UNITED STATES ENVIRONMENTAL PROTECTION AGENCY WASHINGTON, D.C. 20460

Jamin Huang Bayer CropScience 2 T.W. Alexander Drive P.O. Box 12014 Research Triangle Park, NC 27709
Dear Dr. Huang: Subject: Conditional Data; Pollinator Study Poncho 600 EPA Registration No. 264-789 Date Submitted: August 8, 2006 OFFICE OF PREVENTION, PESTICIDES "'-'-' AND TOXIC SUBSTANCES



### 6

The Agency has completed its review of the field test for pollinator study report submitted as a condition of registration. A copy of that review is enclosed. If you have any questions regarding this letter, please call me at (703) 306-0415.

Enclosure Sincerely yours, 

# \/**~-·--·-~**..•.

## _"···········~

# A~..::::::::::·:::::::::·._.._ _) Kable Bo DaVIS**-~** Entomologist

### :~ Insecticide-Rodenticide Branch Registration Division (7505P)UNITED STATES ENVIRONMENTAL PROTECTION AGENCY WASHINGTON D.C., 20460

OFFICE OF**:;~:** PREVENTION, PESTICIDES AND TOXIC SUBSTANCES 

### MEMORANDUM DATE: November 16,2007
SUBJECT: .FROM:

THRU: Review ofData Package DP336888 for**Clot~!ani~~~** 

### PC_ Code 044309 Allen Vaughan, Biologist

# ~~~:**r.~** EnvironmentalRisk BranchV 11 

### Environmental Fate and Effects Division (7507P) 

### II 

# /30j0

# 7 Mah Shamim, Branch Chief

## Mllf}~ EnvironmentalRiskBranch V //**n~** 

### EnvironmentalFate and Effects Division (7507P) TO: Kable Davis, RiskManager Reviewer John Hebert, RiskManager (RM 07) Insecticide/Rodenticide Branch Registration Division (7505P) EFED has reviewed the following study submitted for clothianidin (PC Code 044309). The completed DER for this studyis attached: Cutler, C. 2006. An Investigation ofthe Potential Long Term Impact ofClothianidin SeedTreated Canola onHoneyBees,Apismellifera L. Laboratory Repmi ID: 2005- CSD-EBTIX064. MRID 46907801 (with addendum 46907802). This studyis scientifically sound and satisfies the guideline requirements for a field toxicitytest with honeybees (OPP Gdln. No. 141-5; OPPTS 850.3040). Overall, there was no difference between colonies from clothianidin-treated and control fields. Although sporadic treatment or site differences were found on various dates, essentiallyno differences in worker or drone mortality, workerlongevity, or brood development occurred during the study. Colonies in treated fields had similar weight gains and honeyyields as those in control fields. Qualitative assessments, made the following springbyexperienced beeresearchers, confirmed that colonies from clothianidin-treated fields were as strong and healthy as those from control fields. Itwas concluded that honeybees that forage on clothianidin seed-treated canola will be exposed to clothianidin residues iri pollen, nectar, and honey; however, exposure concentrations arebelow thoserequired to elicit acute and sublethal effects.

### DATA EVALUATION RECORD HONEY BEE- FIELD TESTINGFORPOLLINATORS §141-5 (OPPTS 850. 3040)

__ 1. 

### CHEMICAL: Clothianidin PC Code No.: 044309 

### 2. TESTMATERIAL: 1) Prosper FL 2) Poncho 600 FS 

### 3. CITATION: Author: Cutler, C. Purity: 1) 9.49% 2) 48.0%

Title: AnInvestigation ofthe PotentialLong-Term Impact of Clothianidin Seed Treated Canola onHoney Bees,Apis 

### mell?fera L. Study CompletionDate: August 1, 2006 Laboratory: Department ofEnvironmental Biology University ofGuelph Guelph, Ontario, NlG 2Wl Sponsor: Bayer CropScience P.O. Box 12014, 2 T.W. Alexander Drive Research Triangle Park, NC 27709 Laboratory Report ID: 2005-CSD-EBTIX064 DP Barcode: D336888

MRID No.: 469078-01 

### 4. REVIEWED BY: Christie E. Padova, StaffScientist, Dynamac Corporation 

### Signature: Date: 9118/07 

### APPROVED BY: Teri S. Myers, Ph.D., Senior Scientist, Cambridge Environmental Inc. 

### Signature:

## ~A'\.'

# 5/l'Jyl/}-'v5. APPROVED BY: Allen Vaughan, Biologist, ERB

- V 

### Signature:

# ~/.U.

# u+- Date: 9/26/07



### Date: 

# o(tG.jo-:t-DP Barcode: D336888 MRlD No. 469078-01 (with 469078-02)



### 6. DISCLAIMER: This documentprovides guidance for EPA and PMRA reviewers onhow to complete a data evaluation record after reviewing a scientific study concerning the longtenn,toxicity ofa pesticide to honeybees following an actual-use field exposure. It is not intended to prescribe conditions to any external party for conductingthis studynor to establish absolute criteriaregardingthe assessment ofwhether the study is scientifically sound and whetherthe study satisfies any applicable data requirements. Reviewers are expected to review and to determine for each study, on a case-by-casebasis, whether it is scientifically sound and provides sufficient infonnation to satisfy applicable data requirements. Studies that fail to meet any ofthe conditions maybe accepted, if appropriate; similarly, studies that meet all ofthe conditions mayberejected, if appropriate. In sum, thereviewer is to take into account the totality offactors related to the test methodology and results in determining the acceptability ofthe study. 

### 7. STUDY PARAMETERS: Scientific Name ofTest Organism: Apis mellifera L. 

### Age or Size ofTest Organism atTestInitiation: Queens in all colonies wereofthe same lineage and ca. the same age.



### 8. CONCLUSIONS:Definitive StudyDuration: 130 days (approximately 2 complete life cycles); 21-day exposureperiod (duringpeakbloom) followed bya 109-daypost-exposureperiod.

In a 130-day study (21-day exposure followed by 109-daypost-exposureperiod), the longterm toxicity ofclothianidin-treated seed was examined in the honeybee,Apismellifera L., under open field conditions at four test sites. Each site contained one 1-ha field planted with canola seed, Brassica napus var. Hyola420, thathad beentreated with the end-use products Prosper® FL at 1250 mL/100 kg seed and Poncho® 600 FS at 417 mL/100 kg seed, delivering clothianidin at 400 g ai/100 kg seed, thehighest commercial rate for use in Canada. In addition, each site contained one 1-ha control field planted with canola seed that had been treated at the samerate with specially-prepared Blank Prosper FL and Poncho 600 FS formulations. Each treated and control field were separated byat least 250m. Fourhoneybee colonies wereplaced in the middle ofeach field (n=32) during a 3- weekbloom period (Day 1 =July 1, 2005), and thereafter moved to a fall apiary for the remainder ofthe study (Day 130 =November 7, 2005). Throughout the study, colonies were assessed for beemortality, workerlongevity, and brood development. In addition, samples ofhoney, beeswax, and worker-gathered pollen and nectar were regularly analyzed 2-·: DP Barcode: D336888 MRID No. 469078-01 (with 469078-02)
for clothianidinresidues. Colonyweight gain while inthe canola fields and honeyyield per colonywas also determined. Overall, there was no differencebetween colonies from clothianidin-treated and control fields. Although sporadic treatment or site differences were found on various dates, essentiallyno differences in worker ordrone mortality, worker longevity, orbrood -development occurred during the study. Colonies in treated fields had similar weight gains and honey yields as those in control fields. Qualitative assessments byexperienced bee researchers confirmed that colonies from**clothianidin~treated** field were as strong and healthy as those from control fields. Themajority ofsamples collected (>75%) for residue analysis had no detectable levels of
·clothianidinresidues (LOQ = 0.5 ng/g). Themaximum concentrations ofclothianidin detected inhoney, nectar, pollen, and beeswax samples were 0.928, 2.24, 2.59, and <0.5 

### ng!g, respectively. These levels were approximately 8-fold below the reported field relevant NOAEC of20ppb. Itwas concluded that honeybees that forage on clothianidin seed-treated canola will be exposed to clothianidin residues in the form ofpollen, nectar, and honey; however, exposure concentrations are below those required to elicit acute and sublethal effects. In a study addendum (MRID 469078-02), the status of29 ofthe original 32 over-wintered colonies was assessed onApril 19-20, 2006. Observations included the presence/absence ofthe queen, presence/absence ofeggs and larvae, area ofsealed brood, number offrames ofworkers, and overall health ofthe colony. The spring assessment found no significant difference in the healthbetween the treated and control colonies. Two treated colonies and two control colonies did not survive the winter. Ofthe 25 colonies that did survive the winter, a healthyqueen was found in 21, and thepresence ofeggs and larvae in the remaining four indicated that these colonies were queen-right. There was no statistical difference between treated and control colonies inthe amount ofsealed brood or in the A number offrames ofworkers. Collectively, 24 colonies were classified "healthy", one was classified "weak" (<4 frames oflivebees), and four were "dead". This study is scientifically sound and satisfies EFED concerningthe guideline requirements for a field toxicity test with honeybees (OPP Gdln. No. 141-5; OPPTS 850.3040). 

### 9. ADEQUACY OFTHE STUDY: A. 

### Classification: Acceptable 

### B. Rationale: N/ A 3DP Barcode: D336888 MRlD No. 469078-01 (with 469078-02)

C. 

### Repairability: N/A

10. 

### GUIDELINE DEVIATIONS: N/A

11. 

### SUBMISSION PURPOSE: This studywas submitted to provide data onthe toxicity of clothianidinto honeybees in a field test for the**purpos~** ofchemical registration (new use). Specifically, the test was conducted inresponse to a request bythe Canadian PMRA and the U.S. EPA; as a condition for Poncho® registration in these countries, Bayer CropScience was asked to investigate the long-tenn toxicity ofclothianidin-treated canola to foraging honeybees. 

### 12. MATERIALS AND METHODS: A. Test Orgamsms Guideline Criteria Reported Information. Species: Species ofconcern 

### (Apis**mell~fera,** 

### Apis mell?fera L. 

### Megachile rotundata, or

### Nomia melanderi) Colony description at beginning oftest: Each colony consisted ofa single brood chamber (24 em deep, 10 frames per super) below a shallowhoney super (originally empty, 16.5 em deep, 9 frames per super).



### Pre-testhealth:

4Queens in all colonies were ofthe same lineage and approximatelythe same age. A queen excluder was placed between the brood chamber and honey super to retain the queen in the brood chamber. Colonies were adjusted for strength to establish similar quantities offood stores (pollen and nectar), brood in all stages of development, and adults in each. Colonies were assessed for presence ofVarroaDP Barcode: 0336888 MRID No. 469078-01 (with 469078-02)



### - Guideline Criteria Reported Inforn'tidion mite, tracheal mite, and infectious honeybee diseases (American Foulbrood, European Foulbrood, and Chalkbrood) priorto placement in canola and throughout the study.

- Colonies were also treated with Checkmite® prior to placement in canola. 

### Supplier Priorto field testing, the honeybee colonies were held at a spring apiarynear the Townsend House Bee Research Facility, University of Guelph, Ontario. 

### All bees from the same source? Yes 

### B T tS t



## . es ;ys em Guideline Criteria Reported Information Exposure Site Location and Establishment: The four test sites were located in Elora, Ontario, Canada, at the University ofGuelph, Elora Research Station (sites El and E2), and two neighboring farms ownedbyAllan and Phillip Wallace (sites W3 and W4).
East test site consisted oftwo 1-hectare fields; one planted with clothianidin-treated canola seed and the otherplanted with control seed, giving a total ofeight fields. Fields at each site were separated by at least 250 m. Planting ofthe canola seed occurred onMay 20-21, 2005. Seeds were sown to a depth of4 em at the highest recommended rate of 15-20 seeds/m (8.0 kg/ha).

5DP Barcode: D336888

. MRID No. 469078-01 (with 469078-02)
·'·': 

### . GuidelineCriteria . ·. 

### ReportedInforni1ition :· 

### Site Preparation: All fields received a pre-plant treatment with Treflan EC® (43% trifluralin) at 2.0 Llha, and with fertilizer (ammonium nitrate, 34-0-0) at 100 kg Nlha according to Ontario canola

- production recommendations. Prior to introduction ofthe colonies, a 10 m x 10 m clearing was mowed inthe middle of each canola field to accommodate four colonies. 

### Number ofReplicates/Treatment: Four colonies per field, with 1treated and 1 control field per site, and four sites (32 total colonies) 

### Post-exposure Site Location: The fall apiary was located at the fonner University ofGuelph, Cambridge Research Station, Ontario, Canada. 

### Lighting: Natural; not further described. 

### Precipitation: Dailyprecipitationranged from 0.0 to 18.8 mm during the exposureperiod (data obtained from the Canadian weather website cited in the studyreport; refer to Reviewer's Comments section). The maximum rainfall event occurred onJuly 16 and 17, when 18.8 and 10.4rnm precipitation occurred, respectively. Total precipitation during the exposure period was 35.4 mm. 

### Temperature: Mean daily temperatures ranged from 15.2 to 26.7°C during the exposure period. 

### Relative humidity: Not reported


### C. TestDesi n Guideline Criteria ReportedInformation indingtest? None reported

6DP Barcode: D336888 MRID No. 469078-01 (with 469078-02)



### - Guideline Criteria Reported Information Reference toxicant tested? No 

### Duration ofExposure Period 21 days, during canola bloom period 

### Duration ofPost-exposure Period 109 days in the fall apiary

- 

### Test 9ubstance(s): Prosper FL Formulation Type: flowable suspension Batch No.: 312065M Ai: 9.64% clothianidin +the fungicides thiram, carboxin, and metalaxyl at ca. 9, 4, and 0.3%, respectively (refer to Reviewer's Comments section) Source: Gustafson, McKinney, TX Poncho 600 FS Formulation Type: flowable suspension BatchNo: 407483M Ai: 48.0% clothianidin Source: Bayer CropScience, Kansas City, MO 

### Control Substance(s): ProsperFLBlank Lot No.: TAM113:70-l Ai: thiram, carboxin, and metalaxyl Source: Gustafson, McKinney, TX Poncho 600 Blank Lot No.: TAM113:67-1A Source: Gustafson, McKinney, TX 

### Canola Seed: Variety A: Hyola 420, supplied from Interstate Payco Seed Company, West Fargo, ND

7

## .. . •,·. DP Barcode: D336888


### Guideline Criteria Application Rate:

-



### Verification ofApplication Rate:
Method ofSeed Coating:

Colony Introduction:

I MRID No. 469078-01 (with 469078-02)

-- 

### Reported Information Treatment 1: Prosper FL at 1250 mL/100 kg and Poncho 600 FS at 417 mL/100 kg, to deliver clothianidin at the rate of400 g ai/100 kg seed. Treatment 2: Blank Prosper FL (containing thiram, carboxin, and metalaxyl) and Blank Poncho 600 FS at the same ratios as used for Treatment 1. Mean (n=3) of4175 ppm or417 g ai/100 kg seed Forboth treatments, slurries werepreparedby combining the appropriate quantities ofeach fonnulation. The slurries were applied to the seed using the Gustafson CBT-50 seed treater. Due to the large quantity ofseed (100 kg per treatment), each treatment was divided into four 25-kg batches for treatment. The colonies were moved to the canola fields over a two-night period (June 27/28 and June 29/30), when approximately one-quarter to two-thirds ofcanola blooms in the test fields had opened (detennined byvisual estimation). All colonies were positioned so that the entrances faced approximately south. June 30, 2005 was identified as Day 0 ofthe 3-week exposureperiod. Honey supers were removed from and added to colonies as needed throughout the study

.



### 8DP Barcode: D336888 MRID No. 469078-01 (with 469078-02)

·.· 

### Guideline Criteria ReportedInformation I .

.· . ..•. · . ... 

### Post-exposure: Colonies were moved at night from the canola fields to the fall apiary on July 20/21, when approximately 20% bloom remained in each field. Colonies remained there until study

- tennination (Day 130; November 7). Control colonies were separated from those from the clothianidin-treated fields byat least 30m. No other colonies werepresent at the fall apiary. 

### D B" l . lA


## . 10 

### og1ca ssessments Guideline Criteria ReportedInformation Canola:

- Seedling emergence rates (detennined on June 3 and June 7/8)

- Developmentrates

- Crucifer flea beetle (Phyllotreta cruciferae Goeze) and striped flea beetle (Phyllotreta striolata (F.) damage 

### Weight Gain: Colonies were weighed on Days -1 (the night oftranspo1i to the canola fields) and Day21 (the night colonies were moved to the fall apiary). 

### HoneyYield: Honeyyield percolonybyweight. 

### AdultMortality: Dead workers and drones were collected and counted ca. every7 days from Days 0 to 130. Mortality was assessed using Gary Dead Bee Traps (DBT) or 1 x 2m white sheets placed on the ground extending out from the hive entrance. As only eight DBT units were available, one randomly selected colony at each field was fitted with a DBT, while the entrance sheet method was used for the remaining three colonies.

9DP Barcode: D336888 MRID No. 469078-01 (with 469078-02)


### Guideline Criteria Reported Information I

### Brood: The area ofsealed brood was determined on Days -2, 1/2, 14/15, 33/34, and ca. every 14 days up to Day 98 (refer to Reviewer's Comments section).

- The area ofsealedbrood was estimatedby placing an emptytemplate brood frame that was divided into six quadrants over each test brood frame, and estimating the percent sealed. Estimates were performed on both sides of each frame, for all 10 frames of**ea~h** colony. 

### Worker Longevity: Tagged workerbees were counted on Days 5 and 9.(post-introduction assessments), 14 and 15, and thereafter at ca. 14-dayintervals up to Day98. On Day4 (allowing for a 3-day colony acclimation to the canola fields), newlyemerged (<24 hours) workerbees (from spare colonies maintained at the Townsend House Bee Research Facility) were marked with Opalith® colored/numbered thoracic tags (Graze, Bienenzuchtgera.te), and 50 marked workers were then introduced to each colony. Assessments onDay 5 indicated unsuccessful introductions in six colonies (three control and three treatment), and therefore areintroduction was perfonned on Day 8. On Day 70, a second set oftagged workers was added to all colonies. At that time, 25 colonies had no tagged workers left, four colonies had one tagged worker, one colonyhad five tagged workers, and one colony still had 12 tagged workers. Following each reintroduction of tagged workers, those from subsequent introductions were disregarded during data collection.

10DP Barcode: D336888 MRID No. 469078-01 (with 469078-02)



### -- Guideline Criteria Reported Information Queen Assessments: Queen assessments were conducted at the time ofbrood assessment, i.e., Days -2, 1/2, 14115, 33/34, and ca. every 14 days up to Day 98.

- Queens were located and visuallyinspected to ensure nonnal physical health and behavior. When queens were not located, thepresence of eggs confirmed the presence ofa laying queen in the colony within the last 3 days. Inspections were also conducted at these times for queen supercedure cells (elongate cells in which a new queen is reared). Most often, these cells were opened to verifythe presence ofa larva, and then destroyed. Ifthe queen was absent in a colony, however, in some cases supercedure cells were left to allow a new queen to be reared bythe colony. In other cases, marked queens were collected from spare colonies and introduced to the experimental colonies (refer to Reviewer's Comments section for further detail).
E R "d A l .



# . es1 ue natySIS Guideline Criteria Reported Information

. Nectar Collection: A 5-g pooled sample ofnectar (when available) was collected from colonies at each field on Days -3/-1, 7, 14/15, 42, and thereafter at ca. 21-dayintervals up to Day 83. Nectar was extracted from cells using a disposable syringe, or removed by gently shaking abrood frame over a sheet ofwaxed paper.

11
·:' \.DP Barcode: .Q336888 MRID No. 469078-01 (with 469078-02)

-. 

### Guideline Criteria Reported Inforh:lation·

. ·.·· . •. 

### Honey Collection: A 5-gpooled sample ofhoney was collected from colonies at each field using a small disposable spatula on Days -3/-1, 7, 13, 40, and thereafter at ca. 21-day intervals up to Day

- 102. 

### Pollen Collection: A 10-g pooled sample ofpollen was collected fiom colonies at each field on Days -3/-1,7, 14/15, 42, and thereafter at ca. 21-dayintervals up to Day 106. Pollen was collected over a 24-hourperiod using an OAC pollen trap. Approximately 5 g ofeach sample was analyzed under a light microscope to confirm the bees foraged on canola. The remainder was used for residue analysis. 

### Beeswax Collection: A 3-cm2 pooled sample ofbrood and food-free beeswax was collected from colonies at each field on Days -3/-1, 7, 13, 40, and thereafter at ca. 21-day intervals.

12DP Barcode: D336888


### Guideline Criteria Storage ofSamples:

Extraction/ Analysis:

..t
·.**'~** MRID No. 469078-01 (with 469078-02) 

### Reported Inforrilation All samples collected for residue analysis were held in a freezer at -20°C until shipment to the laboratory, and were shipped on dryice. Conditions ofstorage (samples and extracts) once at the laboratory were not reported. Intervals ofstorage (reviewer-determined): Honey: 157 days priorto extraction, and 49 days prior to analysis. Nectar: 283 days priorto extraction, and 26 days prior to analysis. Pollen: 212 days priorto extraction, and 44 days priorto analysis. Beeswax: 273 days prior to extraction, and 22 days prior to analysis. 

### Storage stability assessments were apparentlynotperformed. The residue method for clothianidin in pollen, honey, and nectar was based on BayerMethod 00554 with minormodifications. Forwax, a short summary supplied byRalfSchoning of Bayer for imidacloprid was used as the basis for extraction. For all matrices, concentrations ofclothianidin were detennined using LC/MS/MS, and the limit ofquantitation was 0.5 ppb (ng/g)
.

13DP Barcode: D336888 MRID No. 469078-01 (with 469078-02)


### 13. REPORTED RESULTS:

Guideline Criteria Reported Information 

### Quality assurance and GLP compliance statements were Yes (see Reviewer's Comments section for 

### includedin the report? details regarding non-GLP sections). 

### Raw data included? No, raw datawere generallynot provided for any measured parameter. Summarized data tables (means) were provided for worker and drone mortality, and worker longevity. 

### Signs oftoxicity (ifany) were described? Yes
CanolaEmergence: Canola emergence, development, and flea beetle damage were compared bytreatment and site. There were significantlymore emerged plants permeter in clothianidin seed-treated fields than in untreated fields onbothJune 3 and June 8 (p<O.OOOl). Comparison among treated sites indicated that emergence was greatest at site E2 and lowest at site W 4 at bothintervals, although the difference was only significant onJune 3 (p = 0.0029). Onboth sampling days, however, a significant site-treatment interaction was found, with generally greater emergence in treated fields (p 

### = 0.026 on June 3 and p 

### = 0.0025 on June 8). Althoughthere were generallymore emerged canolaplants permeterin clothianidin-treated fields than control fields, development ofemerged plants was the same inboth. The growth stage ofemergedplants did not differ with treatment or site, and there was no significant interaction ofthose effects. Fleabeetle damage was significantly greater in control fields onbothJune 3 and June 8 (p<O.OOOl). Although flea beetle damage did not varywith site on June 3, a differencebetween sites was found on June 8 (p = 0.014). Weight Gain: Therewas no significant difference inweight gain ofcolonies from control and clothianidintreated fields (Figure 1 ). In both treatments, colony weights increased approximately 23-24kg during the 3-week exposureperiodin canola fields. In addition, differences in colonyweight gain were not significant among sites, and there was no significant treatment-site interaction.
14DP Barcode: D336888 MRID No. 469078-01 (with 469078-02)


### Figure 1: Mean (±SEM)honey bee colony weightgain andhoneyyieldafter exposure to clothianidin-treated (n=16) andcontrol (n=15) canola. Colonies were in canolafieldsfor21 days during bloom (Days 0-21), andthereafter movedto a fall apimy, approximately 35 km away, where they were maintainedforanother 109 days. 

### 60**-r-··-··-···-·-······················-····~** ...................................................................___ ...._ ................................................................................................**~**............**~**............., 

## I Treatment 50 · 

# I""· '"'''·'I 

## Controi: t 

### '

HoneyYield: 

## CoronyWelght G·aln (Day**0~21}** 

## Honey**Yield/Co~ony** 

### ·(Day

# o ..130)

There was no significant difference in honey yield from colonies from control and clothianidintreated fields (Figure 1). A mean of45.3 kg and 44.7 kg ofhoneywas harvested from treated and control fields, respectively, overthe 130 days ofthe experiment. Values were comparable to the 2005 Ontario honey yield average of46.6 kg. In addition, differences in honey yield were not significant among sites, and there was no significant treatment-site interaction for honeyyield.

15DP Barcode: D336888 MRID No. 469078-01 (with 469078-02)
Adult Mortality: In analyzing changes inthe number ofdead workers ordrones over time, DBT (dead bee trap) and sheet data were analyzed separately, and the GLM incorporated effects ofday, treatment, and the interaction oftheseterms. For analyses onindividual days, the GLM inmost cases was able to simultaneouslyincorporate analysis ofeffects ofsite, treatihent, and method ofdead bee collection. In some cases, however, inclusion ofone ofthese parameters resulted in a significant LacliofFit (LOF) in the model, while itselfnot contributing significantlyto the model. In such cases, the parameter causing the LOF was removed, resultir:g in a simplerbut more robust model (Table 1). There were significant changes over time inthe number ofdead workers recovered from colonies withboth theDBT (trap) and the sheet methods (p<O.OOl). However, regardless ofthe dead bee collectionmethod used, there was no significant difference in dead workers due to treatment or the day-treatment interaction. Although the recoveryofdead drones changed over time with the entrance sheetmethod (p<O.OOl), no change overtime was found with DBT. As with workers, regardless ofthe dead bee collection method used, treatment and the day-treatment interaction had no significant effect onthe number ofdead drones found. Table 1: Honey bee mortality in colonies located in clothianidin-treated (n=16) andcontrol (n

### =16) canol afields. Ateach offoursites (one treatment, one control fieldpersite) were three colonies equippedwith a white entrance sheet, andone colony fitted with a dead bee trap (DBT). Effects of site, treatment, dead bee assessment method, andtheir interaction were determined usinga general linear modelplatform (SASInstitute 2003). Statistically-significant effects (a = 0.05) are in bold. Day Dead Workers Dead Drones Effects Test Statistics Effects Test Statistics 

### i Site p = 0.45 Site P = 0.1 I Treatment p = 0.75 Treatment p = 0.44 Site*Trt p = 0.51 Site*Tti p = 0.45 13 Site2 p = 0.40 Site p = 0.69 Treatment p = 0.13 Treatment p = 0.61 Site*Trt p = 0.18 Method p = 0.92 Site*Trt p = 0.39 Site*Method p = 0.56 Trt*Method p = 0.58

16DP Barcode: D336888 MRID No. 469078-01 (with 469078-02)

"" oO Day Dead Workers Dead Drones !!-"c.-:: Effects Test Statistics Effects Test Statistics 18 Treatment3 p = 0.07 Treatmene p = 0.18 Method p < 0.001 Method p = 0.06 Trt*Method P=O.l3 Trt*Method p = 0.12

- 21 Colonies moved from canola fields to fall apiary

. 28 Site p = 0.77 Site p = 0.97 Treatment p = 0.31 Treatment p = 0.38 Method p = 0.34 Method p = 0.32 Site*Trt p = 0.71 Site*Trt p = 0.57 Site*Method p = 0.27 Site*Method p = 0.31 Trt*Method p = 0.03 Trt*Method p = 0.70 35 1 Site p = 0.51 Site p = 0.41 Treatment p = 0.35 Treatment p = 0.88 Site*Trt p = 0.06 Site*Trt p = 0.45 

### 4i Site p = 0.20 Site p = 0.59 Treatment p = 0.46 Treatment p = 0.35 Site*Trt p = 0.50 Site*Trt p = 0.67 49 Treatmene p = 0.15 Site p = 0.93 Method p = 0.09 Treatment p = 0.60 Trt*Method p = 0.002 Method p = 0.32 Site*Trt p = 0.57 Site*Method p = 0.81 Trt*Method p = 0.71 64 Site p = 0.96 Site p = 0.87 Treatment p = 0.31 Treatment p = 0.23 Method p = 0.32 Method p = 0.97 Site*Trt p = 0.56 Site*Trt p = 0.26 Site*Method p = 0.42 Site*Method p = 0.37
17DP Barcode: D336888 MRID No. 469078-01 (with 469078-02)
Day Dead Workers Dead Drones ·5-...::_ Effects Test Statistics Effects Test Statistics Trt*Method p = 0.03 Trt*Method p = 0.90 71 Site p = 0.39 Site p = 0.67

- Treatment p = 0.07 Treatment p = 0.22 Method p = 0.52 Method p = 0.84 Site*Trt p = 0.33 Site*Trt p = 0.28 Site*Method p = 0.13 Site*Method p = 0.95 Trt*Method p = 0.79 Trt*Method p = 0.73 77/79 Site p = 0.002 Site p = 0.72 Treatment p =0.02 Treatment p =0.97 Method p < 0.001 Method p = 0.82 Site*Trt p = 0.98 Site*Trt p = 0.54 Site*Method p = 0.004 Site*Method p = 0.68 T!i*Method p = 0.004 Trt*Method p = 0.47 89 Site p = 0.50 Site p = 0.99 Treatment p = 0.62 Treatment p = 0.47 Method p = 0.46 Method p = 0.29 Site*Trt p =0.37 Site*Trt p = 0.12 Site*Method p = 0.91 Site*Method p = 0.88 Trt*Method P=O.l8 Trt*Method p = 0.70 92 Site p = 0.03 Site p =·0.86 Treatment p < 0.001 Treatment p = 0.57 Method p <0.001 Method p = 0.89 Site*Trt p = 0.04 Site*Trt p = 0.99 Site*Method p =0.002 Site*Method p = 0.35 T1i*Method p =0.004 Trt*Method p = 0.87

18DP Barcode: D336888 MRID No. 469078-01 (with 469078-02)



### - Day Dead Workers Dead Drones**~~:** Effects Test Statistics Effects Test Statistics 99 Site4 P=0.19 Site p = 0.16 Method p < 0.001 Treatment p = 0.47 Site*Method p = 0.17 Method p = 0.04

- Site*Trt p = 0.06 Site*Method p = 0.03 Trt*Method p = 0.96 106 Site p =0.07 Treatment3

. p = 0.07

-· Treatment p = 0.54 Method p = 0.08 Method p = 0.38 Trt*Method p = 0.10 Site*Trt p = 0.49 Site*Method p = 0.08 Trt*Method p = 0.89 112 Site p < 0.001 Treatment3 p = 0.07 Treatment p = 0.13 Method p = 0.05 Method p < 0.001 Trt*Method p = 0.07 Site*Trt p = 0.81 Site*Method p < 0.001 Trt*Method p = 0.02 120 Treatment3 p = 0.008 Treatment3 p = 0.20 Method p < 0.001 Method p < 0.001 Trt*Method p = 0.009 Trt*Method p = 0.24

19DP Bai·code: D336888 MRID No. 469078-01 (with 469078-02)

·- Day Dead Workers Dead Drones**~-c-.**•. Effects Test Statistics Effects Test Statistics 127 Site p < 0.001 Site p < 0.001 Treatment p =0.82 Treatment p =0.34

- Method p < 0.001 Method p < 0.001 Site*Trt p = 0.22 Site*Trt p = 0.60 Site*Method p < 0.001 Site*Method p < 0.001 Trt*Method p =0.26 Trt*Method p =0.72 1 DBT data unsmtable for analysis. 2 Methodparameterresulted in a significant Lack ofFit and therefore was omitted from the model. 3 Site parameterresulted in a significant Lack ofFit and therefore was omitted from the model. 4 Treatmentparameter resulted in a significant Lack ofFit and therefore was omitted from the model. On various collection dates, there were significant differences due to site, dead bee recovery method (DBT or sheet), treatment, and the interaction ofthese terms. However, there were no consistent trends in effect ofthese variables dmingthe experiment. Recovery ofdead bees using DBTwas usually significantly greaterthan that with entrance sheets, although a significant method-site interaction was often found, indicatinghigh variabilityin the number ofdead bees recovered amongthe colonies fitted with a DBT. From a total of 18 samplingdates over 130 days, onlyone data set (Day 56) showed a statistically significant increase in workermortality in treated colonies, whereas onthree sampling dates (Days 77/79, 92, and 12) mortality from control colonies was statisticallyhigher, Thus, there was overall no relevant differencebetween treatment and control colonies in workermotiality. In general, more dead workers than drones were recovered throughout the experiment, regardless ofthe collection method (Table 2). Effects on the number ofdead drones recovered were minimal, but were occasionally found near the end ofthe experiment. As expected, the number ofdead workers increased near the end ofthe experiment (e.g., Day 99) as colonies prepared for over-wintering.

20,. DP Barcode: D336888 MRID No. 469078-01 (with 469078-02)


### Table 2: 

### Honey bee worker and

### dro~e mortality in colonies located in clothitimidin-treated (n=I6) andcontrol (n=l6) canola.fields. Ateach offoursites (one treatment, one control.fleld persite) H"ere three colonies equipped with a white entrance sheet, andone colony.fittedwith a d db t 

### (DBT)

ea ee rap Day Mean No. Dead per Colony Dead bee trap (DBT) Entrance Sheet

- Treated Control Treated Control WorkerMortality 7
-- -- 43.1 39.8 13 70.5 4.0 22.1 14.1 18 213.5 123.3 26.4 18.8 28 66.8 17.8 18.7 37.8 35
-- 

### -- 34.1 44.9 42 

### -- -- 24.9 34.4 49 174.5 54.8 47.6 95.5 56 128.5 40.0 29.9 35.6 64 72.3 27.8 28.1 44.8 71 19. I 63.0 11.6 44.8 77 117.5 188.9. 37.2 29.0 89 54.8 107.5 72.8 47.8 92 24.7 86.8 15.7 20.9 99 356.3 333.5 79.8 113.0 106 255.3 225.3 208.3 160.8 112 117.3 148.0 16.7 9.4 120 141.0 291.8 4.8 6.3 127 87.3 109.6 18.1 2.9

' DroneMortality 7 

### -- -- 4.2 5.4 13 10.8 3.0 7.1 7.3 18 56.8 14.3 6.0 8.8
21DP Barcode: D336888 MRID No. 469078-01 (with 469078-02)

- 

### -. Day Mean No. Dead per Colony**~~:** Dead bee trap (DBT) Entrance Sheet Treated Control Treated Control 28 0.2 5.8 6.3 8.5 35

-- 

### -- 8.5 7.8

- 42

-- 

### -- 2.7 5.0 49 7.3 9.3 15.5 27.3 56 6.8 12.0 14.8 19.0 64 4.5 14.5 5.8 13.8 71 1.8 4.0 1.4 5.3 77 6.0 13.7 11.6 3.2 89 6.9 36.3 45.2 54.1 92 3.4 4.8 3.3 5.8 99 42.0 38.0 29.7 25.0 106 9.5 143.8 6.2 11.9 112 4.0 93.8 0.3 0.3 120 4.3 2.3 0.3 0.2 127 37.8 36.0 2.0 1.2 There was a noticeable spike in the number ofdead workers recovered in DBT on Day 18 (Table 2). In the week preceding collection ofdead bees on Day 13, the mean maximum temperature was 30.9°C. Underthese conditions, it is possible that DBT (essentially large metal boxes covering the colony entrance) caused poor ventilation and over-heating, resulting in an increased number ofdead workers on this day. Brood Assessment: Although brood assessments were to be conducted up to Day 130, it was evident byDay 112 that there was no or minimal sealed brood in colonies in preparation for over-wintering. Therefore, the final brood assessment was conducted onDay 97/98. The amount ofsealed brood per colony changed significantly over time in colonies from control and clothianidin-treated canola fields (p<0.0001). However, on most days there was no effect of site, treatment, and/or sampler (the individual detenniningthe amount ofsealed brood), orthe 22

## ,. DP Barcode: D336888 MRID No. 469078-01 (with 469078-02)
interaction ofthese tenns (Table 3). On Day 1/2, the amount ofsealed-brood:Fler colonydiffered significantly across sites and on Day 33/34, the amount ofsealed brood differed with the sampler. At no time duringthe experiment did the amount ofsealed brood in colonies from clothianidin-treated field differ significantlyfrom that found in colonies from control fields. 

### Table 3: 

### Mean area o.fsealedbrood in honey bee colonies in clothianidin-treated (n=16) and control (n=16) canolafields. Colonies wereplaced in canolafields on July 1 (Day 1) andwere movedto a fall apiary on July 21 (Day 21)forthe remainder of the experiment (to Day 130). Effects of site, treatment, sampler (individual determining tjw amount ofsealedbrood), and their interaction were determined usingageneral linear modelplatform (SASInstitute 2003).**Statistically-sign~ficant** 

### effects (a = 

### 0.05) are in bold. Day Mean Area (cm2) Sealed Broodper Colony Effect Statistics Treated Control 11 5304.2 5104.3 Site p = 0.007 Treatment p = 0.06 Site*Trt p = 0.09 14 5126.14 4814.7 Site p = 0.32 Treatment p =0.61 Sampler p = 0.27 Site*Trt p =0.48 Site*Sampler p =0.39 Trt*Sampler p = 0.56 33/342 4816.8 4762.0 Sampler p = 0.05 Treatment p = 0.84 Sampler*Trt p = 0.88 48/492 4975.0 4780.3 Sampler p = 0.36 Treatment p = 0.69 Sampler*T1i P=0.16 63/642 4612.1 4238.6 Sampler P=0.17 Treatment p = 0.93 Sampler*Trt p = 0.14

23DP Barcode: D336888
Day Mean Area (cm2 ) Sealed Brood per Colony Treated Control 76/84 1 4597.4 4917.6

- 97/98 1 1179.9 819.6

1Could not incorporate 'sampler' effect into model. 2 Could not incorporate 'site' effect into model. Worker Longevity: MRID No. 469078-01 (with 469078-02)

-. Effect ".:Statistics

Site p = 0.53 Treatment p = 0.49 Site*Trt p = 0.79 Site p = 0.54 Treatment p = 0.15 Site*Trt p = 0.07

The number oftagged workers decreased overtime in colonies from both clothianidin-treated and control canola fields (p<O.OOOl). The GLM found no significant effect ofsite or treatment on longevity oftagged workers. There also was no significant date*site, date*treatment, or site*treatment interaction ofthe tenns. Throughout the experiment, there was no significant difference in the nm11ber oftagged workers found in colonies from clothianidin-treated and control fields on any give day (Table 4). That is, workers lived as long in colonies in treated fields as in control fields. 

### Table4: Honey bee worker longevity in colonies in clothianidin-treated (n=J6) and control (n=16) canolajields. Workers (n=50) taggedwith colored/numberedthoracic tags were added to each colony on Day4 (July 4); a secondtaggedworkedintroduction (n=50) was made onDay 70 (Sept. 8). Effects of site, treatment, and their interaction were determined using a general linear model**pla~form** (SASInstitute 2003). Day Mean No. Tagged Workers Effect Statistics Treated Control 5/9 40.7 42.6 Site p = 0.87 Treatment p = 0.30 Site*Trt p = 0.12 14/15 30.3 30.3 Site p = 0.81 Treatment p = 0.98 Site*Trt p = 0.34

24.**~.**



### ...

.**:·~** : DP Barcode: D336888 MRJD No. 469078-01 (with 469078-02)



### -- Day Mean No. Tagged Workers Effect Statistics Treated Control 33/34 9.7 8.2 Site p = 0.54 Treatment p = 0.48

- Site*Trt p = 0.08 48149 1.7 1.4 Site p = 0.72 Treatment p = 0.70 Site*Trt p = 0.26 63/64 0.5 0.1 Site p = 0.77 Treatment p = 0.22 Site*Trt p = 0.55 76/77 24.8 NA 1 Site

-- Treatment
-- Site*Trt

-- 84 NA 1 12.4 Site


### -- Treatment


### -- Site*Trt


### -- 97/98 5.8 5.1 Site p = 0.71 Treatment p = 0.62 Site*Trt p = 0.27 1Data not collected. Disease: Incidence ofdisease was low throughout the study. Colonies were treated with Checkmite® priorto placement in canola. As a result, Varroa and tracheal mite incidence was very low throughout the study; in the majority ofcolonies, no Vanoaor tracheal mites were detected. American Foulbrood and European Foulbrood were not found in any colonies during the study. Chalkbrood was sporadically detected at very low levels (i.e., 5-10 cells/colony throughout the study. However, as workers routinelyremove chalkbrood mummies, the disease never affected the overall health ofcolonies.

25DP Barcode: D336888 MRID No. 469078-01 (with 469078-02) Queen Losses and Overall ColonyHealth: Thepresence ofeggs and larvae were observed in colonies throughout the study. Due to losses ofthe queen, some colonies at some observations had low numbers ofeggs and larvae. Loss of queens from colonies was expected given the intense amount ofdata collection, movement of colonies, and large number ofcolonies in the study. Duringthe experiment, personnel replaced**que~ns** in eight colonies (Table 5). In five colonies (E2Cc, W3Cc, W3Cd, E2Tb, and E2Tc), original marked queens that died or were killed were naturallyreplaced by a virgin queen (i.e., the colonyreplaced the original queen on its own). Colonies W3Cc and E2Tc wereboth artificially and naturallyre-queened. Therefore, a total ofsix colonies from clothianidin-treated fields and five colonies from control fields were naturally and/or artificiallyre-queened during the study. ColonyW3Ta was problematic throughout the study. 

### It was found to be queenless on July 8 (likelykillea during the move ofcolonies to canola fields), and subsequentlydid not accept artificiallyre-introduced queens. However, the colony was found to be successfully naturallyre-queened on Day 63 and thereafter. T bl 5 

### R

a e : oney b l J

. 

### h ee co ames to w uc newqueens were zntrod d uce . July 8 August 4 August 18 Treated Colonies E2Ta X E2Tc X W3Ta X X X W4Tc X X W4Td X Control Colonies W3Cc X W4Ca X W4Cb X Three colonies (W3Cc, W4Cb, and W4Td) were classified as "dead" pmi way through the study. These colonies were artificially or naturallyre-queened during the experiment, but failed to successfully establish a queen. Although data from these colonies mayhave inadvertently been collected (prior to status was realized), these data were omitted from some statistical analyses, e.g., sealed brood analyses nearthe end ofthe experiment. Given that adequate data were collected from these colonies through much ofthe experiment, and that there were a large number ofreplicates in total, the loss ofthese colonies had no impact on the study overall.
26DP Barcode: D336888 MRID No. 469078-01 (with 469078-02)

As a general observation, experienced beekeepers/researchers qualitatively assessed colonies from clothianidin-treated and untreated canola fields throughout the study and found no differences in overall colonyhealth and vigor. Residue Analysis: Clothianidin was detected in treated seed at the prescribed level at an average of417 g ai/100 kg seed. In-phase recovery(± SD) ofclothianidin residues fro;m spiked samples ofhoney, nectar, pollen, and beeswax was 93 ± 13.0, 89 ± 13.3, 87 ± 13.8, and 104 ± 21.6%, respectively. Themajorityofsamples (>75%) collected had no detectable levels ofclothianidinresidues (LOQ 

### = 0.5 ng/g), whetherfrom colonies in treated or control fields (Table 6). Themaximum concentration ofclothianidin detected in honey, nectar, pollen, and beeswax samples was 0.928, 2.24, 2.59, and <0.5 ng/g, respectively. No clothianidinresidues were detected in honey, pollen, orbeeswax samples collected from control fields, although analyses conducted in January2006 detected residues inthree nectar samples from control colonies (field E1C, July 7; field W3C, <' July 7; and field W3C, August 11). Subsequent analyses ofback-upnectar samples detected residues in two control colonies (field ElC, July7; and field W3C, July 7), suggesting that workers in control colonies mayhave foraged on clothianidin-treated canola. This mayhave occurred because the separationbetween some pairs ofcontrol and treated fields was insufficient orbecause the forage in some control fields was oflower quality (due to insectdamage and lower rates plant emergence), which mayhave lured workers from control fields to the treated fields. Clothianidin was also detected in two nectar samples when the colonies were not in canola fields (field W3T, June 27; field W3C, August 11). 

### Table 6: Clothianidin residues in honey, nectar,pollen, andbeeswax collectedfrom honey bee colonies in clothianidin-treatedandcontrol canolajields. Pooledsamples were collectedat each l 21 d 

### site approxunatety every ays. Matrix Treatment Total No. Samples Samples with Residue Detected Residues Detected (ng ai/g) Honey Clothianidin 28 W4T,July07 0.501 W3T, July07 0.647 E2T, July07 0.510 EIT, July 07 0.928 W3T, July 07 0.507 Control 28


### -- <0.5 1

27DP Barcode: D336888 MRID No. 469078-01 (with 469078-02)

.. 

### - Matrix Treatment Total No. Samples Samples with**-~** Residue Detected Residues Detected (ng ai/g) Nectar2 Clothianidin 15 (Jan.) EIT, July 07 0.521 E2T, July 07 0.979 W3T, July07 1.17 W4T, July07 0.855

. 23 (Mar.) W3Td, July 27 0.693 E1T, July 07 2.24 .E2T, July 07 0.717 W3T, July07 1.74 W4T,July07 1.14 Control 15 (Jan.) EIC,July07 0.535 W3C, July07 0.670 W3C, August 11 0.969 23 (Mar.) ElC, July 07 0.691 W3C, July07 0.922 Pollen Clothianidin 19 E1T, July07 1.05 E2T, July 07 0.698 W3T, July 07 2.59 E1T, July 14 1.40 Control 19


### -- <0.5 Beeswax Clothianidin 0


### -- <0.5 Control 0


### -- <0.5 'I - 0.5 ng/g

- LOQ
. 2 Residue analyses conducted in January 2006 unexpectedly detected clothianidin residues in nectar samples collected from control colonies. Therefore, back-up nectar samples were sent to the laboratory in March 2006 for reanalysis. Reported Statistical Results: Plant emergence, development, and flea beetle damage were compared among treatments and sites usi;ng a general linear model (GLM) platform (SAS Institute, 2003). Colony weight gain during exposure, honeyyield during exposure, worker and drone m01iality, brood area per colony, and tagged worker longevity (based on the number oftagged workers recorded each collection day) were also compared overtime using a GLMplatfonn. 

### 14. SUPPLEMENTAL ASSESSMENT OF OVERWINTERED COLONIES: A supplemental report documenting further assessments ofthe colonies during over-wintering was concun·ently-submitted [MRID 469078-02; Cutler, C., and C. Scott-Dupree. 2006. Spring
28DP Barcode: D336888 MRID No. 469078-01 (with 469078-02)
2006 Assessment ofOverwintered Colonies Studied in an Investigation ofthe"Potential LongTenn Impact ofClothianidin Seed Treated Canola on HoneyBees,Apis mellifera L. Unpublished report conducted bythe University ofGuelph, Ontario, Canada, and sponsored by Bayer CropScience, Research Triangle Park, NC. Report submitted July 12, 2006]. Data presented in the addendum repmi were not collected in accordance with GLP requirements, and raw data were not submitted. As the colonies preparedfor over-winteringbeginning in late October, each colony was administered ca. 30 g ofa mixture ofoxytetracycline and icing sugar. Colonies were then provided access to 150 L ofa sucrose:water (2: 1) solution. In mid-November, colony entrances were reduced, an upper entrance was provided, and insulation was placed between the inner cover and the colony lid. On April 19-20, 2006, the status ofover-wintered colonies was assessed for the presence/absence and health ofqueen, presence/absence ofeggs and larvae, area ofsealed brood, number offi·ames ofworkers, and overall health based on a collective assessment ofall dataper colony. Colonies were classified as "healtl)_y" ifthey had 2:4 frames of livebees, and "weak" with <4 frames oflivebees. Overall, the spring assessment found no significant differences in the health oftreated versus control colonies. Ofthe initial 32 colonies, three were classified as "dead" at the end ofthe fall 2005 data collection, and an additional four colonies (two from treated fields and two from control fields) did not survive the winter. 

### It was reported that a loss of 10-15% ofcolonies in an apiary over winter is not uncommon in Canada. Ofthe 25 colonies that survived winter, a healthy queen was found in 21. The presence ofeggs and larvae, however, confinned that the remaining four colonies were queen-right. There was no difference between control and clothianidin-treated colonies in amount ofsealed brood (p = 0.56) or in the number offrames of workers (p 

### = 0,95). Collectively, 24 colonies were classified "healthy", one was classified "weak", and four. were "dead". 

### 15. REVIEWER'S VERIFICATION OF STATISTICAL RESULTS: Replicate data were not provided to statisticallyverify the results ofthis study. Thereviewer visuallyverified the reported results and agrees with the study author's assessments.

29DP Barcode: D336888 MRID No. 469078-01 (with 469078-02) 

### 16. REVIEWER'S COMMENTS: Thereviewer=s conclusions agreed with the studyauthor=s. Although sporadic differences between treatment and control colonies were found on various dates, essentiallyno differences in beemortality, worker longevity, or brood development occun-ed throughout the study. In addition, colonies in clothianidin-treated field gained as much weight and yielded as much honey as those in control fields. No residues ofclothianidin were detected (LOQ = 0.5 ppb) in the majority (>75%) ofsamples (honey, nectar, pollen, andbeeswax) collected for residue analysis. The maximum concentration ofclothianidin in any samplewas 2.59 ng/g in pollen on Day7. Based on the reported nectar/pollen oral NOAEC for honey bees of20 ng/g (Schmuck and Keppler, 2003), the maximum concentration ofclothianidin detected in any sample during this studywas nearly 8-fold below the repmied oral NOAEC, indicating a high margin ofsafety. The flowable suspension Prosper FL nominally contains 9.49% clothianidin, 9.49% thiram, 4.43% carboxin, and 0.316% metalaxyl. The respective CAS Numbers are 210880-92-5, 137-26- 8, 5234-68-4, and 57837-19-1. A Certificate ofAnalysis was not provided for this test substance, and onlythe actual percentage ofthe active ingredient ofinterest, i.e., clothianidin at 9.64%, was reported in the appendix (in "Appendix 4- Seed Treatment Phase Report"). The actual percentages ofthe other active components were not repmied. To the knowledge ofthe study author, no other flowering crops or com grown from seed treated with clothianidin wereplanted within a 1-km radius ofthe canola test plots. The availability to bees ofalternative forage within 1km oftheir colonies while situated in canola fields was also minimal. Although potential forage crops (e.g., soybean, com, alfalfa) were within 1km ofsome fields, none ofthese were in bloom while honeybee colonies were in the canola fields. Itwas reported that brood assessments required opening the colony supers for ca. 60 minutes, sometimes under veryhot, no-shade conditions, and that this procedure was very stressful for the bees. Coupled with the additional stress ofmoving the colonies to the fall apiary (which took several hours), it was decided not to conductbrood assessments on the day ofcolony removal from the canola fields. Furthennore, it was decided to wait a week after the move before continuing with the brood assessments, to allow the colonies to acclimatize to their new sun-oundings. Originally, brood assessments were to include the presence/absence ofeggs, unsealed larvae, and sealed brood for each colony. However, the study author reported it was apparent duringthe Day 1 assessment that it would notbepossible to make all assessments for all 32 colonies [due to lack oftime and/or adequate sunlight (fall apiary assessments only)]. Therefore, it was decided to determinethe amount ofsealed brood only, which would reflect development ofegg and larval stages. It was repmied that since normal, healthy, unsealed brood eventually are sealed by workers, effects ofclothianidin on brood development would still be detected.
30**;'~** ... DP Barcode: D336888 MRID No. 469078-01 (with 469078-02)

It was reported that queens were lost in some colonies duringthe experiment, e.g., theywere accidentallykilled duringdata collection, moving colonies, orrejected bythe colony overtime. In such cases, marked queens from the same/similarlineage were collected from spare colonies at theTownsend House BeeResearch Facilityand introduced to the experimental colonies. In other cases, a new queen was allowed to emerge from a supercedure cell to replace the old queen. Swan11 cells (queen cells usually found onthebottom ofthe combs before swam1ing) when found were destroyed to prevent swarming. While the entrance sheet method ofcollecting dead bees resulted inno technical difficulties or inadequate data, the operation ofthe DBT (traps) was occasionallyunreliable during the experiment. For example, on various collection dates, traps were left partially open, cameloose from the colony, orbecamepartially filled with water afterheavyrainfall events. Another colony (ElTc) was mistakenlythought to be "dead" on September 27-30. Therefore, adult mortality data from some colonies fitted with a DBT was notused on some data collection dates. Data were excluded ifthe number ofdeadbees in the DBT were unusually low (i.e., 0-1 dead bees) because, for example, the trap was not tightly fitted to the colony, or ifthe DBT was partially filled with water due to rain, which mayhave caused some livebees to drown. One dates where there were less than three treated and three control colonies with DBT from which data could be used, analysis comparing dead bee collection methods were not perfonned. In such cases, the "method"parameter (DBT vs. entrance sheet) was not used in the GLM. A total ofn=l8 collection dates incorporated entrance sheet dead bee collection data, while n=15 collection dates incorporated DBT collection data. Forthe 3 years priorto the field studies, the sites had beenplanted with alfalfa, com, soybean, barley, and wheat in 2002; soybean, barley, and com in 2003; and soybean, com white bean, and corn/barley in 2004.

31DP Barcode: D336888 MRID No. 469078-01 (with 469078-02)
The studytimetable was as follows: ·**~..__·** Seed Treatment Phase Experiment Start: April 27, 2005 Test and Control Item Receipt: May4, 2005 Report Completion: November22, 2005 Field StudyPhase Seed planted May20-21,2005

.. Experiment Start (Day 1): July 1, 2005

- Experiment Completion (Day 130): November 7, 2005 Final Report Completion: August 1, 2006 Residue Analysis Phase Experiment Start (first samples received): August 30, 2005 Analytical InitiationDate: December 14, 2005 Analytical Completion: May 15, 2006 Repmi Completion: July 5, 2006 The GPS Coordinates for the canola fields were -80.4 X-coordinate and 43.6 Y-coordinate (combined and reduced to three significant figures), and for the fall apiary were -80.3 Xcoordinate and 43 .4 Y-coordinate. Signed and dated GLP, QualityAssurance and No Data Confidentiality statements were provided. The test was conducted in compliance with the OECD and EPA Principles ofGood LaboratoryPractice. However, the following field studyphase data were non-GLP compliant: " Seed storage- beforeplanting, seed was stored for ca. 2 weeks in a 10°C walk-in refrigerator at 39% relative humidity. Although no fluctuations in temperature orrelative humidity were observed, the refrigerator maintenance and data logging'was non-GLP compliant. " Planting and maintenance (fertilizer and herbicide application) offields

. • GPS coordinates oftreatment (canola fields), pre-treatment (Airport apiary), and posttreatment (fonnerUniversity ofGuelph- CambridgeResearch Station) sites. • Ground truthing. • Weather data were obtained from Environment Canadaweather stations in close proximity to sites at which colonies were maintained. Data are available on-line at http://www.climatc.wcathcrofficc.cc.gc.ca/climatcData/canada c.html.

o Samplerefrigeration temperatures.

o Statistical Analysis -the software used for statistical analysis (JMP Version 5.1, SAS Institute) was not GLP validated.

32DP Barcode: D336888 MRID No. 469078-01 (with 469078-02)


### 16. REFERENCES: Mailvaganam, S. 2005. Estimated Number ofBeekeepers, Colonies ofBees, Production and Value ofHoney, Ontario, 1982 to 2005. Ontario Ministry ofAgriculture, Food and Rural f'. 

### Affairs. httpJ/\vww.omatl:a.gov.on.ca/Enl2.1ish/stats/hort/honey.htmi. (Accessed 20/02/06). OECD Principles ofGood Laboratory Practice (OECD Series on Principles ofGood Laboratory Practice and Compliance Monitoring, Number 1),**E~V/MC/CHEM** 

### (98)17. SAS Institute. 2003. JMP Statistical Software, Release 5.1. SAS Institute, Cary, NC. Schmuck, R., and J. Keppler. 2003. Clothianidin: Ecotoxicological profile and risk assessment. Pflanzenschutz-Nachrichten Bayer 56: 26-58. U.S. Environmental Protection Agency, Good Laboratory Practice Standards, 40 CFR Part 160 PP-300165A; FRL-3518-2, RIN 2070-AB68, Federal Insecticide, Fungicide and RodenticideAct (FIFRA), October 16, 1989.

33