1
4.2
29.5
30.1
7.9
88.9

695

5-OH
12.0
2.1
3.8
185.8
0.3
0.8
1.6
3.0
13.9
9.2

Fine Textured Soil
Olefin
IMI
12.0
12.0
6.8
13.5
14.9
12.8
220.8
95.0
0.2
2.8
0.7
7.3
5.7
19.0
14.2
28.4
52.1
45.9
30.4
60.6

Total
12.0
22.2
20.9
94.2
5.4
16.7
27.1
47.2
76.2

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 49662101

CDPR IMI Apple DER

Table S-8. Exact probability levels for the Wilcoxon rank sum and Median tests for the difference inimidacloprid and total imidacloprid residue distribution between coarse and fine-textured soils.

Probability Level for Non-Parametric Test
Between Soils
Plant
SampleImidacloprid

Total Residue

Wilcoxon

MedianWilcoxon

MedianLeaves

0.00340.00680.00120.008

Nectar

0.7

0.4

0.5

0.5

Pollen

0.43

0.27

0.9

1

696

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49662101

CDPR IMI Apple DER

Figure S-1. Leaf Concentration: Comparison of imidacloprid and total imidacloprid residue between
data collected in 2014 and 2015.
A. Imidacloprid
Distribution of logimid
2014

Percent

30

20

10

0
50

2015

Percent

40
30
20
10

year

0

2014
2015
-5

0

5

10

logimid
NormalKernelB. Total imidacloprid residue.
Distribution of logtot

Percent

30

2014

20

10

0

Percent

30

2015

20

10

year

0
2014
2015
-5

5

0

logtotNormal697

Kernel10

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49662101

CDPR IMI Apple DER

Figure S-2. Nectar Concentration: Comparison of imidacloprid and total imidacloprid residue
between data collected in 2014 and 2015.
A. Imidacloprid
Distribution of logimid
40

2014

Percent

30

20

10
0

Percent

60

2015

40

20

year

0
2014

2015
-2

-4

2

0

logimid
NormalKernelB. Total imidacloprid residue.
Distribution of logtot
50

2014

Percent

40

30

20

10

0

Percent

40

2015

30
20
10

year

0
2014

2015
-2

2

0

logtotNormal698

Kernel4

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49662101

CDPR IMI Apple DER

Figure S-3. Pollen Concentration: Comparison of imidacloprid and total imidacloprid residue
between data collected in 2014 and 2015.
A. Imidacloprid
Distribution of logimid

Percent

40

2014

30

20

10
0
40

2015

Percent

30
20
10

year

0

2014
2015
-2

0

2

4

6

logimid
NormalKernelB. Total imidacloprid residue.
Distribution of logtot

Percent

40

2014

30

20

10
0
40

2015

Percent

30

20

10

year

0
2014

2015

-2

0

2

4

logtotNormal699

Kernel6

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49662101

CDPR IMI Apple DER

Figure S-4. Leaves: comparison of imidacloprid and total imidacloprid residue between coarse andfine textured soil derived data.
A. Imidacloprid
Distribution of logimid
Coarse80

Percent

60

40

20
0
80

Fine

Percent

60

40

20

Soil

0
Coarse

Fine

-5

-10

0

5

10

logimid
NormalKernelB. Total imidacloprid residue.
Distribution of logtot
80

CoarsePercent

60
40

20

0

Percent

80

Fine

60
40
20

Soil

0

Coarse
Fine
-5

5

0

logtotNormal700

Kernel10

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49662101

CDPR IMI Apple DER

Figure S-5. Nectar: comparison of imidacloprid and total imidacloprid residue between coarse andfine textured soil derived data.
A. Imidacloprid
Distribution of logimid

Percent

40

Coarse3020
100Percent

40

Fine

30
20
10

Soil

0
Coarse
Fine
-2

0

2

4

6

logimid
NormalKernelB. Total imidacloprid residue.
Distribution of logtot
30

CoarsePercent

25
20
15
10
5

0

Percent

60

Fine

40

20

Soil

0
Coarse

Fine

-2

0

2

logtotNormal

701

Kernel4

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 49662101

CDPR IMI Apple

Figure S-6. Pollen: comparison of imidacloprid and total imidacloprid residue between coarse andfine textured soil derived data.
A. Imidacloprid
Distribution of logimid

Percent

30

Coarse

20
10
0
50

Fine

Percent

40
30
20
10

Soil

0
Coarse
Fine
-4

-2

0

2

4

logimid
NormalKernelB. Total imidacloprid residue.
Distribution of logtot

Percent

30

Coarse20
100Percent

40

Fine

30
20
10

Soil

0
Coarse
Fine
-2

0

2

4

logtotNormal34702

Kernel6

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 49662101

CDPR IMI Apple

9. REFERENCES

1. Brungardt, J. 2010. An Analytical Method for the Determination of Residues of Imidacloprid
and its Metabolites Imidacloprid Olefin and 5-Hydroxy Imidacloprid in Bee Relevant Matrices
Using LC/MS/MS. Bayer Method No. NT-005-P10-01.
2. Miller, A. 2014. An Analytical Method for the Determination of Residues of Imidacloprid and its
Metabolites Imidacloprid Olefin and 5-Hydroxy Imidacloprid in Bee Relevant Matrices Using
LC/MS/MS. Bayer CropScience Report Number: NT-006-A13-01.
3. Office of Pesticide Programs, US EPA. 2000. Assigning values to nondetected/nonquantified
pesticide residues in human health food exposure assessments. EPA Docket #OPP-00570A.
4. Noland, P.A. 1992. Imidacloprid and metabolites – freezer storage stability study in crops.
Bayer CropScience Report No. 103237. MRID 42556135.
5.

Noland, P.A. 1993. Imidacloprid and metabolites – freezer storage stability study in crops.
Bayer CropScience Report No. 103237-1. MRID 42810311.

6. Noland, P.A. 1994. Imidacloprid and metabolites – freezer storage stability study in crops.
Bayer CropScience Report No. 103237-2. MRID 43197203.
7. Noland, P.A. 1994. Imidacloprid and metabolites – freezer storage stability study in crops.
Bayer CropScience Report No. 103237-3. MRID 43487302.
8. Lenz, C.A. 1993. Addendum 1. Imidacloprid and metabolites – freezer storage stability study incrops (wheat matrices, tomatoseed, tomato, cauliflower, and lettuce). Bayer CropScience
Report No. 103949-1. MRID 42810313.
9.

Lenz, C.A. 1993. Addendum 2. Imidacloprid and metabolites – freezer storage stability study incrops (wheat matrices, tomatoseed, tomato, cauliflower, and lettuce). Bayer CropScience
Report No. 103949-2. MRID 43197201.

10. Lenz, C.A. 1993. Addendum 3. Imidacloprid and metabolites – freezer storage stability study incrops (wheat matrices, tomatoseed, tomato, cauliflower, and lettuce). Bayer CropScience
Report No. 103949-3. MRID 43487301.
11. Schöning, R. 2014. Storage stability of imidacloprid and its 5-Hydroxy and olefin metabolite
in/on plant matrices for 36 Months. Bayer CropScience Report No. P642094733 Amendment
No. 1.

35703

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 49819401

CDPR IMI Stone Fruit

Reference
Gould, T., and Jerkins, E. (2016) Determination of the Residues of Imidacloprid, 5-Hydroxy
Imidacloprid, and Imidacloprid Olefin in Bee Relevant Matrices Collected from Stone Fruit Trees
following Application of Imidacloprid over Two Successive Years: Final Report. Project Number:
EBNTN013. Unpublished study prepared by Bayer Cropscience. 466p. MRID 49819401, CDPR Study ID
289102, Data Volume 51950-0904, Tracking ID# 273965

1. STUDY INFORMATION
Chemical:Test Material:
Study Type:

Sponsor:Sponsor Study
ID:
Study
Completion
Date:
GLP Status:

Imidacloprid
PC Code129099Admire Pro Systemic
Percent Active
42.8%
Protectant
Ingredient:
Field residue study on Stone Fruit to establish imidacloprid and metabolite levels
in nectar, pollen and leaves in 9 trial site locations following one soil and two
foliar applications of Admire Pro® Systemic Protectant in two successive years.
Bayer CropScience
2T.W. Alexander Drive
Experiment Start and
September 13, 2013 –Research Triangle Park, NC
End Date:
July 16, 2015
USA 27709
Nine trial sites thatEBNTN013
included cherry, plum,
apricot and peachStudy Locations:located in California.
January 13, 2016
GLP; protocol reviewed by CDPR.
[CDPR Study ID 289102, Data Volume 51950-0904, Tracking ID# 273965]

2. REVIEWER INFORMATION
Study Reviewed by:
California Departmentof Pesticide Regulation

Richard Bireley, Sr. Environmental Scientist (Specialist)
John Troiano, Ph.D., Research Scientist III
Alexander Kolosovich, Sr. Environmental Scientist (Specialist)
Brigitte Tafarella, Environmental Scientist
Denise Alder, Sr. Environmental Scientist (Specialist)
Russell Darling, Sr. Environmental Scientist (Specialist)

3. EXECUTIVE SUMMARY
A total of nine field trials were conducted to measure the magnitude of imidacloprid residues in stone
fruit (cherry, plum, apricot and peach) nectar, pollen and leaves following one soil and two foliar
applications of Admire Pro® Systemic Protectant in two successive years. Admire Pro SystemicProtectant is a suspension concentrate formulation containing 550 g/L of imidacloprid.
One soil and two foliar applications were made in 2013 and 2014, post-bloom. Across both years,
individual soil application (drip irrigation) rates were 0.38 lb imidacloprid/acre. The interval between thesoil application and the first foliar application was 3 to 7 days. For all foliar applications (airblasts),
individual rates ranged from 0.058 to 0.064 lb imidacloprid/acre. The interval between the first and704

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 49819401

CDPR IMI Stone Fruitsecond foliar application was 7 to 11 days. Application volumes ranged from 13,000 to 16,600 gal/acre(GPA) for the soil applications and 53 to 90 GPA for the foliar applications. Total annual application rates
ranged from 0.50 to 0.51 lb imidacloprid/acre, which is the maximum labeled amount allowed. In 2013,
all applications were made after stone fruit harvest at BBCH growth stages 91 to 99. In 2014, soil
applications were targeted for 21 days prior to stone fruit harvest and made at BBCH growth stages 77
to 81. The two foliar applications were targeted such that the last would occur 7 days prior to fruit
harvest, with sprays made at BBCH growth stages 76 to 89.
All applications were made using ground-based equipment. The adjuvant Dyne-Amic was used in all
foliar applications at a rate of 0.25% v/v, except in trial NT027-13ZA, when a rate of 0.025% v/v wasused.
Stone fruit flowers (blossoms) and leaf samples were collected once in the spring of 2014, following thepost-harvest fall 2013 applications, and once in the spring of 2015, following the pre-harvest fall 2014
applications. In 2014, flower samples were collected at 133 to 160 days after the last application (DALA),
and leaf samples were collected at 155 to 188 DALA. In 2015, flower samples were collected at 211 to309 DALA, and leaf samples were collected at 230 to 323 DALA.
Single composite samples of cherry, plum, apricot, or peach flowers and leaves were collected from thecontrol plot of each trial on the same days that samples were collected from the treated plot.
The residues of Admire Pro Systemic Protectant (imidacloprid, 5-hydroxy imidacloprid, and imidacloprid
olefin) were quantitated by high performance liquid chromatography/triple stage quadrupole mass
spectrometry (LC/MS/MS) and LC/high resolution mass spectrometry (LC/HRMS) using stable
isotopically labeled internal standards. The individual analyte residues were summed to give a total
imidacloprid residue.

4. STUDY VALIDITY

Guideline Followed:
Guideline Deviations:
Other Deviations:Classification:
Rationale:
Reparability:

Protocol was reviewed by CDPR
N/A
N/A
ACCEPTABLE
N/A
N/A

5. MATERIALS AND METHODS

Test item:

Description:
CAS #:

Test Material Characterization
Admire Pro Systemic
Percent Active
Protectant
Ingredient:
EPA Reg. No.
Suspension Concentrate
Density (20°C):
138261-41-3
Solubility:705

42.8% A.I.
264-827
1.41 - 1.54 g/mL
0.51 to 0.61 g/L

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 49819401

CDPR IMI Stone Fruit

5A. STUDY DESIGNThis study requirement was part of the imidacloprid special review at the California Department of
Pesticide Regulation (CDPR). The study design and protocol were approved by the CDPR prior to study
initiation. This study was conducted using GLP standards and following an approved protocol. The study
initiation date was August 02, 2013. The experimental start date was September 13, 2013 (first field
application) and the experimental end date was July 16, 2015 (last sample analysis). Nine trials wereconducted for this study in multiple locations in California, representing each of the three soil texture
categories (fine, medium, and coarse). Each trial includes one untreated control plot (UTC) and one
treated (TRTD) plot to be treated in each of two consecutive years.
Stone fruit varieties representing those commonly grown in the area of the trials and agronomic
practices typical for commercial production of cherry, plum, apricot, and peach were used at all trial
locations.
Homogenization and analysis of the leaf, nectar, and pollen samples from this study were performed byBayer CropScience in Research Triangle Park (RTP), NC. Final report preparation was performed byCritical Path Services, LLC, located in RTP, NC.
5B. APPLICATION TIMING AND RATESIn 2013, applications were made post-harvest. In 2014, the soil application was made 21 days before
harvest and the last foliar application was made 7 days before harvest, which is in compliance with the
pre-harvest intervals on the label. Across both years, individual soil application rates were 0.38 lb
imidacloprid/acre, which is the maximum allowed for soil applications. The interval between the soil
application and the first foliar application was 3 to 7 days. For all foliar applications, individual rates
ranged from 0.058 to 0.064 lb imidacloprid/acre. The interval between the first and second foliar
application was 7 to 11 days. Application volumes ranged from 13,000 to 16,600 gallons per acre (GPA)
for the soil applications and 53 to 90 GPA for the foliar applications; 16,600 gallons per acre is equivalent
in volume to approximately one-half inch of water covering one acre. Total annual application rates
ranged from 0.50 to 0.51 lb imidacloprid/acre, which the label states is the maximum annual rate
permitted regardless of formulation or method of application.
In 2013, all applications were made after the stone fruit was harvested; at BBCH growth stages 91 to 99(BBCH 91: shoot growth completed, foliage still fully green; BBCH 99: harvested product). In 2014, soil
applications were targeted for 21 days prior to stone fruit harvest and made at BBCH growth stages 77
to 81 (BBCH 77: fruit about 70% of final size; BBCH 81: beginning of fruit coloring); the two foliar
applications were targeted such that the last would occur 7 days prior to fruit harvest, with sprays madeat BBCH growth stages 76 to 89 (BBCH 76: fruit about 70% of final size; BBCH 89: fruit ripe for
consumption, fruit have typical taste and firmness).

706

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 49819401

CDPR IMI Stone Fruit

5C. STUDY SITE LOCATION AND CHARACTERISTICS
The CDPR required a variety of soil types to be included in the study design. Bayer CropScience
conducted the study in three soil texture types (fine, medium, and coarse) based on Soil Survey
Geographic (SSURGO) Database mapping units. There are nine trial sites in this study design: three infine texture soils, three in medium, and three in coarse (Table 1).
Table 1. Study Site Location and Characteristics
Trial (Field)
Trial
OM pH
CEC
%
Identification
Location(%)(meq/1 Sand
(City, State)
00g soil)NT019-13ZA
Stockton,
2.3 7.5
16.8
46
California
NT020-13ZA
Merced,
1.6
6.7
21.2
36
California
NT021-13ZA
Yuba City,
1.5
6.5
17.2
44
California
NT022-13ZA
Yuba City,
1.6
6.6
19.0
26
California
NT023-13ZA
Yuba City,
1.7
5.7
11.5
52
California
NT024-13ZA
Kerman,0.50 6.4
5.2
84
California
NT025-13ZA
Kerman,0.21 8.3
5.8
84
California
NT026-13ZA
Sanger,
1.3
6.3
6.2
60
California
NT026-13ZA
Kingsburg,
1.7
7.2
11.6
56
California

%
Silt

%
Clay

Soil Types

Rainfall Temperature(in)Range (°F)

30

24

Loam

16.4

29 – 92

28

36

Clay loam

11.5

25 – 96

28

28

Clay loam

20.7

26 – 92

36

38

Clay loam

20.7

26 – 92

26

22

20.7

26 – 92

14

2

Sandy clay
loam
Loamy sand

7.5

30 – 98

14

2

Loamy sand

7.2

29 – 98

34

6

Sandy loam

10.3

31 – 97

32

12

Sandy loam

10.3

31 - 97

5D. SAMPLE COLLECTION, HANDLING, PROCESSING
Stone fruit flower (also called blossom) and leaf samples were collected once in the spring of 2014,
following the post-harvest fall 2013 applications, and once in the spring of 2015, following the preharvest fall 2014 applications. Each TRTD plot was divided into two subplots. At each sampling interval,
two composite flower samples (one from each subplot) of cherry, plum, apricot, or peach flowers (to behand-processed to obtain nectar and pollen) were collected by hand when the stone fruit trees were atbloom, BBCH 65 (BBCH 65: full flowering, at least 50% of flowers open, first petals falling). Two
composite leaf samples were also collected for (one from each subplot) cherry, plum, apricot, or peachleaves after bloom, once the leaves had expanded at BBCH 69 to 75 (BBCH 69: end of flowering, allpetals fallen; BBCH 75: fruit about half final size) or at BBCH 19 (first leaves fully expanded). In 2014,
flower samples were collected at 133 to 160 days after the last application (DALA), and leaf samples
were collected at 155 to 188 DALA. In 2015, flower samples were collected at 211 to 309 DALA, and leaf
samples were collected at 230 to 323 DALA.
707

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 49819401

CDPR IMI Stone Fruit

Single composite samples of stone fruit flowers and leaves were collected from the control plot of eachtrial on the same days that samples were collected from the treated plot. Stone fruit flowers and leaves
were collected by hand into Ziplock bags. Each composite flower sample contained a minimum of 125 g(minimum 250 flowers), which was collected from at least 12 different areas of the plot, avoiding theedges (except in trial NT026-13ZA, when unfavourable weather caused the 2015 samples to beunderweight). Each composite leaf sample contained a minimum of 100 g.
After collection, flowers were hand-processed at a facility near the field site to obtain the bee-relevant
matrices of nectar and pollen. Nectar processing began the same day as flower collection. Nectar fromthe floral nectary was removed by micropipette and placed into a pre-weighed glass collection vial. The
flowers were then allowed to dry overnight at room temperature to desiccate the pollen. The next day,pollen was removed from the flowers either by vacuum aspiration with collection in filter tips or bytapping the pollen from the flowers onto wax paper with collection of the accumulated pollen into avial. All resulting nectar and pollen samples were labelled and placed in the freezer immediately after
they were generated. After processing was completed, the flowers were discarded.
The samples of stone fruit leaves, nectar, and pollen were placed into labelled (study number andsample number) containers for shipment. All leaf, nectar, and pollen samples were placed in frozen
storage within 4 hours of collection with the exception of the 2014 leaf samples from trial NT020-13ZA,
which were frozen in 5 hours and 15 minutes, and the 2015 pollen samples from trial NT027-13ZA,
which were frozen in 6 hours and 10 minutes. Samples remained frozen until receipt at Bayer
CropScience in RTP, NC.
Sample Storage.
Upon arrival at Bayer CropScience, all leaf, nectar, and pollen samples were immediately transferred tofrozen storage. The leaf samples were homogenized with dry ice using a Robot Coupe chopper and thenwere returned to frozen storage immediately following homogenization. Pollen and nectar were usedwithout further preparation. All samples remained frozen at all times except during subsampling foranalysis.
Stability studies have indicated that imidacloprid residues are stable (<30% decomposition) for 24months (728 to 769 days) of freezer storage in the following representative crops: an oilseed (tomatoseed), a non-oily grain (wheat), a leafy vegetable (lettuce), a root crop (potato), a tree fruit (apple), anda fruiting vegetable (tomato)9-15. An additional stability study has indicated that imidacloprid residues
are stable (<30% decomposition) for 36 months of freezer storage in wheat (grain), orange (fruit),
tomato (fruit), bean (seed), and rape (seed)16. Demonstrated freezer stability in all of the above crops isrepresentative of the freezer stability of imidacloprid, 5-hydroxy imidacloprid, and imidacloprid olefin
residues to be expected for cherry, plum, apricot, and peach leaves from this study. The leaves analyzed
in this study were held in frozen storage for a maximum of 561 days (18 months) prior to extraction.
To demonstrate that imidacloprid, 5-hydroxy imidacloprid, and imidacloprid olefin were stable in stone
fruit nectar and pollen, samples of a nectar surrogate and commercial pollen were fortified with amixture of imidacloprid, 5-hydroxy imidacloprid, and imidacloprid olefin at a level of 100 or 200 ppb.
These transit stability samples were shipped to the field site at the start of each study year and were
subsequently stored with the study samples; the exception is trial NT025-13ZA, when the field site did
not receive 2014 stability samples. The transit stability samples were analyzed after all sample analyses
were complete. The transit stability analyses indicate that the imidacloprid residues are stable (<30%708Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 49819401

CDPR IMI Stone Fruitdecomposition) during concurrent transit and freezer storage with the nectar and pollen samples fromthis study.
5E. ANALYTICAL METHODS
The analytical methods6-7 used in this study measured the residues of imidacloprid, 5-hydroxyimidacloprid, and imidacloprid olefin in stone fruit leaves, nectar, and pollen. All neat analyticalreference standards were stored frozen prior to dilution. All reference standard solutions were preparedin parent equivalents and corrected for purity during initial preparation.
For stone fruit leaves6, a 2.5 g sample was weighed into a 50-mL polypropylene conical centrifuge tube,
and 10 mL of HPLC-grade water was added. The tube was mixed manually for 1 minute, followed byadding 20 mL of acetonitrile and mechanical shaking (HS501 digital) for an additional 1 minute. Then, 3 gof MgSO4 and 1.5 g of NaCl were added. The sample was amended with a mixed internal standard
solution and manually mixed for 1 minute. The sample was centrifuged and 20 mL of organic
supernatant was transferred into a separate 50-mL polypropylene conical centrifuge tube containing 0.3
g of Bondesil-PSA and 1.8 g of MgSO4. The sample was again manually mixed for 1 minute. The sample
extract was centrifuged, and a 1.25 mL aliquot of supernatant was transferred into a clean culture tube.
The sample aliquot was evaporated to near dryness on a Turbo-Vap. The extract was reconstituted with1.25 mL of 9:1 water/methanol containing 10 mM NH3HCO3 by vortexing, then transferred into a 2-mL
sample vial for high performance liquid chromatography/triple stage quadrupole mass spectrometry(LC/MS/MS) analysis.
For stone fruit nectar7, a 0.1-mL sample was weighed into a 20 x 150 culture tube and dissolved in 4 mLof water. If the total nectar sample was less than 0.1 mL, the entire sample was weighed and extracted.
The mixture was amended with isotopically labeled internal standards, and the resultant solution was
mixed well and applied to an Agilent BondElut Solid Phase Extraction (SPE) cartridge (50 mg resin;previously conditioned with methanol then water). The cartridge was washed with 1 mL ofwater/methanol (19:1 v/v), and the combined eluates were discarded. The analytes were extracted fromthe cartridge with 0.5 mL of water/methanol (4:1 v/v). The eluate was collected into a 2 mL sample vial
for analysis by LC/MS/MS.
For stone fruit pollen7, a 0.1-g sample was weighed into a small Precellys vial containing 2.8 mm steel
balls. If the available pollen sample amount was not sufficient for an analysis, samples of the sameinterval and trial site were composited and analyzed. The composite sample was weighed and extracted.
A 1-mL portion of water/methanol (3:1 v/v) was added, and the mixture was homogenized at 5000
beats/minute for 1 minute on a Precellys homogenizer. The isotopically labeled internal standards wereadded and mixed, and the mixture was centrifuged at 12,000 rpm for 2 minutes. The supernatant wastransferred into a clean culture tube containing 2.5 mL of water and was evaporated to an aqueous
remainder, then applied to a 3-mL ChemElut SPE cartridge. After 10 to 15 minutes, the cartridge waswashed with 4 mL of hexane/ethyl acetate (1:1 v/v) three times into a clean culture tube. The combined
eluates were evaporated to dryness. The analytes were dissolved in 0.5 mL of water/methanol (4:1 v/v).
The solution was transferred into a 2 mL sample vial for analysis by LC/high resolution mass
spectrometry (LC/HRMS).
Quantitation of each analyte was based on the daughter ion transitions of the analyte and therespective internal standard analog. The responses of the LC/MS/MS and LC/HRMS systems to eachanalyte and its internal standard were measured in samples and in standards, and a relative response709

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 49819401

CDPR IMI Stone Fruit

was calculated (as the ratio of the analyte and the stable isotopically labeled internal standard
responses). The relative response of the analyte in each sample was compared to the relative responseof the analyte in the standards.
The relative responses of imidacloprid and its analytes were measured over the range of 0.12 to 2000
ppb. The analyte relative responses were fit to a linearity curve calculated using linear regression
analysis with 1/x weighting. Correlation coefficients were calculated with the same software. All data
are reported in parent equivalents, and the individual measured residues of imidacloprid, 5-hydroxyimidacloprid, and imidacloprid olefin are summed to give a total imidacloprid residue.
5F. QUALITY ASSURANCE RESULTS
No total imidacloprid residue was calculated for the UTC samples, so individual analyte levels aredescribed. Imidacloprid (parent) residues in UTC stone fruit pollen ranged from below the analyte LOD
to 29 ppb (trial NT023-13ZA, peach). Imidacloprid residues in UTC stone fruit nectar ranged from below
the analyte LOD to 1.0 ppb (trial NT024-13ZA, cherry). Imidacloprid residues in UTC stone fruit leaves
ranged from below the analyte LOD to 1.0 ppb (trial NT023-13ZA, peach). 5-Hydroxy imidacloprid
residues in UTC stone fruit pollen ranged from below the analyte LOD to 32 ppb (trial NT023-13ZA,
peach). 5-Hydroxy imidacloprid residues in UTC stone fruit nectar ranged from below the analyte LOD to
31 ppb (trial NT026-13ZA, apricot). 5-Hydroxy imidacloprid residues in UTC stone fruit leaves were all
below the analyte LOD. Imidacloprid olefin residues in UTC stone fruit pollen ranged from below theanalyte LOD to 190 ppb (trial NT022-13ZA, plum). Imidacloprid olefin residues in UTC nectar and leaves
were all below the analyte LOD.
All recoveries were corrected for any interferences in corresponding controls. The overall means of therecoveries for each matrix at each fortification level were within the range of 70 to 120%, and thestandard deviation values were below 20%.
The limit of quantitation (LOQ) is defined as the lowest fortification level of an analyte at which
acceptable recovery has been achieved. The LOQ for a total residue is the highest of the LOQ values
assigned to the individual analytes for a particular matrix.
The limit of detection (LOD) is defined as the lowest concentration of an analyte that can be determined
to be statistically different from a blank. The LODs were determined from method validation dataobtained from control samples fortified at the respective analyte LOQs. The LODs were calculated bymultiplying the standard deviation of recovery measurements at the LOQ by t0.99 [where t0.99 is the onetailed t-statistic at the 99% confidence level for the number of replicates (n)].8 The LOD for the total
imidacloprid residue in each matrix is the highest LOD value of any one individual analyte for thatparticular matrix.

710Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 49819401

Table 2. Summary of LOQs and LODs
MatrixAnalyteImidacloprid
Cherry, plum,
5-hydroxy Imidacloprid
apricot, andImidacloprid olefin
peach leaves
Total Imidacloprid
Cherry, plum,
Imidacloprid
apricot, and5-hydroxy Imidacloprid
peach nectar
Imidacloprid olefin
Total Imidacloprid
Cherry, plum,
Imidacloprid
apricot, and5-hydroxy Imidacloprid
peach pollen
Imidacloprid olefin
Total Imidacloprid

CDPR IMI Stone Fruit
LOQ (ppb, parent equivalents)
5.0
5.0
5.0
5.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0

LOD (ppb, parent equivalents)
0.5
0.4
1.6
1.6
0.3
0.7
0.6
0.7
0.4
0.5
0.3
0.5

6. RESULTS:
The imidacloprid residue data for cherry, plum, apricot, and peach (stone fruit) leaves, nectar, andpollen are provided in Table 3. Only residue values above the respective analyte LODs are reported. Any
residue value that was below the LOD is reported as less than the LOD (<LOD). The total imidacloprid
residue is a sum of the analyte residue values that are greater than the respective analyte LODs. If theanalyte value was less than the LOD, a default value equal to half of the analyte LOD (half-LOD) was
added into the sum. For samples with a reported total residue of less than LOD, the sum of the analytehalf-LOD values for the respective matrix was used to calculate the average residue values.

Imidacloprid Olefin
Residues (ppb)

Imidacloprid Residue
(ppb)

1.6
8.6
2.8

0.5
164.1
40.3

CherryBrooksCoarse

0.50

711

323

Total Imidacloprid Residue(ppb)

5-Hydroxy Imidacloprid
Residue (ppb)

Coarse

0.4
26.4
6.8

Total Rate, lb. a.i./acre

CherryBrooks

LODs (ppb):
0.50
174

Soil TypeDAA (Days After the Last
Application)

Stone Fruit Leaves
NT024-13ZA
Kerman,California
Region 10,
2014NT024-13ZA
Kerman,California
Region 10,
2015

Crop and Variety

Location (City, State,
NAFTA Region, SamplingYear)

Trial Identification

Table 3. Imidacloprid Residue Data from Cherry, Plum, Apricot and Peach

1.6
199.1
49.9
Avg.: 124.5

25.6
25.9

12.8
13.4

148.8
148.3

187.1
187.6
Avg.:
187.35

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

NT021-13ZA

NT022-13ZA

NT022-13ZA

NT026-13ZA

Total Imidacloprid Residue(ppb)

Coarse

0.50

168

17.7
22.0

9.4
9.9

204.4
245.5

CherryBrooksCoarse

0.50

292

21.0
22.6

14.1
18.2

146.2
160.0

CherryBrooks

Fine

0.50

182

<LOD
<LOD

<LOD
<LOD

2.6
4.2

Plum
French

Fine

0.50

249

1.6
2.3

5.3
5.6

13.8
19.7

20.7
27.6
Avg.: 24.15

Plum
French

Fine

0.50

180

1.0
1.9

2.1
3.6

2.3
6.4

5.4
11.9
Avg.: 8.65

Plum
French

Fine

0.50

243

1.9
3.4

6.7
8.2

9.2
18.8

17.7
30.4
Avg.: 24.05

Plum
FrenchMedium

0.50

180

0.5
<LOD

<LOD
<LOD

5.6
0.6

6.9
1.6
Avg.: 4.25

Plum
FrenchMedium

0.50

243

0.8
0.4

4.7
<LOD

4.8
4.5

10.4
5.7
Avg.: 8.05

Apricot
Castlebrite

Coarse

0.50

174

3.0
2.1

8.4
7.7

28.7
20.0

40.1
29.8
Avg.: 34.95

Soil Type

CherryBrooksCrop and Variety

Imidacloprid Residue(ppb)

NT021-13ZA

Imidacloprid Olefin
Residues (ppb)NT020-13ZA

5-Hydroxy Imidacloprid
Residue (ppb)NT020-13ZADAA (Days After the Last
Application)

NT025-13ZA

Kerman,California
Region 10,
2014
Kerman,California
Region 10,
2015
Merced,California
Region 10,
2014
Merced,California
Region 10,
2015
Yuba City,
California
Region 10,
2014
Yuba City,
California
Region 10,
2015
Yuba City,
California
Region 10,
2014
Yuba City,
California
Region 10,
2015
Sanger,California
Region 10
2014

CDPR IMI Stone Fruit
Total Rate, lb. a.i./acreNT025-13ZA

Location (City, State,
NAFTA Region, SamplingYear)

Trial Identification

MRID 49819401

712

231.4
277.5
Avg.:
254.45
181.3
200.8
Avg.:
191.053.6
5.2
Avg.: 4.4

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

Imidacloprid Olefin
Residues (ppb)

Imidacloprid Residue(ppb)

Total Imidacloprid Residue(ppb)

5-Hydroxy Imidacloprid
Residue (ppb)

Coarse

0.50

309

0.8
1.1

3.6
5.4

12.5
15.5

16.8
21.9
Avg.: 19.35

Peach
Flavor
Crest

Fine

0.50

155

0.5
0.5

4.6
5.2

18.6
23.0

23.6
28.6
Avg.: 26.1

Peach
Flavor
Crest

Fine

0.50

230

1.2
0.7

7.7
6.2

4.0
4.1

12.9
11.1
Avg.: 12

Peach
BountyMedium

0.50

159

<LOD
<LOD

<LOD
<LOD

6.4
11.2

7.4
12.2
Avg.: 9.8

Peach
BountyMedium

0.50

230

0.6
0.5

3.3
2.5

6.2
4.8

10.0
7.8
Avg.: 8.9

Peach Late
Ross

Medium

0.51

188

16.4
8.9

21.8
13.2

56.0
27.5

94.2
49.6
Avg.: 71.9

Peach Late
Ross

Medium

0.51

264

5.6
9.5

10.7
18.7

17.8
28.5

34.1
56.7
Avg.: 45.4

CherryBrooksCoarseLODs (ppb):
0.50
160

0.7
1.6
0.9

0.6
<LOD
<LOD

0.3
5.5
3.9

0.7
7.4
5.1
Avg.: 6.25

CherryBrooksCoarse

0.50

1.1
0.9

<LOD
0.8

5.9
7.1

7.3
8.8
Avg.: 8.05

Soil Type

Apricot
Castlebrite

Crop and Variety

DAA (Days After the Last
Application)

Sanger,California
Region 10
2015
NT019-13ZA Stockton,
California
Region 10,
2014NT019-13ZA Stockton,
California
Region 10,
2015NT023-13ZA Yuba City,
California
Region 10,
2014NT023-13ZA Yuba City,
California
Region 10,
2015NT027-13ZA Kingsburg,
California
Region 10
2014NT027-13ZA Kingsburg,
California
Region 10
2015
Stone Fruit Nectar
NT024-13ZA
Kerman,California
Region 10,
2014NT025-13ZA
Kerman,California
Region 10,

CDPR IMI Stone Fruit
Total Rate, lb. a.i./acreNT026-13ZA

Location (City, State,
NAFTA Region, SamplingYear)

Trial Identification

MRID 49819401

713

152

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

NT022-13ZA

NT022-13ZA

NT026-13ZA

NT026-13ZA

Total Imidacloprid Residue(ppb)

Coarse

0.50

279

1.2
1.5

<LOD
<LOD

5.9
8.9

7.4
10.7
Avg.: 9.05

PlumFrench

Fine

0.50

156

<LOD
<LOD

<LOD
<LOD

<LOD
0.3

0.8
1.0
Avg.: 0.9

Plum
French

Fine

0.50

231

<LOD
<LOD

<LOD
<LOD

<LOD
<LOD

0.8
0.8
Avg.: 0.8

PlumFrench

Fine

0.50

155

<LOD
<LOD

<LOD
<LOD

0.4
<LOD

1.1
0.8
Avg.: 0.95

PlumFrench

Fine

0.50

223

<LOD
<LOD

<LOD
<LOD

<LOD
<LOD

0.8
0.8
Avg.: 0.8

PlumFrenchMedium

0.50

155

<LOD
<LOD

<LOD
<LOD

0.4
0.3

1.0
1.0
Avg.: 1.0

Plum
FrenchMedium

0.50

223

<LOD
<LOD

<LOD
<LOD

<LOD
<LOD

0.8
0.8
Avg.: 0.8

Apricot
Castlebrite

Coarse

0.50

152

28.7
28.8

0.8
1.4

2.5
3.4

31.9
33.6
Avg.: 32.75

Apricot
Castlebrite

Coarse

0.50

291

<LOD
<LOD

<LOD
0.7

1.0
0.8

1.7
1.8
Avg.: 1.75

Soil Type

CherryBrooksCrop and Variety

Imidacloprid Residue(ppb)

NT021-13ZA

Imidacloprid Olefin
Residues (ppb)NT021-13ZA

5-Hydroxy Imidacloprid
Residue (ppb)NT020-13ZADAA (Days After the Last
Application)

NT020-13ZA

2014
Kerman,California
Region 10,
2015
Merced,California
Region 10,
2014
Merced,California
Region 10,
2015
Yuba City,
California
Region 10,
2014
Yuba City,
California
Region 10,
2015
Yuba City,
California
Region 10,
2014
Yuba City,
California
Region 10,
2015
Sanger,California
Region 10
2014
Sanger,California
Region 10

CDPR IMI Stone Fruit
Total Rate, lb. a.i./acreNT025-13ZA

Location (City, State,
NAFTA Region, SamplingYear)

Trial Identification

MRID 49819401

714

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

5-Hydroxy Imidacloprid
Residue (ppb)

Imidacloprid Olefin
Residues (ppb)

Imidacloprid Residue(ppb)

Total Imidacloprid Residue(ppb)

DAA (Days After the Last
Application)

Peach
Flavor
Crest

Fine

0.50

133

7.5
5.5

0.8
0.7

1.2
0.6

9.6
6.7
Avg.: 8.15

Peach
Flavor
Crest

Fine

0.50

214

<LOD
<LOD

3.5
<LOD

<LOD
<LOD

4.0
0.8
Avg.: 2.4

Peach
BountyMedium

0.50

141

<LOD
<LOD

0.7
0.8

0.6
0.8

1.6
1.9
Avg.: 1.75

Peach
BountyMedium

0.50

211

<LOD
<LOD

<LOD
<LOD

<LOD
<LOD

0.8
0.8
Avg.: 0.8

Peach Late
Ross

Medium

0.51

145

2.3
1.9

0.9
0.7

0.6
0.5

3.8
3.0
Avg.: 3.4

Peach Late
Ross

Medium

0.51

246

1.0
0.7

0.8
<LOD

<LOD
<LOD

1.9
1.2
Avg.: 1.55

CherryBrooksCoarseLODs (ppb):
0.50
160

0.5
1.2
3.0

0.3
0.4
1.3

0.4
11.8
12.0

0.5
13.4
16.6
Avg.: 15

CherryBrooksCoarse

0.50

309

<LOD
<LOD

0.6
<LOD

5.1
5.4

5.9
5.8
Avg.: 5.85

CherryBrooksCoarse

0.50

152

3.9
1.3

2.9
0.7

18.5
18.6

25.3
20.5

Soil Type

Total Rate, lb. a.i./acre2015NT019-13ZA Stockton,
California
Region 10,
2014NT019-13ZA Stockton,
California
Region 10,
2015NT023-13ZA Yuba City,
California
Region 10,
2014NT023-13ZA Yuba City,
California
Region 10,
2015NT027-13ZA Kingsburg,
California
Region 10
2014NT027-13ZA Kingsburg,
California
Region 10
2015
Stone Fruit Pollen
NT024-13ZA
Kerman,California
Region 10,
2014NT024-13ZA
Kerman,California
Region 10,
2015NT025-13ZA
Kerman,California

CDPR IMI Stone Fruit

Crop and Variety

Location (City, State,
NAFTA Region, SamplingYear)

Trial Identification

MRID 49819401

715

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

NT025-13ZA

NT020-13ZA

NT020-13ZA

NT021-13ZA

NT021-13ZA

NT022-13ZA

NT022-13ZA

NT026-13ZA

NT026-13ZA

Region 10,
2014
Kerman,California
Region 10,
2015
Merced,California
Region 10,
2014
Merced,California
Region 10,
2015
Yuba City,
California
Region 10,
2014
Yuba City,
California
Region 10,
2015
Yuba City,
California
Region 10,
2014
Yuba City,
California
Region 10,
2014
Sanger,California
Region 10
2014
Sanger,California

Total Imidacloprid Residue(ppb)

Imidacloprid Residue(ppb)

Imidacloprid Olefin
Residues (ppb)5-Hydroxy Imidacloprid
Residue (ppb)

Soil Type

Total Rate, lb. a.i./acre

DAA (Days After the Last
Application)

CDPR IMI Stone Fruit

Crop and Variety

Location (City, State,
NAFTA Region, SamplingYear)

Trial Identification

MRID 49819401

Avg.: 22.9
CherryBrooksCoarse

0.50

279

0.8
1.4

0.9
1.9

8.3
12.4

10.0
15.7
Avg.: 12.85

PlumFrench

Fine

0.50

156

2.1
4.0

0.6
1.7

53.6
108.7

56.3
114.4
Avg.: 85.35

Plum
French

Fine

0.50

231

<LOD
<LOD

<LOD
<LOD

1.9
28.0

2.3
28.4
Avg.: 15.35

Plum
French

Fine

0.50

155

1.3

0.5

33.5

35.3
Avg.: 35.3

Plum
French

Fine

0.50

223

0.5
1.1

0.7
4.0

3.2
7.5

4.4
12.6
Avg.: 8.5

PlumFrenchMedium

0.50

155

1.1

0.3

29.6

31.0
Avg.: 31.0

PlumFrenchMedium

0.50

223

<LOD

<LOD

3.1

3.5
Avg.: 3.5

Apricot
Castlebrite

Coarse

0.50

152

1.7
1.4

1.3
1.0

49.4
27.3

52.5
29.8
Avg.: 41.15

Apricot
Castlebrite

Coarse

0.50

291

<LOD

<LOD

2.7

3.1
Avg.: 3.1

716

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

NT027-13ZA

Total Imidacloprid Residue
(ppb)

Fine

0.50

133

9.4
3.7

3.9
2.0

328.0
127.7

Peach
Flavor
Crest

Fine

0.50

214

2.4
0.8

3.7
1.8

38.4
52.3

Peach
BountyMedium

0.50

141

1.0

1.3

54.7

57.0
Avg.: 57.0

Peach
BountyMedium

0.50

211

2.6
2.8

14.3
40.3

122.1
144.7

139.0
187.8
Avg.: 163.4

Peach Late
Ross

Medium

0.51

145

3.7

2.1

27.8

33.6
Avg.: 33.6

Peach Late
Ross

Medium

0.51

246

1.9
<LOD

3.3
2.7

3.2
3.8

8.4
6.7
Avg.: 7.55

Soil Type

Peach
FlavorCrest

Crop and Variety

Imidacloprid Residue(ppb)

NT027-13ZA

Imidacloprid Olefin
Residues (ppb)NT023-13ZA

5-Hydroxy Imidacloprid
Residue (ppb)NT023-13ZADAA (Days After the Last
Application)

NT019-13ZA

Region 10
2015
Stockton,
California
Region 10,
2014
Stockton,
California
Region 10,
2015
Yuba City,
California
Region 10,
2014
Yuba City,
California
Region 10,
2015
Kingsburg,
California
Region 10
2014
Kingsburg,
California
Region 10
2015

CDPR IMI Stone Fruit
Total Rate, lb. a.i./acreNT019-13ZA

Location (City, State,
NAFTA Region, SamplingYear)

Trial Identification

MRID 49819401

717

341.3
133.4
Avg.:
237.3544.5
54.9
Avg.: 49.7

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 49819401

CDPR IMI Stone Fruit

7. STATISTICAL ANALYSIS

Study Objectives and Design
The objective of the study was to determine the concentration of imidacloprid and its degradation
products, 5-Hydroxy imidacloprid and imidacloprid olefin, in leaves, nectar, and pollen of stone fruit
trees in response to one soil and two foliar applications of a imidacloprid pesticide product applied in
the previous year. The two-year study had applications made in 2013 and 2014 and plant matrices
harvested in the respective following years of 2014 and 2015. Trees received one soil application applied
at 0.38 lbs imidacloprid per acre and two foliar applications ranging between 0.058 and 0.064 lbs
imidacloprid per acre. The first foliar application was made 3 to 7 days after the soil application and thesecond foliar spray was applied 7 to 11 days after the first foliar application. In 2014, flower samples
were collected 133 to 160 days after the last foliar application and in 2015, flower samples werecollected 211 to 309 days after the last foliar application. In 2014, leaf samples were collected 155 to
188 days after the last foliar application and in 2015, sampling ranged from 230 to 323 days after thelast foliar application. As specified in the data call-in, the study was conducted at 9 separate sites withthe study being replicated at each site in the next year. Two composite samples were collected fromeach treated plot for each plant matrix. Untreated control plots were also included at each site with only
one composite sample taken at the same time sampling was occurring for treated plots.
Non-parametric statistical tests were used to test for differences in distribution of concentrations
between years, untreated control to treated plants, extra floral nectar concentration between samplingintervals, and between soil type. Non-parametric tests do not require tests for normality as they arerobust to differences in distribution and they are also robust for experimental designs with low
replicates (Helsel and Hirsch, 2002). The PROC NPAR1WAY procedure in the Statistical Analysis System(SAS) statistical package was used to conduct Wilcoxon-Mann –Whitney (Wilcoxon), Median nonparametric, and Kuiper tests. A significant result from the Wilcoxon test indicates differences in the
shape of distributions; A significant result from the Median test indicates differences in the location of
the medians between distributions; and A significant result from the Kuiper test indicates differences inthe empirical distributions between two groups. The Exact option for each statistic was implemented as
it provides permutation testing, a statistical method that minimizes the effect of sample size anddistributional differences. Using the Exact option, the Monte Carlo procedure was also implemented
which provided 10,000 separate runs for each statistic to produce the permutation distributions. The
test for potential differences in soil type had 3 levels so the DSCF option in PROC NPAR1WAY, which
invokes the Dwass, Steel, Critchlow-Fligner multiple comparison test, was used to provide pairwise tests
for two-sample rankings.
Additional procedures used for descriptive statistics were PROC MEANS to calculate mean values fromthe replicates at each site, PROC CAPACITY to produce cumulative statistics, and PROC BOXPLOT toproduce comparative graphics. Data from treated sites were averaged to test for the effects of years,
soil type, and to compare to untreated control plots where only 1 replicate sample was available.
Graphical comparisons are presented with data transformed to a natural logarithm scale, providing
clearer contrasts between the distributions. Although both limits of detection (LOD) and limits ofquantification (LOQ) were indicated, only data less than the LOD were indicated as <LOD in the data set.
Values were provided when samples were between the LOD and LOQ. For statistical analyses, values
noted as below the limit of detection (LOD) were assigned half the value of the respective detection
limit (Table 2). Values between the LOD and LOQ were used as reported. Results were reported in ppm
on a wet weight/weight basis. The distribution of concentrations in bee relevant plant matrices was718

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 49819401

CDPR IMI Stone Fruitcalculated using all the raw data because these values represent the actual range of exposure to bees
and other organisms that feed off the nectar and pollen of plants.

Detection rate noted for each plant matrix: Counts for the number of samples reported below therespective detection limit for each matrix are presented in Table 4 for treated plants and untreatedcontrol plants. The LOQ for the leaf matrix was at 5 ng/g (ppb) and at 1 ng/g (ppb) for nectar and pollen
matrices. For treated plants, the majority of concentrations for parent imidacloprid were above the LOQ
for leaves and pollen, but not for nectar. The percent of values above the LOQ for imidacloprid were
75% for leaves, 100% for pollen, and 26% for nectar. None of the olefin imidacloprid values were above
the LOQ, whereas, 5-Hydroxy imidacloprid values were measured above the LOQ but at lower frequency
than for the parent: 33%, 60%, and 29% for leaves, pollen and nectar, respectively. Figure 1 provides agraphical comparison for the relative range in concentrations measured between the residues in treatedplants. A few imidacloprid residues were reported above the LOQ in untreated control plants in leaf
(11%) and nectar (12%) matrices but nearly all samples for pollen were above the LOQ at 89% of thetotal number of samples taken. Except for pollen, results for the imidacloprid olefin and 5-Hydroxyimidacloprid metabolites were below the LOD. For pollen, no residues above the LOQ were reported for
imidacloprid olefin but 33% of the values were above the LOQ for the 5-Hydroxy imidacloprid
metabolite. Figure 2 provides a graphical comparison for the relative range in concentrations measured
between the residues in untreated control plants.
Comparison between years: Potential difference between years was measured to indicate the presenceof carry-over effects of residues between years for treated plants. Except for imidacloprid residue
measured in pollen, there was no statistical difference between years (Table 5, Difference Between
Years for Treated Plants heading; Figure 3). The significant result for imidacloprid residues in pollen
show a greater range in concentrations measured in the first year of the study. This pattern indicates nopotential for carry-over of residues due to the pattern of application used in this study.
Comparison between treated and untreated control plants: The distribution statistics for all
treatments are presented for leaves, nectar, and pollen in Table 6. Non-parametric tests conducted on
the mean of replicate samples for foliar treated plots indicated a significantly greater range inimidacloprid residues for treated plants compared to untreated control plants for leaf and pollen
matrices but not for nectar samples (Table 5, Treated vs UTC heading; Figure 4). The results for the
metabolites did not indicate a consistent difference between treated and untreated plants (Figures 5
and 6). The inconsistency was caused by the lower range in residues reported for the metabolites
coupled with a few detections in untreated plants that were above the LOQ. For example, a value of
190 ng/g was reported for imidacloprid olefin in pollen sampled from an untreated plant. The highest
value in treated plants was 40.3 ng/g. This disparity resulted in a wider range in values noted for
untreated plants.
Comparison of distribution between soil types: As reported in Table 3B of the report, the sites were
distributed among the three requested coarse, medium, and fine soil texture categories. There were 3
sites in each category. Results of non-parametric tests indicated that the range in concentrations forparent imidacloprid in leaves and nectar was greater in plants grown in coarse-textured soils than in the719

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 49819401

CDPR IMI Stone Fruitother two soil categories (Table 7; Figure 7). No other significant effects were observed for parent
imidacloprid in pollen samples or for the two metabolites in the plant matrices (Figures 7 and 8).

Magnitude of residues in bee-relevant matrices: The observed distributions derived from the individual
analyses ostensibly determines the expected range in concentrations of imidacloprid residues in bee
relevant plant samples for the combination of plant species and application scenario tested in this study(Table 6 Treated Plants heading). The median and maximum values for total residue in nectar were 1.7
and 33.6 ng/g, respectively, on a wet weight basis. For pollen, median and maximum values were higher
at 26.9 and 341.3 ng/g, respectively.
8. STUDY STRENGTHS, LIMITATIONS AND CONCLUSIONSIn the context of documenting the magnitude of imidacloprid residues in bee-related matrices of stone
fruit, the following strengths are observed with this study.
1.
Data provide quantitative values of total imidacloprid residues expected in pollen, nectar, andleaves of various varieties of stone fruit.
2.
The study was replicated over two years with measurements in plant samples taken at a meanof 158 days after the last application in 2014 and at a mean of 251 days after the last application in
2015.
Limitations noted in this study include:
1.
Samples were taken from a variety of stone fruits (cherry, peach, plum, or apricot). Since theeffect of different varieties on distribution of residues is unknown, the results will reflect generalobservations made to all stone fruits.
2.
Leaf and flower samples were collected at different times, so establishing correlations between
concentrations in leaves and flowers (nectar and pollen) is not possible.
Overall, considering the strengths and limitations of this study, the following conclusions can be drawn:
1.
Bee-relevant matrices: Significant concentrations of imidacloprid residues were measured inpollen samples taken in the year following one soil and two foliar spray applications. For example, theaverage concentration of total imidacloprid residues in pollen at one of the sites was 160 ng/g forsamples taken 211 days after the last application made in the previous year. Maximum concentrations inpollen was measured at 341 ng/g with a median value of 27 ng/g for 30 samples. Concentrations in
nectar were lower with many samples reported at the LOD for each residue. The maximum nectar
concentration was 34 ng/g with a median value of 2 ng/g for 34 samples.
2.
Soil type: An effect of soil type was measured but it was inconsistent between the plant
matrices: Higher concentrations were indicated for leaves and nectar of plants sampled from coarsetextured soil sites but no effect was observed for pollen. The range in concentrations for the residues
was similar between plants grown in medium and fine-textured soil sites.
720

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 49819401

CDPR IMI Stone Fruit

3.
The untreated control plots were contaminated with imidacloprid residues. Elevatedconcentrations of imidacloprid and metabolite residues were measured in untreated control plants. For
example, two pollen samples collected in 2015 from untreated plots had higher total imidacloprid
residue values than from their corresponding treated plot. In site NT020-13ZA-H004, which was a plumorchard, the total imidacloprid residue in the untreated plot in 2015 was 246.4 ng/g (32.1 IMI-5-OH,
187.3 IMI Olefin, and 27.0 ng/g parent imidacloprid), whereas total residue from the replicate samples
from the treated plot was 31.0 ng/g (0.3 IMI-5-OH, 1.1 IMI Olefin, and 29.6 ng/g parent imidacloprid)and 3.5 ng/g (0.15 IMI-5-OH, 0.25 IMI Olefin, and 3.1 ng/g parent imidacloprid). It is unclear how thiscontamination occurred.

9. STUDY VALIDITY/CLASSIFICATIONClassification/Utility for Bee Risk Assessment. This study is classified as acceptable. It provides anaccurate assessment of Imidacloprid (and its metabolites) residues in leaves, pollen, and nectar during
bloom for stone fruit trees under the exposure and cultural conditions used in this study. The study was
conducted using the maximum annual application rate (0.5 lbs ai/A) and residue values presented
should be considered reliable. The label for Admire Pro Systemic Protectant prohibits soil applications tostone fruit pre-bloom, during bloom, or when bees are foraging, but this restriction does not specify theamount of time before bloom that applications are prohibited.
Temporal Variability in Residues. This study was not designed for temporal analysis of declining
concentrations, but rather, to provide an annual snapshot of residue concentrations during flowering.
Only one sample per year of each matrix was collected and analyzed so there is no way to know the rate
at which concentrations were decreasing.
Spatial Variability in Residues. All nine sites were located in the Central Valley of California. The
southernmost sites were located just north of Visalia (Kingsburg) and the northernmost sites were in
Yuba City. Climatic conditions were similar across all nine sites. Peaches, of the varieties Flavor Crest,
Bounty, and Late Ross were grown in Stockton (NT019-13ZA), Yuba City (NT023-13ZA), and Kingsburg(NT027-13ZA), respectively. Brooks variety cherries were grown in two sites in Kerman (NT025-13ZA andNT024-13ZA). French variety plums were grown in fine soil in Merced (NT020-13ZA) and in fine (NT02113ZA) and medium soil (NT022-13ZA) in Yuba City. Castlebright variety apricots were grown in Sanger(NT026-13ZA). Concentrations of total Imidacloprid in nectar were highest in Apricots grown in Sanger in2014 (33 µg/g), but in 2015 the same trees had concentrations of only 2 ng/g. Concentrations of total
Imidacloprid in pollen in the same trees (Apricots in Sanger) followed a similar pattern, with an averageof 41 µg/g in 2014 and an average of 3 µg/g in 2015. The study authors did not offer any explanations as
to why total imidacloprid concentrations would vary so widely in the same trees in two consecutive
years. The interval from the time of the last application to the time that samples were collected was
longer in 2015, but it the difference in these intervals was similar for the other sites and none of theother sites had such a drastic change in residue levels from year to year. This introduces significant
uncertainty to the results. Total Imidacloprid residues in pollen, nectar, and leaves in other sites weresimilar to each other, and all of the other nectar samples in all other stone fruits and locations contained
10.7 µg/g or less of total imidacloprid. The highest concentration in pollen was 341 ng/g from peaches
grown in Stockton (NT019-13ZA), but that was only one individual sample, and all the other samples
contained less than 200 µg/g, with most of those below 100 ng/g. Leaf samples were collected at
different times than flower (i.e., pollen and nectar) samples, so the concentrations in leaves cannot becorrelated with concentrations in pollen and nectar.
721

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 49819401

CDPR IMI Stone Fruit

Table 4. Counts of chemical analytical results for parent imidacloprid and 5-Hydroxy imidacloprid and imidacloprid olefin
metabolites that were indicated as above the LOQ, between the LOQ and LOD, and below the LOD (Table 2) for treated anduntreated control plants.

Comparison of Total Number of Samples Reported Above the LOQ, Between the LOQ and LOD, and Below the LOD

Treatment
andPlant
SampleTotal
NumberImidacloprid
NumberNumberAbove
Between
LOQ
LOD and
LOQ

Number
Below
LOD

Total
Number5-Hydroxy Imidacloprid
NumberNumberNumberAbove
BetweenBelow
LOQ
LOD and
LOD
LOQ

Total
NumberOlefin Imidacloprid
NumberNumberAbove
Between
LOQ
LOD and
LOQ

NumberBelow<LOD

Treated Plants: Foliar Application
Leaf
36
27
Nectar34
9Pollen
30
30

9
11

0
14

36
34

12
10

19
4

5
20

36
34

0
0

29
12

7
22

0

0

30

18

4

8

30

0

25

5

Untreated Control Plants
Leaf
18
Nectar17
Pollen
18

2
2

2
4

14
11

18
17

0
1

0
0

18
16

18
17

0
0

0
0

18
17

16

2

0

18

6

4

8

18

0

7

11

722

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 49819401

CDPR IMI Stone Fruit

Table 5. Statistical results for test of differences in concentration of parent imidacloprid and 5-Hydroxy imidacloprid andimidacloprid olefin metabolites measured between replicate years for treated plants and between treated and untreatedcontrol plants.

Exact Probability Levels for Wilcoxon, Median, and Kuiper Tests
Comparing Concentrations Between Years
Imidacloprid
Comparison and
Plant Matrix
Wilcoxon Median Kuiper
Difference Between Years for Treated Plants
Leaves1
0.98
0.89
Nectar
0.4
0.16
0.04
Pollen
0.03
0.06
0.26
Treated vs Untreated Plants
Leaves
0.001
0.01
0.003
Nectar
0.16
0.18
0.95
Pollen
0.001
0.02
0.08

5-Hydroxy Metabolite
Wilcoxon Median Kuiper

Olefin Metabolite
Wilcoxon Median Kuiper

0.37
0.14
0.02

1
0.33
0.09

0.57
0.93
0.66

0.34
0.4
0.98

1
0.63
1

0.89
0.91
0.26

0.001
0.04
0.02

0.001
0.04
0.09

0.003
0.53
0.08

0.001
0.003
0.14

0.001
0.003
0.09

0.008
0.3
0.03

723

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 49819401

CDPR IMI Stone Fruit

Table 6. Distributional statistics for concentration of parent imidacloprid (IMI) and 5-Hydroxy imidacloprid (5-OH) andimidacloprid olefin (Olefin) metabolites and total residue (Total) in leaves, nectar, and pollen of stone fruit trees treated withimidacloprid in the previous year and in untreated control plants. Acronyms in the table are; N=NUMBER OF OBSERVATIONS;SD=STANDARD DEVIATION; CV= COEFFICIENT OF VARIATION.
Leaves5-OH
Olefin

Total

36.0
6.9
5.5
80.0
0.8

Statistic
IMI
Treated Plants
N
36.0
36.0
Mean (ng/g)
45.4
6.5
SD (ng/g)
66.8
9.0
CV (%)
148.0
138.0
Min (ng/g)
0.6
0.2
Median(ng/g)
16.7
1.8
75th (ng/g)
34.5
9.2
90th (ng/g)
160.0
22.6
95th (ng/g)
204.4
25.9
Max (ng/g)
245.5
26.4
% of Total
77.1
11.0
Untreated Control Plants
N
18
18
Mean (ng/g)
1.3
0.2
SD (ng/g)
2.90.0
CV (%)
220.0
0.0
Min (ng/g)
0.3
0.2
Median(ng/g)
0.3
0.2
75th (ng/g)
0.3
0.2
90th (ng/g)
6.4
0.2
95th (ng/g)
11.2
0.2
Max (ng/g)
11.2
0.2% of Total
56.5
8.7

IMI

Nectar5-OH Olefin

IMI

Pollen5-OH Olefin

Total

Total

36.0
58.9
78.4
133.0
1.6

34
1.6
2.4
153.0
0.2

34
2.7
6.8
255.0
0.4

34
0.6
0.6
104.0
0.3

34
4.8
7.7
161.0
0.8

30.0
44.8
66.8
149.0
1.9

30.0
1.8
1.9
105.0
0.3

30.0
3.2
7.5
236.0
0.2

30.0
49.7
71.7
144.0
2.3

5.5
9.7
14.1
18.7
21.8
11.7

22.9
53.3
199.1
231.5
277.4

0.4
1.2
5.9
7.1
8.9
32.3

0.4
1.2
5.5
28.7
28.8
55.4

0.3
0.7
0.8
1.4
3.5
11.7

1.7
6.8
9.5
32.0
33.6

23.0
52.3
124.9
144.7
328.0
90.1

1.3
2.6
3.8
4.0
9.4
3.6

1.3
2.7
4.0
14.3
40.3
6.4

26.9
54.9
136.2
187.8
341.3

18
0.8
0.0
0.0
0.8

18
2.3
2.9
125.0
1.3

17
0.9
1.8
196.0
0.2

17
2.1
7.4
345.0
0.4

17
0.3
0.0
0.0
0.3

17
3.4
7.6
227.0
0.8

18
6.5
8.5
131.0
0.6

18
4.2
9.8
234.0
0.3

18
22.5
60.6
269.0
0.2

18
33.2
73.8
222.0
1.0

0.8
0.8
0.8
0.8
0.8
34.8

1.3
1.3
7.4
12.2
12.2

0.2
0.5
4.8
6.2
6.2
26.5

0.4
0.4
0.4
30.9
30.9
61.8

0.3
0.3
0.3
0.3
0.3
8.8

0.8
1.2
6.9
32.1
32.1

3.1
6.5
27.0
28.9
28.9
19.6

0.6
1.7
30.1
32.1
32.1
12.7

0.2
2.7
187.3
190.0
190.0
67.8

5.7
14.8
220.9
246.4
246.4

724

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 49819401

CDPR IMI Stone Fruit

Table 7. Statistical results for test of differences in concentration of parent imidacloprid and5-Hydroxy imidacloprid (5-Hydroxy) and imidacloprid olefin (Olefin) metabolites measured
between plants grown in coarse, medium, and fine-textured soils.

Treatment,
Plant Matrix, andSpecific Soil
Contrasts
Treated Plants
Leaves - Overall
Fine vs. Medium
Fine vs. Coarse
Medium vs.
CoarseNectar - Overall
Fine vs. Medium
Fine vs. Coarse
Medium vs.
CoarsePollen - Overall
Fine vs. Medium
Fine vs. Coarse
Medium vs.
CoarseUntreated Plants
Leaves - Overall

Exact Probability Levels for Nonparametric Tests of Differences
Between Soil Type
Imidacloprid 5-Hydroxy
OlefinWilcoxon
Wilcoxon
Wilcoxon
0.001

0.02
1.00
0.064

0.22
0.94
0.13

0.002
0.96
0.016

0.064
0.15
0.91
0.21

0.50
0.93
0.96
0.92

0.016
0.22
0.70
0.18

0.28
0.66
0.92
0.70

0.99
0.57
0.97
0.60

0.60

0.75

0.65

0.27
0.58

1.00
1.00
1.00

1.00
1.00
1.00

1.00
0.29
1.00
0.52

1.00
1.00
1.00
1.00

0.50
0.92

0.52
0.75
1.00
0.82

1.00
0.47
1.00
0.55

0.50

0.73

0.43

0.88
0.03450.0431

0.25

Fine vs. Medium
Fine vs. Coarse
Medium vs.
CoarseNectar - Overall

0.48

0.32

Fine vs. Medium
Fine vs. Coarse
Medium vs.
CoarsePollen - Overall
Fine vs. Medium
Fine vs. Coarse
Medium vs.
Coarse

0.33
0.55
0.87
0.43

725

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
Figure 1. Treated Plants: Comprison of the relative range in concentrations for parent imidacloprid
and 5-Hydroxy imidacloprid and imidacloprid olefin metabolite residues in leaves, nectar, and pollen
samples from stone fruit trees exposed to imidacloprid treatments in the previous year. Values weretransformed to natural logarithms.

Page 23 of 33
726

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
Figure 2. Untreated Control Plants: Comparison of the relative range in concentrations for parent
imidacloprid and 5-Hydroxy imidacloprid and imidacloprid olefin metabolite residues in leaves, nectar,
and pollen samples from stone fruit trees grown in untreated control plots. Values were transformed
to natural logarithms.

Page 24 of 33
727

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
Figure 3. Treated Plants: Comparison of the relative range in concentrations between replicate years
for midacloprid residues in leaves, nectar, and pollen samples obtained from stone fruit trees treatedwith imidacloprid in the previous year. Values were transformed to natural logarithms.

Page 25 of 33
728

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
Figure 4. Treated vs Untreated Control Plants: Comparison of the relative range in imidacloprid
concentrations between treated and untreated control plants for residues measured in leaves, nectar,
and pollen samples obtained from stone fruit trees treated with imidacloprid in the previous year.
Values were transformed to natural logarithms.

Page 26 of 33729

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
Figure 5. Treated vs Untreated Control Plants: Comparison of the relative range in 5-Hydroxyimidacloprid metabolite concentrations between treated and untreated control plants for residues
measured in leaves, nectar, and pollen samples obtained from stone fruit trees treated withimidacloprid in the previous year. Values were transformed to natural logarithms.

Page 27 of 33
730

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
Figure 6. Treated vs Untreated Control Plants: Comparison of the relative range in imidacloprid olefin
metabolite concentrations between treated and untreated control plants for residues measured inleaves, nectar, and pollen samples obtained from stone fruit trees treated with imidacloprid in theprevious year. Values were transformed to natural logarithms.

Page 28 of 33
731

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
Figure 7. Soil Type: Comparison of the relative range in imidacloprid concentrations between plants
grown in coarse, medium, and fine-textured soil types. Residues were measured in leaves, nectar, andpollen samples obtained from stone fruit trees treated with imidacloprid in the previous year. Values
were transformed to natural logarithms.

Page 29 of 33732

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
Figure 8. Soil Type: Comparison of the relative range in 5-Hydroxy imidacloprid metabolite
concentrations between plants grown in coarse, medium, and fine-textured soil types. Residues were
measured in leaves, nectar, and pollen samples obtained from stone fruit trees treated withimidacloprid in the previous year. Values were transformed to natural logarithms.

Page 30 of 33733

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
Figure 9. Soil Type: Comparison of the relative range in imidacloprid olefin metabolite concentrations
between plants grown in coarse, medium, and fine-textured soil types. Residues were measured inleaves, nectar, and pollen samples obtained from stone fruit trees treated with imidacloprid in theprevious year. Values were transformed to natural logarithms.

Page 31 of 33734

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 49819401

CDPR IMI Stone Fruit

10. REFERENCES

1. Brungardt, J. 2010. An Analytical Method for the Determination of Residues of Imidacloprid
and its Metabolites Imidacloprid Olefin and 5-Hydroxy Imidacloprid in Bee Relevant Matrices
Using LC/MS/MS. Bayer Method No. NT-005-P10-01.
2. Helsel, D.R. and R.M. Hirsch. 2002. Chapter A3: Statistical Methods in Water Resources.
Techniques of Water-Resources Investigations of the United States Geological Survey Book 4,
Hydrologic Analysis and Interpretation. United States Geological Survey.
3.

Hoelscher, A. 1998. Physical and chemical properties of the active ingredient imidacloprid.
Bayer CropScience Report No. MO-03-006885, M-105510-01-1.

4. Krohn, J. 1993. Vapour Pressure Curve of Imidacloprid. Bayer CropScience Laboratory Project
ID 14 200 0804. Report No. M-004042-01-1.
5. Lenz, C.A. 1993. Addendum 1. Imidacloprid and metabolites – freezer storage stability study incrops (wheat matrices, tomatoseed, tomato, cauliflower, and lettuce). Bayer CropScience
Report No. 103949-1. MRID 42810313.
6. Lenz, C.A. 1993. Addendum 2. Imidacloprid and metabolites – freezer storage stability study incrops (wheat matrices, tomatoseed, tomato, cauliflower, and lettuce). Bayer CropScience
Report No. 103949-2. MRID 43197201.
7. Lenz, C.A. 1993. Addendum 3. Imidacloprid and metabolites – freezer storage stability study incrops (wheat matrices, tomatoseed, tomato, cauliflower, and lettuce). Bayer CropScience
Report No. 103949-3. MRID 43487301.
8. Miller, A. 2014. An Analytical Method for the Determination of Residues of Imidacloprid and its
Metabolites Imidacloprid Olefin and 5-Hydroxy Imidacloprid in Bee Relevant Matrices Using
LC/MS/MS. Bayer CropScience Report Number: NT-006-A13-01.
9.

Noland, P.A. 1992. Imidacloprid and metabolites – freezer storage stability study in crops.
Bayer CropScience Report No. 103237. MRID 42556135.

10. Noland, P.A. 1993. Imidacloprid and metabolites – freezer storage stability study in crops.
Bayer CropScience Report No. 103237-1. MRID 42810311.
11. Noland, P.A. 1994. Imidacloprid and metabolites – freezer storage stability study in crops.
Bayer CropScience Report No. 103237-2. MRID 43197203.
12. Noland, P.A. 1994. Imidacloprid and metabolites – freezer storage stability study in crops.
Bayer CropScience Report No. 103237-3. MRID 43487302.
13. Office of Pesticide Programs, US EPA. 2000. Assigning values to nondetected/nonquantified
pesticide residues in human health food exposure assessments. EPA Docket #OPP-00570A.
32
735

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 49819401

CDPR IMI Stone Fruit

14. Schöning, R. 2014. Storage stability of imidacloprid and its 5-Hydroxy and olefin metabolite
in/on plant matrices for 36 Months. Bayer CropScience Report No. P642094733 Amendment No.
1.
15. Talbott, T.D. 1991. Product Chemistry of BAY NTN 33893 Technical. Bayer CropScience Report
No. MRID M004227-01-1.
16. USEPA, PMRA and CDPR. 2012. White Paper in Support of the Proposed Risk Assessment
Process for Bees. Submitted to the FIFRA Scientific Advisory Panel for Review and Comment
September 11-14, 2012. Published by USEPA, PMRA and CDPR. 275 pages.
17. USEPA, PMRA and CDPR. 2014. Guidance for Assessing Pesticide Risk to Bees. Published by
USEPA, PMRA, and CDPR June 2014. 59 Pages.

33736

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 49090502

CDPR IMI Strawberry

Reference
Gould, T.; Dallstream, K.; Beedle, E. (2012) Determination of the Residues of Imidacloprid and its
Metabolites 5-Hydroxy Imidacloprid and Imidacloprid Olefin in Bee Relevant Matrices Collected from
Strawberries, Grown at Locations Treated with Imidacloprid at Least Once Per Year During Two
Successive Years. Project Number: EBTNL056/04, M/445207/01/2. Unpublished study prepared byBayer Cropscience LP and California Agricultural Research Inc. 186p. MRID 49090502, CDPR Study ID
268742, Data Volume 51950-0812, Tracking ID# 256590

1. STUDY INFORMATION
Chemical:Imidacloprid
PC Code1290991) Admire Pro
Percent Active 1) 42.8%
Test Material:2) Alias 4F
Ingredient:2) 40%
Non-Guideline field residue study to establish imidacloprid and metabolite levelsin blossoms, anthers, pollen and leaves from strawberry in site locations that
Study Type:have been previously treated with imidacloprid at least once for two successive
years.
Bayer CropScience
2T.W. Alexander Drive
Experiment Start and
September 2011 –Sponsor:Research Triangle Park, NC
End Date:
December 13, 2012
USA 27709
Sponsor StudyEBNTL056-04
ID:
Seven treated fields
Study Locations:sites within California
Studythat consist of either
December 27, 2012
Completion
sand or loam soil.
Date:
GLP Status:

Non-GLP; protocol reviewed by CDPR.
[CDPR study ID 260012, Data Volume 51950-0791, Tracking ID# 247269]

2. REVIEWER INFORMATION

Primary Reviewers:
California
Department of
Pesticide Regulation

John Troiano, Ph.D., Research Scientist III, Environmental Monitoring
BranchRichard Bireley, Senior Environmental Scientist (Specialist),
Ecotoxicology Group, Pesticide Registration Branch
Denise Alder, Senior Environmental Scientist (Specialist), Lead
Reevaluation Coordinator, Pesticide Registration Branch
Russell Darling, Environmental Scientist, Reevaluation Coordinator,
Pesticide Registration Branch

3. EXECUTIVE SUMMARY
Blossom and leaf samples were collected from seven treated field sites in Santa Barbara County,
California to determine the residues of imidacloprid and its metabolites (5-hydroxy imidacloprid andimidacloprid olefin) in blossoms, anthers, pollen and leaves collected from strawberry plants grown atlocations treated with imidacloprid at least once per year for two years. The site locations consisted ofeither a sand soil (3 sites, “light”) or a loam soil (4 sites, “medium”), which had all previous received
1737Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 49090502

CDPR IMI Strawberry

applications of either Alias 4F or Admire Pro at a rate of 0.5 lb. a.i./acre in the prior year as well as anapplication of imidacloprid in 2010.
Duplicate composite samples of strawberry blossoms for direct analysis, strawberry blossoms for anthersamples, strawberry blossoms for pollen samples and strawberry leaves were collected at a BBCH
ranging from 61 to 69 (flowering) at each field site.
The residues of imidacloprid, 5-hydroxy imidacloprid, and imidacloprid olefin were quantitated by highperformance liquid chromatography/triple stage quadrupole mass spectrometry (LC/MS/MS) using
stable isotopically labeled internal standards. The individual analyte residues were summed to give a
total imidacloprid residue.
4. STUDY VALIDITY
Guideline Followed:
Guideline Deviations:
Other Deviations:Classification:
Rationale:
Reparability:

Non-guideline study (protocol was reviewed by U.S. EPA/PMRA/CDPR)
N/A
N/A
Acceptable
N/A
N/A

5. MATERIALS AND METHODS

Test item:
Description:
CAS #:

Test Material Characterization
Admire Pro, Alias andpH (24°C)
Unknown Formulations
Unknown
Density (20°C):
138261-41-3
Solubility:

7.8
1.54 g/mL
0.51 to 0.61 g/L

5A. STUDY DESIGN
Seven treated field sites in California representing two soil categories classified as either 'light,' generally
sand soil, or 'medium,' generally loam soil, were selected based on previous application(s) of
imidacloprid according to grower interviews. Classification of the soils was obtained from the Soil Survey
Geographic (SSURGO) Database provided by the Natural Resources Conservation Service. All test sites
had received applications of imidacloprid in the preceding years (2011, 2010).
The field sampling phase of this study occurred in September, 2011, and was not conducted under GLP.
The GLP experimental start date was August 22, 2012 (leaf sample homogenization), and theexperimental end date was December 13, 2012 (last analysis). The field sampling phase of the study was
conducted by California Agricultural Research, Inc. (CAR).
The sampling of blossoms and leaves, as well as the separation of the pollen and anthers, were
performed at the test site. Homogenization of the leaves and blossoms for direct analysis from thisstudy were performed at the Bayer Research Park (BRP) located in Stilwell, KS. Sample analysis andreport preparation was performed at Bayer CropScience located in Research Triangle Park, NC. All raw
data associated with this study are retained along with the protocol, protocol amendments and the final
report under Notebook Number EBNTL056-04 at locations specified by Bayer CropScience.
2738Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 49090502

CDPR IMI Strawberry

5B. APPLICATION TIMING AND RATESIn 2011, one application of either Alias 4F or Admire Pro was made to the soil at a rate of 0.5 lb. a.i./acrein each of the seven field sites. In 2010, one soil application of an imidacloprid product (the subject
products were not identified for four out of seven of the sites) was made at a rate of 0.5 lb. a.i./acre ineach of the seven field sites. Method, timing or growth stage, and spray volume of these applications
were not reported by the study authors. Thus, it is uncertain when the sampling was conducted inrelation to the imidacloprid applications.
The test substance use pattern for imidacloprid in 2011 and 2010 for each site is described in Table 1. All
site locations were in Santa Barbara county in California, NAFTA Region 10. For the applications in 2011,
either Alias 4F or Admire Pro were applied at a rate of 0.5 lb ai/A.
Table 1. Study Use Pattern for 2011 Application of Imidacloprid on Strawberry.
Trial
LocationYear
End UseTiming/Grow Spray
Identification
Product ath Stage
Volume b
NT031-11ZA
Santa
2011
Alias 4F
Unknown
Unknown
Maria, CA2010
Alias 4F
Unknown
Unknown
Region 10
NT032-11ZA
Santa
2011
Alias 4F
Unknown
Unknown
Maria, CA2010
Alias 4F
Unknown
Unknown
Region 10
NT033-11ZA
Santa
2011
Alias 4F
Unknown
Unknown
Maria, CA2010
Alias 4F
Unknown
Unknown
Region 10
NT035-11ZA
Santa
2011
Alias 4F
Unknown
Unknown
Maria, CA2010
Not Available c
Unknown
Unknown
Region 10
NT037-11ZA
Santa
2011
Admire Pro
Unknown
Unknown
Maria, CA2010
Not Available c
Unknown
Unknown
Region 10
NT038-11ZA
Santa
2011
Admire Pro
Unknown
UnknowncMaria, CA2010
Not Available
Unknown
Unknown
Region 10
NT039-11ZA
Santa
2011
Admire Pro
Unknown
UnknowncMaria, CA2010
Not Available
Unknown
Unknown
Region 10

Rate (lb.
a.i./A) a
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5

a

imidacloprid use was obtained from verbal communications with the growers or pest control advisors.
Rates and spray volumes that were provided in the application order/recommendation. See Appendix 2 of thestudy report for field trial summary data.
c
imidacloprid use was confirmed by the grower, but no documentation was available .
b

5C. STUDY SITE LOCATION AND CHARACTERISTICS
The trial site conditions including soil characteristics are listed in Table 2. All sites used agronomic
practices typical for commercial production of strawberries. A chronological listing of significant study
dates is given in Appendix 1 of the study report.
3739

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 49090502

CDPR IMI Strawberry

Temperatures and rainfall recorded during the field phase of the study were similar to average historical
records, with no significantly unusual weather conditions that would affect the conclusions of the study.
Temperature and precipitation data for each trial were obtained from the nearest National Climatic Data
Center (NCDC) weather station. Application, sampling and climatic data are located in Appendix 2 of thestudy report.

Table 2. Trial Site Conditions for Imidacloprid on Strawberry.
Trial Identification
Trial Location
Soil Type a

NT031-11ZA
NT032-11ZA
NT033-11ZA
NT035-11ZA
NT037-11ZA
NT038-11ZA
NT039-11ZA
aSanta Maria, CA
Region 10
Santa Maria, CA
Region 10
Santa Maria, CA
Region 10
Santa Maria, CA
Region 10
Santa Maria, CA
Region 10
Santa Maria, CA
Region 10
Santa Maria, CA
Region 10

Batteravia Loamy
Sand, Light
Oceano Sand,
Light
Batteravia Loamy
Sand, Light
Sorento Sandy
Loam, Medium
Sorrento Loam,
MediumSorrento Loam,
MediumSorento Sandy
Loam, Medium

Rainfall (in) b
0.1

Temperature
Range (°F) b
58-78

0.1

58-78

0.1

58-78

0.1

58-78

0.1

58-78

0.1

58-78

0.1

58-78

Classification of the soils was obtained from the Soil Survey Geographic (SSURGO) Database provided by the
Natural Resources Conservation Service.
b
Data is for the interval of the month of sampling. Meteorological data were obtained from nearby government
weather stations.

5D. SAMPLE COLLECTION, HANDLING, PROCESSING
At each sampling interval, duplicate samples of strawberry blossoms, strawberry blossoms for anthersamples, strawberry blossoms for pollen samples, and strawberry leaves were collected from thetreated sites during flowering.
Strawberry blossoms and leaves were placed into coolers with dry ice directly after sampling. Strawberry
blossoms for anther samples were processed by removing the anthers from the blossom withtweezers/small scissors and transferred to a sampling jar and frozen. Strawberry blossoms for pollen
samples were allowed to dry overnight, and the pollen was removed by vacuum and frozen.
Untreated (control) blossom and leaf samples used for validation of the analytical method and fortransit stability samples were obtained from a nearby farm that was believed to have no recent
imidacloprid application.

4740

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 49090502

CDPR IMI Strawberry

All samples were placed in frozen storage at CAR Samples were shipped to BRP frozen via Agricultural
Chemicals Development Services (ACDS) and were placed in frozen storage upon receipt and remained
frozen throughout the storage period at BRP.
The anther and pollen samples, along with the transit stability samples, were shipped frozen to Bayer
CropScience, Research Triangle Park (RTP), NC, via ACDS. Homogenization of the leaf and blossom
samples was conducted at BRP and shipped frozen via FedEx to RTP. During storage at RTP, the antherand pollen samples, along with the transit stability samples, reached room temperature forapproximately three days due to freezer failure. Transit stability samples accompanied the field samples
during all sample handling and preparation steps to ensure the validity of the sampling practices.
Sample Storage.
Upon arrival at BRP, all samples were immediately transferred to frozen storage. The leaf samples were
homogenized with dry ice using a Robot Coupe chopper (Jackson, MS). The leaf samples were returned
to frozen storage immediately following homogenization. Samples were transferred to Bayer
CropScience, Research Triangle Park, NC, for analysis. Samples remained frozen at all times except
during preparation for analysis A summary of collection, shipment, and homogenization dates for thetrials is given in Appendix 1 of the study report.
Additionally, freezer storage stability studies have indicated the imidacloprid residues are stable (<30%decomposition) for 24 months (728 to 769 days) of freezer storage in the following representative crops:
an oilseed (cottonseed), a non-oily grain (wheat), a leafy vegetable (lettuce), a root crop (potato), a tree
fruit (apple), and a fruiting vegetable (tomato). 7-11
Demonstrated freezer stability in the transit stability samples from this study, as well as stability
demonstrated in the above crops is representative of the freezer stability of imidacloprid residues to be
expected in the blossoms, anthers, pollen, and leaves collected in this study.
5E. ANALYTICAL METHODS
The analytical method used in this study measured the residues of imidacloprid, 5-hydroxy imidacloprid,and imidacloprid olefin in strawberry blossoms, anthers, pollen and leaves.4
All neat analytical reference standards were stored frozen when not in use and all solutions were stored
refrigerated or frozen. Analytical standard solutions used in this study were not stored for longer thanapproximately 2 months and have been shown to be stable for this period of time. 5 All reference
standard solutions were prepared in parent imidacloprid equivalents and corrected for purity duringinitial preparation.
Samples of anthers, blossoms, leaves, and pollen were analyzed for residues of imidacloprid,imidacloprid olefin, and imidacloprid 5-hydroxy. For anthers, blossoms, and leaves, a sample was
extracted by blending samples with 2:1 acetonitrile/water (ACN/H20). The mixture was amended with
MgS04, NaCI, and isotopically labeled internal standards, mixed well, and centrifuged. An aliquot of thesupernatant was transferred to a 15-mL centrifuge tube containing MgS04 and Bondesil-PSA. The
contents of the tube were mixed well and centrifuged. About 1.2 ml of the supernatant was percolated
through a C18 SPE cartridge (50 mg Bond Elut) into a 20x150mm culture tube. The eluate was5741Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 49090502

CDPR IMI Strawberry

evaporated to dryness and the residue was reconstituted with 1:9 methanol/water (MeOH/H20) foranalysis by high performance liquid chromatography/tandem mass spectrometry (LC/MS/MS).

For pollen a sample was extracted by shaking with water for 10 minutes, adding ACN and shaking for an
additional 55 minutes. The mixture was amended with isotopically labeled internals standards, mixed
well and centrifuged. The supernatant was drawn off, evaporated to an aqueous remainder, amended
with water and applied to a Chern Elute cartridge (3-ml). The analytes were extracted from the cartridgewith 3 x 5 ml of hexane/ethyl acetate (1:1 v/v). The eluate was evaporated to dryness and the residue
was reconstituted with 1:9 methanol/water (MeOH/H20) for analysis by LC/MS/MS.
Quantitation of each analyte was based on the daughter ion transitions of the analytes and therespective internal standard analogs. The responses of LC/MS/MS system to each analyte and its
internal standard were measured in samples and in standards, and a relative response was calculated as
the ratio of the analyte and the stable isotopically labeled internal standard responses. The relative
response of the analyte in each sample was compared to the relative response of the analyte in thestandards (Appendix 3 of the study report).
The relative responses of imidacloprid, 5-hydroxy imidacloprid, and imidacloprid olefin in solvent were
measured over the range of 3 ppb to 3000 ppb for each analyte. The coefficients of determination (r2)were calculated using linear regression analysis (Microsoft Excel 2010; Appendix 3 of the study report).
The total imidacloprid residue is the sum of the individual measured residue values of imidacloprid, 5hydroxy imidacloprid, and imidacloprid olefin in parent equivalents. For the purpose of calculating total
imidacloprid residues where individual analyte residue values were less than the limit of detection(LOD), the residues were assigned a finite value of ½ the value of the respective LOD. This value is only
an estimate of potential residue in a sample, not a measured value.
Blossoms and leaves were collected from the control test site and were used for validation, LOD
determination, and concurrent recoveries. Commercially obtained bee pollen was also used forvalidation and recovery. Because only small, untreated control samples of anthers and pollen weregenerated in the field phase of this study, surrogate anther and pollen controls were used for validation,
LOD determination, and concurrent recoveries. Blossom validation LOD data was used for anthers while
the control strawberry blossoms were used for anther concurrent recoveries. Commercial bee pollen
was obtained from a local health food store and stored at room temperature. The bee pollen was usedwithout any preparation. The commercial pollen was used for pollen validation, LOD determination, andfor concurrent recoveries.
The method for determining imidacloprid, 5-hydroxy imidacloprid, and imidacloprid olefin residues
in/on strawberry blossoms, anthers, pollen and leaves was validated by measuring the recovery of theseanalytes from control matrices fortified at their respective LOQs.
Additional recoveries at higher fortification levels validated the method for the highest residues
observed in individual matrices. Concurrent recoveries of imidacloprid, 5-hydroxy imidacloprid, andimidacloprid olefin were measured with each set of samples to verify method performance.
5F. QUALITY ASSURANCE RESULTS

6742

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 49090502

CDPR IMI Strawberry

The responses of the LC/MS/MS system to imidacloprid, 5-hydroxy imidacloprid, and imidacloprid olefin
were linear in solvent over the range of 3 ppb to 3000 ppb for each analyte. The coefficients ofdetermination were >0.99.
All recoveries were corrected for any interferences in corresponding controls. The overall mean valuesof the recoveries for each matrix were within the acceptable range of 70% to 120%, and the standard
deviation values were below 20%, except for 5-hydroxy imidacloprid recovery from blossoms which had
a percent recovery standard deviation of 24%.
The limit of quantitation (LOQ) is defined as the lowest fortification level of an analyte at which
acceptable recovery has been achieved. The LOQ for total residue is the highest of the LOQ values
assigned to the individual analytes for a particular matrix.
The limit of detection (LOD) is defined as the lowest concentration of an analyte that can be determined
to be statistically different from a blank. The LODs were determined from method validation dataobtained from control samples fortified at the respective analyte LOQs. The LODs were calculated bymultiplying the standard deviation of recovery measurements at the LOQ by t0.99 [where t0.99 is the onetailed t-statistic at the 99% confidence level for the number of replicates (n)] 6. The LOD for the total
imidacloprid residue in each matrix is the highest LOD value of any individual analyte for that particularmatrix.
The LOQs and LODs are summarized in the table below.
Summary of LOQs and LODs
MatrixAnalyteStrawberry
Imidacloprid
Blossom
5-hydroxy Imidacloprid
Imidacloprid olefin
Total Imidacloprid
Strawberry
Imidacloprid
Anthers
5-hydroxy Imidacloprid
Imidacloprid olefin
Total Imidacloprid
Strawberry
Imidacloprid
Pollen5-hydroxy Imidacloprid
Imidacloprid olefin
Total Imidacloprid
Strawberry
Imidacloprid
Leaves5-hydroxy Imidacloprid
Imidacloprid olefin
Total Imidacloprid

LOQ (ppb, parent equivalents)
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
10
10
10
10
10
10
10
10

7
743

LOD (ppb, parent equivalents)
0.87
3.9
1.3
3.9
0.87
3.9
1.3
3.9
1.1
2.6
2.2
2.6
2.2
3.0
4.6
4.6

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 49090502

CDPR IMI Strawberry

Any individual analyte or total residue measured to be less than the Limit of Quantitation (LOQ) was
reported as <5 ppb for blossoms and anthers and <10 ppb for leaves and pollen. In Appendix 3 of thestudy report, any residue value that was below the LOD was reported as less than the LOD andmeasured residue values between the LOQ and LOD are also provided.
6. RESULTS:
Summary statistics for concentration of total imidacloprid in strawberry blossoms, anthers, pollen andleaves are contained in Table 3. The individual analyte residues were summed to give a total
imidacloprid residue. The study authors stated that when individual analyte residue values were lessthan the LOD, the residues were assigned a finite value of half the value of the respective LOD.

Table 3. Magnitude of total imidacloprid residues in strawberry blossoms, anthers, pollen, and leaves.

Total Imidacloprid Residue Levels (ppb)ba

Commodity
Strawberry Blossoms
Strawberry Anthers
Strawberry Pollen
Strawberry Leaves
Strawberry Blossoms
Strawberry Anthers
Strawberry Pollen
Strawberry Leaves

Soil Texture
Categorya
CoarseCoarseCoarseCoarseMediumMediumMediumMedium

N
6
6
6
6
8
8
8
8

HighestStandard
Avg. Site
Max Residue Median Mean Deviation

Min
210
81
78
1700
<5.0
11
<10.0
<10.0

530
500
300
250
320
280
2800 2400
31
18
33
23
<10.0 <10.0
18
17

380
200
210
2100
6.4
13
<10.0
11

360
180
190
2200
9.4
18
<10.0
11

130
82
95
410
9.1
7.9
<10.0
<10.0

Classification of the soils was obtained from the Soil Survey Geographic (SSURGO) Database provided by the
Natural Resources Conservation Service and converted to soil texture categories.
b
Abbreviations used are as follows: Min is the lowest treated residue value; Max is the highest treated residue
value; Median is the geometric median of the treated residue values; Mean is the mathematical average of thetreated residue values; Standard Deviation is the standard deviation for a small population of “N” samples.

The total imidacloprid residue data for strawberry blossoms, anthers, pollen and leaves are provided inTable 4.
Table 4. Imidacloprid Residue Data from Strawberry.

8744Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

Total Imidacloprid Residue(ppb)

Imidacloprid Residue (ppb)5-Hydroxy Imidacloprid
Residue (ppb)

Imidacloprid Olefin Residue(ppb)

Residue Sample Collection
DAT (Days after 2010

Rate, lb a.i./A

Plot Name

Commodity

CDPR IMI Strawberry

Location (City, State,
Region)Trial Identification

MRID 49090502

a

Blossoms
NT031-1ZA
Santa Maria,
CA, Region 10

Residue (ppb)
46
470
33
420

Sand 1

Blossom

0.5

NA

10
12

NT032-1ZA

Santa Maria,
CA, Region 10

Sand 2

Blossom

0.5

NA

8.7
8.2

31
27

340
350

NT033-1ZA

Santa Maria,
CA, Region 10

Sand 3Blossom

0.5

NA

8.5
10

38
40

160
170

NT035-1ZA

Santa Maria,
CA, Region 10

Loam 1

Blossom

0.5

NA

<5.0
<5.0

7.0
<5.0

22
<5.0

NT037-1ZA

Santa Maria,
CA, Region 10

Loam 3Blossom

0.5

NA

<5.0
<5.0

7.4
<5.0

<5.0
<5.0

NT038-1ZA

Santa Maria,
CA, Region 10

Loam 4

Blossom

0.5

NA

<5.0
<5.0

5.1
6.8

<5.0
<5.0

NT039-1ZA

Santa Maria,
CA, Region 10

Loam 5

Blossom

0.5

NA

<5.0
<5.0

<5.0
<5.0

<5.0
<5.0
a

Anthers
NT031-1ZA
Santa Maria,
CA, Region 10

Sand 1

Anthers

0.5

NA

19
9.5

NT032-1ZA

Santa Maria,
CA, Region 10

Sand 2

Anthers

0.5

NA

7.5
9.5

24
35

160
180

NT033-1ZA

Santa Maria,
CA, Region 10

Sand 3

Anthers

0.5

NA

5.5
7.6

26
20

64
54

9
745

Residue (ppb)
43
240
26
170

530
470
Average
500
380
380
Average
380
210
220
Average
220
31<5.0
Average18
11
6.0
Average
8.6
6.7
8.8
Average
7.7
<5.0
<5.0
Average
<5.0
300
210
Average250
190
220
Average
210
96
81
Average
89

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

5-Hydroxy Imidacloprid
Residue (ppb)

Anthers

0.5

NA

<5.0
<5.0

13
7.6

38
<5.0

NT037-1ZA

Santa Maria,
CA, Region 10

Loam 3

Anthers

0.5

NA

<5.0
<5.0

13
22

<5.0
<5.0

NT038-1ZA

Santa Maria,
CA, Region 10

Loam 4

Anthers

0.5

NA

<5.0
8.8

8.7
23

<5.0
<5.0

NT039-1ZA

Santa Maria,
CA, Region 10

Loam 5

Anthers

0.5

NA

<5.0
<5.0

11
11

<5.0
<5.0

Plot Namea

PollenNT031-1ZA
Santa Maria,
CA, Region 10

Sand 1

Pollen

0.5

NA

17
14

NT032-1ZA

Santa Maria,
CA, Region 10

Sand 2

Pollen

0.5

NA

14
10

32
25

190
150

NT033-1ZA

Santa Maria,
CA, Region 10

Sand 3Pollen

0.5

NA

<10
<10

21
18

57
54

NT035-1ZA

Santa Maria,
CA, Region 10

Loam 1

Pollen

0.5

NA

<10
<10

<10
<10

<10
<10

NT037-1ZA

Santa Maria,
CA, Region 10

Loam 3Pollen

0.5

NA

<10
<10

<10
<10

<10
<10

NT038-1ZA

Santa Maria,
CA, Region 10

Loam 4

Pollen

0.5

NA

<10
<10

<10
<10

<10
<10

Sand 1

Leaves

0.5

NA

14
17

LeavesNT031-1ZA
Santa Maria,
CA, Region 10

Residue (ppb)
42
260
33
200

a

10
746

Residue (ppb)
50
1600
64
2200

Total Imidacloprid Residue(ppb)

Imidacloprid Olefin Residue(ppb)

Loam 1Imidacloprid Residue (ppb)

Residue Sample Collection
DAT (Days after 2010

Santa Maria,
CA, Region 10

Location (City, State,
Region)NT035-1ZA

Trial Identification

Rate, lb a.i./A

CDPR IMI Strawberry

Commodity

MRID 49090502

19
11
Average15
14
25
Average
19
13
33
Average23
12
13
Average13
320
250
Average
280
240
190
Average
210
87
78
Average83<10<10
Average<10<10<10
Average<10<10<10
Average
<10
1700
2300
Average
2000

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

5-Hydroxy Imidacloprid
Residue (ppb)

Leaves

0.5

NA

27
18

53
48

2300
1900

NT033-1ZA

Santa Maria,
CA, Region 10

Sand 3

Leaves

0.5

NA

42
25

100
70

2700
1800

NT035-1ZA

Santa Maria,
CA, Region 10

Loam 1

Leaves

0.5

NA

<10
<10

<10
<10

14
<10

NT037-1ZA

Santa Maria,
CA, Region 10

Loam 3

Leaves

0.5

NA

<10
<10

<10
<10

<10
<10

NT038-1ZA

Santa Maria,
CA, Region 10

Loam 4

Leaves

0.5

NA

<10
<10

<10
<10

<10
<10

NT039-1ZA

Santa Maria,
CA, Region 10

Loam 5

Leaves

0.5

NA

<10
<10

<10
<10

<10
<10

Plot NameaTotal Imidacloprid Residue(ppb)

Imidacloprid Olefin Residue(ppb)

Sand 2

Imidacloprid Residue (ppb)

Residue Sample Collection
DAT (Days after 2010

Santa Maria,
CA, Region 10

Location (City, State,
Region)NT032-1ZA

Trial Identification

Rate, lb a.i./A

CDPR IMI Strawberry

Commodity

MRID 49090502

2400
1900
Average
2200
2800
1900
Average
2400
18
16
Average
17<10
10
Average<10
12
13
Average
13<10<10
Average<10

See Appendix 3 of the study report for analytical data summaries and refined data used to prepare this summary.
Total
imidacloprid is the sum of imidacloprid, 5-hydroxy imidacloprid, and imidacloprid olefin .

In coarse-textured soils, mean residues of total imidacloprid were 360, 180, 190, and 2200 ppb inblossoms, anthers, pollen, and leaves, respectively and, maximum residues were 530, 300, 320, and2800 ppb in blossoms, anthers, pollen, and leaves respectively. In medium-textured soils, mean residuesof total imidacloprid were 9.4, 18, <0.010 (LOQ), and 11 ppb in blossoms, anthers, pollen, and leaves,respectively, and maximum residues were 31, 33, <0.010 (LOQ), and 18 ppb in blossoms, anthers, pollen,
and leaves respectively. Thus, in all matrices (blossoms, anthers, pollen, and leaves) mean
concentrations of total imidacloprid were approximately ten times higher in strawberries grown incoarse-textured soil than in medium-textured soil.
7. STUDY VALIDITY/CLASSIFICATION AND STUDY LIMITATIONSClassification/Utility for Bee Risk Assessment. The study is ACCEPTABLE for considering the level ofresidues of imidacloprid in pollen after soil application in strawberry. The level of imidacloprid residues
appeared to be much higher in strawberries grown in coarse-textured sandy soil than in medium11
747

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 49090502

CDPR IMI Strawberry

textured loamy soil. In sandy soil, soil application at 0.5 lb ai/A of imidacloprid for two consecutive years
resulted in maximum total imidacloprid residues of 320 ppb (mean ± SEM, 190 ± 95), 300 ppb (180±82),
530 ppb (360±130) and 2800 ppb (2200±410) respectively in strawberry pollen, anthers, blossoms andleaves. Residues in strawberry nectar, another bee-relevant matrix, were not measured. Anotherconcern is that the date of the last soil application was not provided, thus the duration from the soil
application to the collection of the samples could not be estimated.
Temporal Variability in Residues. This study was not designed to measure temporal variability inresidues. All samples were taken in 2011, within five days of each other. Thus, this study was designed
to analyze imidacloprid residues at a single time point. In addition, time of sampling in relation to theimidacloprid applications, is unknown.
Spatial Variability in Residues. All seven sites for this strawberry study were located in Santa Maria, CA.
As expected, reported weather conditions (temperature and precipitation) were the same across allseven sites. As a result of the close proximity of trial sites, this study provides very limited information
on how differences in environmental conditions across different areas of the US may affectaccumulation of total imidacloprid in bee-relevant matrices. However, because there are different soil
types represented (3 sand soil sites and 4 loam soil sites), this study may offer insight to how soil typemay affect accumulation of total imidacloprid in bee-relevant matrices. In all matrices (blossoms,
anthers, pollen, and leaves) mean concentrations of total imidacloprid were higher in strawberriesgrown in sandy soil than in loam soil.
Pesticide Carryover. This study was not designed to measure pesticide carryover. All samples weretaken in 2011, within five days of each other. This this study is designed to analyze imidacloprid residues
at a single time point.
8. CONCLUSION
Samples were obtained from 7 field sites where soil in 3 of the sites was classified as coarse-textured
and the other 4 classified as medium-textured. The cultivar of strawberry was not reported that wasgrown in each field. Parent imidacloprid and 2 breakdown products, the 5-Hdroxy and Olefin residues,
were measured. Concentrations were measured for whole blossoms, anthers and pollen separated fromthe blossom, and leaves. Chemical analysis was conducted on samples stored for more than 1 year.
Samples were purported to store well over this period but storage data were only referenced and notprovided.
In coarse-textured soils, mean residues of total imidacloprid were 360, 180, 190, and 2200 ppb inblossoms, anthers, pollen, and leaves, respectively and, maximum residues were 530, 300, 320, and2800 ppb in blossoms, anthers, pollen, and leaves respectively. In medium-textured soils, mean residuesof total imidacloprid were 9.4, 18, <0.010 (LOQ), and 11 ppb in blossoms, anthers, pollen, and leaves,respectively, and maximum residues were 31, 33, <0.010 (LOQ), and 18 ppb in blossoms, anthers, pollen,
and leaves respectively. Thus, in all matrices (blossoms, anthers, pollen, and leaves) mean
concentrations of total imidacloprid were approximately ten times higher in strawberries grown incoarse-textured soil than in medium-textured soil.
9. REFERENCES
12748

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 49090502

CDPR IMI Strawberry1.

Hoelscher, A. 1998. Physical and chemical properties of the active ingredient imidacloprid.
Bayer CropScience Report No. M0-03-006885, M-105510-01-1.

2.

Talbott, T. D. 1991. Product Chemistry of BAY NTN 33893 Technical. Bayer CropScience Report
No. MRID M004227-01-1.

3.

Krohn, J. 1993. Vapour Pressure Curve of lmidacloprid. Bayer CropScience Laboratory Project ID
14 200 0804. Report No. M-004042-01-1.

4.

Brungardt, Jaime 201 0. An Analytical Method for the Determination of Residues of lmidacloprid
and its Metabolites lmidacloprid Olefin and 5-Hydroxy lmidacloprid in Bee Relevant Matrices
Using LC/MS/MS. Bayer Report No. NT-005-P10-01.

5. Schoning, R. 1999. Residue Analytical Method for the Determination of Residues of lmidacloprid,
Hydroxy-lmidacloprid, Olefin-lmidacloprid, YRC 2894, YRC 2894-Amide, and 4-Hydroxy-YRC
2894-Amide in Plant Material by HPLC with Electrospray MS/MS Detection. Method No. 00573.
Report No. MR-853/98.
6. Office of Pesticide Programs, US EPA. 2000. Assigning values to non-detected/nonquantified
pesticide residues in human health food exposure assessments. EPA Docket #OPP-00570A.
7. Noland, P.A. 1992. lmidacloprid and metabolites- freezer storage stability study in crops. Bayer
CropScience Report No. 103237. MRID 42556135.
8. Noland, P.A. 1993. lmidacloprid and metabolites- freezer storage stability study in crops. Bayer
CropScience Report No. 103237-1. MRID 42810311.26. Noland, P.A. 1994. lmidacloprid andmetabolites -freezer storage stability study in crops. Bayer CropScience Report No. 103237-2.
MRID 43197203.
9. Lenz, C.A. 1993. Addendum 1. lmidacloprid and metabolites- freezer storage stability study incrops (what matrices, cottonseed, tomato, cauliflower, and lettuce). Bayer CropScience Report
No. 103949-1. MRID 42810313.
10. Lenz, C.A. 1993. Addendum 2. lmidacloprid and metabolites- freezer storage stability study incrops (what matrices, cottonseed, tomato, cauliflower, and lettuce). Bayer CropScience Report
No. 103949-2. MRID 43197201.
11. Lenz, C.A. 1993. Addendum 3. lmidacloprid and metabolites- freezer storage stability study incrops (what matrices, cottonseed, tomato, cauliflower, and lettuce). Bayer CropScience Report
No. 103949-3. MRID 43487301.

13749

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49665202

CDPR IMI Soil and Foliar Cotton Study

Reference
Fischer, D.R., and Jerkins, E. (2015) Determination of the Residues of Imidacloprid, 5-Hydroxy
Imidacloprid, and Imidacloprid Olefin in Bee Relevant Matrices Collected from Cotton During Two
Successive Years: Final Report. Project Number: EBNTN011. Unpublished study prepared by Bayer
CropScience 632pg. MRID 49665202, CDPR study ID 285681, Data Volume 51950-0900,
Tracking ID# 270950

1. STUDY INFORMATION
Chemical:Test Material:
Study Type:

Sponsor:Sponsor Study
ID:
Study
Completion
Date:
Date of
Amendment:
GLP Status:

Imidacloprid
PC Code129099Admire Pro Systemic
Purity:43.50% a.i. w.w.
Protectant (SC)
Non-Guideline field residue study on cotton to establish imidacloprid andmetabolite levels in pollen, nectar (floral and extrafloral), and leaves following
four applications in each of two successive years and three different soil types(fine, medium, and coarse).
Bayer CropScience
2T.W. Alexander Drive
Experiment Start andApril 12, 2013 Research Triangle Park, NC
End Date:
April 9, 2015
USA 27709
EBNTN011
Study Locations:

June 19, 2015
April 13, 2016

9 Field Trials in thecities of:Davis, Fresno, Kerman,
Sanger, Wheatland, andYuba City, California

GLP-compliant; protocol reviewed by EPA, PMRA, CDPR, CDPR study ID 266879,
Data Volume 51950-0808, Tracking ID# 254696.]

2. REVIEWER INFORMATION

Primary Reviewers:
California Departmentof Pesticide
Regulation

John Troiano, Ph.D., Research Scientist III, Environmental Monitoring
BranchRichard Bireley, Senior Environmental Scientist (Specialist), Ecotoxicology
Group, Pesticide Registration Branch
Denise Alder, Senior Environmental Scientist (Specialist), Lead Reevaluation
Coordinator, Pesticide Registration Branch
Russell Darling, Environmental Scientist, Reevaluation Coordinator,
Pesticide Registration BranchSecondary Reviewer:

TBD

3. EXECUTIVE SUMMARY
A total of nine (9) field trials were conducted to measure the magnitude of imidacloprid residues in beerelevant cotton pollen, nectar, and in/on leaves following four applications of Admire Pro SystemicProtectant, EPA Reg. No. 264-827 in each of two successive years. Admire Pro Systemic Protectant is a
750

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49665202

CDPR IMI Soil and Foliar Cotton Study

suspension concentrate formulation containing 550 g/L imidacloprid. Three (3) trials have one year ofdata and six (6) trials have two years of data.
Treated plots received one soil (in-furrow) spray application of Admire Pro at planting (BBCH 00: dry seed)followed by 3 equivalent Admire Pro foliar spray applications per planting season. Individual soil
application rates ranged from 0.37 to 0.38 kg imidacloprid/ha per application (0.33 to 0.34 lb/A), andspray volumes were 13 to 15 gal/A. The interval between the soil and first foliar application was 75 to
99 days. Individual foliar application rates ranged from 0.063 to 0.067 kg imidacloprid/ha per application(0.056 to 0.060 lb/A). Foliar and soil applications were made using ground-based equipment. Also, theadjuvant Dyne-Amic (0.25% v/v) was used in all foliar applications. Moreover, all foliar applications weremade between BBCH growth stages 61 and 72 (BBCH 61: beginning of flowering; BBCH 72: about 20% ofbolls have attained their final size). The interval between foliar applications was 6 to 7 days. The foliar
spray volumes ranged from 14 to 20 gal/A. Total seasonal application rates ranged from 0.56 to 0.57 kg
imidacloprid/ha (0.50 to 0.51 lb/A).
Cotton leaf and flower samples were collected at three sampling intervals: 4 to 5 days prior to the first
foliar application (70 to 95 days after the soil application), 4 to 5 days after the last foliar application, and
12 to 14 days after the last foliar application. At each sampling interval, duplicate composite samples (twoseparate runs through the plot) of cotton flowers and cotton leaves were collected from the treated plots
when the plants were at bloom, BBCH 61 (begin flowering, early bloom) to BBCH 73 (about 30% of bolls
have attained their final size). Single composite samples of cotton leaves and flowers were collected fromthe control plot of each trial on the same days that samples were collected from the treated plots.
After their collection, cotton flowers were hand-processed at the field site to obtain the bee-relevantsamples of cotton pollen, floral nectar, and extra floral nectar. The processed flowers were discarded. The
residues of Admire Pro Systemic Protectant (imidacloprid, 5-hydroxy imidacloprid, and imidacloprid
olefin) were quantitated by high performance liquid chromatography/triple stage quadrupole mass
spectrometry (LC/MS/MS) and LC/high resolution mass spectrometry (LC/HRMS) using stable isotopicallylabeled internal standards. The individual analyte residues were summed to give a total imidacloprid
residue.
Storage stability studies indicate that the imidacloprid residues would have been stable during frozen
storage for at least 1,080 days (36 months) in cotton leaves prior to analysis. Transit spikes showed thatimidacloprid residues were stable in pollen and nectar for the duration of the study. The maximum
storage period of frozen samples in this study for Admire Pro Systemic Protectant was 569 days for cotton
leaves, 226 days for cotton pollen, and 211 days for cotton floral and extrafloral nectar.

4. STUDY VALIDITY

Guideline Followed:
Guideline Deviations:
Other Deviations:Classification:
Rationale:
Reparability:

Non-guideline study (protocol was reviewed by EPA/PMRA/CDPR
N/A
N/A
Quantitative
The data from the study will provide a basis for developing aquantitative assessment of exposure levels to bees that can be used in arisk assessment scenario.
N/A

751

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49665202

CDPR IMI Soil and Foliar Cotton Study

5. MATERIALS AND METHODS
Test Material Characterization
Test item:
Description:
Lot No./Batch No.
Material Source:
Material Receipt
Date:
Expiration Date:

Storage of Test
Material:

Admire Pro Systemic Protectant(Imidacloprid) 550 g a.i./L SC
Suspension concentrate (SC)
Batch No. NK41CX0578
Bayer CropScience
Not Reported
12/03/2014
Ambient (35-86ºF)
except trials NT001-13ZB,
NT002-13ZB, and NT005-13ZB
when the temperature briefly
reached 94ºF.

CAS #:
Purity:Density:
Cert. #
Analysis
Date:
Solubility:

138261-41-3

SampleStorage:-47ºC to -11ºC-116ºF to -52ºF

43.50% w/w
1.41 – 1.54 g/mL
213CJ2446
12/03/2012
0.51 to 0.61 g/L

5A. STUDY DESIGNThis study requirement was part of the imidacloprid special review at the California Department of
Pesticide Regulation (CDPR). The study design and protocol were approved by the CDPR prior to study
initiation. This study was conducted using GLP standards and following an approved protocol. The study
initiation date was April 12, 2013. The experimental start date was May 10, 2013 (first application), andthe experimental end date was January 26, 2016 (last analysis).
Two plots were included in each trial, to be planted and treated in each of two consecutive years. Only
the first year of trial NT002-13ZB could be completed and reported because the plot location was nolonger available. Trials NT001-13ZB and NT005-13ZB were restarted in 2014 and ran for a two year time
period.
5B. APPLICATION TIMING AND RATESThe full study report provides (1) Chronological listing of significant study dates (Appendix 1); (2) Field
report summaries for each trial detailing the actual amount of test substance applied, plot sizes, dates oftreatment, dates of sample collection, maintenance chemicals, climatic data, and irrigation data
(Appendix 2); and (3) Quality assurance statements for each trial (Appendix 3). Information on application
timing is provided in Table 1. Soil and meteorological characteristics of the study sites are provided inTable 2 and Table 3. Table 4 provides the sampling dates and cotton developmental stages.
BBCH or Biologische Bundesanstalt, Bundessortenamt und CHemische Industrie, identifies the specificphenological development stages of cotton. Plot TRTD received one soil (in-furrow) spray application of
Admire Pro at planting (BBCH 00: dry seed) followed by 3 equivalent Admire Pro foliar spray applications
per planting season. Individual soil application rates ranged from 0.35 to 0.38 kg imidacloprid/ha perapplication (0.32 to 0.34 lb/A), and spray volumes were 13 to 15 gal/A. The interval between the soil andfirst foliar application was 75 to 99 days. Individual foliar application rates ranged from 0.063 to 0.067 kg
imidacloprid/ha/application (0.056 to 0.060 lb/A). All foliar applications were made between BBCH
growth stages 61 and 72 (BBCH 61: beginning of flowering; BBCH 72: about 20% of bolls have attained
their final size). The interval between foliar applications was 6 to 8 days. The foliar spray volumes ranged752

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49665202

CDPR IMI Soil and Foliar Cotton Study

from 14 to 20 gal/A. Total seasonal application rates ranged from 0.55 to 0.57 kg imidacloprid/ha(0.49 to 0.51 lb/A).
Temperature and precipitation data were recorded for each trial and are summarized in Appendix 2 ofthe study report EBNTN011. Temperatures recorded during the field phase of the study were similar to
average historical records. Recorded rainfall was lower than historical records across all trials, but thiswould not affect the conclusions of the study. Irrigation supplemented normal rainfall as needed in sometrials. CDPR requested that the trial sites be distributed as three coarse, three medium, and three finetextured soils [per USDA’s Soil Survey Geographic database (SSURGO) mapping units]; however, due tovarious issues, the final trials contained four coarse, three medium and two fine textured sites (in somecases, the plots were shifted a short distance within the designated field, but this placed the plot in acoarse texture mapping unit instead of the intended medium texture area). The two-year study includes
one year of residue data from each of two fine texture fields, one to two years of data from the three
medium texture fields, and one to two years of data from four coarse texture fields. See Table 3 for moresite specific soil information.

NT00113ZB

NT00213ZBc

Yuba City, CA
Region 10

AdmirePro

Wheatland, CA Admire
Region 10
Pro

Soil
Spray
Foliar2014 Spray
FoliarSpray
Foliar
Spray
TRTD
Soil
Spray

00

Foliar2015 Spray
FoliarSpray
Foliar
Spray
Soil
Spray
Foliar
TRTD 2014 Spray
FoliarSpray
Foliar
Spray

61

753

61
65
65
00

61
65
00
65
69
72

14
(133)
16
(150)
16
(147)
16
(148)
14
(134)

0.328
(0.368)0.058(0.065)0.057(0.064)0.057(0.064)0.316(0.354)

20
(188)
20
(188)
20
(187)
15
(142)
15
(142)
15
(144)
15
(140)

0.057
(0.064)0.057(0.064)0.057(0.064)0.331(0.371)
0.057(0.064)0.058(0.065)0.057(0.064)93

0.50(0.56)

7
7
308
88

0.49
(0.55)

7
8
NA
92
7
7

0.50
(0.56)

Adjuvant

NAb

Total Rate, lb a.i./A(kg a.i./ha)Retreatment
Interval (days)

Timing/GrowthStage (BBCH)
Actual Spray
Volume, GPA(L/ha)
Rate, lb a.i./A (kga.i./ha)MethodYear

Plot Name

Applicationa

Formulation

Location (City, State,
NAFTA Region)

Trial Identification

Table 1. Summary of foliar and soil application rates and timing*.

NADyne-Amic
0.25 % v/v
Dyne-Amic
0.25 % v/v
Dyne-Amic
0.25 % v/v
NADyne-Amic
0.25 % v/v
Dyne-Amic
0.25 % v/v
Dyne-Amic
0.25 % v/v
NADyne-Amic
0.25 % v/v
Dyne-Amic
0.25 % v/v
Dyne-Amic
0.25 % v/v

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

NT00313ZA

NT00413ZA

NT00513ZBCDPR IMI Soil and Foliar Cotton Study

Fresno, CA
Region 10

Davis, CA
Region 10

Yuba City, CA
Region 10

AdmirePro

AdmirePro

Admire
Pro

754

65
65
65
00
61
65
65
00
61
61
61
00
61
61
61
00
61
65
65
00
61

0.330
(0.370)0.058(0.066)0.058(0.065)0.058(0.066)0.330(0.370)0.058(0.065)0.059(0.066)0.060(0.067)0.332(0.372)0.058(0.065)0.058(0.065)0.057(0.064)0.327(0.367)0.057(0.064)0.057(0.064)0.057(0.063)0.337(0.377)0.056(0.063)0.057(0.064)0.057(0.064)0.333(0.3740.057(0.064)

NA
81

0.51
(0.57)

7
7
254
77

0.51
(0.57)

7
7
NA
91

0.51
(0.57)

7
7
253
99

0.50
(0.56)

7
7
NA
91

0.51
(0.57)

7
7
307
91

0.50
(0.56)

Adjuvant

14
(132)
15
(142)
15
(140)
15
(142)
14
(132)
15
(141)
15
(142)
16
(145)
14
(127)
15
(142)
15
(140)
14
(136)
15
(139)
14
(135)
17
(156)
16
(148)
15
(143)
15
(142)
15
(144)
15
(142)
15
(141)
20
(187)

Total Rate, lb a.i./A(kg a.i./ha)00

Retreatment
Interval (days)

Soil
Spray
FoliarSpray2013 Foliar
Spray
Foliar
Spray
TRTD
Soil
Spray
FoliarSpray2014 Foliar
Spray
Foliar
Spray
Soil
Spray
FoliarSpray2013 Foliar
Spray
Foliar
Spray
TRTD
Soil
Spray
FoliarSpray2014 Foliar
Spray
Foliar
Spray
Soil
Spray
FoliarSpray2014 Foliar
Spray
Foliar
Spray
TRTD
Soil
Spray
Foliar2015 Spray

Timing/GrowthStage (BBCH)
Actual Spray
Volume, GPA(L/ha)
Rate, lb a.i./A (kga.i./ha)MethodYear

Plot Name

Applicationa

Formulation

Location (City, State,
NAFTA Region)

Trial Identification

MRID 49665202

NA

Dyne-Amic
0.25 % v/v
Dyne-Amic
0.25 % v/v
Dyne-Amic
0.25 % v/v
NADyne-Amic
0.25 % v/v
Dyne-Amic
0.25 % v/v
Dyne-Amic
0.25 % v/v
NADyne-Amic
0.25 % v/v
Dyne-Amic
0.25 % v/v
Dyne-Amic
0.25 % v/v
NADyne-Amic
0.25 % v/v
Dyne-Amic
0.25 % v/v
Dyne-Amic
0.25 % v/v
NADyne-Amic
0.25 % v/v
Dyne-Amic
0.25 % v/v
Dyne-Amic
0.25 % v/v
NADyne-Amic
0.25 % v/v

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

NT00613ZA

NT00713ZACDPR IMI Soil and Foliar Cotton Study

Sanger, CA
Region 10

Fresno, CA
Region 10
2013

AdmirePro

Admire
Pro

755

65
00
61
61
65
00
61
65
65
00
61
65
65
00
61
61
65

0.057
(0.064)0.057(0.064)0.328(0.368)0.058(0.065)0.058(0.065)0.058(0.065)0.332(0.372)0.058(0.065)0.058(0.065)0.057(0.064)0.331(0.372)0.057(0.064)0.057(0.064)0.057(0.064)0.330(0.369)0.057(0.064)0.057(0.064)0.057(0.064)

7
7

NA
95

0.50
(0.56)

7
6
249
77

0.51
(0.57)

7
6
NA
81

0.50
(0.56)

7
7
262
75
7
7

0.50
(0.56)

Adjuvant

20
(187)
20
(187)
13
(118)
16
(146)
15
(142)
15
(144)
13
(125)
19
(179)
19
(181)
20
(185)
14
(132)
15
(141)
15
(141)
15
(140)
14
(132)
15
(140)
15
(140)
15
(141)

Total Rate, lb a.i./A(kg a.i./ha)61

Retreatment
Interval (days)

FoliarSpray
Foliar
Spray
Soil
Spray
FoliarSpray2013 Foliar
Spray
TRTD
Foliar
Spray
Soil
Spray
FoliarSpray2014 Foliar
Spray
Foliar
Spray
Soil
Spray
FoliarSpray2013 Foliar
Spray
Foliar
Spray
Soil
TRTD
Spray
Foliar2014 Spray
FoliarSpray
FoliarSpray

Timing/GrowthStage (BBCH)
Actual Spray
Volume, GPA(L/ha)
Rate, lb a.i./A (kga.i./ha)MethodYear

Plot Name

Applicationa

Formulation

Location (City, State,
NAFTA Region)

Trial Identification

MRID 49665202

Dyne-Amic
0.25 % v/v
Dyne-Amic
0.25 % v/v
NADyne-Amic
0.25 % v/v
Dyne-Amic
0.25 % v/v
Dyne-Amic
0.25 % v/v
NADyne-Amic
0.25 % v/v
Dyne-Amic
0.25 % v/v
Dyne-Amic
0.25 % v/v
NADyne-Amic
0.25 % v/v
Dyne-Amic
0.25 % v/v
Dyne-Amic
0.25 % v/v
NADyne-Amic
0.25 % v/v
Dyne-Amic
0.25 % v/v
Dyne-Amic
0.25 % v/v

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

CDPR IMI Soil and Foliar Cotton Study

Adjuvant

Total Rate, lb a.i./A(kg a.i./ha)Retreatment
Interval (days)

Timing/GrowthStage (BBCH)
Actual Spray
Volume, GPA(L/ha)
Rate, lb a.i./A (kga.i./ha)MethodYear

Plot Name

Applicationa

Formulation

Location (City, State,
NAFTA Region)

Trial Identification

MRID 49665202

Soil 00 14
0.328
NA
0.50
NA
Spray
(130) (0.368)(0.56)
Foliar 65
150.057 83
Dyne-Amic
Spray
0.25 % v/v(141) (0.064)
2013 Foliar 65 15
0.057
7
Dyne-Amic
Spray(141) (0.064)
0.25 % v/v
Foliar 65 15
0.058
7
Dyne-Amic
Spray(142) (0.065)
0.25 % v/v
Soil 00 14
0.326 226 0.50
NANT008Kerman, CA Admire TRTD
Spray(130) (0.365)(0.56)
13ZA
Pro
Region 10
Foliar 61 15
0.056
83
Dyne-Amic
Spray(138) (0.063)
0.25 % v/v
2014 Foliar 61 15
0.057
7
Dyne-Amic
Spray(141) (0.064)
0.25 % v/v
Foliar 65
7Dyne-Amic
15
0.057
Spray
0.25 % v/v(140) (0.064)
Soil 00 14
0.331
NA
0.50
NA
Spray
(132) (0.371)(0.56)
Foliar 61 15
0.056
83
Dyne-Amic
Spray(139) (0.063)
0.25 % v/v
2013 Foliar 65 15
0.057
7
Dyne-Amic
Spray(141) (0.064)
0.25 % v/v
Foliar 65 15
0.057
7
Dyne-Amic
Spray(141) (0.064)
0.25 % v/v
NT009Kerman, CA Admire TRTD
Soil 00NA
140.330 225
0.50
13ZA
Pro
Region 10
Spray(131) (0.369)(0.56)
Foliar 61 15
0.057
83
Dyne-Amic
Spray(141) (0.064)
0.25 % v/v
2014 Foliar 61 15
0.057
7
Dyne-Amic
Spray(141) (0.064)
0.25 % v/v
Foliar 65 15
0.057
7
Dyne-Amic
Spray(141) (0.064)
0.25 % v/v
aValues for spray volume, rate, and total rate have been rounded using values provided in Appendix 2of the study report.
b
NA = Not applicable.
c
Only the first year of trial NT002-13ZB will be reported because the plot location was no longer
available.
*Table 4 of the study report756

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 49665202

CDPR IMI Soil and Foliar Cotton Study

Table 2. Soil and meteorological characteristics of the study sites*.

Trial
IDa

Trial Location (City,Country/State, Year, GPS
Coordinatesb)

Soil Characteristicsc
CEC
(meq/
%
%
100 g %OM (%) pH soil) Sand Silt Clay

NT001Yuba City, CA, 2014
13ZB (38°59.055’ N, 121°36.115’ W) 2.0
NT002Wheatland, CA, 2014
13ZB
(39°00.22’ N, 121°27.59 W) 1.5
NT003- Fresno, CA, 2013-2014
13ZA
1.0(36.73628, -119.87515)
NT004- Davis, CA, 2013-2014
13ZA
1.6(38.5337, -121.7793)
NT005Yuba City, CA, 2014
13ZB
(39°02.24’ N, 121°37.63’ W) 2.7
NT006- Sanger, CA, 2013-2014
13ZA
0.38
(36.69982, -119.46196)
NT007- Fresno, CA, 2013-2014
13ZA
0.81
(36.73916, -119.87599)
NT008- Kerman, CA, 2013-2014
13ZA
0.51
(36.79552, -120.05406)
NT009Kerman, CA, 201313ZA 2014(36.79516, -120.05676) 0.34

6.1

19

19

46

Meteorological Datad
Total

RainfallType (in)

Silty
clay
35 loam 18.92

Temp. Range(°F)

Variety

Pima
41

97
Pima

14

39

36

7.2 14

67

18

6.5

7.0 1939

36

25

Loam 0.92
Sandy
15 Loam 3.92

7.4 1263

28

5.7 4.573

26

25 Loam 14.93
Clay
31 loam 18.92
Sandy
7
Loam 5.69
Sandy
9
Loam 3.92
Loamy
1sand 2.74

5.6 4.5

85

14

1

19

25

44

5.9 5.7

69

24

6.5

a
b
c

Loamy
sand 2.74

47

97

30

102

DP 358 RF32

93

PHY 804 RF

Pima
Pima

41

97

31

99

Pima
PHY 802 RF
30

102

29

99

29

99

Delta Pine
358 RF
Delta Pine
358 RFPHY 802 RF

Site conditions listed are for the TRTD plot. For UTC plot conditions, see Appendix 2 of the study report.
GPS coordinates are in the form (latitude, longitude).
Soil characteristics are based on analyses of composite soil samples colle3cted from within the treated plot. The
central area of the treated plot was identified (e.g., the central 100 by 200 ft. section from within a 200 by 400ft. plot), and soil subsamples were collected from the four corners of that central area. The four subsamples
were composited into one sample for analysis. Abbreviations used: %OM = percent organic matter; CEC = cationexchange capacity.
d Data is for the interval of the month of first application through the month of last sampling.
Meteorological data were obtained from nearby government weather stations or on-site weather stations.
* Combined table from Table 3A and Table 8 of the study report.

757

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49665202

CDPR IMI Soil and Foliar Cotton Study

Table 3. SSURGO soil characteristics of the study sites*.
Trial ID

Component
Drainage
Hydrologic Runoff
General Texture CDPR
Group
%a
Class
Description
Texture(SSURGO)
Category
Yuba City, Marcum-GridleyFine
45 / 40 Moderately
C/D
Low /
CA
Well Drained
MediumClay Loam,
0-1% slopes
Wheatland, Columbia Fine
Coarse85
Somewhat
A
Very LowCA
Sandy Loam,
Poorly Drained
0-1% slopes(~10% KimballLoam)
Fresno, CA Ramona Loam
Medium80
Well DrainedC
Low

Particle
Size

NT00413ZA

Davis, CA

NT00513ZB

Yuba City,CA

NT00113ZB
NT00213ZB

NT00313ZA

NT00613ZA
NT00713ZA
NT00813ZA
NT00913ZA

Trial
Location

Reiff Very Fine
Sandy Loam

Marcum-Gridley
Clay Loam,
0-1% slopes
Sanger, CA Hanford finesandy loam,
gravelly
substratum
Fresno, CA Greenfield
Sandy Loam,
0-3% slopes
Kerman, CA Hanford Coarse
Sandy Loam
Kerman, CA Hanford Sandy
Loam, silty
substratum

Fine
Coarseloamy

Fineloamy

Coarse85

Well DrainedA

Very Low

Coarseloamy

Fine

45 / 40

Moderately
Well Drained

C/D

Low /
MediumFine

Coarse85

Well DrainedA

Very Low

Coarseloamy

Coarse85

Well DrainedA

Very Low

Coarseloamy

Coarse85

Well DrainedA

Very Low

Coarseloamy

Coarse85

Well DrainedA

Very Low

CoarseloamyaMajor component(s) of the soil as a percentage of total soil; for Marcum-Gridley: Marcum is45%, hydrologic group C, and Low runoff; Gridley is 40%, hydrologic group B, and Medium runoff.
* From Table 3B of the study report.
5D. SAMPLE COLLECTION, HANDLING, PROCESSING
Cotton Plant Matrices.
Cotton leaf and flower samples were collected at three sampling intervals. The first samples werecollected at early bloom, prior to any foliar sprays, to measure residues in bee-relevant matrices as aresult of the at-plant soil application. The second and third sampling intervals measured residues thatwere a result of the soil application at planting plus three additional at-bloom sprays. The first, second,
and third sampling intervals corresponded to 4 to 5 days prior to the first foliar application (70 to 95 days
after the soil application), 4 to 5 days after the last foliar application, and 10 to 14 days after the last foliar
application, respectively (Table 4). At each sampling interval, duplicate composite samples (two separate
runs through the plot) of cotton flowers and cotton leaves were collected from the treated plots when theplants were at bloom, BBCH 61 (begin flowering, early bloom) to BBCH 73 (about 30% of bolls have758

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 49665202

CDPR IMI Soil and Foliar Cotton Study

attained their final size). Single composite samples of cotton leaves and flowers were collected from thecontrol plot of each trial on the same days that samples were collected from the treated plots.
Cotton flowers and leaves were collected by hand into Ziplock bags. Each composite flower sample
contained a minimum of 125 g (minimum 250 blossoms) collected from at least 12 different areas of theplot, avoiding the edges. Exceptions occurred in trials NT001-13ZB and NT005-13ZB, when flower sample
weights were below the minimum or were not recorded, however sufficient flowers were collected fromprocessing. Each composite leaf sample contained a minimum of 100 g. All samples were protected fromsunlight and placed in field coolers containing ice or ice substitute or in portable freezers.
After their collection, cotton flowers were hand-processed at the field site to obtain the bee-relevantsamples of cotton pollen, floral nectar, and extrafloral nectar; all available matrices were collected.
Processing occurred the same day as flower collection, except during the last 2015 sampling interval intrial NT001-14ZB, when nectar samples were collected same day and then the flowers were allowed to air
dry overnight, with pollen samples being collected the following day. Extrafloral nectar from the subbracteal and inner bracteal nectaries was removed using a micropipette and placed into a pre-weighed
amber glass collection vial. Nectar from the floral nectary was removed by micropipette and placed in a
separate vial. Pollen was removed from the cotton blossoms either by vacuum aspiration with collection
in filter tips or by tapping the pollen from the blossoms onto wax paper and collection of the accumulated
pollen into a vial. All resulting nectar and pollen samples were labeled and placed in the frozen storage(via freezer or dry ice) immediately after they were generated. After processing was completed, theflowers were discarded.
Figures 4 through 6 provide trends in total imidacloprid residue measured in extrafloral nectar, pollen,
and leaf tissue at each site. Figure 7 depicts the relationship between concentration of total imidacloprid
residues measured in leaves and floral nectar parsed out by soil type.

759

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49665202

CDPR IMI Soil and Foliar Cotton Study

Table 4. Sampling dates and cotton developmental stages for plant residue analysis*
Year 1
†
‡
Dates
BBCH
61
61
61
65
67
67
65
65
67
65
65
67
65
65
67

Pollen, Nectar, Extrafloral Nectar, and Leaf SamplingYear 2
Year 1
†
†
‡
‡
Dates
Dates
BBCH
BBCH

Davis 1 = NT004-13ZA
86 DASA,
95 DASA,61-5 DA1FA-4 DA1FA
5 DA3FA
65
4 DA3FA
13 DA3FA67
13 DA3FA
Fresno 1 = NT003-13ZA
76 DASA,
72 DASA,61-5 DA1FA-5 DA1FA
5 DA3FA
65
5 DA3FA
14 DA3FA
67
14 DA3FA
Fresno 2 = NT007-13ZA
76 DASA,
70 DASA,61-5 DA1FA-5 DA1FA
5 DA3FA
65
5 DA3FA
14 DA3FA
67
14 DA3FA
Kerman 1 = NT008-13ZA
78 DASA,
78 DASA,61-5 DA1FA-5 DA1FA
4 DA3FA
65
4 DA3FA
14 DA3FA
67
12 DA3FA
Kerman 2 = NT009-13ZA
78 DASA,
78 DASA,61-5 DA1FA-5 DA1FA
4 DA3FA
65
5 DA3FA
14 DA3FA
67
14 DA3FA

61
65
65
65
69
73
61
61
61
61
3

XX
67

Year 2
‡
Dates
BBCH
†

Sanger 1 = NT006-13ZA
90 DASA,
73 DASA,61-5 DA1FA-4 DA1FA
4 DA3FA
65
4 DA3FA
14 DA3FA
67
14 DA3FA
Wheatland = NT002-13ZB
88 DASA,
1NCNC-4 DA1FA
5 DA3FANC
NC
14 DA3FA
NC
NC
Yuba City 1 = NT001-13ZB
88 DASA,
2TBC
TBC-5 DA1FA
5 DA3FA
TBC
TBC
14 DA3FA
TBC
TBC
Yuba City 2 = NT005-13ZB
86 DASA,
TBC
TBC-5 DA1FA
5 DA3FA
TBC
TBC
14 DA3FA
TBC
TBC

†

BBCH = Biologische Bundesanstalt, Bundessortenamt und CHemische Industrie growth stage scale for cotton.
DASA = Days after the soil (in-furrow) application; DA1FA = days after the first foliar application; DA3FA = days after
the third foliar (and last) application. A negative number designates days prior to the indicated application.
1
NC = Not collected; no year 2 samples from trial NT002-13ZB can be collected.
2
TBC = To Be Collected; the samples will be collected when the second year of the trial is performed.
3
BBCH XX indicates no growth stage reported in the field data summary.
*Combination of Appendix 1 and Appendix 4 (Section 6) of the study report.
‡

Sample Storage.
Composite samples of cotton leaves, pollen, floral nectar, and extrafloral nectar were placed into labeled(study number and sample number) containers for shipment. All samples were frozen within
4 hours of collection with the exception of certain samples in trial NT004-13ZA, which were frozen within
9 hours and 1 minute, and 2015 samples from trials NT001-13ZB and NT005-13ZB took up to 7 hours and
20 minutes to be stored frozen (in a freezer or on dry ice). Samples remained frozen until receipt at Bayer
CropScience in RTP, NC.

760

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49665202

CDPR IMI Soil and Foliar Cotton Study

Stability studies have indicated that imidacloprid residues are stable {<30% decomposition) for 24 months(728 to 769 days) of freezer storage in the following representative crops: an oilseed (cottonseed), a nonoily grain (wheat), a leafy vegetable {lettuce), a root crop {potato), a tree fruit {apple), and a fruiting
vegetable {tomato)4-10. An additional stability study has indicated that imidacloprid residues are stable(<30% decomposition) for 36 months of freezer storage in wheat (grain), orange {fruit), tomato (fruit),
bean {seed), and rape (seed)11. Demonstrated freezer stability in all of the above crops is representative of
the freezer stability of imidacloprid, 5-hydroxy imidacloprid, and imidacloprid olefin residues to be
expected for cotton leaves from this study. The cotton leaves analyzed in this study were held in frozen
storage for a maximum of 569 days (19 months) prior to extraction.
Based on the available storage stability data,4-11 the imidacloprid residues would be representative of theresidues to be expected after the use of Admire Pro Systemic Protectant on the tested crops.
5E. ANALYTICAL METHODS
The analytical methods1-2 used in this study measured the residues of imidacloprid, 5-hydroxyimidacloprid, and imidacloprid olefin in cotton leaves, floral nectar, extra floral nectar, and pollen. These
data are reported in Appendix 4 of the full study report titled, “Analytical Report for EBNTN011
Determination of the Residues of Imidacloprid, 5-Hydroxy Imidacloprid, and Imidacloprid Olefin in Bee
Relevant Matrices Collected from Cotton During Two Successive Years.”
For the cotton leaves, a 2.5-g sample was weighed into a 50-mL polypropylene conical centrifuge tube,
and 10 mL HPLC-grade water was added. The tube was mixed manually for 1 minute, followed by adding
20 mL of acetonitrile and shaking for an additional 1 minute. Then, 3 g of MgSO4 and 1.5 g of NaCl wereadded. The sample was amended with a mixed internal standard solution and mixed manually for 1
minute. For leaf samples which were found to contain high residues of imidacloprid (>2 ppm), as
determined by an initial run in which the response exceeded the calibration curve, the sample wasamended with a 10X mixed internal standard solution before the salts were added. The sample wascentrifuged. For samples containing low levels of imidacloprid residue, 20 mL of organic supernatant wastransferred into a separate 50-mL polypropylene conical centrifuge tube containing 0.3 g of Bondesil-PSA
and 1.8 g of MgSO4. For samples containing high levels of imidacloprid residue, 2.0 mL of organic
supernatant and 18.0 mL of acetonitrile were transferred into a separate 50-mL polypropylene conicalcentrifuge tube containing 0.3 g of Bondesil-PSA and 1.8 g of MgSO4, which was manually mixed for 1
minute. The sample extract was centrifuged, and a 1.25 mL aliquot of supernatant was transferred into aclean culture tube. The sample aliquot was evaporated to near dryness using a Turbo-Vap (Biotage,
Charlotte, NC). The solid was reconstituted with 1.25 mL of 9:1 water/MeOH containing 10 mM NH4HCO3
by vortexing, and the resulting solution was transferred into a 2-mL sample vial for LC/MS/MS analysis.
For nectar, a 0.1-mL sample was weighed into a 20 x 150 mm culture tube and dissolved in 4 mL of water.
If the total sample volume was less than 0.1 mL, the entire sample was weighed and recorded. The
mixture was amended with isotopically labeled internal standards, mixed well, and applied to an Agilent
BondElut SPE cartridge (50 mg resin; previously conditioned with methanol then water). The cartridge waswashed with 1 mL of MeOH/H2O (1:19 v/v), and the combined eluates were discarded. The analytes were
eluted from the cartridge with 0.5 mL of MeOH/H2O (1:4 v/v). The eluate was collected in a 2 mL sample
vial for analysis by LC/MS/MS.
For pollen, a 0.1-g sample was weighed into a 2-mL centrifuge tube containing 2.8 mm steel balls. If an
individual sample volume was not sufficient for analysis, the two samples collected at the same interval
761

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49665202

CDPR IMI Soil and Foliar Cotton Study

were composited and analyzed. The composite sample was weighed and recorded. A 1-mL portion of
methanol/water (3:1 v/v) was added, and the mixture was homogenized with a bead mixer at 5000
beats/min for 1 minute on a Precellys homogenizer (Bertin Technologies, Rockville, MD). The isotopicallylabeled internal standards were added and mixed, and the mixture was centrifuged at 12,000 rpm for 2
minute. The supernatant was transferred into a clean culture tube containing 2.5 mL of water. The sample
was evaporated to an aqueous remainder and applied to a 3-mL ChemElut SPE cartridge. After 10 to 15
minutes, the cartridge was washed three times with 4 mL of hexane/ethyl acetate (1:1 v/v), and theeluates were collected in a clean culture tube. The combined eluates were evaporated to dryness. The
analytes were dissolved in 0.5 mL of MeOH/H2O (1:4 v/v), and the resulting solution was transferred intoa 2 mL sample vial for analysis by LC/high resolution mass spectrometry (LC/HRMS).
Quantitation of each analyte was based on the daughter ion transitions of the analyte and the respective
internal standard analog. The responses of the LC/MS/MS and LC/HRMS systems to each analyte and its
internal standard were measured in samples and in standards, and a relative response was calculated (asthe ratio of the analyte and the stable isotopically labeled internal standard responses). The relative
response of the analyte in each sample was compared to the relative response of the analyte in thestandards provided in Appendix 4 of the study report.
The relative responses of imidacloprid were measured over the range of 0.00012 to 4 ppm. The
correlation coefficients (R) were calculated using linear regression analysis with 1/x weighting.
All data are reported in parent equivalents, and the individual measured residues of imidacloprid,
5-hydroxy imidacloprid, and imidacloprid olefin are summed to give a total imidacloprid residue.
5F. QUALITY ASSURANCE RESULTS
The responses of the LC/MS/MS and LC/HRMS systems to imidacloprid, 5-hydroxy imidacloprid, andimidacloprid olefin were linear in solvent over the range of 0.00012 to 4 ppm. The correlation coefficientswere >0.99. The response data and analytical data summaries are located in Appendix 4.
Control interferences for cotton matrices are discussed in this paragraph; no total imidacloprid residue
was calculated for the UTC samples, so the levels of imidacloprid as an individual analyte are described.
Imidacloprid (parent) residues in UTC cotton floral nectar ranged from below the analyte LOD to
0.067 ppm. Imidacloprid residues in UTC cotton extrafloral nectar ranged from below the analyte LOD to
0.053 ppm. Imidacloprid residues in UTC cotton pollen ranged from below the analyte LOD to
0.017 ppm. Imidacloprid residues in UTC cotton leaves ranged from below the analyte LOD to
0.052 ppm.
All recoveries were corrected for any interferences in corresponding controls. The overall means of therecoveries for each matrix at each fortification level were within the acceptable range of 70 to 120%, andstandard deviation values were below 20%.
The limit of quantitation (LOQ) is defined as the lowest fortification level of an analyte at which
acceptable recovery has been achieved. The LOQ for a total residue is the highest of the LOQ values
assigned to the individual analytes for a particular matrix. The limit of detection (LOD) is defined as thelowest concentration of an analyte that can be determined to be statistically different from a blank. The
LODs were determined from method validation data obtained from control samples fortified at therespective analyte LOQs. The LODs were calculated by multiplying the standard deviation of recovery
762

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49665202

CDPR IMI Soil and Foliar Cotton Study

measurements at the LOQ by t0.99 [where t0.99 is the one-tailed t-statistic at the 99% confidence level forthe number of replicates (n)].3 The LOD for the total imidacloprid residue in each matrix is the highest LOD
value of any one individual analyte for that particular matrix.
Limits of quantification and detection for imidacloprid and metabolites*.
MatrixAnalyteLOQ (ppb)

LOD (ppb)

Imidacloprid

5.0

1.2

5-Hydroxy Imidacloprid

5.0

0.7

Imidacloprid Olefin

5.0

0.8

Total Imidacloprid

5.0

1.2

Imidacloprid

1.0

0.3

5-Hydroxy Imidacloprid

1.0

0.7

Imidacloprid Olefin

1.0

0.6

Total Imidacloprid

1.0

0.7

Imidacloprid

1.0

0.3

5-Hydroxy Imidacloprid

1.0

0.7

Imidacloprid Olefin

1.0

0.6

Total Imidacloprid

1.0

0.7

Imidacloprid

1.0

0.4

5-Hydroxy Imidacloprid

1.0

0.5

Imidacloprid Olefin

1.0

0.3

Total Imidacloprid* From page 20 of the study report.

1.0

0.5

Cotton Leaves

Cotton Extrafloral
Nectar

Cotton Floral Nectar

Cotton Pollen

6. RESULTS:
6.A. COMPARISON OF CONCENTRATIONS MEASURED FOR PARENT AND DEGRADATESComparison of the relative concentrations measured for parent imidacloprid and degradation products infloral nectar, extrafloral nectar, pollen, and leaves are presented in Table 6-3 through Table 6-6.
Concentrations were reported as ppm on a weight/weight basis. The LOD of values were low, ranging
from 0.3 ug/g to 1.2 ug/g (ppb), so data reported as below the LOD were assigned one-half the LOD value.
Comparison of the contribution of each chemical to the total residue indicates that parent imidacloprid
represented the majority of the total residue measured in each plant part. For floral nectar, extrafloral
nectar, and pollen (bee relevant tissue), parent imidacloprid comprised over 90% or greater of the total
for most comparisons. When the levels of residues are low the ratios could be affected by the insertion of½ the LOD where the tendency would be for overestimation of the contribution of a degradation product.
Since the degradation products (considered as toxic as parent imidacloprid) are comprised of a small
portion of the measured residue, the following discussions will focus on comparing total imidacloprid
residue measured in plant tissues. Statistical procedures used in the Statistical Analysis System (SAS)software to provide distribution statistics and statistical tests were PROC CAPABILITY, PROC SHEWHART,
PROC TTEST, PROC UNIVARIATE, and PROC NPAR1WAY. Figure 6-1 illustrates the statistical aspects
relayed in the Box-and-Whisker plots used to compare the distribution of concentrations calculated for
total imidacloprid residue at each sampling interval. For each represented data set, the box graphic

763

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49665202

CDPR IMI Soil and Foliar Cotton Studypresents values for the mean, median, minimum, maximum, and 25th and 75th percentiles of the
distribution.
6.B. Potential for Yearly Carry-over of Residues
Site specific curves provide comparison between years for mean concentrations of total imidacloprid
residue measured at each sampling interval for 8 of the 9 sites (Figure 6-2 through Figure 6-5). The study
was conducted only one year at the Wheatland site (NT002-13ZB). Some curves are very similar between
the years, such as floral nectar at the Sanger site and leaves at the Kerman and Davis sites. But someindicate a greater concentration measured at sampling interval 2 in 2013 than in 2014, such as floral
nectar at the Davis site and Kerman site 1 and leaves at Fresno site 1. In addition, mean values at the firstsampling interval were very similar between years at each site for floral nectar, extrafloral nectar, and leaf
samples (Table 6-1). The similarity in starting values for each year and the variation in patterns observed
between years indicated no consistent effect for a carry-over of residues. These observations wereconfirmed by lack of a significant difference between years using either Wilcoxon’s Signed Rank Test for
zero as the location for the difference in value between the 2 years or Student’s paired t-Test (Table 6-2).
Most sites reported 2 replicate samples so tests were conducted on mean values calculated for each siteat each sampling interval within each year.
For pollen samples, there was a noticeable difference in the magnitude of concentrations and trend
measured between 2013 and 2014 data (Figure 6-4). Concentrations in pollen samples at all sites and at
all sampling intervals were lower in the first year of the study compared to when measured in the second
year of the study. This pattern was not consistent with all other patterns measured for that year in theother plant tissues or for the pattern in pollen residues measured in the second year. Since the abnormal
pattern noted for pollen was limited to the first year of the study, this potentially indicates problems
caused by sampling procedures and/or chemical analyses for pollen in 2013. One might argue that theeffect could be caused by climatic or site differences, but if true, then the patterns would also be
expected in the other plant samples as they were obtained at the same sites and at the same samplinginterval. Therefore, yearly comparisons for pollen appear to be compromised due to experimental
anomalies encountered in the analyses for the first year.
6.C. MAGNITUDE OF RESIDUES IN BEE-RELEVANT MATRICES
Based on the lack of differences measured between years, data were pooled from both years todetermine the expected distributional properties for concentrations in plant samples. General patterns
for total imidacloprid residues in plant samples are illustrated in Figure 6.6.
Floral Nectar. Comparison of overall statistics for total imidacloprid residue indicated much greater
concentrations in all plant parts at the second sampling interval than at the other two sampling intervals(Figure 6-6). The first soil application occurred at planting. Sampling for the first interval occurred at amean of 81 days after the soil application with a range of values from 70 to 95 days (Table 4 above). The
median total imidacloprid residue at the first sampling interval was 9 ppb in floral nectar with a maximum
value of 127 ppb and 90th percentile value of 50 ppb (Table 6-3). The second sampling interval was
conducted after 3 foliar applications of imidacloprid and was approximately 23 days after the firstinterval. Concentrations in floral nectar at the second sampling interval increased to a median of 70 ppb
with a maximum value of 171 ppb and a 90th percentile value of 144 ppb. The third sampling interval
764

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49665202

CDPR IMI Soil and Foliar Cotton Study

occurred approximately 9 days after the second interval with the total imidacloprid residue concentration
dropping to a median of 35 ppb with a maximum value of 117 ppb and a 90th percentile value of 113 ppb.
Extrafloral Nectar. Although a similar increase in concentration was observed at sampling interval 2 forextrafloral nectar, the increase in concentration measured was relatively much larger than observed forfloral nectar. The median value at the second sampling interval was 276 ppb, which was approximately 4times greater than the value measured for floral nectar (Table 6-4). At the second interval the maximum
value was 2775 ppb and the 90th percentile value was 1882 ppb.
Pollen. Even though the pattern in the first year indicated potential analytical problems, the trend forpooled pollen data was similar to floral and extrafloral nectar where increased concentrations wereobserved at sampling interval 2. The median value at the first sampling interval was 1 ppb which
increased to 46 ppb at the second sampling interval (Table 6-5). At the second interval the maximum
value was 2906 ppb and the 90th percentile value was 409 ppb.
6.D. MAGNITUDE OF RESIDUES IN LEAVESAs expected, direct foliar applications of imidacloprid to plants between the first and second samplinginterval greatly increased the magnitude of residues of total imidacloprid in leaves measured at thesecond sampling interval (Table 6-6). At the first sampling interval that occurred around 81 days after thesoil application, the median total residue in leaves was 24 ppb. The median concentration at samplinginterval 2 was 80 times greater at 1956 ppb. At interval 3, the levels in leaves were decreased with themedian value down to 441 ppb, a value that was still greater than residues measured at the first interval.
6.E. SITE SPECIFIC TRENDS
Temporal patterns in residue concentrations for the 9 separate sites are depicted in Figure 6-2 throughFigure 6-5 for floral nectar, extrafloral nectar, pollen, and leaves, respectively. The general pattern noted
above where concentrations rise steeply at the second sampling interval is reflected in floral nectar
samples with 13 of the 15 curves (Figure 6-2). Curves at three sites (Fresno site 1 in 2013, and both Yuba
City sites in 2015) reflect a different pattern where a rise in concentration over time is noted.
Concentrations at a fourth site, Kerman site 2 in 2013 were stable over time. The cause for thesedifferences is unknown but uncertainties in sampling methodology may cause this variation. All othersites, though, indicated that there should be sharp increases in concentrations caused by foliar
applications. Decreases in concentration from interval 2 to 3 were predominant but there were subtle
differences in slopes where some indicated deep decreases in concentrations between the intervals whileothers indicated only a shallow, slight decrease in concentration over time. Data in 2014 for the two
Kerman sites (green lines) exemplify the potential differences in slopes measured between intervals 2 and
3 where a large decrease is indicated at Kerman1 (solid dots) but a small decrease is indicated at Kerman2(open squares).
Trends in total imidacloprid residue measured in extrafloral nectar also reflected a large increase inconcentration at sampling interval 2 followed by reductions at sampling interval 3 (Figure 6-3). The
exception was data for Fresno site 1 in 2013. Data in 2014 reflected the pattern measured at the rest of
the sites. The increase at some sites was rather large where, for example at Davis site 1 in 2013 theaverage concentration was 1629 ppb.
For pollen samples, there were noticeable differences in the trend and magnitude of concentrations
measured between 2013 and 2014 data as previously indicated (Figure 6-4). Concentrations in pollen at765

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49665202

CDPR IMI Soil and Foliar Cotton Study

all sites in the first year of the study were lower than when measured in the second year of the study. The
pattern for the second year of the study is consistent with the general trend observed for all other plant
samples in both years where foliar application greater increased the measured concentrations.
Lastly, all curves for leaf concentrations exhibited an increase at interval 2 and a sharp decline at interval
3 (Figure 6-5). The sharp decrease in concentrations between intervals 2 and 3 does not appear to be due
to washoff from rainfall or irrigation. For example, at the Fresno sites and the Sanger sites, there was norecorded rainfall during this time interval. Also, the irrigation systems used had a low potential for wetting
the leaves because drip irrigation was used at the Fresno sites and a combination of drip and furrow
methods at the Sanger site.
6.F. LEAF AND NECTAR CONCENTRATION IN RELATION TO SOIL TYPE
Originally, the proposed study design suggested sampling sites located in coarse, medium, and finetextured soils with 3 replicates assigned to each soil type. There is an inherent difficulty in fulfilling the
proposed design when the study is conducted after fields have been planted and then growers arecontacted in an effort to procure their cooperation. The reported soils in this study were biased towards
coarse soils where in 2013 five of six sites were located in coarse soil, one in medium textured soils andnone in the fine textured category. In 2014, two sites were located in the fine textured category, one site
in medium, and six sites in the coarse textured category. Then in 2015, there were only 2 sites in finetextured soil. Analysis for the effect of soil is not possible in light of the noted differences measured inconcentration between years and the uneven replication in soil type between years.

7. STUDY STRENGTHS, LIMITATIONS AND CONCLUSIONSIn the context of documenting the magnitude of imidacloprid residues in bee-related matrices of cotton,
the following strengths are observed with this study.
1. Data provide quantitative values of total imidacloprid residues expected in floral nectar,
extrafloral nectar, pollen, and leaves of cotton.
2. Measurements were taken at 3 time intervals in an attempt to quantify levels expected in planttissues: The first interval reflected concentrations following a period of time after a soil
application at planting, and the second and third sampling intervals expected concentrations
expected in plants after three additional foliar applications.
Limitations noted in this study include:
1. The values most likely do not reflect a maximal exposure to bee relevant matrices because
sampling did not occur directly after foliar application. Sampling after the third foliar application
averaged 5 days. Substantial decreases at nearly an order of magnitude were noted in residues
from plants sampled between the 2rd and 3th foliar application where the sampling interval
averaged 9 days. Since there was no potential for redistribution of residues due to water
movement from either irrigation or rainfall, the steep dissipation indicates that concentrations
would most likely have been highest if samples were taken directly after foliar applications.

766

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49665202

CDPR IMI Soil and Foliar Cotton Study

2. The study did not follow the residue data protocol calling for three replicates each in fine,medium and coarse soils resulting in uncertainty in regards to the effects of soil type on
imidacloprid concentration in cotton flower parts and leaves.
3. An inadequate sample of extrafloral nectar to analyse was collected following the soil applicationand prior to the foliar application in replicate NT003-13ZA.
4. Pollen analysis appeared problematic in the first year of the study, which was initiated in 2013.
Patterns for all other plant tissues and for analyses of pollen in 2014 indicated a very large
increase in total imidacloprid concentration for interval 2 due to foliar application, an effect thatwas extended to the third sampling interval. This apparent anomalous pattern was evident at all
experimental locations. Since the other plant samples were obtained simultaneously, effects ofclimate or site should be minimal. This condition indicates that an experimental condition such as
an analytical problem was most likely the cause for lower concentrations measured in the firstyear.
5. Data are inadequate to compare concentrations in cotton matrices between soil types because
data from medium and fine soil types were not adequately represented.
Overall, considering the strengths and limitations of this study, the following conclusions can be drawn:
1. Imidacloprid residues were measured in bee-relevant matrices from soil application: Maximum
concentrations of total imidacloprid residues at approximately 81 days after soil application of
0.34 lb ai/A were 127, 36, and 43 ppb in floral nectar, extrafloral nectar, and pollen, respectively.
Median concentrations were 9, 3, and 1 ppb in floral nectar, extrafloral nectar, and pollen,
respectively.
2. Imidacloprid residues that were measured in bee-relevant matrices increased from additional
foliar sprays: The distribution of total imidacloprid residues resulting from 3 additional foliar
sprays each at 0.056 – 0.067 lb ai/A increased the maximum values to 170, 2775, and 2906 ppb, infloral nectar, extrafloral nectar, and pollen, respectively. Median concentrations were 70, 276,
and 46 ppb in floral nectar, extrafloral nectar, and pollen, respectively.
3. Concentrations in bee-relevant matrices generally decreased overtime following the foliar
applications: Subsequent sampling 14 days after the 3rd foliar application resulted in maximum
total imidacloprid residues of 117, 136, and 182 ppb in nectar, extrafloral nectar, and pollen,
respectively. Median concentrations were 35, 27, and 15 ppb in floral nectar, extrafloral nectar,
and pollen, respectively.
4. No evidence for carry-over effects between years: As indicated in the discussion, comparison of
the starting values between years and the patterns over time compared between years for eachsite for leaf, floral and extra-floral nectar indicated no consistent evidence for carry-over effects.
Comparison between years for pollen samples appeared compromised so comparisons between
years were not worthwhile.

8. STUDY VALIDITY/CLASSIFICATION

The data from this study provide an expected distribution of the concentrations of imidacloprid residues
that bees are exposed to in nectar, pollen and extrafloral nectaries of cotton plants grown under actual
agronomic practices in California. Relating concentrations measured in flower parts to bee health is767

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 49665202

CDPR IMI Soil and Foliar Cotton Study

possible by comparing the concentrations measured in bee relevant plant parts to target values thatdefine acute or chronic exposure scenarios. These data, however, would represent a minimal exposure
assessment to foliar applications because samples were not taken during the period of foliar application.
Therefore, there is uncertainty as to how reflective the values obtained at the 3 sampling intervals
represent maximum exposure scenarios. The study is considered scientifically sound and useful for risk
assessment purposes. The study is classified as ACCEPTABLE for quantitative use in risk assessment.

768

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49665202

CDPR IMI Soil and Foliar Cotton Study

9. REFERENCES

1. Brungardt, J. 2010. An Analytical Method for the Determination of Residues oflmidacloprid and its Metabolites lmidacloprid Olefin and 5-Hydroxy lmidacloprid in Bee
Relevant Matrices Using LC/MS/MS. Bayer Method No. NT-005-P10-01.

2. Miller, A. 2014. An Analytical Method for the Determination of Residues of lmidacloprid
and its Metabolites lmidacloprid Olefin and 5-Hydroxy lmidacloprid in Bee Relevant
Matrices Using LC/MS/MS. Bayer CropScience Report Number: NT-006-A13-01.
3. Office of Pesticide Programs, US EPA. 2000. Assigning values to nondetected/
nonquantified pesticide residues in human health food exposure assessments.
EPA Docket #OPP-00570A.
4. Noland, P.A. 1992. lmidacloprid and metabolites- freezer storage stability study incrops. Bayer CropScience Report No. 103237. MRID 42556135.
5. Noland, P.A. 1993. lmidacloprid and metabolites- freezer storage stability study incrops. Bayer CropScience Report No. 103237-1. MRID 42810311.
6. Noland, P.A. 1994. lmidacloprid and metabolites- freezer storage stability study incrops. Bayer CropScience Report No. 103237-2. MRID 43197203.
7. Noland, P.A. 1994. lmidacloprid and metabolites- freezer storage stability study incrops. Bayer CropScience Report No. 103237-3. MRID 43487302.
8. Lenz, C.A. 1993. Addendum 1. lmidacloprid and metabolites- freezer storage stability
study in crops (wheat matrices, cottonseed, tomato, cauliflower, and lettuce).
Bayer CropScience Report No. 103949-1. MRID 42810313.
9. Lenz, C.A. 1993. Addendum 2. lmidacloprid and metabolites- freezer storage stability
study in crops (wheat matrices, cottonseed, tomato, cauliflower, and lettuce).
Bayer CropScience Report No. 103949-2. MRID 43197201.
10. Lenz, C.A. 1993. Addendum 3. lmidacloprid and metabolites- freezer storage stability
study in crops (wheat matrices, cottonseed, tomato, cauliflower, and lettuce).
Bayer CropScience Report No. 1 03949-3. MRI D 43487301.
11. Schoning, R. 2014. Storage stability of imidacloprid and its 5-Hydroxy and olefine
metabolite in/on plant matrices for 36 Months. Bayer CropScience Report No.
P642094733 Amendment No. 1.

20769

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49665202

CDPR IMI Soil and Foliar Cotton Study

Table 6-1. Comparison of cumulative distributional statistics for concentration of total imidacloprid residues between the first year of thestudy (Year 1) and the second year of the study (Year 2) in floral nectar (ppb). Acronyms in the table are: Total Imidacloprid Residue = Sum ofparent and degrades; N=Number of paired observations; SD=Standard Deviation; CV=Coefficient of Variation. Numbered Interval denotes
timing of sampling where interval 1 was approximately 81 days after the first soil application and intervals 2 and 3 were at 5 and 14 days
after a third foliar spray coinciding with 91 and 100 days after the soil application.
Total Imidacloprid Residue

StatisticN
Mean (ug/L)SD (ug/L)
CV (%)Min (ug/L)Median (ug/L)
Max (ug/L)
StatisticN
Mean (ug/L)SD (ug/L)
CV (%)Min (ug/L)Median (ug/L)
Max (ug/L)Interval 1
Year 1
Year 2
8
8
27.3
19.6
34.6
16.1
127.0
82.1
1.7
1
6.2
18.5
83.1
53.4

Floral Nectar
Interval 2
Year 1
Year 2
8
8
91.4
65.6
49.0
36.5
53.6
55.7
20.9
17.9
83.1
72.3
153.3
113.4

Interval 3
Year 1
Year 2
8
8
37.7
44.0
24.6
28.5
65.2
64.8
10.5
17.6
31.2
37.6
78.8
103.9

Interval 1
Year 1
Year 2
6
6
5.1
13
3.4
13.7
67.0
105.5
1.2
0.9
4.9
10.2
9.7
35.9

Extrafloral Nectar
Interval 2
Year 1
Year 2
8
8
528.0
651.5
650.0
580.6
123.0
89.1
54.3
225.2
194.8
373.1
1951.51629.6

Leaves
8
41.5
63.6
153.2
1.4
19.7
192.9

8
53.3
48.9
91.7
5.6
44.6
140.1

8
1532.4
715.6
46.7
800.9
1417.93098.28
1882.5500.7
26.6
1196.81762.62860.3

Interval 3
Year 1
Year 2
8
8
38.5
42.3
21.1
33.3
54.7
78.8
7.6
16.2
34.8
26.3
63.1
111.2

Pollen
8
318.5
176.1
55.3
103.8
261.1
698.6

21
770

8
434.4
122.0
28.1
293.7
420.7
616.5

8
0.9
0.5
50.7
0.6
0.8
2.0

8
10.8
13.4
124.3
0.6
6.9
41.1

8
21.0
16.1
76.5
4.2
19.5
45.4

8
524.0
757.6
144.6
45.6
231.1
2316.8

8
8.1
6.6
82.0
3.2
5.8
22.8

8
97.1
45.7
47.1
17.2
105.5
153.3

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49665202

CDPR IMI Soil and Foliar Cotton Study

Table 6-2. Statistical results for test of differences in concentration measured between years 1 and 2
for concentration of total imidacloprid residue.

Total Imidacloprid Residue
Interval
Probability Level
SampledforPlant Sample
Extrafloral Nectar

Floral Nectar

LeavesPollen

1
2
3
1
2
3
1
2
3
1
2
3

22
771

T-Test
0.15
0.75
0.79
0.44
0.29
0.56
0.64
0.16
0.21
0.08
0.10
0.00

Sign Rank
0.16
0.84
0.95
0.55
0.38
0.64
0.25
0.25
0.25
0.03
0.01
0.01

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49665202

CDPR IMI Soil and Foliar Cotton Study

Table 6-3. Floral Nectar: Cumulative distributional statistics for concentration of imidacloprid and related metabolites in cotton floral nectar(ppb). Acronyms in the table are: IMI=Imidacloprid; Olefin=Imidacloprid Olefin; 5-OH=5 Hydroxy Imdacloprid; Total Imidacloprid Residue (inbold) = Sum of parent and degrades; N=Number of observations; SD=Standard Deviation; CV=Coefficient of Variation. Numbered Interval
denotes timing of sampling where interval 1 was approximately 81 days after the first soil application and intervals 2 and 3 were at 5 and 14
days after a third foliar spray coinciding with 91 and 100 days after the soil application.

Statistic

Floral Nectar: Distribution of Imidacloprid Residues by Interval Sampled
Interval 1
Interval 2Interval 3
5-OH Olefin
IMI
Total 5-OH Olefin
IMI
Total 5-OH Olefin
IMI

N
Mean (ppb)
SD (ppb)
CV (%)Min (ppb)
Median (ppb)
75th (ppb)
90th (ppb)
95th (ppb)
Max (ppb)% of Mean Total

32
0.6
0.4
71.4
0.4
0.4
0.8
1.4
1.5
1.8
2.8

32
0.6
0.5
87.2
0.3
0.3
0.8
1.4
1.8
2.0
2.8

32
20.0
26.6
133.2
0.3
8.6
34.1
47.4
71.0
123.4
94.3

32
21.2
27.4
129.4
1.0
9.2
35.2
50.2
74.0
127.0

33
2.1
1.2
54.9
0.4
1.9
2.7
3.5
4.6
4.7
2.8

23
772

33
1.3
0.9
67.4
0.3
1.2
1.4
2.8
3.1
3.7
1.7

33
71.2
41.1
57.8
11.5
66.3
93.8
128.8
139.0
164.0
95.4

33
74.6
42.8
57.4
12.2
69.6
97.7
134.9
144.0
170.6

33
1.2
0.7
57.2
0.4
1.0
1.5
2.0
3.0
3.2
3.1

33
1.1
0.8
69.6
0.3
1.0
1.4
1.9
2.1
4.5
2.7

33
38.1
26.2
68.8
9.0
32.4
45.9
66.0
109.2
112.5
94.3

Total
33
40.4
27.2
67.2
10.2
35.1
50.1
70.1
113.1
117.3

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49665202

CDPR IMI Soil and Foliar Cotton Study

Table 6-4. Extrafloral Nectar: Cumulative distributional statistics for concentration of imidacloprid and related metabolites in cotton
extrafloral nectar (ppb). Acronyms in the table are: IMI=Imidacloprid; Olefin=Imidacloprid Olefin; 5-OH=5 Hydroxy Imdacloprid; Total
Imidacloprid Residue (in bold) = Sum of parent and degrades; N=Number of observations; SD=Standard Deviation; CV=Coefficient of
Variation. Numbered Interval denotes timing of sampling where interval 1 was approximately 81 days after the first soil application andintervals 2 and 3 were at 5 and 14 days after a third foliar spray coinciding with 91 and 100 days after the soil application.

StatisticN
Mean (ppb)
SD (ppb)
CV (%)Min (ppb)
Median (ppb)
75th (ppb)
90th (ppb)
95th (ppb)
Max (ppb)% of Mean Total

ExtraFloral Nectar: Distribution of Imidacloprid Residues by Interval Sampled
Interval 1
Interval 2Interval 3
5-OH Olefin
IMI
Total 5-OH Olefin
IMI
Total 5-OH Olefin
IMI
28.0
28.0
28.0
28.0
33.0
33.0
33.0
33.0
33.0
33.0
33.0
0.5
0.5
5.6
6.6
15.2
2.9
543.0
561.5
1.6
0.6
37.4
0.3
0.4
7.9
8.1
16.5
3.3
603.2
622.2
1.1
0.7
31.6
69.0
79.7 140.0 122.2 108.8 117.4 111.0
110.1 70.5 115.5 84.5
0.4
0.3
0.2
1.1
3.2
0.8
43.8
47.3
0.4
0.3
5.2
0.4
0.3
1.9
3.3
8.3
1.5
266.9
276.4
1.3
0.3
25.1
0.4
0.7
6.8
7.9
20.8
2.7
584.0
607.9
1.9
0.7
43.2
1.0
1.2
17.9
19.3
36.9
6.0
1426.5 1470.4 3.5
1.1
85.6
1.4
1.6
18.3
19.3
53.9
8.5
1819.7 1881.5 4.1
2.0
110.5
1.6
1.9
34.1
35.9
77.0
17.5 2680.0 2774.5 5.0
4.1
130.4
7.1
7.9
84.8
2.7
0.5
96.7
4.0
1.5
94.2

24
773

Total
33.0
39.7
32.7
82.5
7.6
26.7
45.6
90.8
113.2
136.1

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49665202

CDPR IMI Soil and Foliar Cotton Study

Table 6-5. Pollen: Cumulative distributional statistics for concentration of imidacloprid and related metabolites in cotton pollen (ppb).
Acronyms in the table are: IMI=Imidacloprid; Olefin=Imidacloprid Olefin; 5-OH=5 Hydroxy Imdacloprid; Total Imidacloprid Residue (in bold) =Sum of parent and degrades; N=Number of observations; SD=Standard Deviation; CV=Coefficient of Variation. Numbered Interval denotes
timing of sampling where interval 1 was approximately 81 days after the first soil application and intervals 2 and 3 were at 5 and 14 days
after a third foliar spray coinciding with 91 and 100 days after the soil application.

StatisticN
Mean (ppb)
SD (ppb)
CV (%)Min (ppb)
Median (ppb)
75th (ppb)
90th (ppb)
95th (ppb)
Max (ppb)% of Mean Total

Pollen: Distribution of Imidacloprid Residues by Interval Sampled
Interval 1
Interval 2
5-OH Olefin
IMI
Total 5-OH Olefin
IMI
Total 5-OH
34.0
34.0
34.0 34.0
34.0
34.0
34.0
34.0
34.0
0.3
0.3
5.0
5.6
4.0
1.6
252.4
258.0
0.9
0.2
0.6
10.1 10.2
9.5
3.4
568.2
580.9
1.0
76.3 209.5 203.6 184.1 237.6 206.6 225.1
225.1 120.5
0.3
0.2
0.2
0.6
0.3
0.2
3.4
3.8
0.3
0.3
0.2
0.5
1.0
0.9
0.2
41.9
45.5
0.3
0.3
0.2
3.9
5.3
2.7
1.5
197.5
200.9
1.2
0.3
0.2
14.7 15.8
5.5
3.9
401.4
409.0
2.2
0.8
8.0
37.9 38.9
34.5
10.6 1682.2 1727.3
3.7
1.5
3.3
42.5 43.4
44.4
15.5 2846.3 2906.2
4.0
5.7
4.6
89.3
1.6
0.6
97.8
1.8

25
774

Interval 3
Olefin IMI
34.0
34.0
0.6
48.8
0.7
57.0
115.5 116.9
0.2
2.5
0.2
14.0
0.8
92.9
1.6
146.9
2.3
160.0
2.3
175.9
1.2
97.2

Total
34.0
50.2
58.4
116.3
2.9
14.6
94.4
150.0
165.5
182.2

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49665202

CDPR IMI Soil and Foliar Cotton Study

Table 6-6. Leaves: Cumulative distributional statistics for concentration of imidacloprid and related metabolites in cotton leaves (ppb).
Acronyms in the table are: IMI=Imidacloprid; Olefin=Imidacloprid Olefin; 5-OH=5 Hydroxy Imdacloprid; Total Imidacloprid Residue (in bold) =Sum of parent and degrades; N=Number of observations; SD=Standard Deviation; CV=Coefficient of Variation. Numbered Interval denotes
timing of sampling where interval 1 was approximately 81 days after the first soil application and intervals 2 and 3 were at 5 and 14 days
after a third foliar spray coinciding with 91 and 100 days after the soil application.

Statistic

Leaves: Distribution of Imidacloprid Residues by Interval Sampled
Interval 1
Interval 2
5-OH Olefin IMI
Total 5-OH Olefin
IMI
Total 5-OH

N
Mean (ppb)
SD (ppb)
CV (%)Min (ppb)
Median (ppb)
75th (ppb)
90th (ppb)
95th (ppb)
Max (ppb)% of Mean Total

34.0
3.4
4.8
142.8
0.4
1.9
4.2
9.3
12.0
24.3
7.6

34.0
2.5
3.7
146.2
0.4
0.7
3.5
4.7
10.7
17.1
5.6

34.0
39.1
59.5
151.9
0.6
20.7
39.2
103.5
132.5
316.5
86.9

34.0
45.0
67.6
150.1
1.4
23.9
46.5
119.3
154.3
357.9

34.0
103.1
35.1
34.0
53.9
91.6
121.1
172.3
173.0
196.6
6.3

34.0
41.3
16.7
40.3
16.0
22.0
42.3
47.6
77.1
79.7
2.5

26
775

34.0
1493.4
649.7
43.5
401.4
1352.91776.02554.82869.43043.991.2

34.0
1637.941.5
481.5
1497.01956.12745.72992.73203.7

34.0
43.4
16.7
37.9
14.7
42.5
56.6
64.6
70.5
88.7
12.1

Interval 3
Olefin IMI
34.0
26.9
10.5
38.9
5.4
28.7
32.6
37.0
45.2
50.8
7.5

Total

34.0
34.0
287.0 357.9
167.0 51.5
58.2
56.8
38.0 321.4
233.1 440.4
378.3 3651.9
561.9 708.3
620.9 719.3
625.6
80.2

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49665202

CDPR IMI Soil & Foliar Cotton Study

Figure 6-1. Explanation of statistical meaning of the Box-and-Whisker plots.

Maximum Observation

75% Quartile
Mean ValueMedian Value (50%)
25% Quartile
Minimum Observation

27
776

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49665202

CDPR IMI Soil & Foliar Cotton Study.
igure 6-2. Floral Nectar: Trend in total imidacloprid residue measured in floral nectar at each site.
Within each panel, markers denote the site. For example, Fresno site 1 are solid circles and Fresno site 2
are open squares and Wheatland site 1 are open triangles. Years are reflected by the color and style oflines where black small-dashed lines are 2012, green solid lines are 2014 data, and blue large-dashedlines are 2015 data.
200

200

Fresno1 - 2013
Fresno1 - 2014
Fresno2 - 2013
Fresno2 - 2014
Wheatland1 - 2014

Total Imidacloprid Residue (ppb)

150

150

100

100

50

50

0

0

200

200

Davis1 - 2013
Davis1 - 2014
Sanger1 - 2013
Sanger1 - 2014

150

100

50

50

60

70

80

Yuba City1 - 2014
Yuba City1 - 2015
Yuba City2 - 2014
Yuba City2 - 2015

150

100

0

Kerman1 - 2013
Kerman1 - 2014
Kerman2 - 2013
Kerman2 - 2014

90

100

110

120

0

60

70

80

90

100

110

120

Days After Imidacloprid Soil Application
Specific Site Information:
Fresno1=NT003-13ZA; Fresno2=NT007-13ZA
Wheatland=NT002-13ZB;
Davis1=NT004-13ZA; Sanger1=NT006-13ZA

28
777

Specific Site Information:
Kerman1=NT008-13ZA; Kerman2=NT009-13ZA
Yuba City1=NT001-13ZB; Yuba City2=NT005-13ZB

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49665202

CDPR IMI Soil & Foliar Cotton Study

Figure 6-3. Extrafloral Nectar: Trend in total imidacloprid residue measured in extrafloral nectar at eachsite. Within each panel, markers denote the site. For example, Fresno site 1 are solid circles and Fresno
site 2 are open squares and Wheatland site 1 are open triangles. Years are reflected by the color andstyle of lines where black small-dashed lines are 2012, green solid lines are 2014 data, and blue largedashed lines are 2015 data.
2000

1500

Total Imidacloprid Residue (ppb)2000

Fresno1 - 2013
Fresno1 - 2014
Fresno2 - 2013
Fresno2 - 2014
Wheatland1 - 2014

1500

1000

1000

500

500

0

0

2000

2000

Davis1 - 2013
Davis1 - 2014
Sanger1 - 2013
Sanger1 - 2014

1500

1000

500

500

60

70

80

Yuba City1 - 2014

Yuba City1 - 2015

Yuba City2 - 2014

Yuba City2 - 2015

1500

1000

0

Kerman1 - 2013
Kerman1 - 2014
Kerman2 - 2013
Kerman2 - 2014

90

100

110

120

0

60

70

80

90

100

110

120

Days After Imidacloprid Soil
Application
Specific Site Information:
Fresno1=NT003-13ZA; Fresno2=NT007-13ZA
Wheatland=NT002-13ZB;
Davis1=NT004-13ZA; Sanger1=NT006-13ZA

Specific Site Information:
Kerman1=NT008-13ZA; Kerman2=NT009-13ZA
Yuba City1=NT001-13ZB; Yuba City2=NT005-13ZB
29
778

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49665202

CDPR IMI Soil & Foliar Cotton Study

Figure 6-4. Pollen: Trend in total imidacloprid residue measured in pollen at each site. Within eachpanel markers denote the site, for example Fresno site 1 are solid circles and Fresno site 2 are open
squares and Wheatland site 1 are open triangles. Years are reflected by the color and style of lines
where black small-dashed lines are 2012, green solid lines are 2014 data, and blue large-dashed lines
are 2015 data. Note the larger scale in the plots for the Yuba City sites.

500

Total Imidacloprid Residue (ppb)400

Fresno1 - 2013

Fresno1 - 2014

Fresno2 - 2013

Fresno2 - 2014

500

400

Wheatland1 - 2014

300

300

200

200

100

100

0

0

Kerman1 - 2013

Kerman1 - 2014

Kerman2 - 2013

Kerman2 - 2014

2500

500

400

Davis1 - 2013

Davis1 - 2014

Sanger1 - 2013

Sanger1 - 2014

Yuba City1 - 2014
Yuba City1 - 20152000

Yuba City2 - 2014
Yuba City2 - 2015

300

1500

200

1000

100

500

0

60

70

80

90

100

110

120

0

60

70

80

90

100

110

120

Days After Imidacloprid Soil Application

Specific Site Information:
Kerman1=NT008-13ZA; Kerman2=NT009-13ZA
Yuba City1=NT001-13ZB; Yuba City2=NT005-13ZB

Specific Site Information:
Fresno1=NT003-13ZA; Fresno2=NT007-13ZA
Wheatland=NT002-13ZB;
Davis1=NT004-13ZA; Sanger1=NT006-13ZA
30
779

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49665202

CDPR IMI Soil & Foliar Cotton Study

Figure 6-5. Leaves: Trend in total imidacloprid residue measured in leaves at each site. Within eachpanel, markers denote the site. For example, Fresno site 1 are solid circles and Fresno site 2 are open
squares and Wheatland site 1 are open triangles. Years are reflected by the color and style of lines
where black small-dashed lines are 2012, green solid lines are 2014 data, and blue large-dashed lines
are 2015 data. Note the larger scale in the plots for the Davis site.
2500

2000

Total Imidacloprid Residue (ppb)

2500

Fresno1 - 2013
Fresno1 - 2014
Fresno2 - 2013
Fresno2 - 2014
Wheatland1 - 2014

Kerman1 - 2013

Kerman1 - 2014

Kerman2 - 2013

Kerman2 - 2014

2000

1500

1500

1000

1000

500

500

0

0
2500

3500

Yuba City1 - 2014
Yuba City1 - 2015
Yuba City2 - 2014
Yuba City2 - 2015

Davis1 - 2013
Davis1 - 2014

3000

2000

Sanger1 - 2013
Sanger1 - 2014

2500

1500

2000
1500

1000

1000
500

500
0

60

70

80

90

100

110

120

0

60

70

80

90

100

110

120

Days After Imidacloprid Soil Application
Specific Site Information:
Kerman1=NT008-13ZA; Kerman2=NT009-13ZA
Yuba City1=NT001-13ZB; Yuba City2=NT005-13ZB

Specific Site Information:
Fresno1=NT003-13ZA; Fresno2=NT007-13ZA
Wheatland=NT002-13ZB;
Davis1=NT004-13ZA; Sanger1=NT006-13ZA
31
780

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49665202

CDPR IMI Soil & Foliar Cotton Study

Figure -6-6. Relative distribution of concentration of total imidacloprid residues compared between intervals as measured in (A) Floral Nectar;(B) Extrafloral Nectar; (C) Pollen; and (D) Leaves. Data were averaged over all sites.

B. Extrafloral Nectar

A. Floral Nectar

Interval 1

Interval 2Interval 1

Interval 3

C. Pollen

Interval 1

Interval 2Interval 3

D. LeavesInterval 2Interval 1

Interval 3

32781

Interval 2Interval 3

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

U.S. EPA Data Evaluation Reports (Imidacloprid):
U.S. EPA. (2016). Data evaluation report: determination of the residues of imidacloprid and its metabolites 5-hydroxy imidacloprid andimidacloprid olefin in bee relevant matrices collected from blueberries following soil application of imidacloprid over two successive years.
Washington, D.C.: Author. Laboratory Report Number EBNTY006.
U.S. EPA. (2016). Data evaluation report: determination of the residues of imidacloprid and its metabolites 5-hydroxy imidacloprid andimidacloprid olefin in bee relevant matrices collected from cherry trees following foliar application of imidacloprid over two successive years.
Washington, D.C.: Author. Laboratory Report Number EBNTY008.
U.S. EPA. (2016). Data evaluation report: admire pro - magnitude of the residues of imidacloprid and its metabolites 5-hydroxy imidacloprid andimidacloprid olefin in bee relevant matrices collected from citrus trees following foliar applications of imidacloprid over two successive years.
Washington, D.C.: Author. Laboratory Report Number EBNTY007.
U.S. EPA. (2016). Data evaluation report: determination of the residues of imidacloprid and its metabolites 5-hydroxy imidacloprid andimidacloprid olefin in bee relevant matrices collected from treated cotton during two successive years and in white clover planted after treatedcotton. Washington, D.C.: Author. Laboratory Report Number EBNTY010.

782

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

Clothianidin Data Evaluations (begin on next page)783

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49602801

CDPR Clothianidin Cucurbit DER

Reference
Rose, A. (2015) Clothianidin: Quantitation of Residues of Clothianidin, TZNG, and TZMU in Leaves,
Nectar, Pollen and Soil Following Soil Application of Belay Insecticide to Cucurbits: Final Report.
Project Number: VP-38263. Unpublished study prepared by Valent U.S.A. Corporation and Rose
Consulting. 1103. MRID 49602801, CDPR Study ID 283866, Data Volume 52884-0245,
Tracking ID# 269547

1. STUDY INFORMATION
Chemical:

Clothianidin

Test Material:

Belay Insecticide

Study Type:

Field residue study on pumpkin crops to meansure clothianidin and its metabolite
levels in soil, leaves, nectar and pollen after a single soil application per year for
three years.

Sponsor:Valent U.S.A. Corporation
1600 Riviera Ave., Suite 200
Walnut Creek, U.S.A. 94596

Sponsor Study
ID:
Study
Completion
Date:
GLP Status:

PC CodePercent Active
Ingredient:

23.0%

Experiment Start andEnd Date:

June 12, 2012 –December 12, 2014

Study Locations:

A total of 12 pumpkin
trial sites in multiple
locations throughout
California.

VP-38263
March 16, 2015

44309

Non Good Laboratory Practice; protocols reviewed by CDPR.
[CDPR Study ID 265308, Data Volume 52884-0174, Tracking ID#253142
REVISED CDPR Study ID 266052, Data Volume 52884-0175, Tracking ID# 254177]

2. REVIEWER INFORMATION
Study Reviewed by:Richard Bireley, Sr. Environmental Scientist (Specialist)
California
John Troiano, Ph.D., Research Scientist III
Department of
Alexander Kolosovich, Environmental Scientist
Pesticide Regulation
Brigitte Tafarella, Environmental Scientist
Russell Darling, Environmental Scientist
Denise Alder, Sr. Environmental Scientist (Specialist)

Study Reviewed by:United States
Environmental
Protection Agency

Michael Wagman, Biologist, EPA/EFED/ERB6
Amy Blankinship, Senior Scientist, EPA/EFED/ERB6
EPA Reviewer Comments: EPA considers the study to bescientifically sound and it is classified as Acceptable1784

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49602801

CDPR Clothianidin Cucurbit DER

3. EXECUTIVE SUMMARY
The objective of this study was to quantify the extent to which insect pollinators may be exposed toclothianidin and its degradates thiazolylnitroguanidine (TZNG) and thiazolylmethylurea (TZMU) following
applications of Belay® Insecticide to cucurbits. This was accomplished by measuring residue
concentrations of these chemicals in leaves, nectar and pollen from pumpkin (a cucurbit) flowers. The
study continued for a total of 3 years to examine possible year-over-year accumulation of clothianidin
and clothianidin-related residues in soil and their possible uptake by subsequent (second and third year)
cucurbit crops.
Belay® Insecticide (active ingredient, clothianidin) was applied with pumpkin (Cucurbita pepo L.) seeding
to 9 field sites in California. At 3 of the 9 sites a second use pattern (application to pumpkin plants at
BBCH growth stage 201-229) was tested. Single applications were applied to the soil (chemigation or infurrow) of all trial sites at the maximum product label-allowed rate of 0.2 lb. a.i./acre. Plants weregrown following local agronomic practice. When the plants were in full bloom, around BBCH growthstage 605, leaf punches and male flowers were collected. Floral nectar and pollen were collected andprocessed from the flowers. In study years 2 and 3, soil cores (0-12 inch soil horizon) were collected. All
samples were stored and remained frozen pending residue analysis.
Leaf punches, nectar, pollen and soil were analyzed by LC-MS/MS using validated analytical methods.

4. STUDY VALIDITY

Guideline Followed:
Guideline Deviations:
Other Deviations:Classification:
Rationale:
Reparability:

Non-guideline study (protocol was reviewed by CDPR)
N/A
N/A
ACCEPTABLE
N/A
N/A

5. MATERIALS AND METHODS
Test Material Characterization
Test item:
Description:
Material Source:
CAS #:

Percent Active Ingredient:
Molecular Formula:
Molecular Weight:
Valent Lot Number:

Belay Insecticide
Soluble Concentrate (SC)Valent U.S.A. Corporation
210880-92-5

23.0% A.I.
C6H8ClN5O2S
205.68 g/mol
AS 2351a

5A. STUDY DESIGN
Belay® Insecticide is currently registered for foliar and soil use on cucurbits in California. Residue datafor clothianidin in nectar and pollen collected from cucurbit plants and clothianidin in soil was requested

2785Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49602801

CDPR Clothianidin Cucurbit DER

by California Department of Pesticide Regulation (CDPR). Interim reports (report dates May 17, 2013
and February 27, 2014) were submitted to CDPR. In addition, portions of this study were published1.
Pumpkin (Cucurbita pepo L.) seeds were purchased by the field Principal Investigators and planted at thetime of the Belay® Insecticide application or planted for a later season application (plants at BBCH
growth stage 201 to 229; side shoots visible on main stem). Although at the same field site, separateplots were used for the at-planting and late season applications of Belay® Insecticide. Seeds were either
hand planted (Sites 1, 2, 5, 6, 7, and 9) or planted by a single row planter (sites 3, 4, and 8).
Pumpkin plants were thinned as necessary so that the distance between plants was 2 to 3 feet. Plants
were irrigated as necessary to maintain a healthy crop. Irrigation was applied by subsurface drip tape or
by furrow flood irrigation. Occasionally some insect pests were observed and were controlled byapplication of non-neonicotinoid insecticides such as Sevin, Pristine, or Asana.
Mature pumpkins were removed from the plot and irrigation stopped. The vines were allowed todesiccate in the plot, and then the plot was cultivated to a depth of about 6 inches. Cultivation mixed
the soil and likely distributed clothianidin residues more evenly across the plot. Plots remained fallow
pending planting of the same pumpkin variety in 2013 and 2014.
5B. APPLICATION TIMING AND RATESBelay® Insecticide was applied by chemigation (subsurface drip tape) at sites 1, 2, 5, 6, 7, and 9 on thesame day that the pumpkin seeds were planted. At sites 3, 4, and 8 seeds were planted in a furrow,
Belay® Insecticide sprayed into the furrow (using a C02 canister powered single, hand-held flat fan
nozzle), then the furrow covered with soil. At 3 of the 9 sites a second use pattern application to
pumpkin plants at BBCH growth stage 201-229 was tested.
Belay® Insecticide was applied in a single application per year at an application rate of 0.2 lb. a. i./acre at
all sites.
Table 1a. Application Method and Application Dates for At-Planting Belay Insecticide on Pumpkins.
Year 1

Application Date
Year 2

Year 3Chemigation - subsurface drip

7/12/2012

8/9/2013

6/20/2014

2

Chemigation - subsurface drip

7/13/2012

7/31/2013

6/23/20143

In-furrow spray

6/26/2012

6/20/2013

5/15/20144

In-furrow spray

6/26/2012

6/20/2013

5/15/2014

5

Chemigation-subsurface drip

7/11/2012

7/12/2013

6/2/2014

6

Chemigation - subsurface drip

7/6/2012

7/12/2013

6/24/2014

7

Chemigation - subsurface drip

7/5/2012

7/12/2013

6/23/20148

In-furrow spray

7/6/2012

6/26/2013

5/14/2014

9

Chemigation - subsurface drip

7/6/2012

7/9/2013

6/23/2014

Site Number

Application Method

1

3
786

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49602801

CDPR Clothianidin Cucurbit DER

Table 1b. Application Method and Application Date for Late Season Belay Insecticide Applications.
Year 1

Application Date
Year 2

Year 3Chemigation - subsurface drip

8/15/2012

9/12/2013

7/20/20144a

In-furrow subsurface shank

7/27/2012

8/9/2013

7/11/20147a

Chemigation - subsurface drip

7/25/2012

8/23/2013

7/17/2014

Site Number

Application Method

1a5C. STUDY SITE LOCATION AND CHARACTERISTICS
Belay® Insecticide (active ingredient, clothianidin) was applied concomitant with pumpkin (Cucurbita
pepo L.) seeding to 9 field sites in California. The soil characterization data and field site location aresummarized in Table 2.
Table 2. Field Site Locations and Soil Series, Characterization and Organic Matter.
Site
Field Site
Soil
Soil Series & Texture Soil Characterization
NumberCountyTexture(%Sand/Silt/Clay)12FresnoFresnoMediumCoarse345

CoarseCoarseMedium6
7
8

MaderaMaderaSan LuisObispoTulareTulareMadera9

TulareMediumCoarseFine
CoarseRamona Loam
Hanford Coarse
Sandy Loam
Awater Loamy Sand
Atwater Sandy Loam
Nacimieno-Los Osos
complex, Loam
Nord Sandy Loam
Centerville Clay
Grangeville Loamy
Sand
Centerville Sandy
Clay Loam

65/24/11
84/13/3

Percent
Organic
Matter0.43
0.34

85/10/5
73/16/11
45/31/24

0.81
1.08
1.6

69/24/7
31/25/44
77/16/7

1.2
2.1
1.5

50/17/33

3.7

5D. SAMPLE COLLECTION, HANDLING, PROCESSING
Collection of Soil Coarse
Soil cores were collected prior to Belay® Insecticide applications in study years 2 and 3. For soil coring,each treatment plot was divided into 3 approximately equal size subplots (Subplots 1, 2, and 3) theneach of the subplots divided into 16 approximately equal size sectors (numbered 1 through 16). Seven
sectors were randomly selected for coring. Soil cores (0-12 inch horizon) in plastic sleeves were takenwith a slide hammer or tractor-mounted Giddings hydraulic soil probe. The seven soil cores from eachsector were combined in the field to give a total of three replicates per plot (field site). Soil cores werestored in temperature-monitored freezers at the field site until they were shipped frozen to theAnalytical Laboratory.
Collection of Leaf Punches and Flowers
4787

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49602801

CDPR Clothianidin Cucurbit DER

Leaf punches and male flowers were collected when the field was in full bloom, generally around BBCH
growth stage 605 (also referred to as stage 61; 51h flower open on main stem). Approximately 100 male
flower buds were indiscriminately selected the afternoon before anthesis occured and covered withpaper bags in order to prevent visits from insects. Male flowers can be easily distinguished from female
flowers by their much longer and thinner styles. The following day, on the day of sampling, duplicate
leaf punch samples were collected. Two punches were collected from leaves near 25 of the 100 covered
male flowers (total 50 leaf punches; except 55 leaf punches Site 2 in year 1). Leaf punches were placed
into tared, labeled Ziploc plastic bags and the gross weight recorded. Leaf punch samples weretransported to the laboratory in coolers containing Blue Ice (or equivalent).
Duplicate samples of covered flowers were collected and transported to the laboratory in coolers
containing Blue Ice (or equivalent). The number of flowers collected and their weights were recorded. In
general, each field site indiscriminately collected 35 to 75 flowers per replicate.
Nectar and Pollen Samples
Flowers were processed on the day they were harvested from the field plot. Each flower was cut aroundthe sepals, then the sepals and petals were removed (discarded) to expose the receptacle and nectary
and the single pollen-laden stamen. Nectar was collected from the cavity within the receptacle with aglass pipette. The nectar was transferred into a label and tared glass vial. Nectar extraction frommultiple flowers continued until at least 0.5 g of nectar had been collected. The weight of the vial, plus
the nectar, was recorded. A scalpel or razor blade was used to scrape pollen from the filament. Pollen
was collected from the same flowers from which nectar was collected. Pollen was transferred to a tared,label vial and the weight of the vial, plus the pollen, was recorded. The number of flowers processed wasrecorded. The flower dissection, nectar extraction and pollen collection was repeated with the second
replicate of flowers. After processing, the flowers were discarded.
Sample Storage
Soil cores were placed into coolers in the field and stored in freezers at the field sites pending shipment
to the Analytical Laboratory. Leaf punch samples were hand carried to the laboratory in coolers. Once inthe laboratory, the samples were placed into freezers (temperature <0°F) at each field site.
Immediately after final weights were recorded, the labeled sample vials containing nectar and pollen
were placed into a freezer (temperature <0°F) at each field site where they remained until they wereshipped to the analytical laboratory.
Samples were packaged for shipment by the field Principal Investigators. Chain of Custody forms wereprepared and accompanied the samples. Samples were shipped by freezer truck (Agricultural Chemical
Delivery Services, Inc. (ACDS, Inc.)) or by overnight delivery (United Parcel Service or FedEx). Samples forovernight delivery were packaged in a cooler containing Blue Ice to keep the samples cold.
Once at the Valent Technical Center, samples were placed into a temperature monitored, walk-in
freezer until analyzed.

5788

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49602801

CDPR Clothianidin Cucurbit DER

5E. ANALYTICAL METHODS
Methods used in this analytical study were Valent U.S.A. Corporation's methods RM-39N-l, RM-39Pl,
RM-39S-l, RM-39S-2, RM-39L-l, RM-39L-2.
Samples of pumpkin nectar were dissolved in methanol/water (40:60, v/v) acidified with 0.05% formic
acid and analyzed by LC/MS-MS after spiking with isotopically labeled internal standards (d3 clothianidin,
13 15
C/ N-TZNG, and d3TZMU) to compensate for matrix effect (method RM-39N-l). The limit of detection(LOD) was 0.2 ppb, and the limit of quantitation (LOQ) was 1 ppb for clothianidin and its metabolites,
TZNG and TZMU and reported in Table 3 below.
Samples of pumpkin pollen were extracted with water and acetonitrile followed by adding sodium
chloride and anhydrous magnesium sulfate salts. The acetonitrile extract was partitioned with n-hexane,
and the acetonitrile phase was collected and concentrated to dryness. Residues were re-dissolved inmethanol/water (40:60, v/v) acidified with 0.05% formic acid and analyzed by LC/MS-MS after spiking
with isotopically labeled internal standards (d3 clothianidin, 13C,15N-TZNG, and d3 TZMU) to compensate
for matrix effect (method RM-39P-l). Using this method, the LOD was 0.25 ppb, and the LOQ was 1 ppb
for clothianidin and its metabolites, TZNG and TZMU.
Residues in leaves were measured using two methods. The first method measured clothianidin
concentration in leaves. Leaf samples were extracted with methanol and water (40:60, v/v) acidified
with 0.05% formic acid and analyzed using an accurate mass UPLC/Q-TOF MS-MS after spiking withisotopically labeled internal standard (d3 clothianidin) to compensate for matrix effect(method RM39L-l). The LOD in this method was 2.5 ppb, and the LOQ was 5 ppb for clothianidin. Thismethod was superseded following discussions with regulatory authorities to include determination ofclothianidin and its major metabolites, TZNG and TZMU in leaves. Leaf samples were extracted withwater acidified with 0.05% formic acid and acetonitrile followed by adding sodium chloride andanhydrous magnesium sulfate salts. An aliquot of the acetonitrile extract was cleaned through a Strata
Cl8-E column and concentrated to dryness. Residues were re-dissolved in water/methanol (75:25, v/v)
acidified with 0.05% formic acid and analyzed by accurate mass UPLC/Q-TOF MS-MS after spiking withisotopically labeled internal standard (d3 clothianidin, 13C,15N-TZNG, and d3 TZMU) to compensate formatrix effect (method RM-39L-2). Using this method, the LOD was 1.3 ppb, and the LOQ was 5 ppb for
clothianidin and its metabolites, TZNG and TZMU.
To measure “total” clothianidin and its major metabolites, TZNG and TZMU in soil, soil samples wereextracted with water and methanol (75:25, v/v) acidified with 0.05% formic acid and analyzed using an
accurate mass UPLC/Q-TOF MS-MS after spiking with isotopically labeled internal standards (d3clothianidin, 13C,15N-TZNG, and d3 TZMU) to compensate for matrix effect. The LOD was 1.3 ppb, and the
LOQ was 5 ppb for clothianidin, TZNG and TZMU in this method.
To estimate the "bioavailable" concentration of clothianidin, soil samples with total clothianidin
concentration greater than the LOQ were shaken with 0.01 M calcium chloride solution for 24 hours,
and an aliquot of the sample was analyzed using an accurate mass UPLC/Q-TOF MS-MS after spiking
with isotopically labeled internal standard (d3 clothianidin) to compensate for matrix effect. The LOD is
0.3 ppb, and the LOQ is 5 ppb for clothianidin in this method.

6789

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49602801

CDPR Clothianidin Cucurbit DER

A total of 72 pumpkin floral nectar samples, 72 pumpkin pollen samples, 58 leaf samples and 72 soil
samples were analyzed for clothianidin and its metabolite residues; 48 leaf samples were analyzed only
for clothianidin residues and 54 soil samples were analyzed for “bioavailable” clothianidin residues. A
total of 7 soil transit stability samples were analyzed for clothianidin, 16 nectar and 16 pollen transit
stability samples were analyzed for clothianidin, TZNG and TZMU.
Table 3. Summary of Limit of Quantitation and Limit of Detection.
MatrixAnalyteLOQ(ppb, parent
equivalents)
Pumpkin Soil
Clothianidin, TZNG and TZMU
5.0
Pumpkin Pollen
Clothianidin, TZNG and TZMU
1.0
Pumpkin Nectar
Clothianidin, TZNG and TZMU
1.0
Pumpkin Leaves
Clothianidin, TZNG and TZMU
5.0

LOD
(ppb, parent
equivalents)
1.3
0.25
0.2
1.3

5F. QUALITY ASSURANCE RESULTS
Quality assurance measures taken during the analytical phase of this study included, but were notlimited to the following:
All analytical standards used in this study were kept at reduced temperature in a refrigerator or in afreezer at all times when not in use.
At least five different standard concentrations were injected within each analytical set. The
concentration (ng/mL) of clothianidin and its metabolites detected in sample extracts was interpolated
from the standard calibration curve. The LC/MS-MS and accurate mass UPLC/Q-TOF MS-MS systems
were calibrated for each set of samples by analyzing these calibrating standard concentrations, withthese standards interspersed within the analytical sequence. A second-order polynomial fit (weighted
relative to 1/concentration) was then calculated from the concentrations and the detector response of
the calibration standards. To verify performance, the percent difference between the actual
concentration and the calculated concentration for each of the calibration standards (based on the
curve) was also calculated. Each of the standards were required to be within 15% of the theoreticalconcentration and the coefficient of determination (r2) of the weighted polynomial calibration curve was
required be greater than or equal to 0.99. Minor exceedance of these criteria for the calibration
standards were accepted for the lowest standards in some cases, however the coefficient ofdetermination (r2) of the weighted polynomial calibration curve was always greater than or equal to
0.99.
The reproducibility of the LC/MS-MS and accurate mass UPLC/Q-TOF MS-MS systems was verified bycomparison of instrument responses obtained from the repeated analysis of a continuing standard (amid-level calibration standard) analyzed with the study samples. The continuing calibration standards
were interspersed within the samples in the analytical sequence, and the analytical sequence began andended with a continuing calibration standard. For an analytical set (injection sequence) to be acceptable,
the coefficient of variation (CV) of these responses was required to be 10% or less. Minor exceedance of
these criteria for the continuing calibration standards were accepted in some cases.

7790

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49602801

CDPR Clothianidin Cucurbit DER

Laboratory fortification samples were analyzed concurrently with each analytical set to demonstrate
method performance. Laboratory fortification samples were prepared using artificial nectar andcommercially available pollen when not enough untreated control pumpkin nectar or pollen wereavailable, while untreated control soil from pumpkin fields and pumpkin leaf samples were used for soil
and leaf laboratory fortification samples. Each sample set included at least one untreated control (UTC)and two laboratory fortification samples. Fortifications ranged from 1 to 10 ppb for nectar and pollen
samples and 5 to 50 ppb for leaf and soil samples. Generally, concurrent fortification recoveries for
clothianidin, TZNG and TZMU in the laboratory fortified samples were in the range of 70 to 120. In somecases, minor exceedance of these criteria was accepted.

6. RESULTS:

6.1 Soil Results
Table 4. Mean Concentrations of Total Clothianidin, TZNG and TZMU Residues in Pumpkin Soil following
Soil Applications of Belay Insecticide At-Planting.
Mean Concentration (ppb)1
Year 2
Year 3Site
NumberClothianidin
TZNG
TZMU
Clothianidin
TZNG
TZMU
2
2
2
1
6.5
[0.7]
[0.7]
15.8
[0.7]
[0.7]22(2.5)3[0.7]2[0.7]2(3.7)3[0.7]2[0.7]2
2
2
2
3
2
3
[0.7]
[0.7]
[0.7]
(4.6)
[0.7]
[0.7]24(2.7)3[0.7]2[0.7]2
6.7[0.7]2[0.7]2
2
2
2
5
13.4
[0.7]
[0.7]
9.3
[0.7]
[0.7]26[0.7]2[0.7]2[0.7]2
6.6[0.7]2[0.7]2
2
2
2
2
7
11.2
[0.7]
[0.7]
[0.7]
[0.7]
[0.7]28
13.3[0.7]2[0.7]2
11.1[0.7]2[0.7]2
2
2
2
9
5.9
[0.7]
[0.7]
17.5
[0.7]
[0.7]2
1
LOQ= 5 ppb; LOD= 1.3 ppb
2Values in square brackets are <LOD and entered as ½ LOD
3Values in parenthesis are between the LOQ and the LOD
Table 5. Mean Concentrations of “Bioavailable” Clothianidin Residues in Pumpkin Soil following Soil
Applications of Belay Insecticide At-Planting.
Mean Concentrations of Clothianidin (ppb)1
Site #
Year 2
Year 3
2
1
(4.0)
10.8
2
NE3
NE3
3
NE3
(3.0)23
4NE(2.3)2
5
6.7
6.3
6
NE3
(2.9)2
7
6.1
NE3
8
5.0
5.2
2
9
(3.2)
9.9
1
LOQ= 5 ppb; LOD= 1.3 ppb
2Values in parenthesis are between the LOQ and the LOD

8791

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49602801
3

CDPR Clothianidin Cucurbit DER

NE, not extracted because total clothianidin residues were <LOQ

Table 6. Mean Total Concentrations of Clothianidin, TZNG and TZMU in Pumpkin Soil following Soil
Applications of Belay Insecticide at Growth Stage BBCH 201-299.
Mean Concentrations (ppb)1
Year 2
Year 3Site
NumberClothianidin
TZNG
TZMU
Clothianidin
TZNG
TZMU
2
2
2
1a
7.1
[0.7]
[0.7]
10.6
[0.7]
[0.7]24a8.0[0.7]2[0.7]210.5[0.7]2[0.7]2
2
2
2
7a
8.7
[0.7]
[0.7]
39.9
[0.7]
[0.7]2
1
LOQ= 5 ppb; LOD= 1.3 ppb
2Values in square brackets are <LOD and entered as ½ LOD
Table 7. Mean Bioavailable Concentrations of Clothianidin in Pumpkin Soil following Soil Applications of
Belay Insecticide at Growth Stage BBCH 201-299.
Mean Concentrations of Clothianidin (ppb)1
Site
NumberYear 1
Year 2
2
1a
(4.4)
6.6
4a
(3.6)26.7
27a(3.4)`
19.4
1
LOQ= 5 ppb; LOD= 1.3 ppb
2Values in parenthesis are between the LOQ and the LOD
6.2 Leaf Results
Table 8. Mean Concentration of Clothianidin, TZNG and TZMU in Pumpkin Leaves following Soil
Applications of Belay Insecticide At-Planting.
Mean Concentration (ppb)1
Year 1
Year 2
Year 3Site
Number Clothianidin TZNG TZMU Clothianidin TZNG TZMU Clothianidin TZNG
1
16.3
3.6
4.5
63.2
10.1
25.6
111
15.3
2
80.0
14.2
19.4
38.1
5.8
12.5
26.6
3.5
3
15.3
3.2
7.6
16.2
1.6
6.6
45.6
6.6
4
52.0
6.3
28.0
13.0
3.2
4.7
44.1
7.3
5
26.1
4.2
7.3
28.8
4.5
7.4
56.3
6.3
6
13.1
3.6
2.0
26.9
NS2
NS2
71.3
7.2
2
2
2
2
7
150
NS
NS
12.4
NS
NS
28.8
2.1
8
50.5
5.5
12.5
32.5
6.0
8.0
31.0
4.9
2
2
2
2
9
18.1
NS
NS
19.5
NS
NS
20.4
3.2
1
LOQ= 5 ppb; LOD=1.3 ppb
2NS, No sample remaining for analysis using method RM-39L-2

9
792

TZMU
20.5
8.4
9.0
11.9
9.1
7.0
3.9
3.2
5.2

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49602801

CDPR Clothianidin Cucurbit DER

Table 9. Mean Concentrations of Clothianidin, TZNG and TZMU in Pumpkin Leaves Following Soil
Applications of Belay Insecticide at Growth Stage BBCH 201-299.
Mean Concentration (ppb)1
Year 1
Year 2
Year 3Site
Number Clothianidin TZNG TZMU Clothianidin TZNG TZMU Clothianidin TZNG TZMU
1a
137
20.6
27.1
431
27.1
77.4
80.5
10.0
11.1
4a
103
19.8
27.6
72.9
10.8
22.8
111
16.5
40.7
2
2
2
2
7a
26.5
NS
NS
7.9
NS
NS
14.5
1.5
2.9
1
LOQ= 5 ppb; LOD=1.3 ppb
2NS, No sample remaining for analysis using method RM-39L-2
6.3 Nectar Residues
Table 10. Mean Concentrations of Clothianidin, TZNG and TZMU in Pumpkin Floral Nectar following Soil
Applications of Belay Insecticide At-Planting.
Mean Concentration (ppb)1
Year 1
Year 2
Year 3Site
Number Clothianidin TZNG TZMU Clothianidin TZNG TZMU Clothianidin TZNG TZMU
1
2.06
[0.1]2 [0.1]2
4.1(0.3)3 (0.6)3
4.9(0.4)3 (0.4)3
3
2
3
3
2
5.44
(0.43) [0.1]
2.0
(0.2)
(0.6)
1.3
[0.1]2 [0.1]23(0.7)3[0.1]2 [0.1]2(0.7)3[0.1]2 (0.3)3
2.0[0.1]2 [0.1]2
2
3
3
2
2
4
1.7
[0.1]
(0.2)
(0.7)
[0.1] [0.1]2.1[0.1]2 [0.1]2
51.0(0.4)3 [0.1]2
1.5
1.4(0.3)32.2
1.1(0.2)3
2
2
2
3
2
6
1.9
[0.1]
[0.1]
2.0
[0.1] (0.2)
4.0
[0.1]
(0.5)375.8(0.3)3(0.3)
1.1[0.1]2 [0.1]2
4.6[0.1]2 (0.2)3
3
2
3
3
8
2.9
(0.2)
[0.1]
3.2
(0.3)
(0.3)
1.1
[0.1]2 [0.1]292.2(0.3)3 [0.1]2
1.4[0.1]2 [0.1]2
1.6[0.1]2 [0.1]2
1
LOQ= 1 ppb; LOD= 0.2 ppb
2Values in square brackets are <LOD and entered as ½ LOD
3Values in parenthesis are between the LOQ and the LOD
Table 11. Mean Concentrations of Clothianidin, TZNG and TZMU in Pumpkin Floral Nectar following Soil
Applications of Belay Insecticide at Growth Stage BBCH 201-299.
Mean Concentration (ppb)1
Year 1
Year 2
Year 3Site
Number Clothianidin TZNG TZMU Clothianidin TZNG TZMU Clothianidin TZNG TZMU
1a12.8
(0.7)3 (0.6)3
17.0(0.9)32.0
11.1(0.9)3 (0.9)3
3
3
3
3
4a
5.4
(0.5)
(0.5)
3.1
(0.2)
(0.4)
4.94
(0.4)3 (0.7)3
7a
4.2(0.4)3 (0.2)32.1[0.1]2 (0.4)3(0.9)3[0.1]2 [0.1]2
1
LOQ= 1 ppb; LOD= 0.2 ppb
2Values in square brackets are <LOD and entered as ½ LOD
3Values in parenthesis are between the LOQ and the LOD

10793

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49602801

CDPR Clothianidin Cucurbit DER

6.4 Pollen Residues
Table 12. Mean Concentrations of Clothianidin, TZNG and TZMU in Pumpkin Pollen following Soil
Applications of Belay Insecticide At-Planting.
Mean Concentration (ppb)1
Year 1
Year 2
Year 3Site
Number Clothianidin TZNG TZMU Clothianidin TZNG TZMU Clothianidin TZNG TZMU
1
3.9
(0.5)2 (0.5)2
9.4(0.9)2
1.1
14.2
1.9
1.2
2
2
2
2
2
2
7.5
(0.7)
(0.6)
6.2
(0.6)
(0.7)
3.8
(0.4)
(0.3)2
2
2
3
3
2
3
2.8
(0.3)
(0.3)
1.3
[0.1]
[0.1]
4.6
(0.5)
(0.4)2
2
2
3
3
4
4.3
(0.6)
(0.8)
1.9
[0.1]
[0.1]
11.6
1.0
1.7
5
2.1
(0.8)2 [0.1]3
4.2
2.6(0.9)24.7
2.8(0.8)26
4.0(0.5)2 (0.5)2
3.9(0.3)2 (0.3)2
7.7(0.7)2 (0.9)2
7
15.5
1.3
1.0
2.2
[0.1]3 (0.4)2
10.0(0.6)2 (0.5)2
2
2
2
8
6.4
(0.5)
(0.9)
7.3
(0.6)
1.1
2.0
(0.2)2 [0.1]3
9
7.3
1.1
(0.8)2
5.4(0.7)2 (0.9)2
5.0(0.8)2 (0.5)2
1
LOQ= 1 ppb; LOD= 0.2 ppb
2Values in parenthesis are between the LOQ and the LOD
3
Values in square brackets are <LOD and entered as ½ LOD
Table 13. Mean Concentrations of Clothianidin, TZNG and TZMU in Pumpkin Pollen following Soil
Application of Belay Insecticide at Growth Stage BBCH 201-299.
Mean Concentration (ppb)1
Year 1
Year 2
Year 3Site
Number Clothianidin TZNG TZMU Clothianidin TZNG TZMU Clothianidin TZNG TZMU
1a
33.3
2.2
2.5
27.4
1.5
1.3
37.9
3.9
2.7
2
2
4a
18.3
1.3
1.6
9.8
(0.8)
(0.8)
18.2
1.6
2.0
7a
9.8
1.1
1.1
1.8
[0.1]3 (0.3)2
1.5[0.1]3 [0.1]3
1
LOQ= 1 ppb; LOD= 0.2 ppb
2Values in parenthesis are between the LOQ and the LOD
3
Values in square brackets are <LOD and entered as ½ LOD

7. STUDY VALIDITY/CLASSIFICATION AND STUDY LIMITATIONSClassification/Utility for Bee Risk Assessment. This study is classified as acceptable. It provides acomprehensive overview of clothianidin concentrations in soil, as well as pumpkin leaves, pollen, andnectar during bloom. The residue values presented should be considered to be fully reliable. However, adecline curve cannot be constructed because samples were only collected from each matrix at one time
point in each year.
Temporal Variability in Residues. This study was conducted over a three year period. Soil applications of
0.2 lb ai/acre were made annually for three years at planting, or, approximately one month later, at
BBCH 201 (first shoots visible) to 229 (ninth leaf unfolded on main stem). Samples were collected during
bloom, approximately two months after planting. Concentrations in soil, leaves, pollen, and nectar were
all significantly higher following applications at BBCH 201-229, compared to applications at planting.

11794

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49602801

CDPR Clothianidin Cucurbit DER

Coarse soils had significantly higher concentrations of clothianidin in nectar and pollen than fine soils
following applications at BBCH 201-229 (Tables 18 and 20).
Spatial Variability in Residues. Eight of the nine sites were located in the San Joaquin Valley of California(Fresno, Madera, and Tulare Counties), and one site was located in California’s Central Coast Region (SanLuis Obispo County). Temperatures were similar across all sites. There were no apparent differences inresidue concentrations based on location.
Pesticide Carryover. The study authors stated that a two-way ANOVA showed that there was asignificant effect on concentrations in soil based on year of sampling (p = 0.04), but that the year ofsampling did not have significant effects on concentrations in leaves, pollen, or nectar. The average
number of days between the last application and sample collection was also slightly shorter during Year
3 (331 days for the At-Planting applications, and 314 days for the BBCH 201-229 applications), thanduring year 2 (368 days for the At-Planting applications, and 359 days for the BBCH 201-229
applications).
Table 14. Summary Statistics for Soil Grouped by Year and Time of Application.
MatrixApplicationTime

At Planting
Soil
BBCH 201-299
a

Year
2
3
2
3

Mean ± SDa(µg a.i./kg ww)
6.4 ± 5.9
8.4 ± 5.8
7.9 ± 3.2
20 ± 16

N
27
27
9
9

Median(µg a.i./kg ww)
4.9
7.4
6.9
18

90thPercentile(µg a.i./kg ww)
14
16
11
39

Standard Deviation

Table 15. Results of a Two-Way Analysis of Variance Testing the Effects of Application Timing and Yearon Clothianidin Residues in Soil.
Type III Sum
Degrees of
Mean
Sourceof Squared
Freedom
Squares
Application Timing
1.59
1
1.59
(Main Effects)
Year of Sampling
0.82
1
0.82
(Main Effect)
Application Timing x Year
0.04
1
0.04
(Interaction Effect)
Error
13.12
68
0.19
Note: Data were log-transformed prior to analysis to improve data normality

F-Ratio
8.26

P
0.01

4.25

0.04

0.18

0.67

Relationships between Leaf and Nectar/Pollen Residues. The study authors determined that there
were significant relationships between clothianidin concentrations in pumpkin leaves compared tonectar (n = 36, r2 = 0.66, p<0.001) and pollen (n = 36, r2 = 0.67, p<0.001) collected from plants grownunder the same conditions. The relationship between clothianidin concentrations in leaves and pollen isrepresented by the following equation: CN = 0.17(CL)0.75, where CN is the concentration in nectar and CL isthe concentration in leaves. The relationship between clothianidin concentrations in leaves and pollen isrepresented by the following equation: CP = 0.31(CL)0.81, where CP represents the concentration in pollen
and CL represents the concentration in leaves. The data used in these equations is not transformed. The

12795

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49602801

CDPR Clothianidin Cucurbit DER

study authors stated that there is a large degree of uncertainty in these relationships, because it isunclear how these relationships would apply to pumpkins grown in other locations, or to other use
patterns. Therefore these equations should not be considered reliable until more data is acquired, sothey can be tested and refined. Future studies should attempt to generate similar equations so thatthese relationships can be better understood.
Magnitude of Residues. Descriptive statistics for concentrations of Clothianidin, TZNG, and TZMU
residues in nectar, pollen, soil, and leaves are presented in Table 16. The study authors conductedstatistical analyses and found that, except for soil (Table 14), there were no significant differences
between years, but that there were significant differences associated with the timing of applications for
all matrices (i.e., at planting vs. at BBCH 201-229).
Table 16. Summary Statistics for Pumpkin Nectar, Pollen, Leaves and Pollen Grouped by Year and Timeof Application.

MatrixApplicationTime
At Planting

Nectar
BBCH 201- 229

At Planting
PollenBBCH 201- 229

At Planting
Leaves
BBCH 201 - 229
a

Year
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3

n
18
18
18
6
6
6
18
18
18
6
6
6
18
18
18
6
6
6

Mean ± SD(µg ai/kg
ww)
2.6 ± 2.3
1.9 ± 1.2
2.7 ± 1.5
7.5 ± 4.5
7.4 ± 7.5
5.7 ± 4.6
6 ± 5.3
4.7 ± 2.7
7.1 ± 4.2
20 ± 1213 ± 1219 ± 1747 ± 4428 ± 18
48 ± 32
89 ± 57
170 ± 200
69 ± 54aMedian (µgai/kg ww)
1.9
1.6
2.2
6.8
3.5
4.9
4.8
4.2
6
20
9.8
18
26
23
42
90
73
68

90th
Percentile(µg ai/kg
ww)
5.1
3.1
4.5
13
17
11
7.8
7.9
12
33
27
38
100
38
70
150
430
120

Standard Deviation

The registrant collected samples from pumpkins grown in different soil types, but did not analyze theeffects that different soil types have on clothianidin concentrations in bee-relevant matrices, so DPR
conducted independent analyses (Tables 17-20). Site 7 was classified as fine soil, Sites 5 and 9 wereclassified as medium soils, and Sites 1, 2, 3, 4, 6, and 8 were classified as coarse soils. The study authors
also calculated consumption and exposure (ng/bee) based on mean concentrations and 90th percentile
residue values.

13796Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49602801

CDPR Clothianidin Cucurbit DER

Table 17. Clothianidin Concentrations in Nectar in Different Soil Types Resulting from At-Planting
Applications (all units in ng/g).
Soil Type
Fine
MediumCoarseMean (± SD)
3.85 (± 3.30)
1.68 (± 0.54)
2.39 (± 1.55)

Median
2.89
1.52
1.97

Maximum
9.58
2.91
6.36

Table 18. Clothianidin Concentrations in Nectar in Fine and Coarse Soils Resulting from the BBCH 201229 Applications (all units in ng/g).
Soil Type
Mean (± SD)
MedianMaximumFine
2.43 (± 2.00)
1.87
5.98
Coarse9.06 (± 5.26)*
9.04
18.01
* Significantly higher concentrations resulted from application at BBCH 201-229 in coarse soil compared to fine soil
(Welch’s t-test, p = 0.002)

Table 19. Clothianidin Concentrations in Pollen in Different Soil Types Resulting from At-Planting
Applications (all units in ng/g).
Soil Type
Fine
MediumCoarseMean (± SD)
9.24 (± 8.86)
4.76 (± 1.76)
5.72 (± 3.59)

Median
7.07
4.81
4.85

Maximum
25.81
8.27
17.03

Table 20. Clothianidin Concentrations in Pollen in Fine and Coarse Soils Resulting from the BBCH 201229 Applications (all units in ng/g).
Soil Type
Mean (± SD)
MedianMaximum
Fine
4.37 (± 4.79)
1.86
13.41
Coarse24.14 (± 11.20)*
25.29
44.47
* Significantly higher concentrations resulted from application at BBCH 201-229 in coarse soil compared to fine soil
(Mann-Whitney-Wilcoxson test, p = 0.0023)

8. STATISTICAL ANALYSIS
1. Table S-1 summarizes the number of plant samples that were obtained from each trial site for eachyear with an indication of the soil texture at each site, the method of chemical application used at eachsite, and the timing of applications. Note that there is confounding in the experimental design withrespect to method of application and soil type because not all sites received the same method of
application. Also, three of the sites were split across method of application to test timing of application.
Site 4, which received a furrow application, had a second plot where the application occurred 1 monthafter planting. Then for chemigation applications, Site 1 and 7 were similarly treated. The analysis of thedata was conducted to provide guidance for these questions:
1. Was there bias in analyses of clothianidin residues over years?
2. Was there a difference in chemical concentration in plant samples due to timing of application?
3. Was there a difference in chemical concentration in plant samples due to method of application?

14797

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49602801

CDPR Clothianidin Cucurbit DER

4. Was there a difference in chemical concentration due to soil type?
When no bias was measured, this indicated that the data could potentially be pooled over the factor
tested to proceed on to the next test. For example, bias between years was tested. If no difference indistribution between years was measured then data for years at a treatment level were pooled. Two
methods of chemical analysis were indicated. One denoted L-1, which only reported on concentration ofparent and the other denoted L-2, which reported on parent and degradation products. Only data from
L-2 are presented in this analysis. Values were reported that were below the limit of detection (LOD).
These values were substituted with ½ LOD stated for the year and plant sample. Total clothianidin
residue was determined as a simple addition of all analytes.
2. Bias in concentration between years. Analyses were conducted to determine if there weresignificant differences in distribution of clothianidin residue concentration between years in the plant
samples. Tables S-2 through S-4 contain a comparison of the distributions for each analyte between
years for leaves, nectar, and pollen, respectively. Non-parametric tests were conducted to measurepotential differences in the concentration between the 3 years. A significant Wilcoxon test provides anindication of general differences in the distribution, whereas, the Median test provides an indication if
differences in the median values that are present between the distributions. The tests were run using
the exact option in Proc Npar1way in the SAS program with the Monte Carlo option where 10,000
iterations of the tests were run. No significant differences in distributions between years were indicatedfor chemical analysis in any plant sample. Figures S-1 contains an example for the comparison of the
distribution of clothianidin concentration in leaves between years.
3. Bias in concentration due to timing of applications. Based on the results of the analysis comparing
distribution between years, the data were pooled over years to test the potential effect of timing of
application. Tables S-6 through S-8 compare the distributions for application at planting to applications
made 30 days after planting for leaves, nectar, and pollen, respectively. The analyses were conducted
on data from trial sites 1, 4 and 7 because these were sites with the split application treatments.
Statistical results indicated that the distribution of values measured at the later application was shifted
toward higher values (Table S-9). The shift in distribution was highly significant for leaf samples (FigureS-2). For nectar, the Wilcoxon test indicated a significant difference for all chemicals but only theMedian test indicated a trend. Graphical comparison indicates there is a trend for higher concentrations
for the later applications (Figure S-3). Results for pollen were similar with graphical comparison
indicating a shift toward higher values at the later application data (Figure S-4). Median total
clothianidin values for all plant samples were 2 to 3 times greater for the later applications andmaximum values were 2.5, 2, and 1.8 times greater in leaves, nectar, and pollen samples.
4. Bias in concentration between methods of application. This aspect of the study was confounded
whereby effects of method of application were unevenly distributed amongst the soil types (Table S-1).
For example, only coarse soils were located in furrow application treatments so potential effects due tothe other soil types were not represented. Owing to the presence of confounding effects, thesecomparisons are presented for informational purposes only. Table S-10 compares the distribution for
total clothianidin residues between furrow and chemigation methods of application. Based on
differences measured for timing of application, separate statistical analyses were conducted for atplanting and 1 month later application comparisons. The majority of results indicated no difference inthe overall distribution and in the median values for both methods of application (Table S-11 andFigures S-5 through S-7). The only significant effects were indicated at planting for Wilcoxon test for15
798

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49602801

CDPR Clothianidin Cucurbit DER

distributions of TZNG and total residue in nectar: Tests for location of the median value were notsignificant.
5. Bias in concentration due to soil type. As for comparisons between methods of application, thedesign of the study was confounded with respect to effects of soil type with method of application(Table S-1). Again, these comparisons are presented for informational purposes only. Since there
appeared to be no consistent differences in distribution of residue concentrations between furrow or
chemigation methods of application, these data were combined and the distributions compared
between soil types. Tables S-12 through S-14 contain the distributions by each soil type as measured forleaves, nectar, and pollen, respectively. The highest total clothianidin residue nectar value was 20.8
ppb, which was measured in a medium-textured soil plot for an application made 1 month after
planting. The second highest value was 10.3 ppb, which was measured in a fine-textured soil for an
application made at planting. For pollen, the pattern was similar with 51.6 ppb being the highest value
measured at the fine-textured site for the 1 month later application.
6. Concentration distribution of residue in bee relevant matrices. Tables S-7 and S-8 contain the
distribution of residues measured in nectar and pollen, respectively, for the trial sites combined within
the two timings of application treatments. For nectar, the maximum and median values were 10.3 and2.3 ppb for at planting treatments, and 29.4 and 6.7 ppb for treatments made 1 month after planting.
Pollen values tended to be higher with maximum and median values of 20.8 and 6.1 ppb for at planting
treatments, and 51.6 and 18.7 ppb for treatments made 1 month after planting.
7. Relationship in clothianidin concentrations between plant samples. Figure S-9 shows therelationship measured in all replicate samples for concentrations of clothianidin between leaves andnectar (A), leaves and pollen (B), and nectar and pollen (C) samples. The R-square for leaves and nectar
is 0.68 and the relationship indicates a good relationship where nectar values increase with increase inleaf concentration. The R-square for leaves and pollen is lower for all data at 0.39, but removal of theone potential outlier increases the R-square value to 0.56. The general response is similar to thatobserved for nectar where concentration in pollen tends to increase with increased concentration inleaves. The relationship is not as clear cut between nectar and pollen concentrations as there is
obviously more scatter associated with that graph.
8. Soil concentration. Based on the analyses for plant samples, the most relevant comparison is the
distribution of soil concentrations between applications made at planting or 1 month after planting.
Table S-15 contains the statistics for the comparison of these two distributions where soil data was
combined over 2013 and 2014 for each treatment. Non-parametric statistical tests indicated that the
Wilcoxon test was significant at P=0.021 but the test for location of the Median values was not
significant with P=0.41. In Figure S-8, concentrations for applications made 1 month after planting
indicate a skew towards higher concentrations where maximum and median values for the 1 month
later applications are 1.9 and 1.5 times greater than at planting values, respectively.
Conclusion:
1. For bee relevant matrices, the response of pumpkin to the pattern of application used in this study
indicated higher concentrations for total clothianidin residues measured in pollen than in nectar
samples. The highest pollen value was 51.6 ppb compared to 29.4 ppb for nectar. Potential forbiological significance relies upon comparison to chronic feeding benchmark values, but they have notyet been established.
16799

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49602801

CDPR Clothianidin Cucurbit DER

2. The study design was confounded by too many instances of adding a treatment factor, but at theexpense of producing an incomplete treatment design matrix. For example, applications at some trialsites were made to furrows rather than by chemigation. A trial site represented a soil type so adding the
furrow application method without also applying chemigation at that site confounded comparisons ofsoil type. Effects could have been caused by method of application and not due to soil type. Thus it was
not possible to conclude potential effects of soil type.
3. A comparison was made for effect and timing of application on concentrations measured in planttissues. Delaying application from at planting to 1 month after planting resulted in higher
concentrations, especially significant for leaf samples. Analysis of soil sampled between these two dates
of application indicated potentially higher concentrations for applications made 1 month after planting.
The measurement of increased concentration in plant samples may in part be due to higher soil
concentrations as well as a decreased time interval between application and plant sampling.
4. Increasing concentrations of clothianidin leaf tissue resulted in concomitant increases inconcentration measured in nectar and pollen samples.
5. Lack of difference in concentration between years indicated no carry-over effect from the application
rates and methods used in the studies: i.e. rate at 0.2 lbs/acre applied either as a chemigation treatment
or furrow injection, and applied either at planting or 30 days after planting.

17800

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49602801

CDPR Clothianidin Cucurbit DER

Table S-1. Summary of the number of samples taken at each year and for each sample type at each trialsite. The method used was indicated as L-2 that measured parent and degradation products. Note that
there were different methods of application and different timing of applications distributed across thetrial sites and across the soil categories.
Application
Timing, Trial
Site Number,and Soil Texture
At Planting

Furrow/Soil Application
LeavesNectarPollen

Soil

2012

2013

2014

2012

2013

2014

2012

2013

2014

2013

2014

2
2

2
2

2
2

2
2

2
2

2
2

2
2

2
2

2
2

3
3

3
3

2

2

2

2

2

2

2

2

2

3

3

6

6

6

6

6

6

6

6

6

9

9

Month Delay

2012

2013

2014

2012

2013

2014

2012

2013

2014

2013

2014

4A-Coarse

2

2

2

2

2

2

2

2

2

3

3

2

2

2

2

2

2

2

2

2

3

3

1-Medium
2-Coarse
3-Coarse
4-Coarse
5-Medium
6-Coarse
7-Fine8-Coarse
9-Medium
Total

Total

Chemigation
At Planting
1-Medium
2-Coarse
3-Coarse
4-Coarse
5-Medium
6-Coarse
7-Fine8-Coarse
9-Medium
Total
Month Delay
1A-Medium
7A-Fine
Total

Leaves
2012 2013 2014
2
2
2
2
2
2

Nectar2012 2013
2
2
2
2

2014
2
2

Pollen2012 2013
2
2
2
2

2014
2
2

Soil
2013
2014
3
3
3
3

2
2
0

2
0
0

2
2
2

2
2
2

2
2
2

2
2
2

2
2
2

2
2
2

2
2
2

3
3
3

3
3
3

0

0

2

2

2

2

2

2

2

3

3

8

6

12

12

12

12

12

12

12

18

18

2012
2
0

2013
2
0

2014
2
2

2012
2
2

2013
2
2

2014
2
2

2012
2
2

2013
2
2

2014
2
2

2013
3
3

2014
3
3

2

2

4

4

4

4

4

4

4

6

6

18
801

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49602801

CDPR Clothianidin Cucurbit DER

Table S-2. Leaves: Comparison of statistics for the distribution for years 2012, 2013, and 2014 for concentration of clothianidin, its degradation
products and total residue in leaves of pumpkin.

Clothianidin
Statistic

TZNG

TZMU

Total Residue

2012

2013

2014

2012

2013

2014

2012

2013

2014

2012

2013

2014

N
Mean
SD

18
65.2
51.7

16
91.1
130.3

24
53.4
38.4

18
9.0
7.5

16
8.6
8.0

24
7.1
5.2

18
15.1
10.7

16
20.6
24.4

24
11.1
11.3

18
89.3
68.5

16
120.4
161.6

24
71.5
52.9

CV (%)MinMedian
75th
90th
95th
Max

79.3
15.2
51.8
95.7
171.3
184.4
184.4

142.9
11.8
39.9
90.3
409.8
423.6
423.6

71.8
13.1
42.5
66.4
118.1
129.4
156.4

83.2
2.6
4.6
13.5
25.0
26.0
26.0

92.1
0.7
5.8
10.4
24.1
30.1
30.1

74.1
1.3
6.2
7.8
16.2
16.8
21.4

71.1
0.7
12.5
22.8
33.2
33.9
33.9

118.2
4.4
9.3
22.8
73.3
81.5
81.5

102.4
2.8
8.1
11.4
31.5
32.2
49.2

76.7
20.7
70.2
133.6
231.2
239.3
239.3

134.2
17.0
54.3
128.8
515.4
527.0
527.0

73.9
17.4
58.4
82.7
166.5
169.7
209.3

% of Total

73.0

75.7

74.7

10.1

7.2

9.9

16.9

17.1

15.4

19
802

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49602801

CDPR Clothianidin Cucurbit DER

Table S-3. Nectar: Comparison of statistics for the distribution for years 2012, 2013, and 2014 for concentration of clothianidin, its degradation
products and total residue in nectar of pumpkin flowers.

Clothianidin
Statistic

TZNG

TZMU

Total Residue

2012

2013

2014

2012

2013

2014

2012

2013

2014

2012

2013

2014

N
Mean
SD
CV (%)MinMedian
75th
90th
95th
Max

24
3.9
3.6
92.6
0.6
2.4
5.3
9.6
12.3
13.3

24
3.3
4.4
135.1
0.7
1.9
3.0
5.4
15.9
18.0

24
3.4
2.9
83.6
0.6
2.4
4.3
6.3
10.6
11.6

24
0.3
0.2
63.7
0.1
0.3
0.5
0.6
0.7
0.7

24
0.3
0.4
118.1
0.1
0.2
0.3
0.9
1.4
1.5

24
0.3
0.4
110.1
0.1
0.1
0.3
0.8
0.9
1.5

24
0.2
0.2
82.7
0.1
0.1
0.3
0.6
0.6
0.8

24
0.4
0.5
114.5
0.1
0.3
0.5
1.7
7.9
2.0

24
0.3
0.3
86.8
0.1
0.2
0.5
0.8
0.9
1.0

24
4.4
3.9
88.5
0.8
2.8
6.0
10.3
13.5
14.7

24
4.0
5.1
125.7
0.9
2.8
3.7
6.5
18.9
20.8

24
4.0
3.3
81.1
0.8
3.3
5.0
7.2
12.3
13.4

% of Total

87.5

80.7

84.6

7.0

8.4

7.9

5.2

10.9

7.7

20
803

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49602801

CDPR Clothianidin Cucurbit DER

Table S-4. Pollen: Comparison of statistics for the distribution for years 2012, 2013, and 2014 for concentration of clothianidin, its degradation
products and total residue in pollen of pumpkin flowers.

Clothianidin
Statistic

TZNG

TZMU

Total Residue

2012

2013

2014

2012

2013

2014

2012

2013

2014

2012

2013

2014

N
Mean
SD
CV (%)MinMedian
75th
90th
95th
Max

24
9.6
9.7
101.3
1.7
5.7
9.5
26.0
28.9
37.6

24
6.7
7.1
104.5
1.2
4.8
8.0
10.6
24.6
30.3

24
10.1
10.2
100.8
1.4
6.8
12.6
19.9
31.2
44.5

24
0.9
0.7
75.0
0.1
0.7
1.2
2.0
2.1
2.4

24
0.7
0.7
99.8
0.1
0.6
0.9
1.6
2.4
2.9

24
1.2
1.2
95.7
0.1
0.7
1.6
3.1
3.7
4.2

24
0.9
0.7
77.0
0.1
0.6
1.1
2.1
2.3
2.6

24
0.7
0.4
63.4
0.1
0.7
1.0
1.2
1.3
1.4

24
1.0
0.8
88.9
0.1
0.6
1.2
2.5
2.6
2.9

24
11.4
11.0
96.0
2.6
6.6
11.7
29.9
33.5
42.3

24
8.1
7.7
95.0
1.5
6.9
9.8
12.9
27.2
33.3

24
12.3
11.9
96.9
1.6
9.2
15.8
23.6
37.5
51.6

% of Total

84.1

83.0

82.4

8.0

8.9

9.8

8.0

8.1

7.8

21
804

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49602801

CDPR Clothianidin Cucurbit DER

Table S-5. Results of non-parametric statistical tests for measuring differences in distributions for years
2012, 2013, and 2014 clothianidin, its degradation products and total residue in leaves nectar andpollens samples of pumpkin.
Non-Parametric Test Significance Levels For Differences Between Years
Clothianidin
Plant Sample

TZNG

TZMU

Total Residue

Wilcoxon

MedianWilcoxon

MedianWilcoxon

MedianWilcoxon

MedianLeavesNectar

0.95
0.39

0.76
0.73

0.86
0.50

0.94
0.41

0.19
0.17

0.40
0.25

0.86
0.62

0.76
0.93

Pollen

0.25

0.56

0.32

0.95

0.67

0.91

0.33

0.42

Table S-6. Leaves: Comparison of statistics between timing of application for the distribution ofclothianidin, its degradation products and total residue in leaves of pumpkin.
Clothianidin

TZNG

TZMU

At
Planting

30 Days
After
Planting

Total Residue

Statistic

At
Planting

30 Days
After
Planting

N

14

14

14

14

14

14

14

14

Mean

50.1

143.1

6.8

15.3

14.2

29.9

71.1

188.3

SD

39.3

126.5

5.1

8.9

13.1

23.9

55.3

156.4

CV (%)

78.5

88.4

74.8

58.6

92.7

79.8

77.8

83.1

Min

14.0

13.1

1.9

1.3

3.1

2.8

21.4

17.4

Median

38.6

111.2

5.9

15.4

8.7

25.1

55.3

157.8

75th

65.7

171.3

8.7

24.1

22.7

33.9

84.3

231.2

90th

98.5

409.3

11.4

26.0

33.2

73.3

153.1

515.4

95th

156.4

423.6

21.4

30.1

43.2

81.5

209.3

527.0

Max

156.4

423.6

21.4

30.1

43.2

81.5

209.3

527.0

% of Total

70.5

76.0

9.6

8.1

19.9

15.9

22
805

At
Planting

30 Days
After
Planting

At
Planting

30 Days
After
Planting

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49602801

CDPR Clothianidin Cucurbit DER

Table S-7. Nectar: Comparison of statistics between timing of application for the distribution ofclothianidin, its degradation products and total residue in nectar of pumpkin flowers.
Clothianidin

TZNG

TZMU

Total Residue

At
Planting

30 Days
After
Planting

At
Planting

30 Days
After
Planting

At
Planting

30 Days
After
Planting

At
Planting

30 Days
After
Planting

N
Mean
SD

18
3.0
2.4

18
6.8
5.4

18
0.2
0.1

18
0.5
0.3

18
0.2
0.2

18
0.7
0.6

18
3.5
2.6

18
8.0
6.2

CV (%)
Min

78.3
0.7

79.3
0.6

56.5
0.1

66.8
0.1

75.2
0.1

85.4
0.1

74.4
0.9

77.8
0.8

Median
75th
90th
95th

2.4
3.8
6.3
9.6

4.9
11.6
15.9
18.0

0.2
0.3
0.3
0.5

0.4
0.7
0.9
0.9

0.2
0.3
0.5
0.7

0.5
0.8
1.9
2.0

2.8
4.2
7.2
10.3

6.1
13.4
18.9
20.8

Max

9.6

18.0

0.5

0.9

0.7

2.0

10.3

20.8

% of Total

87.0

85.5

6.1

5.8

6.9

8.1

Statistic

Table S-8. Pollen: Comparison of statistics between timing of application for the distribution ofclothianidin, its degradation products and total residue in pollen of pumpkin flowers.
Clothianidin

TZNG

TZMU

At
Planting

30 Days
After
Planting

Total Residue

Statistic

At
Planting

30 Days
After
Planting

N
Mean

18
8.1

18
17.6

18
0.8

18
1.4

18
0.8

18
1.4

18
9.7

18
20.3

SD
CV (%)

6.3
77.4

13.4
76.4

0.7
86.1

1.2
83.5

0.6
69.5

1.0
70.2

7.3
75.2

15.4
75.5

Min
Median
75th
90th
95th
Max

1.6
6.8
11.1
17.0
25.8
25.8

1.4
15.0
28.9
37.6
44.5
44.5

0.1
0.6
0.9
2.1
2.4
2.4

0.1
1.4
2.0
3.7
4.2
4.2

0.1
0.6
1.2
1.4
2.5
2.5

0.1
1.3
2.1
2.6
2.9
2.9

1.9
8.0
12.9
20.6
29.4
29.4

1.6
18.7
33.3
42.3
51.6
51.6

% of Total

83.7

86.3

7.9

6.9

8.4

6.7

23
806

At
Planting

30 Days
After
Planting

At
Planting

30 Days
After
Planting

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49602801

CDPR Clothianidin Cucurbit DER

Table S-9. Results of non-parametric statistical tests for measuring differences in distributions for years
2012, 2013, and 2014 clothianidin, its degradation products and total residue in leaves nectar andpollens samples of pumpkin.
Non-Parametric Test Significance Levels For Differences Between
Applications at Planting or Made One Month Later
LeavesNectarPollenClothianidin
Residue

Wilcoxon

MedianWilcoxon

MedianWilcoxon

MedianTZNG

0.00670.11300.01000.05500.12000.3100TZMU

0.09600.05600.00310.01580.14000.1300

Clothianidin

0.00840.00740.01900.10000.13000.3200Total Residue

0.02000.00640.01900.09300.07000.090024807

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49602801

CDPR Clothianidin Cucurbit DER

Table S-10. Total Clothianidin Residue: Comparison of statistics between applications applied either tothe furrow or through chemigation for the distribution of total clothianidin residues leaves, nectar, andpollen of pumpkin plants.
Application at Planting
LeavesNectarPollenStatistic

Chemigation

FurrowChemigation

FurrowChemigation

Furrow

N
Mean

26
64.7

18
51.8

36
3.3

18
2.0

36
8.1

18
5.8

SD
CV (%)MinMedian
75th
90th
95th

45.4
70.2
20.7
52.9
81.0
133.9
153.1

25.4
49.0
17.0
54.7
64.7
82.0
115.0

2.0
61.4
1.3
2.8
4.1
6.5
7.2

1.2
59.1
0.8
1.5
3.0
4.3
4.4

5.2
63.5
2.6
7.4
9.3
12.9
20.6

4.3
73.5
1.5
4.6
8.7
11.2
17.6

Max

209.3

115.0

10.3

4.4

29.4

17.6

Application One Month After Planting
LeavesStatisticN
MeanSD
CV (%)MinMedian
75th
90th
95th
Max

NectarPollenChemigation
8
209.7
207.5
99.0

Furrow
6
159.8
40.7
25.5

Chemigation
12
9.2
7.2
78.2

Furrow
6
5.4
2.1
38.7

Chemigation
12
21.4
18.3
85.3

Furrow
6
18.1
7.7
42.4

17.2
154.8
377.4
527.0
527.0
527.0

114.0
155.0
169.7
231.2
231.2
231.2

0.8
9.6
14.1
18.9
20.8
20.8

2.9
5.1
6.7
8.8
8.8
8.8

1.6
22.3
35.5
42.3
51.6
51.6

10.5
16.3
23.6
29.9
29.9
29.9

25
808

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49602801

CDPR Clothianidin Cucurbit DER

Table S-11. Results of non-parametric statistical tests conducted to compare the distribution ofclothianidin, its degradation products, and total residue between chemigation and furrow application
methods that were made either at planting or 1 month after planting. Concentrations are compared for
leaf, nectar, and pollens samples of pumpkin plants.
LeavesApplication at Planting
NectarWilcoxon
MedianPollenWilcoxon
MedianChemicalWilcoxon

MedianTZNG
TZMU
Clothianidin
Total Residue

0.63000.65000.40000.57001.00000.37000.76001.0000ChemicalApplication One Month After Planting
LeavesNectarPollenWilcoxon
MedianWilcoxon
MedianWilcoxon
MedianTZNG

0.88001.00000.53000.61000.95001.0000TZMU

0.41000.59000.94000.62000.89001.0000

Clothianidin0.75000.59000.55000.63000.89001.0000Total Residue

1.00001.00000.61000.62000.90001.00000.03000.33000.01700.005826809

0.14000.77000.39000.14000.00300.31000.07500.03900.00600.68000.39000.1540Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49602801

CDPR Clothianidin Cucurbit DER

Table S-12. Leaves: Comparison of distribution statistics for concentration of clothianidin and total residue in leaves of pumpkin grown in eachsoil type. Note that data were pooled for furrow and chemigation applications within a soil type when indicated (see Table S-1). Separate
analyses are presented for applications made at planting or 30 days after planting.

Application At Planting
Clothianidin

Application 1 Month After Planting

Total Residue

Clothianidin

Total Residue

Statistic

CoarseMediumFine

CoarseMediumFine

CoarseMediumFine

CoarseMedium

Fine

N
Mean
SD
CV (%)MinMedian
75th

28
41.5
23.0
55.4
11.8
39.6
51.8

14
49.4
37.9
76.8
15.7
36.7
58.6

2
28.9
6.3
21.8
24.4
28.9

28
57.1
31.1
54.4
17.0
55.1
73.0

14
67.5
52.6
77.9
23.2
50.7
74.2

2
34.9
7.7
22.1
29.4
34.9

6
113.7
30.6
27.0
82.0
106.0
118.1

6
215.5
163.4
75.8
31.6
156.9
409.8

2
14.5
2.0
13.6
13.1
14.5

6
159.8
40.0
72.0
25.5
114.0
155.0

6
273.2
202.1
74.0
42.1
200.2
515.4

2
19.0
2.2
11.6
17.4
19.0

90th
95th
Max

75.5
88.7
95.7

98.5
156.4
156.4

33.3

115.0
147.6
133.9

153.1
209.3
209.3

171.3
171.3
171.3

423.6
423.6
423.6

15.9

169.7
231.2
231.2

527.0
527.0
527.0

% of Total

72.5

73.2

82.8

71.2

78.9

76.5

40.3

27
810

20.5

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49602801

CDPR Clothianidin Cucurbit DER

Table S-13. Nectar: Comparison of distribution statistics for concentration of clothianidin and total residue in nectar of pumpkin plants grown ineach soil type. Note that data were pooled for furrow and chemigation applications within a soil type when indicated (see Table S-1). Separate
analyses are presented for applications made at planting or 30 days after planting.

Application At Planting
Clothianidin

Application 1 Month After Planting

Total Residue

Clothianidin

Total Residue

Statistic

CoarseMediumFine

CoarseMediumFine

CoarseMediumFine

CoarseMedium

Fine

N
Mean
SD
CV (%)MinMedian
75th
90th
95th
Max

30
2.2
1.4
65.2
0.6
1.8
2.6
4.1
4.5
6.4

18
2.4
1.5
61.5
0.9
1.7
2.9
5.4
6.3
6.3

6
0.4
3.3
85.8
1.1
2.9
5.6
9.6
9.6
9.6

30
2.5
1.5
61.0
0.8
2.1
3.1
4.6
5.0
7.2

18
3.1
1.6
53.3
1.3
3.0
3.6
6.5
7.2
7.2

6
4.2
3.5
82.4
1.3
3.2
6.1
10.3
10.3
10.3

6
4.5
1.8
39.5
2.5
4.2
5.3
7.5
7.5
7.5

6
13.6
2.8
20.7
10.6
12.8
15.9
18.0
18.0
18.0

6
2.4
2.0
82.3
0.6
1.9
3.2
6.0
6.0
6.0

6
5.4
2.1
38.7
2.9
5.1
6.7
8.8
8.8
8.8

6
15.6
3.4
22.1
12.3
14.1
18.9
20.8
20.8
20.8

6
2.9
2.3
81.0
0.8
2.1
3.9
7.0
7.0
7.0

% of Total

86.1

76.3

9.0

82.9

87.4

85.0

28
811

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49602801

CDPR Clothianidin Cucurbit DER

Table S-14. Pollen: Comparison of distribution statistics for concentration of clothianidin and total residue in pollen of pumpkin plants grown ineach soil type. Note that data were pooled for furrow and chemigation applications within a soil type when indicated (see Table S-1). Separate
analyses are presented for applications made at planting or 30 days after planting.

Application At Planting
Clothianidin

Application 1 Month After Planting

Total Residue

Clothianidin

Total Residue

Statistic

CoarseMediumFine

CoarseMediumFine

CoarseMediumFine

CoarseMedium

Fine

N
Mean
SD
CV (%)MinMedian
75th
90th
95th

30
5.0
2.9
56.9
1.2
4.3
7.4
7.7
9.5

18
6.3
3.8
60.0
1.7
5.3
8.3
11.5
17.0

6
9.2
8.9
95.9
2.2
7.1
11.1
25.8
25.8

30
6.1
3.6
58.3
1.5
5.7
8.8
9.4
11.2

18
8.4
4.3
51.3
2.6
7.5
10.1
14.1
20.6

6
10.5
10.0
95.1
2.6
8.0
12.3
29.4
29.4

6
15.4
6.6
42.9
9.1
13.6
19.9
26.0
26.0

6
32.9
7.1
21.6
24.6
30.7
37.6
44.5
44.5

6
4.4
4.8
109.7
1.4
1.9
6.2
13.4
13.4

6
18.1
7.7
42.4
10.5
16.3
23.6
29.9
29.9

6
37.6
8.5
22.6
27.2
35.5
42.3
51.6
51.6

6
5.3
6.2
117.2
1.6
2.1
6.7
17.4
17.4

Max

13.8

17.0

25.8

17.6

20.6

29.4

26.0

44.5

13.4

29.9

51.6

17.4

% of Total

82.3

74.8

87.8

85.1

87.5

82.3

29
812

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49602801

CDPR Clothianidin Cucurbit DER

Table S-15. Soil: Comparison of statistics for the distribution concentration of clothianidin in soil for
applications made at planting compared to those made 1 month after planting.
Clothianidin

Statistic

At Planting

1 Month After Planting

N

54

18

Mean

7.4

14

SD

5.9

12.7

CV (%)

79.6

89.6

Min

0.7

3.6

Median

5.6

8.5

75th

11.0

18.3

90th

15.8

38.4

95th

18.8

43.1

Max

22.2

43.1

30
813

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49602801

CDPR Clothianidin Cucurbit DER

Figure S-1. Leaf Samples: Comparison of box plots for the distribution of clothianidin residues
between data collected in 2012, 2013, and 2014.
Box Chart for Clothianidin
3σ Limits

Clothianidin Residue Concentration (ppb)

500

400

300

200

UCL

100

Z=67.474137931
LCL
0
2012

2013

2014

Subgroup Index (yearcat)

31814

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49602801

CDPR Clothianidin Cucurbit DER

Figure S-2. Leaf Samples: Comparison of the distribution of clothianidin concentration measured for
applications made at planting or one month after application. Data has been pooled for all years.
Distribution of totcloth60

At Planting

Percent

50
40

30

20
10
0
60

Month Later

Percent

50
40

30

20
10

appdelay0
At Planting
Month Later

-200

0

200

400

totcloth
Normal32
815

Kernel600

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49602801

CDPR Clothianidin Cucurbit DER

Figure S-3. Nectar Samples: Comparison of the distribution of total clothianidin residue
concentration measured for applications made at planting or one month after application. Data has
been pooled for all years.
Distribution of totcloth
At Planting

Percent

30

20
10
0

Percent

40

Month Later

30
20
10

appdelay0

At Planting
Month Later

-10

0

10

20

totcloth
Normal33
816

Kernel30

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49602801

CDPR Clothianidin Cucurbit DER

Figure S-4. Pollen Samples: Comparison of the distribution of total clothianidin residue
concentration measured for applications made at planting or one month after application. Data has
been pooled for all years.
Distribution of totcloth40

At Planting

Percent

30
20
10
0

Percent

30

Month Later20

10appdelay0

At Planting
Month Later

-25

25

0

50

totcloth
Normal34
817

Kernel75

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49602801

CDPR Clothianidin Cucurbit DER

Figure S-5. Leaf Samples: Comparison of the distribution of total clothianidin residue concentration
measured for applications made either directly to the furrow or through the chemigation system forapplication made at planting. Data has been pooled for all years.
Figure S-5 (A). Application At Planting
Distribution of totcloth

Percent60

Chemigation

40

20

0

Percent

30

Furrow

20
10

AppType0
Chemigation
Furrow

-100

100

0

200

totcloth
NormalKernelFigure S-5 (B). Application 1 Month After Planting
Distribution of totcloth40

Chemigation

Percent

30

20

10
0

Percent

30

Furrow

20

10

AppType0
Chemigation
Furrow
-500

-250

250

0

500

totcloth
Normal35
818

Kernel750

1000

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49602801

CDPR Clothianidin Cucurbit DER

Figure S-6. Nectar Samples: Comparison of the distribution of total clothianidin residue
concentration measured for applications made either directly to the furrow or through thechemigation system for application made at planting. Data has been pooled for all years.
Figure S-6 (A). Application At Planting
Distribution of totcloth

Percent

40

Chemigation

30

20
10

0
50

Furrow

Percent

40
30
20
10

AppType0
Chemigation
Furrow0

5

10

totcloth
NormalKernelFigure S-6 (B). Application 1 Month After Planting
Distribution of totcloth

Percent

40

Chemigation

30
20
10
0

Percent

30

Furrow

20
10

AppType0
Chemigation
Furrow
-10

0

10

20

totcloth
Normal36
819

Kernel30

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49602801

CDPR Clothianidin Cucurbit DER

Figure S-7. Pollen Samples: Comparison of the distribution of total clothianidin residue
concentration measured for applications made either directly to the furrow or through thechemigation system for application made at planting. Data has been pooled for all years.
Figure S-7 (A). Application At Planting
Distribution of totcloth50

Chemigation

Percent

40
30

20

10

0

Percent

40

Furrow

30

20

10

AppType0
Chemigation
Furrow

-10

0

10

20

30

totclothNorm alKernelFigure S-7 (B). Application 1 Month After Planting
Distribution of totcloth

Percent

40

Chemigation

30
20
10
0
50

Furrow

Percent

40
30
20
10

AppType0
Chemigation
Furrow
-25

0

25

50

totcloth
Normal37820

Kernel75

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49602801

CDPR Clothianidin Cucurbit DER

Figure S-8. Soil Samples: Comparison of the distribution of clothianidin residue concentration
measured for applications made either at planting or 1 month after planting. Data has been pooled
over 2013 and 2014 years.
Distribution of logcloth
25

At Planting

Percent

20
15
10
5
0
40

Month Later

Percent

30
20
10

appdelay0
At Planting
Month Later
-2

0

2

4

logcloth
Normal38
821

KernelAppendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49602801

CDPR Clothianidin Cucurbit DER

Figure S-9. Relationship of concentration of clothianidin measured between A) Leaves and Nectar; B)
Leaves and Pollen; C) Nectar and Pollen.
Figure S-9 (A). Leaves and Nectar20

y = 0.0406x + 0.9596
R² = 0.6792
Nectar Concentration (ppb)

15

10

5

0

0

50

100

150

200

Leaves Concentration (ppb)
Figure S-9 (B). Leaves and Pollen

Pollen Concentration (ppb)60

y = 0.0763x + 4.2102
R² = 0.3861
40

20

0

0

50

100

Leaves Concentration (ppb)

39
822

150

200

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49602801

CDPR Clothianidin Cucurbit DER

Figure S-9 (C). Nectar and Pollen
60

Pollen Concentration (ppb)

y = 1.4452x + 3.2489
R² = 0.4274
40

20

0

0

5

10

Nectar Concentration (ppb)

40
823

15

20

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49602801

CDPR Clothianidin Cucurbit DER

9. REFERENCES
1.

Bondarenko, S., Rose, A., Ansolabehere, M., and Allen, R. 2014. “Clothianidin residues in pollen andnectar of cucurbits following different use patterns”, Abstract of Papers, 248th ACS National
Meeting & Exposition, San Francisco, CA, August 10-14, 2014. Poster: AGR0-602.

2. Citation for soil texture. URL: <http://www.tucson.ars.ag.gov/agwa/manual/version15/fao.htm>
3. Vidal, M. G., Jong, D., Wien, H. C. and Morse, R. A. 2006. Nectar and pollen production in pumpkin(Cucurbita pepo L.). Revista Brasil. Bot. 29(2), 267-273.
4. Jones, A., Harrington, P., Turnbull, G. 2014. Neonicotinoid concentrations in arable soils after seedtreatment applications in preceding years. Pest Manag. Sci. 70, 1780-1784.
5. U.S. Environmental Protection Agency. 2008. OPPTS Harmonized Test Guidelines, No. 835.1230
Adsorption/Desorption (Batch Equilibrium), EPA 712-C-08-009.
6. OECD Guidelines for Testing of Chemicals. 2000. No. 106 Adsorption Desorption Using a Batch
Equilibrium Method. DOl: 10.1787/20745753.
7. Rortais, A., Arnold, G., Halm, M-P., Touffet-Briens, F. 2005. “Modes of honeybees exposure tosystemic insecticides: estimated amounts of contaminated pollen and nectar consumed by different
categories of bees,” Apidologie 36, 71-83.
8. EPA-HQ-OPP-2012-0543-0004. “White Paper in Support of the Proposed Risk Assessment for Bees,”
Submitted to the FIFRA Scientific Advisory Panel for Review and Comment. September 11-14, 2012.
9. U.S. Environmental Protection Agency, Office of Pesticide Programs. "Guidance for Assessing
Pesticide Risks to Bees", June 19, 2014. URL: <http://www2.epa.gov/pollinatorprotection/pollinator-risk-assessment-guidance>.
10. U.S. Environmental Protection Agency, 2003, Data Evaluation Record for Study No. MRID 45422430.
Wilkins, P. “TI-435 Metabolite TZNG: Acute Oral Toxicity to Honey Bees (Apis me/litera)”; National
Bee Unit, England, Report No. 110057, 2000.
11. U. S. Environmental Protection Agency, 2003, Data Evaluation Record for Study No. MRID
45422429. Wilkins, P. “TI-435 Metabolite TZMU: Acute Oral Toxicity to Honey Bees (Apisme/litera)”; National Bee Unit, England, Report No. 110055, 2000.
12. U.S. Environmental Protection Agency, 2003, Data Evaluation Record for Study No. MRID 45422426.
Weyman, G. S. “TI-435 Technical: Acute Contact and Oral Toxicity to Honey Bees”; CovanceLaboratories, Ltd., Report No. 110049, 1998.
13. Bondarenko, S. “Freezer storage stability of Clothianidin, TZNG, and TZMU in surrogate nectar,”
Valent study number VP-38484. Ongoing.

41824

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50154302

CDPR Cloth Almond

Reference
Bodarenko, S. (2017) Clothianidin: Quantitation of Residues of Clothianidin, TZNG and TZMU in
Nectar, Pollen and Leaves Following Foliar Post Bloom Application of Belay Insecticide to AlmondTrees. Project ID: VP-38473. Unpublished study prepared by Valent U.S.A. Corporation. 861 pp. MRID
50154302, CDPR Study ID 298000, Data Volume 52884-0279, Tracking ID# 280318

1. STUDY INFORMATION
Chemical:Test Material:

Study Type:

Sponsor:Sponsor Study
ID:
Study
Completion
Date:

GLP Status:

PC Code044309Percent
Belay Insecticide
Active20%
Ingredient:
Residue study to measure the magnitude of Clothianidin and its major
metabolites, TZNG and TZMU, in almond leaves, pollen, and nectar following
foliar applications.
Valent U.S.A. Corporation
Experiment Start and March 24, 2014 –
6560 Trinity Court
End Date:
November 14, 2016
Dublin, California 94568
Clothianidin

43411B104
Study Locations:February 23, 2017

Nine (9) trial sites
including

TBD; protocol reviewed by CDPR.
[CDPR Study ID 298000, Data Volume 52884-0279, Tracking ID# 280318]

2. REVIEWER INFORMATION
Study Reviewed by:Richard Bireley, Sr. Environmental Scientist (Specialist)
California Department
John Troiano, Ph.D., Research Scientist III
of Pesticide Regulation
Alexander Kolosovich, Sr. Environmental Scientist (Specialist)
Brigitte Tafarella, Environmental Scientist
Denise Alder, Sr. Environmental Scientist (Specialist)
Russell Darling, Sr. Environmental Scientist (Specialist)

1825

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50154302

CDPR Cloth Almond

3. EXECUTIVE SUMMARY

The study was conducted to measure residues of clothianidin and its metabolites, TZNG and TZMU, in
nectar, pollen, and leaves collected from almond trees treated with Belay Insecticide (active ingredient
clothianidin) over two years. The treatment regimen (two post-bloom foliar applications of Belay
Insecticide at BBCH growth stage 7.5 and 21 days before harvest in 2014 and 2015) was evaluated atnine trials located in California in 2014–2016. An NIS (nonionic surfactant) adjuvant (0.20 % v/v) wasused in all foliar applications. The total amount of clothianidin applied to almond trees each year was at
the maximum annual application rate of 0.2 lb a.i./Acre.
Each trial consisted of a single treated plot with a minimum of 9 almond trees. Each treated plot
consisted of three subplots. In the 2014 blooming season, control samples of nectar, pollen, leaves, andsoil were collected at the field sites during evaluation of the sampling method and the same field plots
were then used as treated plots in the study. Samples of almond flowers were collected by hand fromeach subplot during the blooming period in 2015 and 2016. The flower collection was done after 139
days (shortest) and 252 days (longest) after the last Belay Insecticide application. The collected flowers
were then processed to nectar and pollen. The leaves were collected at BBCH growth stage ca. 6.7 in2015 and 2016. Soil cores were collected from each trial to characterize clothianidin and its metabolites
background in soil before treatment if no pesticide history was available. Soil cores were also used toobtain an estimate of the residues in the root zone of almond trees on the day of/day after the secondand forth applications of Belay Insecticide in 2014 and 2015 and during the 2015 and 2016 bloom
samplings. Nectar, pollen, anthers, leaves, and soil were analyzed for clothianidin, TZNG, and TZMU
residues using liquid chromatography mass spectrometry (LC-MS/MS).
4. STUDY VALIDITY
Guideline Followed:
Guideline Deviations:
Other Deviations:Classification:
Rationale:
Reparability:

Protocol was reviewed and approved by CDPR
N/A
See Section 6
ACCEPTABLE
N/A
N/A

5. MATERIALS
Test Material Characterization for Foliar Application End Use Product
Test item:
Belay Insecticide
Percent A.I.:
23% A.I.
Formulation Type:
Soluble Concentrate (SC)
Lot/Batch Number:
V13C-1742-2,V15C-1742-1
CAS #:
210880-92-5
Expiration Date:
1/25/2015, 3/3/2016

5A. STUDY DESIGNThe objective of this study is to determine residues of clothianidin and its metabolites, TZNG and TZMU,
in almond nectar, pollen, and leaves collected following post-bloom foliar applications of Belay
Insecticide to almond trees over two years. One treated plot received two post-bloom foliar applicationsof Belay Insecticide to almond trees in 2014 and two post-bloom foliar applications of Belay Insecticide
to almond trees in 2015. The first foliar application of Belay Insecticide was made at the application rateof 0.1 lb a.i./Acre at BBCH growth stage 7.5 (fruit at half size), and the second foliar application of Belay
2826

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50154302

CDPR Cloth AlmondInsecticide was made at the application rate of 0.1 lb a.i./Acre at 21 days before harvest. An NIS(nonionic surfactant) adjuvant (0.20 % v/v) was used in all foliar applications. The total amount ofclothianidin applied in each year is equivalent to the maximum annual use rate of 0.2 lb a.i./A (224 ga.i./ha).

Each field trial consisted of one treated plot of a minimum of 9 almond trees and each treated plot
comprised of three individual subplots. Standard agronomic practices for growing almonds were usedon the treated plots.
5B. STUDY SITE LOCATION AND CHARACTERISTICS
The nine field trials were located in the United States of America in California, a commercial area forgrowing almonds, on either coarse-textured or medium-textured soils. The trial location and soil
characterization information is presented in appendix 3 through appendix 12 of the study report, and issummarized in the table below:
Table 1. Site Locations and Cotton Varieties
Trial
Site
Site
Identification
Trial Location

1
2
3
4
5
6
7
8
9

V-38473-A
V-38473-B
V-38473-C
V-38473-D
V-38473-E
V-38473-F
V-38473-G
V-38473-H
V-38473-I

Height ofTrees atBloom(ft)
16
12
13
14-16
18-20
18
15-20
16
18

Dos Palos, Merced
Kerman, Fresno
Madera, Madera
Strathmore, Tulare
Dinuba, Tulare
Lost Hills, Kern
Shafter, Kern
Arbuckle, Colusa
Winters, Yolo

Table 2. Trial Site Conditions
Site
Identification
Sand % Silt %V-38473-A
53
23
V-38473-B
77
19
V-38473-C
86
9
V-38473-D
53
31
V-38473-E
71
25
V-38473-F
48
21
V-38473-G
56
25
V-38473-H
39
29
V-38473-I
47
29

Clay %
24
4
5
16
4
31
19
32
24

USDA Textural
Class
Sandy Clay Loam
Loamy Sand
Loamy Sand
Sandy Loam
Sandy Loam
Sandy Clay Loam
Sandy Loam
Clay Loam
Loam

3
827

Almond Variety

Butte
Monterey
Nonpareil
Fritz
SonoraNonpareil/Monterey
Nonpareil/Monterey/Fritz
Winters
Mission

CEC
Meq/100g
20.9
19.3
6.4
12.7
7.3
22.9
14.6
22.1
17.0

Organic
Matter %
1.4
1.5
0.58
2.5
1.5
1.2
0.80
1.9
1.4

Plot
Area
(Acres)0.27270.25000.16970.12120.12120.76510.43090.29620.3182

Soil pH
7.6
7.1
8.0
7.3
7.9
8.1
6.1
7.1
5.7

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50154302

CDPR Cloth Almond

5C. APPLICATION TIMING AND RATESOne treated plot received two post-bloom foliar applications of Belay Insecticide to almond trees in2014 and two post-bloom foliar applications of Belay Insecticide to almond trees in 2015. The first foliar
application of Belay Insecticide was made at the application rate of 0.1 lb a.i./Acre at BBCH growth stage
7.5 (fruits at half size), and the second foliar application of Belay Insecticide was made at the application
rate of 0.1 lb a.i./Acre at 21 days before harvest with exceptions for trials V-38473-F and V38473-G. For
V-38473-F trial, the second and fourth applications of Belay Insecticide were made at 32 and 44 days
before harvest, respectively. For V-38473-G trial, the second application of Belay Insecticide was done
after almond harvest. The total clothianidin amount applied in each year is equivalent to the maximum
annual use rate of 0.2 lb a.i./A (224 g a.i./ha). Belay Insecticide was sprayed using an orchard air blast toboth sides of the tree rows. Non-ionic surfactant at 0.20% (v/v) was applied with each application

3/28/14
10/2/14
3/27/14
9/29/14
4/2/14
7/21/14

V-38473-D
V-38473-D
V-38473-E
V-38473-E
V-38473-F
V-38473-F
V-38473-G
V-38473-G
V-38473-H
V-38473-H
V-38473-I
V-38473-I

1
2
1
2
1
2
1
2
1
2
1
2

4/8/14
6/12/14
4/9/14
6/11/14
4/4/14
8/5/14
3/24/14
9/23/14
4/30/14
7/16/14
5/9/14
7/16/14

4
828

150.5
147.7
150.7
150.7
149.8
150.2

0.101
0.099
0.101
0.101
0.099
0.099

0.200

125.7
122.8
125.3
119.1
124.9
118.1
104.1
114.6
127.1
101.0
138.2
110.0

0.101
0.100
0.101
0.100
0.118
0.096
0.103
0.100
0.101
0.101
0.101
0.100

0.201

0.202
0.198

0.201
0.213
0.203
0.202
0.200

% of Target Applied

Application
rate (lbsai./Acre)

Spray Volume

BBCH Growth Stage

Year: 2014
7.5
8.9
7.5
8.9
7.5
21 days
PHIa
Foliar7.5
Foliar8.1
Foliar7.5
Foliar8.1
Foliar7.5
Foliar8.5c
Foliar7.5
Foliar
9.1d
Foliar7.5
Foliar8.5
Foliar7.5
Foliar8.5
FoliarFoliarFoliarFoliarFoliarFoliar

Total

1
2
1
2
1
2

Spray

V-38473-A
V-38473-A
V-38473-B
V-38473-B
V-38473-C
V-38473-C

Application Type

Application Date

Application Number

Trial ID

Table 3. Study Use Pattern for Clothianidin

101
99
101
101
98
99
101
100
101
100
118b
96
103
100
101
101
101
100

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

4/9/15
8/14/15
4/8/15
8/25/15
4/6/15
7/14/15

V-38473-D
V-38473-D
V-38473-E
V-38473-E
V-38473-F
V-38473-F
V-38473-G
V-38473-G
V-38473-H
V-38473-H
V-38473-I
V-38473-I

3
4
3
4
3
4
3
4
3
4
3
4

5/6/15
6/12/15
5/7/15
6/12/15
3/30/15
8/9/15
3/31/15
7/24/15
4/16/15
6/12/15
4/16/15
6/12/15

a

151.6
148.5
148.8
150.4
152.1
150.9

0.102
0.100
0.100
0.101
0.100
0.099

0.201

122.7
126.4
122.1
125.9
110.8
108.3
112.3
112.7
121.6
124.8
133.0
137.3

0.100
0.101
0.100
0.100
0.103
0.097
0.101
0.099
0.100
0.103
0.100
0.103

0.200

0.201
0.199

0.200
0.200
0.200
0.203
0.202

% of Target Applied

Application
rate (lbsai./Acre)

Spray Volume

BBCH Growth Stage

Year: 2015
7.5
8.9
7.5
8.9
7.5
21 days
PHIa
Foliar7.5
Foliar8.1
Foliar7.5
Foliar8.1
Foliar7.5
Foliar8.5c
Foliar7.5
Foliar8.5
Foliar7.5
Foliar8.5
Foliar7.5
Foliar8.5
FoliarFoliarFoliarFoliarFoliarFoliar

Total

3
4
3
4
3
4

Spray

V-38473-A
V-38473-A
V-38473-B
V-38473-B
V-38473-C
V-38473-C

Application TypeCDPR Cloth AlmondApplication Date

Application Number

Trial ID

MRID 50154302

101
100
100
101
100
100
100
101
100
101
103
97
101
99
101
103
100
103

Re-treatment interval (Number of days between applications). Not applicable= n/a.
b Application rate outside the acceptable range, see deviation;c For trial V-38473-F, applications 2 and 4 were made at 32 and 44 days before harvest, respectively, instead of at
21 days before harvest as specified in the protocol;d Application occurred after the harvest.

5D. SAMPLE COLLECTION, HANDLING, PROCESSING
Soil
The soil core samples were collected at each trial to characterize soil properties and to measureclothianidin residue background from previous agricultural activities when no data were provided aboutclothianidin use. Typically, three soil cores of 2 inches (5.1 cm) were sampled using a soil auger or aprobe to a depth of 12 inches (30 cm) from each plot area. The three soil cores (one from each subplot)
were composited together to generate one sample per plot.

5829

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50154302

CDPR Cloth Almond

The soil core samples were also collected at each site from three subplots on the day of/day after thesecond application of Belay Insecticide in 2014 and 2015 and during the 2015 and 2016 bloom
samplings, except at trials V-38473-D and V-38473-E. These sites soil samples were not collected after
the second application in 2014. To collect a soil sample, two random adjacent almond trees wereselected from each subplot. Five (5) soil cores were then collected between the two trees to a depth of
12 inches (30 cm) and 2 inches (5.1 cm) in diameter using a hand held device. The only exception was fortrial V-38473-F where soil cores from the 2015 bloom sampling were collected to a depth of 18–23 cm.
This sampling technique was used to obtain an estimate of the residues in the root zone of the almondtree.
Once collected, soil samples were placed into coolers with blue ice or wet ice, transported to the field
facility, and stored in a temperature-monitored freezer pending shipment to the analytical laboratory.
Aliquots of composited soil samples from each plot were shipped to AGVISE Laboratories for soil
characterization analysis when no characterization data were provided.
Nectar, Pollen, and Leaf
A single composite flower sample and leaf sample was taken from each replicate subplot resulting incollection of three samples of each matrix for each treated plot. The flower samples were collectedduring the blooming period in 2015 and 2016 and the leaf samples were collected at BBCH growth stage
6.7 in 2015 and 2016. The flower sampling was done when there were enough flowers in the field toobtain sufficient amounts of pollen and nectar for residue analysis. The leaf sampling was done when
first leaves were unfolded. Single composite samples of almond flowers and leaves were collected fromeach site in the 2014 blooming season and used as control samples. Sample collection dates are
summarized in the table below.
Table 4. Sampling Events and Timing.
Sampling
FlowerEvent
TimingCollection
Date
Trial V-38473-A
2015
Bloom
2/18/2015
2016
Bloom
2/18/2016
Trial V-38473-B
2015
Bloom
2/20/152016
Bloom
2/22/16
Trial V-38473-C
2015
Bloom
2/18/152016
Bloom
2/17/16
Trial V-38473-D
2015
Bloom
2/17/152016
Bloom
2/17/16
Trial V-38473-E251
2015
Bloom
2/18/152016
Bloom
2/18/16

DALA(Days)

Timing(BBCH)

LeavesCollection
Date

139
188

7.2
7.2

3/17/15
3/16/16

166
215

144
181

7.2
7.2

3/20/15
3/17/16

172
205

212
218

6.7
6.7

3/2/15
3/4/16

224
234

250
250

6.7
6.7

3/18/15
3/3/16

279
265

252
251

6.7
6.7

3/19/15
3/3/16

281
265

a

6
830

DALAa
(Days)

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50154302

Sampling
Event

Trial V-38473-F
2015
2016
Trial V-38473-G
2015
2016
Trial V-38473-H
2015
2016
Trial V-38473-I
2015
2016

CDPR Cloth AlmondTimingFlowerCollection
Date

Timing(BBCH)

LeavesCollection
Date

DALA
(Days)DALAa(Days)

Bloom
Bloom

2/18/15
2/20/16

197
195

6.7
6.7

3/4/15
2/29/16

211
204

Bloom
Bloom

2/16/15
2/16/16

146
207

6.7
6.7

2/26/15
2/29/16

156
220

Bloom
Bloom

2/10/15
2/17/16

209
250

6.7
6.7

3/13/15
3/21/16

240
283

Bloom
Bloom

2/19/15
2/17/16

218
250

6.7
6.7

3/13/15
3/16/16

240
278

a

Flowers from the same subplot were randomly collected from each quadrant of the tree at the middle ofthe current season’s terminal shoots of the lower, middle, and upper lateral branches of the tree andcomposited in a 1-gallon bag. The collected flowers were placed in a cooler containing blue ice or wet
ice and transported to the field facility for processing into pollen and nectar samples. The bag(s) withthe flowers were left in a cooler for 1–3 hours (for conditioning), except at trial V-38473-C. The
conditioning allowed a pool of nectar to form in the flower nectaries. Nectar and pollen processed fromindividual flowers collected from the same subplot were composited to generate one sample of nectar
and one sample of pollen. Nectar was collected at the base of the flower filament using a glass
microcapillary and transferred through pre-split septa into a 1.8-mL pre-labeled vial at the field facility
with exceptions for trials V-38473-A and V-38473-B where nectar was collected in the field from flowers.
After extraction of nectar, the blossoms were placed on a dry, clean surface and allowed to dry forseveral hours. The drying allowed the release of pollen from the anthers. The released pollen was“vacuumed” into a pipette tip containing a barrier, using a vacuum system. After pollen processing wascomplete, the pipette tips were transferred into a vial.
Immediately after final weights were recorded, the labeled sample vials containing nectar and pollen
were placed into a freezer where they remained until they were shipped to the analytical laboratory.
After processing was completed, the flowers were discarded.
New emerged leaves were sampled from the middle of the current season’s terminal shoots of thelower, middle, and upper lateral branches of the tree and transferred into a pre-labeled container. Leaf
samples were collected and placed in a cooler containing blue or wet ice and transported to the field
facility where they were stored in a temperature-monitored freezer pending shipment to the analyticallaboratory.
Sample Storage.
In the field facility, all collected samples were stored in a temperature-monitored freezer pending
shipment to the analytical laboratory. Samples were shipped by a freezer truck (Agricultural Chemical
Delivery Services, Inc.) or by FedEx on dry ice accompanied by the chain of custody documents. Once
7831

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50154302

CDPR Cloth Almondsamples were received at the analytical laboratory, they were placed into a temperature monitored,
walk in freezer pending analysis.

The almond nectar and pollen samples generated during the study were stored up to 176 and 196 days
before analysis, respectively. Valent U.S.A. Corporation is conducting freezer storage stability studies ofclothianidin, TZNG, and TZMU in surrogate nectar1 and corn pollen2. Interim analyses after 1 year offrozen storage indicate that these chemicals are stable in both matrices.
The almond leaf samples generated during the study were stored up to 152 days before analysis.
Clothianidin residues have been shown to be stable on a variety of leafy vegetable crops when stored
frozen for up to 242 days3 and 4; therefore, a storage stability study on almond leaves was not conductedwith this study. Lettuce leaf samples from the head lettuce residue study were stored for up to 242 days,
and turnip top leaves and mustard greens from the rotational crop study were stored for 309 days.
The soil samples generated during the study were stored up to 334 days before analysis except only
three soil samples from V-38473-C trial were stored up to 489 days. Clothianidin, TZNG, and TZMU
residues have been shown to be stable in soil when stored frozen for up to 356 days5; therefore, astorage stability study on soil was not conducted with this study.
5E. ANALYTICAL METHODS
Method Summary for Analyzing Almond Nectar Samples
The method used to analyze samples of nectar in this study was Valent Method RM-39N-1, and it
allowed the quantitative determination of residues of clothianidin and its metabolites, TZNG and TZMU.
A copy of the method is included in appendix 13 of the study report. Generally, 0.100 g of nectar sample
was weighed into an autosampler vial and dissolved in 1.0 mL of methanol/water solution (40:60, v/v)
acidified with 0.05% formic acid. The sample was spiked with isotopically labeled internal standards(clothianidin-d3, TZNG-13C-15N, and TZMU-d3), filtered through a Whatman 0.2 µm nylon membrane
syringe filter, if particles were present, and then analyzed by LC-MS/MS.
Due to the small sample size of some nectar samples, the entire sample was used for the analysis.
Method Summary for Analyzing Almond Pollen and Anther Samples
The method used to analyze pollen and anthers in this study was Valent Method RM-39P-1, and it
allowed the quantitative determination of residues of clothianidin, TZNG, and TZMU. A copy of themethod is included in appendix 14 of the study report. Generally, 0.100 g of pollen/anther sample wasweighed into a 50-mL polypropylene centrifuge tube and extracted with water (10.0 mL), andacetonitrile (10.0 mL) followed by adding sodium chloride (1.0 g) and anhydrous magnesium sulfate (2.0g) salts. Further, the upper acetonitrile phase (9.0 mL) was removed, partitioned with n-hexane (5 mL)
containing magnesium sulfate (0.5 g), then collected (8.0 mL) and concentrated to dryness. Residues
were re-dissolved in 1.0 mL of methanol/water (40:60, v/v) acidified with 0.05% formic acid, filteredthrough a Whatman 0.2 µm nylon membrane syringe filter, and analyzed by LC-MS/MS after spiking
with isotopically labeled internal standards (clothianidin-d3, TZNG-13C-15N, and TZMU-d3) to compensate
for matrix effect.

8832

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50154302

CDPR Cloth AlmondDue to the small sample size of some pollen or anther samples, the entire sample was used for the
analysis and the final volume of the sample was adjusted to either 0.2 or 0.5 mL, depending on thesample size. For these samples, instead of filtering, the sample extract was centrifuged to remove any
particles before analysis.
Method Summary for Analyzing Almond Leaf Samples
The method used to analyze almond leaf samples in this study was Valent Method RM-39L-2 and it
allowed quantitative determination of clothianidin, TZNG, and TZMU. A copy of the method is included
in appendix 15 of the study report. Generally, 2.0 g of homogenized leaf sample was weighed into a 50mL polypropylene centrifuge tube and extracted with 10 mL of water acidified with 0.05% formic acidand acetonitrile (10 mL), followed by adding sodium chloride (2.0 g) and anhydrous magnesium (4.0 g)
sulfate salts. Further, an aliquot (1.0 mL) of the acetonitrile extract was cleaned through a Strata C18-E
column and concentrated to dryness. Residues were re-dissolved in 1.0 mL of water/methanol (75:25,v/v) acidified with 0.05% formic acid, filtered through a Whatman 0.2 µm nylon membrane syringe filter
into a vial, and analyzed by accurate mass UPLC/Q-TOF MS-MS after spiking with isotopically labeledinternal standards (clothianidin-d3, TZNG-13C-15N, and TZMU-d3) to compensate for matrix effect.
Method Summary for Analyzing Soil Samples
The method used to analyze soil samples in this study was Valent Method RM-39S-2, and it allowed
quantitative determination of clothianidin, TZNG, and TZMU. A copy of the method is included inappendix 16 of the study report. Typically, a 20.0-g soil sample was weighed into a 50-mL polypropylene
centrifuge tube and extracted with 25.0 mL of methanol/water (25:75, v/v) acidified with 0.05% formic
acid. The sample was shaken on a shaker for 1 hour and then centrifuged. Extraction was repeated again
with a fresh portion of solvent (25.0 mL). The supernatants were combined and then spiked withisotopically labeled internal standards (clothianidin-d3, TZNG-13C-15N, and TZMU-d3) to compensate formatrix effect. Then an aliquot was filtered through a Whatman 0.2 µm nylon membrane syringe filter
directly into an autosampler vial and then analyzed by accurate mass UPLC/Q-TOF MS-MS.
For the site selection when no appropriate documentation was provided that clothianidin andthiamethoxam had not been used in the last year at the trial site, collected soil samples were analyzed
for clothianidin residues using Valent Method RM-39S-1. This method allowed quantitative
determination of clothianidin. A copy of the method is included in appendix 16 of the study report.
Typically, a 20.0-g soil sample was weighed into a 50-mL polypropylene centrifuge tube and extractedwith 25.0 mL of methanol/water (40:60, v/v) acidified with 0.05% formic acid. The sample was shaken
on a shaker for 1 hour and then centrifuged. Extraction was repeated again with a fresh portion ofsolvent (25.0 mL). The supernatants were combined and then spiked with the isotopically labeledinternal standard (clothianidin-d3) to compensate for matrix effect. Then an aliquot was filtered througha Whatman 0.2 µm nylon membrane syringe filter directly into an autosampler vial and analyzed byaccurate mass UPLC/Q-TOF MS-MS.
5F. QUALITY ASSURANCE RESULTS
Quality control measures taken during the analytical phase of this study included, but were not limited
to, the following:

9833

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50154302

CDPR Cloth Almond

All analytical standards used in this study were kept at reduced temperature in a refrigerator or in afreezer at all times when not in use. The temperatures of all refrigerators and freezers used to store
samples and standards at Valent Technical Center for this study were continuously monitored using a
datalogger. Data from the datalogger are printed on a regular schedule and archived at the ValentTechnical Center. Valent certified the analytical reference standards used in this study prior to use.
Certification documents are included in this report in appendix 2 of the study report. Certification data
and retain samples of these materials are archived at the Valent Technical Center. All raw data
generated from this study will be archived at the Valent Technical Center.
At least five different standard concentrations were injected within each analytical set. The
concentration (ng/mL) of clothianidin and its metabolites detected in sample extracts was interpolated
from the standard calibration curve. The LC/MS-MS and accurate mass UPLC/Q-TOF MS-MS systems
were calibrated for each set of samples by analyzing these calibrating standard concentrations, withthese standards interspersed within the analytical sequence. A second-order polynomial fit (weighted
relative to 1/concentration) was then calculated from the concentrations and the detector response of
the calibration standards. To verify performance, the percent difference between the actual
concentration and the calculated concentration for each of the calibration standards (based on the
curve) was also calculated. Each of the standards was required to be within 15% (20% for method RM39S-2) of the theoretical concentration and the coefficient of determination (r2) of the weighted
polynomial calibration curve was required to be greater than or equal to 0.99. No exceedance of thesecriteria for the calibration standards was observed in the study.
The reproducibility of the LC/MS-MS and accurate mass UPLC/Q-TOF MS-MS systems was verified bycomparison of instrument responses obtained from the repeated analysis of a continuing standard (amidlevel calibration standard) analyzed with the study samples. The continuing calibration standards
were interspersed within the samples in the analytical sequence, and the analytical sequence began andended with a continuing calibration standard. For an analytical set (injection sequence) to be acceptable,
the coefficient of variation (CV) of the back calculated concentration of the continuing standards was
required to be 10% (method RM-39N-1 and RM-39P-1) and 15% (method RM-39L-2 and RM-39S-2). No
exceedance of these criteria for the continuing calibration standards was observed in the study.
At least one control sample and two fortified samples were analyzed with each set of the study samples
to verify method performance. Fortifications were made at 1.00 and 10.0 ng/g for nectar and pollen
samples, and at 5.0 and 50.0 ng/g for leaves and soil. For an analytical run to be acceptable, method
recoveries were required to be between 70 and 120%. Recoveries of the concurrent laboratory fortified
samples were within this range. Generally, recoveries of the concurrent laboratory fortified samples
were within this range. In some cases, minor exceedance of these criteria was accepted.
Method verification for each matrix was conducted for clothianidin, TZNG, and TZMU. Method RM 39N1 was verified at 1.00 ng/g (LOQ) and 10.0 ng/g (10× LOQ) using almond nectar or artificial nectar.
Method RM-39P-1 was verified at 1.00 ng/g (LOQ) and 10.0 ng/g (10× LOQ) using commercially available
pollen. Methods RM-39L-2 and RM-39S-2 were verified at 5.0 ng/g (LOQ) and 50.0 ng/g (10× LOQ) levels
using untreated control almond leaves and untreated control soil.
Table 5. Summary of LOQs and LODs

10834

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50154302
MethodMatrixAnalyte

RM-39N-1

Nectar

RM-39P-1

PollenRM-39P-1

Anthers

RM-39L-2

LeavesRM-39S-1

Soil

Clothianidin, TZNG andTZMUClothianidin, TZNG andTZMUClothianidin, TZNG andTZMUClothianidin, TZNG andTZMUClothianidin

RM-39S-2

Soil

Clothianidin, TZNG and
TZMU

LOD
(ppb, parent
equivalents)
0.20

CDPR Cloth Almond

LOQ(ppb, parent
equivalents)
1.0

0.25

1.0

0.25

1.0

1.3

5.0

1.3

5.0

1.3

5.0

6. DEVIATIONS DURING FIELD PHASE
During the field portion of this study the following deviations occurred:
Trial V-38473-C:
Several weather data points were not collected for the trial period because Weather Station CIMIS #188
had some gaps in recording daily temperatures.
The 2015 pollen sample size did not meet protocol requirements because a significant amount of pollen
was diluted/washed off by heavy fog and could not be collected. Also, flowers were not placed in acooler with blue ice for the 1- to 3-hour conditioning period during sampling events.
Trial V-38473-F:
During the first application of Belay Insecticide to the almond plot, a spray error was made resulting in
the actual application rate to the center (sample) row was likely 90.8% of the target rate. This deviation
was due to a technical error by the field trial personnel and the sprayer malfunction during application.
Due to excessively moist soil, soil samples were collected 2 days after flower collection in 2015 insteadof on the day of/day after flower collection. Also, the soil cores were collected to a depth of 18–23 cm(7– 9 inches) instead 30 cm (12 inches.).
The timing of the second application in 2014 was 32 days before harvest and the timing of the fourth
application in 2015 was 44 days before harvest, instead of 21 days before harvest as specified in the
protocol. Almond harvest timing is variable from season to season, so despite efforts to meet the 21-day
requirement through communication with the grower, the actual timings were longer than desired.
Transport temperatures for the test substance were not monitored between the field test facility
storage and the field site, as required by Valent SOP VP-203.

11835

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50154302

CDPR Cloth Almond

Trial V-38473-G:

The second application of Belay Insecticide to almond trees was conducted after the final almondharvest instead of 21 days pre-harvest as required by the protocol. The orchard had three varieties ofalmonds with different harvest dates, so the application was delayed.
Transport temperatures of the test substance were not monitored from the field test facility storage tothe field site, as required by Valent SOP VP-203.
Flower, pollen, and nectar samples were not collected in 2014 because the almond bloom was too far
along to collect these samples. This sampling was only to be used to evaluate the sampling method
during the 2014 bloom.
No weight was recorded for the leaf sample V-38473-G-5 because the Field Residue Data Book forms
had not yet been received and field personnel neglected to record the weight. This deviation had no
negative impact on the study. Additionally, this sample was collected at BBCH 7.2, instead of at BBCH 6.7
as required by the protocol. This sampling was only to be used to evaluate the sampling method and use
the collected control sample for method verification and QC samples.
No soil core sample was collected from subplot 3 (2014, second application) as required by the protocol
because the soil core sampler handle broke during sampling.

12836Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50154302

CDPR Cloth Almond

7. RESULTS:
Raw data as reported for concentrations of Clothianidin, TZNG, and TZMU are reproduced in Tables 6through 11 where Table 6 contains data for almond nectar, Table 7 for almond pollen, Table 8 foralmond anthers, Table 9 for almond leaves, Table 10 for soil samples taken at bloom, and Table 11 forsoil samples taken after applications 2 and 4. Summary statistics for total clothianidin residues are
reproduced in Table 12.
Table 6. Clothianidin, TZNG and TZMU Residues in Almond Nectar
Trial
Sample ID
DALAa
Clothianidin
TZNG
ppbb
Average
ppbb
Average2015
V-38473-A-15
<0.20
<0.20
A
139
<0.20
<0.20
V-38473-A-16
<0.20
<0.20
V-38473-A-17
<0.20
<0.20
V-38473-B-15(0.34)
<0.20
B
144
0.67
<0.20
V-38473-B-16
1.28
<0.20
V-38473-B-17(0.40)<0.20
V-38473-C-15
<0.20
<0.20
C
212
<0.20
<0.20
V-38473-C-16
<0.20
<0.20
V-38473-C-17(0.21)<0.20
V-38473-D-15(0.70)
<0.20
D
250
0.70
<0.20
V-38473-D-16(0.56)<0.20
V-38473-D-17(0.84)<0.20
V-38473-E-15(0.73)
<0.20
E
252
0.50
<0.20
V-38473-E-16(0.47)<0.20
V-38473-E-17(0.30)<0.20
V-38473-F-15
<0.20
<0.20
F
197
<0.20
<0.20
V-38473-F-16
<0.20
<0.20
V-38473-F-17
<0.20
<0.20
V-38473-G-15
<0.20
<0.20
G
146
0.24
<0.20
V-38473-G-16(0.40)<0.20
V-38473-G-17(0.23)<0.20
V-38473-H-15
<0.20
<0.20
H
209
0.20
<0.20
V-38473-H-16
<0.20
<0.20
V-38473-H-17
<0.20
<0.20
V-38473-I-15
<0.20
<0.20
I
218
0.20
<0.20
V-38473-I-16
<0.20
<0.20
V-38473-I-17
<0.20
<0.20
Minimum: <0.20<0.20
Maximum: 1.28
<0.20
c
Average: (0.28)cMedian: <0.20c90th Percentile: (0.71)

13
837

ppbb
<0.20
<0.20
<0.20
<0.20
(0.53)(0.20)<0.20<0.20(0.22)
<0.20
<0.20
<0.20
<0.20
<0.20
<0.20
<0.20
<0.20
<0.20
<0.20
<0.20
<0.20
<0.20
<0.20
<0.20
<0.20
<0.20
<0.20
<0.20
(0.53)ccc

TZMU
Average

<0.20

0.28

<0.20

<0.20

<0.20

<0.20

<0.20

<0.20

<0.20

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50154302

CDPR Cloth Almond

2016
A

B

C

D

E

F

G

H

I

V-38473-A-33
188
V-38473-A-34
V-38473-A-35
V-38473-B-33
181
V-38473-B-34
V-38473-B-35
V-38473-C-33
218
V-38473-C-34
V-38473-C-35
V-38473-D-33
250
V-38473-D-34
V-38473-D-35
V-38473-E-33
251
V-38473-E-34
V-38473-E-35
V-38473-F-33
195
V-38473-F-34
V-38473-F-35
V-38473-G-33
207
V-38473-G-34
V-38473-G-35
V-38473-H-33
250
V-38473-H-34
V-38473-H-35
V-38473-I-33
250
V-38473-I-34
V-38473-I-35
Minimum:
Maximum:
Average:
Median:90th Percentile:(0.87)
1.15
2.04
<0.20
<0.20
<0.20
<0.20
<0.20
(0.37)
<0.20
<0.20
<0.20
(0.33)
1.09(0.29)(0.29)
<0.20
<0.20
<0.20
(0.41)(0.26)
<0.20
<0.20
<0.20
<0.20
<0.20
<0.20
<0.20
2.04
0.33
<0.20
0.96

1.35

<0.20

<0.20

<0.20

0.57

<0.20

0.26

<0.20

<0.20

<0.20
<0.20
<0.20
<0.20
<0.20
<0.20
<0.20
<0.20
<0.20
<0.20
<0.20
<0.20
<0.20
<0.20
<0.20
<0.20
<0.20
<0.20
<0.20
<0.20
<0.20
<0.20
<0.20
<0.20
<0.20
<0.20
<0.20
<0.20
<0.20
c

a

<0.20

<0.20

<0.20

<0.20

<0.20

<0.20

<0.20

<0.20

<0.20

<0.20
<0.20
<0.20
<0.20
<0.20
<0.20
<0.20
<0.20
<0.20
<0.20
<0.20
<0.20
<0.20
<0.20
<0.20
<0.20
<0.20
<0.20
<0.20
<0.20
<0.20
<0.20
<0.20
<0.20
<0.20
<0.20
<0.20
<0.20
<0.20

<0.20

<0.20

<0.20

<0.20

<0.20

<0.20

<0.20

<0.20

<0.20

c

c

c

c

c

DALA= days after last application
Values in parenthesis are between the LOQ and LOD
cDescriptive statistics were not calculated because >50% of the results are below the LOD
In calculating the average concentration and descriptive statistics, values below the LOD are substituted
with half of the LOD value. LOD= 0.20 ppb and LOQ= 1.00 ppb for Clothianidin, TZNG and TZMU.
b

14
838

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50154302

CDPR Cloth Almond

Table 7. Clothianidin, TZNG and TZMU Residues in Almond Pollen
Trial
Sample ID
DALAa Clothianidin
TZNG
ppbb
Average ppbb
Average2015
V-38473-A-18
3.06
8.19
A
140
2.73
2.86
V-38473-A-19
1.90
<0.25
V-38473-A-20
3.22(0.25)
V-38473-B-18
4.58(0.27)
B
145
5.30(0.41)
V-38473-B-19
7.08(0.64)
V-38473-B-20
4.26(0.32)
V-38473-C-18
12.7(0.83)
C
214
13.4(0.64)
V-38473-C-20
14.0(0.44)F

G

H

I

V-38473-F-18
198
V-38473-F-19
V-38473-F-20
V-38473-G-18
147
V-38473-G-19
V-38473-G-20
V-38473-H-18
210
V-38473-H-19
V-38473-H-20
V-38473-I-18
219
V-38473-I-19
V-38473-I-20
Minimum:
Maximum:
Average:
Median:90th Percentile:(0.77)
1.60
1.10
2.21
1.29
2.23
10.4
13.3
11.0
7.45
8.26
20.0
(0.77)
20.0
6.52
4.42
13.4

V-38473-A-36
V-38473-A-37
V-38473-A-38
V-38473-B-36
V-38473-B-37
V-38473-B-38
V-38473-C-36
V-38473-C-37
V-38473-C-38
V-38473-F-36
V-38473-F-37
V-38473-F-38

5.42
3.83
5.23
3.04
2.76
3.82
11.7
4.54
7.20
1.04
(0.62)(0.60)

1.16

1.91

11.5

11.9

<0.25
1.90
<0.25
<0.25
<0.25
<0.25
2.48
<0.25
2.39
(0.90)(0.26)(0.51)
<0.25
8.19
1.01
(0.30)
2.40(0.72)

<0.25

1.66

(0.56)

TZMU
ppbb
22.8
(0.98)
2.78
1.70
3.59
3.09
3.63
1.73
(0.56)
3.74(0.49)(0.30)(0.25)
<0.25
5.85
2.03
7.37
2.23
(0.95)
1.58
<0.25
22.8
3.29
1.88
6.00

Average

8.86

2.79

2.68
1.60

<0.25

5.08

1.59

2016
A

B

C

F

189

182

219
220
221
196

4.82

3.21

7.80

(0.75)

15
839

<0.25
<0.25
<0.25
<0.25
<0.25
<0.25
(0.54)
<0.25
<0.25
<0.25
<0.25
<0.25

<0.25

<0.25

(0.26)<0.25(0.45)(0.38)(0.47)(0.44)(0.80)(0.59)
1.25(0.63)(0.45)
<0.25
<0.25
<0.25

(0.43)(0.61)(0.77)<0.25

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50154302

Trial

G

H

I

Sample ID

DALAa

V-38473-G-36
208
V-38473-G-37
V-38473-G-38
V-38473-H-36
251
V-38473-H-37
V-38473-H-38
V-38473-I-36
251
V-38473-I-37
V-38473-I-38
Minimum:
Maximum:
Average:
Median:90th Percentile:

Clothianidin
ppbb
Average
1.15(0.90)
1.00(0.55)
8.81
11
13.8
10.4
5.98
4.92
4.32
4.46
(0.55)
13.8
4.77
4.32
10.4

a

TZNG
ppbb
<0.25
<0.25
<0.25
(0.32)(0.51)(0.40)
<0.25
<0.25
<0.25
<0.25
(0.54)<0.25<0.25(0.40)CDPR Cloth Almond

Average<0.25(0.41)

<0.25

TZMU
ppbb
<0.25
<0.25
<0.25
(0.98)
1.19(0.81)(0.56)(0.45)(0.49)<0.25
1.25(0.51)(0.45)(0.98)

Average<0.25(0.99)(0.50)

DALA= days after last application
Values in parenthesis are between the LOQ and the LOD
In calculating the average concentration and descriptive statistics, values below the LOD are substituted
with half of the LOD value
LOD= 0.25 ppb and LOQ= 1.00 ppb for clothianidin, TZNG and TZMU

b

16
840

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50154302

CDPR Cloth Almond

Table 8. Clothianidin, TZNG and TZMU Residues in Almond Anthers
Trial
Sample ID
DALAa Clothianidin
TZNG
ppbb
Average ppbb
Average2015
V-38473-D-18
23.1
11.0
D
250
43.4
4.25
V-38473-D-19
88.1
1.42
V-38473-D-20
19.2(0.35)
V-38473-E-18
15.2
1.04
E
252
18.7
(0.83)
V-38473-E-19
27.0(0.75)
V-38473-E-20
13.9(0.70)
Minimum: 13.9
(0.35)
Maximum: 88.1
11.0
Average: 31.1
2.54
Median: 21.1
(0.89)
90th Percentile: 57.5
6.21

TZMU
ppbb
43.3
4.57
1.70
(0.84)(0.72)(0.50)(0.50)
43.3
8.61
1.27
23.9

Average

16.5(0.69)

2016
DE

V-38473-D-36
250
V-38473-D-37
V-38473-D-38
V-38473-E-36
251
V-38473-E-37
V-38473-E-38
Minimum:
Maximum:
Average:
Median:90th Percentile:

1.38(0.75)
1.04
9.34
2.19
(0.35)(0.35)
9.34
2.51
1.21
5.77

1.06

3.96

a

<0.25
<0.25
<0.25
(0.45)(0.32)(0.31)<0.25(0.45)<0.25<0.25(0.38)<0.25(0.36)

<0.25
<0.25
<0.25
(0.28)
<0.25
<0.25
<0.25
(0.28)
<0.25
<0.25
<0.25

<0.25

<0.25

DALA= days after last application
Values in parenthesis are between the LOQ and the LOD
In calculating the average concentration and descriptive statistics, values below the LOD are substituted
with half of the LOD value
LOD= 0.25 ppb and LOQ= 1.00 ppb for clothianidin, TZNG and TZMU

b

17
841

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50154302

CDPR Cloth Almond

Table 9. Clothianidin, TZNG and TZMU Residues in Almond Leaves
Trial
Sample ID
DALAa Clothianidin
TZNG
ppbb
Average ppbb
Average2015
V-38473-A-21(3.01)<1.3
A
166(3.09)<1.3
V-38473-A-22(3.08)<1.3
V-38473-A-23(3.18)<1.3
V-38473-B-21
<1.3
<1.3
B
172
5.62
(1.55)
V-38473-B-22(3.64)<1.3
V-38473-B-23
12.6(3.35)
V-38473-C-21
5.57
<1.3
C
224
10.1
<1.3
V-38473-C-22
9.39
<1.3
V-38473-C-23
15.4
<1.3
V-38473-D-21
<1.3
<1.3
D
279
<1.3
<1.3
V-38473-D-22
<1.3
<1.3
V-38473-D-23
<1.3
<1.3
V-38473-E-21
10.0
5.64
E
281
8.43
(4.36)
V-38473-E-22
8.29(3.72)
V-38473-E-23
6.97(3.73)
V-38473-F-21
<1.3
<1.3
F
211
<1.3
<1.3
V-38473-F-22
<1.3
<1.3
V-38473-F-23
<1.3
<1.3
V-38473-G-21(2.41)<2.61)
G156(2.61)(2.30)
V-38473-G-22(2.01)(2.01)
V-38473-G-23(3.39)(2.28)
V-38473-H-21
<1.3
<1.3
H
240
(1.15)<1.3
V-38473-H-22(2.15)<1.3
V-38473-H-23
<1.3
<1.3
V-38473-I-21(2.16)<1.3
I
240(1.43)<1.3
V-38473-I-22(1.47)<1.3
V-38473-I-23
<1.3
<1.3
Minimum: <1.3
<1.3
Maximum: 15.4
5.64
Average: (3.75)(1.35)Median: (2.16)<1.3
th
90 Percentile: 9.64
(3.50)

2016
A

B

V-38473-A-39
V-38473-A-40
V-38473-A-41
V-38473-B-39
V-38473-B-40
V-38473-B-41

215

205

(3.99)(3.94)
5.02<1.3(1.38)
5.12(4.32)(2.38)

18
842

<1.3
<1.3
<1.3
<1.3
<1.3
(1.95)<1.3(1.08)

TZMU
ppbb(1.94)(1.56)(1.48)
<1.3
<1.3
<1.3
(1.38)(1.54)(1.65)
<1.3
<1.3
<1.3
<1.3
<1.3
<1.3
<1.3
<1.3
<1.3
<1.3
<1.3
<1.3
<1.3
<1.3
<1.3
<1.3
<1.3
<1.3
<1.3
(1.94)<1.3<1.3(1.55)(1.45)(1.49)
<1.3
<1.3
<1.3
<1.3

Average(1.66)<1.3(1.53)

<1.3

<1.3

<1.3

<1.3

<1.3

<1.3

(1.20)<1.3

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50154302

Trial

C

D

E

F

G

H

I

Sample ID

DALAa

V-38473-C-39
234
V-38473-C-40
V-38473-C-41
V-38473-D-39
265
V-38473-D-40
V-38473-D-41
V-38473-E-39
265
V-38473-E-40
V-38473-E-41
V-38473-F-39
204
V-38473-F-40
V-38473-F-41
V-38473-G-39
220
V-38473-G-40
V-38473-G-41
V-38473-H-39
283
V-38473-H-40
V-38473-H-41
V-38473-I-39
278
V-38473-I-40
V-38473-I-41
Minimum:
Maximum:
Average:
Median:90th Percentile:

Clothianidin
ppbb
Average(1.58)(1.81)(2.04)(1.82)<1.3(1.26)(1.56)(1.58)(3.72)(4.71)
5.94(4.45)(3.43)(2.68)(2.82)(1.79)(4.30)(4.49)(3.87)
5.31(2.62)(3.55)(2.67)
5.35(3.72)
5.10(4.62)
6.96
<1.3
6.96
(3.37)(3.72)
5.33

a

TZNG
ppbb
<1.3
<1.3
<1.3
<1.3
<1.3
<1.3
(2.83)(3.52)(3.67)(1.79)(2.08)(1.54)(2.77)(2.89)(3.20)
<1.3
<1.3
<1.3
<1.3
<1.3
<1.3
<1.3
(3.67)(1.38)<1.3(3.01)CDPR Cloth Almond

Average<1.3<1.3(3.34)(1.80)(2.95)

<1.3

<1.3

TZMU
ppbb
<1.3
<1.3
<1.3
<1.3
<1.3
<1.3
<1.3
<1.3
<1.3
<1.3
<1.3
<1.3
<1.3
<1.3
<1.3
<1.3
<1.3
<1.3
<1.3
<1.3
<1.3
<1.3
(1.49)
<1.3
<1.3
<1.3

Average
<1.3

<1.3

<1.3

<1.3

<1.3

<1.3

<1.3

DALA= days after last application
Values in parenthesis are between the LOQ and the LOD
In calculating the average concentration and descriptive statistics, values below the LOD are substituted
with half of the LOD value
LOD= 1.3 ppb and LOQ= 5.0 ppb for clothianidin, TZNG and TZMU

b

19
843

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50154302

CDPR Cloth Almond

Table 10. Clothianidin, TZNG and TZMU Residues in Soil Collected during Bloom
Trial
Sample ID
DALAa Clothianidin
TZNG
TZMU
ppbb
Average ppbb
Average ppbb
2015
V-38473-A-9
32.8
<1.3
<1.3
A
139
45.0
<1.3
V-38473-A-10
38.3
<1.3
<1.3
V-38473-A-11
64.0
<1.3
(1.48)
V-38473-B-9
20.1
<1.3
<1.3
B
144
19.0
<1.3
V-38473-B-10
17.0
<1.3
<1.3
V-38473-B-11
19.8
<1.3
<1.3
V-38473-C-9
6.15
<1.3
<1.3
C
212
7.04
<1.3
V-38473-C-10
10.4
<1.3
<1.3
V-38473-C-11(4.59)<1.3<1.3
V-38473-D-9
6.88
<1.3
<1.3
D
250
11.5
<1.3
V-38473-D-10
16.3
<1.3
<1.3
V-38473-D-11
11.3
<1.3
<1.3
V-38473-E-9(3.90)
<1.3
<1.3
E
252
11.7
(1.09)
V-38473-E-10
25.8(1.97)<1.3
V-38473-E-11
5.49
<1.3
<1.3
V-38473-F-9
6.74
<1.3
<1.3
F
199
(4.75)<1.3
V-38473-F-10(2.60)<1.3<1.3
V-38473-F-11(4.93)<1.3<1.3
V-38473-G-9
10.6
<1.3
<1.3
G
146
8.40
<1.3
V-38473-G-10
6.96
<1.3
<1.3
V-38473-G-11
7.61
<1.3
<1.3
V-38473-H-9
6.78
<1.3
<1.3
H
209
6.81
<1.3
V-38473-H-10
8.27
<1.3
<1.3
V-38473-H-11
5.37
<1.3
<1.3
V-38473-I-9
5.02
<1.3
<1.3
I
218
10.3
<1.3
V-38473-I-10
11.8
<1.3
<1.3
V-38473-I-11
14.0
<1.3
<1.3
Minimum: (2.60)
<1.3
<1.3
Maximum: 64.0
(1.97)(1.48)cc
Average: 13.8ccMedian: 8.27
cc
90th Percentile: 28.6

Average(0.93)

<1.3

<1.3

<1.3

<1.3

<1.3

<1.3

<1.3

<1.3

2016
A

B

V-38473-A-27
V-38473-A-28
V-38473-A-29
V-38473-B-27
V-38473-B-28
V-38473-B-29

188

181

44.0
42.7
49.9
9.65
10.8
12.5

45.5

11.0

20
844

<1.3
<1.3
<1.3
<1.3
<1.3
<1.3

<1.3

<1.3

(1.78)<1.3(1.50)
<1.3
<1.3
<1.3

(1.31)<1.3

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50154302

Trial

C

D

E

F

G

H

I

Sample ID

DALAa

V-38473-C-27A
218
V-38473-C-28B
V-38473-C-29C
V-38473-D-27
250
V-38473-D-28
V-38473-D-29
V-38473-E-27
251
V-38473-E-28
V-38473-E-29
V-38473-F-27
195
V-38473-F-28
V-38473-F-29
V-38473-G-27
207
V-38473-G-28
V-38473-G-29
V-38473-H-27
250
V-38473-H-28
V-38473-H-29
V-38473-I-27
250
V-38473-I-28
V-38473-I-29
Minimum:
Maximum:
Average:
Median:90th Percentile:

Clothianidin
ppbb
Average
7.86(4.87)(3.55)(3.21)(4.76)
6.48
9.53
5.16
(3.43)
5.45(4.16)
8.74(4.43)
5.90
8.47(4.78)
9.13
11.6
10.0
15.6
22.2
27.1
28.3
30.9
16.7
25.9
23.3
37.8
(3.21)
49.9
16.0
9.65
39.8

a

TZNG
ppbb
<1.3
<1.3
<1.3
<1.3
<1.3
<1.3
<1.3
<1.3
<1.3
<1.3
<1.3
<1.3
<1.3
<1.3
<1.3
<1.3
<1.3
<1.3
<1.3
<1.3
<1.3
<1.3
<1.3
c

CDPR Cloth Almond

Average
<1.3

<1.3

<1.3

<1.3

<1.3

<1.3

<1.3

TZMU
ppbb
<1.3
<1.3
<1.3
<1.3
<1.3
<1.3
<1.3
<1.3
<1.3
<1.3
<1.3
<1.3
<1.3
<1.3
<1.3
<1.3
<1.3
<1.3
<1.3
<1.3
<1.3
<1.3
(1.78)

Average
<1.3

<1.3

<1.3

<1.3

<1.3

<1.3

<1.3

c

c

c

c

c

DALA= days after last application
Values in parenthesis are between the LOQ and the LOD
cDescriptive statistics were not calculated because >50% of the results are below the LOD
Reported concentration is based on dry weight
In calculating average concentration and descriptive statistics, values below the LOD are substituted
with half of the LOD value
LOD= 1.3 ppb and LOQ= 5.0 ppb for clothianidin, TZNG and TZMU
b

21
845

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50154302

CDPR Cloth Almond

Table 11. Clothianidin, TZNG and TZMU Residues in Soil Collected after Applications 2 and 4
Trial
Sample ID
DALAa Clothianidin
TZNG
TZMU
ppbb
Average ppbb
Average ppbb
Average2014
V-38473-A-6
39.5
<1.3
<1.3
A
0
34.0
<1.3
<1.3
V-38473-A-7
42.2
<1.3
<1.3
V-38473-A-8
20.3
<1.3
<1.3
V-38473-B-6
32.1
<1.3
<1.3
B
0
29.6
<1.3
<1.3
V-38473-B-7
26.9
<1.3
<1.3
V-38473-B-8
29.7
<1.3
<1.3
V-38473-C-6
20.5
<1.3
<1.3
C
0
19.5
<1.3
<1.3
V-38473-C-7
20.2
<1.3
<1.3
V-38473-C-8
17.7
<1.3
<1.3
V-38473-F-6
11.0
<1.3
<1.3
F
0
10.4
<1.3
<1.3
V-38473-F-7
14.5
<1.3
<1.3
V-38473-F-8
5.79
<1.3
<1.3
V-38473-G-6
20.7
<1.3
<1.3
G
0
15.0
<1.3
<1.3
V-38473-G-7
9.23
<1.3
<1.3
V-38473-H-6
41.5
<1.3
<1.3
H
0
28.0
<1.3
<1.3
V-38473-H-7
21.6
<1.3
<1.3
V-38473-H-8
21.0
<1.3
<1.3
V-38473-I-6
21.1
<1.3
<1.3
I
0
19.7
<1.3
<1.3
V-38473-I-7
17.0
<1.3
<1.3
V-38473-I-8
21.1
<1.3
<1.3
Minimum: 5.79
<1.3
<1.3
Maximum: 42.2
<1.3
<1.3
c
c
Average: 22.7ccMedian: 20.8cc90th Percentile: 39.7

2015
A

B

C

D

V-38473-A-24
V-38473-A-25
V-38473-A-26
V-38473-B-24
V-38473-B-25
V-38473-B-26
V-38473-C-24
V-38473-C-25
V-38473-C-26
V-38473-D-24
V-38473-D-25
V-38473-D-26

0

0

0

0

91.0
86.3
99.8
58.8
59.7
57.2
58.2
51.3
96.5
14.7
20.4
8.09

(1.50)
<1.3
<1.3
<1.3
<1.3
<1.3
<1.3
<1.3
<1.3
<1.3
<1.3
<1.3
22
846

(0.93)

<1.3

<1.3

<1.3

(3.40)(2.04)(2.13)
<1.3
<1.3
<1.3
<1.3
<1.3
<1.3
<1.3
<1.3
<1.3

(2.52)

<1.3

<1.3

<1.3

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50154302

Trial

E

F

G

H

I

Sample ID

DALAa

V-38473-E-24
0V-38473-E-25
V-38473-E-26
V-38473-F-24
0V-38473-F-25
V-38473-F-26
V-38473-G-24
0V-38473-G-25
V-38473-G-26
V-38473-H-24
0
V-38473-H-25
V-38473-H-26
V-38473-I-24
0
V-38473-I-25
V-38473-I-26
Minimum:
Maximum:
Average:
Median:th
90 Percentile:

Clothianidin
ppbb
Average(4.36)
11.3(2.69)
15.8
16.8
16.1
24.3
36.1
20.1
23.2
19.6
19.8
54.6
65.2
70.5
(2.69)
99.8
40.8
24.3
88.2

a

TZNG
ppbb
<1.3
<1.3
<1.3
<1.3
<1.3
<1.3
<1.3
<1.3
<1.3
<1.3
<1.3
<1.3
<1.3
<1.3
<1.3
<1.3
(1.50)c

CDPR Cloth Almond

Average
<1.3

<1.3

<1.3

<1.3

<1.3

TZMU
ppbb
<1.3
<1.3
<1.3
<1.3
<1.3
<1.3
<1.3
<1.3
<1.3
<1.3
<1.3
<1.3
<1.3
<1.3
<1.3
<1.3
(3.40)

Average
<1.3

<1.3

<1.3

<1.3

<1.3

c

c

c

c

c

DALA= days after last application
Values in parenthesis are between the LOQ and the LOD
cDescriptive statistics were not calculated because >50% of the results are below the LOD
Reported concentration is based on dry weight
In calculating average concentration and descriptive statistics, values below the LOD are substituted
with half of the LOD value
LOD= 1.3 ppb and LOQ= 5.0 ppb for clothianidin, TZNG and TZMU
b

23
847

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50154302

CDPR Cloth Almond

Table 12. Summary Statistics for Clothianidin Residues in Almond Nectar, Pollen, Anthers, Leaves, andSoil Collected at Bloom
Minimum Maximum Mean±SD
Median
90th
(ppb)
(ppb)
(ppb)
(ppb)
Percentile
Matrix
Year
N
(ppb)
a
a
a
2015
27
<0.20
1.28
a
a
a
Nectar
2016
27
<0.20
2.04
Combined
54
<0.20
2.04
(0.31)±0.38<0.20(0.81)
2015
20(0.77)
20.0
6.52±5.45
4.42
13.4
Pollen2016
21(0.55)
13.8
4.77±3.79
4.32
10.4
Combined41(0.55)
20.0
5.62±4.70
4.32
12.7
2015
6
13.9
88.1
31.1±28.3
21.1
57.5
Anthers2016
6(0.35)
9.34
2.51±3.41
1.21
5.77
Combined
12(0.35)
88.1
16.8±24.3
11.6
26.6
2015
27
<1.30
15.4
(3.75)±4.07(2.16)
9.64
Leavesb
2016
27
<1.30
6.96
(3.37)±1.69(3.72)
5.33
Combined
54
<1.30
15.4
(3.56)±3.09(2.91)
6.97
a
a
a
2015
27
(2.60)
64.0
a
a
a
Soil
2016
27
(3.21)
49.9
aaa
Combined54(2.60)
64.0
aDescriptive statistics were not calculated because >50% of the results are below the LOD
bAlmond leaves were not present at bloom; they were collected later ca. 1 month after bloom
Values in parenthesis are between the LOQ and the LOD
In calculating the average concentration and descriptive statistics, values below the LOD are substituted
with half of the LOD value.
For nectar, LOD= 0.20 ppb and LOQ= 1.00 ppb for clothianidin, TZNG, and TZMU
For pollen and anthers, LOD= 0.25 ppb and LOQ= 1.00 ppb for clothianidin, TZNG, and TZMU
For leaves and soil, LOD= 1.3 ppb and LOQ= 5.0 ppb for clothianidin, TZNG, and TZMU

24
848

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50154302

CDPR Cloth Almond

8. STATISTICAL ANALYSIS
Study Objectives and Design
The study was conducted to determine the concentration of clothianidin and its metabolites TZNG, and
TZMU in anthers, nectar, leaves, and pollen of almond trees in response to a previous year’s foliar
applications of a clothianidin pesticide product. Two applications of Belay were made post bloom where
the first foliar application was made at an application rate of 0.1 lb a.i./Acre (BBCH growth stage 7.5,
fruits at half size), and the second foliar application of Belay Insecticide was made at an application rateof 0.1 lb a.i./Acre at 21 days before harvest. In the second year, flower parts and leaves were harvested
and analyzed for clothianidin and its degradation products. The crops received a second set of foliar
treatments after bloom in the second year and the same sampling scheme was then followed at bloomin the third year. Soil samples were taken after the second application in each year and again whensamples were taken the next year at bloom.
Non-parametric statistical tests were used to test for differences in distribution of concentrations
between years and between soil type. Non-parametric tests do not require tests for normality as they
are robust to differences in distribution and experimental designs with low replicates (Helsel and Hirsch,2002). The PROC NPAR1WAY procedure in the Statistical Analysis System (SAS) statistical package wasused to conduct Wilcoxon-Mann –Whitney (Wilcoxon), Median non-parametric, and Kuiper tests. A
significant result from the Wilcoxon test indicates differences in the shape of distributions. A significant
result from the Median test indicates differences in the location of the medians between distributions;and a significant result from the Kuiper test indicates differences in the empirical distributions between
two groups. The Exact option for each statistic was implemented as it provides permutation testing, astatistical method that minimizes the effect of sample size and distributional differences. Using the Exact
option, the Monte Carlo procedure was also implemented that provided 10,000 separate runs for eachstatistic to produce the permutation distributions. The test for potential differences due to soil type had
3 levels so the DSCF option in PROC NPAR1WAY, which invokes the Dwass, Steel, Critchlow-Fligner
multiple comparison test was used to provide pairwise tests for two-sample rankings. Additional
procedures used for descriptive statistics were PROC MEANS to calculate mean values from thereplicates at each site, PROC CAPACITY to produce cumulative statistics, and PROC BOX plot to produce
comparative graphics. Statistical analysis for effect of years and soil type were conducted on the meanof the replicate samples taken from each site. Graphical comparisons are presented on datatransformed to a natural logarithm scale, providing clearer contrasts between the distributions. Values
indicated as less than the limit of detection (LOD) were assigned ½ their respective LOD value (Table 5).
Values were reported between the limit of detection and limit of quantification (LOQ) in parentheses sothese were used as reported. For determination of the potential distribution of concentrations in bee
relevant plant matrices, the distribution of the raw data is presented as these values represent theactual range of exposure to bees and other organisms that feed off the nectar and pollen of plants.
Detection rate noted for each plant matrix: Counts for the number of samples reported below
respective detection limits for each matrix are presented in Table 13. Parent clothianidin residues wereabove the LOD for all samples except for nectar where 55% of the samples were below the LOD. Except
for pollen samples, the majority of the other analyses for degradation products were reported below
the LOD. Statistical analyses were not conducted for plant and soil matrices where the majority of
results were reported below the LOD. In addition, since summation of the residues would be highly

25
849

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50154302

CDPR Cloth Almondbiased due to the large amount of substitution, statistical analyses were not conducted on total
residues.

Comparison of distribution between years: Potential difference between years was measured for two
reasons. First, greater concentrations measured in year 2 would indicate potential for carry-over effects
between years. Second, if there was no effect of years then the data could be pooled for subsequent
tests between soil types. The result for analyses conducted on plant matrices with sufficient detections
indicated no significant difference in the distribution of concentration of residues between years, asbased on exact probability levels for a two-sided test (Table 14). Graphical comparisons between years
indicated significant overlap in the distribution of concentration for clothianidin residues between years
in the plant samples taken at bloom (Figure 1) and for soil samples (Figure 2). The data for anthers
indicated a higher distribution for the first year of the study, but samples were only taken from a subsetof two sites in each year so this difference could be due to the low number of replicates. The result for
clothianidin residues indicated that the data for both years can be combined in further tests forcomparison of effect of soil type.
Comparison of distribution between soil types: Based on the soil characteristics provided in the Table
embedded in section ‘3.2 Test Sites’ in the report, the sites were classified as: coarse-textured sites are
B, C, and E; medium-textured sites are D, G, and I; and moderately fine-textured sites are A, F, and H.
Although the soil description at sites A,F, and H were not strictly indicated as fine-textured, theirclassification into a moderately fine-textured classification provided for potential distinction within themedium-textured classification. These categories are based on the USDA classification of soils (Soil
Science Division Staff, 2017, see Table 3.1). Results of the non-parametric test indicated no difference inthe distribution between the three soil types for parent clothianidin (Table 15). Graphic comparisonsindicated significant overlap in the distributions for all sampled matrices (Figures 3 and 4).
Data for bee relevant matrices: The distribution derived from the individual analyses ostensibly
determines the expected range in concentrations of clothianidin and TZNG, and TZMU degradation
product residues in bee relevant plant samples for this combination of plant species and application
scenario (Table 16). Also, although the number of samples noted below the LOD was problematic for
conducting meaningful statistical tests, the presence of parent clothianidin indicated a potential for
degradation products to be present. Therefore, total residues were calculated as the addition of allresidues with results indicated as <LOD set at one-half the respective LOD for each matrix. For nectar,
most concentrations were below the LOD so the total residue concentration was low with a maximum
value estimated at 2.2 ng/g and the median value at 0.3 ng/g. Concentrations in pollen were higher
where the maximum total residue value was estimated at 34.1 ng/g and the median value at 6.0 ng/g.
The number of samples taken for anthers was small since only two of the plots were monitored in eachyear. The distribution for total residue concentration appeared to be higher than measured for pollen
with the maximum value at 94.1 ng/g and the median value at 12.6 ng/g.

26
850

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50154302

CDPR Cloth AlmondLongevity of residues in soil: The distribution of soil concentration measured after the second foliar
application in each year and then at bloom the next year is presented in Table 17. The mean soil
concentration at bloom was 14.9 ng/g compared to 32.7 ng/g after application. The length of time
between the last foliar application and sampling at bloom in the next year varied at each site, ranging
from 139 to 252 days. Estimates of the dissipation half-life can be calculated from the initialconcentration measured after application and at bloom according to Equation 1:
Equation 1

Half-life (days) = ln(2) / ((ln(Co)-ln(Cn)/DALA)In Equation 1, Co is the concentration at application, Cn is the concentration at bloom, and DALA is thedays after the last application. Basing the calculation on the mean values in Table 17, Co is 32.7 and Cn is14.9 ng/g. At an average DALA of 208 days, the terrestrial field dissipation half-life estimate is 162 days.
This is a comparatively large value with respect to dissipation of pesticide residues in soil and indicatesthat the residues are long-lived in the soil.
Conclusions
1. Utility of the data: The study followed the design as indicted in the data call-in where the study was
replicated in two years at 9 sites. The 9 sites appeared to be evenly distributed amongst the soil types
with 3 replicates in coarse, medium, and fine- textured soils.
2. Concentrations in Bee Relevant Matrices: By default, the distributions reported in Table 16 represent
the expected concentrations in bee relevant matrices that result from foliar clothianidin treatments
applied to almond trees in the previous growing season. Median and maximum values for total
clothianidin residues in pollen are 6.0 and 34.1 ng/g on wet weight basis and 0.3 and 2.2 ng/g for nectar,
respectively.
3. No carry-over effect between years: Concentrations measured in plant matrices were similar
between the two years of the study, indicating low potential for carry-over effects due to sequential
foliar applications at the concentrations and timing used in this study.
4. No effect of Soil Type: Concentrations in plant matrices at bloom were similar between plants grownin the 3 soil types, indicating that foliar sprays produce similar results regardless of the soil condition.
5. Residues are long-lived in soil: The estimated dissipation half-life for clothianidin in soil was 162 days,a value that indicates slow dissipation in the soil environment.
9. STUDY STRENGTHS, LIMITATIONS AND CONCLUSIONSIn the context of documenting the magnitude of clothianidin residues in bee-related matrices of almondtrees, the following strengths are observed with this study.
1. Data provide quantitative values of total clothianidin residues expected in pollen, nectar, andleaves of almond trees when measured at bloom in response to foliar applications made in theprevious growing season.

27
851

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50154302

CDPR Cloth Almond

2. The study was replicated over two years with measurements in plant samples taken at a meanof 197 days after the last application in the first year of the study and at a mean of 221 days
after the last application in the second year of the study.

3. The 9 sites were evenly replicated over the requested 3 soil texture categories that reflectedstratification between coarse, medium, and moderately fine-textured classifications.
Limitations noted in this study include:
1. In this study, the treatment applications were not conducted according to the “worst case”
scenario permitted by the product label. The product label allows foliar applications to be madeafter bloom and up to 21 days before harvest, with a minimum reapplication interval of 10 days.
Thus, the “worst case” application schedule permitted by the label would have been one
application at 31 days before harvest and a second application at 21 days before harvest.
However, in this study, plots received two applications anywhere from one to six months apart,and some plots received the second application at 32 or 44 days before harvest. In addition,some applications were conducted at harvest or after harvest which is not permitted by the
product label. It is unclear how these deviations from the “worst case” application schedule
permitted by the label may have affected the residues measured in bee-relevant matrices thenext season.
Classification/Utility for Bee Risk Assessment. This study is classified as acceptable. It provides asnapshot of Clothianidin, TZNG and TZMU residues in nectar, pollen, and leaves collected from almondtrees treated with Belay Insecticide (active ingredient clothianidin) over two years. The residue values
presented should be considered to be fully reliable.
Temporal Variability in Residues. This study was not designed for temporal analysis of declining
concentrations, but rather, to provide a snapshot of residue concentrations during flowering. Samples
were collected at only one time point per year and so there is no way to know if concentrations wereincreasing or decreasing.
Overall, considering the strengths and limitations of this study, the following conclusions can be drawn:
Bee-relevant matrices: Clothianidin residues were measured in pollen sampled in the year following
applications to plants of two foliar sprays made in the previous year. Most of the analyses for nectar
indicated concentrations below the LOD of 0.1 ng/g on a wet weight basis. Therefore, exposure to bees
from nectar is minimal; whereas, exposure to pollen requires comparison of the measured distribution
to target values that define acute or chronic exposure scenarios.
Spatial Variability in Residues. Concentrations in plant matrices at bloom were similar between plants
grown in the 3 soil types, indicating that foliar sprays produce similar results regardless of the soil
condition.
No carry-over effects of years: Concentrations measured in plant matrices were similar between the
two years of the study indicating low potential for carry-over effects due to sequential foliar applications
at the concentrations and timing of applications used in this study.

28852

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50154302

CDPR Cloth Almond

10. STUDY VALIDITY/CLASSIFICATION
The data from this study provide an expected distribution of the concentrations of clothianidin residues
that bees are exposed to in nectar and pollen of almond trees under actual agronomic practices in
California. Relating concentrations measured in flower parts to bee health is possible by comparing theconcentrations measured in bee relevant plant parts to target values that define acute or chronic
exposure scenarios. The study is considered scientifically sound and useful for risk assessment purposes.
The study is classified as ACCEPTABLE for quantitative use in risk assessment.

29853

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50154302

CDPR Cloth Almond

Table 13. Counts of chemical analytical results for clothianidin and TZNG, and TZMU degradation products comparing the total number ofsamples collected for each matrix and the number of analyses where the concentration was indicated below respective detection limits.

Comparison of Total Number of Samples Reported Above the LOQ, Between the LOQ and LOD,
and Below the LOD
Clothianidin

TZNG

TZMU

Total
NumberNumber>LOQ

Number<LOQ

Number<LOD

Total
NumberNumber>LOQ

Number<LOQ

Number<LOD

Total
NumberNumber>LOQ

Number<LOQ

Number
<LOD

47

45

2

0

47

0

2

45

47

0

3

44

54
54

43
13

11
29

0
12

54
54

0
1

1
16

53
37

54
54

0
0

3
8

51
46

NectarPollen

54
42

4
38

18
4

32
0

54
42

0
6

0
12

54
24

54
42

1
17

2
18

51
7

Anthers

12

10

2

0

12

3

6

3

12

3

4

5

Plant Sample
Soil: After
Application
Soil: At Bloom
Leaves30854Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50154302

CDPR Cloth Almond

Table 14. Effect of Year: Exact probability levels for Wilcoxon, Median, and Kuiper non-parametrictests for differences in the distribution of clothianidin concentrations between years. NA=Not
Analyzed.

Nonparametric Test Exact
Probability Levels: Effect ofYear
Clothianidin
SourcePollenNectarLeavesSoil at Application
Soil at Bloom

Wilcoxon
0.38
NA
0.65
0.47
1

Median
1
NA
1
1
1

31
855

Kuiper
0.73
NA
0.26
0.68
0.57

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50154302

CDPR Cloth Almond

Table 15. Effect of Soil Type: Exact probability levels for Wilcoxon and Median non-parametric tests
for differences in the distribution of clothianidin clothianidin and TZNG, and TZMU degradation
products between soil types. NA=Not Analyzed.

SourceLeavesNectarPollenSoil at Application
Soil at Bloom

Wilcoxon Nonparametric Test for
Effect of Soil: Exact Probability
Level
Clothianidin TZNG
TZMU
0.15
NA
NA
0.42
NA
NA
0.48
0.33
0.27
0.84
NA
NA
0.78
NA
NA

32
856

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50154302

CDPR Cloth Almond

Table 16. Distribution of clothianidin and TZNG and TZMU degradation products measured in nectar, pollen, and anthers sampled fromalmond trees that were exposed to two applications of clothianidin in the year previous to bloom. Samples were combined from twoconsecutive years of study.

StatisticN
Mean (ng/g)
SD (ng/g)
CV (%)Min (ng/g)
Median (ng/g)
75th (ng/g)
90th (ng/g)
95th (ng/g)
Max (ng/g)% of Total

Clothianidin
54
0.3
0.4
123
0.1
0.1
0.4
0.8
1.2
2.0
57.7

Nectar
TZNG
TZMU
54
54
0.1
0.1
0
0.06
0
55
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.2
0.1
0.5
19.2
19.2

Total
54
0.52
0.41
78
0.3
0.3
0.6
1.0
1.4
2.2

Plant Sample
Pollen
TZNG
TZMU
41
41
0.59
1.87
1.34
37.1
228
199
0.1
0.1
0.1
0.6
0.4
1.7
0.9
3.6
2.4
5.9
8.2
22.8
7.3
23.1

Clothianidin
41
5.62
4.7
83.6
0.6
4.3
8.3
12.7
13.8
20.0
69.6

33
857

Total
41
8.08
7.22
89
0.8
6.0
11.3
17.2
20.8
34.1

Clothianidin
12
16.8
24.4
145
0.4
11.6
21.2
27.0
88.1
88.1
74.3

Anthers
TZNG
TZMU
12
12
1.4
4.4
3.1
12.3
219
281
0.1
0.125
0.4
0.39
0.9
1.27
1.4
4.57
11.0
43.3
11.0
43.3
6.2
19.5

Total
12
22.6
31.1
138
0.8
12.6
24.9
77.4
94.1
94.1

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50154302

CDPR Cloth Almond

Table 17. Soil concentrations: Distribution of soil concentration of clothianidin for samples measured
directly after the second foliar application in each year and then at bloom in the following year.

StatisticN
Mean (ng/g)
SD (ng/g)
CV (%)Min (ng/g)
Median (ng/g)
Max (ng/g)

Clothianidin Soil
Concentration
At
Application At Bloom
16
18
32.7
14.9
24.7
12.9
75.4
86.4
6.1
4.8
23.9
10.6
92.4
45.5

34
858

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50154302

CDPR Cloth Almond

Figure 1. Year Comparison for plant samples: Comparison of the distribution of clothianidin residues
expressed as natural logarithms between a sequential study replicated over two years. Yearlycomparisons are made for leaf, nectar, pollen, and anther samples taken at bloom when sampled in
the year following respective foliar applications.

35859

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50154302

CDPR Cloth Almond

Figure 2. Year comparison for soil samples: Comparison of the distribution of clothianidin residues insoil samples expressed as natural logarithms between a sequential study replicated over two years. In
the graphic ‘At App’ indicates samples taken directly after the second foliar application and ‘At Bloom’
indicates samples taken in the next year when trees were blooming.

36860

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50154302

CDPR Cloth Almond

Figure 3. Comparison between soil type: Comparison of the distribution of clothianidin residues
expressed as natural logarithms between a sequential study replicated over two years. Yearlycomparisons are made for leaf, nectar, pollen, and anther samples taken at bloom when sampled in
the year following respective foliar applications.

37861

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50154302

CDPR Cloth Almond

Table 4. Comparison between sequential replicate studies of the distribution of clothianidin residues
measured between plants grown in 3 different soil types for soil samples taken either after full
treatment application or at bloom in the year following respective foliar applications. Soil-App
indicates samples taken directly after the second foliar application and Soil-Bloom indicates samples
taken in the next year when trees were blooming.

38862

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50154302

CDPR Cloth Almond

8. REFERENCES:
1. Bondarenko, S. Freezer Storage Stability of Clothianidin, TZNG, and TZMU in Surrogate Nectar.
Study Number VP-38484; Valent U.S.A. Corporation. Ongoing.
2. Bondarenko, S. Freezer Storage Stability of Clothianidin, TZNG, and TZMU in Corn Pollen. Study
Number VP-38489; Valent U.S.A. Corporation. Ongoing.
3. Duah, F.K. TI-435 600 FS – Magnitude of the Residue in Field Rotational Crops. Bayer Report No.
109880. MRID 45422619; Bayer Corporation Agricultural Division: February 6, 2001.
4. Helsel, D.R. and R.M. Hirsch. 2002. Chapter A3: Statistical Methods in Water Resources.
Techniques of Water-Resources Investigations of the United States Geological Survey Book 4,
Hydrologic Analysis and Interpretation. United States Geological Survey.
5. Schramel, O. Determination of the Storage Stability of TI-435 and of the Metabolites TZNG,
TZMU, TMG and MNG in Soil – Interim Report. Bayer Report No. P 64190023. MRID 45422612;
Bayer AG, Agrochemical Division: October 25, 2000.
6. Soil Science Division Staff. 2017. Soil survey manual. C. Ditzler, K. Scheffe, and H.C. Monger(eds.). USDA Handbook 18. Government Printing Office, Washington, D.C.
7. Stewart, E. Clothianidin Residues on Head Lettuce from Trials Conducted in the United States in2007. Study Number SARS-07-74. MRID 47471609; Stewart Agricultural Research Services, Inc.:
January 16, 2008.

39863

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49733302

CDPR Clothianidin Foliar Cotton Study

Reference
Rose, A. (2015) Clothianidin: Quantitation of Residues of Clothianidin in Leaves and Clothianidin, TZNG, and
TZMU in Extrafloral Nectars, Floral Nectar, and Pollen from Cotton Plants: Final Report. Project Number: VP38259. Unpublished study prepared by Valent U.S.A. Corporation and California Agricultural Research Inc.
559p. MRID 49733302, CDPR Study ID 287359, Data Volume 52884-0251, Tracking ID# 272757

1. STUDY INFORMATION
Chemical:

Clothianidin

Test Material:

Belay Insecticide

Study Type:

Non-Guideline field residue study on cotton plants to establish clothianidin andmetabolite levels in extrafloral nectars, floral nectar, pollen and leaves in site
locations that were treated with two foliar applications.

Sponsor:Valent U.S.A. Corporation
1600 Riviera Ave., Suite 200
Walnut Creek, U.S.A. 94596

Sponsor Study
ID:
Study
Completion
Date:
GLP Status:

PC CodePercent Active
Ingredient:

44309
23.0%

Experiment Start andEnd Date:

May 29, 2012 –May 9, 2013

Two trial sites that
included Prima andAcala Cotton located inStudy Locations:Fresno and Kerman,
September 29, 2015
California.
All phases of study were conducted under Good Laboratory Practice; protocol
reviewed by CDPR.
[CDPR study ID 264408, Data Volume 52884-0173, Tracking ID# 252080]
VP-38259

2. REVIEWER INFORMATION
Study Reviewed by:Richard Bireley, Sr. Environmental Scientist (Specialist)
California
John Troiano, Ph.D., Research Scientist III
Department of
Alexander Kolosovich, Environmental Scientist
Pesticide Regulation
Brigitte Tafarella, Environmental Scientist
Russell Darling, Environmental Scientist
Denise Alder, Sr. Environmental Scientist (Specialist)

Study Reviewed by:United States
Environmental
Protection Agency

Michael Wagman, Biologist, EPA/EFED/ERB6
Amy Blankinship, Senior Scientist, EPA/EFED/ERB6

Date:
Date:

EPA Reviewer Comments: EPA considers the study to bescientifically sound and it is classified as Acceptable

3. EXECUTIVE SUMMARY
Two field trials were conducted to quantify the extent to which insect pollinators may be exposed toclothianidin and its degradates thiazolylnitroguanidine (TZNG) and thiazolylmethylurea (TZMU) following
two foliar applications of Belay® Insecticide to cotton. When applying Belay® Insecticide to field-grown

1864

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49733302

CDPR Clothianidin Foliar Cotton Study

cotton, leaf nectar, nectar and pollen from cotton flowers and concentrations of clothianidin in or on
leaves were measured.
Cotton (Gossypium hirsutum variety Acala - Site 1 or Gossypium barbadense variety Pima – Site 2) was
grown from seed at two field trial sites in California. The first foliar application of Belay® Insecticide(active ingredient, clothianidin) was applied approximately 7 days prior to floral bloom. The second
Belay® Insecticide application occurred at floral bloom, 7 days after the first application. Each spray
application of clothianidin was applied “over-the-top” at a rate of 0.1 lb. a.i./acre per application. No
products containing clothianidin or thiamethoxam had been applied to either trial site in the three years
prior to this study. Starting 21 days after planting and continuing until the second Belay® Insecticide
application, treatment plots were surveyed for the presence of honey bees and, if present, their
numbers quantified. In addition, plants were surveyed for the presence of main stem leaf nectar.
Leaves for residue analysis were collected from day 1 to day 28 after the last Belay® Insecticide
application. Main stem leaves were collected from the upper portion of the plant where flowers werecollected and received Belay® Insecticide applications. Leaf nectar was collected from 3 days after the
first Belay® Insecticide application until 28 days after the second application.
First day open flowers were harvested and transported into the laboratory where they wereimmediately processed into subbracteal nectar, floral nectar and pollen. Subbracteal nectar wascollected from Acala cotton beginning 5 days after the last Belay® Insecticide application but could notcollect any subbracteal nectar from Pima cotton. Floral nectar and pollen was collected from the same
flowers used to collect subbracteal nectar.

4. STUDY VALIDITY

Guideline Followed:
Non-guideline study (protocol was reviewed by CDPR)
Guideline Deviations: N/A
Other Deviations:
No deviations were made during the analytical portion. Protocol wasamended.
Classification:
ACCEPTABLE
Rationale:
N/A
Reparability:
N/A

5. MATERIALS AND METHODS
Test Material Characterization
Test item:
Description:
Material Source:
CAS #:

Percent Active Ingredient:
Molecular Formula:
Molecular Weight:
Valent Lot Number:

Belay Insecticide
Soluble Concentrate (SC)Valent U.S.A. Corporation
210880-92-5

23.0% A.I.
C6H8ClN5O2S
205.68 g/mol
AS 2351a

5A. STUDY DESIGN
The experimental start date was May 29, 2012 and experimental end date was May 9, 2013 (last datacollection). The field sampling phase of the study was conducted by California Agricultural Research, Inc.

2865

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49733302

CDPR Clothianidin Foliar Cotton Study

Data was collected from two cotton trials conducted in outdoor plots owned by California Agricultural
Research, Inc. and located in Fresno and Kerman, California. The soil types for these two locations aredescribed as Sandy Loam and Loamy Sand. Treatment plot dimensions varied but, in general, wereabout 0.3 acres in area. Control plots were up to 0.3 acres in area. The site 1 control plot was about 200
feet from the treatment plot. This control plot was converted to a treatment plot (protocol amendment
2) and then subsequently abandoned (protocol amendment 3). The site 2 control plot was >200 feetfrom the treatment plot. Drip irrigation was installed in all plots. Bee hives were located about ¼ milefrom site 2 and adjacent (within about 100 feet) to the treatment plot at site 1.
5B. APPLICATION TIMING AND RATESBelay® Insecticide was applied to the foliage of the mature cotton plants using a tractor mounted boom
containing flat fan Teejets. Belay® Insecticide was applied twice at application rates of 0.1 lb. a.i./acre
and a spray interval of 7 days. The first foliar application of Belay® Insecticide (active ingredient,clothianidin) was applied approximately 7 days prior to floral bloom (about BBCH plant growth stage51). The second Belay® Insecticide application occurred at floral bloom (defined in this study as when
the site contained approximately 500 blossoms), 7 days after the first application.
Induce (0.25%, v/v), a non-ionic surfactant, was added to each tank mix prior to application. Key
application information is collated in Table 1:
Table 1. Application Method and Application Dates for Belay Insecticide on Cotton
Site Number
1
1
2
2

Application Method
Foliar- Over the Top
Foliar- Over the Top
Foliar- Over the Top
Foliar- Over the Top

Application Date
July 16, 2012
July 23, 2012
July, 13, 2012
July 20, 2012

Application Rate(lb. a.i./acre)
0.1
0.1
0.1
0.1

5C. STUDY SITE LOCATION AND CHARACTERISTICS
Both field sites were in Fresno County located in California, where surface soil samples were collectedfrom each treatment plot prior to planting. Agvise Laboratories (Northwood, ND) analysed soils for
textures and percent organic matter. The soil characterization data and field site location aresummarized in Table 2:
Table 2. Field Site Locations and Soil Series, Characterization and Organic Matter
Soil
Percent
Site
Field Site
GPS
Soil Series Characterization
Organic
NumberCountyCoordinates 1 & Texture (%Sand/Silt/Clay)
MatterRamona
N36.73709
1
FresnoSandy
56/29/15
1.2
W-119.87587
Loam
Hanford
N36.79409
Loamy
84/13/3
0.34
2
FresnoW-120.05673
Sand
1
Northwest Corner of field treatment plot

3866

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49733302

CDPR Clothianidin Foliar Cotton Study

5D. SAMPLE COLLECTION, HANDLING, PROCESSING
Leaf nectar was collected from 3 days after the first Belay® Insecticide application until 28 days after thesecond application. Leaf nectar was collected in the field on the same days that flowers were collected.
Leaf nectar was collected using hand-held glass microcapillary tubes. Main stem leaves near the top of
the plant were turned over to expose the single gland on the mid-rib of the leaf. If nectar was observed,
the microcapillary was brought into contact with the gland and the nectar extracted by capillary action.
Many leaves contained no nectar and those that did contained much less than 1 uL. Hundreds of leaves
were examined in order to collect the amount (about 0.1 g) required for chemical analysis. The
microcapillary was inserted into a tared, labeled glass vial and the leaf nectar expelled by closing thehole in the end of the bulb holder and gently squeezing the bulb. The vials were placed into a cooler
containing Blue Ice in the field and then the cooler was hand carried to the field laboratory. The vials
were placed into freezer storage on the day of collection. Due to extensive plant manipulation andhandling during field collection, it is likely that leaf nectar was contaminated. In addition, evaporation ofwater during sample collection could artificially increase leaf nectar residue concentrations.
Flowers were hand carried in coolers to the field laboratory where they were processed on the day they
were harvested from the field plot. Subbracteal nectar was collected by inserting a glass microcapillaryon or into the subbracteal nectary located on the outside of the flower at the base of the bract. Mostoften the nectary was dry and no nectar could be collected. Subbracteal nectar was not present at any
sampling time in Pima cotton (site 2). Subbracteal nectar extraction continued until the microcapillary
tube contained a reasonable amount of visible nectar. The microcapillary was then inserted into a tared,labeled vial and the nectar expelled by closing the hole in the end of the bulb holder and gently
squeezing the bulb. This process was continued with additional flowers until at least 0.1 g of subbracteal
nectar was collected. For at least two samplings (days 14 and 21), this amount was not feasible.
A microcapillary tube was used to collect floral and/or inner bracteal nectar from the same flowers fromwhich subbracteal nectar was collected. Occasionally, nonsubbracteal nectar extracted flowers were
processed. The microcapillary was inserted under the lip of the calyx and into the floral nectary. Floral
nectar was present in most of the flowers. The microcapillary was then inserted into a tared, labeled
glass vial and the nectar expelled by closing the hole in the end of the bulb holder and gently squeezing
the bulb.
Pollen was collected from the same flowers from which subbracteal and floral nectars were collected.
When less than the minimum amount was collected, pollen was collected from untouched flowers. The
petals were folded back to expose the pollen laden anthers. A plastic barrier pipette was connected to asmall diaphragm vacuum pump by flexible tygon tubing. The small end of the barrier pipette was cut toenlarge the opening. The barrier pipette was then brushed over the tips of the anthers and the pollen,
and likely dislodged parts of the anther, was retained within the pipette by the barrier. Pollen present at
the base of the stigma was, if possible, also vacuum collected. The barrier pipette was replaced with aclean one as needed. The pipettes were placed into a tared, labeled glass vial. Flowers were discarded
after pollen collection.
A total of 72 cotton floral nectar and extrafloral nectar samples, 29 cotton pollen samples, 30 cotton leaf
samples and 28 stability samples were analyzed for clothianidin and its metabolites residues.
Sample Storage.

4867

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49733302

CDPR Clothianidin Foliar Cotton Study

Leaves for residue analysis and flowers for processing were hand carried to the field laboratory incoolers with Blue Ice. Once in the laboratory, the leaf samples were placed into a freezer (temperature<0°F).
Pollen, nectar and leaf samples were stored in monitored freezers at the field sites pending shipment to
the Valent Technical Center. Samples were shipped frozen and upon arrival at the Valent TechnicalCenter samples were placed into a temperature monitored, walk-in freezer until analyzed. Pollen
samples were stored (sampling to extraction) for a maximum of 122 days, nectar samples for up to
56 days, and leaves for up to 193 days. Transit stability samples for pollen were stored up to 283 days
and for nectars were stored for up to 91 days prior to extraction for analysis.
The nectar stability samples were stored for up to 91 days, and pollen stability samples were stored up
to 283 days. Clothianidin residues have been shown to be stable on a variety of leafy vegetable crops
when stored frozen for up to 242 days1, therefore a storage stability study on cotton leaf tissues was notconducted with this study.
5E. ANALYTICAL METHODS
Analyses were conducted in a total of 16 sets. Each set included at least 6 standards for the calibration
curve, at least one untreated sample, two laboratory fortification samples, and 7-15 samples.
Instrument software was used to integrate the analyte and internal standard peak response (areaintegration) for each injection. Excel was used to calculate analyte concentrations in sample extracts
based on peak area ratios (area analyte/area internal standard) and the standard curve (second orderpolynomial). Excel was also used to calculate sample residue concentrations based on the extract
concentration, the extract volume, the dilution factor (if any) and the sample weight. Some samples
were collected before the second application of Belay® Insecticide. Although these samples wereanalyzed, only samples analyzed after the second pesticide application are presented in this report.
Leaf samples were extracted with methanol and water (40:60, v/v) acidified with 0.05% formic acid andanalyzed using an accurate mass UPLC/Q-TOF MS-MS after spiking with isotopically labeled internal
standard (d3-clothianidin) to compensate for matrix effect (method RM-39L-1). The method wasvalidated at 5 ppb and 50 ppb. Subsequently, the method was re-validated at 5,000 ppb and 25,000 ppb.
Frozen leaf samples were homogenized in the presence of Dry Ice. After sublimation of the Dry Ice, anaccurately weighed (about 2 g) leaf subsample was extracted in a mixture of acidified methanol andwater. The solids were allowed to settle and an aliquot of the mother liquor was filtered through a nylon
syringe filter. An aliquot of the filtered liquid was fortified with isotopically labeled clothianidin (internal
standard) and analyzed by accurate mass UPLC/ Q-TOF MS-MS. The LOD in this method was 2.5 ppb,
and the LOQ was 5.0 ppb for clothianidin.
Samples of cotton floral nectar or extrafloral nectar were dissolved in methanol/water (40:60, v/v)
acidified with 0.05% formic acid and analyzed by LC/MS-MS after spiking with isotopically labeledinternal standards (d3-clothianidin, 13C, 15N-TZNG, and d3-TZMU) to compensate for matrix effect(method RM-39N-1). The method was validated at 1 ppb and 10 ppb for each analyte before using it toanalyze study samples. Subsequently, the method was validated at higher levels (250 ppb and5,000 ppb) using artificial nectar. Nectar validation was conducted using artificial nectar that contained
11% sucrose, 40% fructose, and 49% glucose in water. Sugar content of the artificial nectar was 36%(36 ºBrix). Nectar samples were removed from freezer storage on the day of chemical analysis andallowed to warm to room temperature. An accurately weighed nectar subsample (about 0.1 g) was

5868Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49733302

CDPR Clothianidin Foliar Cotton Study

dissolved in a mixture of methanol and water, each containing 0.05% formic acid, fortified withisotopically labeled internal standards then an aliquot of each sample was filtered through a nylon
syringe filter, if particles were present, into an autosampler vial prior to analysis by HPLC/MS-MS. The
limit of detection (LOD) was 0.2 ppb, and the limit of quantitation (LOQ) was 1.0 ppb for clothianidin,
TZNG and TZMU in this method.
Samples of cotton pollen were extracted with acetonitrile and water followed by adding sodium chloride
and anhydrous magnesium sulfate salts. The acetonitrile extract was partitioned with n-hexane, andacetonitrile phase was collected and concentrated to dryness. Residues were re-dissolved inmethanol/water (40:60, v/v) acidified with 0.05% formic acid and analyzed by LC/MS-MS after spiking
with isotopically labeled internal standards (d3-clothianidin, 13C, 15N-TZNG, and d3-TZMU) tocompensate for matrix effect (method RM-39P-1). The method was validated at 1 ppb and 10 ppb foreach analyte before using it to analyze study samples. Using this method, the LOD was 0.25 ppb, and the
LOQ was 1.0 ppb for clothianidin, TZNG and TZMU.
Subsequently, the method was validated at higher levels (100 ppb and 500 ppb) using commerciallyavailable bee pollen. The barrier pipettes were removed from the storage vial and the cotton pollen
dislodged. The pollen was collected into a vial and homogenized by stirring. An accurately weighedpollen subsample (about 0.1 g) was removed and extracted in a mixture of acetonitrile and water.
Sodium chloride and magnesium sulfate was added to increase the ionic strength and effect a clean
phase separation during centrifugation. An aliquot of the organic phase (upper acetonitrile) wasremoved then partitioned against hexane (discarded). An aliquot of the acetonitrile solution wasconcentrated to dryness under a stream of nitrogen. Residues were dissolved in a mixture of acidified
methanol and water, filtered through a nylon syringe filter into an autosampler vial then fortified withisotopically labeled internal standards. Extracts were analyzed by HPLC/MS-MS.
5F. QUALITY ASSURANCE RESULTS
Six to eight different standard concentrations were injected within each analytical set. The
concentration (ng/mL) of clothianidin and its metabolites detected in sample extracts was interpolated
from the standard calibration curve. The LC/MS-MS system was calibrated for each set of samples byanalyzing these calibrating standard concentrations, with these standards interspersed within theanalytical sequence. A second-order polynomial fit (weighted relative to 1/concentration) was thencalculated from the concentrations and the instrument response of the calibration standards. To verify
performance, the percent difference between the actual concentration and the calculated concentrationfor each of the calibration standards (based on the curve) was also calculated. Each of the standards was
required to be within 15% of the theoretical concentration and the coefficient of determination (r2) of
the weighted polynomial calibration curve was required to be greater than or equal to 0.99. Minor
exceedance of these criteria for the calibration standards were accepted for the lowest standards insome cases, however the coefficient of determination (r2) of the weighted polynomial calibration curve
was always greater than or equal to 0.99.
The reproducibility of the LC/MS-MS system was verified by comparison of instrument responses
obtained from the repeated analysis of a reference standard (a continuing calibration standard)
analyzed with the study samples. The continuing calibration standards were interspersed within thesamples in the analytical sequence, and the analytical sequence began and ended with a continuingcalibration standard. For an analytical data set (injection sequence) to be acceptable, the coefficient of6869

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49733302

CDPR Clothianidin Foliar Cotton Studyvariation (CV) of these responses was required to be 10% or less. Minor exceedance of these criteria forthe continuing calibration standards were accepted in some cases.
One control sample and at least one fortified sample were analyzed with each set of study samples toverify method performance. Fortifications were made at 1.0 and 10.0 ppb for nectar and pollen samples,and 5.0 ppb and 50.0 ppb for leaves. For an analytical run to be acceptable, method recoveries wererequired to be between 70 and 120%. Generally, recoveries of the concurrent laboratory fortified
samples were within this range. In some cases, minor exceedance of these criteria was accepted.

6. RESULTS:
6.1 LEAF RESIDUESNo clothianidin was detected (LOD, 2.5 ppb) in cotton leaves collected prior to the first Belay®
Insecticide application or in control leaf samples. Although the same application equipment was used atboth field sites, mean clothianidin leaf concentrations were higher in Pima cotton (site 2) than in Acala
cotton (site 1). In both varieties, residues were highest immediately after the last application anddeclined thereafter. One day after the last application of Belay® Insecticide, clothianidin concentrations
were 11,100 ppb in Pima cotton and 9,820 ppb in Acala cotton. Twenty-eight days after the application,leaf residue concentrations declined to 57.5 ppb in Pima cotton and 25.7 ppb in Acala cotton.
6.2 NECTAR ANALYSES
Mean concentrations of clothianidin in Acala (site 1) leaf nectar were highest (3,390 ppb) 1 day after thelast application of Belay® Insecticide, declining to 14.6 ppb on day 28. Leaf nectar residue concentrationsof clothianidin were highest in Pima cotton 1 day after the last Belay® Insecticide application (210 ppb),
declining to 24.4 ppb on day 21. Only one replicate sample of Pima leaf nectar was collected on day 28
and the concentration of clothianidin in it were higher (57.0 ppb) than in the day 21 sample. Residue
concentrations of metabolites were lower than parent concentrations (except TZMU in Pima at day 21)
in both cotton varieties with TZMU concentrations higher than TZNG concentrations.
Clothianidin residues in subbracteal nectar were highest and approximately equal (about 620 ppb) in
Acala on days 5 and 7 after the last application of Belay® Insecticide. Clothianidin concentrations
declined thereafter and were 3.4 ppb at 28 days post application. Except for the last interval, TZMU
concentrations were higher than TZNG concentrations in subbracteal nectar but always much lower
than parent concentrations. Metabolite concentrations declined over time and were below the LOQ at
day 28.
Floral nectar concentrations were initially higher in Acala cotton (site 1) than in Pima (site 2) cotton.
Mean residue concentrations were highest 1 day after the last Belay® Insecticide application in Acala
cotton (142 ppb) but in Prima cotton, the highest residue came 7 days after the last application (95.8
ppb). The unexpected high residue value in one replicate suggests that it was inadvertently
contaminated, either during sample collection, sample processing, or during work-up for analysis.
Clothianidin residues in floral nectar dissipated to concentrations below the LOQ by day 28 in bothcotton varieties.
6.3 POLLEN ANALYSES

7870

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49733302

CDPR Clothianidin Foliar Cotton Study

In general, clothianidin residues were highest 1 to 5 days after the last Belay® Insecticide application andthen declined over the subsequent 21 days. In Acala cotton (site 1), mean clothianidin residues werehighest (300 ppb) 1 day after the last pesticide application declining to <LOQ at day 28. Mean
clothianidin residues were highest 3 days (130 ppb) and 5 days (123 ppb) after the last Belay® Insecticide
application in Pima cotton (site 2). Mean residue concentrations then declined and were below the LOQ
by day 28.
For the first 14 days after the last pesticide application, mean residue concentrations of TZNG and TZMU
were about 10 to 100 times lower than clothianidin concentrations in Acala cotton pollen. TZNG and
TZMU concentrations were usually higher in Pima (site 2) cotton pollen than in Acala (site 2) cotton
pollen and sometimes (TZMU only) concentrations were higher than that of clothianidin. In Pima cotton,mean residues of TZNG increased following the last Belay® Insecticide application and were highest(45.9 ppb) on day 7; TZNG residue concentrations declined thereafter and were below the LOQ on
day 28. Mean TZMU concentrations in Pima pollen also increased after the last pesticide application andwere highest (109 ppb) 7 days post-application. TZMU mean concentrations declined thereafter andwere 1.2 ppb at day 28.
6.4 SITE SUMMARIES
Table 3. Site 1 (Acala cotton variety): Average residues of clothianidin, TZNG and TZMU detected in floral
nectar, extrafloral nectar, pollen and leaf tissues samples collected after the second Belay® Insecticide
application are presented below:

MatrixFloral Nectar

Subbracteal
NectarLeaf Nectar

Days After Last
Application (DALA)
1
3
5
7
14
21
28
Half-Life:
1
3
5
7
14
21
28
Half-Life
3a
1
3
5
7
14

Clothianidin
Concentration
(ppb)
142
51.4
22.5
27.5
11.4
(0.48)b
(0.40)b
3.2 Days
621
623
171
15.9
(3.4)b
2.9 Days
2288
3393
1386
340
126
64.6
8
871

TZNG
Concentration
(PPB)
20.9
22.0
16.6
15.6
2.8
2.8
3.4

TZMU
Concentration
(ppb)
4.6
5.0
2.5
3.5
1.9
1.5
(0.34)b

8.2
6.6
2.5
(0.89)b
(0.63)b

41.9
37.8
17.0
2.0
(0.51)b

32.9
28.3
30.5
23.2
19.2
15.0

208
67.6
215
60.5
20.2
23.7

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49733302

CDPR Clothianidin Foliar Cotton Study

Clothianidin
TZNG
TZMU
Days After Last
Concentration
Concentration
Concentration
MatrixApplication (DALA)
(ppb)
(PPB)
(ppb)
21
16.2
2.8
5.2
28
14.6
2.3
4.1
Half-Life
3.6 Days
1
300
3.4
4.6
3
125
2.0
8.6
5
419
1.5
7.5
7
79.9
1.9
5.4
Pollen
14
15.4
1.1
2.1
b
21
1.5
(0.92)(0.22)bbb
28(0.45)(0.43)<0.25
Half-Life
2.9 Days
1
9823
3
5747
5
2818
7
1546
Leaf Tissue
14
202
21
41.1
28
25.7
Half-Life
3 Daysa
Three days after the first Belay Insecticide application and 4 days before the second Belay Insecticide
application.
b
Values in parenthesis are between the LOQ and the LOD.

Residues of clothianidin, TZNG and TZMU were detected in floral nectar, extrafloral nectar, pollen andleaf tissues of Acala cotton plants. Average concentrations of clothianidin, TZNG and TZMU in floral
nectar ranged from 0.40 to 142 ppb; 2.8 to 22.0 ppb; and 0.34 to 5.0 ppb, respectively. Average residuesof clothianidin, TZNG, and TZMU in subbracteal nectar varied from 3.4 to 623 ppb; 0.63 to 8.2 ppb; and0.51 to 41.9 ppb, respectively. In leaf nectar, average residues ranged from 14.6 to 3,393 ppb; 2.3 to
32.9 ppb; and 4.1 to 208 ppb for clothianidin, TZNG and TZMU, respectively. Average concentrations ofclothianidin, TZNG and TZMU in pollen ranged from 0.45 to 419 ppb; 0.43 to 3.4 ppb; and below <0.25
to 8.6 ppb, respectively. High clothianidin concentration detected in pollen samples at 5 days after thelast application may be a result of pollen sample contamination during field sample collection. Residuesof clothianidin in leaf tissues ranged from 25.7 to 9,823 ppb.
Table 4. Site 2 (Pima cotton variety): Average residues of clothianidin, TZNG and TZMU detected in floral
nectar, leaf nectar, pollen and leaf tissue samples collected after the second Belay® Insecticide
application are presented below:

MatrixFloral Nectar

Days After Last
Application(DALA)
1
3

Clothianidin
Concentration
(ppb)
32.6
18.2

9
872

TZNG
Concentration
(PPB)
28.8
22.3

TZMU
Concentration
(ppb)
5.3
4.7

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49733302

CDPR Clothianidin Foliar Cotton Study

Days After Last
Clothianidin
TZNG
TZMU
Application
Concentration
Concentration
Concentration
Matrix(DALA)
(ppb)
(PPB)
(ppb)
5
53.4
51.8
8.1
7
95.8
36.4
8.2
b
14
11.8
(0.99)
3.7
21
1.2
2.5
1.2
b
28
(0.46)
2.8(0.82)b
Half-Life
4.2 Days
a
3
138
24.6
46.8
1
210
29.6
45.8
3
162
42.5
78.9
5
147
31.5
61.2
Leaf Nectar
7
92.9
35.8
68.1
14
49.6
11.5
23.3
21
24.4
17.9
32.5
28
57.0
13.4
29.7
Half-Life
6.2 Days
1
19.2
1.3
2.0
3
130
9.9
20.0
5
123
27.4
65.5
7
94.6
45.9
109
Pollen
14
28.6
17.7
38.0
21
1.2
1.0
1.7
28
(0.8)b(0.54)b
1.2
Half-Life
2.8 Days
1
11,076
3
7,300
5
5,200
7
3,888
Leaf Tissue
14
709
21
162
28
57.5
Half-Life
3 Daysa
Three days after the first Belay Insecticide application and 4 days before the second Belay Insecticide
application.
b
Values in parenthesis are between the LOQ and the LOD.

Residues of clothianidin, TZNG and TZMU were detected in floral nectar, extrafloral nectar, pollen andleaf tissues of Pima cotton plants. Average concentrations of clothianidin, TZNG and TZMU in floral
nectar ranged from 0.46 to 95.8 ppb; 0.99 to 51.8 ppb; and 0.82 to 8.2 ppb, respectively. In leaf nectar,
average residues ranged from 24.4 to 210 ppb; 11.5 to 42.5 ppb; and 23.3 to 78.9 ppb for clothianidin,
TZNG and TZMU, respectively. Average concentrations of clothianidin, TZNG and TZMU in pollen ranged
from 0.80 to 130 ppb; 0.54 to 45.9 ppb; and 1.2 to 109 ppb, respectively. Residues of clothianidin in leaf
tissues ranged from 57.5 to 11,076 ppb.

10873

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49733302

CDPR Clothianidin Foliar Cotton Study

7. STUDY VALIDITY/CLASSIFICATION AND STUDY LIMITATIONSClassification/Utility for Bee Risk Assessment. This study is classified as acceptable. It provides
quantitative information regarding the magnitude of clothianidin, TZNG and TZMU residues in floral
nectar, extrafloral nectar, pollen and leaf tissues in cotton after two foliar applications of Belay®
Insecticide (active ingredient, clothianidin). Applications were made at the maximum labeled rate, andthus the results are relevant to the current bee risk assessment. Residues were sampled over a period of
1 to 28 days after the last application and thus provide information regarding the temporal variability ofresidues after application. The study protocol was reviewed by CDPR before study initiation. It is
important to note that the study authors stated that it is likely that leaf nectar was contaminated and, in
addition, evaporation of water during sample collection could have artificially increased leaf nectar
residue concentrations. Thus the results for residue concentrations in leaf nectar may not be reliable.
Temporal Variability in Residues. Sampling was conducted from 1 to 28 days after the last Belay®
Insecticide application. In general, average residues of clothianidin, TZNG and TZMU detected in floral
nectar, leaf nectar, pollen and leaf tissue samples were highest between 1 to 7 days after the second
application and then declined thereafter.
Spatial Variability in Residues. Both field sites were in Fresno County, located in California. The soil was
described as Ramona Sandy Loam at site 1 (Acala cotton) and Hanford Loamy Sand at site 2 (Pimacotton). Small variations in average residue concentrations were noted between the two study sites.
Mean clothianidin leaf concentrations were higher in Pima cotton (site 2) than in Acala cotton (site 1).
TZNG and TZMU concentrations were usually higher in Pima cotton (site 2) pollen than in Acala cotton(site 2) pollen as well.
Pesticide Carryover. This study was not designed to measure the extent to which prior year applicationsof clothianidin contributed to year-to-year carryover. Therefore, the effects of pesticide carryover incotton are unknown.
Magnitude of Residues. Average residues of clothianidin, TZNG and TZMU detected in floral nectar,
extrafloral nectar, pollen and leaf tissues samples collected after the second Belay® Insecticide
application are presented for each study site (Tables 3 and 4).
Although the same application equipment was used at both field sites, mean clothianidin leaf
concentrations were higher in Pima cotton (site 2) than in Acala cotton (site 1). In both varieties,
residues were highest immediately after the last application and declined thereafter. One day after thelast application of Belay® Insecticide, clothianidin leaf concentrations were 11,100 ppb in Pima cotton
and 9,820 ppb in Acala cotton. Twenty-eight days after the application, leaf residue concentrations
declined to 57.5 ppb in Pima cotton and 25.7 ppb in Acala cotton. The decline of clothianidin on leaves
was first-order with a half-life of about 3 days at both sites.
Mean concentrations of clothianidin in Acala leaf nectar were highest (3,390 ppb) 1 day after the last
application of Belay® Insecticide, declining to 14.6 ppb on day 28. Leaf nectar residue concentrations ofclothianidin were highest in Pima cotton 1 day after the last Belay® Insecticide application (210 ppb),
declining to 24.4 ppb on day 21. Residue concentrations of metabolites were lower than parent
concentrations (except TZMU in Pima at day 21) in both cotton varieties with TZMU concentrations

11874

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49733302

CDPR Clothianidin Foliar Cotton Study

higher than TZNG concentrations. The decline in clothianidin in leaf nectar was first-order with a half-lifeof 3.6 and 6.2 days in Acala and Pima cotton, respectively.
Clothianidin residues in subbracteal nectar were highest and approximately equal (about 620 ppb) in
Acala on days 5 and 7 after the last application of Belay® Insecticide. Clothianidin concentrations
declined thereafter and were 3.4 ppb at 28 days post application. Except for the last interval, TZMU
concentrations were higher than TZNG concentrations in subbracteal nectar but always much lower
than parent concentrations. Metabolite concentrations declined over time and were below the LOQ at
day 28. The decline in clothianidin in subbracteal nectar was first-order with a half-life of 2.9 days.
Floral nectar concentrations were initially higher in Acala cotton than in Pima cotton. Mean residue
concentrations were highest 1 day after the last Belay® Insecticide application in Acala cotton (142 ppb)
but in Prima cotton, the highest residue came 7 days after the last application (95.8 ppb). Clothianidin
residues in floral nectar dissipated to concentrations below the LOQ by day 28 in both cotton varieties.
The first-order half-life of clothianidin in floral nectar was 3.2 and 4.2 days in Acala and Pima cotton,
respectively.
In Acala cotton, mean clothianidin residues in pollen were highest (300 ppb) 1 day after the last
pesticide application declining to <LOQ at day 28. In Pima cotton, mean clothianidin residues in pollen
were highest 3 days (130 ppb) and 5 days (123 ppb) after the last Belay® Insecticide application. Mean
residue concentrations then declined and were below the LOQ by day 28. The first-order half-life ofclothianidin in pollen was 2.9 and 2.8 days in Acala and Pima cotton, respectively.
For the first 14 days after the last pesticide application, mean residue concentrations of TZNG and TZMU
were about 10 to 100 times lower than clothianidin concentrations in Acala cotton pollen. TZNG and
TZMU concentrations were usually higher in Pima cotton pollen than in Acala cotton pollen. In Pima
cotton, mean residues of TZNG and TZMU increased following the last Belay® Insecticide application andwere highest on day 7; residue concentrations declined thereafter.

8. STATISTICAL ANALYSIS
Clothianidin was applied in two foliar sprays to cotton plants at two sites in Fresno County, CA in 2012.
The first application was made 7 days prior to floral bloom and the second application was made atfloral bloom. Soil at site 1 was a Ramona sandy loam, which is a medium-textured soil with 56% sandcontent, whereas, the soil at site 2 was a Hanford sandy loam, which is a coarse-textured soil with 84%
sand content. Potential comparisons of the effect of soil were compromised because different cultivars
were planted at each site where ‘Acala’ was planted at site 1 and ‘Pima’ was planted at site 2. Thus,
effects from soil type could be confounded by differences due to cultivar used. Since the sprays wereapplied to foliage at bloom, effects of soil were not expected and not analyzed. The objective of thestudy design was to describe the degradation rate of residues of clothianidin and its transformation
products after foliar application to cotton plants. An analysis of the potential distribution of clothianidin
residues measured at a specific sampling time interval was restricted because only two replicates wereobtained for each plant sample from only two experimental sites. The distribution of data pooled overall sampling dates at each site provided guidance on the maximum values that were measured.
The analysis of the data was conducted to provide guidance for the following questions:

12875Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49733302

CDPR Clothianidin Foliar Cotton Study

A. What was the overall distribution of clothianidin, TZNG and TZMU concentrations for data
pooled over the sampling intervals?
i.
Sampling intervals analyzed were at 1, 7, 14(15), 21 and 28 days after the second foliar
application.
B. Was there a temporal pattern in residue concentrations over time?
C. Was there a relationship between residues in the plant tissues?
A. Distribution of concentration data pooled over sampling intervals. Tables S-1 through S-5 containthe distributional statistics for leaf, foliar nectar, leaf nectar, subbracteal nectar and pollen,
respectively, for each site. For leaves, only parent residue was measured where the medianconcentration was 1,812.0 ppb and the maximum value at 11,127.0 ppb. For floral nectar, the medianconcentration of total residue was 40.9 ppb with a maximum at 239.9 ppb. Median and maximum
values were higher in leaf nectar at 185.5 and 4,267.9 ppb, respectively. Subbracteal nectar samples
were not measured at site 2 but samples at site 1 had median and maximum concentrations at 190.9
and 694.2 ppb, respectively. Pollen concentrations for median and maximum values were 38.5 and771.4 ppb. The magnitude and range in these values potentially are biologically significant and should
be compared to biological benchmarks, when established.
B. Temporal Pattern in Concentration of Residues. Figures S-1 through S-5 present residue
concentrations measured over time for leaves, floral nectar, leaf nectar, subbracteal nectar, and pollen,
respectively. For leaves, the raw data indicated decreased concentration over time with concave
curvature in the response. A graph of data transformed to natural, base E, logarithms resulted in ahighly significant straight line: R-square values were very high at 0.96 and 0.99 for sites 1 and 2,
respectively. The transformation was also successful in linearizing the regressions for the other plant
samples. Testing for significance of regression using the PROC REG procedure in the SAS analysis
software similarly indicated highly significant regressions over time in the other plant samples, except
for TZNG and TZMU residues in pollen. Equations based on natural log transformed data are used toprovide estimates of dissipation half-lives with examples for determination of half-lives for data
generated in aerobic and terrestrial field dissipation studies.
The logarithmic regression is expressed as in Equation 1:
Equation 1
Ln(Concentration) = Ln(Initial Concentration) + b(Time)The half-life is determined as Equation 2
Equation 2
T1/2 = Ln(.5)/b
In Equation 2, Ln(.5) is the determination made at 50% of dissipated residues and b is the coefficient
determined from fit of Equation 1 to the observed dissipation data. Table S-6 (A) compares thecoefficients from the linear regression of the transformed data between the plant samples and between
the two sites. Table S-6 (B) compares the half-live values calculated according to Equation 2. Addition of13876Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49733302

CDPR Clothianidin Foliar Cotton Study

degradation products tended to increase the estimate of the half-life where, for example, at site 1 thehalf-life in floral nectar increased from 3.2 for parent clothianidin to 5.5 days when total residue wasanalyzed.
These regression equations also could be used to estimate the amount of time a residue would beabove a benchmark value. For floral nectar, the mean total clothianidin value for the 3 samples takenfrom both sites 1 day after the second application was 100.3 ppb. If a chronic value was set at 25 ppb,
then according to Equation 1:
Ln(Concentration) = 25, the target value
Ln(Initial Concentration) = 100.3, the mean of the 3 samples
b = 0.1285, the mean of the coefficients for total clothianidin for floral nectar in Table S-6 (A)x = time
Equation 3
Ln(25) = Ln(100.3) + (-0.1285)(x)3.219 = 4.608 - .1285(x)x = 10.8 days
Substituting and then solving Equation 3 results in an estimate of around 11 days above the 25 ppb
benchmark value. Table S-7 contains the estimated number of days above the assumed benchmark
values of 15, 20, 25 and 30 for floral nectar, leaf nectar, subbracteal nectar and pollen samples. The
initial values were an average from the two sites as were the coefficient values for b. Individual analyses
at site 2 would have resulted in longer estimated time intervals because concentrations were generally
larger at that site. The averaged initial values in leaf nectar were large, which resulted in estimatesgreater than 30 days for the amount of time above the benchmark values.
C. Relationship between Residue in Plant Tissue. Figure S-6 contains the relationship for measured
clothianidin concentration between leaf and floral nectar samples (A) and between leaf and pollen
samples (B). Both graphs indicate a general relationship for increasing concentrations in floral nectar
and pollen in response to increases in measured concentration leaves. However, the observed varianceamongst the points only result in a low measure of R-square and thus a low accuracy in equations
derived to describe the relationship.
Conclusion:
1. Foliar applications to cotton plants during bloom resulted in high concentration of clothianidin and its
degradation products in bee relevant samples of nectar and pollen. Maximum and median values for
total clothianidin residues are as followed: 239.9 and 40.9 ppb in floral nectar, respectively; 694.2 and190.9 ppb in subbracteal nectar, respectively; 4,267.9 and 185.5 ppb in leaf nectar, respectively; and771.4 and 38.5 ppb in pollen, respectively. These values will require additional analysis to determine
their significance when related to derived benchmark values.
2. Significant regressions were measured for each of the residues in each of the plant samples whereresidues were observed to decrease within the 28 day sampling interval, as measured after the second
clothianidin application. The derived equations can be used to calculate the days above which

14877Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49733302

CDPR Clothianidin Foliar Cotton Study

concentrations would be expected to be higher than relevant benchmark values. An example is given
for floral nectar where based on the mean of the initial values measured at the 2 sites in this study,
concentrations would be expected to be greater than an assumed benchmark value of 25 ppb for
approximately 11 days. Table S-7 contains a summary of estimated days above benchmark values of 15,
20, 25, and 30 ppb for floral nectar, subbracteal nectar, leaf nectar, and pollen. Values were calculated
according to Equation 3.
3. There was a general relationship noted in graphs for increasing concentrations of clothianidin
measured in leaf tissue to result in concomitant increases in concentration measured in nectar andpollen samples. The graphs, however, had a large amount of variability so resultant equations
describing the relationships would have had low accuracy.
4. The applications were made to the foliage so effects due to soil were expected to be minimal.
Potential differences due to soil type were not investigated because only one test site was investigatedfor each soil category and different cultivars were used at each site.

15878Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49733302

CDPR Clothianidin Foliar Cotton Study

Table S-1. Leaves: Comparison of statistics for the distribution of year 2012 concentrations ofclothianidin in cotton leaves where data have been pooled over all sampling dates (ranging from 1 to 28
days after the last foliar application to cotton plants). Note that the degradation products were notmeasured in leaf tissue.

StatisticN
MeanSD
CV (%)MinMedian
75th
90th
95th

Distribution in Leaves (ng/g)
Clothianidin
28
3,471.1
3,759.2
108.3
23.1
1,812.0
5,746.5
9,917.0
11,026.0

Table S-2. Floral Nectar: Comparison of statistics for the distribution of year 2012 concentrations ofclothianidin in cotton floral nectar where data have been pooled over all sampling dates (ranging from 1
to 28 days after the last foliar application to cotton plants).

StatisticN
MeanSD
CV (%)MinMedian
75th
90th
95th
Max
% of Total

Clothianidin
27
29.5
43.2
146.4
0.2
17.4
32.0
79.4
142.0
182.0
182.0

Distribution in Floral Nectar (ng/g)
TZNG
TZMU
27
27
16.5
3.6
15.8
2.6
96.2
72.5
0.9
0.2
14.2
3.1
23.5
5.3
40.9
7.5
47.0
8.8
62.7
10.9
33.2
7.3

16
879

Total
27
49.6
56.3
113.4
2.7
40.9
75.2
127.8
167.5
239.9

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49733302

CDPR Clothianidin Foliar Cotton Study

Table S-3. Leaf Nectar: Comparison of statistics for the distribution of year 2012 concentrations ofclothianidin in cotton leaf nectar where data have been pooled over all sampling dates (ranging from 1
to 28 days after the last foliar application to cotton plants).

StatisticN
MeanSD
CV (%)MinMedian
75th
90th
95th
Max
% of Total

Clothianidin
27
448.5
946.9
211.2
9.9
104.0
213.0
1,692.0
2,624.0
4,163.0
85.6

Distribution in Floral Nectar (ng/g)
TZNG
TZMU
27
27
22.0
53.4
12.4
60.4
56.6
113.0
2.3
3.6
20.0
39.1
32.9
67.7
38.9
90.0
39.5
110.0
45.6
320.0
4.2
10.2

Total
27
523.9
977.1
186.5
15.9
185.5
338.9
2,035.8
2,710.9
4,267.9

Table S-4. Subbracteal Nectar: Comparison of statistics for the distribution of year 2012 concentrationsof clothianidin in cotton subbracteal nectar where data have been pooled over all sampling dates(ranging from 1 to 28 days after the last foliar application to cotton plants). Note that no subbracteal
nectar samples were obtained from Site 2.

StatisticN
MeanSD
CV (%)MinMedian
75th
90th
95th
Max
% of Total

Clothianidin
10
286.9
295.6
103.1
2.0
171.5
598.0
647.0
651.0
651.0
92.4

Distribution in Floral Nectar (ng/g)
TZNG
TZMU
10
10
3.8
19.8
3.3
18.4
87.2
92.9
0.3
0.5
2.5
17.0
7.0
39.4
8.3
41.9
8.4
43.7
8.4
43.7
1.2
6.4

17
880

Total
10
310.4
316.9
102.1
2.8
190.9
649.8
692.9
694.2
694.2

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49733302

CDPR Clothianidin Foliar Cotton Study

Table S-5. Pollen: Comparison of statistics for the distribution of year 2012 concentrations ofclothianidin in cotton pollen where data have been pooled over all sampling dates (ranging from 1 to 28
days after the last foliar application to cotton plants).

StatisticN
MeanSD
CV (%)MinMedian
75th
90th
95th
Max
% of Total

Clothianidin
27
88.1
156.4
177.6
0.2
19.7
123.0
246.0
300.0
760.0
75.7

Distribution in Floral Nectar (ng/g)
TZNG
TZMU
27
27
8.4
19.9
18.2
41.0
217.2
205.8
0.2
0.1
1.5
4.6
3.8
17.3
28.8
58.6
30.7
79.1
87.9
199.0
7.2
17.1

18
881

Total
27
116.4
175.4
150.7
1.0
38.5
771.4
308.0
456.9
771.4

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49733302

CDPR Clothianidin Foliar Cotton Study

Table S-6. Regression results measuring the change in observed concentrations in each plant sample over time. Table S-6 (A) contains thecoefficient of the linear regression of the data transformed to natural logarithms, denoted as b, and the R-square value. Table S-6 (B) contains
the estimated half-lives from each regression as calculated according to Equation 2. Except for TZNG and TZMU residue measures in pollen,
regressions were highly significant.
Table S-6 (A) Regression Results
Regression Results for Linear Coefficient (b) and R-square Values (R2)Clothianidin
Site 1
Plant Sample
LeavesFloral Nectar
Leaf Nectar
Subbracteal Nectar
PollenTZNG

Site 2TZMU

Site 1Site 2

Total Residue

Site 1Site 2Site 1Site 2

b

R2

b

R2

b

R2

b

R2

b

R2

b

R2

b

R2

b

R2

-0.233-0.216-0.193-0.243-0.253

0.96
0.90
0.85
0.97
0.93

-0.202-0.173-0.080-0.182

0.99
0.78
0.67
0.66

-0.086-0.102-0.127-0.062

0.88
0.90
0.91
0.80

-0.121-0.044-0.087

0.59
0.60
0.21

-0.088-0.126-0.200-0.173

0.84
0.81
0.96
0.92

0.081
-0.037-0.044

0.81
0.47
0.06

-0.130-0.180-0.233-0.223

0.92
0.86
0.97
0.92

-0.127-0.060-0.105

0.78
0.70
0.37

Table S-6 (B) Estimated Half-lives
Estimated Half-lives for Each Plant Sample at Each Site (Days)
Clothianidin

TZNG

TZMU

Total Residue

Plant Sample

Site 1Site 2Site 1Site 2Site 1Site 2Site 1Site 2

LeavesFloral Nectar
Leaf Nectar
Subbracteal Nectar

2.97
3.21
3.59
2.85

3.43
4.01
8.66
-

8.06
6.79
5.46

5.73
15.75
-

7.88
5.50
3.47

8.56
18.73
-

5.33
3.85
2.97

5.46
11.55
-

Pollen

2.74

3.81

11.18

NS

4.01

NS

3.11

6.60

19
882

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49733302

CDPR Clothianidin Foliar Cotton Study

Table S-7. Estimated time interval that concentrations measured in bee relevant cotton plant samples
would be above assumed benchmark values. Data were pooled from the 2 sites and initial values were
determined as the average from both sites for those measured 1 day after the second foliar application.
Values for days above assumed benchmark calculated according to Equation 3.

Plant Sample

InitialValue(ng/g)

Averaged
Coefficient(b)Floral Nectar

100.30

Leaf Nectar

Estimated Days Over Assumed Benchmark Value
Value (ng/g)
15.0

20.0

25.0

30.0

-0.129

14.8

12.5

10.8

9.4

1885.90-0.120

40.3

37.9

36.0

34.5

Subbracteal Nectar

670.65-0.233

16.3

15.1

14.1

13.3

Pollen117.30-0.164

12.5

10.8

9.4

8.3

20
883

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49733302

CDPR Clothianidin Foliar Cotton Study

Figure S-1. Leaves: Concentration of clothianidin residues measured in cotton leaves over a 28 day
period. Initial samples are indicated as those taken 1 day after the second foliar application to plants inbloom. Figure S-1 (A) contains raw data and Figure S-1 (B) displays the transformed data to natural
logarithms.
Figure S-1 (A) Raw Data
Site1

Leaf Concentration (ppb)

15,000Site 2

12,000
9,000
6,000
3,000
0

0

5

10

15

20

25

30

Days After Last Application

Figure S-1 (B) Transformed to Natural Logarithms
Site 110Site 2Site 1: y = -0.2329x + 9.103
R² = 0.9609

Leaf Concentration (ppb)8Site 2: y = -0.202x + 9.5228
R² = 0.9926

6

4

2

0

0

5

10

15

Days After Last Application
21
884

20

25

30

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49733302

CDPR Clothianidin Foliar Cotton Study

Figure S-2. Floral Nectar: Concentration of clothianidin residues measured in floral nectar of cotton
plants over a 28 day period. Initial samples are indicated as those taken 1 day after the second foliar
application to plants in bloom. Figure S-2 (A) contains raw data and Figure S-2 (B) displays thetransformed data to natural logarithms.
Figure S-2 (A) Raw Data

Site1

Floral Nectar Concentration (ppb)

100

Site2

80
60
40
20
0

0

5

10

15

20

25

30

Days After Last Application

Figure S-2 (B) Transformed to Natural Logarithms
Site 1

FloralNectarConcentration(ppb)
FloralNectarConcentration

5

Site 2Site 1: y = -0.2165x + 4.6619
R² = 0.9036
Site 2: y = -0.1731x + 4.1784
R² = 0.7776

4
3
2
1
0
-1

0

5

10

15

Days After Last Application
22
885

20

25

30

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49733302

CDPR Clothianidin Foliar Cotton Study

Figure S-3. Leaf Nectar: Concentration of clothianidin residues measured in leaf nectar of cotton plants
over a 28 day period. Initial samples are indicated as those taken 1 day after the second foliar
application to plants in bloom. Figure S-3 (A) contains raw data and Figure S-3 (B) displays thetransformed data to natural logarithms.
Figure S-3 (A) Raw Data

Site1

Leaf
NectarConcentration
(ppb)
Leaf
NectarConcentration
(ppb)
Leaf
NectarConcentration
(ppb)

5000
5,000

Site2

4000
4,000
3000
3,000
2000
2,000
1000
1,000
0

0

5

10

15

20

25

30

Days After Last Application

Figure S-3 (B) Transformed to Natural Logarithms
Site 1

Leaf Nectar Concentration (ppb)

10Site 2Site 1: y = -0.1925x + 7.2401
R² = 0.8462

8

Site 2: y = -0.0756x + 4.6666
R² = 0.1893

6
4
2
0

0.0

5.0

10.0

15.0

20.0

Days After Last Application
23
886

25.0

30.0

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49733302

CDPR Clothianidin Foliar Cotton Study

Figure S-4. Subbracteal Nectar: Concentration of clothianidin residues measured in subbracteal nectar of
cotton plants over a 28 day period. Initial samples are indicated as those taken 1 day after the second
foliar application to plants in bloom. Figure S-4 (A) contains raw data and Figure S-4 (B) displays thetransformed data to natural logarithms.

Subbracteal Nectar Concentration (ppb)

Figure S-4 (A) Raw Data

Site1

1,000
800
600
400
200
0

0

5

10

15

20

25

30

Days After Last Application

Subbracteal
NectarConcentration
Subbracteal
NectarConcentration(ppb)

Figure S-4 (B) Transformed to Natural Logarithms
Site 110

y = -0.2426x + 8.0156
R² = 0.9704

8
6
4
2
0

0

5

10

15

Days After Last Application
24
887

20

25

30

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49733302

CDPR Clothianidin Foliar Cotton Study

Figure S-5. Pollen: Concentration of clothianidin residues measured in pollen of cotton plants over a 28
day period. Initial samples are indicated as those taken 1 day after the second foliar application to plants
in bloom. Figure S-5 (A) contains raw data and Figure S-5 (B) displays the transformed data to natural
logarithms.
Figure S-5 (A) Raw Data

Site1

Pollen Concentration (ppb)

300

Site2

250
200
150
100
50
0

0

5

10

15

20

25

30

Days After Last Application

Figure S-5 (B) Transformed to Natural Logarithms
Site 1Site 2

PollenPollen Concentration
Concentration (ppb)(ppb)
PollenConcentration
(ppb)

9

Site 1: y = -0.2527x + 6.0898
R² = 0.9346

7

Site 2: y = -0.1825x + 4.7073
R² = 0.6639

5
3
1
-1

0

5

10

15

20

Days After Last Application
25
888

25

30

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49733302

CDPR Clothianidin Foliar Cotton Study

Figure S-6. Relationship measured for concentration of clothianidin residues. Figure S-6 (A) displays therelationship of clothianidin between leaf and floral nectars and Figure S-5 (B) displays the relationship ofclothianidin between leaf and pollen.
Figure S-6 (A) Leaf and Floral Nectar

Floral Nectar Concentration (ppb)

250

Site1

Site 2

6,000

9,000

200
150
100
50
0

0

3,000

12,00015,00012,00015,000

Leaf Concentration (ppb)

Figure S-6 (B) Leaf and Pollen

Pollen Concentration (ppb)

400

Site1

Site 2

6,000

9,000

300

200

100

0

0

3,000

Leaf Concentration (ppb)26889

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49733302

CDPR Clothianidin Foliar Cotton Study

9. REFERENCES
1. Hoelscher, A. 1998. Physical and chemical properties of the active ingredient imidacloprid.
Bayer CropScience Report No. MO-03-006885, M-105510-01-1.
2. Talbott, T.D. 1991. Product Chemistry of BAY NTN 33893 Technical. Bayer CropScience Report
No. MRID M004227-01-1.
3. Krohn, J. 1993. Vapour Pressure Curve of Imidacloprid. Bayer CropScience Laboratory Project
ID 14 200 0804. Report No. M-004042-01-1.
4. U.S. EPA, PMRA and CDPR. 2012. White Paper in Support of the Proposed Risk Assessment
Process for Bees. Submitted to the FIFRA Scientific Advisory Panel for Review and Comment
September 11-14, 2012. Published by USEPA, PMRA and CDPR. 275 pages.
5. U.S. EPA, PMRA and CDPR. 2014. Guidance for Assessing Pesticide Risk to Bees. Published by
U.S. EPA, PMRA, and CDPR June 2014. 59 Pages.
6. Brungardt, J. 2010. An Analytical Method for the Determination of Residues of Imidacloprid
and its Metabolites Imidacloprid Olefin and 5-Hydroxy Imidacloprid in Bee Relevant Matrices
Using LC/MS/MS. Bayer Method No. NT-005-P10-01.
7. Miller, A. 2014. An Analytical Method for the Determination of Residues of Imidacloprid and its
Metabolites Imidacloprid Olefin and 5-Hydroxy Imidacloprid in Bee Relevant Matrices Using
LC/MS/MS. Bayer CropScience Report Number: NT-006-A13-01.
8. Office of Pesticide Programs, US EPA. 2000. Assigning values to nondetected/nonquantified
pesticide residues in human health food exposure assessments. EPA Docket #OPP-00570A.
9. Noland, P.A. 1992. Imidacloprid and metabolites – freezer storage stability study in crops.
Bayer CropScience Report No. 103237. MRID 42556135.
10. Noland, P.A. 1993. Imidacloprid and metabolites – freezer storage stability study in crops.
Bayer CropScience Report No. 103237-1. MRID 42810311.
11. Noland, P.A. 1994. Imidacloprid and metabolites – freezer storage stability study in crops.
Bayer CropScience Report No. 103237-2. MRID 43197203.
12. Noland, P.A. 1994. Imidacloprid and metabolites – freezer storage stability study in crops.
Bayer CropScience Report No. 103237-3. MRID 43487302.
13. Lenz, C.A. 1993. Addendum 1. Imidacloprid and metabolites – freezer storage stability study incrops (wheat matrices, tomatoseed, tomato, cauliflower, and lettuce). Bayer CropScience
Report No. 103949-1. MRID 42810313.

27
890

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

Year/Authors/Title
Bondarenko, S. 2016.
Clothianidin: Quantitation of
Residues of Clothianidin,
TZNG and TZMU in Nectar,
Pollen and Leaves Collectedfrom Pumpkins Following Soil
Application of Belay
Insecticide and Foliar
Application of Belay 50 WDG
Insecticide
Valent Technical Cener Study
Number: VP-38971

Study Type
Summary
Notes/Uncertainties
Non-Guideline This study quantified clothianidin, TZNG, and TZMU residues in pumpkin
Nothing that would affect thefield residue(Cucurbita peto L. var pepo) grown in three locations: North Dakota (ND; loam validity of the study.
study onor sandy loam), California (CA; loamy sand or sand), and Oregon (OR; silt loam).
pumpkin toThree replicate plots were used in each location for each treatment. One set of
establish
plots received a soil application at planting at a nominal rate of 0.2 lbs. ai/A,
clothianidin
and another set of plots at each location received a single soil application at
andBBCH stage ca. 14 at a nominal rate 0.2 lbs. ai/A. The final set of plots received
metabolite
a single foliar application BBCH stage ca. 14 at a nominal rate 0.1 lbs. ai/A. The
concentrations soil application rate equals the maximum label rate of 0.2 lbs. ai/A for Belay®
in leaves and 50 WDG, but the foliar application rate exceeded the maximum rate of 0.067
manuallylbs. ai/A. Nectar and pollen were sampled 5 times during the blooming period.
collected
In the plots that received an at-plant soil application, samples of pollen andnectar andnectar were collected 47-75, 42-69, and 52-79 days after the application in ND,
pollenCA, and OR, respectively. In the plots that received a soil or foliar application at
following soil BBCH stage 14, samples of pollen and nectar were collected 25-53, 21-48, andor foliar
22-49 days after the application in ND, CA, and OR, respectively. Analyses ofapplications
fortified samples of pollen (79-102% clothianidin, 88-96 TZNG, and 84-95
TZMU) and nectar (93-109% clothianidin, 88-101 TZNG, and 99-109 TZMU)
were all within acceptable limits. Nectar and pollen samples were manually
collected from flowers. Clothianidin residues in nectar from plots receiving atplant soil applications across all locations were 5.84 ppb or less and decreased
over the course of the sampling, and concentrations for TZNG or TZMU were
low, always less than the LOQ of 1.0 ppb and often less than the LOD of 0.20
ppb. Clothianidin residues in pollen from plots receiving at-plant soil
applications across all locations were 38.3 ppb or less and decreased over thecourse of the sampling in CA and OR but remain fairly constant in ND, andconcentrations for TZNG or TZMU were low, always less than the LOQ of 1.0
ppb except in the initial samples in CA and often less than the LOD of 0.25 ppb.
Clothianidin residues in nectar from plots receiving soil applications at BBCH
stage 14 across all locations were 11.3 ppb or less and decreased over thecourse of the sampling, and concentrations for TZNG or TZMU were low,frequently less than the LOQ of 1.0 ppb or the LOD of 0.20 ppb. Clothianidin
residues in pollen from plots receiving soil applications at BBCH stage 14 acrossall locations were 31.9 ppb or less and decreased over the course of thesampling in CA and OR but increased over time in ND, and concentrations for
TZNG or TZMU were low, always less than the LOQ of 1.0 ppb except in CA andoften less than the LOD of 0.25 ppb. Clothianidin residues in nectar from plots891

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

receiving foliar applications at BBCH stage 14 across all locations wereconsistently greater than other application scenarios at 43.5 ppb or less anddecreased less dramatically over the course of the sampling, andconcentrations for TZNG or TZMU were low, always less than the LOD of 0.20
ppb. Clothianidin residues in pollen from plots receiving foliar applications at
BBCH stage 14 across all locations were always low at 3.03 ppb or less but did
not show a consistent pattern as sampling progressed, and concentrations for
TZNG or TZMU were low, always less than the LOD of 0.25 ppb. Mean residues
for clothianidin in nectar and pollen were generally greater across all sample
periods in CA than in ND or OR.

892

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

Year/Authors/Title

Study Type

Summary

Notes/Uncertainties

Bondarenko, S. 2017.
Clothianidin:
Quantitation of Residuesof Clothianidin, TZNG,
and TZMU in Nectar,
Pollen, and Leaves
Following Foliar Post
Bloom Application of
Belay Insecticide/Clutch
50 WDG Insecticide toApple Trees.

Non-Guidelinefield residue study
on apples toestablish
Clothianidin andmetabolite levels
in nectar, pollen,
and leaves
following foliar
applications

This study quantified Clothianidin residues in apple (Malus pumila)grown in three locations: Ontario, Canada (CAN; loam), Hood River,
Oregon (OR; loam), and Parkdale, OR (OR; sandy loam). One
replicate plot was used in each location, and each plot received apost-bloom foliar application of Belay Insecticide/Clutch 50 WDG
Insecticide to apple trees in 2014 and again in 2015. The post-bloom
foliar application was made at the nominal application rate of 0.1874
lb ai/A at ca. 7 days before harvest in September 2014. The postbloom foliar application was made at the nominal application rate of
0.1874 lb ai/A at ca. 7 days before harvest in August-September
2015. The maximum annual use rate of Clothianidin approved in
Canada is 0.1874 lb ai/A. For foliar applications, the substance wasapplied using an orchard air blast to both sides of the tree rows. Soilsamples were collected using a soil auger or a probe. Pollen andnectar samples were collected from single composite flower samples
during the blooming period in spring 2015 and 2016. Leaf samples
were collected from new emerged whole leaves in the terminal
shoots. In 2015, flowers and whole leaves were collected 231, 218,
and 229 days after the last application in Ontario, Canada, Hood
River, OR, and Parkdale, OR, respectively. In 2016, flowers andwhole leaves were collected 247, 231, and 245 days after the last
application in Ontario, Canada, Hood River, OR, and Parkdale, OR,
respectively. Average recoveries for Clothianidin (82-96%), TZNG
(71-95%), and TZMU (83-100%) in nectar, pollen, leaf, and soil
samples were all within the 70-120% acceptable range. The
maximum measured Clothianidin residues resulting from post-bloom
foliar application in 2015 were 0.71 ng/g in nectar, 57.4 ng/g inpollen, and 2.55 ng/g in leaves. The maximum measured Clothianidin
residues resulting from post-bloom foliar application in 2016 were<0.20 ng/g in nectar, 31.1 ng/g in pollen, and 2.58 ng/g in leaves.
This study is acceptable.

Minimum pollen andnectar sample amounts
were not obtained in 2015or 2016 for all subplot
samples, as the flowers did
not yield the necessaryamounts. This deviation
may lead to a gap in datafor pollen and nectar
results.

MRID: 50154304
Valent Study Number:
VP-38552

893

In 2015 and 2016, poor
quality pollen samples
were collected, as the
samples were contaminatedwith anthers, flower
filaments, and other flower
materials. In both years,
scarce amounts of apple
pollen were collected in alltrial locations. This may
affect the recoveries for
Clothianidin in pollen.

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

Year/Authors/Title

Study Type

Summary

Notes/Uncertainties

Bondarenko, S.
2017. Clothianidin:
Quantitation of
Residues ofClothianidin, TZNG,
and TZMU in
Nectar, Pollen, andLeaves Following
Foliar Post Bloom
Application of Belay
Insecticide to Peach
Trees.

Non-Guidelinefield residue
study on peaches
to establish
Clothianidin andmetabolite levels
in nectar, pollen,
and leaves
following foliar
applications

This study quantified Clothianidin residues in peach (Prunus persica) grown inthree locations: Athens, Georgia (GA; sandy clay loam), Monetta, South
Carolina (SC; sand), and Selma, California (CA; loamy sand). One replicate
plot was used in each location, and each plot received two post-bloom foliar
applications of Belay Insecticide to peach trees in 2014 and again in 2015. The
first post-bloom foliar application in June-July 2014 was made at the nominal
application rate of 0.1 lb ai/A at 35-40 days before harvest (BBCH 73-81). The
second application in 2014 was made at least 10 days after the previous
application, and at least 21 days before harvest (at the same nominal
application rate). The second post-bloom foliar application in June-July 2015
was made at the nominal application rate of 0.1 lb ai/A at 35-40 days before
harvest (BBCH 72-81). The second application in 2015 was made at least 10
days after the previous application, and at least 21 days before harvest (at thesame nominal application rate). The maximum annual use rate of Clothianidin
is 0.2 lb ai/A. For foliar applications, the substance was applied using an
orchard air blast to both sides of the tree rows. Soil samples were collectedusing a soil auger or a probe. Pollen and nectar samples were collected fromsingle composite flower samples during the blooming period in February –March 2015 and 2016. Leaf samples were collected from new emerged leaves
in the terminal shoots at the BBCH growth stage ca. 72 in March – April 2015
and 2016. In 2015, flowers were collected 276, 248, and 234 days after the last
application in Georgia, South Carolina, and California, respectively. In 2015,
leaves were collected 301, 269, and 251 days after the last application in
Georgia, South Carolina, and California, respectively. In 2016, flowers werecollected 280, 245, and 233 days after the last application in Georgia, South
Carolina, and California, respectively. In 2016, leaves were collected 314, 286,
and 254 days after the last application in Georgia, South Carolina, andCalifornia, respectively. Average recoveries for Clothianidin (90-101%),
TZNG (85-102%), and TZMU (84-99%) in nectar, pollen, leaf, and soil
samples were all within the 70-120% acceptable range. The maximum
measured Clothianidin residues resulting from post-bloom foliar application in2015 were 0.21 ng/g in nectar, 6.19 ng/g in pollen, and 12.2 ng/g in leaves.
The maximum measured Clothianidin residues resulting from post-bloom
foliar application in 2016 were 0.30 ng/g in nectar, 130 ng/g in pollen, and13.3 ng/g in leaves. This study is acceptable, but the abnormally high
Clothianidin residue in pollen in 2016 suggests possible contamination.

The collected pollen
sample size in 2015
and 2016 did not meetthe protocol
requirements. This led
to a data gap in thepollen results.

MRID: 50154303
Valent Study
Number: VP-38563

894

In 2015 and 2016, poor
quality pollen samples
were collected at all
three sites. Pollen
samples had
contaminants such as
anthers, flower
filaments, and otherflower materials.
Additionally, scarcepollen was collected at
the California site. This
could have a potential
impact on the integrityof the data collected.
The maximum
Clothianidin residue inpeach pollen in 2016 isabnormally high,suggesting either
contamination during
the field sample or inanalytical sample
processing.

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

Year/Authors/Title

Study Type

Summary

Notes/Uncertainties

Bondarenko, S. 2017.
Clothianidin: Quantitationof Residues ofClothianidin, TZNG, and
TZMU in Pollen andLeaves Collected from
Grapevines Following Soiland Foliar Applications of
Belay 50 WDG Insecticide(Clutch 50 WDG).

Non-Guidelinefield residue study
on grapes toestablish
Clothianidin andmetabolite levels
in pollen andleaves following
soil and foliar
applications

This study quantified Clothianidin residues in grape (Vitis
vinifera) grown in three locations: California (CA; sandyloam), Oregon (OR; loam), and Ontario, Canada (CAN;
sandy loam and loam). Three replicate plots were used ineach location, and each plot received a post-bloom foliar
application, a pre-bloom soil application, or a pre-bloom
foliar application of Belay 50 WDG/Clutch 50 WDG
Insecticide. The post-bloom foliar application was made at
the nominal application rate of 0.1 lb ai/A at BBCH
growth stage ca. 71 (berry swelling) in 2015. The prebloom soil application was made at the nominal
application rate of 0.2 lb ai/A at BBCH growth stage ca.
08 (bud break on grapevines) in 2016. The pre-bloom
foliar application was made at the nominal application rateof 0.1 lb ai/A at BBCH growth stage ca.14 (approximately
4 leaves unfolded) in 2016. For foliar applications, thesubstance was applied using an air blast sprayer. For soil
applications, the substance was applied using either aboom sprayer or drip irrigation. Soil samples werecollected using a small shovel, core sampler, or soil tube.
Pollen samples were collected from grape flower clusters,
and leaf samples were collected using 1 inch leaf-punches.
Average recoveries for Clothianidin (93-116%), TZNG
(84-88%), and TZMU (79-80%) in pollen, leaf, and soil
samples were all within the 70-120% acceptable range.
The maximum measured Clothianidin residues resulting
from pre-bloom foliar application at BBCH ca. 14 were1564 ng/g in pollen and 12781 ng/g in leaves. Maximum
measured Clothianidin residues resulting from pre-bloom
soil application at BBCH ca. 08 were 206 ng/g in pollen
and 417 ng/g in leaves. Maximum measured Clothianidin
residues resulting from post-bloom foliar application at
BBCH ca. 71 were 31.9 ng/g in pollen and 15932 ng/g inleaves. This study is acceptable.

Pollen sample weights required by
the protocol for all samples and alltreatments were not met, as
flowers did not produce sufficientamounts of pollen. No pollen wascollected at late bloom sampling,
due to advanced stage of blooms.
This may lead to a data gap inpollen results.

MRID: 50154305
Valent Study Number: VP38992

895

Abnormally high Clothianidin
residues in soil were observed in
one of the trials after foliar
application to the grapevines, withlow residues in pollen and leaf
samples collected during thebloom. Soil samples were collectedon the same day that nearby plots
received irrigation treatment. Soilsamples from these plots may have
inadvertently been miscollected,
leading to the discrepancy inresults.

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

U.S. EPA Data Evaluation Reports (Clothianidin):
U.S. EPA. (2017). Data evaluation report: clothianidin: quantitation of residues ofclothianidin, tzng, and tzmu in nectar, pollen, and leaves following soil application of belay
insecticide to four different species of cucurbit. Washington, D.C.: Author. Laboratory
Report Number VP-38938.
U.S. EPA. (2017). Data evaluation report: clothianidin: quantitation of residues of clothianidin,
tzng, and tzmu in nectar and pollen following foliar application of Clutch 50 WDG insecticide
to cucurbits. Washington, D.C.: Author. Laboratory Report Number: VP-38313.
U.S. EPA. (2017). Data evaluation report: clothianidin: quantitation of residues of clothainidin,
tzng, and tzmu in pollen and leaves collected from potatoes follwing soil and foliar applicationsof Belay insecticide. Washington, D.C.: Author. Laboratory Report Number VP-38985.

896

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

Thiamethoxam Data Evaluations (begin on next page)897

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 49550801

CDPR THX Cucumber

Reference
Hampton. R. (2013) Thiamethoxam 75 SG (A9549C) - Magnitude of the Residues in Leaves, Flowers,
Pollen, and Nectar of Cucumbers, Representative Commodity of Cucurbit Vegetables, EPA Crop Group9, in California: Final Report. Project Number: TK0024668. Unpublished study prepared by Syngenta
Crop Protection, LLC. 67. MRID 49550801, CDPR Study ID 269320, Data Volume 52691-0466, Tracking
ID# 269320

1. STUDY INFORMATION
Chemical:Test Material:

PC CodePercent
ActiveIngredient:

Thiamethoxam
Platinum 75SG

60109
75%

Study Type:

Fild residue study on cucumber crops to measure the magnitude ofThiamethoxam and CGA322704 on the leaves, pollen and nectar of the plantfollowing an in-furrow treatment at cucumber seeding.

Sponsor:Syngenta Crop Protection, LLC
410 Swing Road
Greensboro, North Carolina
27409

Sponsor Study
ID:
Study
Completion
Date:
GLP Status:

Experiment Start
andEnd Date:

May 6, 2011 –November 30, 2012

Study Locations:

3 trial sites of
cucumber which werelocated in Fresno,
California and San Luis
Obispo, California.

TK0024668
January 18, 2013

Non-GLP; protocol reviewed by CDPR.
[CDPR Study ID 269320, Data Volume 52691-0466, Tracking ID# 269320]

2. REVIEWER INFORMATION
Study Reviewed by:Richard Bireley, Sr. Environmental Scientist (Specialist)
California Department
John Troiano, Ph.D., Research Scientist III
of Pesticide Regulation
Alexander Kolosovich, Environmental Scientist
Brigitte Tafarella, Environmental Scientist
Denise Alder, Sr. Environmental Scientist (Specialist)
Russell Darling, Environmental Scientist

898

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 49550801

CDPR THX Cucumber

3. EXECUTIVE SUMMARY

A two-year study was conducted in 2011 and 2012 to determine the magnitude of thiamethoxam and
CGA322704 residues in cucumber leaves, flowers, pollen, and nectar. The study consisted of three trials
that were located in California and each consisted of an untreated control plot and three replicatedtreated plots. The trials were conducted on coarse-, medium- and fine-textured soils, which werecharacterized as two sandy loam sites (9% clay and 14% clay) and one clay loam site (38% clay),respectively. Platinum® 75SG (active ingredient, thiamethoxam) was applied as an in-furrow treatment
at cucumber seeding at a target rate of 0.172 lb ai/acre in Years 1 and 2. Composite samples of leaves,female flowers, male flowers, pollen, and nectar were collected for residue analysis from the untreatedplot and treated plots at 43 to 57 days after planting in Year 2.

4. STUDY VALIDITY

Guideline Followed:
Guideline Deviations:
Other Deviations:Classification:
Rationale:
Reparability:

TBD; (protocol was reviewed by CDPR)
N/A
N/A
ACCEPTABLE
N/A
N/A

5. MATERIALS AND METHODS

Test item:
Formulation Type:
CAS #:

Test Material Characterization
Platinum 75SG
Percent A.I.:
Soluble Granule, SG
Batch Number:
153719-23-4
Expiration Date:

75.0% A.I.
592641
April 30, 2013

5A. STUDY DESIGN
Residue data for thiamethoxam (CGA293343) and its major plant metabolite, CGA322704, in the pollen
and nectar of cucurbit vegetables were requested by the California Department of Pesticide Regulations(CDPR) as part of the re-evaluation of the nitroguanidine class of neo-nicotinoid insecticides (Article 8,
Subchapter 1, Chapter 2, Division 6 of Title 3 of the California Code of Regulations).
The purpose of this two-year study was to determine the amount of thiamethoxam and CGA322704 in
cucurbit pollen and nectar from plants grown in fields after an at-plant soil application of Platinum®
75SG in two successive years. The effect of soil type on thiamethoxam uptake and resulting residues inpollen and nectar also was investigated by conducting trials on coarse-, medium-, and fine-textured
soils. The choice of soils represented a range of soil types on which cucurbits may be growncommercially. The goal was for this study to provide realistic measurements of thiamethoxam and
CGA322704 residues to which bees may be exposed to under typical growing conditions in California.

899

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 49550801

CDPR THX Cucumber

5B. APPLICATION TIMING AND RATESThe target application rate for cucurbit vegetables is 0.172 lb ai/acre (3.67 oz of product/acre) as per the
Platinum® 75SG label. For the applications in 2011 and 2012 at each trial site, the maximum amount of
test substance was added to the volume of water needed to cover the plot area (plus overage).
A CO2-pressurized, single-nozzle sprayer was calibrated with water prior to each application of test
substance. To calibrate, the sprayer system was charged, and the volume of water discharged during 30
seconds was collected and recorded. The procedure was repeated 2 additional times, and the mean
output was calculated. The acceptance criterion required that the three collections fell in the targetedrange of 15 gallons per (GPA) ± 5% (or 140 L/ha) for Trial Sites 1 and 2 and 20 gallons/A ± 5% (or 187L/ha) for Trial Site 3.
The single-nozzle sprayer was attached to the planter, and the spray nozzle directed the test substanceapplication into the furrow prior to furrow closing to cover the seed.
Table 1. Applications of Platinum 75SG per year.
Site #
Year
1
2
3

Test Substance Mass (gof product)
27.7
27.7
27.7
27.7
20.8
20.8

2011
2012
2011
2012
2011
2012

Water Volume(gallons)
4
4
4
4
4
4

5C. STUDY SITE LOCATION AND CHARACTERISTICS
The three trial sites were selected based on the USDA soil survey as the soils at the sites were identified
as a loamy sand, a sandy loam, and a silty clay loam (coarse-, medium-, and fine textured soil,
respectively). Trial Sites 1 and 2 were located in Fresno County on land leased and managed by Eurofins
Agroscience Services, Inc. (EASI, Sanger, CA), and Trial Site 3 was located on the research farm of
California Polytechnical University at San Luis Obispo in San Luis Obispo County.
No maintenance pesticides were applied to the treated plots, with the exception of a single applicationof Baythroid (2.8 fl. oz./A) to Trial Site 3 on July 5, 2012. Irrigation was required at each of the three trialsites from planting and test-substance application to harvest. Approximately 0.33 inches of water wasapplied at Trial Sites 1 and 2 on a ca. 3-day schedule; whereas, approximately 3 inches of water wasapplied at Trial Site 3 on ca. 14-day schedule.
After harvest of cucumbers in 2011 (Year 1), vines were mowed and the crop residue was left on the soil
surface. Plots remained fallow until the plots were prepared (i.e., tilled and rebidded) the following
spring prior to planting in Year 2 (2012).
The results of the soil-characterization analyses are summarized below. Although the soil survey maps
indicated that the textural classes of the soils at Trials Sites 1, 2 and 3 were a loamy sand (‘coarse-’),900

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 49550801

CDPR THX Cucumber

sandy loam (‘medium-’) and silty clay loam (‘fine-’), the soils within the plot areas at the Trial Sites 1 and2 were determined by soil-characterization analyses to be sandy loams, whereas, the soil from Trial Site
3 was classified as a clay loam. The three soils differed in the percentages of clay and sand and in soilsolution pH.
Site #

1
2
3

Table 2. Trial Site Conditions for Melon Grown at Sites Previously Treated with Imidacloprid
Trial
Percent Percent
Percent
Organic
pH
Soil Type
Location
Sand
Silt
Clay
Matter (%)(County,
State)
Fresno,
65
26
9
0.5
5.6
MediumCalifornia
Fresno,
56
30
14
0.7
6.9
MediumCalifornia
San Luis
37
25
38
2.8
7.5
Fine
Obispo,California

Soil
Characteristics
Sandy Loam
Sandy Loam
Clay Loam

5D. SAMPLE COLLECTION, HANDLING, PROCESSING
Samples of leaves, female and male flowers, pollen and nectar were collected from all trial sites in Year2 of the study. In preparation for sampling, the untreated and treated plots were enclosed in tunnels
constructed of PVC® pipe covered by netting of a mesh size suitable for excluding foraging bees. In
addition, plots were irrigated within 24 hours of sample collection to ensure that the plants wereadequately hydrated thereby promoting nectar flow.
At each trial site, plant samples were collected from the untreated and treated plots by separate teams
to minimize the potential for cross contamination. Approximately 400 male and 250 female flowers
were collected, which pollen and nectar samples were extracted from (female and male flowers werecollected on successive days to allow adequate time for extraction of nectar and pollen). The male andfemale flower samples were transported on blue ice in separate, labeled plastic bags to the field
laboratory for pollen and nectar extraction. Leaves were subsequently sampled to confirm uptake of thetest substance.
Female Flowers and Nectar
The nectary of the female flower was exposed by removal of the corolla and calyx using a dissection
scalpel. Once exposed, the nectar was collected by capillary action using a 10-µL microcapillary pipette(preliminary method development indicated that nectar could be extracted from approximately 50% of
the flowers in a given sample; if nectar was present, approximately 2 - 4 µL typically was collected fromeach flower). Approximately 250 female flowers were sampled in order to extract the minimum sample
size of nectar required for analysis (ca. 100 µL). The total number of microcapillary pipettes needed to
extract the sample of ca. 250 flowers was recorded, and the entire set of pipettes was then placed into alabeled, extraction-ready 15-mL centrifuge tube. The total sample weight was recorded, and the weightsof the storage container and the pipettes were subtracted to determine the actual nectar sample mass.
After nectar extraction, a ca. 500-1000 g flower sample was placed into a labeled, sealable plastic bag,and the sample mass was recorded. The nectar and flower samples were placed into the freezer until
shipment.
901

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 49550801

CDPR THX Cucumber

Male Flowers and Pollen
Pollen was extracted from the male flowers using a laboratory vacuum pump; a rubber tube was
attached to the pump and fitted with a filtered 1000-µL Eppendorf pipette tip. The pipette tips
containing the trapped pollen were cut in two using nursery pruners (to facilitate solvent extraction),and the two pieces were placed into a 250-mL extraction bottle. The total sample mass was recorded,
and the weights of the extraction bottle and the pipette tips were subtracted to determine the actual
pollen sample mass. After pollen collection, ca. 500 - 1000 g flower samples were placed into labeled,sealable plastic bags, weighed and stored frozen until shipment.
LeavesRepresentative samples of at least 500 g of leaves were collected from the untreated and treated plots.
Leaves were removed by hand from the proximal (i.e., closest to the root), middle and distal portions of
the cucumber vines.
Sample Storage.
Samples were transported from the field sites to freezers at EASI (Sanger, CA) where they were stored
frozen until shipment to the analytical laboratory. Samples were shipped overnight on dry ice via FedEx
to the analytical laboratory.
All samples were received frozen and in good condition at ABC Laboratories from the field trial sites.
The samples were maintained in frozen condition, excluding periods during which the samples were
removed from the freezer for sample preparation, weighing or residue analysis.
The leaf and flower samples were weighed and ground with dry ice using a Robot Coup; thehomogenized samples were placed into labeled, plastic containers and stored in a freezer (allowing the
dry ice to sublime). Extraction solution was added directly to pollen and nectar samples, which werethen shaken on a mechanical shaker for approximately one hour. After sample preparation, thehomogenized leaf and flower samples were stored in plastic containers and placed in a freezer until they
were sub-sampled for analysis; the pollen and nectar extracts were stored directly in a freezer in theextraction containers. Freezer-storage temperatures were monitored and typically were maintained at 20 ± 5 °C.
5E. ANALYTICAL METHODS
The analytical phase was conducted at ABC Laboratories, Inc. (Columbia, MO). The Principal Analytical
Investigator was Richard Schierhoff. Validated analytical methods were provided by the Sponsor to ABC
Laboratories, Inc. Prior to analysis of field samples, the analytical methods were verified by ABC
Laboratories, Inc. as part of this study.
Leaf and Flower Analysis
Leaf and flower samples were analyzed for thiamethoxam and CGA 322704 based on the analyticalmethod described in Syngenta Method REM 179.06, entitled “Residue Method for the Determination of
Residues of Thiamethoxam (CGA 293343) and CGA 322704 in Lettuce, Tomato, Grape and Tobacco902

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 49550801

CDPR THX Cucumber

Samples. Final Determination by LC-MS/MS”. In summary, residues of thiamethoxam and CGA322704
were extracted with 50:50 methanol/water from 10-g leaf and flower samples using a VirtiShear
homogenizer. Extracts were centrifuged and concentrated via SPE cleanup in preparation for LC-MS/MS
analysis. The Limit of Quantitation (LOQ) for both thiamethoxam and CGA322704, in leaf and flower
matrices, was 10.0 ppb. The Limit of Detection (LOD) was targeted to be 5 ppb in thiamethoxam and
CGA322704.
Pollen and Nectar Analysis
Pollen and nectar samples were analyzed for thiamethoxam and CGA322704 based on the analyticalmethod described in Syngenta Method REM 179.07, entitled “Thiamethoxam: Analytical Method for theDetermination of Residues of Thiamethoxam (CGA 293343) and CGA 322704 in Bee and Hive Products.
Final Determination by LC-MS/MS”. The method is presented in APPENDIX 3. In summary, residues ofthiamethoxam and CGA322704 were extracted with water from 0.05 g pollen and nectar samples. The
extraction was conducted with a VirtiShear homogenizer, and extracts were subsequently centrifuged
and passed through a solid-phase extraction cleanup in preparation for LC-MS/MS analysis. The Limit of
Quantitation (LOQ) for both thiamethoxam and CGA322704, in pollen and nectar matrices, was 1.00
ppb. The Limit of Detection (LOD) was targeted to be 0.5 ppb in thiamethoxam and CGA322704.
The LOQs and LODs are summarized in the table below.
Summary of LOQs and LODs
MatrixAnalyteCucumberThiamethoxam andLeaves andCGA322704
Flowers
CucumberThiamethoxam andPollen and
CGA322704
Nectar

LOQ (ppb, parent equivalents)
10

LOD (ppb, parent equivalents)
5.0

1.0

0.5

5F. QUALITY ASSURANCE RESULTS
For each matrix, at least one method-recovery (QC) sample per analytical set was prepared by fortifying
an untreated control sample with thiamethoxam and CGA322704 at concentrations equal to the method
LOQ or 10xLOQ . These samples were then analyzed concurrently with the treated field samples todemonstrate adequate method performance throughout the study, i.e. recoveries of 70-120%.
Syngenta Methods REM179.06 and REM179.07 were verified successfully at ABC Laboratories prior to
the analysis of field samples. Mean percent recoveries fell within the acceptable range of 70 - 120% withrelative standard deviations <20% between the three replicate analyses (n = 3).

6. RESULTS:
No residues >LOD were found in any untreated plant matrix, excluding a nectar sample from Trial Site 3.
Residues greater than the respective LOQs were found in all plant matrices sampled from the treatedcucumber plots. The residues in nectar ranged from 1.26 ppb to 11.48 ppb and from 1.29 ppb to 8.22
ppb in pollen.
903

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 49550801

CDPR THX Cucumber

Table 3. Residues of Thiamethoxam and CGA322704 for Nectar, Pollen, Flowers and Leaves of Cucumber
Site #
Treatment
MatrixResidue Concentraion
Thiamethoxam
CGA322704

TRT1

1

TRT2

TRT3

TRT1

2

TRT2

TRT3

TRT1

3

TRT2

NectarPollenFemale Flower
Male Flower
Leaf
NectarPollenFemale Flower
Male Flower
Leaf
NectarPollenFemale Flower
Male Flower
Leaf
NectarPollenFemale Flower
Male Flower
Leaf
NectarPollenFemale Flower
Male Flower
Leaf
NectarPollenFemale Flower
Male Flower
Leaf
NectarPollenFemale Flower
Male Flower
Leaf
NectarPollenFemale Flower
Male Flower
Leaf
904

7.77
2.81
11.24
13.25
60.27
7.77
7.98
16.97
24.20
136.97
11.48
2.45
26.54
9.84
110.25
6.41
4.67
14.86
13.33
59.83
9.25
4.10
21.57
14.03
61.43
7.50
8.22
23.65
17.73
87.68
1.26
1.29
3.29
3.77
20.59
1.31
3.86
3.56
4.06
18.06

1.18
0.81
2.12
1.50
10.52
1.61
2.09
1.72
4.46
24.24
1.75
0.94
7.07
2.24
22.65
1.14
1.22
0.00
2.22
10.41
1.65
1.71
6.27
1.78
12.24
1.04
2.58
2.55
2.68
16.69
0.00
0.23
0.00
0.00
0.00
0.00
0.52
0.00
0.00
0.00

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 49550801
Site #CDPR THX Cucumber

Treatment

MatrixResidue Concentraion
Thiamethoxam
CGA322704

TRT3

NectarPollenFemale Flower
Male Flower
Leaf

1.51
5.18
4.80
5.21
25.53

0.00
0.54
0.00
0.50
0.00

7. STUDY VALIDITY/CLASSIFICATION AND STUDY LIMITATIONSClassification/Utility for Bee Risk Assessment. This study is classified as acceptable. It provides asnapshot of Thiamethoxam and CGA322704 residues in leaves, flowers, pollen, and nectar during bloom.
The residue values presented should be considered to be fully reliable. However, it is important to notethat it is unclear if concentrations were increasing or decreasing at the time the samples were collected.
Temporal Variability in Residues. This study was not designed for temporal analysis of declining
concentrations, but rather, to provide a snapshot of residue concentrations during flowering. Only one
sample of each matrix was collected and analyzed from each plot so there is no way to know if
concentrations were increasing or decreasing.
Spatial Variability in Residues. Two sites were in Fresno County, in the San Joaquin Valley, and one site
was in San Luis Obispo County, in the Central Coast region. Climatic conditions were similar, except thatin Fresno County locations summer air temperatures were approximately 20°F warmer than the San Luis
Obispo County location. The locations in Fresno County (Trial Sites 1 and 2) both had sandy loam soil but
one of the sites had slightly coarser soil with more sand and less clay than the other site. The San Luis
Obispo County had fine soil that was classified as clay loam. Residue concentrations were higher in the
Fresno County sites than the San Luis Obispo County sites.
Pesticide Carryover. The extent to which prior year applications of imidacloprid contributed to year-toyear carryover was not a part of the study design. Therefore, the effects of pesticide carryover incucumber are unknown.
Table 4. Magnitude of Thiamethoxam and CGA322704 residues in leaves, flowers, pollen, and nectar(Trial Sites 1 and 2 = Fresno County; Trial Site 3 = San Luis Obispo County).
Trial
Site

1

Plant Matrix
Leaf
Female Flower
Male Flower
NectarPollenThiamethoxam Concentration (ppb)
MeanStandard
Maximum
Residue
Deviation
Residue
102.5
38.9
137.0
18.2
7.7
26.5
15.8
7.5
24.2
9.0
2.1
11.5
4.4
3.1
8.0
905

CGA322704 Concentration (ppb)
MeanStandard
Maximum
Residue
Deviation
Residue
22.4
8.8
28.3
4.2
3.5
8.3
3.2
1.8
5.2
1.8
0.3
2.0
1.5
0.8
2.4

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 49550801
Trial
Site

2

3

Plant Matrix

Leaf
Female Flower
Male Flower
NectarPollenLeaf
Female Flower
Male Flower
NectarPollenThiamethoxam Concentration (ppb)
MeanStandard
Maximum
Residue
Deviation
Residue
69.6
15.6
87.7
20.0
4.6
23.7
15.0
2.4
17.7
7.7
1.4
9.3
5.7
2.2
8.2
21.4
3.8
25.5
3.9
0.8
4.8
4.3
0.8
5.2
1.4
0.1
1.5
3.4
2.0
5.2

CDPR THX Cucumber

CGA322704 Concentration (ppb)
MeanStandard
Maximum
Residue
Deviation
Residue
15.3
3.8
19.5
3.4
3.7
7.3
2.6
0.5
3.1
1.4
0.2
1.6
2.1
0.8
3.0
0.0
0.0
0.0
0.0
0.0
0.0
0.2
0.3
0.6
0.0
0.0
0.0
0.5
0.2
0.6

8. STATISTICAL ANALYSIS
1. Sampling intervals between application and harvest of plant samples was nearly equal between
the trial sites.
2. There were 3 sites where initially they were designated as coarse, medium, and fine-textured
soils. Although the soil mapping unit indicated a loamy sand texture, the analysis of texture from soil
sampled at the site indicated a lower sand content that was more aligned with a determination of
medium texture (Table 2). Data were pooled from the medium-textured sites for comparison to data
generated from the fine-textured site. Three trials were conducted within each site, providing 3
replicate values for each soil texture category. There was no true replication for effect of site.
Results of statistical analysis for soil texture provides an indication of potential soil effects soconclusions are only tentative and require further testing to determine veracity.
3. Thiamethoxam parent residues were approximately 85% of the total residue measured for eachplant sample (Table S-1).
4. The maximum concentrations for total thiamethoxam residues measured were 161 ppb in leaves,13.2 ppb in nectar, and 11 ppb in pollen (Table S-1).
5. The box plots in Figure S-1 compare the range in total thiamethoxam residue concentration forthe plant tissues. Concentrations in leaves were approximately 10x greater than in the other plant
sample.
6. Comparison of the concentration of total thiamethoxam residue indicated a high correlation
between concentrations in leaves and nectar and in leaves and female flowers whereconcentrations increased in direct response to increases in leaves (Figure S-2). The correlation was
not as great between leaves and pollen, though there appeared to be a general positive relationshipbetween concentrations measured in the male flowers and pollen (Figure S-3).
7. Tables S-2 through S-6 compare the distribution of total thiamethoxam residues measured
between the samples from plants grown in medium and fine-textured soils. Figures S-4 though S-6906

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 49550801

CDPR THX Cucumber

compare the distribution of concentrations for total thiamethoxam residue. A test of potential
differences between the soil-texture categories was conducted using the Wilcoxon non-parametricrank sum test using the Exact option with the Monte Carlo procedure (Table S-5). A significant
difference in the distribution was measured for all plant samples except for pollen. The patternindicates that concentrations were higher in plants grown in medium-textured soils: Concentrationsin leaves were approximated 5 times greater, approximately 7 times greater in nectar,
approximately 6 times greater in female flowers, and approximately 4 times greater in male flowers
from plants sampled in medium-textured soils compared to plants grown in the fine-texture soil site.

Conclusion: The pattern of application used in this study resulted in low values for the maximum
concentration of total thiamethoxam residues in nectar and pollen of cucumber: Maximum
concentration measured for total thiamethoxam residue was 13.2 ug/L in nectar and 10.8 ug/L inpollen. Differences in concentration due to soil texture measured where, except for pollen, themagnitude of concentrations measured in plant samples was at least 5 times greater in plants grownin medium-textured soils as compared to those at the fine-textured plot site.

907

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 49550801

CDPR THX Cucumber

Table S-1. Statistical summary and distribution for concentration of thiamethoxam, CGA322704 degradant, and total residues measured in leaves, female
flowers, nectar, male flowers, and pollen of cucumber plants.
LeavesStatisticN
MeanSD
CV (%)MinMedian
75th
90th
95th
Max
% of Total

Thiamethoxam CGA322704
9
9
64.5
10.8
41.1
9.4
63.8
87.5
18.1
0.0
60.3
10.5
87.7
16.7
137.0
24.2
137.0
24.2
137.0
24.2
85.7
14.3

Female Flowers
Total
9
75.3
50.4
70.0
18.1
70.8
104.4
161.2
161.2
161.2

Thiamethoxam CGA322704
9
9
14.1
2.2
8.9
2.7
63.3
123.9
3.2
0.0
14.9
1.7
21.6
2.6
26.5
7.1
26.5
7.1
26.5
7.1
86.5
13.5

Male Flowers
StatisticN
MeanSD
CV (%)MinMedian
75th
90th
95th
Max
% of Total

Thiamethoxam CGA322704
9
9
11.7
1.7
6.8
1.4
58.1
83.8
3.8
0.0
13.3
1.8
14.0
2.2
24.2
4.5
24.2
4.5
24.2
4.5
87.3
12.7

Nectar
Total
9
16.3
11.2
69.2
0.3
14.9
26.2
33.6
33.6
33.6

Pollen
Total
9
13.4
8.2
60.9
3.8
14.8
15.8
28.7
28.7
28.7

Thiamethoxam CGA322704
9
9
4.5
1.2
2.4
0.8
52.3
67.0
1.3
0.2
4.1
0.9
5.2
1.7
8.2
2.6
8.2
2.6
8.2
2.6
79.3
20.7
908

Total
9
5.7
3.0
53.5
1.5
5.7
5.9
10.8
10.8
10.8

Thiamethoxam CGA322704 Total
9
9
9
6.0
0.9
7.0
3.8
0.7
5.0
62.6
79.4
64.6
1.3
0.0
1.3
7.5
1.1
8.5
7.8
1.6
9.4
11.5
1.8
13.2
11.5
1.8
13.2
11.5
1.8
13.2
86.6
13.4

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 49550801

CDPR THX Cucumber

Table S-2 Leaves: Statistical summary and distribution for concentration of thiamethoxam, CGA322704 degradant
and total residues measured in leaves of cucumber plants grown in fine and medium-textured soil.
LeavesMedium Textured Soil
StatisticN
MeanSD
CV (%)MinMedianMax% of Total

Thiamethoxam CGA322704
6
86.1
32.1
37.3
59.8
74.6
137.0
84.2

Fine Textured Soil
Total

6
16.1
6.1
38.0
10.4
14.5
24.2
15.8

6
102.2
38.1
37.3
70.2
89.0
161.2

909

Thiamethoxam CGA322704
3
21.4
3.8
17.8
18.1
20.6
25.5
100.0

3
0.0
0.0
0.0
0.0
0.0
0.0
0.0

Total
3
21.4
3.8
17.8
18.1
20.6
25.5

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 49550801

CDPR THX Cucumber

Table S-3 Nectar and female flowers: Statistical summary and distribution for concentration of thiamethoxam,
CGA322704 degradant, and total residues measured in nectar and female flowers of cucumber plants grown in fineand medium-textured soil.
NectarMedium Textured Soil
StatisticN
MeanSD
CV (%)MinMedianMax% of Total

Thiamethoxam CGA322704
6
8.4
1.8
21.2
6.4
7.8
11.5
85.7

6
1.4
0.3
22.1
1.0
1.4
1.8
14.2

Fine Textured Soil
Total
6
9.8
2.0
20.8
7.6
9.2
13.2

Thiamethoxam CGA322704
3
1.4
0.1
9.7
1.3
1.3
1.5
100.0

3
0.0
0.0
0.0
0.0
0.0
0.0
0.0

Total
3
1.4
0.1
9.7
1.3
1.3
1.5

Female Flowers
Medium Textured Soil
StatisticN
MeanSD
CV (%)MinMedianMax% of Total

Thiamethoxam CGA322704
6
19.1
5.8
30.2
11.2
19.3
26.5
85.2

6
3.3
2.8
84.2
0.0
2.3
7.1
14.7

Fine Textured Soil
Total
6
22.4
8.0
35.8
13.4
22.5
33.6

910

Thiamethoxam CGA322704
3
3.9
0.8
20.7
3.3
3.6
4.8
100.0

3
0.0
0.0
0.0
0.0
0.0
0.0
0.0

Total
3
3.9
0.8
20.7
3.3
3.6
4.8

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 49550801

CDPR THX Cucumber

Table S-4 Pollen and male flowers: Statistical summary and distribution for concentration of thiamethoxam,
CGA322704 degradant, and total residues measured in nectar and female flowers of cucumber plants grown in fineand medium-textured soil.
PollenMedium Textured Soil
Statistic

Thiamethoxam CGA322704

Fine Textured Soil
Total

Thiamethoxam CGA322704

Total

N

6

6

6

3

3

3

Mean
SD
CV (%)MinMedianMax% of Total

5.0
2.5
49.8
2.5
4.4
8.2
76.4

1.6
0.7
44.5
0.8
1.5
2.6
23.6

6.6
3.2
47.9
3.4
5.9
10.8

3.4
2.0
57.5
1.3
3.9
5.2
88.9

0.4
0.2
40.4
0.2
0.5
0.5
11.1

3.9
2.2
55.4
1.5
4.4
5.7

Male Flowers
Medium Textured Soil
StatisticN
MeanSD
CV (%)MinMedianMax% of Total

Thiamethoxam CGA322704
6
15.4
5.0
32.4
9.8
13.7
24.2
86.1

6
2.5
1.1
42.4
1.5
2.2
4.5
13.9

Fine Textured Soil
Total
6
17.9
5.93
33.2
12.1
15.7
28.7

911

Thiamethoxam CGA322704
3
4.4
0.8
17.5
3.8
4.1
5.2
96.5

3
0.2
0.3
173.0
0.0
0.0
0.5
3.8

Total
3
4.5
1.0
23.2
3.8
4.1
5.7

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 49550801

CDPR THX Cucumber

Table S-5. Statistical comparison between concentration of total thiamethoxam residue between soil types
measured in leaves, female flowers, nectar, male flowers, and pollen where C=coarse-textured, M=mediumtextured, and F=fine-textured soil. Wilcoxon rank sum test is a nonparametric test for differences amongst the 3categories with the test run using the Exact option. The T-test is a test for differences when there are two categories
and it is run to provide guidance when the Wilcoxon test indicates a significant difference amongst the three soil
categories.

Probability Level Wilcoxon Test
Soil
Comparison
Medium vs
Fine Textured

LeavesFemale Flowers

Nectar

0.022

0.024

0.024

912

Male

Flowers
0.021

Pollen0.26

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 49550801

CDPR THX Cucumber

Figure S-1. Box plots comparing the distribution of concentration of total thiamethoxam residues in samples of planttissue of cucumber.
Thiamethoxam - Cucumber Sampled 2012

Total Thiamethoxm Residue Concentration (ppb)

200

150

100

50

0
Female_Flower

Leaf

Male_Flower

source913

NectarPollenAppendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 49550801

CDPR THX Cucumber

Figure S-2. Relationship of total thiamethoxam residue measured A) in the leaves and nectar and B) in the female
flowers and nectar.

Nectar Total Thiamethoxam Residue (ppb)

A. Relationship between leaves and nectar

20.00

y = 0.0739x + 1.3967
R² = 0.6874

15.00

10.00

5.00

0.00

0

50

100

150

200

Leaves Total Thiamethoxam Residue(ppb)
B. Relationship between female flowers
and nectar.

Nectar Total Thiamethoxam Residue
(ppb)

20.00

y = 0.3734x + 0.8925
R² = 0.8723

15.00

10.00

5.00

0.00

0

10

20

30

40

Female Flower Total Thiamethoxam Residue
(ppb)
914

50

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 49550801

CDPR THX Cucumber

Figure S-3. Relationship of total thiamethoxam residue measured A) in the leaves and pollen and B) in the male
flowers and pollen.
A. Relationship between leaves and pollen

Pollen Total Thiamethoxam Residue
(ppb)

20.00

y = 0.0345x + 3.0907
R² = 0.3274

15.00

10.00

5.00

0.00

0

50

100

150

200

Leaves Total Thiamethoxam Residue (ppb)

Pollen Total Thiamethoxam Residue (ppb)

B. Relationship between male flowers and pollen.

20.00

y = 0.2911x + 1.7814
R² = 0.6126

15.00

10.00

5.00

0.00

0

10

20

30

40

Male Flowers Total Thiamethoxam Residue
(ppb)
915

50

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 49550801

CDPR THX Cucumber

Figure S-4 Leaves. Comparison of distribution of total thiamethoxam residue for medium, and finetexture soils in leaves of cucumber.
Cucumber Leaves
Distribution of totraw

Percent

60

Fine

40
20
0
50

MediumPercent

40
30
20
10

soilcat0
Fine
Medium
0

50

100

150

totraw
Normal916

Kernel200

250

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 49550801

CDPR THX Cucumber

Figure S-5. Comparison of distribution of total thiamethoxam residue for medium, and fine-texture
soils in female flowers and nectar of cucumber.
A. Female Flowers of Cucumber
Distribution of totraw
Fine

Percent

30
20
10
0

MediumPercent

30
20
10

soilcat0
Fine
Medium0

40

20

totraw
NormalKernelB. Nectar
Distribution of totraw

Percent

60

Fine

40
20
0
50

MediumPercent

40
30
20
10

soilcat0
Fine
Medium0

5

10

totraw
Normal917

Kernel15

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 49550801

CDPR THX Cucumber

Figure S-6. Comparison of distribution of total thiamethoxam residue for medium, and fine-texture
soils in male flowers and pollen of cucumber.
A. Male Flowers of Cucumber
Distribution of totraw

Percent

60

Fine

40
20
0
40

MediumPercent

30
20
10

soilcat0
Fine
Medium
0

10

20

30

totraw
NormalKernelB. Pollen
Distribution of totraw
40

Fine

Percent

30
20
10
0

Percent

30

Medium20
10

soilcat0
Fine
Medium
-5

0

5

10

totraw
Normal21918

Kernel15

20

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 49550801

CDPR THX Cucumber

9. REFERENCES
1. Mair, P. (1998) Stability of Residues of CGA-293343 (2 Years Final Report) and CGA-322704 (1Year Interim Report) in Plant Material under Deep Freezer Conditions, Including Method
Validation (Study No. 504/96 consists of Reports #112/96, 127/97, 103/98) MRID 44703525.
2.

Hohl, J. (1999) Stability of Residues of CGA-322704 in Plant Material and Soil Stored Under Deep
Freezer Conditions (Study No. 779-00) MRID 45108001.

3. Oakes, T. (2002) Stability of CGA-293343 and CGA-322704 in Crops and Processed Fractions
Under Freezer Storage Conditions (Study No. 269-98) MRID 45659205.
4. Anderson, L. (2007) Thiamethoxam (CGA293343) and CGA322704. Validation of Residue
Analytical Method REM 179.07 for the determination of Residues in Bee and Hive Products andStorage Stability in Hive Pollen, Wax and Nectar, stored Deep Frozen for 12 months. (Study No.
05-S508).
5. Crook, S (2004) Residue Method for the Determination of Residues of Thiamethoxam(CGA293343) and CGA322704 in Lettuce, Tomato, Grape and Tobacco Samples. Final
Determination by LC/MS/MS" (Syngenta Method REM179.06)
6. Crook, S (2007) Analytical Method for the Determination of Residues of Thiamethoxam(CGA293343) and CGA322704 in Bee and Hive Products. Final Determination by LC-MS/MS(Syngenta Method REM179.07)22919

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50131102

CDPR THX Citrus

Reference
Lange,B., and Rice, F. (2016) Thiamethoxam 75SG (A9549C) – Magnitude of Residues in Leaves,
Flowers, Pollen, and Nectar of Citrus After Soil Application with Platinum 75 SG in California: Final
Report. Project Number: TK0177221. Unpublished study prepared by Syngenta Crop Protection, LLC.
337p. MRID 50131102, CDPR Study ID 297891, Data Volume 52691-0571, Tracking ID# 280248

1. STUDY INFORMATION
Chemical:Test Material:

Study Type:

Sponsor:Sponsor Study
ID:
Study
Completion
Date:

GLP Status:

PC Code060109Percent
Platinum 75 SG
Active42.8%
Ingredient:
Field residue study on Citrus to establish thiamethoxam and metabolite levels in
nectar, pollen, whole flowers and leaves in 9 trial site locations following two soil
applications Platinum 75 SG in two successive years.
Syngenta Crop Protection, LLC
410 Swing Road
Post Office Box 18300
Experiment Start and September 13, 2013 –Greensboro, North Carolina
End Date:
July 16, 201527419-8300 USA
Thiamethoxam

TK0177221
Study Locations:January 13, 2016

Nine trial sites that
included orange andlemon located in
California.

GLP; protocol reviewed by CDPR.
[CDPR Study ID 297891, Data Volume 52691-0571, Tracking ID# 280248]

2. REVIEWER INFORMATION
Study Reviewed by:
California Departmentof Pesticide Regulation

Richard Bireley, Sr. Environmental Scientist (Specialist)
John Troiano, Ph.D., Research Scientist III
Alexander Kolosovich, Sr. Environmental Scientist
Brigitte Tafarella, Environmental Scientist
Denise Alder, Sr. Environmental Scientist (Specialist)
Russell Darling, Sr. Environmental Scientist (Specialist)

3. EXECUTIVE SUMMARY
Nine (9) field trials were conducted in the United States for the purpose of quantifying residues ofthiamethoxam and its major metabolite, CGA322704, in leaves, flowers, pollen, and nectar from citrus
after soil treatment applications with Platinum® 75SG. This study was conducted and reported to satisfy
data requested by the California Department of Pesticide Regulations (CDPR) as part of the re-evaluationof the nitro-guanidine class of neo-nicotinoid insecticides (Article 8, Subchapter 1, Chapter 2, Division 6
of Title 3 of the California Code of Regulations).

1920

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50131102

CDPR THX Citrus

For this study, thiamethoxam (CGA293343), a 75% soluble granule formulation (75% w/w), was applied
to commercial varieties/cultivars of citrus via soil directed spray application. At all field sites leaves,whole flower, pollen and nectar were collected 45 (± 10) days after the last application to fulfill early
bloom sampling events, with the following exceptions. Year 2 samples for CA-3 were collected 59 days
after the application. Year 1 samples for CA-7 were collected 30 days after the application. Year 2
samples were not collected from CA-7 and CA-8, so these trials were continued a third season, resulting
in three annual applications for the “year 2” samples collected for these trials. Representative soil
samples were collected to confirm soil-textural class.
Leaf, whole flower, pollen, and nectar samples were analyzed by validated methods using HPLC-MS/MS
for thiamethoxam and CGA322704 (see Sections 2.4.4.1 and 2.4.4.2). Performance of analytical methods
were verified using control (UTC) samples of leaf, whole flower, pollen, and nectar prior to any analysis
of field samples by analyzing one UTC and three UTC samples fortified with thiamethoxam and
CGA322704 at the respective LOQs and at 10xLOQs. The Limit of Quantitation (LOQ) was 1 ppb for bothanalytes in leaves, flowers, and pollen. The LOQ was 0.5 ppb for both analytes in nectar.
All matrices were analyzed for thiamethoxam and CGA322704 within 435 days (14 months) after sample
collection. Residues have been shown to be stable for at least 24 months in a range of crops.
Individual recoveries from analyte-fortified samples that were analyzed concurrently with field samples
ranged between 69.5 to 112% for thiamethoxam and 76.4 to 115% for CGA322704. The mean recoveryof each matrix was within 70 to 120%, and the relative standard deviations (RSD) were < 20%, indicating
acceptable performance of the analytical method during the conduct of this study.

4. STUDY VALIDITY

Guideline Followed:
Guideline Deviations:
Other Deviations:Classification:
Rationale:
Reparability:

Protocol was reviewed by CDPR
N/A
N/A
ACCEPTABLE
N/A
N/A

5. MATERIALS AND METHODS

Test item:
Description:
CAS #:

Test Material Characterization
Platinum 75 SG
Percent Active Ingredient:
Soluble granule
Design Code:
153719-23-4
Molecular Weight:

75% w.w A.I.
A9549C
291.715A. STUDY DESIGN
The study included nine geographically separated replicated trials that each consisted of a non-treated
and a treated plot large enough to fulfil sample collection requirements. The treated plots were divided
into 3 replicate sub-plots (A, B, and C). The size of each sub-plot varied at each location, measuring 100
ft x 21 ft (CA-1), 90 ft x 21 ft (CA-2), 60 ft x 28 ft (CA-3), 55 ft x 22 ft (CA-4), 110 ft x 20 ft (CA-5), 100 ft x
20 ft (CA-6), 105 ft x 20 ft (CA-7 and CA-8), and 50 ft x 20 ft (CA-9). At each location, the control plot was
located up-slope and up-wind with regard to the prevailing wind direction and separated by a minimum
2921

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50131102

CDPR THX Citrusof 200 ft. (exception CA-2, 125 ft) from the treated plot to minimize potential cross-contamination by
runoff or pollen transfer.
5B. APPLICATION TIMING AND RATESAll trial locations established plots for a non-treated control plot (01) and a treated plot (02). The
treatment list is presented in table 1 below.
Table 1. Application Rates and Timing

Treatment Application
ID (Plot)
NumberYear

End-Use
Product

Treatment List
Application Volume
Method(GPA)-Platinum
75 SG
A9549C

Control
Soil
Directed
Spray

01

--

--

02

1

1

02

2

2

Platinum
75 SG
A9549C*02

3

3

Platinum
75 SG
A9549C--

NominalRate (lbai/Acre)Per
Application-->50

0.172 lb
ai/AcreSoil
Directed
Spray

>50

0.172 lb
ai/AcreSoil
Directed
Spray

>50

0.172 lb
ai/AcreApplication Notes:
GPA=gallons per acre
*Trials CA-7 and CA-8 only, per amendment 6.

3
922

TimingTotal Rate (lbai/Acre) PerYear-45 (±10days)
BeforeBloom
45 (±10days)
BeforeBloom
45 (±10days)
BeforeBloom-0.172

0.172

0.172

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50131102

CDPR THX Citrus

5C. STUDY SITE LOCATION AND CHARACTERISTICS
Table 2. Study Site Location and Characteristics
Trial (Field)
Trial
OM pH
CEC
%
%
Identification
Location(%)(meq/100 Sand Silt
and Crop(City, State)
g soil)CA-1
Sanger,0.22 7.5
5.3
84
9(Valencia
California
0.13 7.5
3.3
88
7Orange)Not CollectedCA-2
Orange0.53 5.5
7.7
78
15(Valencia
Cove,
0.31 6.6
9.5
78
13
Orange)California
Not Collected
CA-3
San Luis
3.1 7.4
24.8
46
26(Lisbon Lemon)
Obispo,California
1.4 7.4
24.3
48
26

CA-4
(Valencia
Orange)
CA-5(Valencia
Orange)San LuisObispo,California
Navelencia,
California

CA-6(Valencia
Orange)CA-7(Pryor Lemon)

OrangeCove,
California
Porterville,
California

CA-8(Lisbon Lemon)

Porterville,
California

CA-9(Valencia
Orange)Fresno,California%
Clay

Soil Types

Rainfall(in)

Temperature
Range (°F)

75Loamy Sand
Sand

0 to 2.4

26 to 1087
9Loamy Sand
Sandy Loam

0 to 2.4

26 to 108

28

Sandy Clay
Loam
Sandy Clay
Loam
Sandy Loam
Clay Loam
Clay Loam
Clay Loam
Sandy Loam
Loamy Sand
Sandy Clay
Loam
Sandy Loam
Sandy Loam
Sandy Loam
Sandy Clay
Loam
Clay Loam
Clay Loam
Clay
Clay
Clay
Sandy Loam
Sandy Loam

0 to 5.29

29 to 100

0 to 5.29

29 to 100

0 to 2.15

27.3 to 105

0 to 2.15

27.3 to 105

0 to 3.12

21.5 to 108

0 to 3.12

21.5 to 108

0 to 2.51

25.8 to 110

26

0.76
1.8
1.3
1.3
1.10
0.40
0.48

7.5
7.4
7.6
7.7
7.8
7.6
7.5

18.5
28.4
32.7
33.1
9.1
7.7
16.8

66
42
34
42
77
81
51

16
26
30
24
13
9
23

18
32
36
34
10
10
26

1.01
0.57
0.62
2.3

6.7
6.5
6.6
7.5

14.8
16.7
17.4
31.2

59
53
55
46

25
27
27
19

16
20
18
35

0.94
0.59
1.8
1.02
0.54
1.2
1.04

7.6
7.9
7.3
7.7
7.7
8.1
8.0

32.7
31.4
34.8
35.9
37.3
13.3
15.9

44
19
44
19
34
23
30
23
32
23
68
19
66
19
Not Collected

37
37
43
47
45
13
15

Precipitation and air temperature data summarized above are representative of the time period (wholemonths) from first application through final sample collection for each trial. Weather conditions did not
negatively impact the crop growth or development. Irrigation was used to supplement rainfall at all trial
locations and the data are provided in the Field Trial Summaries (Appendix 1 of the study report).

4923

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50131102

CDPR THX Citrus

5D. SAMPLE COLLECTION, HANDLING, PROCESSING

For all trials, the non-treated control plots were sampled first or by different personnel to prevent
contamination. For each matrix, one sample was collected from the control plot and each treatedreplicate plot A, B, and C.
At all field sites, leaves, whole flower, pollen and nectar were collected 45 (± 10) days after the last
application to fulfill early bloom sampling, with the following exceptions: Year 2 samples for CA-3 werecollected 59 days after the application; Year 1 samples for CA-7 were collected 30 days after theapplication; Year 2 samples were not collected from CA-7 and CA-8 due to low flowering so these trials
were continued a third season.
Leaf and Whole Flower
Target weights of 250 g for leaves and flowers were collected, except for CA-3 where the Year 2 flower
samples weighed between 45 to 70 g. Additionally, bulk, non-treated leaves and flowers with target
weights of 500 g each were collected for laboratory verification and concurrent fortifications. Leaves
and flowers were collected directly into labelled, sealable plastic bags and held in separate control andtreatment ice chests on substitute ice until placed into frozen storage. Samples were collected from thelower-, middle- and upper-plant canopy for a representative, composite sample.
Pollen and Nectar
Flowers were collected from the untreated control and the treated sub-plots, bagged and placed in ice
chests with substitute ice then transported to the field laboratory for pollen and nectar extraction.
Pollen samples were extracted manually from flowers using a plastic filtered collection tip which was
attached to a vacuum pump. The tips were weighed before and after pollen extraction and the net
weight between the two represented the sample size. Once the target weight of 30 mg was obtained, or
all flowers available for pollen sampling were used, the plastic tips containing pollen were wrapped inparafilm and placed in labeled plastic bottles. The bottles were sealed, placed in resealable plastic bags,
and transferred immediately into separate freezers for treatment and control samples.
Nectar samples were collected manually. A glass microcapillary pipette was used to extract nectar fromthe inside base of the flower and then to transfer the nectar into a pre-weighed glass vial. Each vial was
weighed before and after nectar extraction, with the net weight between the two representing thesample size. Once the target weight of 100 mg was obtained, or all flowers available for nectar sampling
were used, the vials containing nectar were sealed in individual labeled plastic centrifuge tubes, thenplaced in resealable plastic bags and transferred immediately into separate freezers for treatment andcontrol samples.
Target weights of 30 mg for pollen and 100 mg for nectar were collected, except for CA-3 where theYear 2 nectar and pollen samples did not meet size requirements.
Transit Stability
At trial CA-1 and CA-2, plant matrix samples from the field sites were pre-weighed at the laboratory.
Homogenized control leaves and flower samples were used to prepare single control and triplicate
treated samples of leaves and flowers for fortification at the field site. The pre-weighed leaves and5924Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50131102

CDPR THX Citrus

flower samples and pre-measured vials of fortification solution were shipped to the field site. The
contents of a vial were emptied into a bottle containing the homogenized sample, and then rinsed into
the bottles with samples. The vials were then paced into the sample container. The target fortificationlevel for all samples was 50X LOQ. The sample container was secured with electrical tape, and placed
into an ice chest on substitute ice until placed into frozen storage.
Sample Storage.
All residue samples (leaf, whole flower, pollen, and nectar) were shipped from the test sites to EPL Bio
Analytical Services via ACDS freezer truck.
Three separate storage-stability studies, MRID 44703525 (Reference 4), MRID 45108001 (Reference 5)
and MRID 45659205 (Reference 6), were conducted to determine the stability of thiamethoxam and its
metabolite, CGA322704, in various crop matrices stored under deep-freezer conditions. Storage
stability for pollen and nectar stored under deep-freezer conditions was conducted in Syngenta Study
No. 05-S508 (Reference 7). These studies showed that thiamethoxam and metabolite CGA322704 arestable in leaves, whole flower, pollen, and nectar for up to 24 months when stored frozen. Therefore,
residues of thiamethoxam and CGA322704 in citrus leaf, whole flower, pollen, and nectar samples
should not have been adversely affected by frozen storage during this study.
5E. ANALYTICAL METHODS
Leaves, whole flowers, pollen and nectar were analyzed for thiamethoxam and its metabolite
CGA322704. Leaf samples were analyzed using EPL method 110G747D, entitled “Analytical Method forthe Determination of Residues of Thiamethoxam and CGA322704 in Leaves by LC-MS/MS”, which is anadaptation of Syngenta methods REM179.06 (Reference 1) and REM179.07 (Reference 2). Whole flower
samples were analyzed using EPL method 110G747C, entitled “Analytical Method for the Determinationof Residues of Thiamethoxam and CGA322704 in Flowers by LC-MS/MS”, which is also an adaptation ofSyngenta methods REM179.06 (Reference 1) and REM179.07 (Reference 2). Pollen samples wereanalyzed using EPL method 110G747B (and Revision 1), entitled “Analytical Method for theDetermination of Residues of Thiamethoxam and CGA322704 in Pollen by LC-MS/MS,” which is arevision of Syngenta method REM179.07 (Reference 2). Nectar samples were analyzed using EPL
method 110G747A (and Revision 1), entitled “Analytical Method for the Determination of Residues ofThiamethoxam and CGA322704 in Nectar by LC-MS/MS,” which is based on Syngenta method
REM179.07 (Reference 2). The LOQ was 1 ppb, and the LOD was 0.5 ppb for both analytes in leaves,whole flowers and pollen. The LOQ was 0.5 ppb, and the LOD was 0.25 ppb for both analytes in nectar.
The detailed analytical methods appear in appendix 2 of the study report.
Means, medians, standard deviations, and relative standard deviations were calculated using Microsoft
Excel 2010 where appropriate.
Analysis of Leaf Samples
In summary, residues of thiamethoxam and CGA322704 were extracted with 50:50 methanol:0.2%
formic acid from 0.1 g leaf samples. Extracts were centrifuged and aliquots were diluted with deionized(DI) water and stable isotope labelled internal standards were added. Sample extracts were thenpurified by solid-phase extraction (Oasis HLB) and analyzed by high performance liquid chromatography
with triple quadrupole mass spectrometric detection (LC-MS/MS). The LOQ for both analytes in leaves
6925Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50131102

CDPR THX Citrus

was 1.0 ppb and the LOD was 0.5 ppb.
Analysis of Whole Flower Samples

In summary, residues of thiamethoxam and CGA322704 were extracted with 50:50 methanol:0.2%
formic acid from 0.1 g flower sub-samples. Extracts were centrifuged and aliquots were diluted with DI
water, and stable isotope labelled internal standards were added. Sample extracts were then purified bysolid-phase extraction (Oasis HLB) and analyzed by high performance liquid chromatography with triple
quadrupole mass spectrometric detection (LC-MS/MS). The LOQ for both analytes in whole flowers was
1.0 ppb and the LOD was 0.5 ppb.
Analysis of Pollen Samples
In summary, residues of thiamethoxam and CGA322704 were extracted with 50:50 methanol:0.2%
formic acid from pollen samples. Pollen samples collected in the field were received in plastic pipettetips. To prepare these samples for extraction, the pipette tips containing pollen were cut into 3 pieces,
and as much of the pollen as possible was transferred to a 50 mL plastic centrifuge tube. The filters used
to contain the pollen in the pipette tips were also placed in the centrifuge tube, followed by the pipettetips. The extraction solvent was added to the tube which was then mixed with a vortexing apparatus toachieve extraction of the analytes. Weights of each pollen sample were recorded in the field andprovided to EPL. The weights were needed to determine the residue concentrations and to determine
the final volume of solvent needed following the solid phase extraction cleanup. Extracts were
centrifuged and aliquots were diluted with DI water, and stable isotope labelled internal standards wereadded. Sample extracts were then purified by solid-phase extraction (Oasis HLB) and analyzed by highperformance liquid chromatography with triple quadrupole mass spectrometric detection (LC-MS/MS).
The LOQ for both analytes in pollen was 1.0 ppb and the LOD was 0.5 ppb.
Analysis of Nectar Samples
In summary, residues of thiamethoxam and CGA322704 were extracted with 50:50 methanol:0.2%
formic acid from 0.1 g nectar samples. Extracts were centrifuged and aliquots were diluted with DI
water. Sample extracts were then purified by solid-phase extraction (Oasis HLB) and analyzed by highperformance liquid chromatography with triple quadrupole mass spectrometric detection (LC-MS/MS).
The LOQ for both analytes in nectar was 0.5 ppb and the LOD was 0.25 ppb.
Table 3. Summary of LOQs and LODs
MatrixAnalyteLemon and Orange
Thiamethoxam
LeavesCGA322704
Lemon and Orange
Thiamethoxam
PollenCGA322704
Lemon and Orange
Thiamethoxam
NectarCGA322704
Lemon and Orange
Thiamethoxam
Whole Flower
CGA322704

LOQ (ppb, parent equivalents)
1.0
1.0
1.0
1.0
0.5
0.5
1.0
1.0

5F. QUALITY ASSURANCE RESULTS
7926

LOD (ppb, parent equivalents)
0.5
0.5
0.5
0.5
0.25
0.25
0.5
0.5

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50131102

CDPR THX Citrus

Method verification was performed on fortified leaf, whole flower, pollen, and nectar samples prior to
the analysis of field collected samples. Triplicate fortifications at the LOQ and 10x LOQ concentrations
were made for each analyte in each plant matrix. With only one exception, individual recoveries fellwithin the range of 70-120%. A single whole flower LOQ fortification yielded a CGA322704 recovery of
64.6%. Mean recoveries at each fortification level fell within the range of 70-120% for both analytes inall four plant matrices. Relative standard deviations (RSD) at each level were less than 20% for bothanalytes in all four plant matrices.
For each matrix, two concurrent recovery samples per analytical set were prepared by fortifying an
untreated control sample with thiamethoxam and CGA322704 at concentrations samples todemonstrate acceptable method performance throughout the study. Mean concurrent method
recoveries in the range of 70-120% were used to confirm analytical method performance. With a single
exception, individual recoveries from analyte-fortified samples that were analyzed concurrently withfield collected samples fell within the range of 70-120% for all matrices. One pollen LOQ fortification
yielded a recovery of 69.5% for thiamethoxam. All mean recoveries fell within the range of 70-120%, and
all RSD values were less than 20% for both analytes in all plant matrices.
An Agilent 1290 HPLC system with an AB Sciex Triple Quad 6500 mass spectrometer detector was used
for the separation and quantitation of thiamethoxam and CGA322704. For the quantitation of theanalytes of interest, standard curves were prepared by injecting constant volumes of standard solutions
ranging in concentration from 0.004 - 1.0 ng/mL. Constant volume injections were used for sample
extracts as well. A calibration standard typically was injected every 2-5 sample injections. Linear
regression with 1/x weighting was used.

8927

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50131102

Thiamethoxam Residue
(ppb)

UTC
TRT A
TRT B
TRT C
UTC
TRT A
TRT B
TRT C
UTC
TRT A
TRT B
TRT C
UTC
TRT A
TRT B
TRT C
UTC
TRT A
TRT B
TRT C
UTC
TRT A
TRT B
TRT C
UTC
TRT A
TRT B
TRT C
UTC
TRT A
TRT B
TRT C

<LOD
58.8
84.9
17.0
<LOD
14.0
33.9
15.6
<LOD
3.69
2.36
2.72
<LOD
<LOD
<LOQ
<LOQ
<LOD
2.43
3.02
1.29
<LOD
2.95
3.10
3.14
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
1.10

CGA322704 Residues (ppb)

Treatment CodeYear

Crop and Variety

Location (City, State)

Table 4. Thiamethoxam Residue Data from Orange and Lemon Trees

Trial Identification

6. RESULTS:

CDPR THX Citrus

Citrus Leaves
CA-1

Sanger,California

Orange1

CA-2

OrangeCove,
California

Orange1

CA-3

San LuisObispo,California

Lemon

1

CA-4

San LuisObispo,California

Orange1

CA-5

Navelencia,
California

Orange1

CA-6OrangeCove,
California

Orange1

CA-7

Porterville,
California

Lemon

1

CA-8

Porterville,
California

Lemon

1

9
928

<LOD
42.2
75.6
9.70
<LOD
5.83
16.1
10.3
<LOQ
1.07
<LOQ
<LOQ
<LOD
<LOD
<LOD
<LOD
<LOD
2.16
2.23
1.44
<LOD
2.28
2.72
2.63
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

Fresno,California

Orange1

CA-1

Sanger,California

Orange2

CA-2

OrangeCove,
California

Orange2

CA-3

San LuisObispo,California

Lemon

2

CA-4

San LuisObispo,California

Orange2

CA-5

Navelencia,
California

Orange2

CA-6

OrangeCove,
California

Orange2

CA-7

Porterville,
California

Lemon

2

CA-8

Porterville,
California

Lemon

2

10
929

UTC
TRT A
TRT B
TRT C
UTC
TRT A
TRT B
TRT C
UTC
TRT A
TRT B
TRT C
UTC
TRT A
TRT B
TRT C
UTC
TRT A
TRT B
TRT C
UTC
TRT A
TRT B
TRT C
UTC
TRT A
TRT B
TRT C
UTC
TRT A
TRT B
TRT C
UTC
TRT A
TRT B

<LOD
<LOQ
<LOD
1.07
<LOD
67.3
69.4
40.5
<LOD
31.2
30.8
15.3
<LOD
14.5
5.11
6.49
<LOD
<LOQ
1.56
1.07
<LOD
1.37
2.21
<LOQ
<LOD
3.46
2.48
3.46
<LOD
<LOD
<LOD
<LOQ
<LOQ
2.43
2.58

CGA322704 Residues (ppb)

Thiamethoxam Residue
(ppb)

CA-9

Treatment CodeYear

Trial Identification

Crop and Variety

CDPR THX Citrus

Location (City, State)

MRID 50131102

<LOD
<LOD
<LOD
<LOQ
<LOD
47.1
47.8
29.1
<LOD
33.2
21.0
20.6
<LOD
20.2
2.11
3.38
<LOD
<LOQ
1.07
<LOQ
<LOD
2.33
3.15
<LOQ
<LOD
2.82
2.23
2.95
<LOD
1.07
<LOQ
<LOQ
1.06
1.71
1.37

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

CGA322704 Residues (ppb)

Thiamethoxam Residue(ppb)

OrangeTreatment CodeFresno,California

Year

Trial Identification
CA-9

Crop and Variety

CDPR THX Citrus

Location (City, State)

MRID 50131102

2

TRT C
UTC
TRT A
TRT B
TRT C

2.32
<LOD
<LOD
<LOD
<LOD

1.93
<LOD
<LOD
<LOD
<LOD

UTC
TRT A
TRT B
TRT C
UTC
TRT A
TRT B
TRT C
UTC
TRT A
TRT B
TRT C
UTC
TRT A
TRT B
TRT C
UTC
TRT A
TRT B
TRT C
UTC
TRT A
TRT B
TRT C
UTC
TRT A
TRT B
TRT C
UTC

<LOD
49.0
16.7
31.4
<LOD
10.3
14.9
12.9
<LOD
<LOQ
<LOQ
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
2.02
2.06
<LOQ
<LOD
2.10
1.86
2.23
<LOD
<LOD
<LOQ
<LOD
<LOD

<LOD
<LOD
10.6
4.02
5.84
<LOD
1.74
3.09
2.58
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOQ
<LOQ
<LOD
<LOD
<LOQ
<LOQ
<LOQ
<LOD
<LOD
<LOQ
<LOQ
<LOD

Citrus Whole Flower
CA-1

Sanger,California

Orange1

CA-2

OrangeCove,
California

Orange1

CA-3

San LuisObispo,California

Lemon

1

CA-4

San LuisObispo,California

Orange1

CA-5

Navelencia,
California

Orange1

CA-6OrangeCove,
California

Orange1

CA-7

Porterville,
California

Lemon

1

11
930

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

Porterville,
California

Lemon

1

CA-9

Fresno,California

Orange1

CA-1

Sanger,California

Orange2

CA-2

OrangeCove,
California

Orange2

CA-3

San LuisObispo,California

Lemon

2

CA-4

San LuisObispo,California

Orange2

CA-5

Navelencia,
California

Orange2

CA-6

OrangeCove,
California

Orange2

CA-7

Porterville,
California

Lemon

2

12
931

TRT A
TRT B
TRT C
UTC
TRT A
TRT B
TRT C
UTC
TRT A
TRT B
TRT C
UTC
TRT A
TRT B
TRT C
UTC
TRT A
TRT B
TRT C
UTC
TRT A
TRT B
TRT C
UTC
TRT A
TRT B
TRT C
UTC
TRT A
TRT B
TRT C
UTC
TRT A
TRT B
TRT C

<LOD
<LOD
<LOD
<LOD
<LOQ
<LOD
<LOQ
<LOD
46.9
46.4
59.4
<LOD
37.7
30.5
28.4
<LOD
2.04
1.65
2.16
<LOD
<LOD
<LOQ
<LOQ
<LOD
2.08
1.91
1.03
<LOD
3.09
2.50
1.92
<LOD
<LOD
<LOQ
<LOQ

CGA322704 Residues (ppb)

Thiamethoxam Residue
(ppb)

CA-8

Treatment CodeYear

Trial Identification

Crop and Variety

CDPR THX Citrus

Location (City, State)

MRID 50131102

<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
13.6
12.4
17.3
<LOD
7.57
6.58
7.20
<LOD
<LOQ
<LOQ
<LOQ
<LOD
<LOD
<LOQ
<LOD
<LOQ
<LOQ
<LOQ
<LOD
<LOD
1.32
<LOQ
<LOQ
<LOD
<LOD
<LOD
<LOD

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

Porterville,
California

Lemon

2

CA-9

Fresno,California

Orange2

CGA322704 Residues (ppb)

Thiamethoxam Residue
(ppb)

CA-8

Treatment CodeYear

Trial Identification

Crop and Variety

CDPR THX Citrus

Location (City, State)

MRID 50131102

UTC
TRT A
TRT B
TRT C
UTC
TRT A
TRT B
TRT C

<LOD
<LOQ
1.57
1.38
<LOD
1.64
<LOD
<LOQ

<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD

UTC
TRT A
TRT B
TRT C
UTC
TRT A
TRT B
TRT C
UTC
TRT A
TRT B
TRT C
UTC
TRT A
TRT B
TRT C
UTC
TRT A
TRT B
TRT C
UTC
TRT A
TRT B
TRT C
UTC
TRT A

2.34
51.1
32.9
27.9
16.6
36.0
56.9
3.95
3.12
11.2
1.48
1.57
153
51.8
7.80
13.3
1.28
3.79
3.02
1.69
<LOQ
3.47
4.04
2.66
3.68
1.55

<LOD
36.0
29.4
21.2
<LOD
17.9
16.4
<LOD
1.05
<LOQ
<LOD
<LOD
<LOQ
<LOQ
<LOQ
<LOQ
<LOD
2.68
2.49
1.88
<LOD
2.88
3.27
2.14
<LOD
<LOD

Citrus Pollen
CA-1

Sanger,California

Orange1

CA-2

OrangeCove,
California

Orange1

CA-3

San LuisObispo,California

Lemon

1

CA-4

San LuisObispo,California

Orange1

CA-5

Navelencia,
California

Orange1

CA-6OrangeCove,
California

Orange1

CA-7

Porterville,

Lemon

1
13
932

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

CA-8

Porterville,
California

Lemon

1

CA-9

Fresno,California

Orange1

CA-1

Sanger,California

Orange2

CA-2

OrangeCove,
California

Orange2

CA-3

San LuisObispo,California

Lemon

2

CA-4

San LuisObispo,California

Orange2

CA-5

Navelencia,
California

Orange2

CA-6

OrangeCove,
California

Orange

2

14
933

TRT B
TRT C
UTC
TRT A
TRT B
TRT C
UTC
TRT A
TRT B
TRT C
UTC
TRT A
TRT B
TRT C
UTC
TRT A
TRT B
TRT C
UTC
TRT A
TRT B
TRT C
UTC
TRT A
TRT B
TRT C
UTC
TRT A
TRT B
TRT C
UTC
TRT A
TRT B
TRT C
UTC

1.59
1.75
3.44
5.34
3.41
4.18
2.66
6.66
2.96
<LOQ
49.7
67.2
104
73.6
1.08
2.20
1.24
1.18
37.2
12.0
10.8
36.1
<LOQ
1.83
2.07
2.64
1.75
21.7
5.18
21.5
10.1
2.27
4.73
6.94
1.35

CGA322704 Residues (ppb)

Thiamethoxam Residue(ppb)

California

Treatment CodeYear

Trial Identification

Crop and Variety

CDPR THX Citrus

Location (City, State)

MRID 50131102

<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
1.3
<LOQ
1.36
<LOD
40.6
30.0
61.5
<LOD
<LOQ
<LOD
<LOD
101
13.6
12.3
13.3
<LOD
<LOQ
1.62
1.14
<LOD
3.20
3.59
1.39
<LOD
3.17
<LOQ
3.33
<LOD

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

Porterville,
California

Lemon

2

CA-8

Porterville,
California

Lemon

2

CA-9

Fresno,California

Orange2

CGA322704 Residues (ppb)

Thiamethoxam Residue
(ppb)

CA-7

Treatment CodeYear

Trial Identification

Crop and Variety

CDPR THX Citrus

Location (City, State)

MRID 50131102

TRT A
TRT B
TRT C
UTC
TRT A
TRT B
TRT C
UTC
TRT A
TRT B
TRT C

1.66
1.15
1.25
2.48
44.5
3.14
2.22
1.07
1.71
1.95
2.86

<LOD
<LOD
6.53
<LOD
10.3
1.15
2.28
<LOD
<LOQ
<LOQ
<LOQ

UTC
TRT A
TRT B
TRT C
UTC
TRT A
TRT B
TRT C
UTC
TRT A
TRT B
TRT C
UTC
TRT A
TRT B
TRT C
UTC
TRT A
TRT B
TRT C
UTC
TRT A
TRT B

<LOD
22.5
10.4
9.34
<LOD
5.30
5.03
5.62
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
0.691
0.760
<LOQ
<LOD
1.87
0.952

<LOD
3.19
1.84
1.41
<LOD
0.714
0.535
0.774
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOQ
<LOQ
<LOQ
<LOD
0.776
<LOQ

Citrus Nectar
CA-1

Sanger,California

Orange1

CA-2

OrangeCove,
California

Orange1

CA-3

San LuisObispo,California

Lemon

1

CA-4

San LuisObispo,California

Orange1

CA-5

Navelencia,
California

Orange1

CA-6OrangeCove,

Orange115934

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

CA-7

Porterville,
California

Lemon

1

CA-8

Porterville,
California

Lemon

1

CA-9

Fresno,California

Orange1

CA-1

Sanger,California

Orange2

CA-2

OrangeCove,
California

Orange2

CA-3

San LuisObispo,California

Lemon

2

CA-4

San LuisObispo,California

Orange2

CA-5

Navelencia,
California

Orange2

CA-6

OrangeOrange

2
16

935

TRT C
UTC
TRT A
TRT B
TRT C
UTC
TRT A
TRT B
TRT C
UTC
TRT A
TRT B
TRT C
UTC
TRT A
TRT B
TRT C
UTC
TRT A
TRT B
TRT C
UTC
TRT A
TRT B
TRT C
UTC
TRT A
TRT B
TRT C
UTC
TRT A
TRT B
TRT C
UTC
TRT A

1.56
0.504
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
0.520
<LOD
<LOQ
0.549
20.8
16.9
NA
<LOD
8.69
7.22
5.85
<LOD
NA
<LOQ
<LOQ
<LOD
<LOD
<LOQ
<LOD
<LOD
0.923
0.584
<LOD
<LOD
1.12

CGA322704 Residues (ppb)

Thiamethoxam Residue(ppb)

California

Treatment CodeYear

Trial Identification

Crop and Variety

CDPR THX Citrus

Location (City, State)

MRID 50131102

0.691
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOQ
<LOD
<LOD
<LOD
6.96
4.69
NA
<LOD
1.49
1.14
1.38
<LOD
NA
0.893
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOQ
<LOQ
<LOD
<LOD
0.672

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

CA-7

Porterville,
California

Lemon

2

CA-8

Porterville,
California

Lemon

2

CA-9

Fresno,California

Orange

2

TRT B
TRT C
UTC
TRT A
TRT B
TRT C
UTC
TRT A
TRT B
TRT C
UTC
TRT A
TRT B
TRT C

0.574
0.679
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOQ
<LOD
<LOD

CGA322704 Residues (ppb)

Thiamethoxam Residue
(ppb)

Cove,
California

Treatment CodeYear

Trial Identification

Crop and Variety

CDPR THX Citrus

Location (City, State)

MRID 50131102

<LOQ
<LOQ
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD

Table 5. Statistics of Residues of Thiamethoxam and CGA322704 in Citrus Plant Leaves, Whole Flowers,
Pollen and Nectar.
Total
MatrixMinimum Maximum Median
Mean Standard
Rate (lbandAnalyte
N
(ppb)
(ppb)
(ppb)
(ppb) Deviation
ai/Acre) Timing(ppb)
Year 1 Thiamethoxam
27
<LOD
84.9
2.36
9.67
19.7
0.172
LeavesCGA322704
27
<LOD
75.6
1.00
6.97
16.1

0.172

0.172

0.172
0.172
0.172

Year 2
LeavesYear 1
Whole
Flowers
Year 2
Whole
Flowers
Year 1
PollenYear 2
PollenThiamethoxam
CGA322704
Thiamethoxam
CGA322704

27
27
27
27

<LOD
<LOD
<LOD
<LOD

69.4
47.8
49.0
10.6

2.43
2.11
1.00
1.00

11.5
9.38
5.95
1.81

19.5
14.4
11.1
2.10

Thiamethoxam
CGA322704

27
27

<LOD
<LOD

59.4
17.3

1.65
1.00

10.4
3.19

17.7
4.55

Thiamethoxam
CGA322704
Thiamethoxam
CGA322704

27
27
27
27

<LOQ
<LOD
1.15
<LOD

56.9
36.0
104
61.5

3.95
1.00
2.86
1.62

12.7
5.66
16.2
8.11

17.4
9.59
26.5
14.3

17
936

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50131102

CDPR THX Citrus

Total
Rate (lbai/Acre)0.172

MatrixMinimum Maximum MedianMean
andAnalyte
N
(ppb)
(ppb)
(ppb)
(ppb)
TimingYear 1 Thiamethoxam
27
<LOD
22.5
0.500
2.67
NectarCGA322704
27
<LOD
3.19
0.500
0.720
0.172
Year 2 Thiamethoxam
25
<LOD
20.8
0.500
2.83
NectarCGA322704
25
<LOD
6.96
0.500
1.05
LOQ: Limit of Quntitation. 1 ppb for leaves, whole flowers and pollen, 0.5 ppb for nectar.
LOD: Limit of Detection. 0.5 ppb for leaves, whole flowers and pollen, 0.25 ppb for nectar.

Standard
Deviation
(ppb)
4.83
0.582
5.35
1.50

7. Statistical Analysis

Study Objectives and Design
The study was conducted to determine the concentration of thiamethoxam and its degradation
product CGA322704 in whole flowers, nectar, pollen, and leaves of citrus trees in response to soil
application of a thiamethoxam pesticide product applied 45 days prior to bloom. The rate of applicationof Platinum 75CA9549C was 0.172 lbs a.i./Acre. At approximately 45 days after application flower partsand leaves of orange or lemon trees were harvested and analyzed for thiamethoxam and its
degradation product. The crops received a similar second soil application in the next year and the plants
were sampled again at bloom.
Non-parametric statistical tests were used to test for differences in distribution of concentrations
between years and between soil type. Non-parametric tests do not require tests for normality as they
are robust to differences in distribution and they are also robust for experimental designs with low
replicates (Helsel and Hirsch, 2002). The PROC NPAR1WAY procedure in the Statistical Analysis System(SAS) statistical package was used to conduct Wilcoxon-Mann –Whitney (Wilcoxon), Median nonparametric, and Kuiper tests. A significant result from the Wilcoxon test indicates differences in the
shape of distributions; A significant result from the Median test indicates differences in the location of
the medians between distributions; and a significant result from the Kuiper test indicated differences inthe empirical distributions between two groups. The Exact option for each statistic was implemented as
it provides permutation testing, a statistical method that minimizes the effect of sample size anddistributional differences. Using the Exact option the Monte Carlo procedure was also implemented
which provided 10,000 separate runs for each statistic to produce the permutation distributions. The
test for potential differences due to soil type had 3 levels so the DSCF option in PROC NPAR1WAY, which
invokes the Dwass, Steel, Critchlow-Fligner multiple comparison test was used to provide pairwise tests
for two-sample rankings. Additional procedures used for descriptive statistics were PROC MEANS to
calculate mean values from the replicates at each site, PROC CAPACITY to produce cumulative statistics,
and PROC BOX plot to produce comparative graphics. Statistical analysis for effect of years and soil typewere conducted on the mean of the replicate samples taken from each site. Graphical comparisons arepresented on data transformed to a natural logarithm scale, providing clearer contrasts between the
distribution. Also, for statistical analyses, values noted as below the limit of quantification (LOQ) or limit
of detection (LOD) were assigned half the value of the respective detection limit (Table 3). Distributionof concentrations in bee relevant plant matrices was calculated using all the raw data because thesevalues represent the actual range of exposure to bees and other organisms that feed off the nectar andpollen of plants.

18937

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50131102

CDPR THX Citrus

Detection rate noted for each plant matrix: Counts for the number of samples reported below therespective detection limit for each matrix are presented in Table 6. For untreated control plots, mostanalytical results were below the LOD except for measurements of thiamethoxam in pollen samples
where approximately 90% of pollen samples were above the LOQ. For treated plants, the rate ofdetection was again highest for pollen samples where thiamethoxam was measured in 98% of samples.
The rate of detection above the LOQ for thiamethoxam was 42, 54, and 66% of samples for nectar,
whole flowers, and leaves, respectively. Rate of detection above the LOQ for the CGA322704
degradation product was lower than for parent thiamethoxam in treated plants at 56, 29, 24, and 59%
for pollen, nectar, whole flowers, and leaves, respectively.
Comparison of distribution between years: Potential difference between years was measured for two
reasons. First, greater concentrations measured in year 2 would indicate potential for carry-over effects
between years. Second, if there was no effect of years then the data could be pooled for subsequent
tests between soil types. The result for analyses conducted on plant matrices with sufficient detections
indicated no significant difference in the distribution of concentration of residues between years, basedon exact probability levels for a two-sided test (Table 7). Graphical comparisons between years also
indicated significant overlap in the distribution of concentration for thiamethoxam residues between
years in plant samples taken at bloom (Figure 1). The result for thiamethoxam and CGA322704 residues
indicated that the data for both years could be combined in further tests for comparison between
untreated and treated plants, and for effect of soil type.
Comparison of distribution between untreated and treated plants: Non-parametric tests indicatedsignificantly greater range in the distributions for treated plants for all matrices, except for parent
thiamethoxam residue in pollen samples (Table 8 and Figure 2). There was one extreme value
measured in pollen of untreated plants for parent thiamethoxam at 153 ng/g. Normally, this result
could be determined as an outlier but most of the other samples had detections above the LOQ with
two other detections noted at 49.7 and 37.2 ng/g. The authors of the report indicate that the source ofthe pollen samples could have been from the treated plants. A similar effect for similarity in distribution
between pollen concentration of untreated and treated plants has been noted for other combinationsof neonicotinoid treated trees.
Comparison of distribution between soil types: Mean data from the two years were combined fromthe sites to test for potential differences in distributions due to soil texture. Soil textural information
was supplied in supplemental data submitted after the final report. Based on the supplemental soil
characteristics the sites were classified as follows: coarse-textured soil sites were CA-1, CA-2, and Ca-5;
medium-textured soil sites were CA-6 and CA-9; and fine-textured soil sites were CA-3, CA-4, CA-7, andCA-8. Results of the non-parametric test indicated a significant difference in the distribution of leaves,nectar, and whole flowers between citrus trees grown in the 3 soil types for parent thiamethoxam and
CGA322704 degradation product (Table 9 and Figure 3). Many of the median concentration values fortrees grown in medium and fine textured soils were at or near the values that were the substitutedvalues for one-half the LOQ or LOD detection limit for thiamethoxam and CGA322704 analyses. This
indicatates that many values in these soils were reported at the detection limits. In contrast, themedian values in coarse-textured soils were well above the respective plant matrix LOQ and LOD limits.
Differences in distribution between soil types for pollen were not significant but there was a greater
19938Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50131102

CDPR THX Citrus

range indicated in the graphs for trees grown in coarse-textured soils. For comparison, the contrast
between soil types for untreated control trees is presented and no significant differences were
measured for these comparisons, indicating no initial bias due to soil type (Figure 4).

A potential confounding factor was the location of three lemon trees sites used in the study in 3 finetextured soil sites at CA-3, CA-7, and CA-8. The rest of the sites were planted with Valencia oranges.
Potential differences in uptake due to plant species could have confounded the contrasts between soil
type. The soil analyses were rerun with the soil categories assigned according to the distribution of thetype of citrus. The result was 3 coarse-textured sites with Valencia oranges, 3 fine-textured sites withlemon, and the 3 remaining sites of Valencia oranges with 1 fine-textured and 2 medium-textured soil
categories. The comparison for distribution between the sites was essentially the same as for theprevious analyses where the concentration range in coarse-textured soils was greater than for the othertwo designated categories, as reflected in the comparison of thiamethoxam concentration (Figures 5 vs3). Results of statistical analyses were also similar where specific comparisons for leaves, nectar, andwhole flowers indicated differences between the coarse-textured soil sites and the other two categories(Table 10). Pollen as observed before lacked indication of a significant difference but the range inconcentration was again greater in coarse-textured soil. These results support the overall observationthat for these experimental conditions, plants grown in coarse-textured soils exhibit a greater range inconcentration of thiamethoxam residues in leaves, nectar, and whole flowers. Plants grown in medium
or fine-textured soils apparently do not take up as much residue from the soil. As indicated by theauthors the lack of effect on pollen may be due to its higher potential for aerial movement betweenplots so the analyses are prone to confounding from applications made to adjacent treated plots.
Data for bee relevant matrices: The observed distributions derived from the individual analyses
ostensibly determines the expected range in concentrations of thiamethoxam and CGA322704
degradation product residues in bee relevant plant samples for this combination of plant species andapplication scenario (Table 11). Although many samples were below detection limits, the presence ofparent thiamethoxam in the various plant matrices indicates a potential for degradation products to bepresent. Therefore, the complete data set with values set at one-half the respective LOD for each matrix
was included in the determination of distribution of concentrations. For nectar, although someconcentrations were below the LOD, the maximum total residue concentration was 27.8 ng/g and the
median value at 0.4 ng/g. Concentrations of total residue in pollen were higher where the maximum
total residue value was 107.8 ng/g and the median value at 5.5 ng/g.
Conclusions
1. Utility of the data: The study followed the design as indicted in the data call-in where the study was
replicated in two years at 9 sites. Given the limitations of finding experimental sites in existing fields, the9 sites were reasonable representative of the 3 soil types requested in the data call-in with 3 sites incoarse-textured soils, 2 sites in medium-textured soil, and 4 sites in fine-textured soils.
2. Concentrations in Bee Relevant Matrices: By default, the distributions reported in Table 11 represent
the expected concentrations in bee relevant matrices that result from soil application of thiamethoxam
to citrus trees applied 45 days prior to bloom. Median and maximum values for total thiamethoxam

20939

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50131102

CDPR THX Citrus

residues in pollen are 5.5 and 107.8 ng/g on wet weight basis and for nectar are 0.5 and 27.8 ng/g,
respectively.

3. No carry-over effect between years: Concentrations measured in plant matrices between the twoyears of the study was similar, indicating low potential for carry-over effects due to sequential soil
applications at the concentrations and timing used in this study.
4. Effect of Soil Type: Concentrations in plant matrices at bloom were higher in plants grown in coarsetextured soil. The very low concentrations measured in medium and fine-textured soils indicate muchless uptake from the soil.
8. STUDY STRENGTHS, LIMITATIONS AND CONCLUSIONSIn the context of documenting the magnitude of thiamethoxam residues in bee-related matrices of
citrus trees, the following strengths are observed with this study.
1.
Data provide quantitative values of thiamethoxam residues expected in pollen, nectar, andleaves of citrus trees when measured at bloom in response to one soil application of thiamethoxam
applied approximately 45 days prior to bloom.
2.
The study was replicated over two years with measurements in plant samples taken at bloom
where citrus trees had received a soil application of thiamethoxam approximately 45 days before bloom.
3.

The 9 sites were reasonably replicated over the requested 3 soil texture categories.

Limitations noted in this study include:
1.
Approximately 90% of pollen samples from the untreated control were above the LOQ. This issimilar to the rate of detection in pollen samples of treated plots where thiamethoxam was measured in
98% of samples. There was one extreme value measured in pollen of untreated plants for parent
thiamethoxam at 153 ng/g. Normally, this result could be determined as an outlier but most of theother samples had detections above the LOQ with two other detections noted at 49.7 and 37.2 ng/g. It is
unclear if the source of this control contamination may have an effect on the magnitude of residues inthe treatment plots.
Overall, considering the strengths and limitations of this study, the following conclusions can be drawn:
1. Bee-relevant matrices: Thiamethoxam residues were measured in nectar and pollen sampled 45 days
after a soil application. Median and maximum values for total thiamethoxam residues in pollen are 5.5
and 107.8 ng/g on wet weight basis and for nectar are 0.5 and 27.8 ng/g, respectively. Values in Table 11
indicate the potential range in concentrations that bees are exposed to in the field.
2. No carry-over effect of years: Concentrations measured in plant matrices were similar between the
two years of the study indicating low potential for carry-over effects due to a single soil application at
the concentration and timing of application used in this study.

21940

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50131102

CDPR THX Citrus

3. Effect of soil type: Concentrations in plant matrices were higher in plants grown in coarse-textured
soil. Many analyses were indicated as below detection limits in plants grown in medium or fine-textured
soils indicating much less extraction of residues from these soil types.
4. Temporal Variability in Residues. This study was not designed for temporal analysis of declining
concentrations, but rather, to provide a snapshot of residue concentrations during flowering. Samples
were collected at only one time point during bloom.
9. STUDY VALIDITY/CLASSIFICATIONThe study is classified as ACCEPTABLE for quantitative use in risk assessment. The data from this study
provide an expected distribution of the concentrations thiamethoxam residues that bees are exposed toin nectar and pollen of citrus trees under actual agronomic practices in California. Relating
concentrations measured in flower parts to bee health is possible by comparing the concentrations
measured in bee relevant plant parts to target values that define acute or chronic exposure scenarios.
However, approximately 90% of pollen samples from the untreated control were above the LOQ. This issimilar to the rate of detection in pollen samples of treated plots where thiamethoxam was measured in
98% of samples. The similarity in distributions places a serve limit on comparing differences in
distribution caused by years or varieties because results would be confused with the large amount +of
background variance, i.e. what part of the measured effect is due to actual treatment difference incomparison to background variation. The study is considered scientifically sound and useful for risk
assessment purposes.

22941

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50131102

CDPR THX Citrus

Table 6. Counts of chemical analytical results for thiamethoxam and CGA322704 degradation product comparing the total number of samples
collected for each matrix to the number of analyses below the LOQ or LOD for untreated control and treated citrus trees. Trees had beentreated with a soil application of Platinum 75CA9549C at 0.172 lbs a.i./Acre approximately 45 days prior to sampling at bloom.

Comparison of Total Number of Samples Reported Above and Below Detection Limits
Thiamethoxam
Total
NumberNumber> LOQ

Number<LOQ>LOD

18

0

NectarPollen18
18
18

Treated Plants
LeavesWhole Flowers
NectarPollenTreatment andPlant Matrix
Untreated Plants
LeavesWhole Flowers

Number< LOD

CGA322704
Total
NumberNumber> LOQ

Number<LOQ>LOD

Number
< LOD

1

17

18

1

1

16

0
2
16

0
0
2

18
16
0

18
18
18

0
0
2

1
0
1

17
18
15

54
54

36
29

6
12

12
13

54
54

32
13

8
14

14
27

52
54

22
53

6
1

24
0

52
54

15
30

9
11

28
13

23
942

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50131102

CDPR THX Citrus

Table 7. Effect of Year: Exact probability levels for Wilcoxon, Median, and Kuiper non-parametric tests for differences in the distribution ofthiamethoxam and its degradation product CGA322704 between replicate years for leaves, whole flowers, nectar, and pollen of citrus trees.
Trees had been treated with a soil application of Platinum 75CA9549C at 0.172 lbs a.i./Acre approximately 45 days prior to sampling at
bloom.
Exact Probability Levels for Non-parametric Tests of Differences
Between Years

Treatment
Plant Matrix

Thiamethoxam
Wilcoxon

CGA322704

MedianKuiperWilcoxon

MedianKuiper

Untreated Control Plants
LeavesWhole
Flowers

1

1

1

1

1

1

1

1

1

1

1

1

Nectar

1

1

1

1

1

1

Pollen

0.51

0.34

0.89

1

1

1

Thiamethoxam Treated Plants
LeavesWhole
Flowers

0.69

1

1

0.34

1

0.89

0.24

1

1

0.74

1

1

Nectar

0.78

1

1

0.92

1

1

Pollen

0.61

1

0.88

0.37

1

1

24
943

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50131102

CDPR THX Citrus

Table 8. Untreated vs Treated Plants: Exact probability levels for Wilcoxon, Median and Kuiper non-parametric tests for differences in the
distribution of thiamethoxam and its degradation product CGA322704 between untreated control and treated citrus tree leaf, whole flower,
nectar, and pollen samples. Trees had been treated with a soil application of Platinum 75CA9549C at 0.172 lbs a.i./Acre approximately 45
days prior to sampling at bloom.

Non-paramentric Test Exact Probability Levels for Comparing Concentration
Distribution Between Untreated Control and Treated Plants
Thiamethoxam

CGA322704

Plant Matrix

Wilcoxon

MedianKuiperWilcoxon

MedianKuiperLeavesWhole Flowers
Nectar

0.001
0.001
0.001

0.001
0.001
0.002

0.001
0.001
0.078

0.001
0.002
0.001

0.001
0.003
0.001

0.01
0.18
0.078

Pollen

0.13

0.31

0.59

0.001

0.001

0.001

25
944

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50131102

CDPR THX Citrus

Table 9. Effect of Soil Type: Exact probability levels for Wilcoxon non-parametric tests for differencesin the distribution of thiamethoxam, CGA322704 degradation product, and total residue between soil
types in leaf, whole flower, nectar, and pollen samples of citrus trees. Trees had been treated with asoil application of Platinum 75CA9549C at 0.172 lbs a.i./Acre approximately 45 days prior to samplingat bloom. The Wilcoxon probability level is for the combined analysis of all three soil types for eachplant matrix. The DSCF probability level is for the specific 1-degree of freedom contrasts between soil
types, such as Coarse vs Fine-textured soil sites.

Treatment,
Plant Matrix, andSpecific Soil Contrasts

Exact Probability Levels for Non-parametric
Tests of Differences Between Soil Type

Thiamethoxam

CGA322704

Wilcoxon

Wilcoxon

0.037
0.053

0.010
0.012

0.203
0.905

0.203
0.901

0.002
0.012

0.006
0.013

Coarse vs. Medium

0.203

0.199

Fine vs. Medium

0.163

0.505

0.001

0.002

Coarse vs. Fine

0.004

0.007

Coarse vs. Medium

0.203

0.203

0.023
0.390

0.133
0.180

Coarse vs. Fine
Coarse vs. Medium

0.476
0.535

0.188
0.407

Fine vs. Medium

0.776

0.905

Leaves1

0.32

Whole Flowers

1

0.55

Nectar0.84

1

Pollen0.54

0.17

Treated Plants
LeavesCoarse vs. Fine
Coarse vs. Medium
Fine vs. Medium
Whole Flowers
Coarse vs. Fine

NectarFine vs. Medium
PollenUntreated Plants

26945

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50131102

CDPR THX Citrus

Table 10. Potential Species Confounding for Effect of Soil Type: Exact probability levels for Wilcoxon
non-parametric tests for differences in the distribution of thiamethoxam, CGA322704 degradation
product, and total residue between soil types in leaf, whole flower, nectar, and pollen samples of
citrus trees. Trees had been treated with a soil application of Platinum 75CA9549C at 0.172 lbs
a.i./Acre approximately 45 days prior to sampling at bloom. The Wilcoxon probability level is for the
combined analysis of all three soil types for each plant matrix. The DSCF probability level is for the
specific 1-degree of freedom contrasts where the acronyms are: C-Orange=orange trees in coarsetextured soil; F-Lemon=lemon trees in fine-textured soil; and M-F-Orange=orange trees grown inmedium or fine-textured soils.

Exact Probability Levels for Non-parametric
Tests of Differences Between Soil Type
Thiamethoxam

CGA322704

Total Residue

Plant Sample

Wilcoxon

Wilcoxon

Wilcoxon

Treated Plants
Leaves

0.044

0.013

0.025

C-Orange vs. F-Lemon
C-Orange vs. M-F-Orange

0.133
0.064

0.027
0.064

0.064
0.064

F-Lemon vs. M- F-Orange
Whole Flowers

0.997
0.009

0.985
0.011

0.969
0.011

C-Orange vs. F-Lemon
C-Orange vs. M-F-Orange

0.028
0.064

0.037
0.058

0.028
0.064

F-Lemon vs. M- F-Orange

0.743

0.956

0.912

NectarC-Orange vs. F-Lemon

0.001
0.008

0.006
0.022

0.001
0.013

C-Orange vs. M-F-Orange
F-Lemon vs. M- F-Orange

0.064
0.082

0.062
0.547

0.064
0.120

PollenC-Orange vs. F-Lemon

0.440
0.501

0.180
0.278

0.340
0.406

C-Orange vs. M-F-Orange

0.501

0.244

0.406

F-Lemon vs. M- F-Orange

0.986

0.986

0.986

27
946

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50131102

CDPR THX Citrus

Table 11. Distribution of thiamethoxam, CGA322704 degradate, and total residue concentrations measured in nectar, pollen, whole flowers,
and leaves of citrus trees that were exposed to a soil application of Platinum 75CA9549C at 0.172 lbs a.i./acre approximately 45 days prior to
sampling at bloom. Data are the combined results of individual plant samples obtained from two consecutive years of study.

StatisticN (#)
Mean (ng/g)
SD (ng/G)
CV (%)Min (ng/g)
Median (ng/g)
75th (ng/g)
90th (ng/g)
95th (ng/g)
Max (ng/g)% of Total
StatisticN (#)
Mean (ng/g)
SD (ng/g)
CV (%)Min (ng/g)
Median (ng/g)
75th (ng/g)
90th (ng/g)
95th (ng/g)
Max (ng/g)% of Total

NectarThiamethoxam
52
2.6
5.1
201.5
0.1
0.3
1.3
8.7
16.9
22.5
80.2
Whole Flowers
Thiamethoxam
54
7.9
15.0
189.5
0.3
1.5
2.5
31.4
46.9
59.4
79.8

CGA322704
52
0.6
1.2
192.0
0.1
0.1
0.7
1.4
3.2
7.0
19.8

Total
52
3.2
6.3
196.9
0.3
0.4
2.0
10.2
21.6
27.8

CGA322704
54
2.0
3.8
190.7
0.3
0.4
0.5
7.2
12.4
17.3
20.2

Total
54
9.9
18.7
189.2
0.5
1.7
3.0
37.2
59.6
76.7

28
947

PollenThiamethoxam
54
14.4
22.3
154.4
0.5
3.6
13.3
51.1
67.2
104.0
68.2
LeavesThiamethoxam
54
10.4
19.6
188.4
0.3
2.4
6.5
33.9
67.3
84.9
56.8

CGA322704
54
6.6
12.2
185.3
0.3
1.3
3.6
21.2
36.0
61.5
31.3

Total
54
21.1
33.0
156.8
1.4
5.5
23.1
62.3
107.8
135.1

CGA322704
54
7.9
15.3
193.8
0.3
1.6
3.4
29.1
47.1
75.6
43.2

Total
54
18.3
34.7
189.6
0.5
1.1
9.9
64.4
114.4
160.5

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50131102

CDPR THX Citrus

Figure 1. Comparison between Years: Distribution of natural logarithm of thiamethoxam residues inleaves, nectar, pollen and whole flowers of citrus trees compared between sequential replicate
studies years of the study. Trees were treated with a soil application of Platinum 75CA9549C at 0.172
lbs a.i./Acre approximately 45 days prior to sampling at bloom.

29948

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50131102

CDPR THX Citrus

Figure 2. Untreated vs Treated Plots: Comparison of distribution of natural logarithm ofthiamethoxam concentration between untreated control (UTC) and treated (TRT) plants for leaves,nectar, pollen, and whole flowers of citrus trees. Treated trees received a soil application of Platinum
75CA9549C at 0.172 lbs a.i./Acre approximately 45 days prior to sampling at bloom.

30949

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50131102

CDPR THX Citrus

Figure 3. Comparison of Soil Type for Treated Plants: Comparison of distribution of natural logarithmof thiamethoxam concentration measured between Coarse, Medium, and Fine-textured soil types forleaves, nectar, pollen, and whole flowers of treated citrus trees. Treated trees received a soil
application of Platinum 75CA9549C at 0.172 lbs a.i./Acre approximately 45 days prior to sampling at
bloom.

31
950

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50131102

CDPR THX Citrus

Figure 4. Comparison of Soil Type for Untreated Plants: Comparison of distribution of natural
logarithm of thiamethoxam concentration measured between Coarse, Medium, and Fine-textured soil
type for leaves, nectar, pollen, and whole flowers of untreated control citrus trees.

32951Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50131102

CDPR THX Citrus

Figure 5. Comparison between Plant Species and Soil Type: Comparison of thiamethoxam
concentration distribution in leaves, nectar, pollen, and whole flowers of treated citrus trees
measured between Valencia and Lemon citrus plant species. The three categories indicate distributionof 3 Lemon tree sites located in 3 sites with fine-textured (Fine) soils, 3 sites where Valencia orange
trees were grown in 1 fine-textured and 2 medium-textured (Med) soils, and 3 sites with Valencia
orange trees grown in coarse-textured (Coarse) soils.

33952

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50131102

CDPR THX Citrus

10. REFERENCES
1. Anderson, L. (2007) “Thiamethoxam (CGA293343) and CGA322704. Validation of Residue
Analytical Method REM 179.07 for the determination of Residues in Bee and Hive Products andStorage Stability in Hive Pollen, Wax and Nectar, stored Deep Frozen for 12 months.” (Study No.
05-S508).
2. Crook, S (2004) Residue Method for the Determination of Residues of Thiamethoxam(CGA293343) and CGA322704 in Lettuce, Tomato, Grape and Tobacco Samples. Final
Determination by LC/MS/MS" (Syngenta Method REM179.06).
3. Crook, S (2007) Analytical Method for the Determination of Residues of Thiamethoxam(CGA293343) and CGA322704 in Bee and Hive Products. Final Determination by LC-MS/MS(Syngenta Method REM179.07).
4. Helsel, D.R. and R.M. Hirsch. 2002. Chapter A3: Statistical Methods in Water Resources.
Techniques of Water-Resources Investigations of the United States Geological Survey Book 4,
Hydrologic Analysis and Interpretation. United States Geological Survey.
5. Hohl, J. (1999) Stability of Residues of CGA-322704 in Plant Material and Soil Stored Under Deep
Freezer Conditions (Study No. 779-00) MRID 45108001.
6. Mair, P. (1998) Stability of Residues of CGA-293343 (2 Years Final Report) and CGA-322704 (1Year Interim Report) in Plant Material Under Deep Freezer Conditions, Including Method
Validation (Study No. 504/96 consists of Reports #112/96, 127/97, 103/98) MRID 44703525.
7. Oakes, T. (2002) Stability of CGA-293343 and CGA-322704 in Crops and Processed Fractions
Under Freezer Storage Conditions (Study No. 269-98) MRID 45659205.

34
953

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50096606

CDPR THX Stone Fruit

Reference
Rice, F., Lange, B. (2015) Thiamethoxam 25 WG (A9584C) - Magnitude of Residues in Pollen, Nectar,
Flowers, and Leaves of Stone Fruit After Foliar Application with Actara® 25WG in California: Final
Report. Project Number: TK0177222. Unpublished study prepared by Syngenta Crop Protection, LLC.
644. MRID 50096606, CDPR Study ID 288446, Data Volume 52691-0531, Tracking ID# 273604

1. STUDY INFORMATION
Chemical:Thiamethoxam
Test Material:

Study Type:

Sponsor:Sponsor Study
ID:
Study
Completion
Date:
ReportAmendment
Date:

GLP Status:

PC Code60109
Percent
Actara 25WG
Active25.0%
Ingredient:
Non-Guideline field residue study on Stone Fruit to measure Thiamethoxam and
CGA322704 residue levels in nectar, pollen, anthers, flowers and leaves in site
locations that have been treated with Thiamethoxam for two successive years.
Syngenta Crop Protection, LLC
Experiment Start
410 Swing Road
April 24, 2013 –andGreensboro, North Carolina
September 25, 2015
End Date:
27409

TK0177222
December 17, 2015

Study Locations:

October 21, 2016

10 stone fruit trial sites
including peach, plumand sweet cherry
located throughout
California.

Non-GLP; protocol reviewed by CDPR.
[CDPR Study ID 288446, Data Volume 52691-0531, Tracking ID# 273604]

2. REVIEWER INFORMATION
Study Reviewed by:Richard Bireley, Sr. Environmental Scientist (Specialist)
California Department
John Troiano, Ph.D., Research Scientist III
of Pesticide Regulation
Alexander Kolosovich, Sr. Environmental Scientist (Specialist)
Brigitte Tafarella, Environmental Scientist
Denise Alder, Sr. Environmental Scientist (Specialist)
Russell Darling, Sr. Environmental Scientist (Specialist)

3. EXECUTIVE SUMMARY
A two-year study was initiated in 2013 to determine the magnitude of the residue of thiamethoxam(CGA293343) and its major metabolite, CGA322704, in stone fruit (peach, plum, and sweet cherry)leaves, flowers, anthers, pollen, and nectar following foliar applications with Actara® 25WG (EPA Reg.
No. 100-938). The study consisted of 10 trials located in California, each with an untreated control plot
and a treated plot large enough to ensure adequate plants for collection. Over two consecutive growing
seasons, thiamethoxam, formulated as Actara® 25WG, was applied to treated plots as a broadcast foliar
1954

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50096606

CDPR THX Stone Fruit

spray twice (7-day interval) during each growing season at the maximum labeled-use rate of 5.5 oz
formulated product per acre (0.086 lb ai/acre) for each application. Applications were targeted 21- and14-days before normal harvest of mature fruit. Composite samples of leaves, flowers, anthers, pollen
and nectar were collected for residue analysis from untreated control (UTC) and treated (TRT) plots thefollowing bloom period (the following spring).
This report is amended to include information on collection and analysis of second year samples
collected at site CA 10 and to add further explanation regarding residues in control pollen samples.
4. STUDY VALIDITY
Guideline Followed:
Guideline Deviations:
Other Deviations:Classification:
Rationale:
Reparability:

Deviations Exist; (protocol was reviewed by CDPR)
Site CA2 was terminated, Site CA10 was added
Additional year of samples are being collected
ACCEPTABLE
N/A
N/A

5. MATERIALS AND METHODS

Test item:
Formulation Type:
CAS #:

Test Material Characterization
Actara 25WG
Percent A.I.:
Water Dispersible
pH:
Granule, WG
153719-23-4
Solubility:

25.0% A.I.
9.4 (1% aqueous
solution)
4.1 g/L @ 25°C

5A. STUDY DESIGN
The purpose of this two-year study was to determine the amount of thiamethoxam and CGA322704 instone fruit leaves, flowers, anthers, pollen, and nectar after foliar applications of Actara® 25WG (EPAReg. No. 100-938) in two successive years. The study was initiated on April 24, 2013 and theexperimental termination date was on September 25, 2015.
The two-year study initially included nine trial sites (CA-1 through CA-9) each with an untreated control(UTC) plot and a treated (TRT) plot large enough to ensure adequate plants for collection of sufficientquantities of leaves, flowers, anthers, pollen, and nectar for residue analysis. Per Amendment 6(September 02, 2014), a tenth trial site (CA-10) was added to replace CA-2 which had a plum variety that
did not produce pollen. The test substance was applied to treated plots as a foliar broadcast treatment
twice each year. Representative composite samples of leaves, flowers, anthers, pollen, and nectar werecollected from the UTC plot and each TRT replicate plot for residue analysis.
5B. APPLICATION TIMING AND RATESAirblast sprayers were calibrated prior to test-substance application. Sprayer-pass times were recorded
to confirm application rate accuracy. All trials were within the acceptance criteria range of 95 to 105% of
the target 5.5 oz formulated product per acre application for both years.
Application rates and dates are summarized below in table 1.
2955

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50096606

CDPR THX Stone Fruit

Table 1. Summary of Applications on Stone Fruit
Trial Site
Year
ApplicationDate1
9/4/20131
CA-1
9/11/20131
2
6/20/2014
6/27/20141
5/8/2013
CA-2
5/15/20132
4/28/2014
5/5/20141
5/8/2013
CA-3
5/15/20132
4/28/2014
5/5/20141
4/30/2013
CA-4
5/7/2013
2
5/15/2014
5/22/20141
6/18/2013
CA-5
6/25/20132
7/21/2014
7/28/20141
4/30/2013
CA-6
5/7/2013
2
4/24/2014
5/1/2014
1
4/29/2013
CA-7
5/7/2013
2
5/28/2014
6/4/20141
5/28/2013
CA-8
6/4/2013
2
7/15/2014
7/22/20141
4/29/2013
CA-9
5/7/2013
2
4/28/2014
5/6/20141
8/20/2014
CA-10
8/27/20142
8/28/2015
9/4/2015
1

Target Rate(oz/acre)
5.5
5.5
5.5
5.5
5.5
5.5
5.5
5.5
5.5
5.5
5.5
5.5
5.5
5.5
5.5
5.5
5.5
5.5
5.5
5.5
5.5
5.5
5.5
5.5
5.5
5.5
5.5
5.5
5.5
5.5
5.5
5.5
5.5
5.5
5.5
5.5
5.5
5.5
5.5
5.5

Actual Rate(oz/acre)
5.599
5.470
5.480
5.582
5.532
5.556
5.606
5.506
5.524
5.515
5.342
5.559
5.531
5.632
5.586
5.628
5.591
5.594
5.624
5.562
5.515
5.480
5.548
5.531
5.433
5.467
5.543
5.546
5.516
5.497
5.512
5.570
5.489
5.517
5.665
5.533
5.749
5.493
5.476
5.487

Applications made after harvest of fruit, to avoid making applications too close to harvest.

3956

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50096606

CDPR THX Stone Fruit

5C. STUDY SITE LOCATION AND CHARACTERISTICS
The 10 trial sites were distributed within typical peach-, plum-, and sweet cherry-growing areas of theCentral Valley of California (northern, middle, and southern sections) and represented management
practices and weather conditions under which stone fruit is commercially produced. Stone fruit varieties
and site conditions of the test sites are provided below in Table 2 and Table 3.
At each sampling event, 0-12-inch soil cores were collected from control and treated plots. A minimumof 5 cores were collected randomly from each control plot and a minimum of 10 cores were collected
randomly from all replicates of treated plots. Soil cores were then sectioned into 0-6 inch and 6-12 inch
segments; soil was removed from segmented plastic tubes and placed into labeled plastic bags, anddouble bagged. For each trial, soil was combined into a single control and a single treated sample bydepth.

Trial
ID

CA-1
CA-2
CA-3
CA-4
CA-5
CA-6
CA-7
CA-8
CA-9
CA-10

Table 2. Trial Site Conditions for Stone Fruit
Trial
Location
Crop
Variety
OM (%)(County,
State)King,
Peach
Late Ross
1.6 – 1.8
California
Cling
Madera,PlumApple
1.3 – 1.6
California
Dandy
Fresno,Cherry Washingto
0.52
Californian
San Joaquin,
Peach Flavorcrest 1.8 – 2.4
California
Merced,Prune
French
0.70 – 1.5
California
San Joaquin, Cherry Sweet Tart
1.9 – 2.6
California
Sutter,PeachElegant
2.2 – 2.4
California
Lady
Sutter,Prune
French
1.3 – 1.5
California
Sutter,CherryBing
0.82 – 1.6
California
Tulare,Plum
Angeleno
1.3 – 2.4
California

Soil Types

Temperature
Range (°F) b

7.6 – 7.7

CationExchange
Capacity(meq/100g soil)
11.7 -12.1

Sandy Loam

30.9 – 98.5

6.7 – 6.8

7.9 – 9.4

Sandy Loam

27.8 – 97.2

6.5 – 6.6

8.5 – 9.1

29.6 – 97.0

7.3 – 7.5

18.1 – 18.3

Loamy
Sand/Sandy
Loam

5.0 – 5.7

20.0 – 23.4

28.9 – 91.6

7.2 – 7.5

18.8 – 19.9

Clay
Loam/Loam
Loam

7.0 – 7.5

17.9 – 18.3

Clay Loam

26.2 – 92.1

6.4 – 6.7

15.2 – 15.7

Loam

26.2 – 92.1

6.9

6.4 – 7.7

Sandy Loam

36.6 – 94.7

7.2

16.3 – 17.0

Sandy Loam

35.3 – 95.6

pH

4
957

28.9 – 91.6

28.9 – 91.6

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50096606

CDPR THX Stone Fruit

Table 3. Soil Characterization Results.
Trail Site
Soil Type
CA-1
Sandy Loam

CA-2

Sandy Loam

CA-3

Loamy Sand/Sand

CA-4

Loam

CA-5

Clay Loam/Loam

CA-6

Loam

CA-7

Clay Loam

CA-8

Loam

CA-9

Sandy Loam

CA-10

Sandy Loam

% Sand
67
65
67
65
85
87
40
38
32
36
38
40
28
22
48
46
64
68
60
58

% Silt
24
26
23
23
10
10
35
35
39
37
37
35
39
43
28
28
25
23
24
26

% Clay
9
9
10
12
5
3
25
27
29
27
25
25
33
35
24
26
11
9
16
16

5D. SAMPLE COLLECTION, HANDLING, PROCESSING
Leaf samples were collected from UTC and replicated TRT (A, B, C) plots at each site after the last
application of each season. Whole flower, anther, pollen, and nectar samples were collected at an early
bloom stage (50–75% bloom) in the spring following test substance applications in the fall. Leaf samples
were collected after flowering, when leaves had emerged and were near normal size. Also, atappropriate timings, bulk samples were collected from one site for analytical method development andmethod verification as follows: approximately 500 g of leaf and flower samples, 1g of anther samples,and 500 mg of nectar samples. Bulk pollen for method development/verification was purchased from acommercial source. In all cases, samples were collected from the UTC plot first, then from TRT plots orby different personnel to minimize the potential for cross contamination.
Soil samples were collected during the 2014 growing season and again when flowers were collected inspring 2015. For trial CA-10, soil samples were also collected when flowers were collected in spring of2016.
LeavesRepresentative samples of at least 500 g of leaves were collected from UTC and TRT plots after thesecond application of the test substance during both growing seasons. Samples were collected whenleaves were near normal size after the bloom period was complete. Leaves were removed by hand from
5958

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50096606

CDPR THX Stone Fruitthe upper, middle, and lower portions of the trees. All samples met the protocol minimum sample size
requirement.
Flowers
Flowers were removed by hand from the high, low, and middle portions of the trees. Representative
samples of at least 1000 flowers (500 g target sample size) were collected from UTC and TRT plots. Fromthe 1000 flowers, approximately 400 flowers (200 g target sample size) were used for whole flower
sample analysis and approximately 600 flowers (300 g target sample size) were used for subsequent
collection of nectar and pollen samples. It should be noted that in some cases whole flowers and flowers
for collection of nectar and pollen were not counted, nor did they meet the minimum weights. Flowersthat had not yet opened (popcorn stage) were collected and processed for anther samples. Flowers for
direct analysis were placed into plastic bags then into the freezer until shipment to the analyticallaboratory.
Anthers
Anthers were collected from flowers that were in the popcorn stage and had not yet opened. Target
weight for anther samples was approximately 50 mg from the UTC plots and 100 mg from the TRT plots.
All samples, except sample numbers 369, 377, and 393 from CA-6, met the protocol for the minimum
sample size requirement.
PollenPollen was collected using a vacuum pump that was connected by tubing to a 1000-µL pre-weighed
filtered pipette tip. Pollen was vacuumed directly from the anthers into the pipette tips. After weighing
the samples, the pipette tips were placed into plastic bottles and sealed for additional containment.
Pollen samples were then placed into the freezer until shipment. The minimum pollen sample size
required for analysis was approximately 30 mg for both the UTC and TRT samples. All pollen samples,
except sample numbers 119, 127, and 131 from CA-6, and samples 447, 451, 459, and 467 from CA-10,
met the protocol’s minimum sample size requirement. No pollen samples were collected from CA-2 due
to male-sterile plum variety.
NectarNectar was collected using 10 and 20-µL microcapillary pipettes. Approximately 300 flowers werecollected to extract the minimum sample size of nectar required for analysis (≥30 mg for TRT samples,≥30 mg for UTC samples). Nectar samples were placed into sealed and labelled containers where sample
weights were then recorded. Nectar samples were then placed into the freezer until shipment. All
samples met the protocol’s minimum sample size requirement.
Soil
Soil samples were collected during the 2014 growing season and again when flowers were collected inspring 2015. For trial CA-10, soil samples were also collected when flowers were collected in spring
2016.

6
959

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50096606

CDPR THX Stone FruitAt each sampling event, 0-12 inch soil cores were collected from control and treated plots. A minimumof 5 cores were collected randomly from each control plot and a minimum of 10 cores were collected
randomly from all replicates of treated plots. Soil cores were then sectioned into 0-6 and 6-12 inch
segments; soil was removed from segmented plastic tubes and placed into labelled plastic bags, anddouble bagged. For each trial, soil was combined into a single control and a single treated sample bydepth.
Sample Storage.
Samples were transported from field sites to field facility freezers where they were stored frozen until
shipment to the analytical laboratory. For trials CA-1 through CA-9, anther, pollen, and nectar samples
were shipped to Eurofins Agrosciences, Inc. (EASI) in East Brunswick, NJ. Leaf, whole flower, and soil
samples were shipped to the EASI Sample Processing Laboratory in Forsyth, Georgia. Then the leaf,
whole flower, and soil samples were shipped to the EASI laboratory in East Brunswick, NJ after
processing was completed. In March 2015, first year anther and all year-2 samples were sent from the
EASI laboratory to SynTech Research Laboratory Services (SRLS) in Stilwell, KS for analysis, per
Amendment 7 (Appendix 5). For CA-10, leaves collected post application 1 were shipped to the EASI
Sample Processing Laboratory in Forsyth, Georgia, and after processing, they were shipped to the EASI
Laboratory in East Brunswick, New Jersey. All other CA-10 samples were shipped to SynTech ResearchLaboratory Services (SRLS) in Stilwell, Kansas for analysis.
Previous storage stability studies2,3,4 show thiamethoxam and CGA322704 are stable in a variety ofmatrices for up to 12 months. Therefore, residues of thiamethoxam and CGA322704 in stone fruit leaf
and flower samples should not have been adversely affected by freezer storage during this study. The
maximum freezer storage period for samples was 577 days. A 2 year freezer stability study1(Amendment 7 listed in the study report) shows that thiamethoxam and CGA322704 were stable inplant material for 2 years.
All samples were received frozen and in good condition at EASI or SRLS. Samples were maintained frozen(-20 ± 5 °C), except during periods when samples were removed from the freezer for sample
preparation, weighing, or residue analysis. Leaf and flower samples were weighed and homogenizedwith dry ice using a Robot Coupe; the homogenized samples were placed into labelled plastic containers
and stored in a freezer (allowing the dry ice to sublime) until sub-sampled for analysis. Pollen, antherand nectar samples were stored directly in a freezer until analysis.
5E. ANALYTICAL METHODS
The analytical phase for year 1 samples (except for anthers) and year 2 post-second application leaves
was conducted at EASI. The Principal Analytical Investigator was Chelsea Bonetti. From March 2015 totrial completion, per Amendment 7 listed in the study report, the analytical phase was conducted at
SRLS in Stilwell, KS. The Principal Analytical Investigator was Ying Li. Samples analyzed by SynTech
included first year anthers and all year-2 samples, as well as the CA10 samples.
Leaf and Whole Flower Sample Analysis
At EASI Laboratory, residues of thiamethoxam and CGA322704 were extracted with 50:50
methanol/water from 10-g of leaf and flower samples using a TomTec automatic homogenizer unit.
Extracts were cleaned up with PSA and diluted with water in preparation for LC-MS/MS analysis. The
7960

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50096606

CDPR THX Stone Fruit

Limit of Quantitation (LOQ) for both analytes was 10 ppb in the leaf and 1 ppb in the flower matrices.
The Limit of Detection (LOD) was targeted to be 5.0 ppb and 0.5 ppb in leaf and flowers, respectively.
At SRLS Laboratory, residues of thiamethoxam and CGA322704 were extracted with 50:50 (v:v)
methanol:water containing 0.2% formic acid from approximately 10-g of leaf and flower samples using a
blender. Extracts were centrifuged and diluted with water in preparation for LC-MS/MS analysis. The
LOQ for both analytes was 1 ppb in the leaf and flower matrices. The Limit of Detection (LOD) was
targeted to be 0.5 ppb in both leaf and flower matrices.
Anther, Pollen and Nectar Sample Analysis
At EASI Laboratory, residues of thiamethoxam and CGA322704 were extracted with 50:50
methanol/water containing 0.2% acetic acid from approximately 50 mg of pollen and 100 mg nectar
samples. Extraction was conducted with a MP FastPrep-24 homogenizer; extracts were subsequently
centrifuged and prepared for online SPE-LC-MS/MS analysis. The LOQ for both analytes was 1.0 ppb inpollen matrices and 0.5 ppb in the nectar matrix. The LOD was targeted to be 0.35 ppb and 0.186 ppb inpollen and nectar matrices, respectively. Anther samples were not analyzed at EASI.
At SRLS Laboratory, residues of thiamethoxam and CGA322704 were extracted with 50:50 (v:v)
methanol:water containing 0.2% formic acid from approximately 0.1-g of pollen, nectar, and anthersamples. Extraction solution and matrices were mixed well, centrifuged, diluted with water and cleaned
by solid phase extraction in preparation for LC-MS/MS analysis. The LOQ for both analytes was 1 ppb in
the pollen and anther matrices and 0.5 ppb for nectar matrix. The LOD was targeted to be 0.5 ppb inpollen and anther matrices and 0.25 ppb in a nectar matrix, respectively.
The LOQs and LODs are summarized in table 4 below.
Table 4. Summary of LOQs and LODs
Site Laboratory
MatrixEASI Laboratory

SRLS Laboratory

Leaf
FlowerNectarPollenAnthers
Leaf
FlowerNectarPollenAnthers

LOQ(Total Thiamethoxam PPB)
10
1.0
0.5
1.0
1.0
1.0
0.5
1.0
1.0

LOD
(Total Thiamethoxam PPB)
5.0
0.5
0.186
0.35
0.5
0.5
0.25
0.5
0.5

5F. QUALITY ASSURANCE RESULTSAt EASI Analytical Laboratory: A Shimadzu Nexera X2 HPLC system coupled to an API 4000 massspectrometric detector was used for separation and quantitation of thiamethoxam and CGA322704 forleaf matrices. A Shimadzu Nexera X2 HPLC system coupled to an API QTRAPP 6500 mass-spectrometricdetector was used for separation and quantitation of thiamethoxam and CGA322704 for flower
8961

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50096606

CDPR THX Stone Fruitmatrices. An API QTRAPP 6500 mass spectrometric detector equipped with a Spark Holland Symbiosis
online HPLC-SPE system was used for separation and quantitation of thiamethoxam and CGA322704 forpollen and nectar matrices.
At SRLS Analytical Laboratory: A Waters Acquity UPLC system coupled to an API 5500 AB-Sciex mass
spectrometric detector was used for the separation and quantitation of thiamethoxam and CGA322704
for leaf, flower, pollen, anther and nectar matrices. To quantify analytes of interest, standard curves
were prepared by injecting constant volumes of solvent-based standard solutions at appropriate
concentrations. Constant volume injections were used for sample extracts as well. Linear regression
with 1/X weighting was used.
EASI successfully verified both methods prior to analysis of samples. Control samples of each matrix
were fortified with thiamethoxam and CGA322704 at concentrations equal to the method 1xLOQ and
10xLOQ and analyzed according to the appropriate methods. No additional method verification was
performed at SRLS prior to sample analysis as the methods were in-house for other studies. Concurrent
recoveries were used to demonstrate method performance.
For each matrix, at least one method-recovery (QC) sample per analytical set was prepared by fortifying
an untreated control sample with thiamethoxam and CGA322704 at concentrations equal to the method
LOQ or higher and analyzing concurrently with treated samples to demonstrate adequate method
performance throughout the study. Percent recoveries for QCs analyzed at EASI fell within theacceptable range of 70 to 120% with the exception of one flower and two leaf QCs with recoveries
ranging from 128 to 133%. Percent recoveries for QCs analyzed at SRLS fell within the acceptable rangeof 70 to 120%.
6. RESULTS:
During sample analysis at EASI, no thiamethoxam or CGA322704 residues >LOQ were found in any UTC
leaf matrices. Only thiamethoxam residues >LOQ were found in UTC flower (3 samples, 1.97 ppb max),
pollen (6 samples, 39.1 ppb max) and nectar (4 samples, 2.75 ppb max) samples. Residues greater thanrespective LOQs were found in TRT flowers, pollen, and nectar samples. No residues were found in TRTleaf samples collected near bloom. Anther samples collected in Year 1 were analyzed by SLRS. Residues
greater than LOQ were found in all leaf samples collected post application 2. The following tableprovides a summary of the average residues for samples analyzed at EASI.

9962

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50096606

CDPR THX Stone Fruit

Table 5. Residue Analysis on Leaves and Flowers at EASI
Year

Year 1

TimingMatrix

Post
Application 2

Leaf

Bloom

Crop

Peach
Plum
Sweet Cherry
Peach
Plum
Sweet Cherry
Peach
Plum
Sweet Cherry

Leaf

FlowerAverage Residue Concentration
Thiamethoxam
CGA322704
(ppb)
(ppb)
4956
343
6164
287
3941
279
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
1.84
ND
2.65
<LOD
<LOQ
<LOQ

LOQ= 10 ppb for leaf samples, 1 ppb for whole flower and pollen samples and 0.5 ppb for nectar samples.
LOD= 5 ppb for leaf samples, 0.5 ppb for whole flower, 0.35 ppb for pollen and 0.186 ppb for nectar samples.

Table 6. Residue Analysis on Pollen, Nectar and Leaves at EASI
Year

Year 1

TimingBloom

Matrix

Crop

Peach
Plum
Sweet Cherry
Peach
Plum
Sweet Cherry
Peach
Plum
Sweet Cherry

PollenNectarYear 2

Post
Application 2

Leaf

Average Residue Concentration
Thiamethoxam
CGA322704
(ppb)
(ppb)
16.2
<LOQ
18.9
<LOQ
30.3
1.08
<LOQ
<LOQ
1.11
<LOD
<LOQ
<LOQ
5589
276
8316
356
4684
344

LOQ= 10 ppb for leaf samples, 1 ppb for whole flower and pollen samples and 0.5 ppb for nectar samples.
LOD= 5 ppb for leaf samples, 0.5 ppb for whole flower, 0.35 ppb for pollen and 0.186 ppb for nectar samples.

During sample analysis at SRLS, thiamethoxam and CGA322704 residues >LOQ were found in some UTC
samples of all plant matrices at very low levels with the exception of whole flower samples in which no
residues were found >LOQ. Both thiamethoxam and CGA322704 residues >LOQ were found in UTC
leaves (5.07 and 1.15 ppb max, respectively), pollen (382.04 and 2.70 ppb max), nectar (0.74 and 5.10
ppb max), and anthers (5.75 and 1.83 ppb max) samples. Residues, greater than the respective LOQs,
were found in some TRT samples of all plant matrices. The following table provides a summary of theaverage residues for treated plots post-application 2 analyzed at SRLS. Anther samples collected Year 1
and 2 were analyzed at SLRS.

10963

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50096606

CDPR THX Stone Fruit

Table 7. Residue Analysis on Leaves, Flowers, Pollen and Anthers at SRLS
Average Residue Concentration
Year
TimingMatrixCrop
Thiamethoxam
CGA322704
(ppb)
(ppb)
a
≤ 1.0 Day
Leaf
Plum
11765
299
Leaf
Pluma
4.00
1.79
Flower
Pluma
ND
ND
Year 1
Pollen
Pluma
110
1.55
Bloom
Peach
2.34
2.15
Anther
Pluma
4.39
1.95
Sweet Cherry<LOQ (0.83)
NDa
NectarPlum<LOQ (0.42)
ND≤ 1.0 Day
Leaf
Plum
16132
951
Peach
ND
1.14
Leaf
Plum
1.54
1.21
Sweet Cherry
1.81
3.07
Peach
ND
ND
Year 2
FlowerPlum
1.88
ND
Sweet Cherry
ND
ND
Peach
75.4
1.75
Bloom
Pollen
Plum
25.0
1.91
Sweet Cherry
117
1.04
Peach
2.18
1.58
AntherPlum
ND
2.01
Sweet Cherry
10.4
2.92
Peach
<LOQ (0.44)
ND
NectarPlum
0.58<LOQ (0.45)
Sweet Cherry<LOQ (0.35)<LOQ (0.32)
aYear 1 data is for CA-10 trial that was started to replace CA-2
<LOQ= <1 ppb for leaf, whole flower, pollen and anther samples; and <0.5 ppb for nectar samples.
ND= <LOD= ≤0.5 ppb for leaf, whole flower, pollen and anther samples; and ≤0.25 ppb for nectar
samples
7. STATISTICAL ANALYSIS
Study Objectives and Design
The study was conducted to determine the concentration of thiamethoxam and its metabolite
CGA322704 in flowers, anthers, nectar, and pollen of stone fruit trees in response to previous year’s
foliar application of a thiamethoxam pesticide product. In year 1 of the study, two foliar sprays at an
application rate of 0.086 lbs/acre were applied at 21 to 14 days before harvest of the fruit for a total
application of 0.172 lbs/ai. Leaves were sampled after the second foliar spray and analyzed forconcentrations of thiamethoxam and CGA322404 metabolite. Flower parts and leaves were harvested
in the second year of the study following the foliar applications and also analyzed. The crops at the11964

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50096606

CDPR THX Stone Fruitsame study sites received a second set of foliar treatments in the second year and the same sampling
scheme was then followed with the study due for completion upon sampling at bloom in the third year.

Initially there were 9 sites, denoted as CA-1 through CA-9, chosen for the study with 3 sites planted withplums, 3 sites planted with sweet cherries, and 3 sites planted with peach. The plum variety at site C2
named ‘Apple Dandy’ did not produce sufficient pollen for sampling so in year 2 another site, denotedCA-10, was substituted that was planted to a plum variety that produced sufficient pollen. An amended
final report was submitted that contained data for the first replicate year of site CA-2 and for two yearsof data at site CA-10. Site CA-2 was omitted from further analysis due to incomplete data.
The data call-in specified that the test sites were to be distributed across general soil texture categories
with 3 sites each in coarse, medium, and fine-textured soils. The amended report contains information
on particle size distribution, organic matter content, cation exchange capacity, pH and percent moisture
held at 1/3 bar. Based on this data, sites CA-3 and CA-9 are classified as coarse-textured soil; Sites CA-1,
CA-4, CA-6, CA-8, and CA-10 are classified as medium-textured soil; and Sites CA-5 and CA-7 as finetexture soil.
Chemical analytical results that were below detection limits were reported as less than the limit ofquantification (LOQ), less than the limit of detection (LOD), and ND, an apparent acronym for notdetected. In the footnotes on pages 181 and 448 of the report, the ND designation appears to be thesame as LOD. For example on page 181 the footnote is: ‘ND=<LOD=<5 ng/g for leaf samples’. In thetables, data are indicated as <LOD but are also denoted ND. Both of these acronyms appear to indicate
values below the LOD so they were treated as the same. A different laboratory analyzed the dataobtained from year 2 where results specified as <LOQ also had a value noted in parentheses. For
consistency between years, one-half the value of the LOQ rather than the noted value in parentheses
was assigned to the data. There were a few differences noted in the detection levels between thelaboratories. The LOQ for leaf samples in the first year was at 10ng/g whereas for the second year it was
at 1 ng/g. Leaf samples taken directly after thiamethoxam application were all greater than 10 ng/g so
the difference in LOQ did not affect these analyses. For samples taken at bloom, most of theconcentrations were below either the LOQ or the LOD so substituting different values for each year
would bias comparisons made between years. Subsequently statistical analyses were not conducted ondata for leaf samples taken at bloom. The very low leaf concentration concentrations measured atbloom in the second set of analyses logically indicates lack of thiamethoxam parent or degradate
residues in leaves at this sampling interval. Differences were also noted for the LOD for pollen andnectar samples between years where in the first year the values were slightly lower: For pollen LOD was
at 0.35 ng/g in the first year compared to 0.5 ng/g in the second year; For nectar the LOD was at 0.186
ng/g in the first year compared to 0.25 ng/g in the second year. For consistency, one-half the LOD ofthe value noted in the second year was substituted for data indicated as <LOD or as ND for
thiamethoxam parent and degradate. Detection limits are given in Table 8.
Non-parametric statistical tests were used to test for differences in distribution of concentrations
between years, between soil type and between untreated and treated sites. Non-parametric tests do
not require tests for normality as they are robust to differences in distribution and they are also robust
for experimental designs with low replicates (Helsel and Hersch, 2002). The PROC NPAR1WAY procedurein the Statistical Analysis System (SAS) statistical package was used to conduct Wilcoxon-Mann –Whitney (Wilcoxon), Median non-parametric, and Kuiper tests. A significant result from the Wilcoxon
test indicates differences in the shape of distributions; A significant result from the Median test
indicates differences in the location of the medians between distributions; and A significant result from12965

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50096606

CDPR THX Stone Fruitthe Kuiper test indicates differences in the empirical distributions between two groups. The Exact option
for each statistic was implemented as it provides permutation testing, a statistical method thatminimizes the effect of sample size and distributional differences. Using the Exact option the Monte
Carlo procedure was also implemented which provided 10,000 separate runs for each statistic toproduce the permutation distributions. The test for potential differences due to soil type had 3 levels so
the DSCF option in PROC NPAR1WAY, which invokes the Dwass, Steel, Critchlow-Fligner multiple
comparison test , was used to provide pairwise tests for two-sample rankings. Additional procedures
used for descriptive statistics were PROC MEANS to calculate mean values from the replicates at eachsite, PROC CAPACITY to produce cumulative statistics, and PROC BOX plot to produce comparativegraphics. Statistical analysis for effect of years and soil type were conducted on the mean of thereplicate samples taken from each site. Since many of the data at bloom were indicated below detection
limits, graphical comparisons are presented on data transformed to a natural logarithm scale, providing
clearer contrasts between the distributions. Final presentation of the potential distribution ofconcentrations in bee relevant plant matrices is based on all raw data because these values represent
the actual range of exposure to bees and other organisms that feed off the nectar and pollen of plants.
Detection rate noted for each plant matrix: Counts for the number of samples reported above each of
the noted detection limits are presented in Table 9 where Table 9A contains data for treated plants andTable 9B contains data for untreated plants. For leaf analyses, the data appear as expected where
sampling post application resulted in all concentrations for leaf samples above the LOQ in treated plants
and below the LOQ in untreated plants. For samples taken the next year at bloom, the range inconcentration for leaves between treated and untreated plants was similar where counts of most leaf
concentrations were below the LOQ but above the LOD. Except for pollen samples, the distribution of
detections for the other plant matrices between treated and untreated plants was similarly below
detection limits. For pollen, numerous samples above the LOQ were reported for both treated anduntreated plants where, for example, the overall frequency of detection for thiamethoxam was 100% fortreated plants and 94% for untreated plants (Table 10).
Comparison of distribution between years: Potential difference between years was measured for two
reasons. First, greater concentrations measured in year 2 would indicate potential for carry-over effects
between years. Second, if there was no effect of years then the data could be pooled for subsequent
tests between soil type and to untreated control data. Comparison of the distribution between years
was conducted for treated plants because of the probability for detection of thiamethoxam residues. No
significant difference in the distribution of concentration of residues between years was measured asindicated by the lack of significance in the majority of the non-parametric tests (Table 11). Tests forsome degradation products indicated significance but most of these analyses were below detection
limits so those results are most likely an artifact of the substitution of ½ of the respective detection
limits. Graphical comparisons between years indicate significant overlap in the distribution ofconcentration for total residue between years: Figure 1 illustrates the comparison for leaf samples
between years taken after application and at bloom and Figure 2 illustrates the comparison for samples
taken at bloom from the flowers.
Comparison of distribution between untreated and treated plants: As indicated in the previous
comparison for counts of data above the detection limits, the frequency of detection in pollen, nectar,
anthers, and whole flowers was similar between treated and untreated plants. Non-parametric tests
13966

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50096606

CDPR THX Stone Fruit

indicated no significant difference in distribution between untreated and treated plants (Table 12).
Figure 3 illustrates the comparison for between treated and untreated plants for leaves and Figure 4
illustrates the comparison for bloom plant samples. For all matrices at bloom, except pollen,
concentrations of thiamethoxam were below detection limits in untreated plants. The result for pollen
was not typical because the expectation is for no detection of residues in plants in untreated plots.
Maximum concentrations measured for pollen were similar between treated and untreated plants at
383 ng/g and 382 ng/g, respectively. The effect appears to be localized to the blooms because residues
in leaves from untreated plants were essentially non-detectedComparison of distribution between soil type: The distributions for parent thiamethoxam anddegradation product were similar between the plant matrices (Figures 5 and 6). The only indication of apotential effect was for pollen samples where a comparison between the coarse and fine textured soil
types indicated a trend where concentrations in trees grown in coarse textured soils tended to begreater (Table 13 and Figure 6).
Data for bee relevant matrices: Except for pollen samples, the similarity in distributions for plant
matrices taken at bloom between untreated and treated plants indicate that the data reported fortreated most samples indicate concentrations were below detections limits. Owing to the uncertainty as
to the source for residues measured in untreated plants for pollen, the observed distributions fromtreated plots ostensibly determines the expected range in concentrations of thiamethoxam and
CGS322704 residues in bee relevant plant samples for this combination of plant species and application
scenario (Tables 14A and 14B). For pollen, the median total residue value was 30.5 ng/g with amaximum value measured at 383 ng/g. For nectar, the median total residue value was 0.4 ng/g with amaximum value at 2.6 ng/g. Although many of the values reported for anthers were below the detection
limits, resulting in a low median value at 0.8 ng/g, a maximum total residue value was reported at 91.7
ng/g. In contrast, most of the whole flower concentrations were also low but the maximum total residue
value was low at 5.8 ng/g in flowers. Note that many of these total values were a summation of thesubstituted values inserted for data reported below the detection limits.
2. Concentrations in Bee Relevant Matrices: By default, the distributions reported for treated plants inpollen and nectar in table 14A represent the expected distributions from foliar thiamethoxam
treatments applied to stone fruit trees in the previous growing season. Median and maximum values for
total thiamethoxam residues in pollen are 30.5 and 383 ng/g on wet weight basis and for nectar at 0.4
and 2.6 ng/g, respectively. Of note is that many of the values represent the summation of values
substituted for ½ respective detections limits.
8. STUDY STRENGTHS, LIMITATIONS AND CONCLUSIONSIn the context of documenting the magnitude of thiamethoxam residues in bee-related matrices ofstone fruit (peach, plum, and sweet cherry), the following strengths are observed with this study.
1.
Data provide quantitative values for thiamethoxam and the major degradation product, CGA322704, expected in leaves, flowers, anthers, pollen, and nectar of various stone fruit when measured atbloom in response to foliar applications made in the previous growing season.

14967

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50096606

CDPR THX Stone Fruit

2.
The study was replicated over two years. Each year, whole flower, anther, pollen, and nectar
samples were collected at early bloom stage (50–75% bloom) in the spring following test substanceapplications in the fall.
3.
Sites CA-3 and CA-9 are classified as coarse-textured soil; Sites CA-1, CA-4, CA-6, CA-8, and CA-10
are classified as medium-textured soil; and Sites CA-5 and CA-7 as fine-texture soil, allowing for acomparison of the effect of soil type on concentrations measured in plant samples.
Limitations noted in this study include:
1.
Samples were taken from three types of stone fruit (peach, plum, and sweet cherry). Since
the effect of different varieties on distribution of residues is unknown, the results reflect
general observations made to all planted stone fruit.
2.

The frequency of detection in pollen, nectar, anthers, and whole flowers was similar
between treated and untreated plants with no significant difference in the distributions
between them. The similarity in distributions places a serve limit on comparing differencesin distribution caused by years or varieties because results would be confused with the large
amount +of background variance, i.e. what part of the measured effect is due to actual
treatment difference in comparison to background variation.

Overall, considering the strengths and limitations of this study, the following conclusions can be drawn:
Classification/Utility for Bee Risk Assessment. This study is classified as acceptable. Although the
distribution of thiamethoxam residues in leaves indicated large differences between untreated andtreated plants when sampled soon after the second applications, differences were not measured
between untreated and treated plants when samples were taken at bloom for all matrices, except forpollen. The similarity in distributions places a serve limit on comparing differences in distribution
caused by years or varieties because results would be confused with the large amount of backgroundvariance, i.e. what part of the measured effect is due to actual treatment difference in comparison to
background variation.
Magnitude of Residues in Bee-relevant Matrices. By default, the distributions reported for treatedplants in pollen and nectar in table 14A represent the expected distributions from foliar thiamethoxam
treatments applied to stone fruit trees in the previous growing season. For data from treated plot, themedian and maximum values for total thiamethoxam residues in pollen are 30.5 and 383 ng/g on wet
weight basis and for nectar at 0.4 and 2.6 ng/g, respectively. Of note is that many of the values
represent the summation of values substituted for ½ respective detections limits.
Temporal Variability in Residues. This study was not designed for temporal analysis of declining
concentrations, but rather, to provide a snapshot of residue concentrations during flowering. Samples
were collected at only one-time point during bloom.
Effect of Soil Type. There was generally no difference in the magnitude and distribution ofconcentrations of thiamethoxam and degradate between soils. One trend was indicated for pollen
samples where concentrations tended to be greater in coarse textured soils when compared to finetextured soils. This effect requires more study for confirmation.
15968

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50096606

CDPR THX Stone Fruit

Pesticide Carryover. In general, there was no significant difference in the distribution of concentrationof residues between years. Graphical comparisons between years indicate significant overlap in the
distribution of concentration for total residue between years and, subsequently, no carryover effect.
9. STUDY VALIDITY/CLASSIFICATIONThis study is classified as acceptable. The data from this study provide an expected distribution of theconcentrations of thiamethoxam residues that bees are exposed to in extra-floral nectar, nectar, andpollen of stone fruits grown under actual agronomic practices in California. Relating concentrations
measured in flower parts to bee health is possible by comparing the concentrations measured in bee
relevant plant parts to target values that define acute or chronic exposure scenarios. However,differences were not measured between untreated and treated plants when samples were taken atbloom for all matrices. The similarity in distributions places a serve limit on comparing differences in
distribution caused by years or varieties because results would be confused with the large amount +of
background variance, i.e. what part of the measured effect is due to actual treatment difference incomparison to background variation.

16969

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50096606

CDPR THX Stone Fruit

Table 8. Detection limits reported for thiamethoxam and CGA322704 for each year of the study andfor each plant sample. Note that the for nectar and pollen samples the slightly higher values for onehalf the ND/LOD from the second year were used for substitution.

Detection Limit Noted for Thiamethoxam and CGA322704

Year and Plant Sample
Year 1
Leaf
FlowerNectarPollenYear 2
Leaf
FlowerNectarPollenYear 1 and 2
Anthers

LOQ(ng/g Wet
Weight)

1/2 LOQ(ng/g Wet
Weight)

ND/LOD(ng/g Wet
Weight)1/2 ND/LOD(ng/g Wet
Weight)

10.00
1.00
0.50
1.00

5.00
0.50
0.25
0.50

5.00
0.50
0.186
0.35

2.50
0.25
0.093
0.18

1.00
1.00
0.50
1.00

0.50
0.50
0.25
0.50

0.50
0.50
0.25
0.50

0.25
0.25
0.125
0.25

1.00

0.50

0.50

0.25

17
970

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50096606

CDPR THX Stone Fruit

Table 9A. Treated Plants: Counts of chemical analytical results for thiamethoxam and CGA322704 that were indicated as above the LOQ,
between the LOQ and LOD, and below the LOD.

Year and Plant Sample
Year 1Treated Plants: Comparison of total Number of Samples to Results Reported Above the LOQ,
Between the LOQ and LOD, or Below the ND/LOD
Thiamethoxam
CGA322704
Total
NumberNumberNumber
Total
NumberNumberNumberNumber>LOQ LOQ>LOD <ND/LOD Number>LOQ
LOQ>LOD <ND/LOD

Leaves: After Application
Leaves: At Bloom
Anthers
PollenWhole Flowers
Nectar

27
27
27
23
27
27

27
1
3
23
4
3

0
24
6
0
7
11

0
2
18
0
16
13

27
27
27
23
27
27

27
3
4
9
2
0

0
24
1
8
4
8

0
0
22
6
21
19

Year 2
Leaves: After Application
Leaves: At Bloom
Anthers
PollenWhole Flowers
Nectar

27
27
27
27
27
27

27
6
9
27
8
7

0
3
5
0
2
6

0
18
13
0
17
14

27
27
27
27
27
27

27
17
13
18
1
6

0
6
7
3
3
4

0
4
7
6
23
17

18
971

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50096606

CDPR THX Stone Fruit

Table 9B. Untreated Plants: Counts of chemical analytical results for thiamethoxam and CGA322704 that were indicated as above the LOQ,
between the LOQ and LOD, and below the LOD.

Untreated Plants: Comparison of total Number of Samples to Results Reported Above the LOQ,
Between the LOQ and LOD, or Below the ND/LOD

Year and Plant Sample
Year 1
Leaves: After Application
Leaves: At Bloom
Anthers
PollenWhole Flowers
NectarYear 2
Leaves: After Application
Leaves: At Bloom
Anthers
PollenWhole Flowers
Nectar

Total
NumberThiamethoxam
NumberNumberNumber>LOQ LOQ>LOD <ND/LOD

Total
NumberCGA322704
NumberNumberNumber>LOQ LOQ>LOD <ND/LOD

9
9
9
8
9
9

0
0
5
7
2
4

8
8
0
1
0
3

1
1
4
0
9
2

9
9
9
8
9
9

1
0
1
1
0
0

8
8
0
2
1
3

0
1
8
5
8
6

9
9
9
9
9
9

0
0
2
9
0
0

0
0
1
0
0
1

9
9
6
0
9
8

9
9
9
9
9
9

0
0
5
5
0
2

0
1
2
4
0
1

9
8
2
0
9
6

19
972

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50096606

CDPR THX Stone Fruit

Table 10. Comparison of the frequency of detection of thiamethoxam and CGA322704 in leaves, anthers, Counts of chemical analyticalresults for thiamethoxam and CGA322704 that were indicated as above the LOQ, between the LOQ and LOD, and below the LOD.

Overall Frequency (%) of Samples Reported Above the LOQ,
Between the LOQ and LOD, or Below the LOD

Chemical and Plant Sample
Thiamethoxam
Leaves: After Application
Leaves: At Bloom
Anthers: At Bloom
Pollen: At Bloom
Whole Flowers: At Bloom
Nectar: At Bloom
CGA322704
Leaves: After Application
Leaves: At Bloom
Anthers: At Bloom
Pollen: At Bloom
Whole Flowers: At Bloom
Nectar: At Bloom

Treatment Treated
Proportion
Proportion
Proportion
Above LOQ Below LOQ Below LOD

Untreated Trees
Proportion
Proportion
Proportion
Above LOQ Below LOQ Below LOD

100
13
22
100
22
19

0
50
20
0
17
31

0
37
57
0
61
50

0
0
39
94
11
22

44
44
6
6
0
22

56
56
56
0
89
56

100
37
31
54
6
11

0
56
15
22
13
22

0
7
54
24
81
67

6
0
33
35
0
11

44
50
11
35
6
22

50
50
56
29
94
67

20
973

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50096606

CDPR THX Stone Fruit

Table 11. Effect of Years: Exact probability levels for Wilcoxon and Median non-parametric tests for differences in the distribution ofthiamethoxam and CGA322704 degradate between years.

Exact Probability Levels for Non-parametric Tests of Differences Between Years
Thiamethoxam Treated Plants

Plant Sample
and Interval

Thiamethoxam
CGA322704
Total Residue
Wilcoxon
Median Kuiper Wilcoxon Median Kuiper Wilcoxon Median Kuiper

Leaves: After Application
Anthers
PollenWhole Flowers
Nectar

0.6
0.4
0.09
0.73
0.6

1
0.35
0.35
1
1

0.89
1
0.89
0.89
0.89

0.6
0.02
0.05
0.98
0.48

21
974

1
0.06
0.35
1
1

0.37
0.09
0.57
1
0.89

0.54
0.039
0.1
0.82
0.51

0.63
0.35
0.34
1
0.69

0.97
0.89
0.89
1
0.57

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50096606

CDPR THX Stone Fruit

Table 12. Untreated vs Treated Plants: Exact probability levels for Wilcoxon and Median non-parametric tests for differences in the
distribution in chemical analyses conducted on untreated control plants and plants treated with a foliar spray of thiamethoxam.

Exact Probability Levels for Non-parametric Tests of Differences in Concentration Distribution
Between Untreated Control and Treated Plants
Thiamethoxam
CGA322704
Total Residue
Plant Sample
Anthers
PollenWhole Flowers
NectarWilcoxon
0.89
0.34
0.07
0.5

Median
1
0.34
0.06
1

Kuiper
0.89
0.89
0.57
0.89

Wilcoxon
0.42
0.15
0.21
0.86

Median
1
0.34
0.56
1

22
975

Kuiper0.89
1
1
1

Wilcoxon
0.53
0.35
0.03
0.93

Median
1
0.33
0.06
1

Kuiper
0.89
0.89
0.27
0.89

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50096606

CDPR THX Stone Fruit

Table 13. Effect of Soil: Exact probability levels for Wilcoxon and Median non-parametric tests fordifferences in the distribution of thiamethoxam and CGA322704 degradate between trees grown in
different soil types.
A) Non-Parametric Test Results

Exact Probability Levels for Non-parametric
Tests of Differences Between Soil Type
Plant Sample
and Interval
Leaves: After Application
Anthers
PollenWhole Flowers
NectarThiamethoxam
Wilcoxon
0.039
0.48
0.08
0.64
0.32

CGA322704
Wilcoxon
0.11
0.89
0.78
0.22
0.62

Total Residue
Wilcoxon
0.045
0.7
0.08
0.19
0.41

B) Pollen Pairwise Tests Between Soil Types
Pairwise Two-Sided Multiple Comparison Analysis
Dwass, Steel, Critchlow-Fligner Method
Variable: mthia
soil

Wilcoxon Z

DSCF Value

Pr > DSCF

Medium vs. Coarse

1.69712.4

0.2063Medium vs. Fine

-1.27281.8

0.4106

Coarse vs. Fine

-2.3094

3.266

0.054523976

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50096606

CDPR THX Stone Fruit

Table 14A. Distribution of thiamethoxam and CGA322704 degradate measured in pollen, nectar, and anthers sampled from stone fruit thatwere exposed to two applications of thiamethoxam in the year previous to bloom. Samples were combined from two consecutive years ofstudy.

PollenStatistic

NectarAnthers

Thiamethoxam

CGA322704

Total

Thiamethoxam

CGA322704

Total

Thiamethoxam

CGA322704

Total

N (#)

50

50

50

54

54

54

54

54

54

Mean (ng/g)

54.2

1.1

55.3

0.4

0.2

0.6

2.9

1.6

4.5

SD (ng/g)
CV (%)

75.5
139.3

1.2
104.7

75.5
136.5

0.5
135.7

0.3
117.0

0.6
98.8

12.0
420.4

2.9
175.2

13.1
290.9

Min (ng/g)
Median (ng/g)
75th (ng/g)
90th (ng/g)

0.4
29.6
63.3
132.0

0.2
0.9
1.6
2.4

0.5
30.5
63.6
133.2

0.1
0.2
0.3
1.0

0.1
0.1
0.3
0.5

0.3
0.4
0.5
1.6

0.3
0.3
0.5
4.4

0.3
0.3
2.2
4.4

0.5
0.8
3.5
7.9

95th (ng/g)
Max (ng/g)

181.6
382.0

2.6
5.9

182.1
383.0

1.8
2.4

0.7
1.7

2.0
2.6

7.2
87.5

5.8
15.8

11.7
91.7

% of Total

98.0

2.0

60.7

39.3

63.7

36.3

24
977

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50096606

CDPR THX Stone Fruit

Table 14B. Distribution of thiamethoxam and CGA322704 degradate measured in whole flowers and leaves sampled either after application
or in the next year after application at bloom. Samples are from stone fruit that were exposed to two applications of thiamethoxam in theyear previous to bloom. Samples were combined from two consecutive years of study.

Whole Flowers

Leaves: Post Application

Leaves: At Bloom

Statistic

Thiamethoxam

CGA322704

Total

Thiamethoxam

CGA322704

Total

Thiamethoxam

CGA322704

Total

N (#)

54

54

54

54

51

51

54

54

54

Mean (ng/g)

0.9

0.3

1.2

6741.6351.0

7197.7

1.9

2.1

4.0

SD (ng/G)

1.4

0.2

1.4

3648.8

188.7

388.5

1.9

1.3

2.7

CV (%)

154.9

67.7

114.3

54.1

53.8

53.4

95.6

63.0

68.2

Min (ng/g)

0.3

0.3

0.5

2097.0110.0

2273.0

0.3

0.3

0.5

Median (ng/g)

0.3

0.3

0.8

5257.5308.0

5630.0

2.5

2.5

5.0

75th (ng/g)

0.5

0.3

1.3

9508.0399.09828.0

2.5

2.5

5.0

90th (ng/g)

3.5

0.5

4.2

11723.0478.012006.4

4.4

3.4

5.7

95th (ng/g)

4.9

1.1

5.2

13116.2

766.7

14004.4

4.5

5.3

10.9

Max (ng/g)

5.5

1.2

5.8

20996.5

1232.422229.0

10.4

6.3

12.5

% of Total

71.9

27.3

93.7

4.9

47.5

52.5

25
978

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50096606

CDPR THX Stone Fruit

Figure. 1. Year Comparison for Leaves: Comparison of the distribution between years for leaf
thiamethoxam residues transformed to natural logarithms where sampled were obtained either
directly after foliar application of thiamethoxam denoted ‘PostApp’ or at bloom in the year following
application denoted ‘Bloom’.

Stone Fruit: Natural Logrithm of Leaf Thiamethoxam Concentration
Nat
atur
ural
al Logar
Logariithm Thi
hiam
amet
ethox
hoxam
am Conc
oncententrrat
atiion (ng/
ng/g)
g)

10.0
0
10.

7.5

5.0

2.5

0.0

-2.5

ampl
pliing Int
nter
erv
val
al:: PostApp
Sam

Bloom
Year 1

Year 2

Year 1

Year

26
979

Year 2

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50096606

CDPR THX Stone Fruit

Figure 2. Year Comparison for Bloom Plant Samples: Comparison of the distribution between years for
nectar, pollen, anthers, and whole flowers thiamethoxam residues transformed to natural logarithms.
Samples were taken in the year that followed a previous foliar application.

Natural Log Thiamethoxam Concentration in Stone Fruit Plant Tissue

at uralhiametonc entrrati
at ion (ng/g)
ng/ g)
Natural Log Thiam
ethox
hoxam
am Concent

6

4

2

0

-2

-4

Plant Samampl
ple:
e: Nectar
Year 1

PollenYear 2

Anthers

Year 1

Year 2

Year 1

Year

27
980

Flowers
Year 2

Year 1

Year 2

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50096606

CDPR THX Stone Fruit

Figure 3. Untreated vs Treated Leaf Samples: Comparison of the distribution of total thiamethoxam
residues measured between leaves of untreated control (UTC) and treated (TRT) stone fruit trees
where samples were obtained after the second foliar spray application denoted ‘PostApp’ and then atbloom in the next year denoted ‘Bloom’ .

Stone Fruit: Natural Logrithm of Leaf Thiamethoxam Concentration
Nat
atur
ural
al Logar
Logariithm Thi
hiam
amet
ethox
hoxam
am Conc
oncententrrat
atiion (ng/
ng/g)
g)

10.0
0
10.

7.5

5.0

2.5

0.0

-2.5

ampl
pliing Int
nter
erv
val
al:: PostApp
Sam

Bloom
UTC

TRT

UTC

ntrreateated
ed Cont
ontrrol (UTC) vs Treateatm
ment (TRT) Dis tribut
butiion
Unt

28
981

TRT

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50096606

CDPR THX Stone Fruit

Figure 4. Untreated vs Treated Bloom Plant Samples: Comparison of the distribution of thiamethoxam
residues measured between untreated control (UTC) and treated (TRT) plants A) Pollen and B) nectar,
anthers, and whole flowers. Samples were taken in the year that followed a previous foliar
application.

Natural Logarithm of Thiamethoxam Concentration in Stone Fruit Plant Tissue
Nat
atur
ural
al Logar
Logariithm of Thi
hiam
amet
ethox
hoxam
am Conc
oncententrrat
atiion (ng/
ng/g)
g)

6

4

2

0

-2

-4

ampl
ple:
e: Nectar
Plant SamUTC

PollenTRT

Anthers

UTC

TRT

UTC

Flowers
TRT

UTC

ntrreateated
ed Cont
ontrrol (UTC) vs Treateatm
ment (TRT) Dis tribut
butiion
Unt

29
982

TRT

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50096606

CDPR THX Stone Fruit

Figure 5. Soil Texture Comparison for Leaf Analyses: Comparison of the distribution of thiamethoxam
residues measured between stone fruit trees grown in coarse, medium, and fine-textured soils.
Samples were taken in the year that followed a previous foliar application.

30
983

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50096606

CDPR THX Stone Fruit

Figure 6. Soil Texture Comparison for Flower Analyses: Comparison of the distribution ofthiamethoxam residues measured between stone fruit trees grown in coarse, medium, and finetextured soils. Samples were taken in the year that followed a previous foliar application.

31984

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50096606

CDPR THX Stone Fruit

10. REFERENCES

1. Anderson, L. 2007. Thiamethoxam (CGA293343) and CGA322704. Validation of Residue
Analytical Method REM 179.07 for the determination of Residues in Bee and Hive Products andStorage Stability in Hive Pollen, Wax and Nectar, stored Deep Frozen for 12 months. (Study No.
05-S508).
2. Crook, S. 2004. Residue Method for the Determination of Residues of Thiamethoxam(CGA293343) and CGA322704 in Lettuce, Tomato, Grape and Tobacco Samples. Final
Determination by LC/MS/MS (Syngenta Method REM179.06).
3. Crook, S. 2007. Analytical Method for the Determination of Residues of Thiamethoxam(CGA293343) and CGA322704 in Bee and Hive Products. Final Determination by LC-MS/MS(Syngenta Method REM179.07)
4. Helsel, D.R. and R.M. Hirsch. 2002. Chapter A3: Statistical Methods in Water Resources.
Techniques of Water-Resources Investigations of the United States Geological Survey Book 4,
Hydrologic Analysis and Interpretation. United States Geological Survey.
5. Hohl, J. 1999. Stability of Residues of CGA-322704 in Plant Material and Soil Stored Under Deep
Freezer Conditions (Study No. 779-00). (MRID 45108001).
6. Mair, P. 1998. Stability of Residues of CGA-293343 (2 Years Final Report) and CGA-322704 (1Year Interim Report) in Plant Material under Deep Freezer Conditions, Including Method
Validation (Study No. 504/96 consists of Reports #112/96, 127/97, 103/98). (MRID 44703525).
7. Oakes, T. 2002. Stability of CGA-293343 and CGA-322704 in Crops and Processed Fractions
Under Freezer Storage Conditions (Study No. 269-98). (MRID 45659205).

32
985

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50265502

CDPR THX Strawberry

Reference
Trask, J. (2017) Thiamethoxam 25 WG (A9584C) - Magnitude of Residues in Pollen, Nectar, Flowers,
and Leaves of Strawberry After Foliar Application with Actara 25WG in California: Final Report. Report
Number: TK0177224. Unpublished study prepared by Syngenta Crop Protection, LLC. 346. MRID
50265502, CDPR Study ID 301205, Data Volume 52691-0574, Tracking ID# 282051

1. STUDY INFORMATION
Chemical:Thiamethoxam
Test Material:

Study Type:

Sponsor:Sponsor Study
ID:
Study
Completion
Date:

GLP Status:

PC Code60109
Percent
Actara 25WG
Active25%
Ingredient:
Study to measure Thiamethoxam and CGA322704 residues in strawberry pollen,
nectar, flowers and leaves from nine field trials that received foliar applications ofActara 25 WG for two growing seasons.
Syngenta Crop Protection, LLC
Experiment Start
410 Swing Road
April 28, 2014 – June 1,
andGreensboro, North Carolina
2017
End Date:
27409
Nine strawberry field
TK0177224
trials located in in theCentral Valley andStudy Locations:
Coastal Region ofJune 1, 2017
California.
Non-GLP; protocol reviewed by CDPR.
[CDPR Study ID 301205, Data Volume 52691-0574, Tracking ID# 282051]

2. REVIEWER INFORMATION
Study Reviewed by:Richard Bireley, Sr. Environmental Scientist (Specialist)
California Department
John Troiano, Ph.D., Research Scientist III
of Pesticide Regulation
Alexander Kolosovich, Sr. Environmental Scientist (Specialist)
Brigitte Tafarella, Environmental Scientist
Denise Alder, Sr. Environmental Scientist (Specialist)
Russell Darling, Environmental Scientist
3. EXECUTIVE SUMMARY
A nine-site field trial study on strawberries was conducted in the primary strawberry producing areas ofCalifornia. This study was conducted to measure the levels of thiamethoxam and its major metabolite,
CGA322704, in leaves, flowers, soil, pollen, and nectar of strawberries after foliar treatment applicationsof Actara® 25WG were made at the maximum labeled use rate. Residue data for thiamethoxam and its
major metabolite, CGA322704, in the pollen and nectar of strawberry were requested by the California
Department of Pesticide Regulations (CDPR) as part of the reevaluation of the nitroguanidine class ofneonicotinoid insecticides (Article 8, Subchapter 1, Chapter 2, Division 6 of Title 3 of the California Codeof Regulations), according to the “Data Requirements Regarding Re-evaluation of Certain Neonicotinoid
Products” (Prichard, September 15, 2009) and “Recommendations for Additional Thiamethoxam
1986

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50265502

CDPR THX Strawberry

Residue Studies in Almond, Cotton, and Strawberry” (Bireley and Troiano, October 4, amended
December 7, 2012). This study was designed to satisfy this request.
4. STUDY VALIDATION

Guideline Followed:
Guideline Deviations:
Other Deviations:Classification:
Rationale:
Reparability:

TBD; (protocol was reviewed by CDPR)
N/A
N/A
TBD
N/A
N/A

5. MATERIALS AND METHODS

Test item:
Formulation Type:
CAS #:

Test Material Characterization
Actara 25WG
Percent A.I.:
Water Dispersible
Batch Number:
Granule153719-23-4
EPA Reg. No.

25%697333/731279
100-938

5A. STUDY DESIGN
The study included nine geographically separated replicated trials each consisting of a non-treated and atreated plot large enough to fulfill sample collection requirements. The treated plots were divided into 3
replicate sub-plots (A, B, and C). The size of each sub-plot varied at each location, measuring 130 ft x
36.7 ft (CA-1 season 1), 110 ft x 43 ft (CA-1 season 2), 150 ft x 30 ft (CA-2 season 1), 125 ft x 30 ft (CA-2
season 2), 100 ft x 60 ft (CA-3) , 100 ft x 60 ft (CA-4), 100 ft x 50 ft (CA-5), 150 ft x 32 ft (CA-6), 150 ft x 32
ft (CA-7), 230 ft x 35 ft (CA-8) and 100 ft x 75 ft (CA-9). At each location, the control plot was located upslope and up-wind with regard to the prevailing wind direction and separated by a minimum of 200 ft.
for all sites from the treated plot to minimize potential cross-contamination by runoff or pollen transfer
except CA-4, which was 75 feet due to site constraints.
The original two-year study design included test substance applications made during two growing
seasons with sample collection during bloom in each season. However, after the first season application
at the three central valley locations (CA-1, CA-2 and CA-3), weather conditions resulted in poor flower
production and residues could not be analyzed. Therefore, the study design was modified in thefollowing ways: (1) New plants were re-established at the sites (CA-1, CA-2 and CA-3) in the fall of 2014.
Season 2 applications were made in the spring of 2015, matrices collected, and the resulting residue
data is reported. (2) Two additional Central Valley trial locations were added (CA-4 and CA-5) withmatrices collected after single season applications (spring of 2015). (3) A one-season study design was
also used for the four coastal region trial locations (CA-6 through CA-9) with matrices collected after
applications in the spring of 2015.

2987

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50265502

CDPR THX Strawberry

5B. APPLICATION TIMING AND RATESTreatment specifications, including target application rate, spray timing and volume, is presented in thefollowing table.
Table 1. Thiamethoxam Applications with Actara 25WD.
Treatment List
NominalTRT ID Application Application Volume
Rate
Tank Mix(plot)
NumberMethod(GPA)(+10%) Additives(lb
ai/acre)CA-1*
UTC-Control
---1
52.9
0.063
2
54.8
0.063
TRT
Foliar
None
3
55.9
0.063
Broadcast
4
51.0
0.063
Spray
5
50.7
0.063
6
50.4
0.063
CA-2*
UTC
-Control
---1
56.0
0.063
2
55.3
0.063
TRT
Foliar
None
3
55.5
0.063
Broadcast
4
51.0
0.063
Spray
5
50.9
0.063
6
50.4
0.063
CA-3*
UTC
-Control
---1
65.3
0.063
2
65.4
0.063
TRT
Foliar
None
3
65.3
0.063
Broadcast
4
65.7
0.063
Spray
5
65.8
0.063
6
65.7
0.063
CA-4
UTC
-Control---1
Foliar
65.2
0.063
TRT
Broadcast
None
2
65.7
0.063
Spray
3
65.8
0.063
CA-5
UTC
-Control---1
Foliar
59.8
0.063
TRT
Broadcast
None
2
60.2
0.063
Spray
3
59.6
0.063
3
988

Timing

RTI(days)

Total
Rate (lbai/acre)

-25
15
5
25
15
4

-10
10
-10
11
--

--

-25
15
5
25
16
5

-10
10
-9
11
--

-25
15
5
25
15
5

-10
10
-10
10
--

-25
15
5

-10
10
--

-25
15
5

-10
10
--

0.189

0.189

-0.1890.189-0.1890.189-0.189-0.189Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50265502

CDPR THX Strawberry

Treatment List
NominalTRT ID Application Application Volume
Rate
Tank Mix Timing
RTI
Total
(plot)
NumberMethod(GPA)(+10%) Additives(days) Rate (lb(lbai/acre)ai/acre)CA-6
UTC-Control------1
Foliar
50.0
0.063
Adjuvant
25
10
TRT
Broadcast(Dnye0.189
2
49.8
0.063
15
10
Spray
Amic)
3
49.9
0.063
5
-CA-7
UTC
-Control------1
Foliar
74.9
0.063
Adjuvant
25
9
TRT
Broadcast(Spreader
0.189
2
74.8
0.063
16
11
Spray
90)
3
74.4
0.063
5
-CA-8
UTC
-Control------1
Foliar
69.7
0.063
Adjuvant
25
10
TRT
Broadcast(Kinetic)
0.189
2
69.8
0.063
15
10
Spray
3
70.2
0.063
5
-CA-9
UTC
-Control------1
Foliar
49.8
0.063
Adjuvant
28
12
TRT
Broadcast(Spreader
0.189
2
49.8
0.063
16
9
Spray
90)
3
49.7
0.063
5
-Application Notes:
GPA = gallons per acre; RTI = Retreatment Interval
Adjuvants applied at labeled use rates*Season 1 (2014) applications are application #1-3; season 2 (2015) applications are application#4-6

5C. STUDY SITE LOCATION AND CHARACTERISTICS
Table 2. Trial Site Conditions for Strawberry Trials
Soil Characteristics
Trial
Identificati
Texture Class%Sand %Silt %Clayon (City,
State/Year)

CA-1
Porterville,
California
2014-2015

Clay Loam

37

23

40

4
989

%OM

pH

CEC
(meq/
100g)

Meteorological Data1
Study
OverallMonthly
TemperaRainfall
ture
Ranch
Range
(inches)
(°F)

1.8

6.9

28.9

0.00 to 2.27

22 to 107

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50265502
Trial
Identificati
on (City,
State/Year)

CA-2
Porterville,
California
2014-2015
CA-3
Yuba City,
California
2014-2015
CA-4
Woodland,California
2015
CA-5
Fresno,California
2015
CA-6
Nipomo,California
2015
CA-7
Guadalupe,
California
2015
CA-8
Salinas,California
2015
CA-9
Salinas,California
2015

CDPR THX Strawberry

Texture Class

%Sand

%Silt

%Clay

%OM

pH

CEC
(meq/
100g)

Meteorological Data1
Study
OverallMonthly
TemperaRainfall
ture
Ranch
Range
(inches)(°F)

Loamy Sand

81

13

6

0.72

5.7

14.5

0.00 to 2.27

22 to 107

Sandy Clay Loam

53

25

22

1.9

6.3

13.4

0.00 to 7.92

23 to 106

Loam

47

31

22

1.6

7.9

18.0

0.09 to 7.10

27 to 107

Loamy Sand

82

14

4

0.53

7.7

4.5

0.00 to 1.28

37 to 107

Sandy Loam

71

19

10

1.2

7.8

11.1

0.00 to 4.02

32 to 98

Loamy Sand

77

17

6

0.85

7.9

9.6

0.00 to 4.03

32 to 98

Clay Loam

33

33

34

2.3

7.6

24.5

0.00 to 3.06

30 to 98

Clay Loam

37

33

30

2.1

7.3

21.7

0.00 to 3.06

30 to 98

Soil Characteristics

OM= Organic Matter, CEC= Cation Exchange Capacity
1
Precipitation and air temperature data summarized are representative of the time period (whole months) fromplanting through sample collection for each trial.

The weather conditions (high air temperatures) at CA-1, CA-2 and CA-3 in the spring and summer of2014 resulted in poor flower production for the first season of this study. Therefore, new plants were reestablished in November 2014 to the same plots. Irrigation was used to supplement rainfall at all trial
locations and is presented in the Field Trial Summaries (Appendix 1 of the study report).

5990

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50265502

CDPR THX Strawberry

5D. SAMPLE COLLECTION, HANDLING, PROCESSING
For all trials, the non-treated control plots were sampled first or by separate personnel, to prevent
contamination. For each matrix, one sample was collected from each treated replicate plot A, B, and C,
as well as from the control plot.
Leaf and Whole Flower
At all field sites, leaf and whole flower samples were collected at 5 days after the third application (5DA3A), also referred to as days after last application (DALA) during bloom. In addition, leaf samples werecollected before (0 DA3A) and after bloom (4 and 8 weeks after the last application) to establish adecline curve. Target weights of 250g for leaves and 100g for flowers were collected. Additionally, bulk,
non-treated leaves and flowers with target weights of 500g for leaves and 100g for flowers werecollected for laboratory verification and concurrent fortifications. Leaves and flowers were collected
directly into labelled sealable plastic bags and held in separate control and treated ice chests on wet orblue ice until placed into frozen storage. Samples were collected from the lower, middle and upper plant
canopy for a representative, composite sample; sample weights are presented in the field trial
summaries located in Appendix 1 of the study report.
Pollen and Nectar
At all field sites, pollen and nectar samples were collected at 5 DA3A during bloom. Male and female
flowers were collected from the untreated control and the treated sub-plots, bagged and placed in ice
chests with wet or blue ice and then transported to the field laboratory for pollen and nectar extraction.
Pollen samples were extracted manually from male flowers using a plastic filtered collection tip attached
to a vacuum pump. The tips were weighed before and after pollen extraction and the net weight
represented the sample size. Once the target weight of 100mg was obtained, (or all flowers available forpollen sampling were used), the plastic tips containing pollen were wrapped in parafilm and placed inlabeled plastic bottles. The bottles were sealed, placed in resealable plastic bags, and transferred
immediately into separate freezers for the treated and untreated samples.
Female flowers were left in ice chests with blue ice for several hours after collection to maximize nectar
collection. Nectar samples were collected manually by cutting off the flower petals and sepals withscissors. The remaining flower was placed into a centrifuge tube with a filter insert and mesh screen.
The nectar was then harvested using a micro centrifuge by spinning for approximately five seconds. The
filter insert and mesh screen was removed and the centrifuge tube was weighed. Samples were thenwrapped in aluminum foil and placed into a 60-mL amber HDPE bottle. Once the target weight of 100mg
was obtained, (or all flowers available for nectar sampling were used), the vials were transferred into
separate freezers for the treated and untreated samples. Additionally, 3g of nectar and pollen samples
were collected for analytical method development and verification.
Soil
At all field sites, soil samples were collected prior to planting and after the last leaf sampling, targeting
120 DA3A. At each sampling event, five 0-6-inch soil cores were collected from each plot (treated anduntreated) and composited by plot. The target weight for soil samples was 200g.

6991

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50265502

CDPR THX Strawberry

Soil samples were placed into labelled bags and placed into frozen storage. Freezer-storage
temperatures were monitored and typically were maintained at -20°C.
Sample Storage.

All residue samples (leaf, whole flower, pollen, nectar and soil) were shipped from the test sites inseparate ice chests for treated and untreated with dry ice to SynTech analytical laboratory via ACDS
overnight service.
Three separate storage-stability studies; MRID 44703525 (Reference 5), MRID 45108001 (Reference 6)
and MRID 45659205 (Reference 7) were conducted to determine the stability of thiamethoxam(CGA293343) and its metabolite, CGA322704, in various crop matrices and soil stored under deepfreezer conditions. Storage stability for pollen and nectar stored under deep-freezer conditions was
conducted in Syngenta Study No. 05-S508 (Reference 8). These studies showed that thiamethoxam andmetabolite CGA322704 are stable in leaves, whole flower, pollen, and nectar for up to 12 months whenstored frozen.
Additionally a storage stability study, MRID 47751401 (Reference 9), was conducted on soil stored underdeep-freezer conditions for thiamethoxam and metabolite CGA322704. This study showed thatthiamethoxam and CGA322704 are stable in soil for up to 12 months when stored frozen.
Therefore, residues of thiamethoxam and CGA322704 in strawberry leaf, whole flower, pollen, and soil
samples should not have been adversely affected by frozen storage during this study.
5E. ANALYTICAL METHODS
Analysis of Leaf and Whole Flower
Leaf and whole flower samples were analyzed for thiamethoxam and CGA322704 based on theanalytical method described in Syngenta Method REM 179.06, entitled “Residue Method for theDetermination of Residues of Thiamethoxam (CGA 293343) and CGA 322704 in Lettuce, Tomato, Grapeand Tobacco Samples. Final Determination by LC-MS/MS” (Reference 1). The method is presented in the
Analytical Phase Report located in Appendix 2 of the study report. In summary, residues ofthiamethoxam and CGA322704 were extracted with 50:50 methanol/water from 10g leaf and wholeflower samples using a high-speed homogenizer. Extracts were centrifuged and concentrated via SPE
cleanup in preparation for LC-MS/MS analysis. The Limit of Quantitation (LOQ) for both analytes inleaves and flowers was 1.0 ppb. The Limit of Detection (LOD) was targeted to be ≤0.5 ppb for bothanalytes and matrices. Field samples were diluted for analysis as appropriate, to keep the analyte
response within the initial calibration range.
Analysis of Pollen and Nectar
Pollen and nectar samples were analyzed for thiamethoxam and CGA322704 based on the analyticalmethod described in Syngenta Method REM 179.07, entitled “Thiamethoxam: Analytical Method for theDetermination of Residues of Thiamethoxam (CGA293343) and CGA322704 in Bee and Hive Products.
Final Determination by LC-MS/MS”, with modifications found in EPL_BAS Method No. 110G747B,“Analytical Method for the Determination of Residues of Thiamethoxam and CGA3222704 in Pollen by7992

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50265502

CDPR THX Strawberry

LC-MS/MS” (References 3 and 4). The method is presented in the Analytical Phase Report located inAppendix 2 of the study report.

In summary, residues of thiamethoxam and CGA322704 in pollen samples were extracted withmethanol:0.2% formic acid in deionized (DI) water (50:50 v/v). Aliquots were diluted with DI water andstable isotope labeled internal standards were added. Sample extracts were then purified by solid-phaseextraction (Oasis HLB) and analyzed by high performance liquid chromatography with triple quadrupole
mass spectrometric detection (LC-MS/MS). The Limit of Quantitation (LOQ) for both analytes in pollen
was 1.0 ppb. Residues of thiamethoxam and CGA322704 in nectar were extracted with 50:50
methanol/water:0.2% formic acid (aq). Extracts were centrifuged and passed through a solid-phaseextraction cleanup in preparation for LC-MS/MS analysis. The Limit of Quantitation (LOQ) for bothanalytes in nectar was 0.5 ppb. The Limit of Detection (LOD) was targeted to be ≤0.5 ppb and ≤0.25 ppb,
respectively. Field samples were diluted for analysis as appropriate, to keep the analyte response within
the initial calibration range.
Analysis of Soil
Soil samples were analyzed for thiamethoxam and CGA322704 based on the analytical method
described in Syngenta Method T009171-04, entitled “Analytical Method for the Determination of CGA293343 and its Degradates CGA-322704, CGA-355190, CGA-353042, NOA-404617, NOA-407475, SYN501406 and NOA-459602 in Soil by Direct Injection High Performance Liquid Chromatography with Mass
Spectrometric Detection” (Reference 4). The method is presented in the Analytical Phase Report locatedin Appendix 2 of the study report.
In summary, soil was extracted twice with 100 mL of 20% 10mM ammonium acetate/acetonitrile forthirty minutes at room temperature using mechanical shaking. The sample was centrifuged and filtered.
Solvent was evaporated via turbovap to approximately 20-25 mL. After evaporation, the aqueous
sample is transferred to a centrifuge tube and diluted with 10% methanol adjusted to 0.1% acetic acid toa final volume of 50 mL. The sample was then transferred into an HPLC autosampler vial for analysis by
LC/MS/MS. The Limit of Quantitation (LOQ) for both analytes, in soil, was 1.0 ppb. The Limit of Detection(LOD) was targeted to be ≤0.5 ppb. Soil sample residues were corrected for moisture in the results.
5F. QUALITY ASSURANCE RESULTS

The analytical method used to quantify residues of thiamethoxam and CGA322704 was verified oncontrol samples prior to the analysis of treated samples in this study. The performance of themethod for determination of thiamethoxam and CGA322704 residues in strawberry matrices wasdemonstrated by fortifying at least one non-treated control sample of each matrix with thiamethoxam
and CGA322704 prior to each extraction. These fortified samples were analyzed concurrently with eachanalytical sample set. The concurrent fortification levels bracketed the actual residue levels found intreated samples over the course of the study. Individual recoveries are presented in the Analytical Phase
Report located in Appendix 2 of the study report.
Syngenta analytical methods REM179.06 for leaves and whole flowers, REM179.07 for pollen andnectar, and T009171-04 for soil were used with modifications approved by the study director (Appendix
2 of the study report).

8993

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50265502

CDPR THX Strawberry

The methods were successfully proven by analysis of blank untreated control (UTC) samples fortified
with 1.0 ppb each of thiamethoxam and CGA for leaves, whole flower, pollen, and soil, and 0.5 ppb fornectar. These data support a method limit of quantitation (LOQ) of 1.0 ppb for leaves, whole flowers,
pollen, and soil, and 0.5 ppb for nectar. The LOQ is the lowest fortification level at which acceptable
recovery was achieved.
For the quantitation of the analytes of interest, standard curves were prepared by injecting constantvolumes of solvent-based standard solutions at appropriate concentrations (see Section 3.2). Constantvolume injections were used for sample extracts as well.
Calibration curves and residue values were calculated using Analyst 1.5.1 data handling software using
linear regression with 1/x weighting. Representative calibration curves are presented in Appendix 4.
For each matrix, at least one method-recovery (QC) sample per analytical set was prepared by fortifying
an untreated control sample with thiamethoxam and CGA322704 at concentrations equal to the method
LOQ and 10xLOQ and analyzing concurrently with the treated field samples to demonstrate adequate
method performance throughout the study, i.e. recoveries of 70-120%.

Site Laboratory

MatrixLeavesWhole Flowers

SynTech ResearchLaboratory Services,
LLC

PollenNectarSoil

AnalyteThiamethoxam
CGA322704
Thiamethoxam
CGA322704
Thiamethoxam
CGA322704
Thiamethoxam
CGA322704
Thiamethoxam
CGA322704

LOQ (ppb)
1.0
1.0
1.0
1.0
1.0
1.0
0.5
0.5
1.0
1.0

LOD (ppb)
≤0.5
≤0.5
≤0.5
≤0.5
≤0.5
≤0.5
≤0.25
≤0.25
≤0.5
≤0.5

6. RESULTS:
Table 3. Summary of Residues (ppb) for Thiamethoxam and CGA322704 in Control Samples
SampleThiamethoxam Residue
CGA322704 Residue
Description/Index
MatrixConcentration (ppb)
Concentration (ppb)
Number
24.2015.CA1.01.SB.LV.4WK-218
24.2015.CA1.01.SB.NC.BL.5
24.2015.CA1.01.SB.PO.BL-4
24.2015.CA2.01.SB.NC.BL.29

Leaves<LOQ<LOQ

Nectar

61.91

<LOQ

Pollen8.12

ND

Nectar

6.52

0.9

9
994

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50265502

SampleDescription/Index
Number

CDPR THX Strawberry

Thiamethoxam Residue
Concentration (ppb)

MatrixCGA322704 Residue
Concentration (ppb)

24.2015.CA2.01.SB.PO.BL-28

Pollen44.39

1.8

24.2015.CA3.01.SB.LV.8WK-243
24.2015.CA3.01.SB.NC.BL-53

Leaves

1.06

ND

Nectar

104.911ND

24.2015.CA3.01.SB.PO.BL-52

Pollen669.041

1.73

24.2015.CA4.01.SB.LV.BL-75
24.2015.CA4.01.SB.LV.8WK-255

Leaves0.51<LOQ

Leaves

ND

1.53

24.2015.CA4.01.SB.LV.0DA3A.253
24.2015.CA4.01.SB.NC.BL-77

Leaves

1.15

ND

Nectar

22.42

<LOQ

Pollen249.742ND

24.2015.CA4.01.SB.PO.BL-76
24.2015.CA4.01.SB.SOIL.POST.78
24.2015.CA4.01.SB.SOIL.PRE.73

Soil

ND

<LOQ

Soil

ND

<LOQ

24.2015.CA4.01.SB.WF.BL-74

Whole Flower

0.53

ND

24.2015.CA5.01.SB.NC.BL-101
24.2015.CA5.01.SB.PO.BL-100

Nectar

8.95

ND

Pollen47.333<LOQ

24.2015.CA6.01.SB.LV.0DA3A.277

Leaves<LOQ

ND

24.2015.CA6.01.SB.LV.BL-123
24.2015.CA6.01.SB.LV.8WK-279

Leaves

3.44

ND

Leaves<LOQ

ND

24.2015.CA6.01.SB.NC.BL.125
24.2015.CA6.01.SB.PO.BL-124
24.2015.CA6.01.SB.SOIL.POST-126

Nectar

1.89

ND

Pollen

20.04

<LOQ

Soil

<LOQ

ND

10
995

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50265502

SampleDescription/Index
Number

CDPR THX Strawberry

Thiamethoxam Residue
Concentration (ppb)

MatrixCGA322704 Residue
Concentration (ppb)

24.2015.CA6.01.SB.SOIL.PRE.121

Soil

1.20

<LOQ

24.2015.CA6.01.SB.WF.BL-122
24.2015.CA7.01.SB.LV.0DA3A.289

Whole Flower

5.93

ND

Leaves2.01<LOQ

24.2015.CA7.01.SB.LV.8WK-291

Leaves

1.5<LOQ

24.2015.CA7.01.SB.LV.BL-147
24.2015.CA7.01.SB.NC.BL.149
24.2015.CA7.01.SB.PO.BL-148

Leaves1.7<LOQ

Nectar

1.16

ND

Pollen42.994

3.82

24.2015.CA7.01.SB.SOIL.POST-150
24.2015.CA7.01.SB.SOIL.PRE-145

Soil

5.15

<LOQ

Soil

1.29

<LOQ

24.2015.CA7.01.SB.WF.BL-146

Whole Flower

24.2015.CA8.01.SB.LV.0DA3A-301
24.2015.CA8.01.SB.LV.4WK-302

1

ND

Leaves

41.48

<LOQ

Leaves2254.415

75.46

24.2015.CA8.01.SB.LV.8WK-303
24.2015.CA8.01.SB.LV.BL-171
24.2015.CA8.01.SB.NC.BL-173

Leaves1714.645

8.15

Leaves

18.05

1.05

Nectar4.17

0.64

24.2015.CA8.01.SB.PO.BL-172
24.2015.CA8.01.SB.SOIL.POST-174

Pollen

9.78

ND

Soil

20.62

<LOQ

24.2015.CA8.01.SB.SOIL.PRE-169

Soil

10.92

<LOQ

24.2015.CA8.01.SB.WF.BL-170
24.2015.CA9.01.SB.LV.0DA3A-313

Whole Flower

2.24

ND

Leaves

31.44

2.09

11
996

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50265502

CDPR THX Strawberry

SampleDescription/Index
Number1

Thiamethoxam Residue
Concentration (ppb)

MatrixCGA322704 Residue
Concentration (ppb)

24.2015.CA9.01.SB.LV.4WK-314

Leaves

28.85

1.86

24.2015.CA9.01.SB.LV.8WK-315

Leaves638.335

48.03

24.2015.CA9.01.SB.LV.BL.195

Leaves

30.15

1.35

24.2015.CA9.01.SB.NC.BL-197

Nectar

17.35

ND

24.2015.CA9.01.SB.PO.BL-196
24.2015.CA9.01.SB.SOIL.POST-198

Pollen

12.00

<LOQ

Soil

16.41

1.35

24.2015.CA9.01.SB.SOIL.PRE-193
24.2015.CA9.01.SB.WF.BL-194

Soil

19.03

1.15

Whole Flower

3.39

ND

For CA-3 applications, the wind direction was consistently in the direction of the control plot and were less than 5
miles per hour. Sample weights were also lower than the target weight specified in the protocol.
2
For CA-4 applications, the wind direction was consistently in the direction of the control plot and were less than 5
miles per hour. Sample weights were also lower than the target weight specified in the protocol. The control plot
was also located within 75 feet of the test plots, which is less than the target distance specified in the protocol.
3
For CA-5 applications, the wind direction was consistently in the direction of the control plot.
4
CA-7 sample weight for pollen was lower than the target weight specified in the protocol.
5
At CA-8 and CA-9 test sites, three additional inadvertent applications of the test substance (Actara) were madewithin the study area throughout the 4 week and 8 week sample activities.

Table 4. Thiamethoxam and CGA322704 Residues in Leaves, Nectar, Pollen, Whole Flowers and Soil
Nominal Total Thiamethoxa CGA322704
Trail
MatrixTreatment
TimingTreatmentm Residues
Residues
Number/Sample
Rate
(ppb)
(ppb)
24.2015.CA1.01.SB.LV.BL-3

Leaves

UTC

Bloom

NA

ND

<LOQ

24.2015.CA1.02.SB.LV.BL.A-9

Leaves

TRTD

Bloom

0.189

1736.79

38.54

24.2015.CA1.02.SB.LV.BL.B-15
24.2015.CA1.02.SB.LV.BL.C-21

Leaves

TRTD

Bloom

0.189

1984.57

50.72

Leaves

TRTD

Bloom

0.189

2145.93

46.92

24.2015.CA1.01.SB.LV.4WK218

LeavesUTC

4 Week

NA

<LOQ

<LOQ

12
997

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50265502

CDPR THX Strawberry

Trail
Number/Sample

MatrixTreatment

TimingNominal Total
Treatment
Rate

Thiamethoxa
m Residues(ppb)

24.2015.CA1.02.SB.LV.4WK.A221

LeavesTRTD

4 Week

0.189

192.0413.03

24.2015.CA1.02.SB.LV.4WK.B224

LeavesTRTD

4 Week

0.189

218.2

14.36

24.2015.CA1.02.SB.LV.4WK.C227

LeavesTRTD

4 Week

0.189240.3615.43

24.2015.CA1.01.SB.LV.8WK219

LeavesUTC

8 Week

NA

ND

<LOQ

24.2015.CA1.02.SB.LV.8WK.A222

LeavesTRTD

8 Week

0.189

71.54

10.68

24.2015.CA1.02.SB.LV.8WK.B225

LeavesTRTD

8 Week

0.189

71.34

15.26

24.2015.CA1.02.SB.LV.8WK.C228

LeavesTRTD

8 Week

0.189

61.27

10.48

24.2015.CA1.01.SB.NC.BL.5
24.2015.CA1.02.SB.NC.BL.A.11

Nectar

UTC

Bloom

NA

61.91

<LOQ

Nectar

TRT A

Bloom

0.189

296.17

4.33

24.2015.CA1.02.SB.NC.BL.B.17

Nectar

TRT B

Bloom

0.189

647.2512.76

24.2015.CA1.02.SB.NC.BL.C.23
24.2015.CA1.01.SB.PO.BL-4

Nectar

TRT C

Bloom

0.189

199.37

5.38

Pollen

UTC

Bloom

NA

8.12

ND

24.2015.CA1.02.SB.PO.BL.A10-A

Pollen

TRT A

Bloom

0.189

287.87

7.12

24.2015.CA1.02.SB.PO.BL.B16-A

Pollen

TRT B

Bloom

0.189

169.57

4.10

24.2015.CA1.02.SB.PO.BL.C22-A

Pollen

TRT C

Bloom

0.189

173.32

6.76

13
998

CGA322704
Residues(ppb)

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50265502

CDPR THX Strawberry

Trail
Number/Sample

MatrixTreatment

TimingNominal Total
Treatment
Rate

Thiamethoxa
m Residues(ppb)

24.2015.CA1.01.SB.SOIL.POST.
6

Soil

UTC

Post

NA

ND

ND

24.2015.CA1.01.SB.SOIL.POST.
A.12

Soil

TRT A

Post

0.189

3.29

2.79

24.2015.CA1.02.SB.SOIL.POST.
B.18

Soil

TRT B

Post

0.189

1.17

5.62

24.2015.CA1.02.SB.SOIL.POST.
C.24

Soil

TRT C

Post

0.189

1.77

2.42

24.2015.CA1.01.SB.SOIL.PRE.1

Soil

UTC

Pre

NA

ND

ND

24.2015.CA1.02.SB.SOIL.PRE.A.
7

Soil

TRT A

Pre

0.189

3.48

2.30

24.2015.CA1.02.SB.SOIL.PRE.B.
13

Soil

TRT B

Pre

0.189

3.25

3.72

24.2015.CA1.02.SB.SOIL.PRE.C.
19

Soil

TRT C

Pre

0.189

5.31

3.62

24.2015.CA1.01.SB.WF.BL-2*

Whole
Flower

UTC

Bloom

NA

ND

ND

24.2015.CA1.02.SB.WF.BL.A-8

Whole
Flower

TRT A

Bloom

0.189

373.4741.15

24.2015.CA1.02.SB.WF.BL.B-14
24.2015.CA1.02.SB.WF.BL.C-20

Whole
Flower

TRT B

Bloom

0.189

359.66

10.77

Whole
Flower

TRT C

Bloom

0.189

325.72

9.94

24.2015.CA2.01.SB.LV.BL-27
24.2015.CA2.02.SB.LV.BL.A33

Leaves

UTC

Bloom

NA

ND

ND

Leaves

TRTD

Bloom

0.189

1506.08

32.85

24.2015.CA2.02.SB.LV.BL.B39

Leaves

TRTD

Bloom

0.189

1864.7341.40

24.2015.CA-

Leaves

TRTD

Bloom

0.189

1931.85

44.41

14
999

CGA322704
Residues(ppb)

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50265502

CDPR THX Strawberry

MatrixTreatment

TimingNominal Total
Treatment
Rate

Thiamethoxa
m Residues(ppb)

24.2015.CA2.01.SB.LV.4WK230

LeavesUTC

4 WeekNA

ND

ND

24.2015.CA2.02.SB.LV.4WK.A233

LeavesTRTD

4 Week

0.189

283.1354.84

24.2015.CA2.02.SB.LV.4WK.B236

LeavesTRTD

4 Week

0.189

164.9421.05

24.2015.CA2.02.SB.LV.4WK.C239

LeavesTRTD

4 Week

0.189

286.5719.92

24.2015.CA2.01.SB.LV.8WK231

LeavesUTC

8 WeekNA

ND

ND

24.2015.CA2.02.SB.LV.8WK.A234

LeavesTRTD

8 Week

0.189

13.94

4.20

24.2015.CA2.02.SB.LV.8WK.B237

LeavesTRTD

8 Week

0.189

13.00

5.27

24.2015.CA2.02.SB.LV.8WK.C240

LeavesTRTD

8 Week

0.189

16.1

13.43

24.01.CA2.01.SB.LV.BL.A*

Leaves

UTC

Bloom

NA

ND

ND

24.2015.CA2.01.SB.NC.BL.29
24.2015.CA2.02.SB.NC.BL.A.3
5

Nectar

UTC

Bloom

NA

6.52

0.90

Nectar

TRT A

Bloom

0.189

164.62

4.69

24.2015.CA2.02.SB.NC.BL.B.4
1

Nectar

TRT B

Bloom

0.189

149.38

3.62

24.2015.CA2.02.SB.NC.BL.C.4
7

Nectar

TRT C

Bloom

0.189

129.43

4.97

24.2015.CA-

Pollen

UTC

Bloom

NA

44.39

1.80

Trail
Number/Sample

CGA322704
Residues(ppb)

2.02.SB.LV.BL.C45

15
1000

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50265502

CDPR THX Strawberry

MatrixTreatment

TimingNominal Total
Treatment
Rate

Thiamethoxa
m Residues(ppb)

24.2015.CA2.02.SB.PO.BL.A34

Pollen

TRT A

Bloom

0.189

101.696.23

24.2015.CA2.02.SB.PO.BL.B40

Pollen

TRT B

Bloom

0.189

171.7911.43

24.2015.CA2.02.SB.PO.BL.C46

Pollen

TRT C

Bloom

0.189

249.75

13.62

24.2015.CA2.01.SB.SOIL.POST.
30

Soil

UTC

Post

NA

ND

ND

24.2015.CA2.02.SB.SOIL.POST.
A.36

Soil

TRT A

Post

0.189

ND

ND

24.2015.CA2.02.SB.SOIL.POST.B
.42

Soil

TRT B

Post

0.189

3.36

1.10

24.2015.CA2.02.SB.SOIL.POST.C
.48

Soil

TRT C

Post

0.189

4.74

1.45

24.2015.CA2.01.SB.WF.BL-26
24.2015.CA2.02.SB.WF.BL.A32

Whole
Flower

UTC

Bloom

NA

ND

ND

Whole
Flower

TRT A

Bloom

0.189

482.9912.46

24.2015.CA2.02.SB.WF.BL.B38

Whole
Flower

TRT B

Bloom

0.189

466.22

13.02

24.2015.CA2.02.SB.WF.BL.C44
24.2015.CA3.02.SB.LV.0DA3A.A.
244
24.2015.CA3.02.SB.LV.0DA3A.B.
247
24.2015.CA3.02.SB.LV.0DA3A.C-

Whole
Flower

TRT C

Bloom

0.189

424.28

11.82

Leaves

TRTD

0 Day

0.189

8433.93

45.80

Leaves

TRTD

0 Day

0.189

9057.67

41.66

Leaves

TRTD

0 Day

0.189

7605.97

37.01

Trail
Number/Sample

CGA322704
Residues(ppb)

2.01.SB.PO.BL-28

16
1001

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50265502
Trail
Number/Sample

250
24.2015.CA3.01.SB.LV.BL-51
24.2015.CA3.02.SB.LV.BL.A-57
24.2015.CA3.02.SB.LV.BL.B-63
24.2015.CA3.02.SB.LV.BL.C-69
24.2015.CA3.01.SB.LV.4WK-242
24.2015.CA3.02.SB.LV.4WK.A245
24.2015.CA3.02.SB.LV.4WK.B248
24.2015.CA3.02.SB.LV.4WK.C251
24.2015.CA3.01.SB.LV.8WK-243
24.2015.CA3.02.SB.LV.8WK.A246
24.2015.CA3.02.SB.LV.8WK.B249
24.2015.CA3.02.SB.LV.8WK.C252
24.2015.CA3.01.SB.LV.0DA3A.24
1*
24.2015.CA3.01.SB.NC.BL-53
24.2015.CA3.02.SB.NC.BL.A-59
24.2015.CA3.02.SB.NC.BL.B-65
24.2015.CA3.02.SB.NC.BL.C-71

CDPR THX Strawberry

MatrixTreatment

TimingNominal Total
Treatment
Rate

Thiamethoxa
m Residues(ppb)

CGA322704
Residues(ppb)

Leaves

UTC

Bloom

NA

ND

ND

Leaves

TRTD

Bloom

0.189

2151.76

71.32

Leaves

TRTD

Bloom

0.189

2061.65

68.89

Leaves

TRTD

Bloom

0.189

1923.48

67.20

LeavesUTC

4 WeekNA

ND

ND

LeavesTRTD

4 Week

0.189

185.65

10.47

LeavesTRTD

4 Week

0.189

266.47

14.96

LeavesTRTD

4 Week

0.189

289.27

15.8

LeavesUTC

8 Week

NA

1.06

ND

LeavesTRTD

8 Week

0.189

267.15

14.43

LeavesTRTD

8 Week

0.189

318.21

17.25

LeavesTRTD

8 Week

0.189

342.12

18.75

Leaves

UTC

Day 0

NA

ND

ND

Nectar

UTC

Bloom

NA

104.91ND

Nectar

TRT A

Bloom

0.189

211.95

1.76

Nectar

TRT B

Bloom

0.189

136.22

1.93

Nectar

TRT C

Bloom

0.189

189.62

2.62

17
1002

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50265502

CDPR THX Strawberry

Trail
Number/Sample

MatrixTreatment

TimingNominal Total
Treatment
Rate

Thiamethoxa
m Residues(ppb)

CGA322704
Residues(ppb)

24.2015.CA3.01.SB.PO.BL-52
24.2015.CA3.02.SB.PO.BL.A-58
24.2015.CA3.02.SB.PO.BL.B-64
24.2015.CA3.02.SB.PO.BL.C-70
24.2015.CA3.01.SB.SOIL.POST.54
24.2015.CA3.02.SB.SOIL.POST.A.
60
24.2015.CA3.02.SB.SOIL.POST.B.
66
24.2015.CA3.02.SB.SOIL.POST.C.
72
24.2015.CA3.01.SB.SOIL.PRE.49
24.2015.CA3.02.SB.SOIL.PRE.A.5
5
24.2015.CA3.02.SB.SOIL.PRE.B.6
1
24.2015.CA3.02.SB.SOIL.PRE.C.6
7
24.2015.CA3.01.SB.WF.BL-50
24.2015.CA3.02.SB.WF.BL.A-56
24.2015.CA3.02.SB.WF.BL.B-62
24.2015.CA3.02.SB.WF.BL.C-68
24.2015.CA4.02.SB.LV.0DA3A.A.
256
24.2015.CA-

Pollen

UTC

Bloom

NA

669.04

1.73

Pollen

TRT A

Bloom

0.189

1202.23

18.20

Pollen

TRT B

Bloom

0.189

860.91

14.21

Pollen

TRT C

Bloom

0.189

773.85

20.14

Soil

UTC

Post

NA

ND

ND

Soil

TRT A

Post

0.189

15.61

<LOQ

Soil

TRT B

Post

0.189

28.45

1.78

Soil

TRT C

Post

0.189

23.12

2.48

Soil

UTC

Pre

NA

ND

ND

Soil

TRT A

Pre

0.189

20.09

<LOQ

Soil

TRT B

Pre

0.189

36.37

1.24

Soil

TRT C

Pre

0.189

30.38

1.39

Whole
FlowerWhole
FlowerWhole
FlowerWhole
FlowerLeaves

UTC

Bloom

NA

ND

ND

TRT A

Bloom

0.189

352.87

8.14

TRT B

Bloom

0.189

492.36

10.18

TRT C

Bloom

0.189

479.24

10.19

TRTD

0 Day

0.189

8703.72

24.73

TRTD

0 Day

0.189

9099.27

25.87

Leaves181003

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50265502
Trail
Number/Sample

4.02.SB.LV.0DA3A.B.
259
24.2015.CA4.02.SB.LV.0DA3A.C262
24.2015.CA4.01.SB.LV.BL-75
24.2015.CA4.02.SB.LV.BL.A-81
24.2015.CA4.02.SB.LV.BL.B-87
24.2015.CA4.02.SB.LV.BL.C-93
24.2015.CA4.01.SB.LV.4WK-254
24.2015.CA4.02.SB.LV.4WK.A257
24.2015.CA4.02.SB.LV.4WK.B260
24.2015.CA4.02.SB.LV.4WK.C263
24.2015.CA4.01.SB.LV.8WK-255
24.2015.CA4.02.SB.LV.8WK.A258
24.2015.CA4.02.SB.LV.8WK.B261
24.2015.CA4.02.SB.LV.8WK.C264
24.2015.CA4.01.SB.LV.0DA3A.25
324.2015.CA4.01.SB.NC.BL-77
24.2015.CA4.02.SB.NC.BL.A-83

CDPR THX Strawberry

MatrixTreatment

TimingNominal Total
Treatment
Rate

Thiamethoxa
m Residues(ppb)

CGA322704
Residues(ppb)

Leaves

TRTD

0 Day

0.189

8577.80

28.73

Leaves

UTC

Bloom

NA

0.51

<LOQ

Leaves

TRTD

Bloom

0.189

1242.7

33.40

Leaves

TRTD

Bloom

0.189

1644.59

40.46

Leaves

TRTD

Bloom

0.189

1685.71

34.85

LeavesUTC

4 WeekNA

ND

ND

LeavesTRTD

4 Week

0.189

45.26

6.43

LeavesTRTD

4 Week

0.189

59.29

4.80

LeavesTRTD

4 Week

0.189

135.37

9.75

LeavesUTC

8 Week

NA

ND

1.53

LeavesTRTD

8 Week

0.189

29.47

4.80

LeavesTRTD

8 Week

0.189

31.03

5.05

LeavesTRTD

8 Week

0.189

58.84

8.15

Leaves

UTC

Day 0

NA

1.15

ND

Nectar

UTC

Bloom

NA

22.40

<LOQ

Nectar

TRT A

Bloom

0.189

118.42

2.35

19
1004

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50265502

CDPR THX Strawberry

Trail
Number/Sample

MatrixTreatment

TimingNominal Total
Treatment
Rate

Thiamethoxa
m Residues(ppb)

CGA322704
Residues(ppb)

24.2015.CA4.02.SB.NC.BL.B-89
24.2015.CA4.02.SB.NC.BL.C-95
24.2015.CA4.01.SB.PO.BL-76
24.2015.CA4.02.SB.PO.BL.A-82
24.2015.CA4.02.SB.PO.BL.B-88
24.2015.CA4.02.SB.PO.BL.C-94
24.2015.CA4.01.SB.SOIL.POST.78
24.2015.CA4.02.SB.SOIL.POST.A.
84
24.2015.CA4.02.SB.SOIL.POST.B.
90
24.2015.CA4.02.SB.SOIL.POST.C.
96
24.2015.CA4.01.SB.SOIL.PRE.73
24.2015.CA4.02.SB.SOIL.PRE.A.7
924.2015.CA4.02.SB.SOIL.PRE.B.8
524.2015.CA4.02.SB.SOIL.PRE.C.9
124.2015.CA4.01.SB.WF.BL-74
24.2015.CA4.02.SB.WF.BL.A-80
24.2015.CA4.02.SB.WF.BL.B-86
24.2015.CA4.02.SB.WF.BL.C-92

Nectar

TRT B

Bloom

0.189

214.74

9.86

Nectar

TRT C

Bloom

0.189

51.17

0.96

Pollen

UTC

Bloom

NA

249.74ND

Pollen

TRT A

Bloom

0.189

1477.68

32.23

Pollen

TRT B

Bloom

0.189

633.51

19.38

Pollen

TRT C

Bloom

0.189

1159.00

13.45

Soil

UTC

Post

NA

ND

<LOQ

Soil

TRT A

Post

0.189

11.52

1.46

Soil

TRT B

Post

0.189

17.14

1.20

Soil

TRT C

Post

0.189

8.81

1.27

Soil

UTC

Pre

NA

ND

<LOQ

Soil

TRT A

Pre

0.189

ND

<LOQ

Soil

TRT B

Pre

0.189

ND

<LOQ

Soil

TRT C

Pre

0.189

ND

<LOQ

Whole
FlowerWhole
FlowerWhole
FlowerWhole
Flower

UTC

Bloom

NA

0.53

ND

TRT A

Bloom

0.189

511.72

10.04

TRT B

Bloom

0.189

247.61

5.05

TRT C

Bloom

0.189

305.94

5.03

20
1005

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50265502

CDPR THX Strawberry

Trail
Number/Sample

MatrixTreatment

TimingNominal Total
Treatment
Rate

Thiamethoxa
m Residues(ppb)

CGA322704
Residues(ppb)

24.2015.CA5.01.SB.LV.0DA3A.26
5
24.2015.CA5.02.SB.LV.0DA3A.A.
268
24.2015.CA5.02.SB.LV.0DA3A.B.
271
24.2015.CA5.02.SB.LV.0DA3A.C274
24.2015.CA5.01.SB.LV.BL-99
24.2015.CA5.02.SB.LV.BL.A-105
24.2015.CA5.02.SB.LV.BL.B-111
24.2015.CA5.02.SB.LV.BL.C-117
24.2015.CA5.01.SB.LV.4WK-266
24.2015.CA5.02.SB.LV.4WK.A269
24.2015.CA5.02.SB.LV.4WK.B272
24.2015.CA5.02.SB.LV.4WK.C275
24.2015.CA5.01.SB.LV.8WK-267
24.2015.CA5.02.SB.LV.8WK.A270
24.2015.CA5.02.SB.LV.8WK.C273
24.2015.CA5.02.SB.LV.8WK.C276
24.2015.CA-

Leaves

UTC

0 Day

NA

ND

ND

Leaves

TRTD

0 Day

0.189

6709.22

13.32

Leaves

TRTD

0 Day

0.189

7718.34

16.54

Leaves

TRTD

0 Day

0.189

6851.73

19.04

Leaves

UTC

Bloom

NA

ND

ND

Leaves

TRTD

Bloom

0.189

516.33

12.15

Leaves

TRTD

Bloom

0.189

428.12

10.78

Leaves

TRTD

Bloom

0.189

475.7910.85

LeavesUTC

4 WeekNA

ND

ND

LeavesTRTD

4 Week

0.189

204.73

82.53

LeavesTRTD

4 Week

0.189

154.91

24.86

LeavesTRTD

4 Week

0.189

248.96

22.28

LeavesUTC

8 WeekNA

ND

ND

LeavesTRTD

8 Week

0.189

58.36

9.20

LeavesTRTD

8 Week

0.189

46.48

9.46

LeavesTRTD

8 Week

0.189

56.08

11.22

Nectar

UTC

Bloom

NA

8.95

ND

21
1006

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50265502
Trail
Number/Sample

5.01.SB.NC.BL-101
24.2015.CA5.02.SB.NC.BL.A-107
24.2015.CA5.02.SB.NC.BL.B-113
24.2015.CA5.02.SB.NC.BL.C-119
24.2015.CA5.01.SB.PO.BL-100
24.2015.CA5.02.SB.PO.BL.A-106
24.2015.CA5.02.SB.PO.BL.B-112
24.2015.CA5.02.SB.PO.BL.C-118
24.2015.CA5.01.SB.SOIL.POST.10
2
24.2015.CA5.02.SB.SOIL.POST.A.
108
24.2015.CA5.02.SB.SOIL.POST.B.
114
24.2015.CA5.02.SB.SOIL.POST.C.
120
24.2015.CA5.01.SB.SOIL.PRE.97
24.2015.CA5.02.SB.SOIL.PRE.A.1
03
24.2015.CA5.02.SB.SOIL.PRE.B.1
09
24.2015.CA5.02.SB.SOIL.PRE.C.1
15
24.2015.CA5.01.SB.WF.BL-98
24.2015.CA5.02.SB.WF.BL.A-104

CDPR THX Strawberry

MatrixTreatment

TimingNominal Total
Treatment
Rate

Thiamethoxa
m Residues(ppb)

CGA322704
Residues(ppb)

Nectar

TRT A

Bloom

0.189

177.45

5.39

Nectar

TRT B

Bloom

0.189

143.65

3.74

Nectar

TRT C

Bloom

0.189

108.07

3.79

Pollen

UTC

Bloom

NA

47.33

<LOQ

Pollen

TRT A

Bloom

0.189

334.4919.94

Pollen

TRT B

Bloom

0.189

337.62

21.86

Pollen

TRT C

Bloom

0.189

309.42

22.81

Soil

UTC

Post

NA

ND

ND

Soil

TRT A

Post

0.189

4.52

<LOQ

Soil

TRT B

Post

0.189

7.05

1.68

Soil

TRT C

Post

0.189

1.36

ND

Soil

UTC

Pre

NA

ND

ND

Soil

TRT A

Pre

0.189

ND

ND

Soil

TRT B

Pre

0.189

ND

ND

Soil

TRT C

Pre

0.189

ND

ND

Whole
FlowerWhole
Flower

UTC

Bloom

NA

ND

ND

TRT A

Bloom

0.189

241.31

6.59

22
1007

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50265502

CDPR THX Strawberry

Trail
Number/Sample

MatrixTreatment

TimingNominal Total
Treatment
Rate

Thiamethoxa
m Residues(ppb)

CGA322704
Residues(ppb)

24.2015.CA5.02.SB.WF.BL.B-110
24.2015.CA5.02.SB.WF.BL.C-116
24.2015.CA6.01.SB.LV.0DA3A.27
7
24.2015.CA6.02.SB.LV.0DA3A.A.
280
24.2015.CA6.02.SB.LV.0DA3A.B.
283
24.2015.CA6.02.SB.LV.0DA3A.C286
24.2015.CA6.01.SB.LV.BL-123
24.2015.CA6.02.SB.LV.BL.A-129
24.2015.CA6.02.SB.LV.BL.B-135
24.2015.CA6.02.SB.LV.BL.C-141
24.2015.CA6.01.SB.LV.4WK278*
24.2015.CA6.02.SB.LV.4WK.A281
24.2015.CA6.02.SB.LV.4WK.B284
24.2015.CA6.02.SB.LV.4WK.C287
24.2015.CA6.01.SB.LV.8WK-279
24.2015.CA6.02.SB.LV.8WK.A282
24.2015.CA6.02.SB.LV.8WK.B-

Whole
FlowerWhole
FlowerLeaves

TRT B

Bloom

0.189

283.29

6.14

TRT C

Bloom

0.189

263.98

6.57

UTC

0 Day

NA

<LOQ

ND

Leaves

TRTD

0 Day

0.189

5365.14

25.92

Leaves

TRTD

0 Day

0.189

4751.53

26.75

Leaves

TRTD

0 Day

0.189

3487.2121.24

Leaves

UTC

Bloom

NA

3.44

ND

Leaves

TRTD

Bloom

0.189

1889.24

33.79

Leaves

TRTD

Bloom

0.189

1826.40

34.69

Leaves

TRTD

Bloom

0.189

1483.09

25.84

LeavesUTC

4 WeekNA

ND

ND

LeavesTRTD

4 Week

0.189

144.0038.54

LeavesTRTD

4 Week

0.189

210.05

20.38

LeavesTRTD

4 Week

0.189

67.45

9.54

LeavesUTC

8 Week

NA

<LOQ

ND

LeavesTRTD

8 Week

0.189

106.27

5.67

LeavesTRTD

8 Week0.189

134.08

5.79

23
1008

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50265502
Trail
Number/Sample

285
24.2015.CA6.02.SB.LV.8WK.C288
24.2015.CA6.01.SB.NC.BL.125
24.2015.CA6.02.SB.NC.BL.A.131001
24.2015.CA6.02.SB.NC.BL.B.137001
24.2015.CA6.02.SB.NC.BL.C.143001
24.2015.CA6.01.SB.PO.BL-124
24.2015.CA6.02.SB.PO.BL.A-130
24.2015.CA6.02.SB.PO.BL.B-136
24.2015.CA6.02.SB.PO.BL.C-142
24.2015.CA6.01.SB.SOIL.POST126
24.2015.CA6.02.SB.SOIL.POST.A132
24.2015.CA6.02.SB.SOIL.POST.B138
24.2015.CA6.02.SB.SOIL.POST.C144
24.2015.CA6.01.SB.SOIL.PRE.121
24.2015.CA6.02.SB.SOIL.PRE.A.1
27
24.2015.CA6.02.SB.SOIL.PRE.B.1
33

CDPR THX Strawberry

MatrixTreatment

TimingNominal Total
Treatment
Rate

Thiamethoxa
m Residues(ppb)

CGA322704
Residues(ppb)

LeavesTRTD

8 Week

0.189

162.54

6.55

Nectar

UTC

Bloom

NA

1.89

ND

Nectar

TRT A

Bloom

0.189

183.33

4.04

Nectar

TRT B

Bloom

0.189

228.42

5.14

Nectar

TRT C

Bloom

0.189

175.03

4.14

Pollen

UTC

Bloom

NA

20.04

<LOQ

Pollen

TRT A

Bloom

0.189

2486.10

36.63

Pollen

TRT B

Bloom

0.189

2156.36

31.93

Pollen

TRT C

Bloom

0.189

1935.33

30.42

Soil

UTC

Post

NA

<LOQ

ND

Soil

TRT A

Post

0.189

1.01

<LOQ

Soil

TRT B

Post

0.189

2.59

<LOQ

Soil

TRT C

Post

0.189

4.40

<LOQ

Soil

UTC

Pre

NA

1.20

<LOQ

Soil

TRT A

Pre

0.189

<LOQ

<LOQ

Soil

TRT B

Pre

0.189

<LOQ

ND

24
1009

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50265502

CDPR THX Strawberry

Trail
Number/Sample

MatrixTreatment

TimingNominal Total
Treatment
Rate

Thiamethoxa
m Residues(ppb)

CGA322704
Residues(ppb)

24.2015.CA6.02.SB.SOIL.PRE.C.1
39
24.2015.CA6.01.SB.WF.BL-122
24.2015.CA6.02.SB.WF.BL.A-128
24.2015.CA6.02.SB.WF.BL.B-134
24.2015.CA6.02.SB.WF.BL.C-140
24.2015.CA7.01.SB.LV.0DA3A.28
924.2015.CA7.02.SB.LV.0DA3A.A.
292
24.2015.CA7.02.SB.LV.0DA3A.B.
295
24.2015.CA7.02.SB.LV.0DA3A.C298
24.2015.CA7.01.SB.LV.4WK-290
24.2015.CA7.02.SB.LV.4WK.A293
24.2015.CA7.02.SB.LV.4WK.B296
24.2015.CA7.02.SB.LV.4WK.C299
24.2015.CA7.02.SB.LV.8WK.A294
24.2015.CA7.01.SB.LV.8WK-291
24.2015.CA7.02.SB.LV.8WK.B297
24.2015.CA-

Soil

TRT C

Pre

0.189

<LOQ

<LOQ

Whole
FlowerWhole
FlowerWhole
FlowerWhole
FlowerLeaves

UTC

Bloom

NA

5.93

ND

TRT A

Bloom

0.189

829.3

81.79

TRT B

Bloom

0.189

895.27

25.87

TRT C

Bloom

0.189

973.27

22.12

UTC

0 Day

NA

2.01

<LOQ

Leaves

TRTD

0 Day

0.189

5256.56

25.78

Leaves

TRTD

0 Day

0.189

4772.30

24.39

Leaves

TRTD

0 Day

0.189

4851.20

23.31

LeavesUTC

4 WeekNA

ND

ND

LeavesTRTD

4 Week

0.189

289.29

14.54

LeavesTRTD

4 Week

0.189

151.03

6.26

LeavesTRTD

4 Week

0.189

159.04

6.20

LeavesTRTD

8 Week

0.189

72.17

4.16

LeavesUTC

8 Week

NA

1.50

<LOQ

LeavesTRTD

8 Week

0.189

60.08

3.37

LeavesTRTD

8 Week

0.189

74.05

4.06

25
1010

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50265502
Trail
Number/Sample

7.02.SB.LV.8WK.C300
24.2015.CA7.01.SB.LV.BL-147
24.2015.CA7.02.SB.LV.BL.A-153
24.2015.CA7.02.SB.LV.BL.B-159
24.2015.CA7.02.SB.LV.BL.C-165
24.2015.CA7.01.SB.NC.BL.149
24.2015.CA7.02.SB.NC.BL.A.155
24.2015.CA7.02.SB.NC.BL.B.161
24.2015.CA7.02.SB.NC.BL.C.167
24.2015.CA7.01.SB.PO.BL-148
24.2015.CA7.02.SB.PO.BL.A-154
24.2015.CA7.02.SB.PO.BL.B-160
24.2015.CA7.02.SB.PO.BL.C-166
24.2015.CA7.01.SB.SOIL.POST150
24.2015.CA7.02.SB.SOIL.POST.A156
24.2015.CA7.02.SB.SOIL.POST.B162
24.2015.CA7.02.SB.SOIL.POST.C168
24.2015.CA7.01.SB.SOIL.PRE-145
24.2015.CA7.02.SB.SOIL.PRE.A-

CDPR THX Strawberry

MatrixTreatment

TimingNominal Total
Treatment
Rate

Thiamethoxa
m Residues(ppb)

CGA322704
Residues(ppb)

Leaves

UTC

Bloom

NA

1.70

<LOQ

Leaves

TRTD

Bloom

0.189

1156.66

24.78

Leaves

TRTD

Bloom

0.189

1132.48

24.49

Leaves

TRTD

Bloom

0.189

1059.15

23.34

Nectar

UTC

Bloom

NA

1.16

ND

Nectar

TRT A

Bloom

0.189

215.15<LOQ

Nectar

TRT B

Bloom

0.189

206.77

4.15

Nectar

TRT C

Bloom

0.189

200.6

3.28

Pollen

UTC

Bloom

NA

42.99

3.82

Pollen

TRT A

Bloom

0.189

4238.76

43.7

Pollen

TRT B

Bloom

0.189

5251.74

62.34

Pollen

TRT C

Bloom

0.189

7473.47

66.10

Soil

UTC

Post

NA

5.15

<LOQ

Soil

TRT A

Post

0.189

17.15

2.13

Soil

TRT B

Post

0.189

24.88

2.32

Soil

TRT C

Post

0.189

25.78

2.93

Soil

UTC

Pre

NA

1.29

<LOQ

Soil

TRT A

Pre

0.189

<LOQ

ND

26
1011

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50265502
Trail
Number/Sample

151
24.2015.CA7.02.SB.SOIL.PRE.B157
24.2015.CA7.02.SB.SOIL.PRE.C163
24.2015.CA7.01.SB.WF.BL-146
24.2015.CA7.02.SB.WF.BL.A-152
24.2015.CA7.02.SB.WF.BL.B-158
24.2015.CA7.02.SB.WF.BL.C-164
24.2015.CA8.01.SB.LV.0DA3A301
24.2015.CA8.02.SB.LV.0DA3A.A304
24.2015.CA8.02.SB.LV.0DA3A.B307
24.2015.CA8.02.SB.LV.0DA3A.C310
24.2015.CA8.01.SB.LV.4WK-302
24.2015.CA8.02.SB.LV.4WK.A305
24.2015.CA8.02.SB.LV.4WK.B308
24.2015.CA8.02.SB.LV.4WK.C311
24.2015.CA8.01.SB.LV.8WK-303
24.2015.CA8.02.SB.LV.8WK.A306

CDPR THX Strawberry

MatrixTreatment

TimingNominal Total
Treatment
Rate

Thiamethoxa
m Residues(ppb)

CGA322704
Residues
(ppb)

Soil

TRT B

Pre

0.189

<LOQ

ND

Soil

TRT C

Pre

0.189

<LOQ

ND

Whole
FlowerWhole
FlowerWhole
FlowerWhole
FlowerLeaves

UTC

Bloom

NA

1.00

ND

TRT A

Bloom

0.189

493.67

11.15

TRT B

Bloom

0.189

443.68

9.50

TRT C

Bloom

0.189

468.15

8.36

UTC

0 Day

NA

41.48

<LOQ

Leaves

TRTD

0 Day

0.189

2653.53

32.97

Leaves

TRTD

0 Day

0.189

2290.4232.96

Leaves

TRTD

0 Day

0.189

2266.73

34.49

LeavesUTC

4 Week

NA

2254.41

75.46

LeavesTRTD

4 Week

0.189

1035.89

83.59

LeavesTRTD

4 Week

0.189

1333.77

89.31

LeavesTRTD

4 Week

0.189

1187.42

83.04

LeavesUTC

8 WeekNA

1714.64

8.15

LeavesTRTD

8 Week

0.189

1351.70

77.69

27
1012

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50265502

CDPR THX Strawberry

Trail
Number/Sample

MatrixTreatment

TimingNominal Total
Treatment
Rate

Thiamethoxa
m Residues(ppb)

CGA322704
Residues(ppb)

24.2015.CA8.02.SB.LV.8WK.B309
24.2015.CA8.02.SB.LV.8WK.C312
24.2015.CA8.01.SB.LV.BL-171
24.2015.CA8.02.SB.LV.BL.A-177
24.2015.CA8.02.SB.LV.BL.B-183
24.2015.CA8.02.SB.LV.BL.C-189
24.2015.CA8.01.SB.NC.BL-173
24.2015.CA8.02.SB.NC.BL.A-179
24.2015.CA8.02.SB.NC.BL.B-185
24.2015.CA8.02.SB.NC.BL.C-191
24.2015.CA8.01.SB.PO.BL-172
24.2015.CA8.02.SB.PO.BL.A-178
24.2015.CA8.02.SB.PO.BL.B-184
24.2015.CA8.02.SB.PO.BL.C-190
24.2015.CA8.01.SB.SOIL.POST174
24.2015.CA8.02.SB.SOIL.POST.A180
24.2015.CA8.02.SB.SOIL.POST.B186
24.2015.CA8.02.SB.SOIL.POST.C192

LeavesTRTD

8 Week

0.189

1912.47

83.24

LeavesTRTD

8 Week

0.1892405.98

65.44

Leaves

UTC

Bloom

NA

18.05

1.05

Leaves

TRTD

Bloom

0.189

972.9526.29

Leaves

TRTD

Bloom

0.189

893.71

22.86

Leaves

TRTD

Bloom

0.189

851.27

20.27

Nectar

UTC

Bloom

NA

4.17

0.64

Nectar

TRT A

Bloom

0.189

98.09

1.04

Nectar

TRT B

Bloom

0.189

86.87

2.41

Nectar

TRT C

Bloom

0.189

79.18

1.83

Pollen

UTC

Bloom

NA

9.78

ND

Pollen

TRT A

Bloom

0.189

1489.59

7.89

Pollen

TRT B

Bloom

0.189

656.397.73

Pollen

TRT C

Bloom

0.189

540.42

5.86

Soil

UTC

Post

NA

20.62

<LOQ

Soil

TRT A

Post

0.189

84.92

2.35

Soil

TRT B

Post

0.189

85.19

3.13

Soil

TRT C

Post

0.189

81.02

3.01

28
1013

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50265502

CDPR THX Strawberry

Trail
Number/Sample

MatrixTreatment

TimingNominal Total
Treatment
Rate

Thiamethoxa
m Residues(ppb)

CGA322704
Residues(ppb)

24.2015.CA8.01.SB.SOIL.PRE-169
24.2015.CA8.02.SB.SOIL.PRE.A175
24.2015.CA8.02.SB.SOIL.PRE.B181
24.2015.CA8.02.SB.SOIL.PRE.C187
24.2015.CA8.01.SB.WF.BL-170
24.2015.CA8.02.SB.WF.BL.A-176
24.2015.CA8.02.SB.WF.BL.B-182
24.2015.CA8.02.SB.WF.BL.C-188
24.2015.CA9.01.SB.LV.0DA3A313
24.2015.CA9.02.SB.LV.0DA3A.A316
24.2015.CA9.02.SB.LV.0DA3A.B319
24.2015.CA9.02.SB.LV.0DA3A.C322
24.2015.CA9.01.SB.LV.4WK314*
24.2015.CA9.02.SB.LV.4WK.A317*
24.2015.CA9.02.SB.LV.4WK.B320*
24.2015.CA9.02.SB.LV.4WK.C323*

Soil

UTC

Pre

NA

10.92

<LOQ

Soil

TRT A

Pre

0.189

5.52

<LOQ

Soil

TRT B

Pre

0.189

3.94

<LOQ

Soil

TRT C

Pre

0.189

3.88

ND

Whole
FlowerWhole
FlowerWhole
FlowerWhole
FlowerLeaves

UTC

Bloom

NA

2.24

ND

TRT A

Bloom

0.189

274.95

5.42

TRT B

Bloom

0.189

264.55

5.12

TRT C

Bloom

0.189

286.81

5.63

UTC

0 Day

NA

31.44

2.09

Leaves

TRTD

0 Day

0.189

3920.73

33.73

Leaves

TRTD

0 Day

0.189

4322.57

37.56

Leaves

TRTD

0 Day

0.189

4111.73

45.89

LeavesUTC

4 Week

NA

28.85

1.86

LeavesTRTD

4 Week

0.189

124.95

8.82

LeavesTRTD

4 Week

0.189

170.94

12.59

LeavesTRTD

4 Week

0.189

165.90

12.09

29
1014

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50265502

CDPR THX Strawberry

Trail
Number/Sample

MatrixTreatment

TimingNominal Total
Treatment
Rate

Thiamethoxa
m Residues(ppb)

CGA322704
Residues(ppb)

24.2015.CA9.01.SB.LV.8WK-315
24.2015.CA9.02.SB.LV.8WK.A318
24.2015.CA9.02.SB.LV.8WK.B321
24.2015.CA9.02.SB.LV.8WK.C324
24.2015.CA9.01.SB.LV.BL.195
24.2015.CA9.02.SB.LV.BL.A.201
24.2015.CA9.02.SB.LV.BL.B.207
24.2015.CA9.02.SB.LV.BL.C.213
24.2015.CA9.01.SB.NC.BL-197
24.2015.CA9.02.SB.NC.BL.A-203
24.2015.CA9.02.SB.NC.BL.B-209
24.2015.CA9.02.SB.NC.BL.C-215
24.2015.CA9.01.SB.PO.BL-196
24.2015.CA9.02.SB.PO.BL.A-202
24.2015.CA9.02.SB.PO.BL.B-208
24.2015.CA9.02.SB.PO.BL.C-214
24.2015.CA9.01.SB.SOIL.POST198
24.2015.CA9.02.SB.SOIL.POST.A204
24.2015.CA-

LeavesUTC

8 WeekNA

638.33

48.03

LeavesTRTD

8 Week

0.189

413.45

52.79

LeavesTRTD

8 Week

0.189

531.94

51.25

LeavesTRTD

8 Week

0.189

528.8935.59

Leaves

UTC

Bloom

NA

30.15

1.35

Leaves

TRTD

Bloom

0.189

919.86

34.48

Leaves

TRTD

Bloom

0.189

1177.57

38.45

Leaves

TRTD

Bloom

0.189

1177.37

33.01

Nectar

UTC

Bloom

NA

17.35

ND

Nectar

TRT A

Bloom

0.189

375.96

5.22

Nectar

TRT B

Bloom

0.189

232.96

4.16

Nectar

TRT C

Bloom

0.189

152.5

4.19

Pollen

UTC

Bloom

NA

12.00

<LOQ

Pollen

TRT A

Bloom

0.189

7349.43

61.96

Pollen

TRT B

Bloom

0.189

7444.74

47.89

Pollen

TRT C

Bloom

0.189

5354.97

40.91

Soil

UTC

Post

NA

16.41

1.35

Soil

TRT A

Post

0.189

52.25

1.59

Soil

TRT B

Post

0.189

31.27

2.13

30
1015

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50265502
Trail
Number/Sample

9.02.SB.SOIL.POST.B210
24.2015.CA9.02.SB.SOIL.POST.C216
24.2015.CA9.01.SB.SOIL.PRE-193
24.2015.CA9.02.SB.SOIL.PRE.A199
24.2015.CA9.02.SB.SOIL.PRE.B205
24.2015.CA9.02.SB.SOIL.PRE.C211
24.2015.CA9.01.SB.WF.BL-194
24.2015.CA9.02.SB.WF.BL.A-200
24.2015.CA9.02.SB.WF.BL.B-206
24.2015.CA9.02.SB.WF.BL.C-212

CDPR THX Strawberry

MatrixTreatment

TimingNominal Total
Treatment
Rate

Thiamethoxa
m Residues(ppb)

CGA322704
Residues
(ppb)

Soil

TRT C

Post

0.189

30.94

1.64

Soil

UTC

Pre

NA

19.03

1.15

Soil

TRT A

Pre

0.189

19.45

1.14

Soil

TRT B

Pre

0.189

17.46

1.08

Soil

TRT C

Pre

0.189

15.02

1.02

Whole
FlowerWhole
FlowerWhole
FlowerWhole
Flower

UTC

Bloom

NA

3.39

ND

TRT A

Bloom

0.189

368.60

9.28

TRT B

Bloom

0.189

383.65

6.42

TRT C

Bloom

0.189

296.31

7.90

ND = No Detect, NA = Not Applicable LOQ = Limit of Quantitation* Average of duplicate analyses

7. STATISTICAL ANALYSIS
Study objectives and design
The study was conducted to determine the concentration of thiamethoxam and its metabolite,
CGA322704, in leaves, flowers, nectar, and pollen of strawberry plants in response to foliar applicationsof a thiamethoxam pesticide product applied before bloom. Plants received three foliar sprays at an
application rate of 0.063 lbs./acre, resulting in a total application of 0.189 lbs./ai. The first application
was applied at approximately 28 days before bloom. The second application was made at approximately
18 days before bloom with the third spray applied approximately 7 days prior to harvesting of plant
samples at bloom. Leaves were sampled after the third foliar application and extended over time withsamples being taken at 4 and 8 weeks after bloom. Soil samples were also taken prior to the initiation of
the study and then at the end of the study, after the last sampling of leaves. Contaminated samples
were taken from untreated control plots. Three replicate samples from each matrix were targeted fortreated plots and only one sample from each matrix was obtained from the untreated control plots.
311016

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50265502

CDPR THX Strawberry

Plant and soil samples were analyzed for concentrations of thiamethoxam and its metabolite,
CGA322404. The study was conducted only for 1 year.

Data was submitted for 9 sites, denoted as CA-1 through CA-9. The data call-in specified that the testsites were to be distributed across soil texture categories with 3 sites in each coarse, medium, and finetextured soil. The sites were classified as coarse-textured sites for CA-2, CA5, and CA7; mediumtextured sites for CA-6 and CA-4; and moderately-fine textured sites for CA-1, CA-3, CA-8 and CA-9
based on the reported soil textures. Although not strictly denoted as fine-textured, the moderately-fine
textured soils were clayey in nomenclature, representing a finer-textured contrast to plants grown inloamy textured soil. These categories are based on the USDA classification of soils (Soil Science Division
Staff, 2017, see Table 3.1).
Chemical analytical results that were reported as less than the limit of detection (LOD) and less than thelimit of quantification (LOQ) (Table 3). The LOD was 0.5 ng/g (ppb) for leaves, pollen, whole flowers, soil
and 0.25 ng/g for nectar. The LOQ was twice the LOD at 1.0 ng/g for leaves, pollen, whole flowers, soil
and 0.5 ng/g for nectar. Data reported as <LOD or <LOQ were assigned ½ their respective detection
limits.
Non-parametric statistical tests were used to test for differences in distribution of concentrations
between soil type and between untreated and treated sites. Non-parametric tests do not require tests
for normality as they are robust to differences in distribution and they are also robust for experimental
designs with low replicates (Helsel and Hersch, 2002). The PROC NPAR1WAY procedure in the Statistical
Analysis System (SAS) statistical package was used to conduct Wilcoxon-Mann –Whitney (Wilcoxon),
Median non-parametric, and Kuiper tests. A significant result from the Wilcoxon test indicatesdifferences in the shape of distributions; a significant result from the Median test indicates differencesin the location of the medians between distributions; and a significant result from the Kuiper test
indicates differences in the empirical distributions between two groups. The Exact option for eachstatistic was implemented as it provides permutation testing, which is a statistical method thatminimizes the effect of sample size and distributional differences. Using the Exact option, the Monte
Carlo procedure was also implemented which provided 10,000 separate runs for each statistic toproduce the permutation distributions. The test for potential differences due to soil type had 3 levels so
the DSCF option in PROC NPAR1WAY, which invokes the Dwass, Steel, Critchlow-Fligner multiple
comparison test, was used to provide pairwise tests for two-sample rankings.
Additional procedures used for descriptive statistics were PROC MEANS to calculate mean values fromthe replicates at each site, PROC CAPACITY to produce cumulative statistics, and PROC BOXPLOT toproduce comparative graphics. Most of the previous studies conducted for the data call-in werereplicated over years so the mean from each site were used in the statistical analysis. This study was not
replicated over years so the replicate samples taken within each site were used to provide guidance on
potential effects of soil type and comparison between untreated and treated plants. Some comparison
data was transformed to a natural logarithm scale to provide clear contrasts between distributions
presented in the graphics. Figure 1 provides an explanation of the statistics summarized in Box-andWhisker plots used to compare distributions. Final presentation of the potential distribution ofconcentrations in bee relevant plant matrices is based on all raw data because these values represent
the actual range of exposure to bees and other organisms that feed off the nectar and pollen of plants.
Detection rates in each plant matrix with comparison between treated and untreated control plants:
Counts for the number of samples reported above each detection limit are presented in Table 6 where321017

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50265502

CDPR THX Strawberry

Table 6A contains data for treated plants and Table 6B contains data for untreated plants.
Concentrations of thiamethoxam and CGA322704 in treated plant samples were above the LOQ. Parent
thiamethoxam residues comprised greater than 90% of the total residue with most percentages above
95% (Tables 7 and 8). Concentrations in leaves and pollen matrices were greater than those measured innectar and whole flower matrices (Figure 2). In untreated control plants, residues in nectar and pollen
were reported above the LOQ. Nectar and pollen samples had a higher range in values than leaves andwhole flowers in untreated control plants (Figure 3).
As expected, non-parametric tests comparing the distributions between untreated control plants andtreated plants were significant for all plant tissues (Table 9). Graphical comparisons illustrate the higher
range in concentrations measured in samples taken at bloom in treated plants for parent thiamethoxam(Figure 4) and CGA322704 (Figure 5). Although some thiamethoxam residues were measured above theLOQ in untreated plants, the means were orders of magnitude lower than those in treated plots (Tables
7 and 8).
Comparison of distribution between soil types: There was no effect of soil type on the distribution forparent thiamethoxam or CGA322704 in the plant matrices (Table 9; Figure 6).
Concentration in leaves measured over time: Leaves were sampled over time to determine potential
dissipation of residues (Table 10 and Table 7 for samples taken at bloom). Concentrations for bothparent thiamethoxam and CGA322704 metabolite decreased over time (Figures 7 and 8). Comparison
between treated and untreated control plants at 8 weeks after application indicate that thiamethoxam
residues in leaves remained at elevated levels and had not completely dissipated.
Concentrations measured in soil samples: Soil samples were obtained from treated and untreatedcontrol plots prior to the start of the study and then after completion of the study (Table 11). Nonparametric test comparing the distributions between treated and untreated control plants indicated nosignificant difference in thiamethoxam concentrations in samples taken before the start of the study
where Wilcoxon and Median exact probability values were 0.17 and 0.65, respectively. For samples
taken after the study, the tests indicated elevated thiamethoxam concentration in soil sampled fromtreated plots where Wilcoxon and Median exact probability values were 0.002 and 0.06, respectively(Figure 9).
Concentrations in Bee Relevant Matrices: Additional statistics for the distribution of thiamethoxam and
CGA322704 in pollen and nectar samples of treated plants are given in Table 12. Median and maximum
values for total thiamethoxam residues in pollen were 875 and 7540 ng/g on a wet weight basis and fornectar were 182 and 660 ng/g, respectively.
8. STUDY STRENGTHS, LIMITATIONS AND CONCLUSIONSIn the context of documenting the magnitude of thiamethoxam residues in bee-related matrices ofstrawberry, the following strengths are observed with this study.
1. Data provide quantitative values for thiamethoxam and the major degradation product, CGA-322704,
expected in leaves, flowers, nectar, and pollen of strawberry plants in response to foliar applications
made prior to bloom.

33
1018

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50265502

CDPR THX Strawberry

2. The study was not replicated over two years but samples were obtained from 9 distinct sites. Leaf,
whole flower, nectar, and pollen were collected at bloom.
3. Sites were distributed across requested soil types, allowing for a comparison of the effect of soil typeon concentrations measured in plant samples.
Limitations noted in this study include:
1. Potential for carryover effects could not be determined because the study was conducted in only oneof two requested years.
Overall, considering the strengths and limitations of this study, the following conclusions can be drawn:
Classification/Utility for Bee Risk Assessment. This study is classified as acceptable. Althoughthiamethoxam residue concentrations were reported above the LOQ in plant matrices sampled from
untreated plants, the range in concentrations in treated plants were much greater. Values from treatedplants reflect the range that would be expected from foliar applications.
Magnitude of Residues in Bee-relevant Matrices. The distributions reported for treated plants in pollen
and nectar in Table 12 represent the expected distributions from pre-bloom foliar thiamethoxam
treatments applied to strawberry plants. For data from treated plots, the median and maximum values
for total thiamethoxam residues in pollen were 875 and 7540 ng/g on a wet weight basis and for nectar
at 182 and 660 ng/g, respectively. Total residue values represent predominantly parent thiamethoxam,
as it comprised 98% of the total.
Temporal Variability in Residues. Leaf samples taken over time indicated dissipation of residues overtime. Concentrations in treated plants remained elevated when compared to untreated plants 8 week
after bloom.
Effect of Soil Type. There were no differences in the magnitude and distribution of concentrations ofthiamethoxam and CGA322704 between soils.
Pesticide Carryover. The study was not replicated over years so it was not possible to determine
potential for carryover of residues.
9. STUDY VALIDITY/CLASSIFICATIONThis study is classified as ACCEPTABLE. The data from this study provide an expected distribution of theconcentrations of thiamethoxam residues that bees are exposed to in nectar and pollen of strawberry
plants grown in California under the thiamethoxam exposures used in this study. Relating
concentrations measured in flower parts to bee health is possible by comparing the concentration
measured in bee relevant plant parts to target values that define acute or chronic exposure scenarios.
Potential for carryover effects within a site could not be determined because the study was not
replicated over years.

34
1019

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50265502

CDPR THX Strawberry

10. REFERENCES
1. Crook, S (2004) Residue Method for the Determination of Residues of Thiamethoxam(CGA293343) and CGA322704 in Lettuce, Tomato, Grape and Tobacco Samples. Final
Determination by LC/MS/MS" (Syngenta Method REM179.06).
2. Crook, S (2007) Analytical Method for the Determination of Residues of Thiamethoxam(CGA293343) and CGA322704 in Bee and Hive Products. Final Determination by LC-MS/MS(Syngenta Method REM179.07).
3. EPL_BAS Method No. 110G747B (2007) Analytical Method for the Determination of Residues ofThiamethoxam and CGA322704 in Pollen by LC-MS/MS (based on Syngenta Method
REM179.07).
4. Mayer, T.J. (2005) Analytical Method for the Determination of Residues of CGA-293343, CGA 355190, CGA-353042, NOA-404617, NOA-404465, SYN-501406 and NOA-459602 in Soil by Direct
Injection High Performance Liquid Chromatography with Mass Spectrometric Detection(Syngenta Method T009171-04).
5. Mair, P. (1998) Stability of Residues of CGA-293343 (2 Years Final Report) and CGA-322704 (1Year Interim Report) in Plant Material under Deep Freezer Conditions, Including Method
Validation (Study No. 504/96 consists of Reports #112/96, 127/97, 103/98) MRID 44703525.
6. Hohl, J. (1999) Stability of Residues of CGA-322704 in Plant Material and Soil Stored Under Deep
Freezer Conditions (Study No. 779-00) MRID 45108001.
7. Oakes, T. (2002) Stability of CGA-293343 and CGA-322704 in Crops and Processed Fractions
Under Freezer Storage Conditions (Study No. 269-98) MRID 45659205.
8. Anderson, L. (2007) “Thiamethoxam (CGA293343) and CGA322704. Validation of Residue
Analytical Method REM 179.07 for the determination of Residues in Bee and Hive Products andStorage Stablity in Hive Pollen, Wax and Nectar, stored Deep Frozen for 12 months.” (Study No.
05-S508)
9. Dharmasri, C., Tauber, R., (2009) Stability Determination of Thiamethoxam (CGA293343) andMetabolites CGA322704, CGA355190, CGA353042, NOA404617, NOA407475, SYN501406 andNOA459602 in Soil and Water Under Freezer Storage Conditions (Study No. T004683-06) MRID
4775140135
1020

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50265502

CDPR THX Strawberry

Table 6A. Treated Plants: Counts of chemical analytical results for thiamethoxam and CGA322704 that were indicated as above the LOQ,
between the LOQ and LOD, and below the LOD.

Plant Sample
NectarPollenWhole Flowers
Leaves: Bloom
Leaves: After Third App
Leaves: 4 Wks After Bloom
Leaves: 8 Wks After Bloom
Soil: Pre Study
Soil: Post Study

Treated Plants: Comparison of Total Number of Samples Reported Above the LOQ,
Between the LOQ and LOD, and Below the LOD
Thiamethoxam
CGA322704
Total
Number Number Number
Total
Number Number Number
Number
>LOQ
<LOQ
<LOD
Number
>LOQ
<LOQ
<LOD

27
27
27
27
21
27
27
24
27

27
27
27
27
21
27
27
12
26

0
0
0
0
0
0
0
6
0

0
0
0
0
0
0
0
6
1

36
1021

27
27
27
27
21
27
27
24
27

26
27
27
27
21
27
27
8
20

0
0
0
0
0
0
0
8
2

1
0
0
0
0
0
0
8
5

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50265502

CDPR THX Strawberry

Table 6B. Untreated Control Plants: Counts of chemical analytical results for thiamethoxam and CGA322704 that were indicated as above theLOQ, between the LOQ and LOD, and below the LOD.

Plant Sample
NectarPollenWhole Flowers
Leaves: Bloom
Leaves: After Third App
Leaves: 4 Weeks After Bloom
Leaves: 8 Weeks After Bloom
Soil: Pre Study
Soil: Post Study

Untreated Plants: Comparison of Total Number of Samples Reported Above theLOQ, Between the LOQ and LOD, and Below the LOD
Thiamethoxam
CGA322704
Total
Number Number Number
Total
Number Number Number
Number
>LOQ
<LOQ
<LOD
Number >LOQ
<LOQ
<LOD

9
9
9
10
7
9
9
8
9

9
5
5
5
4
2
4
4
3

0
0
0
0
1
6
1
0
1

0
4
4
5
1
1
4
4
5

37
1022

9
9
9
10
6
9
9
8
9

2
3
0
2
1
2
3
1
1

5
3
9
5
3
1
4
3
5

2
3
0
3
2
6
2
4
3

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50265502

CDPR THX Strawberry

Table 7. Untreated vs Treated Plants: Distribution of thiamethoxam (Parent) and CGA322704(Metabolite) in strawberry plant samples taken at bloom.

Samples Taken at Bloom
Statistic

Untreated Control Plants
ParentMetabolite
Total

Thiamethoxam Treated Plants
ParentMetabolite
Total

10
5.5
10.3
186.0
0.3
0.4
30.0
91.7

10
0.5
0.4
75.0
0.3
0.4
1.4
8.3

10
6.0
10.6
176.3
0.5
0.9
31.5

27
1401
529
38
428
1483
2152
97.5

27
35
16
46
11
34
71
2.4

27
1437
543
38
439
1509
2223

9
1.6
2.0
126.0
0.3
0.5
5.9
88.9

9
0.3
0.0
0.0
0.3
0.3
0.3
13.9

9
1.8
2.0
109.0
0.5
0.8
6.2

27
429
191
44
241
373
973
96.8

27
14
16
116
5
10
82
3.2

27
443
201
45
248
390
995

9
25.5
35.3
138.0
1.2
9.0
105.0
98.8

9
0.3
0.3
95.0
0.1
0.1
0.9
1.2

9
25.8
35.2
136.5
1.3
9.1
105.0

27
192
114
59
51
177
647
98.0

27
4
3
65
0
4
13
2.0

27
196
116
59
52
182
660

9
122.6
218.4
178.0
8.1
43.0
669.0
99.1

9
1.1
1.2
113.0
0.3
0.5
3.8
0.9

9
123.7
218.6
177.0
8.4
46.2
670.8

27
2023
2432
120
102
861
7473
98.8

27
25
19
74
4
20
66
1.2

27
2048
2449
120
108
875
7540

LeavesN (#)
Mean (ng/g)
SD
CV (%)Min (ng/g)
Median (ng/g)
Max (ng/g)% of Total
Whole Flowers
N (#)
Mean (ng/g)
SD
CV (%)Min (ng/g)
Median (ng/g)
Max (ng/g)% of Total
NectarN (#)
Mean (ng/g)
SD
CV (%)Min (ng/g)
Median (ng/g)
Max (ng/g)% of Total
PollenN (#)
Mean (ng/g)
SD
CV (%)Min (ng/g)
Median (ng/g)
Max (ng/g)% of Total

38
1023

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50265502

CDPR THX Strawberry

Table 8. Untreated vs Treated Plants: Exact probability levels for Wilcoxon, Median, and Kuiper nonparametric tests for differences in the distribution in chemical analyses conducted on untreatedcontrol plants and plants treated with a foliar spray of thiamethoxam.

Plant Matrix
Whole Flowers
NectarPollenLeaves: Bloom
Leaves: After Third App
Leaves: 4 Wks After Bloom
Leaves: 8 Wks After Bloom
Soil: Pre Study
Soil: Post Study

Non-parametric Test Exact Probability Levels for
Comparing Concentration Distribution Between UntreatedControl and Treated Plants
Thiamethoxam
CGA322704
Wilcoxon Median Kuiper Wilcoxon Median Kuiper
0.001
0.001
0.001
0.001
0.001
0.001
0.001
0.001
0.002
0.001
0.001
0.001
0.001
0.01
0.009
0.01
0.01
0.01
0.001
0.001
0.001
0.001
0.001
0.001
0.001
0.01
0.003
0.001
0.01
0.003
0.001
0.009
0.001
0.001
0.008
0.001
0.003
0.06
0.001
0.001
0.01
0.09
0.18
0.66
1
0.23
0.3
1
0.02
0.06
0.09
0.001
0.01
0.09

39
1024

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50265502

CDPR THX Strawberry

Table 9. Comparison Between Soil Type: Exact probability levels for Wilcoxon rank sum test fordifferences amongst the 3 soil texture categories on the distribution of thiamethoxam and CGA322704
metabolite residues in strawberry plants exposed to 3 foliar sprays. Samples were taken at bloom.

Treatment,
Plant Matrix, andSpecific Soil Contrasts

Treated Plants
LeavesCoarse vs. MedFine
Coarse vs. Medium
MedFine vs. Medium
Whole Flowers
Coarse vs. MedFine
Coarse vs. Medium
MedFine vs. Medium
NectarCoarse vs. MedFine
Coarse vs. Medium
MedFine vs. Medium
PollenCoarse vs. MedFine
Coarse vs. Medium
MedFine vs. Medium

Exact Probability Levels for Nonparametric Tests of Differences
Between Soil Type
Thiamethoxam
CGA322704
Wilcoxon
Wilcoxon
0.19

0.05
0.295
0.981
0.225

0.20

0.071
0.676
0.225
0.62

0.714
0.292
0.276
0.78

0.580
0.840
0.826
0.97

0.837
0.789
0.970
0.44

0.933
1.000
0.970
0.25

0.670
0.617
0.539

0.330
0.389
0.970

0.51
0.46
0.35
0.53

0.32
1
1
0.17

Untreated Plants
LeavesWhole Flowers
NectarPollen401025

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50265502

CDPR THX Strawberry

Table 10. Distribution of thiamethoxam (Parent) and CGA322704 (Metabolite) in strawberry plant leaf
samples taken over time.

Leaf Residue Concentration Sampled Over Time
Untreated Control Plants
Statistic

ParentAfter Third Foliar Application
N (#)
6
Mean (ng/g)
13.0
SD
19.0
CV (%)
145.0
Min (ng/g)
0.3
Median (ng/g)
1.6
Max (ng/g)
41.5% of Total
97.0
4 Weeks After Bloom
N (#)
9Mean (ng/g)
254.0
SD
750.0
CV (%)
295.0
Min (ng/g)
0.3
Median (ng/g)
0.3
Max (ng/g)
2254.0% of Total
96.7
8 Weeks After Bloom
N (#)
9Mean (ng/g)
262.0
SD
584.0
CV (%)
223.0
Min (ng/g)
0.3
Median (ng/g)
0.5
Max (ng/g)
1715.0% of Total
97.4

Thiamethoxam Treated Plants

Metabolite

Total

ParentMetabolite

Total

6
0.6
0.7
113.0
0.3
0.4
2.1

6
13.4
19.0
142.0
0.5
2.0
42.0

21
5753.02297.040.0
227.0
5257.09099.0

21
29.0
9.0
31.0
13.0
27.0
46.0

21
5782.02297.040.0
2301.05282.09125.0

99.5

0.5

27
297.0
330.0
111.0
45.0
192.0
1334.0

27
26.0
27.0
101.0
5.0
15.0
89.0

92.0

8.0

27
341.0
597.0
175.0
13.0
72.0
2406.0

27
29.0
24.0
116.0
3.0
10.0
83.0

94.2

8.0

4.5
9
8.8
25.0
284.0
0.3
0.3
75.5

9
262.7
775.0
255.0
0.5
0.5
2330.0

3.3
9
7.0
16.0
237.0
0.3
0.5
48.0

9
269.0
591.0
220.0
0.5
1.3
1723.0

2.6

41
1026

27
323.0
352.0
109.0
52.0
205.0
1423.0

27
362.0
617.0
171.0
18.0
82.0
2471.0Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50265502

CDPR THX Strawberry

Table 11. Distribution of thiamethoxam (Parent) and CGA322704 (Metabolite) in soil samples.

Residue Concentration in Soil Samples
Statistic
Before Start of Study

Untreated Control Plants
ParentMetabolite
Total

N (#)
8
Mean (ng/g)
4.2
SD
7.0
CV (%)
168.0
Min (ng/g)
0.3
Median (ng/g)
0.7
Max (ng/g)
19.0% of Total
89.4
After Completion of StudyN (#)
9Mean (ng/g)
4.9
SD
8.0
CV (%)
163.0
Min (ng/g)
0.3
Median (ng/g)
0.3
Max (ng/g)
20.6% of Total
90.7

8
0.5
0.3
60.0
0.3
0.5
1.2

8
4.7
7.3
156.0
0.5
1.2
20.2

10.6
9
0.5
0.4
78.0
0.3
0.3
1.4

9
5.4
8.2
154.0
0.5
0.8
21.1

9.3

42
1027

Thiamethoxam Treated Plants
ParentMetabolite
Total
24
7.0
10.4
148.0
0.3
1.9
36.4

24
0.9
1.0
10.0
0.3
0.5
3.7

88.6

11.4

27
21.2
25.8
121.0
0.3
11.5
85.2

27
1.8
1.2
67.0
0.3
1.6
5.6

92.2

7.8

24
7.9
10.6
134.0
0.5
2.6
37.6

27
23.0
26.3
114.0
0.5
13.0
88.0

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50265502

CDPR THX Strawberry

Table 12. Bee Relevant Plant Matrices: Distribution of thiamethoxam, CGA322704 metabolite, andtotal thiamethoxam residues in nectar and pollen samples of strawberry plants exposed to 3 foliar
sprays of thiamethoxam prior to bloom.

Distribution of Residues
Statistic

Thiamethoxam

CGA322704

Total

27
192
114
59
51
177

27
4
3
65
0
4

27
196
116
59
52
182

214
296
376
647

5
5
10
13

215
301
381
660

98.0

2.0

27
2023
2432

27
25
19

27
2048
2449

120
102
861
2486
7349
7445

74
4
20
37
62
62

120
108
875
2522
7411
7493

7473

66

7540

98.8

1.2

NectarN (#)
Mean (ng/g)
SD (ng/G)
CV (%)Min (ng/g)
Median (ng/g)
75th (ng/g)
90th (ng/g)
95th (ng/g)
Max (ng/g)% of Total
PollenN (#)
Mean (ng/g)
SD (ng/G)
CV (%)Min (ng/g)
Median (ng/g)
75th (ng/g)
90th (ng/g)
95th (ng/g)
Max (ng/g)% of Total

43
1028

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50265502

CDPR THX Strawberry

Figure 1. Explanation of the meaning of graphics presented in a Box-and-Whisker plot.

Maximum Observation

75% Quartile
Mean ValueMedian Value (50%)

25% Quartile
Minimum Observation

44
1029

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50265502

CDPR THX Strawberry

Figure 2. Treated Plants: Comparison of the distribution of thiamethoxam residues measured between
leaf, nectar, pollen, and whole flower samples. Values transformed to natural logarithms.

45
1030

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50265502

CDPR THX Strawberry

Figure 3. Untreated Control Plants: Comparison of the distribution of thiamethoxam residues
measured between leaf, nectar, pollen, and whole flower samples. Values transformed to natural
logarithms.

46
1031

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50265502

CDPR THX Strawberry

Figure 4. Untreated vs Treated Bloom Plant Samples: Comparison of the distribution of thiamethoxam
residues measured between untreated control (UTC) and treated (TRT) plants for leaves, nectar,
pollen, and whole flowers.

47
1032

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50265502

CDPR THX Strawberry

Figure 5. Untreated vs Treated Bloom Plant Samples: Comparison of the distribution of CGA322704
thiamethoxam metabolite residues measured between untreated control (UTC) and treated (TRT)
plants for leaves, nectar, pollen, and whole flowers.

48
1033

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50265502

CDPR THX Strawberry

Figure 6. Comparison Between Soil Type: Distribution of thiamethoxam residues measured in leaves,nectar, pollen, and whole flowers between plants grown in soil at 3 different textures. Plants sampled
at bloom and values were transformed to natural logarithms.

49
1034

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50265502

CDPR THX Strawberry

Figure 7. Thiamethoxam Concentration in Leaves Over Time: Comparison of the distribution ofthiamethoxam residues measured in leaves sampled after the third foliar application, at bloom, andthen at 4 and 8 weeks after bloom. Leaves from untreated control plants (UTC) were sampled at thesame time as treated (TRT) plants. Values were transformed to natural logarithms.

50
1035

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50265502

CDPR THX Strawberry

Figure 8. Thiamethoxam Concentration in Leaves Over Time: Comparison of the distribution of
CGA322704 thiamethoxam metabolite residues measured in leaves sampled after the third foliar
application, at bloom, and then at 4 and 8 weeks after bloom. Leaves from untreated control plants(UTC) were sampled at the same time as treated (TRT) plants. Values were transformed to natural
logarithms.

51
1036

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50265502

CDPR THX Strawberry

Figure 9. Thiamethoxam Concentration in Soil Samples: Comparison of the distribution ofthiamethoxam residues measured in soil sampled prior to the start of the study and then after thecompletion of the study. Soil from untreated control plants (UTC) were sampled at the same time asfrom treated (TRT) plants.

52
1037

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 49686801

THX Cotton DER

Reference
Oakes, T., Mäyer, T., Rice, F., Jacobson, B. Grant, J. (2017) Thiamethoxam 40 WG (A11963C) and
Thiamethoxam FS (A9765N) – Magnitude of Residues in Leaves, Flowers, Pollen, Nectar and Extra
Floral Nectar of Cotton Plants After Foliar Application with Centric® 40WG in California or After
Application as a Seed Treatment with Cruiser® 5FS: Amended Final Report. Project Number:
TK0177223. Unpublished study prepared by Syngenta Crop Protection, LLC. 512. MRID 49686801,
CDPR Study ID 304439, Data Volume 52691-0590, Tracking ID# 283477
1. STUDY INFORMATION
Chemical:Thiamethoxam
Test Material:

Study Type:

Sponsor:Sponsor Study
ID:
Study
Completion
Date:
Amendment
Date:
GLP Status:

PC Code60109
Percent
Centric 40WG
40%
ActiveCruiser 5FS
47.6%
Ingredient:
Non-Guideline field residue study on cotton to establish the magnitude ofThiamethoxam residues in leaves, flowers, pollen, nectar and extra floral nectar
during a two year period.
Syngenta Crop Protection, LLC
410 Swing Road
Experiment Start and May 8, 2013- January 4,
Post Office Box 18300
End Date:2017
Greensboro, North Carolina
27419-8300 USA
TK0177223
Study Locations:

July 17, 2015

Nine trial sites thatwere either foliar orseed applications
located in California.

October 30, 2017
TBD; protocol reviewed by CDPR.
[CDPR Study ID 304439, Data Volume 52691-0590, Tracking ID# 283477]

2. REVIEWER INFORMATION
Study Reviewed by:Richard Bireley, Sr. Environmental Scientist (Specialist)
California Department
John Troiano, Ph.D., Research Scientist III
of Pesticide Regulation
Alexander Kolosovich, Sr. Environmental Scientist (Specialist)
Brigitte Tafarella, Environmental Scientist
Denise Alder, Sr. Environmental Scientist (Specialist)
Russell Darling, Environmental Scientist

1038

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 49686801

THX Cotton DER

3. EXECUTIVE SUMMARY
The reason for the final report amendment is to correct the study title and the analytical phase report toincorporate transit stability data, freezer storage stability data, percent Brix data, reassignment of study
responsibilities, and to clarify and correct sample residues for Year 1 nectar and extra floral samples
from Site 6. The analytical report has also been amended for a more consistent reporting format and to
add clarifications throughout. An extensive list of changes to the Analytical Phase Report can be found in
Appendix 1 of the study report.
The study was designed to include nine trials each consisting of an untreated control plot and a threereplicate treated plots to be conducted on coarse-, medium- and fine-textured soils. Centric® 40WG
(active ingredient, thiamethoxam) was applied as a foliar broadcast spray two times during the growing
season at a target rate of 0.063 lb ai/acre/application for two consecutive years. The interval between
applications was 5 days with the last application targeted 12 days before significant flowering. In thefirst year, three of the nine trials also included a three-replicate plot planted with Cruiser® 5FS (active
ingredient, thiamethoxam) treat seed at a targeted rate of 0.375 mg a.i. per seed.
Samples of leaf, whole flower, pollen, nectar and extra floral nectar were collected from all trial sites inYear 1 (2013) and Year 2 (2014) of the study. The target sampling period at all trials (including seedtreatment trials) was at early bloom stage (50–75% bloom). In the foliar-application trials, sampling wastargeted to occur 12 days after the second (last) test substance application. Additionally for Year 2, at six
trial sites extra floral nectar (EFN) was collected at 3 additional target intervals: 5 days after first
application (5 DA1A), 5 days after second application (5 DA2A), and 24 days after the second application(24DA2A). These samples were collected to characterize residues of thiamethoxam and CGA322704 in
EFN during bloom.
Method verification sets were performed at ABC Laboratories prior to the analysis of field samples.
Methods were verified successfully for thiamethoxam and CGA322704 in leaves and whole flowers, andCGA322704 in nectar. Method verification for pollen resulted in mean percent recoveries below
acceptance criteria for both analytes, and nectar mean percent recovery was below acceptance criteria
for thiamethoxam. Concurrent procedural recovery samples were diluted in subsequent analyses toimprove results and achieve acceptable method performance for these analytes and matrices.
4. STUDY VALIDITY
Guideline Followed:
Guideline Deviations:
Other Deviations:Classification:
Rationale:
Reparability:

TBD; (protocol was reviewed by CDPR)
N/A
N/A
TBD
N/A
N/A

1039

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49686801

THX Cotton DER

5. MATERIALS

Test item:
Formulation Type:
CAS #:

Test Material Characterization for Foliar Application End Use ProductCentric 40 WG
Percent A.I.:
40% A.I.
Wettable Granule, WG
Design Code:
A11963C
3153719-23-4
Specific Gravity:
26 – 34 lb/ft

Test item:
Formulation Type:
CAS #:

Test Material Characterization for Seed Treatment End Use Product
Cruiser 5FS
Percent A.I.:
47.6% A.I.
Flowable Suspension
Concentration:
5.18 lb a.i./gallon
153719-23-4
Specific Gravity:
1.295 g/mL

5A. STUDY DESIGN
The purpose of this two-year study was to determine the amount of thiamethoxam and CGA322704 thatwas present in cotton leaves, whole flowers, pollen, nectar, and extra floral nectar in fields after two
successive years of foliar broadcast spray applications of Centric® 40WG. Also, the two analytes were
measured in plants grown from seed treated with Cruiser® 5FS in the first year in three of the trials. The
effect of soil type on thiamethoxam uptake and resulting residues in pollen and nectar were examinedby conducting trials on coarse-, medium-, and fine textured soils, as available. The study initiation date
was May 8, 2013 and the experimental termination date (analytical phase) was January 4, 2017.
Foliar-Application Trials (CA-1 – CA-9)
The study included nine geographically separated trials in the Central Valley of California. Each trial
consisted of an untreated control (UTC) plot and a treated (TRT) plot (divided into three replicate areas)that were each large enough to collect sufficient quantities of flowers, leaves, pollen, nectar, and extra
floral nectar for residue analysis. The plot size for the UTC and the TRT replicate plot at each location
was a minimum of 0.115 A. The UTC plot was located up-slope and up-wind with regard to the prevailingwind direction and separated by a minimum of 50 ft. from the TRT plot to minimize potential crosscontamination by run-off or pollen transfer.
Representative composite samples of leaves, whole flowers, pollen, nectar, and extra floral nectar werecollected from the UTC plot and each of the treated replicate plots for residue analysis in Year 1 andYear 2 of the study (n = 27 samples per matrix each year).
Seed-Treatment Application Trials (CA-2A, CA-6A, and CA-8A)
One additional treated plot (Year 1 only) was added at three of the foliar application locations. Each trial
consisted of three replicated TRT plots that were approximately the same size (0.115 A) as indicatedabove for the foliar-application trials.
Representative composite samples of leaves, whole flowers, pollen, nectar and extra floral nectar werecollected from each of the treated replicate plots for residue analysis in Year 1 of the study (n = 9
samples per matrix).
1040

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49686801

THX Cotton DER

5B. APPLICATION TIMING AND RATES
Foliar Application Trials:
In Year 1 and Year 2, Centric® 40WG (thiamethoxam formulated) was applied to treated plots as abroadcast foliar spray two times at the maximum labeled-use rate of 2.5 oz formulated product per acre
(0.063 lb ai/acre) for each application. The interval between applications was 5 days and the last
application was scheduled to occur 12 days before significant bloom. The test substance was applied
using a calibrated, boom sprayer in a volume of water that ensured uniform application (20 - 30
gallons/acre). The tables below present the application information for each trial.
All applications were made using ground-based equipment. The adjuvant Dyne-Amic (0.25 % v/v) wasused in all foliar applications.
Table 1. Year 1 (2013)
Trial
NumberDate
1
8/30/20132
8/2/2013
3
9/6/2013
4
8/9/2013
5
7/22/20136
8/2/2013
7
8/2/2013
8
8/5/2013
9
8/10/2013a
Gallons Per Acre
Table 2. Year 2 (2014)
Trial
NumberDate
1
8/8/20142
7/18/20143
7/27/20144
7/18/20145
7/7/2014
6
7/21/20147
7/18/20148
7/1/20149
7/11/2014a
Gallons Per Acre

Application 1
Calibrated
GPAa
25.47
25.50
29.47
30.0
30.0
30.0
20.0
20.0
20.0

Application 1
Calibrated
GPAa
24.89
25.44
26.61
30.0
30.0
30.0
20.0
20.0
20.0

Tank Mix
Volume
13.0
13.0
19.0
14.0
14.0
14.0
10.5
10.1
9.0

Tank Mix
Volume
13.0
13.0
20.6
14.0
14.0
14.0
10.5
11.0
9.0

1041

Date
9/4/2013
8/7/2013
9/11/2013
8/14/2013
7/27/2013
8/7/2013
8/7/2013
8/10/2013
8/15/2013

Date
8/13/2014
7/23/2014
8/1/2014
7/23/2014
7/12/2014
7/26/2014
7/23/2014
7/6/2014
7/16/2014

Application 2
Calibrated
GPAa
25.93
25.66
20.04
30.0
30.0
30.0
20.0
20.0
20.0

Tank Mix
Volume
13.0
13.0
13.46
14.0
14.0
14.0
10.5
10.1
9.0

Application 2
Calibrated
GPAa
24.96
25.79
25.71
30.0
30.0
30.0
20.0
20.0
20.0

Tank Mix
Volume
13.0
14.0
19.9
14.0
14.0
14.0
10.5
11.0
9.0

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49686801

THX Cotton DER

Seed-Treatment Application Trials:
These treated plots were planted with a commercial variety of cotton (Phytogen 499) that was treatedwith Cruiser® 5FS at a rate of 0.375 mg a.i. per seed. The seed was treated at Syngenta Seed Care
Institute, Stanton, MN, using commercial seed treatment procedures. The seed treatment rate wasverified by analysis of the treated seed at SGS NAM GLP Laboratory, Brookings, SD. The actual seedtreatment rate based upon the verification analysis was 93% of target or 0.349 mg a.i. per seed. Treatedcotton seed from a batch of certified seed (0.375 mg a.i. per seed) was provided to each selected facility.
The table below presents the planting date and planting rate (seeds/A) for each of the three seedtreatment trials.
Table 3. Seed Treatment Applications
Trial Number
Application/Planting Date2a5/31/20136a
6/11/20138a
6/6/2013
1
Thiamethoxam treated seed (0.375 mg ai/seed)

Planting Rate1 (Seeds/A)
59,739144,500
36,0005C. STUDY SITE LOCATION AND CHARACTERISTICS
Trial site locations were selected based on soil-survey maps, soil characterization information, site
availability, security, and cotton cultural significance to ensure representation of different soil textures(e.g., loamy sand, sandy loam, and loam, as availability allowed). The table below presents the fieldcooperator facility along with site information that includes county, soil series and textural class for eachtrial.
The test plots (UTC and TRT) were managed to mimic typical commercial cotton production in California.
Plot areas were prepared, planted, and maintained according to local agricultural practice with regard totillage, fertilizer inputs, irrigation, and weed and pest control. Irrigation was applied as needed according
to commercial good agricultural practices for maintaining good crop health and yield.
After harvest of cotton in 2013 (Year 1), the cotton was shredded and the plant matter was disked/tilled
into the ground in preparation for Year-2. Plots remained fallow until the plots were prepared, i.e., tilled
and re-bedded, the following spring prior to planting in Year 2 (2014).
Table 4. Results from the Soil Characterization Analysis
Trial
CationField
Application
Trial
Location Organic Soil Exchange%
Cooperator
Type
Number(County, Matter pH
Capacity
Sand
State)(%)(meq/100g
soil)
Research
Foliar1
Tulare,
1.60
7.8
11.6
48
For Hire
Application
California
2
Tulare,
1.4
6.6
14.6
80
California
1.3
6.4
15.6
76
Seed
2a
Tulare,
0.64
6.5
20.5
78
Treatment
California
0.59
5.7
14.0
80
1042

%
Silt

%
Clay

Soil Types

34

18

15
19
17
15

5
5
5
5

Exeter Loam
(Fine/ Medium)
Tujunga Loamy
Sand (Coarse)
Tujunga Loamy
Sand (Coarse)

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49686801

Field
Application
Cooperator
Type

THX Cotton DER

Trial
NumberTrial
Location(County,
State)

SyntechResearch

Foliar
Application

3

Fresno,California

Excel
Research

Foliar
Application

4

Madera,California
Madera,California
Madera,California
Madera,California
Fresno,California
Fresno,California
Fresno,California
Sanger,California

5
6

Cal AgResearch

Seed
Treatment
Foliar
Application

6a7
8

Eurofins

Seed
Treatment
Foliar
Application

8a9

CationOrganic Soil Exchange%
Matter pH
Capacity
Sand(%)(meq/100g
soil)
0.49
6.1
5.6
60
0.40
6.1
5.5
58
0.77
0.86
0.47
0.43
0.90
0.43
0.90
0.77
0.87
0.61
0.52
0.26
0.39
0.78
1.10
1.05

7.2
6.7
7.2
7.1
7.2
6.7
7.6
7.6
7.6
7.6
5.6
5.8
5.9
6.1
7.6
7.5

6.6
7.0
6.1
5.7
8.4
7.8
7.4
6.7
13.0
14.1
4.7
3.8
4.8
5.3
9.7
11.0

78
78
84
82
78
76
82
82
50
48
78
78
72
72
66
70

%
Silt

%
Clay

Soil Types

32
32

8
10

15
17
9
11
15
11
11
11
32
36
16
16
20
22
24
22

7
5
7
7
7
13
7
7
18
16
6
6
8
6
10
8

Hanford Fine
Sandy Loam
(Medium)
Atwater Loamy
Sand (Coarse)
Chino Loamy
Sand (Coarse)
Atwater Loamy
Sand (Coarse)
Atwater Loamy
Sand (Coarse)
Ramona Loam
(Fine/Medium)Hanford Sandy
Loam (Medium)Hanford Sandy
Loam (Medium)
Ramona Sandy
Loam (Medium)5D. SAMPLE COLLECTION, HANDLING, PROCESSING
Samples of leaf, whole flower, pollen, nectar, and extra floral nectar were collected from all trial sites inYear 1 of the study. In the foliar-application trials, sampling was targeted to occur 12 days after thesecond (last) test substance application. The target sampling period at all trials (including seedtreatment trials) was at early bloom stage (50–75% bloom). All trials were sampled 12 days after thesecond application, except trials CA-3 and CA-9, which were sampled 9 and 14 days after the second
application, respectively. These changes from the targeted 12 day schedule were made in order toobtain enough flowers to meet target sample size requirements.
In Year 2, samples of leaf, whole flower, pollen, nectar, and extra floral nectar were also collected fromall trial sites. At all trials, sampling was targeted to occur 12 days after the second (last) test substanceapplication. The target sampling period at all trials was at early bloom stage (50–75% bloom). All trials
were sampled 12 days after the second application. In Year 2 the CA-1 nectar samples collected at 12
Days after second application (bloom) and the CA-2 extra floral nectar samples collected at 24 days after
second application were lost in shipment to the laboratory.
Additionally for Year 2, at six trial sites (CA-1, CA-2, CA-3, CA-4, CA-6 and CA-7), Extra Floral Nectar (EFN)
was collected at 3 additional target intervals: 5 days after first application (5 DA1A), 5 days after second
application (5 DA2A), and 24 days after second application (24 DA2A). These samples were collected to
be able to characterize residues of thiamethoxam and CGA322704 in EFN over time.

1043

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49686801

THX Cotton DER

At each trial site, samples were collected from the UTC plot before the collection of samples from theTRT plot or by separate teams to minimize potential cross contamination. Gloves were worn andreplaced between flower and leaf sampling and between plots.
Whole Flower and Leaf Samples
Each whole flower sample was a minimum of 500 intact flowers, if available. Flower samples weredivided into 2 bags, one bag for the whole flower sample (200 flowers minimum) and another bag forthe processing flowers (300 flowers minimum) from which pollen and nectar were collected. The only
whole-flower samples that did not have the 200-flower minimum was at CA-9 in Year 2. Each leaf
sample was a minimum of 500 g. Samples were collected from the UTC and each TRT replicate plot (A, B,
and C). Samples were collected from the lower, middle and upper plant canopy for a representative,
composite sample. The UTC and TRT leaf and flower samples were stored and transported in separate,labelled plastic sealable bags to the field laboratory on blue or wet ice for freezer storage or further
processing.
Pollen, Nectar and Extra Floral Nectar Samples
The remaining 300 or more flowers were used for collection of pollen and nectar. An additional 300
flowers, when available, with the receptacle, peduncles, and the calyculus bracteoles were collected forextra floral nectar. At some trials, the 300 flowers for pollen and nectar collection were also used forextra floral nectar collection or the number of flowers processed for extra floral nectar were less than
300 due a limited availability of flowers at sampling.
Extraction approaches entail the use of:(1) a vacuum pump fitted with a disposable 1000-µL filtered pipette tips to vacuum and trap the pollen;and,(2) a 10-µL or larger capillary micro-pipettes to extract nectar from the exposed nectary.
The floral nectar was collected from the internal whorl of epicalyx bract nectaries, which occur on the
inner side of the sepal base. The extra floral nectar was collected from the calyculal nectaria.
The nectar collected in each micro-pipette was expelled into a pre-labelled 2 mL glass vial. Pipette tips
and vials were weighed prior to and immediately after pollen and nectar collection to enable calculationof sample mass. The target pollen and nectar sample weight required for analysis – minus the pipette orvial weight – was >100 mg for TRT plots and >250 mg for UTC plots. Pipette tips (containing pollen) wereplaced directly into labelled vessels suitable for solvent extraction. Pollen and nectar samples werestored frozen until shipment. The target sample weight for all matrices collected in the treated replicate
plots were met except for extra floral nectar (23 samples). The sample weight for some extra floral
nectar samples collected from the treated replicate plots were less than the target (>100mg) at trials
CA-1, CA-3, CA-5 and CA-9 in Year 1 and CA-1 and CA-2 in Year 2.
Transit Stability
For transit and freezer stability data, at least two concurrent recovery samples per analytical set wereprepared by fortifying an untreated control sample with thiamethoxam and CGA322704 at the samelevel as the stability samples (50 ppb) and analyzed concurrently with the stability samples to1044

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49686801

THX Cotton DER

demonstrate adequate method performance. The recoveries for both the fresh fortified samples as wellas the stored samples were corrected for any control residues, prior to calculating percent recovery.
Sample Storage.
The leaf and whole flower samples were weighed and homogenized with dry ice using a Robot Coupe;
the homogenized samples were placed into labeled, plastic containers and stored in a freezer (allowing
the dry ice to sublime). After sample preparation, the homogenized leaf and whole flower samples werestored in plastic containers and placed in a freezer until they were sub-sampled for analysis. Pollen,
nectar, and extra floral nectar extracts needed no homogenization and were stored directly in a freezer.
Freezer-storage temperatures were monitored and typically were maintained at -10 to -25 °C.
For the samples in this study that were analyzed for thiamethoxam and CGA322704, the maximum
frozen storage period experienced for any matrix was 303 days (10 months), from the sampling date oftreated samples through the extraction of treated samples.
Previous storage stability studies show thiamethoxam and CGA322704 are stable in a variety of matrices
for up to 12 months. Therefore, residues of thiamethoxam and CGA322704 in cotton leaf, whole flower,
pollen, and nectar samples should not have been adversely affected by freezer storage during this study.
5E. ANALYTICAL METHODS
The reference standard information, as required by 40 CFR Part 160.185(a)(4), as well as representative
LC-MS/MS chromatograms and typical calibration curves, can be found in the Analytical Phase Reportlocated in Appendix 1 of the final study report.
Analysis of Leaves and Flower Samples
Leaf and whole flower samples were analyzed for thiamethoxam and CGA 322704 based on theanalytical method described in Syngenta Method REM 179.06, entitled “Residue Method for theDetermination of Residues of Thiamethoxam (CGA 293343) and CGA 322704 in Lettuce, Tomato, Grapeand Tobacco Samples. Final Determination by LC-MS/MS” 6. In the subject method, residues ofthiamethoxam and CGA322704 were extracted with 50:50 methanol/water from 10-g leaf and wholeflower samples using a high-speed homogenizer. Extracts were centrifuged and concentrated via SPE
cleanup in preparation for LC-MS/MS analysis. The Limit of Quantitation (LOQ) for both analytes inleaves was 5.0 ppb and the Limit of Detection (LOD) was targeted to be 2.5 ppb. The Limit of
Quantitation (LOQ) for both analytes in whole flowers was 1.0 ppb and the Limit of Detection (LOD) was
targeted to be 0.50 ppb.
Analysis of Pollen, Nectar, and Extra Floral Nectar Samples
Pollen, nectar, and extra floral nectar samples were analyzed for thiamethoxam and CGA322704 basedon the analytical method described in Syngenta Method REM 179.07, entitled “Thiamethoxam:
Analytical Method for the Determination of Residues of Thiamethoxam (CGA 293343) and CGA 322704
in Bee and Hive Products. Final Determination by LC-MS/MS” 7. In summary, residues of thiamethoxam
and CGA322704 were extracted with 50:50 methanol/water: 0.2% formic acid (aq) from 0.05 g pollen
and nectar samples. Pollen extracts were centrifuged and passed through a solid-phase extraction
cleanup in preparation for LC-MS/MS analysis. The Limit of Quantitation (LOQ) for both analytes, in1045

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49686801

THX Cotton DER

pollen, nectar, and extra floral nectar, was 1.0 ppb. The Limit of Detection (LOD) was targeted to be 0.50
ppb.
Summary of LOQs and LODs
MatrixAnalyteTotal Thiamethoxam
Total Thiamethoxam

LOQ(ppb, parent equivalents)
5.0
1.0

LOD
(ppb, parent equivalents)
2.5
0.50

LeavesWhole Flowers
Pollen, Nectar,
and Extra Floral
NectarTotal Thiamethoxam

1.0

0.50

5F. QUALITY ASSURANCE RESULTS
Validated analytical methods were provided by the Sponsor to ABC Laboratories, Inc. Prior to analysis offield samples, the analytical methods were verified by ABC Laboratories, Inc. as part of this study. The
standard (calibration) curve generated for each analytical set was used for the quantitation ofthiamethoxam and CGA322704 in the samples. For this study, the correlation coefficient (r) for eachcalibration curve was equal to or greater than 0.990 (r2 equal to or greater than 0.98).
To verify performance of the validated analytical methods at ABC Laboratories prior to analysis of fieldsamples, UTC samples of each matrix were fortified with thiamethoxam and CGA322704 at
concentrations equal to the method LOQ as well as from 5× to 50×LOQ, and analyzed according to themethods described in the “Analytical Methods” portion of this document.
For each matrix, at least one concurrent recovery sample per analytical set was prepared by fortifying an
untreated control sample with thiamethoxam and CGA322704 at concentrations equal to the method
LOQ and up to 200×LOQ (1000× LOQ for extra floral nectar), and analyzing concurrently with the treatedfield samples to demonstrate adequate method performance throughout the study, i.e. recoveries of 70
to 120%. In light of method verification recoveries that did not meet acceptance criteria, concurrent
procedural recovery samples were diluted in subsequent analyses to effect improved results.
6. RESULTS:
Year 1
Thiamethoxam residues >LOD were found in 15 of 36 UTC plant matrices and CGA322704 residues >LOD
were found in 3 of 36 UTC plant matrices. Residues greater than the LOQ were found in all plant
matrices sampled from the treated cotton plots.

1046

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49686801

THX Cotton DER

Table 5. Summary of Thiamethoxam and CGA322704 Residues in Samples from the Treated Plots of the
Foliar Application Trials Year 1 (2013)
Thiamethoxam Concentrations
CGA322704 Concentration
Soil Texture
Plant
(ppb)
(ppb)
Trial
Matrix
Site
MeanStandard Maximum
MeanStandard Maximum
Residue Deviation Residue
Residue Deviation Residue
Year 1 (Foliar Broadcast Plot)
1Fine/Medium
485
236
667
70.3
18
84.8
2
Coarse
337
99
448
70.4
26
100
3
Medium
420
108
505
50.1
19
70.0
4
Coarse
236
62
307
50.3
4.3
54.0
Leaves5
Coarse
57.3
5.2
61.6
27.4
2.5
29.2
6a
Coarse
98.7
33
126
31.6
18
49.2
7
Fine/Medium
92.1
33
130
24.7
16
42.8
8
Medium
92.7
17
108
41.0
3.4
44.8
9
Medium
162
101
278
52.3
12
65.2
1
2
3
4
5
6a
7
8
9

Fine/Medium
CoarseMediumCoarseCoarseCoarseFine/Medium
MediumMedium

1
2
3
4
5
6a
7
8
9

Fine/Medium
CoarseMediumCoarseCoarseCoarseFine/Medium
MediumMedium

1
2
3
4
5
6a
7
8

Fine/Medium
CoarseMediumCoarseCoarseCoarseFine/Medium
MediumWhole
Flowers

147
173
194
93.3
56.2
87.6
78.5
25.3
28.8

13
16
36
6.9
6.6
6.0
5.5
2.9
1.7

156
185
235
101
61.1
94.4
84.6
28.6
30.7

28.4
35.7
37.8
27.2
10.2
15.1
8.48
16.7
15.3

4.8
3.4
5.7
2.4
0.29
1.3
7.2
1.8
2.6

33.8
37.7
43.8
28.8
10.5
16.5
13.4
18.1
18.1

Pollen

3.23
2.64
5.21
1.31
4.13
24.3
7.50
2.09
1.05

0.73
1.1
2.3
0.49
2.3
19
8.3
2.9
1.4

3.79
3.78
7.72
1.78
6.78
46.1
17.0
5.43
2.66

0.507
ND
0.410
ND
0.623
1.36
0.786
1.01
ND

0.44
N/A
0.28
N/A
0.65
0.81
0.64
1.3
N/A

1.02
ND
0.729
ND
1.37
2.29
1.50
2.54
ND

Nectar

0.983
4.41
3.61
1.84
3.34
2.34
1.33
0.908

0.07
2.1
0.42
1.1
3.1
0.30
0.17
0.13

1.06
5.87
4.07
3.02
6.85
2.67
1.49
1.06

ND
ND
ND
0.37
ND
ND
0.349
0.493

N/A
N/A
N/A
0.20
N/A
N/A
0.2
0.22

ND
ND
ND
0.595
ND
ND
0.548
0.662

1047

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49686801

Trial
Site

Soil Texture

9

MediumTHX Cotton DER

Plant
MatrixThiamethoxam Concentrations
(ppb)
Mean
Standard Maximum
Residue Deviation Residue
0.623
0.10
0.740

CGA322704 Concentration
(ppb)
Mean
Standard Maximum
Residue Deviation Residue
0.362
0.19
0.585

1
Fine/Medium
127
89
228
7.26
6.2
14.3
2
Coarse
48.2
24
75.6
1.56
0.93
2.63
3
Medium
176
36
201
3.95
0.51
4.31
Extra
4
Coarse
26.7
7.7
32.2
1.37
0.20
1.60
Floral5
Coarse
33.1
9.4
42.5
1.11
0.29
1.44
a
Nectar6
Coarse
115
36
153
2.32
0.71
3.01
7
Fine/Medium
37.8
11
44.1
1.18
0.18
1.38
8
Medium
35.7
18
50.2
1.54
0.19
1.74
9
Medium
27.8
18
48.6
2.51
2.2
5.00
a
Site 6 control samples EFN 105 and NC 104, as well as treated samples EFN 110 and NC 109 aresuspected to have been mis-labeled but placed in the correct sample bags. The discrepancy between
labels and bags was discovered at the lab, and the bottles were switched. Residues confirm the samples
were probably mis-labeled and had originally been placed in the correct bags; therefore, results fromthe sample labeled as EFN Sample 105 are reported for NC Sample 104, and EFN Sample 110 arereported for NC Sample 109.
Note: For the purposes of calculations, ND samples were treated as ½ the LOD (0.25 ppb for wholeflowers, pollen and nectar; 1.25 ppb for leaves).
Table 6. Summary of Thiamethoxam and CGA322704 Residues in Samples from the Treated Plots of theSeed Treatment Trials.
Thiamethoxam Concentration
CGA322704 Concentrations
Trial
Soil
Plant
(ppb)
(ppb)
Site
TextureMatrixMeanStandard Maximum
MeanStandard Maximum
Residue Deviation Residue
Residue Deviation Residue
2aMedium
6.17
0.85
7.08
4.88
1.4
6.36
Leaves6a
Coarse
9.81
12
23.5
7.30
3.7
10.4
8a
Coarse
1.56
0.4
1.94
ND
N/A
ND
2a
6a
8a

MediumCoarseCoarse2a6a
8aMediumCoarseCoarse2a6a
8aMediumCoarseCoarseWhole
Flowers

6.38
2.07
ND

1.1
1.5
N/A

7.30
3.78
ND

2.72
0.854
ND

0.52
0.45
N/A

3.06
1.36
ND

Pollen

ND
ND
ND

N/A
N/A
N/A

ND
ND
ND

ND
ND
ND

N/A
N/A
N/A

ND
ND
ND

Nectar

0.559
0.664
0.195

0.27
0.46
0.0

0.759
1.16
0.220

0.849
ND
ND

0.17
N/A
N/A

1.04
ND
ND

1048

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49686801

Trial
Site

Soil
TextureTHX Cotton DER

Plant
MatrixThiamethoxam Concentration
(ppb)
Mean
Standard Maximum
Residue Deviation Residue

CGA322704 Concentrations
(ppb)
Mean
Standard Maximum
Residue Deviation Residue

2aMedium
Extra
0.650
0.69
1.45
ND
N/A
ND
Floral6a
Coarse
0.493
0.21
0.624
ND
N/A
ND
Nectar8a
Coarse
ND
N/A
ND
ND
N/A
ND
Note: For the purpose of calculations, ND samples were treated as ½ the LOD (0.25 ppb for the wholeflowers, pollen and nectar; 1.25 ppb for leaves).
Year 2
Thiamethoxam residues >LOD were found in 18 of 60 untreated control samples and CGA322704
residues >LOD were found in 5 of 60 untreated control samples. Residues greater than the LOQ were
found in all plant matrices sampled from the treated cotton plots .
Table 6. Summary of Thiamethoxam and CGA322704 Residues in Samples from the Treated Plots of the
Foliar Application Trials Year 2 (2014).
Trial
Site

Soil Texture

1
2
3
4
5
6
7
8
9

Fine/Medium
CoarseMediumCoarseCoarseCoarseFine/Medium
MediumMedium

1
2
3
4
5
6
7
8
9

Fine/Medium
CoarseMediumCoarseCoarseCoarseFine/Medium
MediumMediumPlant
MatrixLeavesWhole
Flowers

Thiamethoxam Concentrations
(ppb)
Mean
Standard Maximum
Residue Deviation Residue
Year 2 (Foliar Broadcast Plot)
ND
N/S
ND
173
31
195
22.1
2.1
23.7
73.6
3.4
77.3
29.1
16
40.8
24.9
3.4
26.9
113
20
136
129
60
186
51.1
3.1
53.9
19.0
89.1
32.3
24.8
46.7
16.6
69.6
58.6
14.6

4.3
27
4.8
5.2
4.3
2.6
9.9
3.7
3.4
1049

24.0
119
36.8
28.1
50.0
19.2
80.6
62.5
18.5

CGA322704 Concentration
(ppb)
Mean
Standard Maximum
Residue Deviation Residue
ND
21.7
9.76
26.7
11.0
13.0
13.8
9.40
7.49

N/A
2.4
2.6
0.0
0.82
2.5
3.3
2.7
1.4

ND
24.1
11.8
26.7
11.9
15.9
17.6
11.0
8.93

5.38
18.2
9.63
6.13
11.6
4.30
12.5
11.8
3.30

0.74
7.6
1.5
1.0
0.55
0.56
1.1
0.55
0.65

6.22
26.0
10.9
6.84
12.0
4.94
13.7
12.3
4.03

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49686801

THX Cotton DER

Thiamethoxam Concentrations
(ppb)
Mean
Standard Maximum
Residue Deviation Residue

CGA322704 Concentration
(ppb)
Mean
Standard Maximum
Residue Deviation Residue

Trial
Site

Soil Texture

Plant
Matrix

1
2
3
4
5
6
7
8
9

Fine/Medium
CoarseMediumCoarseCoarseCoarseFine/Medium
MediumMediumPollen

1a
2
3
4
5
6
7
8
9

Fine/Medium Nectar------Coarse
20.9
22
46.2
0.560
0.27
0.774
Medium
1.38
0.16
1.49
1.35
0.29
1.53
Coarse
3.32
1.2
4.70
0.537
0.26
0.746
Coarse
2.22
1.5
3.94
0.460
0.36
0.880
Coarse
1.80
1.4
2.89
0.492
0.22
0.680
Fine/Medium
1.70
0.61
2.22
0.435
0.16
0.540
Medium
3.99
2.1
6.47
0.642
0.16
0.832
NectarMedium
1.05
0.36
1.28
ND
N/A
ND
a
No sample available for analysis.
Note: For the purpose of calculations, ND samples were treated as ½ the LD (0.25 ppb for wholeflowers, pollen and nectar/extra floral nectar; 1.25 ppb for leaves).

ND
5.61
55.0
69.0
1.19
57.5
205
51.2
4.84

N/A
2.7
21
47
0.55
41
130
78
5.2

1050

ND
8.64
79.2
122
1.76
96.4
351
141
10.9

ND
ND
3.74
6.18
ND
3.64
11.4
3.50
0.421

N/A
N/A
1.5
3.5
N/A
2.5
4.6
5.6
0.3

ND
ND
5.24
10.2
ND
6.06
15.4
10.0
0.762

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49686801

Trial
Site

1

2

3

4

5
6

7

8
9

THX Cotton DER

Table 7. Summary of Thiamethoxam and CGA322704 Residues in Extra Floral Nectar Samples
from the Treated Plots of the Foliar Application Trials Year 2 (2014)
Thiamethoxam Concentration
CGA322704 Concentration
Soil Texture
Sample(ppb)(ppb)
TimingMeanStandard Maximum Mean
Standard Maximum
Residue Deviation Residue Residue Deviation Residue
Extra Floral Nectar
Fine/Medium 5DA1A
197
94
303
2.01
1.0
3.16
5DA2Aa------Bloom
24.6
4.7
29.3
0.612
0.32
0.864
24DA2A
0.593
0.59
1.28
ND
N/A
ND
Coarse
5DA1A
68.6
62
122
1.03
0.68
1.53
5DA2A
35.3
40
81.5
0.824
0.68
1.58
Bloom
49.4
64
123
1.73
1.0
2.90
a
24DA2A------Medium
5DA1A
239
71
288
3.59
0.96
4.48
5DA2A
542
197
268
13.1
4.3
18.0
Bloom
39.8
7.8
45.1
1.71
0.33
1.90
24DA2A
4.24
0.96
5.06
0.337
0.15
0.512
Coarse
5DA1A
71.9
10
83.4
1.42
0.32
1.73
5DA2A
47.2
4.6
52.5
1.17
0.25
1.42
Bloom
104
65
178
5.04
2.9
8.40
24DA2A
1.38
0.74
2.23
ND
N/A
ND
Coarse
Bloom
50.5
14
66.3
1.78
0.60
2.47
Coarse
5DA1A
29.4
9.8
40.7
0.735
0.48
1.21
5DA2A
50.4
23
67.2
1.37
0.33
1.63
Bloom
6.02
4.5
10.7
ND
N/A
ND
24DA2A
0.588
0.02
0.612
ND
N/A
ND
Fine/Medium 5DA1A
112
37
154
1.21
0.20
1.43
5DA2A
186
83
253
2.20
0.87
2.99
Bloom
57.6
16
76.5
1.49
0.29
1.82
24DA2A
0.626
0.34
0.922
ND
N/A
ND
Medium
Bloom
33.6
18
54.3
1.14
0.55
1.74
Medium
Bloom
9.75
1.6
10.7
0.360
0.19
0.581
a
No sample available for analysis.
Note: For the purpose of calculations, ND samples were treated as ½ the LOD (0.25 ppb).

7. Statistical Analysis

Study Objectives and Design
The main objective of the study was to determine the concentration of thiamethoxam and its
degradation product CGA322704 in whole flowers, nectar, extra floral nectar, pollen, and leaves ofcotton plants in response to foliar applications of a thiamethoxam pesticide product. Additional test
plots were included in year 1 to measure concentrations of thiamethoxam residues in plant matrices in1051

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 49686801

THX Cotton DER

response to a seed-treatment application. For the foliar spray study, the rate of application of Centric
40WG was 0.063 lbs a.i./Acre. Applications were made twice at 5 day intervals with the second
application scheduled to occur 12 days before significant bloom. For the seed treatment study, treated
seed at approximately 0.375 mg a.i./seed were planted at 3 of the trial sites in year 1 of the study. The
foliar application portion of the study was replicated in the next year whereas the seed treatment
portion was discontinued. Sampling of plant matrices were targeted for 12 days after the second foliar
application, which was denoted as an early bloom period where blooms were at 50 to 70%. In order toprovide enough sample for analysis, sampling commenced a few days later at two sites in year 1. In year
2, extra floral nectar was sampled at additional time intervals to characterize concentration ofthiamethoxam residues over time. Additional extra floral nectar samples were taken at 5 days after the
first foliar application, at 5 days after the second foliar application, and finally at 24 days after thesecond foliar application. An amended study report was submitted to update and correct various
aspects of the previous study report. A complete list of changes is published on pages 59 through 65 of
the amended study report. Data used for this analysis were obtained from pages 128 through 147 of theamended study report.
Non-parametric statistical tests were used to test for differences in distribution of concentrations
between years, untreated control to treated plants, extra floral nectar concentration between samplingintervals, and between soil type. Non-parametric tests do not require tests for normality as they arerobust to differences in distribution and they are also robust for experimental designs with low
replicates (Helsel and Hirsch, 2002). The PROC NPAR1WAY procedure in the Statistical Analysis System(SAS) statistical package was used to conduct Wilcoxon-Mann –Whitney (Wilcoxon), Median nonparametric, and Kuiper tests. A significant result from the Wilcoxon test indicates differences in the
shape of distributions; A significant result from the Median test indicates differences in the location of
the medians between distributions; and A significant result from the Kuiper test indicates differences inthe empirical distributions between two groups. The Exact option for each statistic was implemented as
it provides permutation testing, a statistical method that minimizes the effect of sample size anddistributional differences. Using the Exact option, the Monte Carlo procedure was also implemented,
which provided 10,000 separate runs for each statistic to produce the permutation distributions. The
test for potential differences in extra floral nectar concentrations over time had 4 levels so the DSCF
option in PROC NPAR1WAY, which invokes the Dwass, Steel, Critchlow-Fligner multiple comparison test,
was used to provide pairwise tests for two-sample rankings. Additional procedures used for descriptive
statistics were PROC MEANS to calculate mean values from the replicates at each site, PROC CAPACITY
to produce cumulative statistics, and PROC BOXPLOT to produce comparative graphics. Statisticalanalysis for effects and soil type were conducted on the replicate sample mean taken from each site.
Due to limited site and year data, all replicate data was used to compare seed to foliar treatments (year
1 data) and to compare concentrations of extra floral nectar between sampling intervals (year 2 data).
Some graphical comparisons are presented with data transformed to a natural logarithm scale,providing clearer contrasts between the distributions. Although both limits of detection (LOD) andquantification (LOQ) were indicated, only data less than the LOD were indicated as ND in the data set.
Values were provided between the LOD and LOQ. For statistical analyses, values noted as below thelimit of detection (LOD) were assigned half the value of the respective detection limit (Table 5). Values
between the LOD and LOQ were used as reported. The distribution of concentrations in bee relevant
plant matrices were calculated using all the raw data because these values represent the actual range of
exposure to bees and other organisms that feed off the nectar and pollen of plants.

1052

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49686801

THX Cotton DER

Detection rate noted for each plant matrix: Counts for the number of samples reported below therespective detection limit for each matrix are presented in Table 8A for treated plants and Table 8B for
untreated control plants. For plants treated with a foliar spray, the majority of concentrations forthiamethoxam and CGA322704 metabolite were above the LOQ in leaf and flowers sampled at bloom;
Percent of values above the LOQ ranged from 94% to 100% of respective sample sizes. For nectar andpollen samples, the number of samples above the LOQ was greater for thiamethoxam than for the
CGA322704 metabolite. For nectar, 80% of thiamethoxam values were above the LOQ, whereas only 6%of CGA322704 concentrations were above the LOQ: Values for pollen were 83% and 33%, respectively.
The majority of concentrations in extra floral nectar samples were again above the LOQ for samples
taken before and during bloom. The pattern observed for samples obtained at 24 days after the 2nd
foliar application indicated declining concentrations as the percentage of thiamethoxam samples above
the LOQ decreased from 100% to 40% of samples and none of the samples were above the LOQ for
CGA322704.
The distribution of values in seed treated plants indicated much lower proportions of concentrations
measured above the LOQ with similar distributions for both parent and metabolite. In leaf and flower
samples, the percentage of samples above the LOQ ranged from 33% to 44%. For nectar, pollen, andextra floral nectar samples, the range was from 0% to 13% of concentrations above the LOQ (Table 8A).
Although the majority of concentrations measured in plant matrices in untreated control plants werereported below the LOD, concentrations were reported above the LOQ, especially for thiamethoxam
concentration in pollen samples and in extra floral nectar samples taken at the first sampling interval, 5days after the 1st foliar application (Table 8b).
Comparison of distribution between years: Potential difference between years was measured toindicate the presence of carry-over effects of residues. Results for analyses of thiamethoxam
concentrations for foliar treated plants were mostly non-significant (Table 9). Two significant Wilcoxon
tests were indicated for leaves and flowers but the graphic shows that the values were potentiallygreater in year 1 than in year 2 (Figure 2). This pattern implies no potential for carry-over of residues
due to foliar sprays.
Comparison of distribution between untreated and treated plants: The distribution statistics for all
treatments are presented for nectar, pollen, and extra floral nectar in Table 10 and for leaves and wholeflowers in Table 11. Non-parametric tests conducted on the replicate sample mean for foliar treatedplots indicated a significantly greater range in the distribution for foliar treated plants compared tountreated control plants for all matrices (Table 12; Figures 3 and 4). The exception was CGA322704
concentrations in pollen where detection of residues due to treatment were minimal.
Residue concentrations in seed treated plants were low and essentially similar to the range measured in
untreated control plants with the exception of leaf and whole flower matrices where concentrations
were slightly greater in seed treated plants (Table 12; Figures 5 and 6). Although statistical tests forthiamethoxam in flower samples were not significant, the graphical comparison indicated an elevatedrange in concentrations for seed treated plants.

1053

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49686801

THX Cotton DER

Since concentrations were low in seed treated plants, comparison of concentrations to foliar treatedplants indicated a higher range in residues in most matrices in plants treated with foliar sprays (Table
12; Figures 7 and 8). The results for CGA322704 residues in nectar and pollen indicated no differences
due to minimal detection of residues in treated plants.
Comparison of distribution between soil types: Based on the soil characteristics provided in the study
report, the sites were distributed between coarse and medium textured soil types: No sites were in afine, clayey soil type. Sites C-1, C-3, C-7, and C-9 were classified as medium textured soil and sites C-2,
C-4, C-5, C-6 and C-8 were classified as coarse-textured soil. Results of non-parametric tests show no
significant differences in the distributions in the plant matrices between the two soil types (Table 13;
Figures 9 and 10). There was an indication of a significant difference in thiamethoxam concentrations
for nectar but the values in nectar were minimal and the effect is most likely circumspect.
Concentration in extra floral nectar sampled over time: Additional extra floral nectar samples weretaken to determine concentration over time. This aspect of the study was only conducted in year 2 soanalyses were based on the replicate samples obtained with each plot. Distributional statistics for the
sampling intervals are presented in Table 14. Concentrations at the final sampling date wereapproaching background levels measured in untreated control plants so each contrast to concentrations
at the 3 previous sampling dates were significant (Table 15; Figures 11 and 12). Additional differenceswere shown for thiamethoxam residues where concentrations at the first 2 sampling dates were similar
but both higher in range in concentration than for those at bloom.
Data for bee relevant matrices: The observed distributions derived from the individual analyses
ostensibly determines the expected range in concentrations of thiamethoxam and CGA322704 residues
in bee relevant plant samples for the studies combination of plant species and application scenario(Table 10). The median and maximum values for total residue in nectar were 2.4 and 47 ng/g,
respectively, on a wet weight basis. For pollen, median and maximum values were 4 and 366 ng/g,
respectively. For extra floral nectar median and maximum values at bloom were 43 and 242 ng/g,
respectively. Additional sampling of extra floral nectar after each application indicated that potential
exposure occurs prior to bloom where median and maximum concentrations after the first application
were 92 and 306 ng/g and after the second foliar treatment were 69 and 786 ng/g, respectively.
8. Conclusions
1. Utility of the data: The study followed the design as directed in the data call-in with the study being
replicated in two years at 9 sites. Given the limitations of finding experimental sites in existing fields, the9 sites were reasonable representatives of only 2 of the 3 soil types requested in the data call-in.
2. Concentrations in Bee Relevant Matrices: By default, the distributions reported in Table 10, under
the Foliar Treated heading, represent the expected concentrations in bee relevant matrices that result
from two foliar applications of thiamethoxam to cotton plants where the last application was 12 days
prior to bloom. Median and maximum values for total thiamethoxam residues in plant matrices taken atbloom for nectar were 2.4 and 47 ng/g, for pollen were 4 and 366 ng/g and for extra floral nectar were
43 and 242 ng/g, respectively.
3. Extended Exposure from Extra Floral Nectar: Samples of extra floral nectar were obtained prior tobloom after the second application. Concentrations of residues were higher than when measured atbloom, indicating potential exposure prior to bloom.
1054

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49686801

THX Cotton DER

4. No carry-over effect between years: Concentrations measured in plant matrices between the twoyears of the study were similar, indicating low potential for carry-over effects due to foliar applications
at the concentrations and timing used in this study.
5. No Effect of Soil Type: Data for 2 of the 3 soil types were available, coarse and medium textured soils,
for this study. There were no differences in the range of either thiamethoxam or CGA322704 metabolite
residues between the two soil types.
6. Comparison to Seed Treated Plants: In the first year of the study, a few plots were added to compare
concentrations in plant matrices that result from a seed treatment application. Values measured atbloom indicated low concentrations in bee relevant matrices in seed treatments.
9. STUDY STRENGTHS, LIMITATIONS AND CONCLUSIONSIn the context of documenting the magnitude of thiamethoxam residues in bee-related matrices ofcotton plants, the following strengths are observed with this study.
1. The study provided quantitative values for cotton plant matrices exposed to foliar applicationof thiamethoxam.
2. The study was replicated over two years with measurements in plant samples taken at bloom
after two foliar applications of thiamethoxam to cotton plants. Blooms were sampled
approximately 12 days after the second application.
3. The 9 sites were reasonably replicated over the 2 of the requested 3 soil texture categories.
Limitations noted in this study include:
1. Additional aspects of the study such as seed treatment effects were lacking in replication withrespect to the foliar application.
Overall, considering the strengths and limitations of this study, the following conclusions can be drawn:
1. Bee-relevant matrices: Thiamethoxam residues were measured in nectar, pollen, and extra floral
nectar plant matrices sampled 12 days after a second foliar application to cotton plants. Values in Table
10, under the Foliar Treated heading, indicate the potential range in concentrations that bees are
exposed to in the field.
2. Potential for Extended Exposure to Extra Floral Nectar: Additional samples of extra floral nectar
taken after each application indicate potential for significant exposure prior to the blooming period
through foraging on extra floral nectar. The range in concentrations after an application is greater thanwhen sampled at bloom.
3. No carry-over effect of years: Concentrations measured in plant matrices were similar between the
two years of the study indicating low potential for carry-over effects due to foliar treatments at theconcentration and timing of application used in this study.

1055

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49686801

THX Cotton DER

4. Effect of soil type: No differences were measured in the range of residue concentrations in plant
matrices between plants grown in coarse or medium textured soils.
5. Effect of Seed Treatment: Although the number of replications were low for seed treatments, thedata indicate lower concentrations than from foliar applications. Concentrations in plant matrices fromseed treatment were similar to the range measured in untreated control plants.
10. STUDY VALIDITY/CLASSIFICATION
The data from this study provide an expected distribution of the thiamethoxam residue concentrations
that bees are exposed to in nectar, pollen, and extra floral nectar in cotton plants under actual
agronomic practices in California. Relating concentrations measured in flower parts to bee health ispossible by comparing the concentrations measured in bee relevant plant parts to target values thatdefine acute or chronic exposure scenarios. The study is considered scientifically sound and useful forrisk assessment purposes. The study is classified as ACCEPTABLE for quantitative use in risk assessment.

1056

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49686801

THX Cotton DER

11. REFERENCES
1. Mair, P. (1998) Stability of Residues of CGA-293343 (2 Years Final Report) and CGA-322704 (1Year Interim Report) in Plant Material under Deep Freezer Conditions, Including Method
Validation (Study No. 504/96 consists of Reports #112/96, 127/97, 103/98) MRID 44703525.
2. Hohl, J. (1999) Stability of Residues of CGA-322704 in Plant Material and Soil Stored Under Deep
Freezer Conditions (Study No. 779-00) MRID 45108001.
3. Oakes, T. (2002) Stability of CGA-293343 and CGA-322704 in Crops and Processed Fractions
Under Freezer Storage Conditions (Study No. 269-98) MRID 45659205.
4. Hampton, R. (2012) “Thiamethoxam 75 SG (A9549C) - Magnitude of the Residues in Leaves,
Flowers, Pollen, and Nectar of Cucumbers, Representative Commodity of Cucurbit Vegetables,
EPA Crop Group 9, in California” (Report Number: TK0024668).
5. Anderson, L. (2007) Thiamethoxam (CGA293343) and CGA322704. Validation of Residue
Analytical Method REM 179.07 for the determination of Residues in Bee and Hive Products andStorage Stability in Hive Pollen, Wax and Nectar, stored Deep Frozen for 12 months. (Study No.
05-S508).
6. Crook, S (2004) Residue Method for the Determination of Residues of Thiamethoxam(CGA293343) and CGA322704 in Lettuce, Tomato, Grape and Tobacco Samples. Final
Determination by LC/MS/MS" (Syngenta Method REM179.06).
7. Crook, S (2007) Analytical Method for the Determination of Residues of Thiamethoxam(CGA293343) and CGA322704 in Bee and Hive Products. Final Determination by LC-MS/MS(Syngenta Method REM179.07).
8. Mohan, P. and Khadi, B.M. (2007) Nectar Glands in Gossypium. CICR Technical Bulletin No: 37.
Central Institute of Cotton Research Nagpur.

1057

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49686801

THX Cotton DER

Table 8A. Treated Plants: Counts of chemical analytical results for thiamethoxam and CGA322704 thatwere indicated as above the LOQ, between the LOQ and LOD, and below the LOD.

Plant Sample
Foliar Application
Leaf
FlowerNectarPollenExtra Floral Nectar
5 DA1App
5 DA2AppBloom
24 DA2App
Seed Application
Leaf
FlowerNectarPollenExtra Floral Nectar

Treated Plants: Comparison of Total Number of Samples Reported Above theLOQ, Between the LOQ and LOD, and Below the LOD
Thiamethoxam
CGA322704
Total
Number Number Number
Total
Number Number Number
Number >LOQ<LOQ
<LOD Number >LOQ
<LOQ
<LOD
54
54
50
54

51
54
40
45

0
0
9
3

3
0
1
6

54
54
50
54

51
53
3
18

0
0
17
5

3
1
30
31

18
15
53
15

18
15
53
6

0
0
0
6

0
0
0
3

18
15
53
15

15
11
41
0

1
3
6
1

2
1
6
14

9
9
8
9
9

4
3
1
0
1

1
0
3
0
2

4
6
4
9
6

9
9
8
9
9

3
4
1
0
0

3
2
2
0
0

3
3
5
9
9

1058

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 49686801

THX Cotton DER

Table 8B. Untreated Control Plants: Counts of chemical analytical results for thiamethoxam and
CGA322704 that were indicated as above the LOQ, between the LOQ and LOD, and below the LOD.

Plant Sample
Leaf
FlowerNectarPollenExtra Floral Nectar
5 DA1App
5 DA2AppBloom
24 DA2App

Untreated Control Plants: Comparison of Total Number of Samples Reported
Above the LOQ, Between the LOQ and LOD, and Below the LOD
Thiamethoxam
CGA322704
Total
Number Number Number
Total
Number Number Number
Number >LOQ<LOQ
<LOD Number >LOQ
<LOQ
<LOD
18
18
16
17

1
3
3
4

0
1
1
5

17
14
12
8

18
18
16
17

0
0
0
2

1
1
0
3

17
17
16
12

6
5
16
5

4
1
3
1

1
1
2
0

1
3
11
4

6
5
16
5

0
0
2
0

0
0
1
0

6
5
13
5

1059

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 49686801

THX Cotton DER

Table 9. Statistical results for test of differences in concentrations of thiamethoxam or
CGS322704 metabolite residues measured between years 1 and 2.

SourceLeaf
FlowerNectarPollenExtra Floral Nectar

Nonparametric Test Exact
Probability Levels: Effect of Year
Thiamethoxam
Wilcoxon Median
Kuiper
0.02
0.34
0.57
0.02
0.35
0.57
0.54
1
0.95
0.11
0.34
0.26
0.49
1
0.89

1060

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49686801

THX Cotton DER

Table 10. Distributional statistics for concentrations of thiamethoxam (Parent) andCGA322704 (Metabolite) measured in nectar, pollen, and extra floral nectar sampled from
untreated control, foliar treated or seed treated cotton plants.

Distribution of Thiamethoxam Residue Concentration at Bloom
NectarPollenExtra Floral Nectar
Statistic

Parent

Metab

Total

Parent

Metab

Total

Parent

Metab

Total

Untreated Control Plants
N (#)
16
Mean (ng/g)
1.2
SD (ng/g)
2.7
CV (%)
231
Min (ng/g)
0.3
Median (ng/g)
0.3
75th (ng/g)
0.4
90th (ng/g)
2.0
95th (ng/g)
10.9
Max (ng/g)
10.9% of Total
85.7

16
0.3
0.0
0
0.3
0.3
0.3
0.3
0.3
0.3
17.9

16
1.4
2.7
190
0.5
0.5
0.7
2.3
11.2
11.2

17
1.1
1.5
137
0.3
0.7
0.8
4.6
5.2
5.2
68.8

17
0.5
0.5
95
0.3
0.3
0.6
1.3
1.9
1.9
31.3

17
1.6
1.7
107
0.5
0.9
1.5
4.8
5.9
5.9

16
1.7
3.5
208
0.3
0.3
0.7
8.8
12.3
12.3
63.0

16
1.0
2.4
237
0.3
0.3
0.3
2.6
9.6
9.6
37.0

16
2.7
4.5
168
0.5
0.5
1.5
10.4
14.9
14.9

50
3.3
6.5
201
0.3
1.7
2.9
5.3
6.9
46.2
89.2

50
0.4
0.3
69
0.3
0.3
0.6
0.8
1.0
1.5
10.8

50
3.7
6.6
178
0.8
2.4
3.4
5.8
7.3
47.0

54
27.8
58.7
211
0.3
3.8
17.0
96.4
141.0
351.0
93.3

54
1.9
3.3
170
0.3
0.3
2.2
6.1
10.2
15.4
6.4

54
29.8
61.9
208
0.5
4.3
18.5
102.5
151.0
366.4

53
53.4
50.0
94
1.8
41.1
59.3
123.0
178.0
228.0
96.4

53
2.0
2.3
112
0.3
1.4
2.4
3.8
5.0
14.3
3.6

53
55.4
51.8
94
2.1
42.8
61.9
125.9
186.4
242.3

8
0.5
0.3
64
0.3
0.4
0.7
1.2
1.2
1.2
50.0

8
0.5
0.3
68
0.3
0.3
0.8
1.0
1.0
1.0
50.0

8
1.0
0.5
51
0.5
0.9
1.4
1.8
1.8
1.8

9
0.3
0.0
0
0.3
0.3
0.3
0.3
0.3
0.3
50.0

9
0.3
0.0
0
0.3
0.3
0.3
0.3
0.3
0.3
50.0

9
0.5
0.0
0
0.5
0.5
0.5
0.5
0.5
0.5

9
0.5
0.4
87
0.3
0.3
0.6
1.5
1.5
1.5
71.4

9
0.3
0.0
0
0.3
0.3
0.3
0.3
0.3
0.3
35.7

9
0.7
0.4
56
0.5
0.5
0.9
1.7
1.7
1.7

Foliar TreatedN (#)
Mean (ng/g)
SD (ng/g)
CV (%)Min (ng/g)
Median (ng/g)
75th (ng/g)
90th (ng/g)
95th (ng/g)
Max (ng/g)% of Total
Seed TreatedN (#)
Mean (ng/g)
SD (ng/g)
CV (%)Min (ng/g)
Median (ng/g)
75th (ng/g)
90th (ng/g)
95th (ng/g)
Max (ng/g)% of Total

1061

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49686801

THX Cotton DER

Table 11. Distributional statistics for concentrations of thiamethoxam (Parent) andCGA322704 (Metabolite) measured in leaves and flowers sampled from untreated control,
foliar treated or seed treated cotton plants.

Distribution of Thiamethoxam Residue Concentration at Bloom
Whole Flower
LeavesStatistic

Parent

Metab

Total

Parent

Metab

Total

18
1.5
1.1
73
1.3
1.3
1.3
1.3
5.9
5.9
53.6

18
1.3
0.4
26
1.3
1.3
1.3
1.3
2.7
2.7
46.4

18
2.8
1.4
51
2.5
2.5
2.5
2.5
8.7
8.7

18
0.6
0.7
120
0.3
0.3
0.3
1.8
2.7
2.7
66.7

18
0.3
0.1
40
0.3
0.3
0.3
0.3
0.7
0.7
33.3

18
0.9
0.7
81
0.5
0.5
1.0
2.0
2.9
2.9

54
144.4
150.0
104
1.3
94.3
190.0
307.0
505.0
667.0
83.0

54
29.6
23.0
78
1.3
24.4
45.6
56.4
76.4
100.0
17.0

54
173.9
169.0
97
2.5
116.3
219.1
361.8
553.0
743.4

54
69.7
54.0
78
12.3
58.4
87.7
156.0
180.0
235.0
81.9

54
15.4
10.5
68
0.3
12.2
18.1
32.5
37.6
43.8
18.1

54
85.1
63.7
75
15.1
69.8
110.9
189.8
217.1
278.8

9
5.7
7.1
123
1.3
4.7
6.0
23.5
23.5
23.5
55.9

9
4.5
3.3
73
1.3
3.7
6.4
10.4
10.4
10.4
44.1

9
10.2
9.7
95
2.5
9.1
11.6
33.9
33.9
33.9

9
2.9
2.9
99
0.3
1.3
5.1
7.3
7.3
7.3
69.0

9
1.3
1.2
91
0.3
0.7
2.1
3.1
3.1
3.1
31.0

9
4.2
4.0
97
0.5
1.9
7.3
10.3
10.3
10.3

Untreated Control Plants

N (#)
Mean (ng/g)
SD (ng/g)
CV (%)Min (ng/g)
Median (ng/g)
75th (ng/g)
90th (ng/g)
95th (ng/g)
Max (ng/g)% of Total
Foliar TreatedN (#)
Mean (ng/g)
SD (ng/g)
CV (%)Min (ng/g)
Median (ng/g)
75th (ng/g)
90th (ng/g)
95th (ng/g)
Max (ng/g)% of Total
Seed TreatedN (#)
Mean (ng/g)
SD (ng/g)
CV (%)Min (ng/g)
Median (ng/g)
75th (ng/g)
90th (ng/g)
95th (ng/g)
Max (ng/g)% of Total

1062

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 49686801

THX Cotton DER

Table 12. Statistical results for test of differences in concentrations of thiamethoxam or
CGA322704 metabolite measured between untreated control plants and plants treated with
foliar sprays; between untreated control plants and seed treated plants; and between foliar
treated and seed treated plants.

SourceFoliar Treated vs UTC
Leaf
FlowerNectarPollenExtra Floral Nectar

Nonparametric Test Exact Probability Levels
Thiamethoxam
CGA322704
Wilcoxon Median Kuiper Wilcoxon Median Kuiper
0.001
0.001
0.001
0.001
0.001

0.001
0.001
0.001
0.001
0.001

0.001
0.001
0.003
0.001
0.001

0.001
0.001
0.001
0.03
0.001

0.001
0.001
0.001
0.04
0.001

0.001
0.001
0.03
0.68
0.001

Seed Treated vs UTC
Leaf
FlowerNectarPollenExtra Floral Nectar

0.03
0.18
0.48
0.08
0.15

0.03
0.36
0.61
0.08
0.61

0.57
0.89
0.99
0.99
1

0.01
0.02
0.2
0.21
0.17

0.008
0.05
0.2
0.21
0.17

0.26
0.57
0.99
1
1

Foliar vs Seed Treatments
Leaf
FlowerNectarPollenExtra Floral Nectar

0.001
0.001
0.001
0.001
0.001

0.001
0.001
0.04
0.001
0.001

0.001
0.001
0.02
0.001
0.001

0.001
0.001
0.16
0.07
0.001

0.001
0.001
0.35
0.08
0.001

0.001
0.001
0.9
0.95
0.001

1063

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 49686801

THX Cotton DER

Table 13. Statistical results for test of differences in concentrations of thiamethoxam or
CGS322704 metabolite residues measured between plants grown in coarse or medium
textured soils.

SourceLeaf
FlowerNectarPollenExtra Floral Nectar

Nonparametric Test Exact Probability Levels: Effect of Soil Type
Thiamethoxam
CGA322704
Wilcoxon Median Kuiper Wilcoxon Median
Kuiper
0.96
1
0.75
0.63
0.63
0.88
0.9
1
0.99
0.68
1
0.88
0.03
0.05
0.15
0.31
0.34
0.51
0.9
1
0.96
1
0.65
0.96
0.9
1
0.93
0.83
1
0.93

1064

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 49686801

THX Cotton DER

Table 14. Distributional statistics for concentrations of thiamethoxam (Parent) and CGA322704 (Metabolite) measured in extra
floral nectar sampled over time from untreated control or foliar treated cotton plants.

Distribution of Thiamethoxam Concentration in Extra Floral Nectar Over Time
Bloom
5 Days After 1st App
5 Days After 2nd App
24 Days After 2nd App
Statistic

ParentUntreated Control Plants
N (#)
6
Mean (ng/g)
3.0
SD (ng/g)
2.9
CV (%)
96
Min (ng/g)
0.3
Median (ng/g)
1.9
75th (ng/g)
6.1
90th (ng/g)
7.1
95th (ng/g)
7.1
Max (ng/g)
7.1% of Total
90.9

Metab

Total

Parent

Metab

Total

Parent

Metab

Total

Parent

Metab

Total

6
0.3
0.0
0
0.3
0.3
0.3
0.3
0.3
0.3
7.6

6
3.3
2.9
88
0.5
2.1
6.3
7.4
7.4
7.4

5
0.5
0.4
81
0.3
0.3
0.6
1.2
1.2
1.2
62.5

5
0.3
0.0
0
0.3
0.3
0.3
0.3
0.3
0.3
31.3

5
0.8
0.4
55
0.5
0.5
0.9
1.5
1.5
1.5

16
1.7
3.5
208
0.3
0.3
0.7
8.8
12.3
12.3
63.0

16
1.0
2.4
237
0.3
0.3
0.3
2.6
9.6
9.6
37.0

16
2.7
4.5
168
0.5
0.5
1.5
10.4
14.9
14.9

5
1.0
1.7
169
0.3
0.3
0.3
4.1
4.1
4.1
76.9

5
0.3
0.0
0
0.3
0.3
0.3
0.3
0.3
0.3
19.2

5
1.3
1.7
136
0.5
0.5
0.5
4.4
4.4
4.4

18
1.7
1.1
68
0.3
1.4
1.7
3.7
4.5
4.5
1.4

18
121.3
91.3
75
1.4
91.8
160.6
292.5
306.2
306.2

15
172.2
216.0
125
9.0
67.2
253.0
444.0
768.0
768.0
97.9

15
3.7
5.1
138
0.3
1.5
3.0
11.1
18.0
18.0
2.1

15
175.9
221.0
126
9.7
68.7
256.0
454.1
786.0
786.0

53
53.4
50.0
94
1.8
41.1
59.3
123.0
178.0
228.0
96.4

53
2.0
2.3
112
0.3
1.4
2.4
3.8
5.0
14.3
3.6

53
55.4
51.8
94
2.1
42.8
61.9
125.9
186.4
242.3

15
1.5
1.6
104
0.3
0.9
2.2
4.5
5.1
5.1
83.3

15
0.3
0.1
25
0.3
0.3
0.3
0.3
0.5
0.5
16.7

15
1.8
1.6
91
0.5
1.1
2.5
5.0
5.3
5.3

Foliar TreatedN (#)
Mean (ng/g)
SD (ng/g)
CV (%)Min (ng/g)
Median (ng/g)
75th (ng/g)
90th (ng/g)
95th (ng/g)
Max (ng/g)% of Total

18
119.7
90.3
75
1.1
90.7
158.0
288.0
303.0
303.0
98.7

1065

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 49686801

THX Cotton DER

Table 15. Extra floral nectar concentration over time: Exact probability levels for nonparametric test for changes in concentration over time.

Treatment,
Plant Matrix, andSpecific Soil Contrasts
Treated Plants
Overall EffectExact Probability Levels for Nonparametric Tests of Differences Over Time
Thiamethoxam
CGA322704
Wilcoxon
Wilcoxon
0.001

App 1 vs. App 2

0.99
0.004
0.004
0.001
0.001
0.001

App 1 vs. Bloom
App 2 vs. Bloom
Final vs. App 1
Final vs. App 2
Final vs. Bloom

Untreated Plants
Overall Effect

0.001

0.042

App 1 vs. App 2

1.000
0.20
0.11
0.74
0.31
0.98
0.99

App 1 vs. Bloom
App 2 vs. Bloom
Final vs. App 1
Final vs. App 2
Final vs. Bloom

1066

0.93
0.88
0.54
0.001
0.001
0.001

1.00
0.85
0.88
1.00
1.00
0.88

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 49686801

THX Cotton DER

Figure 1. Explanation of statistical meaning of the Box-and-Whisker plots.

Maximum Observation

75% Quartile
Mean ValueMedian Value (50%)
25% Quartile
Minimum Observation

1067

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 49686801

THX Cotton DER

Figure 2. Comparison between years: Distribution of concentrations of thiamethoxam
residues measured in plant matrices sampled at bloom compared between Year 1 and Year 2of the study. Values were transposed to natural logarithms.

1068

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 49686801

THX Cotton DER

Figure 3. Foliar treated plants compared to untreated controls: Distribution ofconcentrations of thiamethoxam residues measured in plant matrices sampled at bloom.
Concentrations in foliar treated plants (FTRT) are compared to untreated control plants(UTC). Values were transposed to natural logarithms.

1069

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 49686801

THX Cotton DER

Figure 4. Foliar treated plants compared to untreated controls: Distribution ofconcentrations of CGA322704 metabolite residues measured in plant matrices sampled atbloom. Concentrations in foliar treated plants (FTRT) are compared to untreated control
plants (UTC). Values were transposed to natural logarithms.

1070

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 49686801

THX Cotton DER

Figure 5. Soil treated plants compared to untreated controls: Distribution of concentrationsof thiamethoxam residues measured in plant matrices sampled at bloom. Concentrations inseed treated plants (STRT) are compared to untreated control plants (UTC). Values weretransposed to natural logarithms.

1071

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 49686801

THX Cotton DER

Figure 6. Soil treated plants compared to untreated controls: Distribution of concentrationsof CGA322704 residues measured in plant matrices sampled at bloom. Concentrations inseed treated plants (STRT) are compared to untreated control plants (UTC). Values weretransposed to natural logarithms.

1072

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 49686801

THX Cotton DER

Figure 7. Foliar treated plants compared to soil treated plants: Distribution ofconcentrations of thiamethoxam residues measured in plant matrices sampled at bloom.
Concentrations in foliar treated plants (FTRT) are compared to plants that received a soil
treatment at planting (SFTR). Values were transposed to natural logarithms.

1073

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 49686801

THX Cotton DER

Figure 8. Foliar treated plants compared to soil treated plants: Distribution ofconcentrations of CGA322704 metabolite residues measured in plant matrices sampled atbloom. Concentrations in foliar treated plants (FTRT) are compared to plants that received asoil treatment at planting (SFTR). Values were transposed to natural logarithms.

1074

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 49686801

THX Cotton DER

Figure 9. Soil comparison: Distribution of concentrations of thiamethoxam residues
measured in plant matrices sampled at bloom. Concentrations in foliar treated plants(FTRT) grown in either coarse or medium textured soil. Values were transposed to natural
logarithms.

1075

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 49686801

THX Cotton DER

Figure 10. Soil comparison: Distribution of concentrations of CGA322704 metabolite
residues measured in plant matrices sampled at bloom. Concentrations in foliar treatedplants (FTRT) grown in either coarse or medium textured soil. Values were transposed tonatural logarithms.

1076

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 49686801

THX Cotton DER

Figure 11. Extra floral nectar concentration over time: Distribution of concentrations ofthiamethoxam residues measured in extra floral nectar directly after the first foliar
application (App 1), directly after the second foliar application (App 2), at bloom, which was
7 days after the second application, and then at 24 days after the second application (Final).
Concentrations in treated plants (Foliar) are compared to untreated control plants(Control). Values were transposed to natural logarithms.

1077

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID TBDCDPR THX Cotton

Figure 12. Extra floral nectar concentration over time: Distribution of concentrations of
CGA322704 metabolic residues measured in extra floral nectar directly after the first foliar
application (App 1), directly after the second foliar application (App 2), at bloom, which was
7 days after the second application, and then at 24 days after the second application (Final).
Concentrations in treated plants (Foliar) are compared to untreated control plants(Control). Values were transposed to natural logarithms.

41

1078

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

Year/Authors/Title
Lange, B. 2017.
Thiamethoxam (A18481A) Determination of Residues inLeaves, Flowers, Anthers,
Pollen, and Nectar of Soybean
Plants After Foliar Application

Study Type
Summary
Notes/Uncertainties
Non-Guideline This study quantified thiamethoxam and CGA322704 residues in soybean
N/Afield residue(Glycine max) grown in three locations: North Carolina (NC; sandy loam),study onLouisiana (LA; silt loam), and Iowa (IA; loam). Three replicate plots were used insoybeans toeach location. Each plot received foliar applications at 10 and 5 days before
establish
bloom at a nominal rate of 0.063 lbs. ai/A. Nectar was sampled at early-, mid-,thiamethoxam and late-bloom. No pollen samples were collected. Anthers were analyzed inandplace of pollen. Samples of anthers and nectar were collected 10-20, 5-15, andLange Research Study
metabolite
5-17 days after the last application in NC, LA, and IA, respectively. Analyses of
Number: LR16192
concentrations fortified samples of anthers (83.7-91.8% thiamethoxam and 89.2-97.1 forGolden Pacific Study Number: in whole
CGA322704) and nectar (89.2-98.8% thiamethoxam and 103-112 for
160670flowers,
CGA322704) were all within acceptable limits. Nectar samples were collected
Report Number: TK0250070
anthers,
by bees within tunnels. Mean thiamethoxam residues in nectar across allleaves andlocations (3.60 ppb) were comparable to CGA322704 residues (3.21 ppb) in thebee-collected early bloom samples but thiamethoxam residues were less than CGA322704 by
nectarthe late-bloom samples (0.500 ppb vs. 4.45 ppb, respectively). Mean
followingthiamethoxam residues (68.2 ppb) in anthers were notably greater thanfoliarCGA322704 residues (9.38 ppb) in the early bloom samples and thiamethoxam
applications
residues remained greater than CGA322704 through the late-bloom samples(10.6 ppb vs. 3.40 ppb, respectively). Mean residues for thiamethoxam in
nectar were greatest across all sample periods in LA with residues comparable
in NC and IA. Mean residues for thiamethoxam in anthers were greatest acrossall sample periods in LA with residues then greater in IA than in NC. Mean
concentrations of CGA322704 in nectar were greatest in LA and comparable in
NC and IA. Mean concentrations of CGA322704 were comparable in anthers
across all regions.

1079

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

Year/Authors/Title
Study Type
Summary
Notes/Uncertainties
Gilson, L. 2017.
Non-Guideline This study quantified thiamethoxam and CGA322704 residues in tomato
N/A.
Thiamethoxam 75SG (A9549C) field residue(Solanum lycopersicum) grown in three locations: Kansas (KS; silt loam), Illinois– Determination of Residues study on(IL; silt loam), and California (CA; sandy loam). Three replicate plots were usedin Pollen, Flowers, and Leaves tomato toin each location. Each plot received a single soil application at transplanting atof Tomato After Soilestablish
nominal rates of 0.172 lbs. ai/A or 0.125 lbs. ai/A. Pollen and whole flowers
Application with Platinum®
thiamethoxam were sampled at early-bloom, and again at 10 and 20 days later. Samples of
75SG.
and
pollen and whole flowers were collected 40-60, 42-61, and 42-60 days postmetabolite
application in KS, IL, and CA, respectively. Analyses of fortified samples ofSyntech Research Laboratory levels inpollen (83.8-97.5% thiamethoxam and 97.0-105 for CGA322704) and whole
Services Study Number
wholeflower (98.2-105% thiamethoxam and 97.3-101 for CGA322704) were all within
069SRUS16C087
flowers,
acceptable limits. Pollen samples were manually extracted from whole flowers.
Report Number: TK0242072
leaves andAt the maximum application rate, mean thiamethoxam residues (68.9 ppb) inmanuallywhole flowers were notably less than CGA322704 residues (120 ppb) in thecollected
early bloom samples but were less different by the late-bloom samples (38.9pollen, andppb vs. 55.7 ppb, respectively). Mean thiamethoxam residues (46.3 ppb) infollowing apollen were also notably less than CGA322704 residues (93.9 ppb) in the earlysoil
bloom samples but were comparable by the late-bloom samples (31.5 ppb vs.
application
27.1 ppb, respectively).

1080

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

Year/Authors/Title
Study Type
Summary
Notes/Uncertainties
Louque, R. 2017.
Non-Guideline This study quantified thiamethoxam and CGA322704 residues in pumpkin
A table on page 108 of theThiamethoxam 75SG (A9549C) field residue(Cucurbita pepo), muskmelon (Cucumis melo), and summer squash (Cucurbita study report indicates– Determination of Residues study onpepo) grown in three locations: North Carolina (NC; sand), Missouri (MO; loamy different application rates
in Leaves, Flowers, Pollen, and pumpkins,sand), and California (CA; clay loam). Three replicate plots were used in each
(0.0172 lbs ai/A for squash in
Nectar of Pumpkin, Summer
summerlocation. Each plot in NC received a single at-planting application at nominal
NC and 0.0858 lbs ai/A for
Squash, and Muskmelons
squash, andrates of 0.172 lbs. ai/A or 0.125 lbs. ai/A in pumpkins, 0.172 lbs. ai/A or 0.0172 melon in NC) than anywhere
After Soil Application.
muskmelon to lbs. ai/A in squash, and 0.0858 or 0.172 lbs ai/A in muskmelon. The body of the else in the report. It isestablish
report only indicates that rates of 0.125 lbs ai/A or 0.172 lbs ai/A were used;
uncertain if this is a
Smithers Viscient Studythiamethoxam however, this is contradicted by a table in the appendix of the report indicating typographical error, since all
Number 1781.4148
and0.0172 lbs ai/A was applied to squash and 0.0858 lbs ai/A to melon at one site. other application rates
Report Number: TK0222530
metabolite
Each plot in MO or CA received a single at-planting application at nominal rates mentioned are 0.125 lbs ai/Alevels inof 0.172 lbs. ai/A or 0.125 lbs. ai/A in pumpkins, 0.172 lbs. ai/A in squash andor 0.172 lbs ai/A. The rates
manuallymuskmelon. Pollen and nectar was sampled at bloom and subsequently at 5,0.0172 and 0.0858 lbs ai/A docollected
10, 15, and 20 days after bloom. Pumpkin samples were collected 58-79, 49-70, not appear outside of thisnectar, pollen, and 37-57 days post-application in NC, MO, and CA, respectively. Summer
one table. Only data from thewhole flowers squash samples were collected 15-19 (only at bloom and a few 5 day postmaximum rate allowed by theand leaves
bloom samples collected), 36-52, and 41-62 days post-application in NC, MO,label were included infollowing soil and CA, respectively. Muskmelon samples were collected 48-68, 35-57, and 43- independent statisticalapplications
64 days post-application in NC, MO, and CA, respectively. Analyses of fortified analysis by DPR. MO squash
samples of pollen (71.2-115% thiamethoxam and 73.5-120 for CGA322704) and samples were collected at 2nectar (77.3-118% thiamethoxam and 70.4-120 for CGA322704) were all within or 4 day intervals, rather than
the 5-day interval specified.
acceptable limits. Pollen and nectar samples were manually extracted fromwhole flowers. Reported mean thiamethoxam residues in pumpkin nectar (4.58
ppb) were notably greater than CGA322704 residues (1.33 ppb) in the early
bloom samples and remained greater through the late-bloom samples (2.11
ppb vs. 0.554 ppb, respectively). Mean thiamethoxam residues (4.76 ppb) in
pumpkin pollen were also notably greater than CGA322704 residues (2.76 ppb)
in the early bloom samples but were comparable by the final samples (2.02 ppb
vs. 2.31 ppb, respectively). Reported mean thiamethoxam residues in summer
squash nectar (13.9 ppb) were notably greater than CGA322704 residues (1.06
ppb) in the early bloom samples and remained greater through the late-bloom
samples (2.06 ppb vs. 0.696 ppb, respectively). Mean thiamethoxam residues insummer squash pollen (6.15 ppb) were also notably greater than CGA322704
residues (1.69 ppb) in the early bloom samples and remained greater throughthe late-bloom samples (2.72 ppb vs. 1.45 ppb, respectively). Reported mean
thiamethoxam residues in muskmelon (21.4 ppb) were notably greater than
CGA322704 residues (2.37 ppb) in the nectar in the early bloom samples and1081

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

remained greater through the late-bloom samples (5.31 ppb vs. 0.832 ppb,
respectively). Mean thiamethoxam residues in the muskmelon pollen (6.49
ppb) were also notably greater than CGA322704 residues (1.85 ppb) in theearly bloom samples remained greater through the late-bloom samples (23.5
ppb vs. 6.14 ppb, respectively).

1082

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

Year/Authors/Title
Study Type
Summary
Notes/Uncertainties
Loque, R. 2017.
Non-Guideline This study quantified thiamethoxam and CGA322704 residues in pumpkin
N/A.
Thiamethoxam 75SG (A9549C) field residue(Cucurbita pepo) grown in three locations: North Carolina (NC; sand), Missouri- Determination of Residues in study on(MO; loamy sand), and California (CA; clay loam). Three replicate plots were
Leaves, Flowers, Pollen, andpumpkin to
used in each location. Each plot received foliar applications at 10 and 5 days
Nectar of Pumpkin After Foliar establish
before bloom at nominal rates of 0.086 lbs. ai/A or 0.023 lbs. ai/A. Pollen andApplication
thiamethoxam nectar were sampled at early-bloom, and again at 5, 10, 15 and 20 days later.
andSamples of pollen and nectar were collected 5-26, 6-27, and 5-27 day [s afterSmithers Viscient Study
metabolite
the last application in NC, MO, and CA, respectively. Analyses of fortified
Number 1781.4149
concentrations samples of pollen (70.2-109% thiamethoxam and 71.7-119 for CGA322704) andin whole
Report Number: TK0242074
nectar (73.5-107% thiamethoxam and 70.3-119 for CGA322704) were all withinflowers, leaves acceptable limits. Pollen and nectar samples were manually extracted fromand manually- whole flowers. At the higher application rate, mean thiamethoxam residues
collected(10.6 ppb) in nectar were notably higher than CGA322704 residues (3.18 ppb)
nectar, pollen, in the early bloom samples but thiamethoxam residues were less thanand following CGA322704 by the late-bloom samples (0.691 ppb vs. 2.07 ppb, respectively).
foliarMean thiamethoxam residues (15.9 ppb) in pollen were also notably greaterapplications
than CGA322704 residues (3.99 ppb) in the early bloom samples but
thiamethoxam residues were less than CGA322704 by the late-bloom samples(1.76 ppb vs. 2.31 ppb, respectively).

1083

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

Year/Authors/Title
Study Type
Summary
Notes/Uncertainties
Lange, B. 2017.
Non-Guideline This study quantified thiamethoxam and CGA322704 residues in sweet orange N/AThiamethoxam WG (A9584C) field residue(Citrus x sinensis) grown in three locations: Florida (FL; sand; 2 sites) and– Determination of Residues study on
California (CA; sandy clay loam). Three replicate plots were used in eachin Leaves, Flowers, Pollen, and orange tolocation. One set of plots received two foliar applications in the fall, 7 days
Nectar of Sweet Orange After establish
apart. A second set of plots at each location received foliar applications 7 days
Foliar Application
thiamethoxam before pre-bloom and 7 days later. A third set of plots received a single
andapplication at pre-bloom. All applications were made at 0.086 lbs. ai/A. Nectar
Lange Research Study
metabolite
and pollen were sampled at early-, mid-, and late-bloom. Samples of pollen andNumber: LR16203
concentrations nectar were collected 69-78, 56-88, and 104-117 days after the last of the fallin whole
Report Number: TK0250069
applications in FL1, FL2, and CA, respectively. Samples of pollen and nectar
flowers, leaves were collected 34-43, 21-53, and 38-51 days after the last of the pre-bloom
and manually- applications in the two remaining application scenarios in FL1, FL2, and CA,
collected
respectively. Analyses of fortified samples of pollen (73.1-113% thiamethoxam
nectar andand 77.8-112 for CGA322704) and nectar (93.4-114% thiamethoxam and 88.0pollen
110 for CGA322704) were all within acceptable limits. Nectar and pollen
following
samples were manually collected from flowers. Mean thiamethoxam residues
foliarin nectar following fall applications across all locations (0.560 ppb) wereapplications
comparable to CGA322704 residues (0.567 ppb) in the early bloom samples
and through the late-bloom samples (0.506 ppb vs. 0.548 ppb, respectively).
Mean thiamethoxam residues in pollen following fall applications across all
locations (39.6 ppb) were notably greater than CGA322704 residues (4.51 ppb)
in the early bloom samples but were less different by the late-bloom samples(5.60 ppb vs. 3.51 ppb, respectively). Mean thiamethoxam residues in pollen
from plots receiving pre-bloom applications were comparable to CGA322704
residues in the early bloom samples but thiamethoxam residues became
somewhat less than CGA322704 in the late-bloom samples. Mean residues forthiamethoxam in nectar following pre-bloom applications were somewhat
greater than CGA322704 residues in early bloom samples becoming
comparable to possibly greater by the late-bloom samples.

1084

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

Year/Authors/Title
Mitchell, J. 2017.
Thiamethoxam 25WG
(A9584C) - Magnitude of
Residues in Leaves, Flowers,
Pollen, and Nectar of Apple
After Foliar Application
Waterborne Study Number:
796.123
Battelle Report Number:
100078149
Report Number: TK0250071

Study Type
Summary
Notes/Uncertainties
Non-Guideline This study quantified thiamethoxam and CGA322704 residues in apple (Malus The application rate is lower
field residue
domestica) grown in three locations: New York (NY; loamy sand), Virginia (VA; than the maximum rate
study on apple sandy loam) and Washington (WA; loamy sand). Three replicate plots wereallowed for pome fruit. Lowerto establish
used in each location. A single application was made 5 days before bloom at a than acceptable recoveries inthiamethoxam nominal rate of 0.086 lbs. ai/A. Nectar and pollen were sampled at early-, mid-, nectar for thiamethoxam
andand late-bloom. Samples of pollen and nectar were collected 9-14, 5-14, and 5- raises the possibility thatmetabolite
13 days post-application in NY, VA, and WA, respectively. Analyses of fortified nectar residue values might
concentrations samples of pollen (70-120% thiamethoxam and 81-109 for CGA322704) andbe higher than reported.
in whole
nectar (74-109 for CGA322704) were within acceptable limits. Analyses offlowers, leaves fortified samples of nectar (68-97% thiamethoxam) were slightly outside theand manually- acceptable limits of 70-120%. Nectar and pollen samples were manually
collected
collected from flowers. Mean thiamethoxam residues in pollen across all
nectar andlocations (1680 ppb) were notably greater than CGA322704 residues (56.4 ppb)
pollenin the early bloom samples and through the late-bloom samples (858 ppb vs.
following a
108 ppb, respectively). Mean thiamethoxam residues in nectar across all
foliarlocations (280 ppb) were notably greater than CGA322704 residues (6.74 ppb)
applicationin the early bloom samples but were less different by the late-bloom samples(35.6 ppb vs. 2.26 ppb, respectively).

1085Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

Year/Authors/Title
Lange, B. 2017.
Thiamethoxam 25WG
(A9584C) – Determination of
Residues in Leaves, Flowers,
Pollen, and Nectar ofBlueberry After Foliar
Application
Lange Research Study
Number: LR16191
Report Number: TK0250072

Study Type
Summary
Notes/Uncertainties
Non-Guideline This study quantified thiamethoxam and CGA322704 residues in blueberry
N/Afield residue(Vaccinium corymbosum) grown in three locations: California (CA; sand),study onQuebec (QC; loam), and Washington (WA; loamy sand). Three replicate plots
blueberry towere used in each location. One set of plots received three foliar applications
establishat 19, 12, and 5 days before bloom at a nominal rate of 0.063 lbs. ai/A, andthiamethoxam another set of plots at each location received a single foliar application 15 days
andbefore bloom at a nominal rate 0.063 lbs. ai/A. Nectar and pollen were
metabolite
sampled at early-, mid-, and late-bloom. Samples of pollen and nectar wereconcentrations collected 5-22, 5-11, and 12-24 days after the last of the three application inin whole
CA, QC, and WA, respectively. In those plots receiving a single application,flowers, leaves pollen and nectar were collected 14-31, 19-25, and 22-34 days post-applicationand manually- in CA, QC, and WA, respectively. Analyses of fortified samples of pollen (75.1collected
101% thiamethoxam and 78.6-102 for CGA322704) and nectar (77.4-96.9%
nectar andthiamethoxam and 85.8-101 for CGA322704) were all within acceptable limits.
pollenNectar and pollen samples were manually collected from flowers. Mean
followingthiamethoxam residues in nectar from plots receiving repeated applications
foliaracross all locations (118 ppb) were less than CGA322704 residues (142 ppb) inapplications
the early bloom samples but thiamethoxam residues were comparable to
CGA322704 by the late-bloom samples (51.2 ppb vs. 59.1 ppb, respectively).
Mean thiamethoxam residues in pollen from plots receiving repeatedapplications (370 ppb) were notably greater than CGA322704 residues (60.2
ppb) in the early bloom samples and thiamethoxam residues remained greaterthan CGA322704 in the late-bloom samples (156 ppb vs. 48.4 ppb,
respectively). Mean residues for thiamethoxam in nectar were greatest acrossall sample periods in QC with residues comparable in CA and WA. Mean
residues for thiamethoxam in pollen were greatest across all sample periods in
WA with residues greater in QC than in CA. Mean concentrations of
CGA322704 in nectar were greatest in QC with residues comparable in CA andWA. Mean concentrations of CGA322704 in pollen were greatest across all
sample periods in QC with residues greater in WA than in CA.

1086

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

Year/Authors/Title
Trask, J. 2017. Endigo® ZC(A13623Q), Endigo® ZCX(A18481A) and Cruiser® 5S(A9765N) – Magnitude of
Residues in Pollen and Leavesof Corn Plants After
Application as a Seed
Treatment with Cruiser® 5S
and After Foliar Application
with Endigo® ZC or Endigo®
ZCX

Study Type
Summary
Notes/Uncertainties
Non-Guideline This study quantified thiamethoxam and CGA322704 residues in corn (Zea
Foliar applications were madefield residuemays) grown in three locations: Pennsylvania (PA; loam), Iowa (IA; silty clay
to corn grown from treatedstudy on corn loam), and Oklahoma (OK; sandy loam). Three replicate plots were used forseeds.
to establish
each treatment in each location. Seeds treated with Cruiser® 5S were planted
thiamethoxam in all test plots. One set of plots received no foliar applications. The remaining
andplots received two foliar applications at 0.086 lbs. ai/A of either Endigo® ZC, or
metabolite
Endigo® ZCX with the first application at either V8 growth stage or at first silk
concentrations emergence. Each initial foliar application was followed 7 days later with thein leaves and same formulated product. Pollen was sampled at pollen shed in all plots. Pollen
manuallywas also collected in OK 2 days following the second application in those plotscollected
receiving the first application at initial silk emergence. Samples of pollen werepollencollected 58, 57, and 58 days after planting in PA, IA, and OK, respectively. In
following
Waterborne Study Number:
those plots receiving the first application at V8 growth stage, pollen was
796.110
foliarcollected 18, 18, and 15 days after the last application in PA, IA, and OK,
EPL Study Number: 110G1111 applications
respectively. In those plots receiving the first application at silk emergence,
Report Number: TK0258214
and seedpollen was collected 1, 3, and 4 days after the first application in PA, IA, andtreatment
OK, respectively. Analyses of fortified samples of pollen (88.4-119%
thiamethoxam and 81.4-109 for CGA322704) were all within acceptable limits.
Pollen samples were manually collected. Mean thiamethoxam residues fromplots regardless of treatment were comparable to CGA322704 residues inpollen except in those treatments that received foliar applications at silkemergence where thiamethoxam residues were notably greater.

1087

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

U.S. EPA Data Evaluation Reports (Thiamethoxam):
U.S. EPA. (2017). Data evaluation report: Thiamethoxam 25 WG (AC9584C) - magnitude of theresidues in pollen, nectar, flowers, and leaves of cranberry after foliar application. Washington,
D.C.: Author. Laboratory Report Number TK0236307.
U.S. EPA. (2017). Data evaluation report: Thiamethoxam 25 Wg (AC9584C) - magnitude of theresidues in leaves, flowers, pollen, and nectar of cucumber after foliar application. Washington,
D.C.: Author. Laboratory Report Number TK0222532.
U.S. EPA. (2017). Data evaluation report: Thiamethoxam - Thiamethoxam 75 SG (A9549C) magnitude of residues in pollen, flowers, and leaves of pepper after soil application: Final report.
Washington, D.C.: Author. Laboratory Report Number TK0236306.
U.S. EPA. (2017). Data evaluation report: Thiamethoxam - Thiamethoxam 25WG (A9584C) magnitude of residues in pollen, flowers, and leaves of tomato after foliar application.
Washington, D.C.: Author. Laboratory Report Number TK0222531.
U.S. EPA (2017). Data evaluation report: Thiamethoxam 75SG (A9549C) - determination ofresidues in leaves, flowers, pollen, and nectar of strawberry after soil application. Washington,
D.C.: Author. Laboratory Report Number TK0250068.

1088

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

Dinotefuran Data Evaluations (begin on next page)1089

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50198501

CDPR Dino Cotton

Reference
Hummel, R. (2017) Quantitation of Residues of Dinotefuran, DN and UF in Nectar, Extrafloral Nectar,
Pollen and Leaves Following Foliar Treatment of Dinotefuran to Cotton. Study Number: 43411B104.
Unpublished study prepared by Landis International, Inc. 307. MRID 50198501, CDPR Study ID 297894,
Data Volume 52911-0490, Tracking ID# 280249

1. STUDY INFORMATION
Chemical:Test Material:
1Study Type:

Sponsor:Sponsor Study
ID:
Study
Completion
Date:

GLP Status:

PC Code44312
Percent
Dinotefuran 20 SG
Active20%
Ingredient:
Residue study to measure the magnitude of Dinotefuran and its major
metabolites, UF and DN, in cotton leaves, pollen, extrafloral nectar and nectar
following foliar applications.
Landis International, Inc.
P.O. Box 5126
Experiment Start and May 3, 2016 –
3185 Madison Highway
End Date:
November 30, 2016
Valdosta, Georgia 31603-5126
USA
Dinotefuran

43411B104
Study Locations:February 26, 2017

Six trial sites of cotton
located in California.

GLP Compliant; protocol reviewed by CDPR.
[CDPR Study ID 297894, Data Volume 52911-0490, Tracking ID# 280249]

2. REVIEWER INFORMATION
Study Reviewed by:Richard Bireley, Sr. Environmental Scientist (Specialist)
California Department
John Troiano, Ph.D., Research Scientist III
of Pesticide Regulation
Alexander Kolosovich, Sr. Environmental Scientist
Brigitte Tafarella, Environmental Scientist
Denise Alder, Sr. Environmental Scientist (Specialist)
Russell Darling, Sr. Environmental Scientist (Specialist)

11090

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50198501

CDPR Dino Cotton

3. EXECUTIVE SUMMARY

The purpose of this study was to determine the residue concentrations of dinotefuran and its major
metabolites, DN and UF, in floral nectar, extra-floral nectar, pollen and leaves collected following foliar
treatment applications of Dinotefuran 20 SG to cotton.
Six field trials were conducted during the 2016 growing season on cotton in California. Three treatedplots and a non-treated plot were established at each test location. Trial CA 1 was planted with pima
cotton (Gossypium barbadense), and the remaining five trials were planted with upland cotton (G.
hirsutum). In general, pima cotton varieties are grown in western states (e.g., California and Arizona),
while upland cotton varieties are grown throughout the United States.
Dinotefuran 20 SG (containing 20% dinotefuran w/w) was applied to cotton plants in two broadcast
foliar applications at a rate of 0.129 – 0.136 lb ai/Acre/application (144 - 152 g ai/ha/application). Allapplications were made in 14 – 17 gal/A of water (131-159 L/ha) using ground equipment. All sprays
were calibrated prior to each application with the volume/time method and consisted of commercial or
simulated commercial application equipment.
Samples were analyzed for residues of dinotefuran and its metabolites, UF and DN, using Eurofins
analytical method No. RA046. Quantitation of residues in all samples was achieved using an external
calibration curve calculated by linear regression of instrument responses for the reference substances atmultiple concentrations. The performance of the instrument was evaluated during each injection set.
4. STUDY VALIDITY
Guideline Followed:
Guideline Deviations:
Other Deviations:Classification:
Rationale:
Reparability:

Protocol was reviewed and approved by CDPR
N/A
N/A
ACCEPTABLE
N/A
N/A

5. MATERIALS

Test Material Characterization for Foliar Application End Use Product
Test item:
Dinotefuran 20 SG
Percent A.I.:
20% A.I.
Formulation Type:
Water Soluble Granule Date of Issue:
April 15, 2015
CAS #:
165252-70-0
Expiration Date:
March 27, 2017
5A. STUDY DESIGNThis study requirement was part of the dinotefuran special review at the California Department of
Pesticide Regulation (CDPR). The study design and protocol were approved by the CDPR. The study
initiation date was May 3, 2016. The experimental start date was July 6, 2016 and the experimental end
date was November 30, 2016 (last sample injection).

21091

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50198501

CDPR Dino Cotton

Six field trials were conducted during the 2016 growing season on cotton in California. Three treatedplots and one non-treated plot were established at each test location. Dinotefuran 20 SG (containing
20% dinotefuran w/w) was applied to cotton plants in two broadcast foliar applications at a rate of
0.129 – 0.136 lb ai/A/application (144 - 152 g ai/ha/application). All applications were made in 14 – 17
gal/A of water (131-159 L/ha) using ground equipment. All sprays were made using commercial or
simulated commercial application equipment, and all sprayers were calibrated prior to each application
with the volume/time method.
Each trial included a non-treated control plot, from which non-treated samples of leaves, extrafloral
nectar, pollen and floral nectar were collected to provide a relative indication of background levels ofdinotefuran and to give an indication of possible analytical matrix interferences. Each non-treated plot
was located at least 100 feet from the nearest treated plot and was not down-wind during foliar
applications.
Commercially available varieties of cotton were used, and each crop was grown following local
agronomic practices at each test site. Trial CA 1 was planted with pima cotton (Gossypium barbadense),
and the remaining five trials were planted with upland cotton (G. hirsutum). The conditions at each testsite are summarized in Table 1 and Table 2.
5B. STUDY SITE LOCATION AND CHARACTERISTICS
The crops were grown and maintained according to typical agricultural practices for each geographicalregion. The crop varieties selected were typical for commercial production in the area. The actual
temperature and rainfall were within normal parameters during the residue study period with the noted
exceptions. Irrigation was used to supply adequate moisture for vigorous crop growth, as needed. There
were no meteorological abnormalities that occurred during the conduct of the study that had asignificant effect on the cotton crops.
Table 1. Site Locations and Cotton Varieties
Trial
Site
Nearest Town/County
Site
Identification
1
CA 1
Madera, California/Madera
County2
CA 2
Porterville, California/Tulare
County3
CA 3
Porterville, California/Tulare
County4
CA 4
Zamora, California/Yolo County

EPA
Region10

VarietyPrima

Irrigation
Type
Flood

10

Acala

Drip

10

Acala

Drip

10

ST 5115
GLT
ST 5115
GLT
Acala

Drip

5

CA 5

Pearson, California/Yolo County

10

6

CA 6

Fresno, California/Fresno County

10

3
1092

Sprinkler
DripAppendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

Table 2. Trial Site Conditions
Trial Site
Sand %

Silt
%

Clay
%

CA 1
CA 2

89
80

9
16

2
4

CA 3

40

20

40

CA 4

26

38

36

CA 5

30

36

34

CA 6

74

24

2

USDA
Textural
Class
Sand
Loamy
Sand
Clay
Loam
Clay
Loam
Clay
Loam
Loamy
Sand

Soil pH

4.6
7.2

Organic
Matter
%
0.52
0.57

33.2

1.2

8

23.2

1.2

7

18.8

2.3

6.7

6.7

0.79

7.7

RTIa

CDPR Dino Cotton

Application Number

MRID 50198501

CEC
Meq/100g

7.8
7.4

5C. APPLICATION TIMING AND RATES

4
1093

n/a
7
n/a
7
n/a
7
n/a
8
n/a
8
n/a
7

Total Rate(g ai/ha)

BBCH 60 9/16/16 17 159 0.134
150
1BBCH 61 9/23/16 17 159 0.134
150
2
CA 2 Broadcast BBCH 59 7/19/16 15 140 0.131
147
1
BBCH 61 7/26/16 15 140 0.129
144
2
CA 3 Broadcast BBCH 55 7/13/16 15 140 0.1301461BBCH 60 7/20/16 15 140 0.131
147
2
CA 4 Broadcast BBCH 60 8/22/16 15 140 0.135
151
1BBCH 61 8/30/16 15 140 0.136
152
2
CA 5 Broadcast BBCH 60 8/23/16 14 131 0.134
150
1BBCH 61 8/31/16 14 131 0.134
150
2
CA 6 Broadcast BBCH 59
7/6/1615 140 0.134
150
1BBCH 61 7/13/16 15 140 0.133149
2aRe-treatment interval (Number of days between applications). Not applicable= n/a.

Total Rate(lb ai/A)

Rate(g ai/ha)Rate (lb ai/ha)

Application
VolumeL/hagal/ABroadcastTiming

MethodCA 1

Application Timing(Crop Stage)

Trial ID

Table 3. Study Use Pattern for Dinotefuran

0.268

300

0.260

291

0.261

293

0.271

303

0.268

300

0.267

299

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50198501

CDPR Dino Cotton

5D. SAMPLE COLLECTION, HANDLING, PROCESSING
To prevent cross-contamination, the non-treated control plots were sampled first, or separate personnel
sampled the non-treated plot independently from other samples. For each matrix, one sample wascollected from each treated replicate plot A, B, and C, as well as from the control plot, at designatedintervals.
Leaf Samples
Leaf samples were collected prior to the second application, and then at early, mid, and late bloom, andagain following bloom (BBCH 69). Samples consisted of a minimum of 24 leaves collected from at least
12 plants. Target weights of 200 g from untreated and 50 g for treated leaves were collected directly
into labelled sealable plastic bags. The samples were then held in separate ice chests labeled “control”
and “treated” on substitute ice until placed into frozen storage.
Pollen Samples
Pollen samples were collected at early, mid, and late bloom. Flowers were collected from the untreatedcontrol and the treated sub-plots and either bagged or placed in trays to be transported to the field
laboratory for pollen extraction. Pollen samples were extracted manually from flowers using a plastic
filtered collection tip attached to a vacuum pump. The samples were vacuumed directly from theanthers or dislodged manually and then collected either into the filtered vacuum tip or placed directly
into a small vial. The sample containers (tips or vials) were weighed before and after the pollen
extraction where the net weight represented the sample size. Once the target weight of 100 mg was
obtained (or when all flowers available for pollen sampling were used), the plastic tips containing pollen
were wrapped in Parafilm and placed in labelled plastic bottles. The bottles were sealed, placed in resealable plastic bags, and transferred immediately into separate freezers for the treated and untreatedsamples.
Extra-Floral Nectar Samples
Extra-floral nectar samples were collected prior to the second application and then at early, mid, andlate bloom, and again following bloom (BBCH 69). Flowers were collected from the untreated control
and the treated sub-plots and then either bagged or placed in trays for transport to the field laboratoryfor extra-floral nectar extraction. Extrafloral nectar samples were collected from the outer parts ofblossoms (sepals, bracts and pedicels) manually using glass micro capillary pipettes. Nectar was thentransferred into a pre-weighed amber glass vial. Each vial was weighed before and after nectar
collection, with the net weight representing the sample size. Once the target weight of 100 mg was
obtained (or when all flowers available for nectar sampling were used), the vials containing nectar weresealed, placed into individual labelled secondary containers and then placed in re-sealable plastic bags
for immediate transfer into separate freezers for the treated and untreated samples.

51094Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50198501

CDPR Dino Cotton

Floral Nectar Samples

Floral nectar samples were collected at early, mid, and late bloom. Flowers were collected from theuntreated control and the treated sub-plots and then either bagged or placed in trays for transport tothe field laboratory for extra-floral nectar extraction. Floral nectar samples were collected from theinner-blossom nectary structures manually using glass micro capillary pipette or spun out using a filteredcentrifuge tube in a table-top centrifuge. Nectar was then transferred into a pre-weighed amber glassvial. Each vial was weighed before and after nectar collection, with the net weight representing thesample size. Once the target weight of 100 mg was obtained, (or when all flowers available for nectar
sampling were used), the vials containing nectar were sealed, placed into individual labelled secondary
containers and then placed in re-sealable plastic bags for immediate transfer into separate freezers forthe treated and untreated samples.
Sample Storage.
All residue samples (leaf, pollen, extra-floral nectar, and floral nectar) were shipped from the test sites inseparate treated and untreated boxes, or ice chests with dry ice, to Eurofins Agroscience Services, Inc.
via ACDS trucking or Federal Express overnight service. All samples were received frozen from the field
and were stored in freezers (approximately -20 °C) at Eurofins Agroscience Services, Inc.
The leaf samples were homogenized to a consistency appropriate for analysis using a bench-top
industrial food processor with dry ice. Homogenized samples were stored frozen in plastic bags until
analysis. Pollen samples were not homogenized due to their uniform powdery texture. Nectar samples
were not homogenized prior to the extraction.
The maximum frozen storage interval from sample collection to extraction for analysis was 133 days fortreated samples. Critical dates and storage intervals for each sample are presented in Appendix F of thestudy report. The available data from freezer storage stability studies on Dinotefuran and its metabolites
show that each analyte is stable in nectar, pollen and leaf matrices for at least 274 days.
5E. ANALYTICAL METHODS
Samples were analyzed for residues of dinotefuran and its metabolites, UF and DN, using Eurofins
analytical method No. RA046 entitled “Residue Analysis of Dinotefuran, DN and UF in Nectar, Pollen,
Leaves and Soil by LC-MS/MS" (Reference 1, Reference 2).
The method performance was verified during sample analysis by determining the recoveries fromcontrol samples fortified with dinotefuran, UF, and DN at 1, 1, and 3.08 ppb and 100, 100, and 61.6 ppb
for nectar; 2, 2, and 3.08 ppb and 100, 100, and 61.6 ppb for pollen; and 5, 5, and 3.08 ppb and 100,
100, and 61.6 ppb for leaves.
In brief, samples of pollen (0.1 g) were extracted with a mixture of methanol and water using a
Fastprep-24 homogenizer. After centrifugation, the samples were subjected to SPE clean-up with a C18(100mg/1mL) SPE cartridge. After elution, the samples were diluted and analyzed by LC-MS/MS.
To minimize the different matrix effect of cotton pollen and organic pollen (used to prepare curve andquality control samples), all cotton pollen samples were diluted using extract from control organic pollen
and analyzed by LC-MS/MS.
61095

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50198501

CDPR Dino Cotton

Samples of nectar (0.1 g) were extracted with a mixture of methanol and water using a Fastprep-24
homogenizer. After centrifugation (if necessary), the samples were transferred to autosampler vials,
diluted and analyzed by LC-MS/MS in a positive ionization mode.
The mass of several nectar samples was less than the minimum mass specified for standard analysis (0.1g); these samples were extracted by adjusting the extraction solvent proportionally to the sample mass.
Low weight quality control samples (sample mass = 0.05 g) were also prepared and tested for recovery.
Samples of leaves (2.5 g) were extracted with a mixture of methanol and water using a multi-tube
vortexer. After centrifugation, the supernatant was decanted into a clean tube and the samples were
subjected to a second extraction, combining the two supernatants. After the second extraction, sample
extracts were then transferred to autosampler vials, diluted and analyzed by LC-MS/MS in a positive
ionization mode.
Quantitation of all samples was achieved using calibration curves calculated by linear regression of
instrument responses for the reference substances at multiple concentrations. The performance of the
instrument was evaluated during each injection set. The correlation coefficient (r) for each calibration
curve was required to be >0.990. The performance of the analytical method was evaluated during eachsample set by fortifying control matrix with mixed standards of each analyte.
5F. QUALITY ASSURANCE RESULTS
The reference substances were used to generate data for both instrument and method performance.
Quantitation of residues in all samples was achieved using an external calibration curve calculated bylinear regression of instrument responses for the reference substances at multiple concentrations. The
performance of the instrument was evaluated during each injection set.
All control samples of the various matrices were free from interferences above the limit of detection(LOD) at the mass transitions used for quantification of the analytes. The LOD for dinotefuran and UF
correspond to 0.3 ppb in nectar, 0.6 ppb in pollen and 1.5 ppb in leaves. The LOD for DN corresponds to
0.185 ppb in nectar, 0.37 ppb in pollen, and 0.925 ppb in leaves.
The limit of quantitation (LOQ) corresponds to 1 ppb for dinotefuran and UF in nectar, 2 ppb fordinotefuran and UF in pollen, 5 ppb for dinotefuran and UF in leaves, and 3.08 ppb for DN in pollen,
nectar and leaves.
The recoveries for dinotefuran in nectar were between 59% and 108% with an average and RSD of 95% ±10%; the recoveries for dinotefuran in pollen were between 73% and 109% with an average and RSD of
87% ± 11%, the recoveries for dinotefuran in leaves were between 87% and 111% with an average and
RSD of 100% ± 7%.
The recoveries for UF in nectar were between 72% and 107% with an average and RSD of 92% ± 11%;
the recoveries for UF in pollen were between 61% and 85% with an average and RSD of 73% ± 12%, therecoveries for UF in leaves were between 95% and 109% with an average and RSD of 103% ± 4%.

71096

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50198501

CDPR Dino Cotton

The recoveries for DN in nectar were between 83% and 106% with an average and RSD of 97% ± 7%; therecoveries for DN in pollen were between 76% and 93% with an average and RSD of 85% ± 7%, therecoveries for DN in leaves were between 95% and 108% with an average and RSD of 102% ± 4%.
Detailed information regarding the reference substances, including the certificates of analysis, ispresented in Appendix C (Reference Substance Information) of the study report.
Detailed analytical data such as supporting raw data for re-calculations, representative chromatograms,
and example calculations are provided in Appendix E (Analytical Summary Report) of the study report.
Table 4: Summary of reported analytical LOQs and LODs for each analyte in each plant sample.
Laboratory

Eurofins
Agroscience
Services, Inc.

MatrixAnalyteLOD(ppb, parent
equivalents)

LOQ(ppb, parent
equivalents)

NectarPollenLeavesNectarPollenLeavesDinotefuran and UF
Dinotefuran and UF
Dinotefuran and UF
DN
DN
DN

0.3
0.6
1.5
0.185
0.37
0.925

1.0
2.0
5.0
3.08
3.08
3.08

6. RESULTS:
Residue data for concentration of parent dinotefuran and UF and DN degradation products in cotton
leaves, extra-floral nectar, floral nectar, and pollen are reproduced from the report in Tables 5-16.
Residue values below the analytical limit of detection (LOD) were reported as ½ of the LOD value notedin Table 4. There was only 1 missing sample for nectar that occurred in Trial CA6 for replicate 1 at thelate-bloom sampling interval (Tables 11, 12 and 13). Total dinotefuran residue determined during
statistical analysis was the sum of the each value noted for the replicate associated at each trial site. Forleaf and extra-floral nectar samples, sampling intervals occurred prior to and after the blooming period
and they are noted as pre-bloom and post-bloom in Tables and graphs, respectively. Successive samples
taken during bloom are noted as early-bloom, mid-bloom, and late-bloom. Plants at pre-bloom hadreceived only 1 application of dinotefuran whereas plants at early-bloom had received the full 2 foliar
spray treatments of dinotefuran. Sampling intervals occurred at approximately 7 day intervals, which
was designated in regressions to determine dissipation half-life values for residues. Statisticalprocedures used in the Statistical Analysis System (SAS) software were PROC CAPABILITY to provide
distribution statistics, PROC GLM for conducting Analysis of Variance (ANOVA) to test effects of soil typeand sampling time on concentration of residues, PROC REG to produce coefficients for dissipation overtime used to estimate half-lives, and PROC BOXPLOT to produce bar charts for comparing distributions
between treatments and trial sites.
Each trial included a non-treated control plot, from which non-treated samples of leaves, extrafloral
nectar, pollen and floral nectar were collected to provide a relative indication of background levels ofdinotefuran and to give an indication of possible analytical matrix interferences. Each non-treated plot
was located at least 100 feet from the nearest treated plot and was not down-wind during foliar
applications. Non-treated samples of leaves, floral nectar and extra-floral nectar did not containquantifiable levels of dinotefuran, UF or DN. However, non-treated pollen samples from selected trial
81097

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50198501

CDPR Dino Cotton

locations (-01, -04 and -05) contained measurable amounts of dinotefuran, UF and DN. For trial -01, thedinotefuran residue in the non-treated pollen sample (LA16-12) was 782 ppb vs. a mean residue of
17,878 ppb in pollen from the treated plots; for trial -04, dinotefuran residue in the non-treated pollen
sample (LA16-198) was 23.3 ppb vs. a mean residue of 428 ppb from pollen in the treated plots; and fortrial -05, dinotefuran residue in the non-treated pollen sample (LA16-260) was 40.5 ppb vs. a mean
residue of 792 ppb from pollen in the treated plots.

91098

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50198501

CDPR Dino Cotton

Table 5. Dino Residues in Cotton Leaves
Trial
CottonTotal Rate
SampleVariety(lb ai/A)
Timing a

a

CA 1

Prima

0.268

CA 2

Acala

0.260

CA 3

Acala

0.261

CA 4

ST 5115
GLT

0.271

CA 5

ST 5115
GLT

0.268

CA 6

Acala

0.267

Replicate
1
4715
7700
2355
1115
600
2240
2035
415
200
167
2205
2675
590
152
76.0
3210
5350
3310
1965
1485
3430
6050
2425
565
765
2075
3030
424
190
56.0

-2
3
10
17
24
-1
7
15
22
29
-1
9
16
22
29
-1
6
14
20
26
-2
7
14
20
26
-2
5
13
20
27

Number of days after final application.

10
1099

Dinotefuran Residue (ppb)
Replicate
Replicate
2
3
5450
4510
8550
8700
3350
1835
1540
1215
600
409
2550
1715
1785
2330
625
575
220
328
103
230
2430
2025
2585
3975
605
1035
146
191
142
124
3175
4155
8550
6150
3110
3215
1580
2175
1230
1260
4755
3000
6150
6250
2285
2210
2870
1525
1130
670
3575
3570
4025
3625
630
715
164
74.0
70.5
46.5

Mean
4892
8317
2513
1290
536
2168
2050
538
249
167
2220
3078
743
163
114
3513
6683
3212
1907
1325
3728
6150
2307
1653
855
3073
3560
590
143
58

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50198501

CDPR Dino Cotton

Table 6. UF Residues in Cotton Leaves
Trial
CottonTotal Rate
Variety(lb ai/A)

a

CA 1

Prima

0.268

CA 2

Acala

0.260

CA 3

Acala

0.261

CA 4

ST 5115
GLT

0.271

CA 5

ST 5115
GLT

0.268

CA 6

Acala

0.267

SampleTiming a

Replicate
1
1135
1870
2090
1155
705
2190
2315
1230
800
510
2390
4935
3995
2245
1580
1530
3545
5350
4735
4655
140
2290
1985
1135
1690
1925
4360
1350
625
309

-2
3
10
17
24
-1
7
15
22
29
-1
9
16
22
29
-1
6
14
20
26
-2
7
14
20
26
-2
5
13
20
27

Number of days after final application

11
1100

UF Residue (ppb)
Replicate
Replicate
2
3
1680
1700
2220
2360
2325
1830
1480
1895
760
545
2235
1440
2250
2510
1385
1300
610
935
465
530
2545
2000
5150
6650
3970
4285
1700
2120
1300
1020
1880
2770
6300
4765
6600
6450
6000
5700
4855
4700
1635
1290
2175
1845
2185
1950
2195
1640
2515
1765
2610
2800
4485
3500
1465
1995
510
458
311
303

Mean
1505
2150
2082
1510
670
1955
2358
1305
782
502
2312
5578
4083
2022
1300
2060
4870
6133
5478
4737
1442
2103
2040
1657
1990
2445
4115
1603
531
308

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50198501

CDPR Dino Cotton

Table 7. DN Residues in Cotton Leaves
Trial
CottonTotal Rate
Variety(lb ai/A)

a

CA 1

Prima

0.268

CA 2

Acala

0.260

CA 3

Acala

0.261

CA 4

ST 5115
GLT

0.271

CA 5

ST 5115
GLT

0.268

CA 6

Acala

0.267

SampleTiming a

Replicate
1
865
1375
2005
1335
985
2145
2720
1945
2050
1430
2500
5000
5450
3495
3430
1525
3985
6700
6950
8200
2300
3130
3695
2460
4105
1255
4355
3300
3280
2660

-2
3
10
17
24
-1
7
15
22
29
-1
9
16
22
29
-1
6
14
20
26
-2
7
14
20
26
-2
5
13
20
27

Number of days after final application.

12
1101

DN Residue (ppb)
Replicate
Replicate
2
3
1170
1000
1725
1825
2395
1725
1625
1955
1145
775
2205
1390
2805
2980
2470
2675
1245
1990
1955
1680
2675
2210
5050
6750
5900
6100
2930
3375
3210
2740
2030
2575
5500
4530
7500
7400
7750
7400
7250
6950
2350
1990
2730
2535
4070
3075
3505
2960
5650
3845
2005
2105
5350
4615
4270
4495
2675
1965
2940
2375

Mean
1012
1642
2042
1638
968
1913
2835
2363
1762
168
2462
5600
5817
3267
3127
2043
4672
7200
7367
7467
2213
2798
3613
2975
4533
1788
4773
4022
2640
2658

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50198501

CDPR Dino Cotton

Table 8. Dinotefuran Residues in Extra-Floral Cotton Nectar.
Trial
CottonTotal Rate
SampleResidue (ppb)b
Variety(lb ai/A)
Timing a
Replicate
Replicate
Replicate
Mean
1
2
3
-2
1740
1300
5080
2707
3
15500
10500
4890
10297
CA 1
Prima
0.268
10
1920
1800
1580
1767
17
647
197
163
336
24
111
216
119
149
-1
82.9
83.7
20.7
62
7
105
173
203
160
CA 2
Acala
0.260
15
5.06
6.24
6.68
6
22
1.93
1.96
2.43
2
29
0.500
0.500
3.50
2
-1
182
589
162
311
9
164
220
153
179
CA 3
Acala
0.261
16
12.9
3.86
6.78
8
22
1.76
1.41
1.17
1
29
0.500
8.96
0.500
3
-1
375
1530
866
924
7
428
250
290
323
CA 4
ST 5115
0.271
14
60.3
44.2
29.4
45
GLT
20
41.7
10.4
6.46
20
26
0.500
2.42
1.69
2
-2
117
75.5
59.2
84
7
2880
569
2670
2040
CA 5
ST 5115
0.268
14
586
189
236
337
GLT
20
7.51
3.32
5.06
5
26
3.08
2.34
3.29
3
-2
545
1070
1030
882
5
1180
1510
986
1225
CA 6
Acala
0.267
13
490
319
233
347
20
8.74
29.3
17.9
19
27
0.500
4.21
0.500
2
a
Number of days after final applicationb
For the purpose of calculating means, values below the LOQ (1.00 ppb) were set to half of the LOQ(0.500 ppb).

13
1102

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50198501

CDPR Dino Cotton

Table 9. UF Residues in Extra-Floral Cotton Nectar
Trial
CottonTotal Rate
SampleVariety(lb ai/A)
Timing a

a

CA 1

Prima

0.268

CA 2

Acala

0.260

CA 3

Acala

0.261

CA 4

ST 5115
GLT

0.271

CA 5

ST 5115
GLT

0.268

CA 6

Acala

0.267

Replicate
1
225
1390
481
250
73.2
38.1
44.9
5.69
2.23
0.500
57.7
35.6
7.06
3.25
1.51
140
155
28.7
8.05
1.81
37.8
467
459
10.3
3.05
250
759
518
28.5
3.87

-2
3
10
17
24
-1
7
15
22
29
-1
9
16
22
29
-1
7
14
20
26
-2
7
14
20
26
-2
5
13
20
27

UF Residue (ppb)b
Replicate
Replicate
2
3
200
632
1360
996
586
444
87.6
72.5
154
72.2
41.8
10.2
57.4
64.0
7.27
5.76
2.23
2.07
1.11
1.02
110
44.1
70.9
62.9
4.48
17.2
2.84
2.92
11.3
1.13
297
276
70.7
83.2
26.0
24.9
17.7
12.6
4.17
2.62
20.1
18.0
127
441
52.7
130
5.45
9.45
3.08
3.31
536
395
846
451
487
266
115
28.1
17.7
3.71

Mean
352
1249
504
137
100
30
55
6
2
1
71
56
10
3
5
238
103
27
13
3
25
345
214
8
3
394
685
424
57
8

Number of days after final application
For the purpose of calculating means, values below the LOQ (1.00 ppb) were set to half of the LOQ(0.500 ppb).
b

14
1103

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50198501

CDPR Dino Cotton

Table 10. DN Residues in Extra-Floral Cotton Nectar
Trial
CottonTotal Rate
SampleDN Residue (ppb)b
Variety(lb ai/A)
Timing a
Replicate
Replicate
Replicate
Mean
1
2
3
-2
132
217
348
202
3
1080
996
413
830
CA 1
Prima
0.268
10
292
438
257
329
17
151
39.3
18.0
69
24
17.3
33.8
10.1
20
-1
11.9
10.2
5.3
9
7
30.9
42.3
43.6
39
CA 2
Acala
0.260
15
5.99
5.77
5.70
6
22
1.54
1.54
1.54
2
29
1.54
1.54
1.54
2
-1
77.1
93.1
52.2
74
9
23.4
62.8
42.8
43
CA 3
Acala
0.261
16
6.23
4.25
26.4
12
22
5.08
1.54
1.54
3
29
1.54
6.58
1.54
3
-1
113
232
202
182
7
121
62.0
70.4
84
CA 4
ST 5115
0.271
14
26.7
24.4
22.8
25
GLT
20
6.92
67.7
9.74
28
26
1.54
6.89
5.01
4
-2
58.3
14.3
11.2
28
7
303
80.6
376
253
CA 5
ST 5115
0.268
14
795
55.4
197
349
GLT
20
5.96
3.57
7.42
6
26
1.54
1.54
1.54
2
-2
104
281
208
196
5
373
490
260
374
CA 6
Acala
0.267
13
294
282
171
249
20
22.2
216
26.6
88
27
6.15
14.7
3.94
8
a
Number of days after final applicationb
For the purpose of calculating means, values below the LOQ (1.00 ppb) were set to half of the LOQ(0.500 ppb).

15
1104

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50198501

CDPR Dino Cotton

Table 11. Dinotefuran Residues in Floral Cotton Nectar.
Trial
CottonTotal Rate
SampleDinotefuran Residue (ppb)
Variety(lb ai/A)
Timing a
Replicate
Replicate
Replicate
1
2
3
3
346
263
257
CA 1
Prima
0.268
10
55.0
54.0
44.5
17
36.0
27.4
28.8
7
47.9
90.7
109
CA 2
Acala
0.260
15
6.74
7.58
7.75
22
6.09
6.21
6.57
9
27.1
321
24.9
CA 3
Acala
0.261
16
3.84
3.86
5.39
22
7.30
4.08
3.48
7
141
27.5
73.8
CA 4
ST 5115
0.271
14
6.96
149
14.2
GLT
20
2.73
2.51
3.30
7
81.6
175
106
CA 5
ST 5115
0.268
14
9.09
18.5
13.9
GLT
20
6.01
5.19
6.01
5
328
274
309
CA 6
Acala
0.267
13
16.0
56.1
19.4
b
20
-19.2
22.5
a
Number of days after final application.
b
The mass of this sample was too small for analysis. The mean is based on two replicates.

16
1105

Mean
289
51
31
83
7
6
124
4
5
81
57
3
121
14
6
304
31
21

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50198501

CDPR Dino Cotton

Table 12. UF Residues in Floral Cotton Nectar.
Trial
CottonTotal Rate
SampleVariety(lb ai/A)
Timing a

a
b

CA 1

Prima

0.268

CA 2

Acala

0.260

CA 3

Acala

0.261

CA 4

ST 5115
GLT

0.271

CA 5

ST 5115
GLT

0.268

CA 6

Acala

0.267

Replicate
1
49.6
15.2
11.5
12.0
2.81
1.45
16.7
2.80
2.97
40.8
5.53
2.41
10.1
3.95
4.45
198
25.7
--b

3
10
17
7
15
22
9
16
22
7
14
20
7
14
20
5
13
20

UF Residue (ppb)
Replicate
Replicate
2
3
48.1
36.5
15.4
15.0
10.7
12.0
26.2
27.7
3.06
3.13
1.37
1.62
99.4
10.5
2.37
3.00
3.07
2.97
6.75
13.0
73.4
7.04
2.90
3.03
18.5
11.4
6.99
5.59
4.95
3.89
136
140
45.0
19.2
16.9
23.3

Number of days after final application.
The mass of this sample was too small for analysis. The mean is based on two replicates.

17
1106

Mean
45
15
11
22
3
1
42
3
3
20
29
3
13
6
4
158
30
20

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50198501

CDPR Dino Cotton

Table 13. DN Residues in Floral Cotton Nectar.
Trial
CottonTotal Rate
SampleVariety(lb ai/A)
Timing a

a

CA 1

Prima

0.268

CA 2

Acala

0.260

CA 3

Acala

0.261

CA 4

ST 5115
GLT

0.271

CA 5

ST 5115
GLT

0.268

CA 6

Acala

0.267

Replicate
1
10.1
1.54
1.54
1.54
1.54
1.54
4.80
1.54
1.54
51.1
5.24
3.79
7.84
3.75
3.49
64.4
9.26
--c

3
10
17
7
15
22
9
16
22
7
14
20
7
14
20
5
13
20

DN Residue (ppb)b
Replicate
Replicate
2
3
6.90
8.86
1.54
1.54
1.54
1.54
9.28
9.43
1.54
1.54
1.54
4.54
69.7
5.17
1.54
1.54
1.54
1.54
6.11
14.6
44.1
9.61
3.65
5.05
35.3
13.6
5.44
10.2
3.42
4.12
47.1
55.6
16.6
6.92
6.43
5.97

Mean
9
2
2
7
2
2
27
2
2
24
20
4
19
6
4
56
11
6

Number of days after final application.
For the purpose of calculating means, values below the LOQ (3.08 ppb) were set to half of the LOQ(1.54 ppb).
c
The mass of this sample was too small for analysis. The mean is based on two replicates.
b

18
1107

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50198501

CDPR Dino Cotton

Table 14. Dinotefuran Residues in Cotton Pollen.
Trial
CottonTotal Rate
SampleVariety(lb ai/A)
Timing a

a

CA 1

Prima

0.268

CA 2

Acala

0.260

CA 3

Acala

0.261

CA 4

ST 5115
GLT

0.271

CA 5

ST 5115
GLT

0.268

CA 6

Acala

0.267

Replicate
1
14757
8610
8212
370
114
34.2
71.3
15.2
42.2
672
686
401
886
183
23.3
201
132
48.6

3
10
17
7
15
22
9
16
22
8
15
20
8
14
20
5
13
20

Number of days after final application.

19
1108

Dinotefuran Residue (ppb)
Replicate
Replicate
2
3
18200
20676
5722
6073
6968
5817
658
742
92.0
13.8
16.1
42.3
314
374
61.0
148
39.4
26.3
347
265
545
419
781
445
641
849
164
389
82.1
50.8
141
146
31.6
83.1
41.9
20.9

Mean
17878
6802
6999
590
73
31
253
75
36
428
550
542
792
245
52
163
82
37

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50198501

CDPR Dino Cotton

Table 15. UF Residues in Cotton Pollen.
Trial
CottonTotal Rate
SampleVariety(lb ai/A)
Timing a

a

CA 1

Prima

0.268

CA 2

Acala

0.260

CA 3

Acala

0.261

CA 4

ST 5115
GLT

0.271

CA 5

ST 5115
GLT

0.268

CA 6

Acala

0.267

Replicate
1
2354
2759
3693
197
146
48.1
38.2
20.7
49.9
219
473
370
124
70.2
18.5
94.5
200
111

3
10
17
7
15
22
9
16
22
8
15
20
8
14
20
5
13
20

Number of days after final application.

20
1109

UF Residue (ppb)
Replicate
Replicate
2
3
2802
3424
1866
2105
2991
2523
343
664
122
25.2
48.2
42.0
93.5
130
32.8
101
48.2
31.7
137
114
287
175
565
463
116
113
73.2
145
42.9
34.3
73.6
70.4
46.3
80.7
66.7
26.8

Mean
2860
2243
3069
401
98
46
87
52
43
157
312
466
118
96
32
80
109
68

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50198501

CDPR Dino Cotton

Table 16. DN Residues in Cotton Pollen.
Trial
CottonTotal Rate
SampleVariety(lb ai/A)
Timing a

a

CA 1

Prima

0.268

CA 2

Acala

0.260

CA 3

Acala

0.261

CA 4

ST 5115
GLT

0.271

CA 5

ST 5115
GLT

0.268

CA 6

Acala

0.267

Replicate
1
1718
2500
3017
268
229
56.8
47.2
40.8
76.6
288
758
537
128
96.1
21.4
52.3
130
101

3
10
17
7
15
22
9
16
22
8
15
20
8
14
20
5
13
20

DN Residue (ppb)
Replicate
Replicate
2
3
2265
2336
1753
1473
2630
1540
605
488
220
33.6
79.9
68.5
116
166
78.0
253
175
67.5
166
114
479
270
877
803
88.4
111
75.7
167
63.4
40.1
42.3
46.5
27.4
60.0
56.4
24.0

Mean
2106
1909
2396
454
161
68
110
124
106
189
502
739
109
113
42
47
72
60

Number of days after final application.

7. STATISTICAL ANALYSISCOMPARISON OF DISTRIBUTION BETWEEEN PARENT AND DEGRADATION PRODUCTS:
Comparison of the relative mean concentrations and associated distribution statistics for parent
dinotefuran and degradation products are presented in Table 17 for leaf samples, Table 18 for extrafloral nectar samples, Table 19 for floral nectar, and Table 20 for pollen samples. The distributions were
derived from all data that were collected as it reflects the actual distribution measured within andbetween fields. When values were below the analytical limit of detection (LOD) the authors reported
values at one-half the respective LOD.
LEAVES: Mean parent dinotefuran residue at the first pre-bloom sampling interval was less than 50% of
the mean total residue (Table 17 and Figure 1). At pre-bloom, the leaf samples were harvested after the
first application of dinotefuran but prior to the second application. Theoretically, concentrations of UF
and DN degradation products were low in the product that was applied, so their presence at thissampling interval indicates rather rapid dissipation of parent dinotefuran. At the post-bloom samplinginterval, which was approximately 28 days after the second application, parent dinotefuran was only 9%of the total residue; DN degradation product accounted for 62% of the total residue and UF degradation
product was at 29% of total residue. Although the degradation products represented a greater portionof total residue over time, the total amount was also decreasing where, at the post-bloom interval, themean total residue was 45% of the residue value measured at early-bloom. The relative percentage of
DN steadily increased throughout the sampling intervals that succeeded the early-bloom interval; DN
211110

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50198501

CDPR Dino Cotton

residues were at 30% at early-bloom rising to 62% of the total residue at post-bloom sampling. Thiseffect was due to the stability of DN residues as compared to parent dinotefuran and UF degradateconcentrations that were decreasing over time (Figure 1).

EXTRA-FLORAL NECTAR: Mean concentration of parent dinotefuran at the pre- and early-bloom
sampling times was measured at approximately 75% of the mean total residue, which was a greater
percentage than measured in leaf samples (Table 18 and Figure 2). At the subsequent mid-bloom
sampling time, the relative amount of parent dinotefuran dropped to approximately 50% of the total
residue with UF and DN degradates at essentially equal percentages. This pattern was reflected
throughout the rest of the sampling times. Total amount of residue decreased over time where at the
post-bloom interval concentrations had decreased to 2% of the amount measured at the early-bloom
sampling interval. Compared to leaves, the relative distribution of residues appeared stable between thesampling times, but the overall dissipation rate was faster. Similarly, relative concentration between
residues from mid- to post-bloom indicated that all residues rapidly dissipated over time.
FLORAL NECTAR: Many of the analyses for the degradate DN were lower than the limit of detection at
the mid- and late-bloom sampling intervals where all replicates for 3 of the 6 trials were assigned half
the respective LOD values. Even though values were inserted into the data set, the resulting statistics
should reflect the distribution and potential dissipation of residues over time. Parent dinotefuran at thefirst early-bloom sampling period was the major portion of residue measured at 69% of mean total
residue (Table 19 and Figure 3). The relative amount dropped to approximately 55% of total residue atthe mid- and late-bloom sampling periods. The UF degradation residue was nearly twice theconcentration of the DN degradate residue throughout all sampling periods. Mean total residue
concentration at late-bloom had decreased rapidly and was at only 9% of the value measured at earlybloom.
POLLEN: The pattern in pollen was similar to that observed in floral nectar where mean parent
dinotefuran was the major portion of residue at 75% of mean total residue when sampled at early
bloom (Table 20 and Figure 4). The portion of parent residue dropped to approximately 55% of the total
residue at the mid- and late-bloom sampling periods. The pattern for UF and DN degradation products
differed from that observed for leaves in that they were measured at essentially equal portions
throughout the sampling intervals. The decrease of mean total residue concentration was slower thanobserved for nectar where the mean concentration at late-bloom was 55% of the value measured atearly-bloom.
STATISTICAL ANALYSIS FOR EFFECT OF TIME AND SOIL TEXTURE CATEGORY:
The six trial sites were evenly split between coarse- and fine-texture soil categories. Analysis of variance
was conducted to determine if there was an overall difference in concentration of residues between thetwo soil texture categories, to verify the dissipation of residues over time, and to determine if thedissipation rate was similar between plants grown in the two texture categories. Since plants from thepre-bloom sampling interval had been exposed to only 1 application of dinotefuran, this data was
excluded from the analysis. Tests for normality of distributions in each plant sample indicated thattransformation to natural logarithms was effective in providing a shift to a normal distribution.
Estimates of dissipation half-lives were determined from the regression coefficient calculated for thesignificant linear effect of time. When data are expressed as natural logarithms, half-life values are
determined according to Equation 1:
Equation 1:

Half-life (days) = log(2)/R

R=regression coefficient for linear effect of time
221111

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50198501

CDPR Dino Cotton

A general result from the analysis of variances was that there was no indication of interaction between
soil type and dissipation rates. This means that the dissipation rate was similar between plant samples
from coarse- and fine-textured soil, even when an effect of soil type was indicated. Furthermore, only
the linear effect of time was consistently significant so dissipation half-lives were calculated from that
estimated parameter, according to Equation 1. Only results for soil type and the linear effect of time are
discussed (ANOVA results are given in Table 21A and estimated dissipation half-lives in Table 21B). The
degradation of parent residues, the appearance of degradation products, and their subsequentdegradation is complex requiring an understanding of the formation and breakdown rate of separate
residues. For studies that cover short time intervals, such as this study, formation of degradation
products generally indicates increases in concentration for a chemical that is relatively long-lived in theenvironment. Calculation of a dissipation half-life for DN and UF degradation products within the timespan of this study is just an indication that these chemicals are relatively short-lived in the environment.
LEAVES: A significant effect of soil type was indicated for all residues except for parent dinotefuran
where concentrations were greater in plants grown in fine-textured soils (Table 21A and Figure 1). This
result appears opposite to previous reports for other neonicotinoids where plants grown in coarsetexture soil exhibited higher concentrations. Significant dissipation was indicated for parent dinotefuran
and the degradate UF as well as for the total residue. Stability of DN residues was previously indicatedwhere it was noted that its relative portion in total residue appeared to increase over the sampling
periods. The half-life value for total residue was estimated at approximately 20 days with rates of 8 and
17 days and no dissipation for parent dinotefuran, UF ,and DN, respectively (Table 21B) .
EXTRA-FLORAL NECTAR: In contrast to leaves, there was no significant effect of soil type soconcentrations were similar between plants grown in coarse- and fine-textured soils (Table 21A).
Although a graphical comparison indicated potentially greater concentrations in coarse-textured soil,
data generated for site CA1 were extremely high compared to the other sites (Tables 2, 8, 9 and 10 and
Figure 2). Removal of trial 1 resulted in similar graphs between the soil types. Dissipation half-live values
were rapid for all residues, which were measured at 5, 4, 5,and 5 days for total residue, parent
dinotefuran, and UF and DN degradates, respectively (Table 21B).
FLORAL NECTAR: A significant difference in concentration for soil type was indicated only for parent
dinotefuran residues where residues appeared greater in coarse-textured soil (Table 21A). Rapid
dissipation was again measured in all residues with half-life values of 4, 3, 5, and 5 days for total residue,
parent dinotefuran, and UF and DN degradates, respectively (Table 21B).
POLLEN: Results for pollen were in stark contrast to the other residues because no effect was indicatedfor soil type or for dissipation of residues (Table 21A). Again, the graphical comparison between soil typeindicates potentially greater concentrations in coarse-textured soil, but as previously indicated, removalof data from trial CA1 results in similar plots (Tables 14, 15, and 16).
MAGNITUDE OF RESIDUES IN BEE-RELEVANT MATRICES
Concentration of total residues at early bloom at site CA1 were within the range measured at the othersites, indicating that exposure to the dinotefuran applications was not extraordinary at this site (Figure5). The concentrations measured for extra-floral nectar and pollen did indicate greater concentrations at
CA1 site than at the other 5 sites. These patterns were similar at subsequent sampling periods for eachplant sample. Difference in distribution between plant samples could be due to normal variability of sitevariables, or as the authors indicate, could be due to the use of the Pima variety of cotton at site CA1 as
23
1112

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50198501

CDPR Dino Cotton

it may have different phenotypical attributes than the highland varieties planted at the other trial sites.
The distribution of residues collected from all sites will be discussed because the range in values reflectvariance that may be caused by specific site environmental variables or by phenotypic differences due toplant variety. As indicated in the analysis for dissipation rate over time, the concentrations measured atearly-bloom reflected the highest range in expected values for parent and degradation products inleaves, extra-floral nectar, and floral nectar. Analyses for pollen differed from the other plant samples
because analyses indicated stability in residues over the time interval of this study. Figures 6 and 7
present a comparison of the distribution of the triplicate measures for floral nectar and pollen at eachtrial site. For nectar samples, a decrease in concentration of total residue is evident at each site whereas
there is overlap of the bar charts overtime for pollen samples at each trial site. The range in values
measured for each bee-relevant matrix will be indicated for the early-bloom sampling period. Potential
exposure scenarios should factor-in the rapid dissipation previously observed for extra-floral and floral
nectar samples.
EXTRAFLORAL NECTAR: The maximum, 90th, and median values measured for parent dinotefuran
residues at early-bloom were 15500, 10500, and 499 ug/kg, respectively. Based on the mean values,
parent dinotefuran was the predominant residue at this time where it was 73% of the total residue.
Maximum, 90th and median values for the UF degradate were 1390, 1360, and 141 ug/kg, and for the DN
degradate were 1080, 996, and 101, respectively. As indicated, the magnitude of residues in extra-floral
samples decreased overtime, with the degradation products comprising a larger portion of the total
residue. Residues were still measured in samples at post-bloom, which occurred 21 days after the
sampling event at early-bloom.
FLORAL NECTAR: Concentrations of residues were much lower in floral nectar than in extra-floral nectar
samples. The maximum, 90th, and median values measured for parent dinotefuran residues at early
bloom were 346, 328, and 125 ug/kg, respectively. Based on the mean values, parent dinotefuran was
the predominant residue at this time where it was 69% of the total residue. Maximum, 90th and medianvalues for the UF degradate were 198, 140, and 27 ug/kg and for the DN degradate were 70, 64, and 10,
respectively. The concentration and distribution in residues over time also reflected the patternmeasured in extra-floral nectar where concentration decreased overtime, and where the degradation
products comprised a greater portion of total residue over time.
POLLEN: Concentrations of residues measured in pollen samples ranged even higher than thosemeasured for extra-floral nectar. The maximum, 90th, and median values measured for parent
dinotefuran residues at early-bloom were 20676, 18200, and 508 ug/kg, respectively. Similar to thepattern noted in the other bee-relevant samples, parent dinotefuran was the predominant residue atthis time where it was 75% of the total residue. Maximum, 90th and median values for the UF degradate
were 3424, 2802, and 127 ug/kg and for the DN degradate were 2336, 2265, and 147, respectively.
Concentrations in pollen were more stable overtime as reflected by nonsignificant regressions and thelarge amount of overlap in bar charts comparing the distribution of concentration at each trial over the
sampling events (Table 21A and Figure 7). The pattern for distribution of residues did reflect reduction in
the relative amount of parent dinotefuran over time where it was reduced to around 55% of the total
residue at mid- and later-bloom sampling.

24
1113

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50198501

8. STUDY STRENGTHS, LIMITATIONS AND CONCLUSIONSCDPR Dino Cotton

In the context of documenting the magnitude of dinotefuran residues in bee-related matrices of cotton
plants, the following strengths are observed with this study.
1. Data provide quantitative values for parent dinotefuran and the UF and DN degradation
products expected in pollen, extra-floral nectar, floral nectar, and leaves of various varieties ofcotton.
2. Sequential sampling over the blooming period provided a basis to determine potential
dissipation half-live rates for the residues.
3. Although data were generated from only 6 of 9 requested sites, the values indicate substantial
exposure to dinotefuran residues in bee-relevant matrices.
4. Two soil types were evenly distributed among the sites where 3 were located in coarse-texture
soil and 3 in fine-textured soil, allowing for a comparison of the effect of soil type on
concentrations measured in plant samples.
Limitations noted in this study include:
1. Samples were taken from a mixture of plant varieties. Since the effect of different varieties on
distribution of residues is unknown, the results reflect general observations made to all planted
cotton crops.
2. Data for only 6 of 9 requested sites in the DCI were submitted.
3. Three of the six nontreated control pollen samples (trial locations -01, -04 and -05) contained
measurable amounts of dinotefuran, UF and DN. For trial -01, the dinotefuran residue in the
non-treated pollen sample (LA16-12) was 782 ppb vs. a mean residue of 17,878 ppb in pollen
from the treated plots; for trial -04, dinotefuran residue in the non-treated pollen sample (LA16198) was 23.3 ppb vs. a mean residue of 428 ppb from pollen in the treated plots; and for trial 05, dinotefuran residue in the non-treated pollen sample (LA16-260) was 40.5 ppb vs. a mean
residue of 792 ppb from pollen in the treated plots. It is unclear if the nature of the control
contamination may have had an effect on the magnitude of residues in the treated plots.
Overall, considering the strengths and limitations of this study, the following conclusions can be drawn:
Classification/Utility for Bee Risk Assessment. This study is classified as acceptable. It provides thedinotefuran, UF and DN residues in nectar, pollen, extrafloral nectar and leaves collected from cotton
that have received two foliar applications of Dinotefuran 20SG (active ingredient dinotefuran).The
residue values presented should be considered to be fully reliable.
Magnitude of Residues in Bee-relevant Matrices. Significant concentration of parent dinotefuran andUF and DN degradation products were measured in bee-relevant matrices directly following a second
foliar application to cotton. In floral nectar, the maximum, 90th, and median values measured for parent
dinotefuran residues at early bloom were 346, 328, and 125 ug/kg, respectively. In pollen, the

25
1114

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50198501

CDPR Dino Cotton

maximum, 90th, and median values measured for parent dinotefuran residues at early-bloom were20676, 18200, and 508 ug/kg, respectively.

Temporal Variability in Residues. Significant dissipation of residues over the sampling time of the study
indicated relatively rapid dissipation half-lives in extra-floral nectar, floral nectar, and leaves estimatedat 5, 4, and 20 days, respectively. Dissipation was not indicated for residues in pollen samples.
Spatial Variability in Residues. There was no consistent effect of soil type on measured concentrations
in plant samples.
Pesticide Carryover. In this study, samples were collected during a single flowering period, and thus,
was not designed to measure pesticide carryover year to year.
9. STUDY VALIDITY/CLASSIFICATIONThe study is classified as ACCEPTABLE for quantitative use in risk assessment. The data from this study
provide an expected distribution of the concentrations of dinotefuran residues that bees are exposed toin extra-floral nectar, nectar, and pollen of cotton plants grown under actual agronomic practices in
California. Relating concentrations measured in flower parts to bee health is possible by comparing theconcentrations measured in bee relevant plant parts to target values that define acute or chronic
exposure scenarios. Relatively rapid dissipation rates were estimated for residues in extra-floral nectar
and floral nectar, which could be factored into potential exposure scenarios. Dissipation of residues was
not measured in pollen samples. The study is considered scientifically sound and useful for risk
assessment purposes.

26
1115

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50198501

CDPR Dino Cotton

Table 17. Leaves: Distribution of dinotefuran residues in leaves of cotton compared between the sampling intervals. A second foliar
application of dinotefuran occurred in between the pre bloom and early bloom sampling intervals. Sampling intervals were approximately 7days apart.

StatisticN
Mean (ug/kg)
SD (ug/kg)
CV (%)Min (ug/kg)
Median (ug/kg)
75th (ug/kg)
90th (ug/kg)
95th (ug/kg)
Max (ug/kg)% of Total

UF
18
1883
664
35
140
1903
2390
2770
2800
2800

Pre-Bloom
DN Parent Total
18
18
18
1905 3266 7054
560 1097 126629
34
18
865 1715 4545
2068 3193 7038
2300 4155 8190
2575 4755 8740
2675 5450 9500
2675 5450 9500

27

27

46

UF
18
3529
1559
44
1845
3005
4765
6300
6650
6650

LeavesMid-Bloom
Early-Bloom
DN Parent Total UF
DN Parent Total UF
18
18
18
18
18
18
18
18
3720 4973 12222 2874 4176 1651 8701 1997
1506 2387 3330 1780 1917 1133 4201 1719
41
48
27
62
46
69
48
86
1375 1785 6840 1230 1725 415 3590 458
3558 4688 12120 2043 3883 1435 7653 1560
5000 6250 12885 3995 5900 2425 10475 2195
5500 8550 17375 6450 7400 3310 17065 5700
6750 8700 20350 6600 7500 3350 17210 6000
6750 8700 20350 6600 7500 3350 17210 6000

Late-Bloom
DN Parent Total UF
18
18
18
18
3275 901 6172 1584
2016 868 4241 1573
62
96
69
99
1245 74 2075 303
2803 447 4711 890
3495 1540 6125 1765
7400 2175 15275 4700
7750 2870 15330 4855
7750 2870 15330 4855

Post-Bloom
DN Parent Total
18
18
183407 509 5500
2239 484 4172
66
95
76
775
47 17292840 320 3603
4105 765 6560
7250 1260 13335
8200 1485 14340
8200 1485 14340

29

30

53

62

41

33

27
1116

48

19

32

15

29

9

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50198501

CDPR Dino Cotton

Table 18. Extra floral-nectar: Distribution of dinotefuran residues in extra floral nectar of cotton plants compared between the samplingintervals. A second foliar application of dinotefuran occurred in between the pre-bloom and early bloom sampling intervals. Samplingintervals were approximately 7 days apart.

StatisticN
Mean (ug/kg)
SD (ug/kg)
CV (%)Min (ug/kg)
Median (ug/kg)
75th (ug/kg)
90th (ug/kg)
95th (ug/kg)
Max (ug/kg)% of Total

UF
18
185
186
101
10
125
276
536
632
632

Pre-Bloom
DN Parent Total
18
18
18
121 828 1134
104 1197 1441
87
145 1275
21
36
99
460 710208 1070 1717
281 1740 2097
348 5080 6060
348 5080 6060

Extra-Floral Nectar
Early-Bloom
Mid-Bloom
UF
DN Parent Total UF
DN Parent Total
18
18
18
18
18
18
18
18
416 271 2371 3057 197 162 418 777
463 319 4160 4871 228 209 647 991
111 118 176 159 115 130 155 12836
23
105 18144
4
13
141 101 499 740
4141
125 206759 376 2670 3487 459 282 490 1302
1360 996 10500 12856 518 438 1800 2693
1390 1080 15500 17970 586 795 1920 2824
1390 1080 15500 17970 586 795 1920 2824

UF
18
37
63
171
2
10
29
115
250
250

Late-Bloom
DN Parent Total UF
18
18
18
18
33
64
133
20
59
156 25540
179 245 191 202
2
1
6
1
7
7
26
3
27
29
96
11
151 197 360
73
216 647 1048 154
216 647 1048 154

Post-Bloom
DN Parent Total
18
18
18
7
27
53
8
60
107
126 224 202
2
1
3
3
2
8
7
4
27
17
119 20234
216 40434
216 404

16

11

14

28

25

12

73

9

78

25

28
1117

21

54

48

38

50

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50198501

CDPR Dino Cotton

Table 19. Floral Nectar: Distribution of dinotefuran residues in extra floral nectar of cotton plants compared between the sampling intervals.
Plants had received two foliar sprays of dinotefuran prior to bloom. Sampling intervals were approximately 7 days apart.

StatisticN
Mean (ug/kg)
SD (ug/kg)
CV (%)Min (ug/kg)
Median (ug/kg)
75th (ug/kg)
90th (ug/kg)
95th (ug/kg)
Max (ug/kg)% of Total

UF
18
50
56
111
7
27
50
140
198
198

Floral Nectar
Early-Bloom
Mid-Bloom
DN Parent Total UF
DN Parent Total
18
18
18 18
18
18
18
48
23
167
240 14
7
27
62
23
117
182 1810
36100
70
76 130 147 130 128
8
2
25
40 2
2
4
30
10
125
187 6
3
14
61
47
274
406 15
9
45
118
64
328
505 45175670
346
590 7344
149 26770
346
590 7344
149 267

UF
17
6
6
98
1
3
11
17
23
23

Late-Bloom
DN Parent Total
17
17
17
3
11
21
16
2
11
78
55
95
2
3
8
13
3
6
4
19
40
6
29
49
52
6
36
6
36
52

21

10

31

15

69

29

14

56

29
1118

54

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50198501

CDPR Dino Cotton

Table 20. Pollen: Distribution of dinotefuran residues in pollen of cotton flowers compared between the sampling intervals. Plants hadreceived two foliar sprays of dinotefuran prior to bloom. Sampling intervals were approximately 7 days apart.

StatisticN
Mean (ug/kg)
SD (ug/kg)
CV (%)Min (ug/kg)
Median (ug/kg)
75th (ug/kg)
90th (ug/kg)
95th (ug/kg)
Max (ug/kg)% of Total

UF
18
617
1058
171
38
127
343
2802
3424
3424

Pollen
Early-Bloom
Mid-Bloom
DN Parent Total UF
DN Parent Total UF
18
18
18 18
18
18
18
18
503 3351 4470 485 480 1305 2270 621
762 6767 8569 832 705 2594 4115 1156
152 202
192 172 147 199 181 186
73
19
42
71
157 2127
14
49
147 508
840 134 194 156 476
488 849 1606 287 479 545 1311 463
2265 18200 23267 2105 1753 6073 9651 2991
2336 20676 26436 2759 2500 8610 13869 3693
2336 20676 26436 2759 2500 8610 13869 3693

Late-Bloom
DN Parent Total
1818
18
569 1283 2472
919 2670 4720
162 208 191
63
21
16
78
45
179
803 445 1711
2630 6968 12589
3017 8212 14922
3017 8212 14922

14

11

23

75

21

21

30
1119

57

25

52

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50198501

CDPR Dino Cotton

Table 21. Results of regression analyses measuring the linear effect of time and soil type on
concentration of dinotefuran residues in cotton plant samples. Data from the pre-bloom samples
were not included because plants had not received full foliar treatments. Table A reports thestatistical significance measured for each effect and Table B reports the estimated half-lives
determined from the regression coefficient for the linear effect of time, according to Equation 1.

A.
Plant Sample andEffectRegression Probability Levels for EffectTotal Residue

Dinotefuran

UF Degradate

DN Degradate

Soil Type

0.001

NS

0.001

NS

Time - Linear

0.001

0.001

0.001

0.001

Soil Type

NS

NS

NS

NS

Time - Linear

0.001

0.001

0.001

0.001

Soil Type

NS

0.04

NS

NS

Time - Linear

0.001

0.001

0.001

0.001

Soil Type

NS

NS

NS

NS

Time - Linear

NS

NS

NS

NS

LeavesExtra-Floral Nectar

Floral Nectar

PollenB.
Estimated Dissipation Half-life (Days):
Plant Sample

Total Residue

Dinotefuran

UF Degradate

DN Degradate

Leaves

19.8

7.5

16.6

NS

Extra-Floral Nectar

4.6

3.8

5.3

5.3

Nectar

4

3.4

5

5.3

Pollen

NS

NS

NS

NS

31
1120

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50198501

CDPR Dino Cotton

Figure 1. Leaves: Distribution of dinotefuran residues in cotton leaves measured at 5 sampling intervals and in plants grown in coarse or finetextured soil. Total residue is the summation of parent dinotefuran and UF and DN degradation products. A second foliar application ofdinotefuran occurred in between the pre-bloom and early-bloom sampling intervals. Sampling occurred at approximately 7 day intervals.

32
1121

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50198501

CDPR Dino Cotton

Figure 2. Extra-Floral nectar: Distribution of dinotefuran residues in extra-floral nectar of cotton plants measured at 5 sampling intervals andin plants grown in coarse or fine-textured soil. Total residue is the summation of parent dinotefuran and UF and DN degradation products. A
second foliar application of dinotefuran occurred in between the pre-bloom and early-bloom sampling intervals. Sampling occurred at
approximately 7 day intervals.

33
1122

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50198501

CDPR Dino Cotton

Figure 3. Floral nectar: Distribution of dinotefuran residues in floral nectar of cotton plants measured at 5 sampling intervals and in plants
grown in coarse or fine-textured soil. Total residue is the summation of parent dinotefuran and UF and DN degradation products. Plants hadreceived two foliar sprays of dinotefuran prior to bloom. Sampling occurred at approximately 7 day intervals.

34
1123

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50198501

CDPR Dino Cotton

Figure 4. Pollen: Distribution of dinotefuran residues in pollen of cotton plants measured at 5 sampling intervals and in plants grown in coarse
or fine-textured soil. Total residue is the summation of parent dinotefuran and UF and DN degradation products. Plants had received two
foliar sprays of dinotefuran prior to bloom. Sampling occurred at approximately 7 day intervals.

35
1124

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document
MRID 50198501

CDPR Dino Cotton

Figure 5. Comparison of the distribution of total dinotefuran residue between plant samples taken at early-bloom. Each site had 3
replicate samples so the extremes in the bar chart indicate the minimum and maximum values and within the box the diamond is themean and the line is the median of the 3 values.

36
1125

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50198501

CDPR Dino Cotton

Figure 6. Nectar: Comparison of the distribution of total dinotefuran residue measured at each sampling interval for each trial site.
Data are express on natural logarithm scale. Each site had 3 replicate samples so the extremes in the bar chart indicate the minimum
and maximum values and within the box the diamond is the mean and the line is the median of the 3 values.

37
1126

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50198501

CDPR Dino Cotton

Figure7. Pollen: Comparison of the distribution of total dinotefuran residue measured at each sampling interval for each trial site.
Data are express on a natural logarithm scale. Each site had 3 replicate samples so the extremes in the bar chart indicate theminimum and maximum values and within the box the diamond is the mean and the line is the median of the 3 values

38
1127

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

MRID 50198501

CDPR Dino Cotton

10. REFERENCES
1.

Daorong Guo, “Residue Analysis of Dinotefuran, DN and UF in Nectar, Pollen, Leaves and Soil by
LC-MS/M”, Method No. RA046, Eurofins Agroscience Services, Inc. Lancaster, PA, 20 Dec 2016.

2.

Daorong Guo, “Method Validation of Residues of Dinotefuran, DN and UF in Nectar, Pollen,
Leaves and Soil Matrices”, Study No. S16-05692, Eurofins Agroscience Services, Inc. Lancaster,PA, 20 Dec 2016

39
1128

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

Year/Authors/Title
Lamore M. 2016.
Quantification of Dinotefuran
and its Metabolites in Beecollected Nectar, Pollen,
Leaves and Soil Following Soil
Application of Dinotefuran 20
SG to Bell Pepper.
(S16-01167)

Study Type
NonGuidelinefield residue
study on bell
peppers toestablish
dinotefuran
andmetabolite
levels in beecollected
nectar,pollen,leaves, andsoil following
soil
applications

Summary
Notes/Uncertainties
Uncertainties: None that
This study quantified Dinotefuran residues in bell pepper plants(Capsicum anuum) grown in three locations: North Carolina (NC; Sandy Loam), would affect the integrity ofGeorgia (GA; Sand), and California (CA; Sandy Loam). Three replicate plots were the study
used in each location and each plot received two soil applications: one
application at a rate of 0.206 lbs ai/A and the second application, seven days
later, at a rate of 0.330 lbs ai/A. The label for Dinotefuran 20SG prohibits soil
and foliar applications to the same plot. In all three locations, soil and foliar
application rates and reapplication intervals were sufficient to represent a worst
case scenario. Analyses of fortified samples of pollen (80-127%) and nectar (93106%) were all within acceptable limits. Pollen and nectar samples werecollected by bumble bees. Bumble bees were caught using a net and placed in a
jar with dry ice. Nectar was harvested via dissection of the bumble bees honeystomachs. Pollen was collected from bumble bee pollen baskets. If pollen
samples collected from bees were less than 300mg, then those samples were
supplemented with anthers collected from fresh flowers. Mesh tunnel tents
were used to ensure bumble bees did not forage on other crops and that otherpollinators did not contaminate the bell peppers.

1129

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

Year/Authors/Title
Louque, R. 2016. Quantitationof Residues of Dinotefuran in
Nectar, Pollen and Leaves
Following Foliar Application
and Trunk Injection of
Dinotefuran 20 SG to Cherry
Trees.
Smithers Viscient Study
Number 10934.4105

Study Type
NonGuidelinefield residue
study oncherry toestablish
dinotefuran
andmetabolite
levels in
nectar,pollen, andleavesfollowing
foliarapplications
and trunk
injections

Summary
Notes/Uncertainties
This study quantified Dinotefuran residues in cherry (Prunus spp.) grown in
The extremely low recovery
three locations: California (CA; clay loam), New York (NY; sandy loam), andduring analysis of one of theOregon (OR; sandy loam). Three replicate plots were used for each type of
fortified pollen samples
application in each location and each plot received either two foliarpresents uncertainty in terms
applications, or one trunk injection, of Dinotefuran 20SG. The foliar applications of the accuracy of thewere conducted at nominal rates of 0.232 lbs ai/A and 0.304 lbs ai/A. The first
analyses conducted. This low
foliar application was scheduled for late in the season (September), before leaf recovery was noted in one ofdrop, and the second foliar application was made seven days after the first.
the nine fortified pollen
Dinotefuran 20SG is not currently registered for use on cherry trees. The closest samples analyzed as part ofrelated plant that is registered on the label for Dinotefuran 20SG is peach, which the quality control
is in the same genus as cherry (Prunus spp.). The maximum single foliar
procedures. The pollen
application rate for peaches is 0.180 lbs ai/A and the maximum seasonal foliar
samples was fortified to aapplication rate is 0.270 lbs ai/A/season. In the section for peaches, the label
level of 99.7 ppb, but thealso contains the following note: “Regardless of application method, do not
analysis resulted in aapply more than a total of 1.8 lbs. of DINOTEFURAN 20 SG (0.360 lb. a.i.) per
measurement of 4.70 ppb
acre per season.” Trunk injection applications were also conducted late in the(4.72%). If a similar error
season (September), before leaf drop, at a rate of 2 g of product per inch ofoccurred during analysis oftrunk diameter either at breast height or right below the first trunk bifurcation. samples then the values
Samples of pollen and nectar were collected 165-170, 190-199, and 236-243
presented for Dinotefurandays after the last application in OR, CA, and NY, respectively. Analyses ofresidues in pollen might befortified samples of pollen (94.3-118%) and nectar (76.2-113%) were all within
20x higher than reported.
acceptable limits except for one pollen sample in which the analytical results
were only 4.72% of the fortification level. The maximum measured dinotefuran
residues resulting from foliar applications were 200 ppb in pollen (237 days
after the last application) and 25 ppb in nectar (238 days after the last
application). The maximum measured dinotefuran residues resulting from tree
injection applications were 31,688 ppb in pollen (201 days after application) and
17,484 ppb in nectar (237 days after application). This study is acceptable, but
the low recoveries suggest that some of the residues in pollen might be as muchas 20x higher than reported.

1130

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

Year/Authors/Title
Lange, B. 2017.
Thiamethoxam 25WG
(A9584C) – Determination of
Residues in Leaves, Flowers,
Pollen, and Nectar ofBlueberry After Foliar
Application
Lange Research Study
Number: LR16191
Report Number: TK0250072

Study Type
Summary
Non-Guideline This study quantified thiamethoxam and CGA322704 residues in blueberry
field residue(Vaccinium corymbosum) grown in three locations: California (CA; sand),study onQuebec (QC; loam), and Washington (WA; loamy sand). Three replicate plots
blueberry towere used in each location. One set of plots received three foliar applications
establishat 19, 12, and 5 days before bloom at a nominal rate of 0.063 lbs. ai/A, andthiamethoxam another set of plots at each location received a single foliar application 15 days
andbefore bloom at a nominal rate 0.063 lbs. ai/A. Nectar and pollen were
metabolite
sampled at early-, mid-, and late-bloom. Samples of pollen and nectar wereconcentrations collected 5-22, 5-11, and 12-24 days after the last of the three application inin whole
CA, QC, and WA, respectively. In those plots receiving a single application,flowers, leaves pollen and nectar were collected 14-31, 19-25, and 22-34 days post-applicationand manually- in CA, QC, and WA, respectively. Analyses of fortified samples of pollen (75.1collected
101% thiamethoxam and 78.6-102 for CGA322704) and nectar (77.4-96.9%
nectar andthiamethoxam and 85.8-101 for CGA322704) were all within acceptable limits.
pollenNectar and pollen samples were manually collected from flowers. Mean
followingthiamethoxam residues in nectar from plots receiving repeated applications
foliaracross all locations (118 ppb) were less than CGA322704 residues (142 ppb) inapplications
the early bloom samples but thiamethoxam residues were comparable to
CGA322704 by the late-bloom samples (51.2 ppb vs. 59.1 ppb, respectively).
Mean thiamethoxam residues in pollen from plots receiving repeatedapplications (370 ppb) were notably greater than CGA322704 residues (60.2
ppb) in the early bloom samples and thiamethoxam residues remained greaterthan CGA322704 in the late-bloom samples (156 ppb vs. 48.4 ppb,
respectively). Mean residues for thiamethoxam in nectar were greatest acrossall sample periods in QC with residues comparable in CA and WA. Mean
residues for thiamethoxam in pollen were greatest across all sample periods in
WA with residues greater in QC than in CA. Mean concentrations of
CGA322704 in nectar were greatest in QC with residues comparable in CA andWA. Mean concentrations of CGA322704 in pollen were greatest across all
sample periods in QC with residues greater in WA than in CA.

1131

Notes/Uncertainties
Uncertainties: None thatwould affect the integrity ofthe study

Appendix 10. Evaluations of Residue Studies Included in this Risk Determination Document

U.S. EPA Data Evaluation Reports (Dinotefuran):
U.S. EPA. (2016). Data evaluation report: amended final report - quantitation of residues ofdinotefuran in nectar, pollen, and laves after foliar treatment application of Dinotefuran 20 SG tocranberries. Washington, D.C.: Author. Laboratory Report Number 10934.4101.
U.S. EPA. (2016). Data evaluation report: amended final report - quantitation of residues ofdinotefuran in pollen and leaves following soil application of Dinotefuran 20 SG to potato.
Washington, D.C.: Author. Laboratory Report Number 10934.4100.
U.S. EPA. (2016). Data evaluation report: quantitation of residues of dinotefuran in nectar,
pollen and leaves following soil application of Dinotefuran 20 SG to pumpkin. Washington,
D.C.: Author. Laboratory Report Number 10934.4104.
U.S. EPA. (2016). Data evaluation report: amended final report - quantitation of residues ofdinotefuran in pollen and leaves following soil and foliar application of Dinotefuran 20 SG to
Tomato. Washington, D.C.: Author. Laboratory Report Number 10934.4103.

1132

Appendix 11. Descriptive Statistics Derived from the Residue Studies
Included in this Risk Determination Document
The following tables present descriptive statistics derived from the residue studies used to characterize
exposure. Study identification numbers are provided, along with the crop, in the header of each table. For
Imidacloprid and Thiamethoxam, the total concentration values (in the “Total” column) are not simply thesum of the parent molecule and metabolites. Rather, the total values were determined from the summationof the concentrations of the parents and metabolites measured on each individual sample. Since exact
values, such as the maximum value, may not be measured on the same individual sample for eachchemical, the distribution for total concentration will not necessarily match up to simple addition ofconcentrations noted for each individual chemical. Section 2.3 of the main document provides additional
information regarding the toxicity of various metabolites. In addition, for a review of each study, pleaserefer to Appendix 10.

Imidacloprid (IMI):
Tomato (EBNTN012)
PollenStatisticN

5-OH

Olefin

IMI

Total

113.0

113.0

113.0

113.0

Mean (ng/g)
SD (ng/g)

8.4
12.1

2.8
3.7

166.4
268.5

177.7
282.8

CV (%)Min (ng/g)
Median (ng/g)
75th (ng/g)
90th (ng/g)
95th (ng/g)
Max (ng/g)

143.6
0.3
3.6
9.8
24.1
37.2
63.5

129.2
0.2
1.0
4.1
7.7
9.6
24.1

161.3
1.4
57.7
162.3
449.8
632.8
1679.7

159.0
1.8
65.2
174.5
476.9
679.3
1762.51133

Appendix 11. Descriptive Statistics Derived from the Residue Studies
Included in this Risk Determination Document
Citrus (EBNTY007)
NectarPollenStatisticN

5-OH

Olefin

IMI

Total

5-OH

Olefin

IMI

Total

67

67

67

67

53

53

53

53

Mean (ng/g)

7.1

4.4

84.3

95.9

117.4

150.9

1751.62020

SD (ng/g)

3.9

2.4

83.7

89.3

53.5

68.4

940.2

1056

CV (%)

54.5

54.9

99.2

93.1

45.5

45.3

53.6

52.2

Min (ng/g)

1.4

1.1

7.6

10.1

7.4

9.5

67.4

86.5

Median (ng/g)

6.1

3.8

50.4

59.4

132.4

169.6

1752

2061.375th (ng/g)

9

6

107.8

119.8

148.3

196

2324

2629.290th (ng/g)

13.9

8.7

243

267.1

177.6

232.7

2846

3257.995th (ng/g)

15

9.5

253.9

280.9

199.1

238.8

3556

3973.2

Max (ng/g)

19.9

10

408.7

431.2

210.4

252.9

3705

4142.1

Citrus (EBNTL056-07)
NectarStatistic
N

5-OH
95

Olefin
95

IMI
95

Total

Mean (ng/g)

1.6

1.6

7.3

10.6

SD (ng/g)

1.8

1.8

7.5

10.4

CV (%)

111.2

111.4

103.1

98.9

Min (ng/g)

0

0.1

0.1

0.3

Median (ng/g)

0.4

0.8

3.3

4.7

75th (ng/g)

3.2

2.5

12.7

19.4

90th (ng/g)

4.3

4.3

18.6

25

95th (ng/g)

5.4

5.6

20.9

29.1

Max (ng/g)

6.4

6.9

33.8

39.9

95

1134

Appendix 11. Descriptive Statistics Derived from the Residue Studies
Included in this Risk Determination Document
Apple (EBNTN014)
NectarPollenStatisticN

5-OH

Olefin

IMI

Total

5-OH

Olefin

IMI

Total

33

33

33

33

34

34

34

34

Mean (ng/g)

1.1

0.6

0.8

2.6

1.1

4.2

18.7

24.1

SD (ng/g)

4.4

0.5

1.5

6.1

2.3

9.4

22.2

26.5

CV (%)

381.7

90.1

184.8

229.2

200.2

224

118.6

109.8

Min (ng/g)

0.35

0.3

0.15

0.8

0.25

0.15

0.7

1.1

Median (ng/g)

0.35

0.3

0.4

1.25

0.5

0.45

11.7

15.18

75th (ng/g)

0.35

0.8

0.9

2.05

1.1

4.2

20.5

30.05

90th (ng/g)

0.7

1.6

1.2

3.45

2.4

9.5

45.9

58.5

95th (ng/g)

0.9

2

2.8

4.05

3.6

14.7

91.3

102.7

Max (ng/g)

26

2.5

8.9

36.3

13.9

52.1

92.8

103.4

Stone Fruit (EBNTN013)
NectarPollenStatistic
N

5-OH
34

Olefin
34

IMI
34

Total

Olefin
30

IMI
30

Total

34

5-OH
30

Mean (ng/g)

2.7

0.6

1.6

4.8

1.8

3.2

44.8

49.7

SD (ng/g)

6.8

0.6

2.4

7.7

1.9

7.5

66.8

71.7

CV (%)

255

104

153

161

105

236

149

144

Min (ng/g)

0.4

0.3

0.2

0.8

0.3

0.2

1.9

2.3

Median (ng/g)

0.4

0.3

0.4

1.7

1.3

1.3

23

26.9

75th (ng/g)

1.2

0.7

1.2

6.8

2.6

2.7

52.3

54.9

90th (ng/g)

5.5

0.8

5.9

9.5

3.8

4

124.9

136.2

95th (ng/g)

28.7

1.4

7.1

32

4

14.3

144.7

187.8

Max (ng/g)

28.8

3.5

8.9

33.6

9.4

40.3

328

341.3

1135

30

Appendix 11. Descriptive Statistics Derived from the Residue Studies
Included in this Risk Determination Document
Blueberry (EBNTY006)
NectarPollen-Blueberry Flowers

Pollen-Bumble Bee

StatisticN

5-OH Olefin

IMI

Total 5-OH Olefin IMI Total 5-OH Olefin IMI Total

87

87

87

87

51

51

51

51

17

17

17

17

Mean (ng/g)

0.5

0.4

1.1

2

0.4

0.7

7.8

9

0.3

0.2

1.4

1.8

SD (ng/g)

0.3

0.2

2.3

2.6

0.4

0.8

7

7.9

0

0.1

0.9

1

CV (%)

67.2

62.6

85.3

104.4

89.9

88.1

0

46.1

64.8

52.6

Min (ng/g)

0.35

0.3

203.8 130.9
0.15
0.8

0.25

0.15

0.2

0.6

0.25

0.15

0.2

0.6

Median (ng/g)

0.35

0.3

0.15

0.8

0.25

0.4

5.2

5.8

0.25

0.15

1.4

1.8

75th (ng/g)

0.35

0.3

0.8

1.95

0.25

1.1

11.9

14.5

0.25

0.15

1.6

2

90th (ng/g)

0.8

0.7

3.2

4.6

0.9

1.9

15

17.5

0.25

0.4

3

3.65

95th (ng/g)

1.3

0.9

6.2

7.45

1.3

2.5

19

22.2

0.25

0.4

3.5

4.05

Max (ng/g)

2.1

1.5

13.8

16.4

1.6

2.8

38.5

42.4

0.25

0.4

3.5

4.05

Cherry (EBNTY008)
NectarPollenStatistic
N

5-OH
65

Olefin
65

IMI
65

Total

Olefin
53

IMI
53

Total

65

5-OH
53

Mean (ng/g)

0.9

0.5

1.5

2.8

5.1

5.2

108.2

118.5

SD (ng/g)

0.9

0.5

1.3

2

5.6

7.3

182

193.5

CV (%)

106.7

155.3

87.6

70.2

107.9

139.7

168.2

163.2

Min (ng/g)
Median(ng/g)
75th (ng/g)

0.35

0.3

0.15

0.15

0.25

0.15

0.9

1.3

0.35

0.3

1.2

2.05

2.2

1.8

15.8

24.2

0.9

0.3

2

3.55

8.9

7.1

131.7

147.5

90th (ng/g)

2.3

1.1

2.8

5.05

13.8

17.7

361.6

393.8

95th (ng/g)

2.8

1.2

3.7

6.8

16

21.9

523.5

560.9

Max (ng/g)

4.4

4

7.8

10.5

22.7

32.4

965.4

1004.41136

53

Appendix 11. Descriptive Statistics Derived from the Residue Studies Included in this Risk Determination
Document
Strawberry (EBNTL056-04)
AntherBlossoms

Statistic
N

5-OH
14

Olefin
14

IMI
14

Total

Mean (ng/g)

20.7

6.1

SD (ng/g)

10.2

CV (%)

PollenOlefin
14

IMI
14

Total

14

5-OH
14

Olefin
14

IMI
14

Total

14

5-OH
14

66

92.8

18

5.5

139.3

162.7

15.1

7.5

67.9

90.5

4.7

84.2

97.1

16.7

3.7

179.5

197.6

13.3

4.3

91.1

108.4

49

78.1

127.5

104.6

93

67.5

128.9

121.4

88

57.7

134.1

119.7

Min (ng/g)

7.6

2.5

2.5

12.6

2.5

2.5

2.5

7.5

5

5

5

15

Median (ng/g)

21

4

20.25

47.4

7.2

2.5

12.25

21.95

5

5

5

15

75th (ng/g)

26

8.8

160

191.4

33

8.7

340

379.7

25

10

150

185

90th (ng/g)

35

9.5

180

224.5

40

10

420

465

33

14

200

247

95th (ng/g)

43

19

240

302

46

12

470

526

42

17

260

319

Max (ng/g)

43

19

240

302

46

12

470

526

42

17

260

319

14

Cotton (EBNTY010)
Extrafloral Nectar

Floral Nectar

PollenBlossoms

5-OH Olefin IMI Total 5-OH Olefin IMI Total 5-OH Olefin IMI Total 5-OH Olefin
StatisticN
192
192
192 192
211
211
211 211
212
212
212
215
215
212
Mean (ng/g)
0.5
0.4
4.9
0.5
0.5
7.9
0.3
0.2
3.4
1.4
1.3
5.8
8.9
3.8
SD (ng/g)
0.4
0.3
4.8
0.3
0.4
7.2
0.1
0.1
6.9
1.0
0.7
5.1
7.6
7.1
CV (%)
93.4
76.7 98.3 88.8
56.3
78.8 90.2 85.7
49.6
77.3 206.7 186.0 75.7
55.2
Min (ng/g)
0.4
0.3
0.2
0.4
0.3
1.1
0.3
0.2
0.3
1.0
1.0
1.1
1.8
0.7
Median (ng/g)
0.4
0.3
3.3
0.4
0.3
5.5
0.3
0.2
1.2
1.0
1.0
4.0
6.2
1.6
75th (ng/g)
0.4
0.3
6.5
0.4
0.3
10.4 11.5
0.3
0.2
2.8
1.0
1.0
7.3
3.3
90th (ng/g)
0.8
0.7
12.1 13.3
0.9
1.0
17.4 18.4
0.3
0.3
6.2
3.1
2.5
6.6
95th (ng/g)
1.3
1.1
14.8 16.0
1.1
1.3
25.2 27.9
0.3
0.5
16.4 16.8
4.0
2.9
Max (ng/g)
3.6
2.4
27.0 30.3
1.7
2.4
36.9 39.4
2.1
1.6
53.0 56.7
5.4
4.3

1137

IMI

Total

215
6.6
6.4
96.8
0.9
4.8
8.8
15.6
20.5
35.8

215
9.3
7.9
84.7
2.8
7.0
11.0
20.0
26.5
45.1

Appendix 11. Descriptive Statistics Derived from the Residue Studies Included in this Risk Determination
Document
Cotton (EBNTN011)
Floral Nectar

Extrafloral Nectar
StatisticN
Mean (ug/kg)
SD (ug/kg)
CV (%)Min (ug/kg)
Median (ug/kg)
75th (ug/kg)
90th (ug/kg)
95th (ug/kg)
Max (ug/kg)

Pollen5-OH

Olefin

IMI

Total

5-OH

Olefin

IMI

Total

5-OH

Olefin

IMI

Total

94
6
11.8
195.8
0.35
1.6
5.9
14.4
34.3
77

94
1.3
2.2
166.7
0.3
0.7
1.3
2.7
5.7
17.5

94
205.5
433.7
210.9
0.15
30.2
186.2
565.3
1338.4
2680

94
213
447.4
210
0.8
32.8
191.1
578.6
1377.62774.5

98
1.3
1
78
0.35
1.05
1.8
3
3.5
4.7

98
1
0.8
78.9
0.3
0.8
1.4
1.9
2.8
4.5

98
43.3
28.2
88.3
0.3
35.55
62.4
103.2
125.6
164

98
45.6
39.7
87
0.95
36.87
66
107
134
170.6

102
5.6
6.3
329.9
0.25
0.25
1.1
2.9
4
44.4

102
0.8
2
253.1
0.15
0.15
0.5
1.9
3.1
15.5

102
102
343.9
337
0.2
11.55
63.7
175.9
319.9
2846.3

102
104.6
351.8
336.1
0.6
11.95
64.8
182.2
326.2
2906.21138

Appendix 11. Descriptive Statistics Derived from the Residue Studies Included in this Risk Determination
Document(This page intentionally left blank)

1139

Appendix 11. Descriptive Statistics Derived from the Residue Studies Included in this Risk Determination
Document

Thiamethoxam:
Cotton (TK0177223) - Foliar
NectarPollenExtra Floral Nectar

StatisticN (#)
Mean (ng/g)
SD (ng/g)
CV (%)Min (ng/g)
Median (ng/g)
75th (ng/g)

Thiamethoxam

CGA322704

Total

Thiamethoxam

CGA322704

Total

Thiamethoxam

CGA322704

Total

50
3.3
6.5
201
0.3
1.7
2.9

50
0.4
0.3
69
0.3
0.3
0.6

50
3.7
6.6
178
0.8
2.4
3.4

54
27.8
58.7
211
0.3
3.8
17

54
1.9
3.3
170
0.3
0.3
2.2

54
29.8
61.9
208
0.5
4.3
18.5

53
53.4
50
94
1.8
41.1
59.3

53
2
2.3
112
0.3
1.4
2.4

53
55.4
51.8
94
2.1
42.8
61.9

90th (ng/g)

5.3

0.8

5.8

96.4

6.1

102.5

123

3.8

125.9

95th (ng/g)

6.9

1

7.3

141

10.2

151

178

5

186.4

Max (ng/g)

46.2

1.5

47

351

15.4

366.4

228

14.3

242.3

1140

Appendix 11. Descriptive Statistics Derived from the Residue Studies Included in this Risk Determination
Document
Citrus (TK0177221)
NectarStatisticN (#)
Mean (ng/g)
SD (ng/g)
CV (%)Min (ng/g)
Median (ng/g)
75th (ng/g)
90th (ng/g)
95th (ng/g)
Max (ng/g)

PollenThiamethoxam

CGA322704

Total

Thiamethoxam

CGA322704

Total

52
2.6
5.1
201.5
0.1
0.3
1.3
8.7
16.9
22.5

52
0.6
1.2
192
0.1
0.1
0.7
1.4
3.2
7

52
3.2
6.3
196.9
0.3
0.4
2
10.2
21.6

54
14.4
22.3
154.4
0.5
3.6
13.3
51.1
67.2
104

54
6.6
12.2
185.3
0.3
1.3
3.6
21.2
36
61.5

54
21.1
33
156.8
1.4
5.5
23.1
62.3
107.8

27.8

1141

135.1

Appendix 11. Descriptive Statistics Derived from the Residue Studies
Included in this Risk Determination Document
Cucumber (TK0024668)
NectarStatisticN (#)
Mean (ng/g)
SD (ng/g)
CV (%)Min (ng/g)
Median (ng/g)
75th (ng/g)
90th (ng/g)
95th (ng/g)
Max (ng/g)

PollenThiamethoxam

CGA322704

Total

Thiamethoxam

CGA322704

Total

9
6.0
3.8
62.5
1.3
7.5
7.8
11.5
11.5
11.5

9
2.6
1.8
71.8
1.0
1.6
5.0
5.0
5.0
5.0

9
8.6
2.3
26.7
6.3
8.5
9.4
13.2
13.2
13.2

9
4.5
2.4
52.2
1.3
4.1
5.2
8.2
8.2
8.2

9
1.2
0.8
66.9
0.2
0.9
1.7
2.6
2.6
2.6

9
5.7
3.0
53.5
1.5
5.7
5.9
10.8
10.8
10.8

Strawberry (TK0177224)
NectarStatisticN (#)
Mean (ng/g)
SD (ng/g)
CV (%)Min (ng/g)
Median (ng/g)
75th (ng/g)
90th (ng/g)
95th (ng/g)
Max (ng/g)

PollenThiamethoxam

CGA322704

Total

Thiamethoxam

CGA322704

Total

27
191.6
113.6
59.3
51.2
177.5
214.7
296.2
376.0
647.3

27
4.0
2.6
64.8
0.1
4.0
5.0
5.4
9.9
12.8

27
195.6
115.5
59.0
52.1
182.8
215.3
300.5
381.2
660.0

27
2023.02432.0
120.0
102.0
861.0
2486.07349.07445.07473.0

27
25.0
18.5
74.2
4.1
19.9
36.6
62.0
62.3
66.1

27
2048.02449.2
119.5
107.9
875.1
2522.77411.47492.67539.61142

Appendix 11. Descriptive Statistics Derived from the Residue Studies
Included in this Risk Determination Document
Cranberry (TK0236307)
NectarStatisticN (#)
Mean (ng/g)
SD (ng/g)
CV (%)Min (ng/g)
Median (ng/g)
75th (ng/g)
90th (ng/g)
95th (ng/g)
Max (ng/g)

Thiamethoxam
27
312.0
527.2
168.9
28.0
112.1
293.5
834.9
1580.02353.2

PollenCGA322704
27
18.1
31.5
174.2
1.3
5.5
12.3
87.0
92.8
118.8

Total
27
330.1
555.8
168.3
30.1
120.6
301.5
921.9
1698.82446.0Thiamethoxam
27
316.0
564.2
178.5
11.2
49.1
331.0
1149.91564.62226.7

Cucumber (TK0222532)
NectarStatisticN (#)
Mean (ng/g)
SD (ng/g)
CV (%)Min (ng/g)
Median (ng/g)
75th (ng/g)
90th (ng/g)
95th (ng/g)
Max (ng/g)

CGA322704
27
10.4
16.6
159.6
0.3
3.4
13.1
26.0
44.2
76.5

Total
27
326.4
575.9
176.4
11.5
52.5
353.6
1226.41608.82252.7

PollenThiamethoxam

CGA322704

Total

Thiamethoxam

CGA322704

Total

27
87.4
88.5
101.2
8.8
51.4
134.0
263.0
281.0
317.0

27
6.9
7.9
113.5
0.5
3.2
9.4
22.0
25.6
25.8

27
94.3
95.7
101.4
9.3
52.4
144.9
288.6
303.0
342.8

27
314.6
355.1
112.8
3.0
173.0
389.0
1050.01060.01410.0

27
17.7
18.4
103.7
1.5
12.9
21.7
34.6
47.5
92.7

27
332.3
365.2
109.9
4.6
186.7
436.5
1079.91142.71431.31143

Appendix 11. Descriptive Statistics Derived from the Residue Studies
Included in this Risk Determination Document
Strawberry (TK0250068)
NectarStatisticN (#)
Mean (ng/g)
SD (ng/g)
CV (%)Min (ng/g)
Median (ng/g)
75th (ng/g)
90th (ng/g)
95th (ng/g)
Max (ng/g)

PollenThiamethoxam

CGA322704

Total

Thiamethoxam

CGA322704

Total

53
23.5
37.0
157.2
0.7
10.0
30.0
48.0
134.0
188.0

53
2.9
4.7
162.5
0.1
0.9
4.3
6.1
10.0
25.0

53
26.4
41.4
156.7
0.9
13.1
33.8
52.3
145.0
213.0

54
202.0
364.1
180.2
8.3
57.0
222.0
491.0
807.0
1930.0

54
30.7
32.2
105.2
1.3
19.0
41.0
73.0
99.0
158.0

54
202.0
364.1
160.1
12.7
78.5
261.0
541.0
848.0
1947.0Pepper (TK0236306)
NectarStatisticN (#)
Mean (ng/g)
SD (ng/g)
CV (%)Min (ng/g)
Median (ng/g)
75th (ng/g)
90th (ng/g)
95th (ng/g)
Max (ng/g)

Thiamethoxam
15
52.5
136.6
259.9
3.0
11.6
29.0
66.8
542.1
542.1

PollenCGA322704
15
78.7
234.5
297.7
0.6
10.3
29.4
114.1
920.4
920.4

Total
15
131.3
370.8
282.4
3.6
19.9
53.7
180.9
1462.41462.4

1144

Thiamethoxam
17
30.7
32.4
105.6
1.1
25.5
38.6
45.9
142.7
142.7

CGA322704
17
110.5
80.8
73.0
8.5
119.4
173.0
224.5
231.6
231.6

Total
17
141.2
92.1
65.1
9.6
168.5
201.6
259.9
274.3
274.3

Appendix 11. Descriptive Statistics Derived from the Residue Studies
Included in this Risk Determination Document
Tomato (TK0222531)
PollenStatisticN (#)
Mean (ng/g)
SD (ng/g)
CV (%)Min (ng/g)
Median (ng/g)
75th (ng/g)
90th (ng/g)
95th (ng/g)
Max (ng/g)

Thiamethoxam
212292.63939.7
171.8
22.6
651.4
2311.66116.19151.515969.0

Bee Collected Pollen

CGA322704
21
423.0
417.8
98.7
22.9
308.8
558.2
834.2
135.2
1524.9

Total

Thiamethoxam
6
0.8
0.4
43.0
0.5
0.8
1.2
1.2
1.2
1.2

21
2715.64085.0
150.4
45.6
117.0
3152.66519.79637.3
16803.2

CGA322704
6
3.6
1.5
40.7
2.1
3.3
4.8
6.0
6.0
6.0

Total
6
4.5
1.6
36.3
2.6
4.2
5.3
7.2
7.2
7.2

Apple (TK0250071)
NectarStatisticN (#)
Mean (ng/g)
SD (ng/g)
CV (%)Min (ng/g)
Median (ng/g)
75th (ng/g)
90th (ng/g)
95th (ng/g)
Max (ng/g)

Thiamethoxam
44
95.5
149.4
156.3
0.0
57.3
89.2
216.0
354.0
756.0

PollenCGA322704
36
2375.62350.9
184.2
0.1
3.6
9.9
86.0
96.0
118.0

Total
44
113.1
153.5
135.7
0.2
79.0
169.5
225.4
361.5
769.0

1145

Thiamethoxam
43
828.4
758.8
91.5
2.8
928.0
1430.01880.02130.02410.0

CGA322704
43
71.3
64.6
90.6
1.9
57.0
88.5
190.0
213.0
222.0

Total
43
899.7
785.0
87.2
4.7
964.8
1599.01954.72153.32471.3

Appendix 11. Descriptive Statistics Derived from the Residue Studies
Included in this Risk Determination Document
Corn (TK0258214) [Corn Seed + Foliar Treatment]
PollenStatisticN (#)
Mean (ng/g)
SD (ng/g)
CV (%)Min (ng/g)
Median (ng/g)
75th (ng/g)
90th (ng/g)
95th (ng/g)
Max (ng/g)

Thiamethoxam
24
216.8
249.6
115.1
5.3
84.6
394.5
528.0
559.3
993.6

CGA322704
24
6.4
3.6
56.2
1.3
5.5
8.1
12.5
12.8
13.0

Total
24
223.2
251.3
112.0
6.6
97.0
401.6
538.9
565.6
1006.6

Muskmelon (TK0222530)
NectarStatisticN (#)
Mean (ng/g)
SD (ng/g)
CV (%)Min (ng/g)
Median (ng/g)
75th (ng/g)
90th (ng/g)
95th (ng/g)
Max (ng/g)

Thiamethoxam
45
11.4
12.2
106.9
0.6
8.4
13.3
25.0
37.4
61.5

PollenCGA322704
45
1.6
1.1
69.7
0.1
1.3
2.0
3.5
3.7
5.1

Total

1146

45
13.0
12.9
99.1
1.3
9.1
14.5
27.9
39.5
66.4

Thiamethoxam
45
12.9
36.7
284.0
0.3
3.4
5.5
11.2
91.8
192.9

CGA322704
45
13.0
12.9
408.5
0.5
1.4
3.4
26.9
113.2
676.0

Total
45
38.2
120.2
315.5
0.8
5.0
15.1
119.7
157.6
767.8

Appendix 11. Descriptive Statistics Derived from the Residue Studies
Included in this Risk Determination Document
Squash (TK0222530)
NectarStatisticN (#)
Mean (ng/g)
SD (ng/g)
CV (%)Min (ng/g)
Median (ng/g)
75th (ng/g)
90th (ng/g)
95th (ng/g)
Max (ng/g)

Thiamethoxam
34
8.9
10.8
120.1
0.3
3.3
15.1
29.2
31.9
32.4

CGA322704
34
1.0
1.0
101.4
0.1
0.7
1.4
2.9
3.2
3.3

Total
34
9.9
11.5
115.1
0.4
4.3
17.1
31.7
33.7
33.8

PollenThiamethoxam
34
5.4
6.3
116.0
0.3
3.0
7.1
14.1
22.0
27.5

CGA322704
34
1.5
0.9
55.2
0.3
1.6
2.0
2.4
3.4
4.4

Total
34
6.9
6.9
99.4
0.5
4.9
8.6
16.1
24.7
31.9

Pumpkin (TK0222530)
NectarStatisticN (#)
Mean (ng/g)
SD (ng/g)
CV (%)Min (ng/g)
Median (ng/g)
75th (ng/g)
90th (ng/g)
95th (ng/g)
Max (ng/g)

Thiamethoxam
44
3.0
2.4
80.9
0.3
1.8
4.3
5.8
8.3
10.5

PollenCGA322704
44
0.7
0.9
130.5
0.1
0.3
0.8
1.6
2.8
3.9

1147

Total
44
3.7
3.1
84.0
0.4
2.1
5.1
8.1
10.4
13.8

Thiamethoxam
45
3.3
3.5
105.4
0.3
2.1
4.7
6.9
10.6
17.0

CGA322704
45
2.6
1.8
70.7
0.3
1.9
3.1
5.0
5.9
10.6

Total
45
5.9
4.5
76.4
0.5
4.4
7.4
12.2
15.5
22.7

Appendix 11. Descriptive Statistics Derived from the Residue Studies
Included in this Risk Determination Document
Pumpkin (TK0242074)
NectarStatisticN (#)
Mean (ng/g)
SD (ng/g)
CV (%)Min (ng/g)
Median (ng/g)
75th (ng/g)
90th (ng/g)
95th (ng/g)
Max (ng/g)

Thiamethoxam
45
4.1
5.2
124.3
0.1
2.3
5.1
8.6
13.5
24.9

PollenCGA322704
45
3.4
3.5
104.3
0.1
2.2
3.7
10.1
10.2
12.6

Total
45
7.5
6.8
90.6
1.0
5.2
9.8
15.0
21.7
29.6

Thiamethoxam
45
6.0
8.4
140.7
0.3
2.4
8.5
14.7
20.4
45.4

CGA322704
45
2.4
1.5
61.8
0.1
2.4
2.9
3.7
4.1
7.4

Total
45
8.4
9.6
115.1
1.0
5.1
10.9
18.0
24.5
52.3

Tomato (TK0242072)
PollenStatisticN (#)
Mean (ng/g)
SD (ng/g)
CV (%)Min (ng/g)
Median (ng/g)
75th (ng/g)
90th (ng/g)
95th (ng/g)
Max (ng/g)

1148

Thiamethoxam
27
32.5
24.0
73.9
3.8
27.0
53.5
66.6
73.0
85.4

CGA322704
27
49.9
50.6
101.4
15.2
34.4
47.7
87.9
187.5
233.4

Total
27
82.4
69.2
83.9
19.7
57.9
108.5
157.2
251.7
318.8

Appendix 11. Descriptive Statistics Derived from the Residue Studies
Included in this Risk Determination Document
Blueberry (TK0250072)
NectarStatisticN (#)
Mean (ng/g)
SD (ng/g)
CV (%)Min (ng/g)
Median (ng/g)
75th (ng/g)
90th (ng/g)
95th (ng/g)
Max (ng/g)

Thiamethoxam
27
89.9
128.9
143.3
1.4
15.5
186.0
267.0
431.0
459.0

PollenCGA322704
27
106.2
135.8
127.9
3.4
30.9
184.0
375.0
381.0
421.0

Total
27
196.1
247.2
126.1
4.8
51.6
389.0
613.0
708.0
713.0

Thiamethoxam
27
247.6
264.6
106.5
2.6
75.0
462.0
757.0
779.0
828.0

CGA322704
27
58.0
62.3
107.5
0.5
33.8
92.8
172.0
173.0
174.0

Total
27
305.6
319.2
104.4
3.1
124.4
635.0
836.4
947.0
987.0

Sweet Orange (TK0250069)
NectarStatisticN (#)
Mean (ng/g)
SD (ng/g)
CV (%)Min (ng/g)
Median (ng/g)
75th (ng/g)
90th (ng/g)
95th (ng/g)
Max (ng/g)

Thiamethoxam
54
0.6
0.8
188.6
0.1
0.5
0.9
1.3
4.8
9.2

PollenCGA322704
54
0.6
0.8
135.8
0.1
0.3
0.7
0.9
2.6
4.3

1149

Total
54
1.5
2.5
166.9
0.3
0.9
1.6
2.1
7.4
13.4

Thiamethoxam
54
37.2
88.7
238.6
2.2
9.3
14.5
91.2
276.0
497.0

CGA322704
54
32.5
89.0
273.5
0.5
6.2
21.8
48.6
289.0
457.0

Total
54
69.7
175.7
251.9
2.9
18.2
36.8
126.7
565.0
948.0

Appendix 11. Descriptive Statistics Derived from the Residue Studies
Included in this Risk Determination Document
Soybean (TK0250070)
NectarStatisticN (#)
Mean (ng/g)
SD (ng/g)
CV (%)Min (ng/g)
Median (ng/g)
75th (ng/g)
90th (ng/g)
95th (ng/g)
Max (ng/g)

Thiamethoxam
27
0.5
0.8
142.5
0.1
0.3
0.3
1.3
2.2
3.6

AntherCGA322704
27
1.2
1.7
149.5
0.1
0.3
1.5
3.2
4.5
7.7

1150

Total
27
1.7
1.9
115.5
0.3
0.9
2.1
4.7
5.8
8.0

Thiamethoxam
35
13.5
16.0
118.6
0.5
8.9
16.5
34.3
51.1
68.2

CGA322704
35
3.0
2.5
84.7
0.3
2.5
4.6
6.9
7.4
9.4

Total
35
16.5
18.3
110.9
1.0
11.3
22.9
41.2
58.3
77.6

Appendix 11. Descriptive Statistics Derived from the Residue Studies Included in this Risk Determination
Document
Stone Fruit (TK0177222)
NectarStatisticN (#)
Mean (ng/g)
SD (ng/g)
CV (%)Min (ng/g)
Median(ng/g)
75th (ng/g)
90th (ng/g)
95th (ng/g)
Max (ng/g)

PollenAnthers

Thiamethoxam CGA322704 Total Thiamethoxam CGA322704 Total Thiamethoxam CGA322704 Total
54
0.4
0.5
135.7
0.1

54
0.2
0.3
117
0.1

54
0.6
0.6
98.8
0.3

50
54.2
75.5
139.3
0.4

50
1.1
1.2
104.7
0.2

50
55.3
75.5
136.5
0.5

54
2.9
12
420.4
0.3

54
1.6
2.9
175.2
0.3

54
4.5
13.1
290.9
0.5

0.2

0.1

0.4

29.6

0.9

30.5

0.3

0.3

0.8

0.3
1
1.8
2.4

0.3
0.5
0.7
1.7

0.5
1.6
2
2.6

63.3
132
181.6
382

1.6
2.4
2.6
5.9

63.6
133.2
182.1
383

0.5
4.4
7.2
87.5

2.2
4.4
5.8
15.8

3.5
7.9
11.7
91.7

1151

Appendix 11. Descriptive Statistics Derived from the Residue Studies Included in this Risk Determination
Document(This page intentionally left blank)1152

Appendix 11. Descriptive Statistics Derived from the Residue Studies
Included in this Risk Determination Document

Clothianidin:
Potato (VP-38985)
Clothianidin
StatisticN
Mean (ng/g)
SD (ng/g)
CV (%)Min (ng/g)
Median (ng/g)
75th (ng/g)
90th (ng/g)
95th (ng/g)
Max (ng/g)

PollenAnthers

9
61.9
25.9
41.8
27.8
61.0
70.3
113.9
113.9
113.9

27
4.7
5.3
111.6
0.3
2.9
5.7
15.1
15.7
21.2

Pumpkin (VP-38263)
Clothianidin
Nectar18

Pollen18

Mean (ng/g)

3

8.1

SD (ng/g)

2.4

6.3

CV (%)

78.3

77.4

Min (ng/g)

0.7

1.6

Median (ng/g)

2.4

6.8

75th (ng/g)

3.8

11.1

90th (ng/g)

6.3

17

95th (ng/g)

9.6

25.8

Max (ng/g)

9.6

25.8

StatisticN1153

Appendix 11. Descriptive Statistics Derived from the Residue Studies
Included in this Risk Determination Document
Pumpkin (VP-38971)
Clothianidin
StatisticN
Mean (ng/g)
SD (ng/g)
CV (%)Min (ng/g)
Median (ng/g)
75th (ng/g)
90th (ng/g)
95th (ng/g)
Max (ng/g)

NectarPollen

38
3.8
3.8
98.9
0.2
1.5
7.4
9.9
10.6
11.3

45
7.0
8.5
121.0
0.6
4.0
7.9
20.3
31.3
31.9

Cucumber (VP-38938)
Clothianidin
StatisticN

NectarAnthers

5

12

Mean (ng/g)

18.3

20.9

SD (ng/g)

148.3

CV (%)

76.7

39.4

Min (ng/g)

7.5

12.2

Median (ng/g)

10.7

18.7

75th (ng/g)

25.5

29.3

90th (ng/g)

39.6

32

95th (ng/g)

39.6

34.2

Max (ng/g)

39.6

34.2

1154

Appendix 11. Descriptive Statistics Derived from the Residue Studies
Included in this Risk Determination Document
Melon (VP-38938)
Clothianidin
StatisticN
Mean (ng/g)
SD (ng/g)
CV (%)Min (ng/g)
Median (ng/g)
75th (ng/g)
90th (ng/g)
95th (ng/g)
Max (ng/g)

Nectar
4
7.9
4.6
58.5
4.7
6.0
10.8
14.6
14.6
14.6

Anthers
15
11.1
4.6
41.7
4.9
10.7
12.0
18.7
20.7
20.7

Pumpkin (VP-38938)
Clothianidin
Nectar15

Pollen15

Anthers
12

Mean (ng/g)

3.1

9.5

5.2

SD (ng/g)

2

10.1

3.3

CV (%)

65

106.3

63.6

Min (ng/g)

0.8

2

1.8

Median (ng/g)

2.1

4.8

3.8

75th (ng/g)

4.3

11.8

7.1

90th (ng/g)

6.6

21

8.9

95th (ng/g)

7.2

40.1

13.2

Max (ng/g)

7.2

40.1

13.2

StatisticN1155

Appendix 11. Descriptive Statistics Derived from the Residue Studies
Included in this Risk Determination Document
Pumpkin (VP-38313)
Clothianidin
NectarPollen20

20

Mean (ng/g)

2.5

27.1

SD (ng/g)

1.7

33.1

CV (%)

68.4

122.4

Min (ng/g)

0.7

1.6

Median (ng/g)

14.8

17

75th (ng/g)

4

70.4

90th (ng/g)

5

71

95th (ng/g)

6

107.9

Max (ng/g)

6.5

123

StatisticN

Squash (VP-38938)
Clothianidin
StatisticN
Mean (ng/g)
SD (ng/g)
CV (%)Min (ng/g)
Median (ng/g)
75th (ng/g)
90th (ng/g)
95th (ng/g)

Nectar
15
2.8
1.1
39.3
1.4
2.6
4.0
4.4
4.5

Pollen
15
6.3
3.5
56.1
2.3
5.6
8.0
10.7
14.7

Anthers
12
5.3
2.0
38.7
2.4
5.5
6.7
7.2
8.7

Max (ng/g)

4.5

14.7

8.7

1156

Appendix 11. Descriptive Statistics Derived from the Residue Studies
Included in this Risk Determination Document
Peach (VP-38563)
Clothianidin
StatisticN
Mean (ng/g)
SD (ng/g)
CV (%)Min (ng/g)
Median (ng/g)
75th (ng/g)
90th (ng/g)
95th (ng/g)
Max (ng/g)

Nectar
18
0.1
0.1
48.5
0.1
0.1
0.1
0.3
0.3
0.3

Pollen
17
10.6
30.9
292.5
0.3
2.0
5.3
10.0
130.1
130.1

Grape Soil (VP-38992)
Clothianidin
Statistic
N

Pollen24

Mean (ng/g)

55.4

SD (ng/g)

58.3

CV (%)

105.3

Min (ng/g)

3.7Median (ng/g)

28.3

75th (ng/g)

86.9

90th (ng/g)

157.3

95th (ng/g)

168

Max (ng/g)

205.9

1157

Appendix 11. Descriptive Statistics Derived from the Residue Studies
Included in this Risk Determination Document
Almond (VP-38473)
Clothianidin
NectarPollenAnthers

54

41

12

Mean (ng/g)

0.3

5.62

16.8

SD (ng/g)

0.4

4.7

24.4

CV (%)

123

83.6

145

Min (ng/g)

0.1

0.55

0.35

Median (ng/g)

0.1

4.32

11.62

75th (ng/g)

0.37

8.26

21.15

90th (ng/g)

0.84

12.7

27

95th (ng/g)

1.15

13.8

88.1

Max (ng/g)

2.04

20

88.1

StatisticN

Grape Foliar (VP-38992)
Clothianidin
Statistic
N

Pollen24

Mean (ng/g)

632.7

SD (ng/g)

382.2

CV (%)

60.4

Min (ng/g)

116.9Median (ng/g)

540.9

75th (ng/g)

886

90th (ng/g)

1229.895th (ng/g)

1246.8Max (ng/g)

1563.91158

Appendix 11. Descriptive Statistics Derived from the Residue Studies
Included in this Risk Determination Document
Apple (VP-38552)
Clothianidin
NectarPollen99Mean (ng/g)

0.4

11.7

SD (ng/g)

0.3

18.7

CV (%)

72.3

159.2

Min (ng/g)

0.1

0.1

Median (ng/g)

0.4

375th (ng/g)

0.6

18.1

90th (ng/g)

0.7

57.4

95th (ng/g)

0.7

57.4

Max (ng/g)

0.7

57.4

StatisticN

Cotton (VP-38259)
Clothianidin
StatisticN
Mean (ng/g)
SD (ng/g)
CV (%)Min (ng/g)
Median (ng/g)
75th (ng/g)
90th (ng/g)
95th (ng/g)

Nectar
27
29.5
43.2
146.3
0.2
17.4
32.0
79.4
142.0

Extrafloral Nectar
27
448.5
946.9
211.1
9.9
104.0
213.0
1692.02624.0Max (ng/g)

182.0

4163.01159

Appendix 11. Descriptive Statistics Derived from the Residue Studies
Included in this Risk Determination Document(This page intentionally left blank)

1160

Appendix 11. Descriptive Statistics Derived from the Residue Studies
Included in this Risk Determination Document

Dinotefuran:
Cotton (43411B104)
Floral Nectar
53

Dinotefuran
Pollen54

Extrafloral Nectar
90

Mean (ng/g)

69.6

1979.3741.49SD (ng/g)

99.7

4482.12102.3CV (%)

143.3

226.4

283.5

Min (ng/g)

2.51

13.8

0.5

Median (ng/g)

22.5

233

108

75th (ng/g)

81

686

545

90th (ng/g)

81.6

6968

1660

95th (ng/g)

321

14757

2880

Max (ng/g)

346

20676

15500

StatisticN

Pumpkin (10934.4104)
Dinotefuran
StatisticN
Mean (ng/g)

Floral Nectar27
17.9

Pollen27
32.1

SD (ng/g)

17.6

28.5

CV (%)

98.2

88.6

Min (ng/g)
Median (ng/g)

0.5
15.7

4.8
17.7

75th (ng/g)

21.2

48.9

90th (ng/g)

39

88.3

95th (ng/g)

50.8

92.9

Max (ng/g)

84.4

105.3

1161

Appendix 11. Descriptive Statistics Derived from the Residue Studies
Included in this Risk Determination Document
Tomato Foliar (10934.4103)
Dinotefuran
Statistic
N

PollenMean (ng/g)

2421

SD (ng/g)

5477

CV (%)

226

Min (ng/g)

5Median (ng/g)

58

75th (ng/g)

1260

90th (ng/g)

10439

95th (ng/g)

12210

Max (ng/g)

22839

24

Tomato Soil (10934.4103)
StatisticN

Dinotefuran
Pollen24

Mean (ng/g)

1307

SD (ng/g)
CV (%)2667
204

Min (ng/g)

4Median (ng/g)

33

75th (ng/g)
90th (ng/g)

652
5532

95th (ng/g)

7208

Max (ng/g)

9813

1162

Appendix 11. Descriptive Statistics Derived from the Residue Studies
Included in this Risk Determination Document
Cranberry (10934.4101)
Dinotefuran
Floral Nectar

Pollen27

27

Mean (ng/g)

340.8

370

SD (ng/g)

275.6

323.3

CV (%)

80.8

387.3

Min (ng/g)

69.3

37.1

Median (ng/g)

220.3

256.5

75th (ng/g)

438.5

581.7

90th (ng/g)

780.9

763.5

95th (ng/g)

1010.71247.6

Max (ng/g)

1159.21268.7

StatisticN

StatisticN

Potato (10934.4100)
Dinotefuran
Pollen27

Mean (ng/g)
SD (ng/g)
CV (%)Min (ng/g)
Median (ng/g)

22.9
27.3
119.3
0.5
9.3

75th (ng/g)
90th (ng/g)
95th (ng/g)

42.2
56.9
78.3

Max (ng/g)

103.6

1163

Appendix 11. Descriptive Statistics Derived from the Residue Studies
Included in this Risk Determination Document
Cherry Foliar (10934.4105)
Dinotefuran
Floral Nectar

Pollen27

26

Mean (ng/g)

8.4

49.8

SD (ng/g)

4.6

51.5

CV (%)

54.7

103.5

Min (ng/g)

1.2

5.6

Median (ng/g)

7.2

28.7

75th (ng/g)

10.4

95.8

90th (ng/g)

12.5

130.5

95th (ng/g)

15.7

153.7

Max (ng/g)

25.5

171.7

StatisticN

Cherry Trunk (10934.4105)
Dinotefuran
Floral Nectar

Pollen27

27

Mean (ng/g)

5403.39321.9SD (ng/g)

4968

8500.3CV (%)

91.9

91.1

Min (ng/g)

118.4

368.3

Median (ng/g)5233.88699

75th (ng/g)

8456.513626

90th (ng/g)

12090.3

21822

95th (ng/g)

16604.5

23697.6

Max (ng/g)

17483.8

31688.5

StatisticN1164

Appendix 11. Descriptive Statistics Derived from the Residue Studies
Included in this Risk Determination Document
Blueberry (10934.4107)
Dinotefuran
Floral Nectar

Pollen27

27

Mean (ng/g)

231.5

183.2

SD (ng/g)

163.9

188.4

CV (%)

70.7

102.8

Min (ng/g)

30

24.7

Median (ng/g)

198.9

110.1

75th (ng/g)

395.5

233.3

90th (ng/g)

470.8

468.9

95th (ng/g)

484.6

581.7

Max (ng/g)

484.7

770.6

Statistic
N

Bell Pepper (S16-01167)
Dinotefuran
Floral Nectar
26

Pollen24

Anthers
15

Mean (ng/g)

1.8

59.4

93.4

SD (ng/g)

1.7

87.5

93.8

CV (%)

95.2

147.2

100.4

Min (ng/g)

0.1

5.6

17.1

Median (ng/g)

1.3

24

49.9

75th (ng/g)

2.7

67.9

141

90th (ng/g)

4.5

183

238

95th (ng/g)

4.8

212

344

Max (ng/g)

6.5

387

344

StatisticN1165

Appendix 12. Letters Notifying Registrants of Reevaluation

Department of Pesticide Regulation
Mary-Ann Warmerdam
Director

Arnold Schwarzenegger
Governor<Contact Name><Company Name><Address><City, State Zip>
Dear <Salutation>:
Pursuant to Title 3, California Code of Regulations, section 6220, et seq., the Director of the
Department of Pesticide Regulation (DPR) notices her decision to initiate a reevaluation ofcertain pesticide products within the nitroguanidine insecticide class of neonicotinoids containing
the following active ingredients: imidacloprid, clothianidin, dinotefuran, and thiamethoxam,
including the following product(s):Product Brand Name, EPA Reg. No.
<Product Name, EPA Reg. No. 999-88-AA>

Active Ingredient
Imidacloprid

DPR is required to investigate all reported pesticide episodes and information received indicatingthat a pesticide may have caused, or is likely to cause, a significant adverse impact. If the
Director finds from the investigation that a significant adverse effect has occurred or is likely tooccur; the pesticide involved shall be reevaluated. Therefore, certain products within thenitroguanidine insecticide class of neonicotinoids, including the above product(s), are being
reevaluated.
BASIS FOR REEVALUATION
In 2008, DPR received an adverse effects disclosure pursuant to Federal Insecticide Fungicide
and Rodenticide Act (FIFRA) section 6(a)(2) and Food and Agricultural Codesection 12825.5 regarding the active ingredient imidacloprid. The disclosure included twelve
residue and two combination residue, honey, bumble bee studies of imidacloprid use on anumber of ornamental plants. DPR’s evaluation of the data noted two critical findings. One, highlevels of imidacloprid in leaves and blossoms of treated plants, and two, increases in residue
levels over time.
Imidacloprid levels in leaves and blossoms varied depending on the application rate and the typeof plant, but the data indicate that residues in some plants measured higher than 4 parts per
million (ppm). The data also indicate that when using soil application methods, imidacloprid
residues remained relatively low for the first six months after application, followed by a dramaticincrease that remained stable in some cases for more than 500 days after treatment. Where
imidacloprid was applied to the soil, no significant decline in residue levels was observed in anyof the studies, even in studies where residues were tested at 540 days after treatment. DPR found1001 I Street • P.O. Box 4015 • Sacramento, California 95812-4015 • www.cdpr.ca.gov
A Department of the California Environmental Protection Agency
Printed on recycled paper, 100% post-consumer--processed chlorine-free.

1166

Appendix 12. Letters Notifying Registrants of Reevaluation<Contact Name>
Page 2that the treatment rates used in the studies where high imidacloprid residue levels were found inleaves and blossoms, were comparable to application rates found on currently registered labels
for orchards, assuming the orchards were planted at a density of 200 trees per acre or fewer. The
data indicate that use of imidacloprid on an annual basis may be additive, in that significant
residues from the previous use season appear to be available to the treated plant. DPR also
received preliminary information from a University of California at Riverside researcher who isinvestigating imidacloprid residues in eucalyptus nectar and pollen. The researcher’s preliminary
results indicate imidacloprid residues in eucalyptus nectar at levels of up to 550 parts per
billion (ppb).
Based upon data on file, DPR estimates the lethal concentration of imidacloprid needed to kill
50 percent of a test population (LC50) of honey bees is 185 ppb1. In their everyday foraging andpollination activities, honey bees collect both nectar and pollen from flowering plants. If theimidacloprid residue levels in a plant’s nectar and pollen are similar to those found in the leaves
and blossoms of the plants described in the adverse effects data, the levels are well above theestimated LC50 for honey bees. The levels found in some of the plants were more than twenty
times the estimated honey bee LC50 of 185 ppb.
All of the neonicotinoids share many of the same characteristics as imidacloprid. However, thethree other neonicotinoids included in this reevaluation, clothianidin, dinotefuran, andthiamethoxam, are in the same chemical family (nitroguanidines) as imidacloprid. These threeother active ingredients, in particular, have soil mobility characteristics and half-lives that arevery similar to imidacloprid. Based on available data, DPR scientists believe these active
ingredients would have the same potential residue concerns as imidacloprid. Data also indicatethat these active ingredients are similar to imidacloprid in toxicity to honey bees. Due to thechemical and toxicological similarities between imidacloprid and the other neonicotinoids, DPR
is providing those registrants with the option of generating data on their own chemicals orproviding/relying upon data generated using a surrogate nitroguanidine.
DPR exempted the following formulation categories and product types from the reevaluation:

1.
2.
3.
4.
5.
6.
7.

Formulated as a gel or impregnated in a strip;
Termiticide;
Flea control products combined with rodenticide;
Pet spot applications;
Ant and roach baits;
Premise application for control of nuisance pests; or,Manufacturing use only products.

1

The LC50 was estimated by converting the acute oral LD50 (the amount of a material that causes the death of
50 percent of a test population) to a concentration in nectar using the standard consumption model used in bee
feeding studies.
1167

Appendix 12. Letters Notifying Registrants of Reevaluation<Contact Name>
Page 3

DPR exempted the above types of products from the reevaluation because the manner in which
the products are formulated or applied makes it unlikely that the neonicotinoid will move intoplants that bloom or be a source of forage for honey bees and pollinators.
DPR plans to work closely with the United States Environmental Protection Agency’s(U.S. EPA’s) Office of Pesticide Programs throughout the reevaluation process. U.S. EPA’s
Registration Review docket for imidacloprid<http://www.epa.gov/oppsrrd1/registration_review/imidacloprid/index.htm> opened in
December 17, 2008, and the dockets for other neonicotinoids were previously scheduled to open
between 2013 and 2015. In order to better ensure a “level playing field” for the neonicotinoidclass as a whole, and to best take advantage of new research as it becomes available, U.S. EPA
moved the docket openings for the remaining neonicotinoids on the registration review schedule(acetamiprid, clothianidin, dinotefuran, nitrapyrin, thiacloprid and thiamethoxam) tofiscal year 2012.
DATA REQUIREMENTS
DPR will inform you of the data required pursuant to this reevaluation in a separate letter. The
data requirements will be finalized and announced after the April 17, 2009 deadline listed below.
MEET WITH DPR STAFF
DPR has scheduled a meeting with registrants to discuss the neonicotinoid reevaluation datarequirements at the time and place noted below. Enclosed are directions for traveling to the Cal/
EPA Headquarters building and a proposed agenda.

DATE:
TIME:
PLACE:

April 1, 2009
10:00 a.m.
California Environmental Protection Agency (Cal/EPA Headquarters)
Coastal Hearing Room, 2nd Floor
1001 I Street
Sacramento, CA 95814

As part of the reevaluation process, DPR intends to require field-based data on neonicotinoids inorder to better understand their impact on honey bees. DPR plans to require registrants to analyze
residues from nectar and pollen of a representative number of crops grown in California. In
addition, DPR plans to require acute [laboratory] toxicity studies on various honey bee life
stages. Attachment 1 contains the details of DPR’s data requirement proposal.
1168

Appendix 12. Letters Notifying Registrants of Reevaluation<Contact Name>
Page 4

DPR will consider written comments on the proposed data requirements received no later thanthe close of business April 17, 2008. Please address all correspondence regarding thisreevaluation as follows:
Neonicotinoid Reevaluation
Attn: Denise Webster
Department of Pesticide Regulation
1001 I Street, P.O. Box 4015
Sacramento, California 95812-4015
CONTACTS
For information regarding the reevaluation process, please contact either Ms. Denise Webster, by
e-mail at <dwebster@cdpr.ca.gov> or by telephone at (916) 324-3522, or Ms. Alveena Prasad,by e-mail at <aprasad@cdpr.ca.gov> or by telephone at (916) 324-3905.
Sincerely,

Original signed by
Ann M. Prichard, Chief
Pesticide Registration Branch(916) 324-3931

February 26, 2009
Date

Enclosures
cc: Ms. Denise Webster, Program Specialist
Ms. Alveena Prasad, Environmental Scientist<Regulatory Scientist>1169