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 of Data Package DP336888 for Clot~!ani~~~ PC_ Code 044309 

Allen 

Environmental 

Vaughan, 

Risk 

Biologist 

Branch 

~~~: V 1 1 r.~ 

Environmental Fate and Effects Division (7507P) II /30 j 0 7 

Mah Shamim, Branch Chief Mllf}~ Environmental Risk Branch V // n~ 

Environmental Fate and Effects Division (7507P) 

TO: Kable Davis, Risk Manager Reviewer 

John Hebert, Risk Manager (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 study is attached: 

Cutler, C. 2006. An Investigation of the Potential Long Term Impact of Clothianidin 

Seed Treated Canola on Honey Bees, Apis mellifera L. Laboratory Repmi ID: 2005-

CSD-EBTIX064. MRID 46907801 (with addendum 46907802). 

This study is scientifically sound and satisfies the guideline requirements for a field 

toxicity test 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, essentially no differences in worker or drone mortality, worker longevity, or 

brood development occurred during the study. Colonies in treated fields had similar 

weight gains and honey yields as those in control fields. Qualitative assessments, 

made the following spring by experienced bee researchers, confirmed that colonies 

from clothianidin-treated fields were as strong and healthy as those from control 

fields. 

It was concluded that honey bees that forage on clothianidin seed-treated canola will 

be exposed to clothianidin residues iri pollen, nectar, and honey; however, exposure 

concentrations are below those required to elicit acute and sublethal effects. 
DATA EVALUATION RECORD 

HONEY BEE- FIELD TESTING FOR POLLINATORS 

§141-5 (OPPTS 850. 3040) __ 

1. CHEMICAL: Clothianidin PC Code No.: 044309 

2. TEST MATERIAL: 1) Prosper FL 

2) Poncho 600 FS 

3. CITATION: 

Author: Cutler, C. 

Purity: 1) 9.49% 

2) 48.0% 

Title: An Investigation of the Potential Long-Term Impact of 

Clothianidin Seed Treated Canola on Honey Bees, Apis 

mell?fera L. 

Study Completion Date: August 1, 2006 

Laboratory: Department of Environmental Biology 

University of Guelph 

Guelph, Ontario, Nl G 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, Staff Scientist, 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 document provides guidance for EPA and PMRA reviewers on how 

to complete a data evaluation record after reviewing a scientific study concerning the longtenn, toxicity of a pesticide to honey bees following an actual-use field exposure. It is not 

intended to prescribe conditions to any external party for conducting this study nor to 

establish absolute criteria regarding the assessment of whether the study is scientifically 

sound and whether the study satisfies any applicable data requirements. Reviewers are 

expected to review and to determine for each study, on a case-by-case basis, whether it is 

scientifically sound and provides sufficient infonnation to satisfy applicable data 

requirements. Studies that fail to meet any of the conditions may be accepted, if 

appropriate; similarly, studies that meet all of the conditions may be rejected, if 

appropriate. In sum, the reviewer is to take into account the totality of factors related to the 

test methodology and results in determining the acceptability of the study. 

7. STUDY PARAMETERS: 

Scientific Name of Test Organism: Apis mellifera L. 

Age or Size of Test Organism at Test Initiation: Queens in all colonies wereofthe same 

lineage and ca. the same age. 

8. CONCLUSIONS: 

Definitive Study Duration: 130 days (approximately 2 complete 

life cycles); 21-day exposure period 

(during peak bloom) followed by a 

109-day post-exposure period. 

In a 130-day study (21-day exposure followed by 1 09-day post-exposure period), the longterm toxicity of clothianidin-treated seed was examined in the honey bee, Apis mellifera L., 

under open field conditions at four test sites. Each site contained one 1-ha field planted 

with canola seed, Brassica napus var. Hyola 420, that had been treated with the end-use 

products Prosper® FL at 1250 mL/1 00 kg seed and Poncho® 600 FS at 417 mL/1 00 kg 

seed, delivering clothianidin at 400 g ai/1 00 kg seed, the highest 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 same rate with specially-prepared Blank Prosper FL and 

Poncho 600 FS formulations. Each treated and control field were separated by at least 

250m. Four honey bee colonies were placed in the middle of each field (n=32) during a 3-

week bloom period (Day 1 =July 1, 2005), and thereafter moved to a fall apiary for the 

remainder of the study (Day 130 =November 7, 2005). Throughout the study, colonies 

were assessed for bee mortality, worker longevity, and brood development. In addition, 

samples of honey, beeswax, and worker-gathered pollen and nectar were regularly analyzed 

2 
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DP Barcode: D336888 MRID No. 469078-01 (with 469078-02) 

for clothianidin residues. Colony weight gain while in the canola fields and honey yield 

per colony was also determined. 

Overall, there was no difference between colonies from clothianidin-treated and control 

fields. Although sporadic treatment or site differences were found on various dates, 

essentially no differences in worker or drone mortality, worker longevity, or brood 

-development occurred during the study. Colonies in treated fields had similar weight gains 

and honey yields as those in control fields. Qualitative assessments by experienced bee 

researchers confirmed that colonies from clothianidin~treated field were as strong and 

healthy as those from control fields. 

The majority of samples collected (>75%) for residue analysis had no detectable levels of 

· clothianidin residues (LOQ = 0.5 ng/g). The maximum concentrations of clothianidin 

detected in honey, 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 of20 ppb. 

It was concluded that honey bees that forage on clothianidin seed-treated canola will be 

exposed to clothianidin residues in the form of pollen, 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 of the original 32 over-wintered 

colonies was assessed on April 19-20, 2006. Observations included the presence/absence 

of the queen, presence/absence of eggs and larvae, area of sealed brood, number of frames 

ofworkers, and overall health of the colony. The spring assessment found no significant 

difference in the health between the treated and control colonies. Two treated colonies and 

two control colonies did not survive the winter. Of the 25 colonies that did survive the 

winter, a healthy queen was found in 21, and the presence of eggs and larvae in the 

remaining four indicated that these colonies were queen-right. There was no statistical 

difference between treated and control colonies in the amount of sealed brood or in the A 

number of frames of workers. Collectively, 24 colonies were classified "healthy", one was 

classified "weak" (<4 frames oflive bees), and four were "dead". 

This study is scientifically sound and satisfies EFED concerning the guideline requirements 

for a field toxicity test with honeybees (OPP Gdln. No. 141-5; OPPTS 850.3040). 

9. ADEQUACY OF THE STUDY: 

A. Classification: Acceptable 

B. Rationale: N/ A 

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

C. Repairability: N/ A 

10. GUIDELINE DEVIATIONS: N/A 

11. SUBMISSION PURPOSE: This study was submitted to provide data on the toxicity of 

clothianidin to honeybees in a field test for the purpos~ of chemical registration (new use). 

Specifically, the test was conducted in response to a request by the 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 of clothianidin-treated canola 

to foraging honey bees. 

12. MATERIALS AND METHODS: 

A. Test Orgamsms 

Guideline Criteria Reported Information. 

Species: 

Species of concern (Apis mell~fera, Apis mell?fera L. 

Megachile rotundata, or Nomia melanderi) 

Colony description at beginning of test: Each colony consisted of a single brood 

chamber (24 em deep, 10 frames per super) 

below a shallow honey super (originally 

empty, 16.5 em deep, 9 frames per super). 

Pre-test health: 

4 

Queens in all colonies were of the same 

lineage and approximately the 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 of food stores 

(pollen and nectar), brood in all stages of 

development, and adults in each. 

Colonies were assessed for presence of Varroa 
DP Barcode: 0336888 MRID No. 469078-01 (with 469078-02) 

- Guideline Criteria Reported Inforn'tidion 

mite, tracheal mite, and infectious honey bee 

diseases (American Foulbrood, European 

Foulbrood, and Chalkbrood) prior to 

placement in canola and throughout the study. 

- Colonies were also treated with Checkmite® 

prior to placement in canola. 

Supplier Prior to field testing, the honey bee colonies 

were held at a spring apiary near the Townsend 

House Bee Research Facility, University of 

Guelph, Ontario. 

All bees from the same source? Yes 

B T . es tS ;ys t em 

Guideline Criteria Reported Information 

Exposure Site Location and Establishment: The four test sites were located in Elora, 

Ontario, Canada, at the University of Guelph, 

Elora Research Station (sites El and E2), and 

two neighboring farms owned by Allan and 

Phillip Wallace (sites W3 and W4). 

East test site consisted of two 1-hectare fields; 

one planted with clothianidin-treated canola 

seed and the other planted with control seed, 

giving a total of eight fields. Fields at each 

site were separated by at least 250 m. 

Planting of the canol a seed occurred on May 

20-21, 2005. Seeds were sown to a depth of 4 

em at the highest recommended rate of 15-20 

seeds/m (8.0 kg/ha). 

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

·'·': . Guideline Criteria . ·. 

Reported Inforni1ition :· 

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 in the middle of 

each canola field to accommodate four 

colonies. 

Number of Replicates/Treatment: Four colonies per field, with 1 treated 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 of Guelph, Cambridge Research 

Station, Ontario, Canada. 

Lighting: Natural; not further described. 

Precipitation: Daily precipitation ranged from 0.0 to 

18.8 mm during the exposure period (data 

obtained from the Canadian weather website 

cited in the study report; refer to Reviewer's 

Comments section). The maximum rainfall 

event occurred on July 16 and 17, when 18.8 

and 10.4 rnm 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. Test Desi n 

Guideline Criteria Reported Information 

inding test? None reported 

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-

Guideline Criteria Reported Information 

Reference toxicant tested? No 

Duration of Exposure Period 21 days, during canola bloom period 

Duration of Post-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 

Batch No: 407483M 

Ai: 48.0% clothianidin 

Source: Bayer CropScience, Kansas City, MO 

Control Substance(s): Prosper FL Blank 

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 of Application Rate: 

Method of Seed Coating: 

Colony Introduction: 

I 

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

--

Reported Information 

Treatment 1: Prosper FL at 1250 mL/1 00 kg 

and Poncho 600 FS at 417 mL/1 00 kg, to 

deliver clothianidin at the rate of 400 g ai/1 00 

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) of 4175 ppm or 417 g ai/100 kg 

seed 

For both treatments, slurries were prepared by 

combining the appropriate quantities of each 

fonnulation. 

The slurries were applied to the seed using the 

Gustafson CBT -50 seed treater. Due to the 

large quantity of seed (1 00 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 of canola blooms in the test fields 

had opened ( detennined by visual estimation). 

All colonies were positioned so that the 

entrances faced approximately south. June 30, 

2005 was identified as Day 0 of the 3-week 

exposure period. 

Honey supers were removed from and added to 

colonies as needed throughout the study . 

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·.· 

Guideline Criteria Reported Information 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 by at least 30m. No 

other colonies were present at the fall apiary. 

D . B" 10 l . og1ca lA ssessments 

Guideline Criteria Reported Information 

Canola: - Seedling emergence rates ( detennined on 

June 3 and June 7/8) 

- Development rates 

- 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 Day 21 

(the night colonies were moved to the fall 

apiary). 

Honey Yield: Honey yield per colony by weight. 

Adult Mortality: Dead workers and drones were collected and 

counted ca. every 7 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. 

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

Guideline Criteria Reported Information 

I 

Brood: The area of sealed 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 of sealed brood was estimated by 

placing an empty template 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 worker bees were counted on Days 5 

and 9 .(post-introduction assessments), 14 and 

15, and thereafter at ca. 14-day intervals up to 

Day98. 

On Day 4 (allowing for a 3-day colony 

acclimation to the canola fields), newlyemerged (<24 hours) worker bees (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 on Day 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 colony had 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. 

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DP 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 visually inspected to 

ensure nonnal physical health and behavior. 

When queens were not located, the presence of 

eggs confirmed the presence of a 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 verify the presence 

of a larva, and then destroyed. If the queen 

was absent in a colony, however, in some 

cases supercedure cells were left to allow a 

new queen to be reared by the 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 . es1 "d ue A l . natySIS 

Guideline Criteria Reported Information . 

Nectar Collection: A 5-g pooled sample of nectar (when 

available) was collected from colonies at each 

field on Days -3/-1, 7, 14/15, 42, and thereafter 

at ca. 21-day intervals up to Day 83. 

Nectar was extracted from cells using a 

disposable syringe, or removed by gently 

shaking a brood frame over a sheet of waxed 

paper. 

11 

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

-. 

Guideline Criteria Reported Inforh:lation · 

. ·.·· . •. 

Honey Collection: A 5-g pooled sample of honey 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 1 0-g pooled sample of pollen was collected 

fiom colonies at each field on Days -3/-1,7, 

14/15, 42, and thereafter at ca. 21-day intervals 

up to Day 106. 

Pollen was collected over a 24-hour period 

using an OAC pollen trap. Approximately 5 g 

of each 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. 

12 
DP Barcode: D336888 

Guideline Criteria 

Storage of Samples: 

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 dry ice. 

Conditions of storage (samples and extracts) 

once at the laboratory were not reported. 

Intervals of storage (reviewer-determined): 

Honey: 157 days prior to extraction, and 49 

days prior to analysis. 

Nectar: 283 days prior to extraction, and 26 

days prior to analysis. 

Pollen: 212 days prior to extraction, and 44 

days prior to analysis. 

Beeswax: 273 days prior to extraction, and 22 

days prior to analysis. 

Storage stability assessments were 

apparently not performed. 

The residue method for clothianidin in pollen, 

honey, and nectar was based on Bayer Method 

00554 with minor modifications. For wax, a 

short summary supplied by Ralf Schoning of 

Bayer for imidacloprid was used as the basis 

for extraction. For all matrices, concentrations 

of clothianidin were detennined using 

LC/MS/MS, and the limit of quantitation was 

0.5 ppb (ng/g) . 

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DP 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 

included in the report? details regarding non-GLP sections). 

Raw data included? No, raw data were generally not provided for 

any measured parameter. Summarized data 

tables (means) were provided for worker and 

drone mortality, and worker longevity. 

Signs of toxicity (if any) were described? Yes 

Canola Emergence: 

Canola emergence, development, and flea beetle damage were compared by treatment and site. 

There were significantly more emerged plants per meter in clothianidin seed-treated fields than in 

untreated fields on both June 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 both intervals, although 

the difference was only significant on June 3 (p = 0.0029). On both 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). 

Although there were generally more emerged canola plants per meter in clothianidin-treated 

fields than control fields, development of emerged plants was the same in both. The growth 

stage of emerged plants did not differ with treatment or site, and there was no significant 

interaction of those effects. 

Flea beetle damage was significantly greater in control fields on both June 3 and June 8 

(p<O.OOOl). Although flea beetle damage did not vary with site on June 3, a difference between 

sites was found on June 8 (p = 0.014). 

Weight Gain: 

There was no significant difference in weight gain of colonies from control and clothianidintreated fields (Figure 1 ). In both treatments, colony weights increased approximately 23-24 kg 

during the 3-week exposure period in canola fields. In addition, differences in colony weight 

gain were not significant among sites, and there was no significant treatment-site interaction. 

14 
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Figure 1: Mean (±SEM) honey bee colony weight gain and honey yield after exposure to 

clothianidin-treated (n=16) and control (n=15) canola. Colonies were in canolafieldsfor 21 

days during bloom (Days 0-21), and thereafter moved to a fall apimy, approximately 35 km 

away, where they were maintainedfor another 109 days. 

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

I Treatment 

50 · I ""· '"'''·'I Controi: 

t 

' 

Honey Yield: 

Corony Welght 

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 of 45.3 kg and 44.7 kg ofhoney was harvested from treated 

and control fields, respectively, over the 130 days ofthe experiment. Values were comparable to 

the 2005 Ontario honey yield average of 46.6 kg. In addition, differences in honey yield were not 

significant among sites, and there was no significant treatment-site interaction for honey yield. 

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Adult Mortality: 

In analyzing changes in the number of dead workers or drones over time, DBT (dead bee trap) 

and sheet data were analyzed separately, and the GLM incorporated effects of day, treatment, and 

the interaction ofthese terms. For analyses on individual days, the GLM in most cases was able 

to simultaneously incorporate analysis of effects of site, treatihent, and method of dead bee 

collection. In some cases, however, inclusion of one of these parameters resulted in a significant 

Lacli of Fit (LOF) in the model, while itself not contributing significantly to the model. In such 

cases, the parameter causing the LOF was removed, resultir:g in a simpler but more robust model 

(Table 1). 

There were significant changes over time in the number of dead workers recovered from colonies 

with both the DBT (trap) and the sheet methods (p<O.OOl). However, regardless of the dead bee 

collection method used, there was no significant difference in dead workers due to treatment or 

the day-treatment interaction. Although the recovery of dead drones changed over time with the 

entrance sheet method (p<O.OOl), no change over time was found with DBT. As with workers, 

regardless of the dead bee collection method used, treatment and the day-treatment interaction 

had no significant effect on the number of dead drones found. 

Table 1: Honey bee mortality in colonies located in clothianidin-treated (n= 16) and control 

(n = 16) canol a fields. At each offour sites (one treatment, one control field per site) were three 

colonies equipped with a white entrance sheet, and one colony fitted with a dead bee trap (DBT). 

Effects of site, treatment, dead bee assessment method, and their interaction were determined 

using a general linear model platform (SAS Institute 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 

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"" 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 

351 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 

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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 

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-

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 

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·-

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 Method parameter resulted in a significant Lack of Fit and therefore was omitted from the model. 

3 Site parameter resulted in a significant Lack of Fit and therefore was omitted from the model. 4 Treatment parameter resulted in a significant Lack ofF it 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 of these terms. However, there were no 

consistent trends in effect of these variables dming the experiment. Recovery of dead bees using 

DBT was usually significantly greater than that with entrance sheets, although a significant 

method-site interaction was often found, indicating high variability in the number of dead bees 

recovered among the colonies fitted with a DBT. From a total of 18 sampling dates over 130 

days, only one data set (Day 56) showed a statistically significant increase in worker mortality in 

treated colonies, whereas on three sampling dates (Days 77/79, 92, and 12) mortality from 

control colonies was statistically higher, Thus, there was overall no relevant difference between 

treatment and control colonies in worker motiality. In general, more dead workers than drones 

were recovered throughout the experiment, regardless of the collection method (Table 2). Effects 

on the number of dead drones recovered were minimal, but were occasionally found near the end 

of the experiment. As expected, the number of dead workers increased near the end of the 

experiment (e.g., Day 99) as colonies prepared for over-wintering. 

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Table 2: Honey bee worker and dro~e mortality in colonies located in clothitimidin-treated 

(n=I6) and control (n=l6) canola.fields. At each offour sites (one treatment, one control.fleld 

per site) H"ere three colonies equipped with a white entrance sheet, and one colony .fitted with a 

d ea db ee t rap (DBT) 

Day Mean No. Dead per Colony 

Dead bee trap (DBT) Entrance Sheet -

Treated Control Treated Control 

Worker Mortality 

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 

' Drone Mortality 

7 -- -- 4.2 5.4 

13 10.8 3.0 7.1 7.3 

18 56.8 14.3 6.0 8.8 

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- -. 

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 of dead workers recovered in DBT on Day 18 (Table 

2). In the week preceding collection of dead bees on Day 13, the mean maximum temperature 

was 30.9°C. Under these 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 of dead workers on this day. 

Brood Assessment: 

Although brood assessments were to be conducted up to Day 130, it was evident by Day 112 that 

there was no or minimal sealed brood in colonies in preparation for over-wintering. Therefore, 

the final brood assessment was conducted on Day 97/98. 

The amount of sealed 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 detennining the amount of sealed brood), or the 

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interaction of these tenns (Table 3 ). On Day 1/2, the amount of sealed-brood :Fler colony differed 

significantly across sites and on Day 33/34, the amount of sealed brood differed with the 

sampler. At no time during the experiment did the amount of sealed brood in colonies from 

clothianidin-treated field differ significantly from that found in colonies from control fields. 

Table 3: Mean area o.fsealed brood in honey bee colonies in clothianidin-treated (n=16) and 

control (n= 16) canolafields. Colonies were placed in canolafields on July 1 (Day 1) and were 

moved to a fall apiary on July 21 (Day 21)for the remainder of the experiment (to Day 130). 

Effects of site, treatment, sampler (individual determining tjw amount ofsealed brood), and their 

interaction were determined using a general linear model platform (SAS Institute 2003). 

Statistically-sign~ficant effects (a = 0.05) are in bold. 

Day Mean Area (cm2

) Sealed Brood per 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 

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Day Mean Area (cm2

) Sealed Brood per Colony 

Treated Control 

76/841 4597.4 4917.6 

-

97/98 1 1179.9 819.6 

1 Could 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 of tagged workers decreased over time in colonies from both clothianidin-treated 

and control canol a fields (p<O.OOOl ). The GLM found no significant effect of site or treatment 

on longevity of tagged workers. There also was no significant date*site, date*treatment, or 

site*treatment interaction of the tenns. Throughout the experiment, there was no significant 

difference in the nm11ber of tagged 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. 

Table 4: Honey bee worker longevity in colonies in clothianidin-treated (n=J6) and control 

(n= 16) canolajields. Workers (n=50) tagged with colored/numbered thoracic tags were added 

to each colony on Day 4 (July 4); a second tagged worked introduction (n=50) was made on Day 

70 (Sept. 8). Effects of site, treatment, and their interaction were determined using a general 

linear model pla~form (SAS Institute 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 

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--

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 NA1 Site --

Treatment --

Site*Trt --

84 NA1 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 

1 Data not collected. 

Disease: 

Incidence of disease was low throughout the study. Colonies were treated with Checkmite® 

prior to placement in canola. As a result, Varroa and tracheal mite incidence was very low 

throughout the study; in the majority of colonies, no Vanoa or tracheal mites were detected. 

American Foulbrood and European Foulbrood were not found in any colonies during the study. 

Chalk brood was sporadically detected at very low levels (i.e., 5-10 cells/colony throughout the 

study. However, as workers routinely remove chalkbrood mummies, the disease never affected 

the overall health of colonies. 

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Queen Losses and Overall Colony Health: 

The presence of eggs and larvae were observed in colonies throughout the study. Due to losses 

of the queen, some colonies at some observations had low numbers of eggs and larvae. Loss of 

queens from colonies was expected given the intense amount of data collection, movement of 

colonies, and large number of colonies in the study. During the 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 naturally replaced by a virgin queen (i.e., 

the colony replaced the original queen on its own). Colonies W3Cc and E2Tc were both 

artificially and naturally re-queened. Therefore, a total of six colonies from clothianidin-treated 

fields and five colonies from control fields were naturally and/or artificially re-queened during 

the study. Colony W3Ta was problematic throughout the study. It was found to be queenless on 

July 8 (likely killea during the move of colonies to canola fields), and subsequentlydid not 

accept artificially re-introduced queens. However, the colony was found to be successfully 

naturally re-queened on Day 63 and thereafter. 

T a e bl 5 : R oney b ee co l ames J . h to w uc new queens were zntro d 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 naturally re-queened during the experiment, but failed to 

successfully establish a queen. Although data from these colonies may have inadvertently been 

collected (prior to status was realized), these data were omitted from some statistical analyses, 

e.g., sealed brood analyses near the end of the experiment. Given that adequate data were 

collected from these colonies through much of the experiment, and that there were a large 

number of replicates in total, the loss of these colonies had no impact on the study overall. 

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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 colony health and vigor. 

Residue Analysis: 

Clothianidin was detected in treated seed at the prescribed level at an average of 417 g ai/1 00 kg 

seed. In-phase recovery(± SD) of clothianidin residues fro;m spiked samples of honey, nectar, 

pollen, and beeswax was 93 ± 13.0, 89 ± 13.3, 87 ± 13.8, and 104 ± 21.6%, respectively. 

The majority of samples (>75%) collected had no detectable levels of clothianidin residues (LOQ 

= 0.5 ng/g), whether from colonies in treated or control fields (Table 6). The maximum 

concentration of clothianidin detected in honey, nectar, pollen, and beeswax samples was 0.928, 

2.24, 2.59, and <0.5 ng/g, respectively. No clothianidin residues were detected in honey, pollen, 

or beeswax samples collected from control fields, although analyses conducted in January 2006 

detected residues in three nectar samples from control colonies (field E1 C, July 7; field W3C, 

<' July 7; and field W3C, August 11). Subsequent analyses ofback-up nectar samples detected 

residues in two control colonies (field El C, July 7; and field W3C, July 7), suggesting that 

workers in control colonies may have foraged on clothianidin-treated canola. This may have 

occurred because the separation between some pairs of control and treated fields was insufficient 

or because the forage in some control fields was oflower quality (due to insect damage and lower 

rates plant emergence), which may have 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, and beeswax collectedfrom honey bee 

colonies in clothianidin-treated and control canolajields. Pooled samples were collected at 

eac h site approxunatety l every 21 d 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 

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.. -

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, honey yield during exposure, worker and drone m01iality, brood area per 

colony, and tagged worker longevity (based on the number of tagged workers recorded each 

collection day) were also compared over time using a GLM platfonn. 

14. SUPPLEMENTAL ASSESSMENT OF OVERWINTERED COLONIES: 

A supplemental report documenting further assessments of the colonies during over-wintering 

was concun·ently-submitted [MRID 469078-02; Cutler, C., and C. Scott-Dupree. 2006. Spring 

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2006 Assessment of Overwintered Colonies Studied in an Investigation of the"Potential LongTenn Impact of Clothianidin Seed Treated Canol a on Honey Bees, Apis mellifera L. 

Unpublished report conducted by the University of Guelph, 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 prepared for over-wintering beginning in late October, each colony was 

administered ca. 30 g of a mixture of oxytetracycline and icing sugar. Colonies were then 

provided access to 150 L of a 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 of over-wintered colonies was 

assessed for the presence/absence and health of queen, presence/absence of eggs and larvae, area 

of sealed brood, number of fi·ames of workers, and overall health based on a collective 

assessment of all data per colony. Colonies were classified as "healtl)_y" if they had 2:4 frames of 

live bees, and "weak" with <4 frames oflive bees. 

Overall, the spring assessment found no significant differences in the health of treated versus 

control colonies. Of the 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% of colonies in an 

apiary over winter is not uncommon in Canada. Of the 25 colonies that survived winter, a 

healthy queen was found in 21. The presence of eggs and larvae, however, confinned that the 

remaining four colonies were queen-right. There was no difference between control and 

clothianidin-treated colonies in amount of sealed brood (p = 0.56) or in the number of frames 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 statistically verify the results of this study. The reviewer 

visually verified the reported results and agrees with the study author's assessments. 

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16. REVIEWER'S COMMENTS: 

The reviewer=s conclusions agreed with the studyauthor=s. Although sporadic differences 

between treatment and control colonies were found on various dates, essentially no differences in 

bee mortality, 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 of clothianidin were detected (LOQ = 0.5 ppb) in the 

majority (>75%) of samples (honey, nectar, pollen, and beeswax) collected for residue analysis. 

The maximum concentration of clothianidin in any sample was 2.59 ng/g in pollen on Day 7. 

Based on the reported nectar/pollen oral NOAEC for honey bees of20 ng/g (Schmuck and 

Keppler, 2003), the maximum concentration of clothianidin detected in any sample during this 

study was nearly 8-fold below the repmied oral NOAEC, indicating a high margin of safety. 

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 5783 7-19-1. A Certificate of Analysis was not provided for this test substance, 

and only the actual percentage of the active ingredient of interest, i.e., clothianidin at 9 .64%, was 

reported in the appendix (in "Appendix 4- Seed Treatment Phase Report"). The actual 

percentages of the other active components were not repmied. 

To the knowledge of the study author, no other flowering crops or com grown from seed treated 

with clothianidin were planted within a 1-km radius of the canola test plots. The availability to 

bees of alternative forage within 1 km of their colonies while situated in canola fields was also 

minimal. Although potential forage crops (e.g., soybean, com, alfalfa) were within 1 km of some 

fields, none of these were in bloom while honey bee colonies were in the canola fields. 

It was reported that brood assessments required opening the colony supers for ca. 60 minutes, 

sometimes under very hot, no-shade conditions, and that this procedure was very stressful for the 

bees. Coupled with the additional stress of moving the colonies to the fall apiary (which took 

several hours), it was decided not to conduct brood assessments on the day of colony 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 of eggs, unsealed larvae, and 

sealed brood for each colony. However, the study author reported it was apparent during the 

Day 1 assessment that it would not be possible to make all assessments for all 32 colonies [due to 

lack of time and/or adequate sunlight (fall apiary assessments only)]. Therefore, it was decided 

to determine the amount of sealed brood only, which would reflect development of egg and larval 

stages. It was repmied that since normal, healthy, unsealed brood eventually are sealed by 

workers, effects of clothianidin on brood development would still be detected. 

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It was reported that queens were lost in some colonies during the experiment, e.g., they were 

accidentally killed during data collection, moving colonies, or rejected by the colony over time. 

In such cases, marked queens from the same/similar lineage were collected from spare colonies 

at the Townsend House Bee Research Facility and 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 on the bottom of the combs before swam1ing) when 

found were destroyed to prevent swarming. 

While the entrance sheet method of collecting dead bees resulted in no technical difficulties or 

inadequate data, the operation of the DBT (traps) was occasionally unreliable during the 

experiment. For example, on various collection dates, traps were left partially open, came loose 

from the colony, or became partially filled with water after heavy rainfall events. Another colony 

(El Tc) was mistakenly thought to be "dead" on September 27-30. Therefore, adult mortality 

data from some colonies fitted with a DBT was not used on some data collection dates. Data 

were excluded if the number of dead bees 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 if the DBT was partially 

filled with water due to rain, which may have caused some live bees 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 of n=l8 

collection dates incorporated entrance sheet dead bee collection data, while n= 15 collection dates 

incorporated DBT collection data. 

For the 3 years prior to the field studies, the sites had been planted 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. 

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The study timetable was as follows: · ~..__· 

Seed Treatment Phase Experiment Start: April 27, 2005 

Test and Control Item Receipt: May 4, 2005 

Report Completion: November 22, 2005 

Field Study Phase Seed planted May 20-21,2005 

.. 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 Initiation Date: 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, Quality Assurance and No Data Confidentiality statements were 

provided. The test was conducted in compliance with the OECD and EPA Principles of Good 

Laboratory Practice. However, the following field study phase data were non-GLP compliant: 

" Seed storage- before planting, seed was stored for ca. 2 weeks in a 10°C walk-in 

refrigerator at 39% relative humidity. Although no fluctuations in temperature or relative 

humidity were observed, the refrigerator maintenance and data logging' was non-GLP 

compliant. 

" Planting and maintenance (fertilizer and herbicide application) of fields . 

• GPS coordinates of treatment (canola fields), pre-treatment (Airport apiary), and posttreatment (fonner University of Guelph- Cambridge Research Station) sites. 

• Ground truthing. 

• Weather data were obtained from Environment Canada weather stations in close proximity 

to sites at which colonies were maintained. Data are available on-line at 

http://www. cl imatc. wcathcroffi cc.cc. gc. ca/ cl imatcData/ canada c.htm l. 

o Sample refrigeration temperatures. 

o Statistical Analysis -the software used for statistical analysis (JMP Version 5.1, SAS 

Institute) was not GLP validated. 

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

16. REFERENCES: 

Mailvaganam, S. 2005. Estimated Number of Beekeepers, Colonies of Bees, Production and 

Value of Honey, Ontario, 1982 to 2005. Ontario Ministry of Agriculture, Food and Rural 

f'. Affairs. httpJ/\vww.omatl:a.gov.on.ca/Enl2.1ish/stats/hort/honey.htmi. (Accessed 

20/02/06). 

OECD Principles of Good Laboratory Practice (OECD Series on Principles of Good 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 

Rodenticide Act (FIFRA), October 16, 1989. 

33