January 24, 2011

Do cument Processing Desk Office of Pesticide Programs (7505P) U.S. Environmental Protection Agency Room S-4900, One Potomac Yard 2777 South Crystal Drive Arlington, Virginia 22202-4501

ATTENTION : George Jeff Herndon (Associate Director, RD, 7505P)

Donald Brady (Director, EFED, 7507P)

Re: Submission of Two Documents Linked to BCS Comments on Clothianidin EPA EFED Assessments dated December 2, 2010

D ear Mr. Herndon and Mr. Brady,

Bayer CropScience (BCS) is herein submitting two documents that are linked to the 1/14/2011 BCS comments on the 12/2/2010 EFED Assessment entitled

“ Revised Assessment for Clothianidin Registration of Prosper T400 Seed Treatment on Mustard Seed (Oilseed and Condiment) and Poncho/Votivo Seed Treatment on Cotton ”.

On 1/13/2011, Professors Scott-Dupree and Cutler, who are the study authors of the canola honeybee field study and its accompanying overwintering report (MRID # 46907801 and 48907802 ), e-mailed you their comments to the criticism of these two reports in the 12/2/2010 EFED assessment and the study reclassification memo signed on 12/3/2010. Their comments have been formatted per PRN 98-5 and are being submitted as a document report.

The other document being submitted is the Appendix I of our 1/14/2011 comments on the 12/2/2010 EFED Assessment. Each of these two documents is being submitted in triplicate. Availability of their MRID # will allow future citation easier. B ayer CropScience 2 T.W. Alexander Drive P. O. Box 12014 Research Triangle Park, NC 27709 Tel: 919 549-20001

12 January 2011

Re: Response to criticism of study EBTIX064 , “ An Investigation of the Potential Long-Term Impact of Clothianidin Seed Treated Canola on Honey Bees, Apis mellifera L. ”

In 2005, we conducted an investigation of the potential long-term impacts of clothianidin seedtreated canola on honey bees, Apis mellifera L. The study was conducted under the conditions of Good Laboratory Practices (GLP) and was required as a condition of registration of Poncho  in Canada and the US. The EPA reviewed and approved the experimental protocols and study design, the work was initiated in late April 2005, and the study was completed in August 2006. The Final Report was submitted to the EPA, who accepted it as scientifically sound, agreed with the conclusions, and found it to be in accordance with the guideline requirements for a field toxicity test with honey bees. We subsequently published the work in the Journal of Economic Entomology, a highly respected international peer-review entomology journal. After review by two anonymous referees, a Subject Editor and an Editor- in -Chief, the manuscript was accepted in final form in less than three months. In recent weeks the soundness of this study has been questioned. Specifically, we found that three nectar samples (two upon reanalysis) from colonies placed in control fields tested positive for clothianidin, indicating that some bees in the controls foraged on canola planted in a treatment plot(s). The harshest critics in the media claim this renders the stu dy “fatally flawed”, while others suggest the work was “sloppy” or “a bit of a joke”. The EPA has made no such suggestions, but has initiated a re-review of the study and now accepts it only as supplemental. Below we offer points in response the criticisms of the study.

General Comments on the Quality of the Study

a. Protocol acceptance by EPA . The possibility of honey bees foraging outside the experimental plots was acknowledged in our initial discussions of the protocol with the EPA. EPA scientists raised concerns about the proposed inter-plot distance, and we were cognizant of
th e potential for inter-plot foraging based on our knowledge of honey bee foraging. However, as we stressed in those initial discussions, it was not logistically possible, or even necessarily preferable given the even greater agronomic and meteorological differences that would have occurred, for us to implement a study incorporating larger experimental plots separated by > 10 km each. The EPA accepted our reasoning and accepted the protocol. b. Rigor of the study . This was a GLP study that received a high level of scrutiny before and during the experiments. On several occasions the QA commended us for our efforts and the quality of the work for this large and complicated experiment. This does not mean the2

study was perfect, but we can confidently say that we completed a comprehensive and detailed experiment, where we made every attempt to strictly adhere to protocols while maintaining high quality data collection and management. The EPA has remained professional in their conduct the past few weeks, but we do take exception to many of the unfounded and disparaging comments in the media.

c. Final Report acceptance by EPA

. In the Final Report we fully acknowledged that because control nectar samples contained clothianidin, bees in control plots must had foraged in treated plots. We suggested reasons for this but concluded that based on all the data, the overall risk of deleterious exposures and adverse effects was minimal. The EPA agreed with this conclusion at the time. Currently, the EPA states that after another review of our work and “in light of additional information”, the study is no longer acceptable. The results and conclusions of the study were clearly stated and have not changed. It is unclear to us what
additional information has rendered the EPA s current decision. We are not aware of any hypothesis driven, field-based experiments that indicate the proposed use pattern of clothianidin poses a toxicological risk to honey bees.
d. Manuscript acceptance by peer reviewers . The Final Report was submitted in manuscript form to the Journal of Economic Entomology. W e again acknowledged that some control bees must had foraged in treated plots and suggested explanations for this, but concludedthat the total data package and study was strong enough to state that the clothianidin seedtreated canola had no impact on honey bees. Two reviewers, a Subject Editor and Editor in Chief agreed. In fact, the cross-contamination issue was not mentioned at all by either ofthe reviewers or editors. That is, they must have agreed that the detected crosscontamination did not affect or weaken the overall conclusions.

Protocol Considerations to the Controversy

1. We took several steps in an attempt to minimize inter-plot foraging by bees. These were supported by EPA officials.

a. Canola was seeded at the highest recommended rate to ensure ample forage for bees.
b. A canola cultivar was used that was highly attractive to bees.

c. Hives were placed in the middle of fields to ensure dispersal over the entire field by bees. Most studies simply place hives at the edge of a field.
d. Bees were introduced to canola fields at approximately 25% bloom to ensure ample forage at the beginning of the experiment and removed when approximately 25% bloom remained to reduce the possibility of the bees seeking alternative forage when resources started dwindling.

2. Although some bees from two control colonies forged on treated canola, we are confident that foragers generally were very faithful to their designated experimental plots.3

a. We ground-truthed extensively and observed very little alternative forage for bees while they were in canola fields. Potential forage crops (e.g. soybean, corn, alfalfa) were within 1 km of some fields, but none of these were in bloom while honey bee colonies were in canola fields.
b. Whenever we were in fields, we observed lots of bees foraging on the canola. Canola is very nutritious to honey bees and is known to be very attractive to them (again, we used a cultivar we knew the bees would forage upon), as seen in greatly increased yields of canola in fields augmented with honey bees (e.g. Sabbahi et al et al. 2005. Influence of honey bee (Hymenoptera: Apidae) density on the production of Canola (Crucifera: Brassicacae). J Econ Entomol 98: 367-372).

c. We are not aware of any data or reports that suggest honey bees prefer to forage on untreated canola vs. clothianidin-treated canola. If this were the case, it certainly speaks to a poor business plan by Bayer CropScience. In fact, given the more vigorous plants produced from insecticide treatment, we expect that the opposite would actually more often be the case, with bees preferring treated plants. There is no reason to believe that bees would have expended more energy to avoid our treatment plants in favor of a more remote untreated alternative.
d. Although not required in the protocol, we did analyze the pollen collected from pollen traps while bees were in canola fields. The technician who completed the analysis has emphatically stated that her analysis found that when in canola fields, over 75% of pollen collected from pollen traps was canola pollen. We cannot say what portion of this was treated vs. control pollen, but based on the proportion of positive samples from treated and control hives, it is reasonable to conclude that bees placed in treated plots did indeed forage more in the treatment plots than control bees (see 2e); the opposite is also likely true. But like many animals, bees need a variety of pollen sources to obtain all of their amino acid requirements. We were therefore not at all surprised that noncanola pollen (~25%) was collected by bees while they were in experimental plots, and it would be unrealistic to expect 100% fidelity to occur. To be sure, exclusive exposure of honey bees to crop monocultures with a lack of forage options is attributed by many experts to be a primary cause of poor honey bee health in North America.

e. When one breaks-down the origins of samples that contained clothianidin residues, in the initial screening 13 samples were from treated fields (all treated fields had positive samples), while only 3 were from two control fields. On reanalysis, 14 samples from treated fields (all treated fields had positive samples) and only 2 from two control fields
had clothianidin residues. If one logically assumes that incidence of positive samples corresponds to forging in clothianidin-treated fields, then one has to conclude that when compared to bees in control plots, a far greater proportion of bees placed in treated plots must had foraged on treated plants.4

f. As per point 2b, we always observed lots of bee foraging on canola, but we did not quantify foraging activity (this was not requested by the EPA in their protocol review). However, we can use a previous similar study to derive some important estimates. Scott-Dupree and Spivak (2001) 1 used the same size test plots and quantified numbers of foraging bees per square meter. We believe these counts provide good evidence that bees did not forage to any great extent away from the canola plots at which they were placed, but again we acknowledge that bees need a variety of floral resources. Our logic is as follows:

i. Scott-Dupree and Spivak (2001) reported in their 1-ha plots an average of 2.4-7.0 bees per square meter per minute, over the blooming period. The average across the 6 test plots in their study was 4.6 bees per square meter. For the two clothianidin test plots, the average was slightly higher (4.85).

ii. Since there are 10,000 square meters per ha, the above figure translates into 24,000 to 70,000 bees foragers per minute in the 1-ha plot, with an overall mean value of 46,000.

iii. The study hives were described as having adult bees covering "at least 10 frames". A frame has approximate ly 17 square decimeters of surface area and approximately 172 bees occupy 1 square decimeter of frame (D. Rogers, pers.

co mm.). 2 Therefore a hive with bees covering 10 frames would have 17*172*10 = 29,240 bees, or approximately 30,000 bees. This is what one would conside r to be a typical hive, and reflects the number of bees we estimated were in each of our hives (P. Kelly, pers. comm.). 3 Assuming one-third of bees in the hive are foragers (D. Rogers, pers. comm.), the four hives in each experimental plot would give a total of ~40,000 forager bees.
iv. The forager counts recorded by Scott-Dupree and Spivak (2001) (46,000 foragers per plot based on their 4.6 bees per square meter average) suggest that a 1-ha canola plot should have easily been able to accommodate our four hives that gave a total of ~40,000 foragers, and that a very high percentage of the bee s would indeed be foraging in their respective test site. Doing the actual math, the calculation ranges from 60 to 175% and averages 115%, suggesting strong site fidelity was likely on the part of forager bees.

1 Scott-Dupree, C. D., and M. S. Spivak. 2001. The impact of Gaucho and TI -435 seed-treated canola on honey bees, Apis mellifera L. Unpublished Study Report 110405. University of Guelph/University of Minnesota

2 Dick Rogers, Apicultural Research Scientist, Ecotoxicology, Bayer CropScience, 2 T.W. Alexander Drive, Research Triangle Park, NC 27709

3 Paul Kelly, Apiary Technician, School of Environmental Sciences, University of Guelph, Guelph, ON, N1G 2W1; Mr. Kelly was actively involved in our study5

v. This supports our expert opinions that: (1) if you place hives next to or within a planting of highly attractive forage plants, bees will use them rather than forage elsewhere; and (2) a 1-ha canola plot can easily support four 10 -frame hives without the bees needing to forage heavily elsewhere. In our study we seeded

ca nola at the highest recommended rate, precisely in an attempt to maximize fidelity to the plot

. vi. Additional thoughts: The calculation should not in theory exceed 100%, but in practice it could if there is some turnover in the cohort of bees within the square meter counting area during the one minute observation period, i.e. some bees leave and others arrive during the 1 minute count interval so the total cumulative count exceeds the number that were present at an average point in time. The other way to achieve counts greater than 100% of the bees placed at the site is if bees from other hives visit the site. This may have happened as well in the Scott-Dupree and Spivak (2001) study, although it is unlikely since their test plots were further apart and there were no detectable residues of pesticides (<0.5 ppb LOQ) from their control samples. Thus, while one cannot use these sorts of counts to precisely quantify the percentage of bees that foraged on site, we suggest that they do provide a good estimate, and in this case that estimate is in the neighborhood of 100%.

3. We believe we can explain why bees from two control plots, E1C and W3C, forged in clothianidin plots. There was no contamination in the other two control plots, E2C and W4C.

a. As stated in the Final Report and the Journal of Economic Entomology publication, there was a seeding problem in a large portion of plot E1C which was not detected for approximately two weeks until plant emergence was recorded. These drills were then reseeded but by this time a stagger in bloom of plants in this field was inevitable. Thus, there was less canola forage available to bees in this plot at any given time period. Based on the calculations above, this could explain why bees in this plot sought forage in a distant (treated) plot.
b. Simply based on different histories of agronomic practices at the Wallace farm (W plots) and the Elora Research Station (E plots), soil and growing conditions differed at these sites. Along with the control seed’s lack of protection from herbivorous insects (which resulted in poorer plants and forage overall), this led to some differences in crop development among sites. Using photos taken during a field tour by Bayer CropScience personnel in mid-July 2005, we can show that while many test plots were in full bloom and hives were still in the canola fields, there was less bloom in plot W3C at this time (Appendix 1). Thus, bees from this field likely forged in treated plots because few blossoms remained in their own plots.6

In conclusion, we wish to emphasize that we agree with the opinion that it was unfortunate, from an experimental design perspective, that a few control samples were contaminated. As scientists, we obviously would have preferred that this did not happen and are disappointed that the experiment did not turn out watertight. Nonetheless, no field study is perfect and there are limits to what is possible in terms of large scale hypothesis-driven field study design s and conduct. For example, it would desirable to have had study sites 10+km from each other to lower the likelihood bee inter-plot visits, but this would have been very impractical for the field crew collecting the data, and would likely introduce greater variation on and surrounding the test sites (different farms and farming practices, possible rainfall events hitting some sites and not others, etc.). Despite some inter-plot foraging, we are confident that bees overall were highly faithful to their test plots. We stand behind our findings of no treatment effects on bees during the foraging season or overwinter. Based on our results and the work of others using honey bees and other bee species, we believe that properly prepared clothianidin seed-treated canola presents negligible risk to honey bee s.

Sincerely,

Chris Cutler, PhD Cynthia Scott-Dupree, PhD Assistant Professor Professor Nova Scotia Agricultural College University of Guelph7 Appendix 1

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Field Site Tour with Baer CropScience Personnel2

MRID # __________________

" Comments from Professors Scott-Dupree and Cutler to the 12/2/2010 EPA EFED Clothianidin Assessment and the MRID # 46907801/ 46907802 Reclassification Memo”

MRID # __________________

" Prospective Multifactorial Study: Effect of microbial and parasitic agents and pesticide residues on the evolution of domestic bee colonies under natural conditions”

Please contact me at jamin.huang@bayer.com or at 919-549-2634 if you have any questions regarding this submission.

Sincerely,

Jamin Huang Senior Regulatory Manager

Copy:

David Fischer, Bayer CropScience