# APPENDIX A.

**OPINION OF PROF. CHRISTIAN KRUPKE**1 Prepared by : Christi a n H. Krupke, Ph.D Associate Professor of Entomology, Purdue University , June 7 2012. A r esponse to a recent review of publications on neonicotinoid s and pollinators prepared by Heintzelman, Kelly, Fischer and Maus ( dated 23 May 2012)

, focusing on work of Krupke et al. (1). Comments of Heintzelman et al., appear in italics , response from lead author of the study (C . Krupke ) below each comment

.

“ Krupke et al. (2012) report the findings of a study on potential routes of exposures for honeybees to pesticides, especially to neonicotinoids, conducted in a corn growing region in USA in 2010. The study was initiated in response to reports of bee kills at Indi ana apiaries in spring of 2010 which coincided with the corn planting period in the area and which were believed to be related to neonicotinoid seed treatment products ”

. The study was conducted in both 2010 and 2011

– there were reports of bee kills both years. There are currently a number of similar reports in 2012 from Indiana, Minnesota, Ohio, and the provinces of Ontario and Quebec in Canada. The investigations into th e causes of these bee kills are ongoing.

All dying bees exhibited the convulsions, uncoordinated movements and tremors that are typical of neurotoxic insecticide exposure. An analysis of the bees using LC

- MS/MS revealed that the neonicotinoid clothianidi n was present in each case (range of 3.8

– 13.3 ppb) ; these were the only insecticides found in th os e samples. Note that thiamethoxam is rapidly metabolized in insects to clothianidin;

so it is not possible to identify which of these compounds honey bees w ere initially exposed to. Appar ently healthy bees collected from the same apiary contained no traces of any insecticides. These data suggest that there is a strong likelihood that the neonicotinoids thiamethoxam and/or clothianidin were responsible for the bee kills we investigated.

Corn and soybean seed treatments are the only major use of clothianidin and thiamethoxam in our study area. All reports of bee kills occurred when corn was being planted nearby . Therefore

, the remainder of our study focused up on itemizing the potential routes of honeybee exposure to these compounds and quantifying the concentrations of a range of pesticides in each potential exposure route

.

“ This study does not provide any fundamentally new evidence about honeybee exposure to neonicotinoid seed treatment products. The exposure levels reported in soil, pollen and nectar are generally consistent with previous research and were not high enough to represent a significant risk for honey bees. ”

While anecdotal reports have persisted for years, there wa s no documentation prior to our study (including

, but not limited to

, peer

- reviewed publications) of the potential causes of bee deaths in association with the planting of neonicotinoid treated seed s in North America.2 Our study took the approach of quantifying pesticide levels in a variety of materials that honey bees may be exposed to near plantings of treated seed. We found seed treatment insecticides at a wide range of concentrations in virtually every sample type we investigated (field soils, dandelions near fields, bee

collected pollen stored in cells and collected from pollen traps , maize pollen, and exhaust talc from planters). The only sample type that showed no trace of neonicotinoids was nectar collected from w ithin the hive

. The concentrations found in soil samples varied (2.1

- 9.6 ppb), and these results reflect the persistence of these compounds in field soils (2) ; we f ound neonicotinoids 2 full growing seasons after the treated seed was planted.

The spuri ous claim that t he concentratio n s of neonicotinoids we reported in stored pollen (2.9

- 10. 7 ppb clothianidin and 6.2

- 20.4 ppb thiamethoxam , found

co

- occurring ) were “not high enough to represent a significant ris k for honey bees” is not supported by d ata

. S ublethal effects of these compounds remain an active area of research and what constitutes a “significant risk” is far from well

defi ne d. Using figures from our study provide a case in point: a bee fed pollen containing 20.4 p p b of thiamethoxam for the 10 d period it spends as a nurse bee would be exposed to an equivalent of ca. 50% of the oral LD50 o f 2.8 ng/bee ( 3 )

. While the effects of such levels of dietary exposure level on individual bees (and colonies) remain unclear, dismissing these results out of hand and without supporting documentation reflects a gross oversimplification of a complex problem

.

“ Higher concentrations found in waste talc collected from inside pneumatic equipment post

- planting represent an intrinsic h azard to honey bees; however actual exposure of bees to this material was not demonstrated and would appear to be preventable. ”

The concentrations found in waste talc we re extremely high (up to 1 5

, 030 ppm
c lothianidin , up to 13

, 240 ppm thiamethoxam )

. These concentrations reflect that the treatments applied to t he seed are partially removed by and become part of the used talc /seed debris that constitutes planter exhaust material

. The material we removed from the planter for our analysis is the s ame mate rial that ultimately enter s the environment. Most of th is material is exhausted while planti ng: either with the seed and into the seed furrow or into the air behind and above the plante r via e xhaust fans. The remaining material in planters is removed durin g routine cleaning operations. Thus, th is material represents a hazard to honey bees and other non

- target insects that encounter it , because all of it (by design) leaves the planter at some point.

W hile neonicotinoids were found on dead bees, stored poll e n, dandelions etc., our methods did not allow us to determine how the materials came to be there. However, given the timing and location of bee kills (near corn fields during corn3 planting) and the extremely high toxicity and potential mobility of waste t alc, our findings lead us to conclude that this planter exhaust has a role in distributing seed treatment pesticides to areas where foraging bees will encounter them. The findin g s of Tapparo et al. ( 4 ), published sh ortly after our work, provides direct sup port for this hypothesis. Th is research group manipu l ated honey bee forager flight paths s o that they flew behind corn planters s o wing neonicotinoid treated seeds. The f oragers returning to the hive were found to be coated with insecticides found in the se ed treatments, significantly above the published lethal levels of 2 2

- 44 ng/bee (5,6)

. Clearly, this exposure route may be preventable

. However, t he c urrent situation is that there are no recommended methods for minimizing or preventing exposu r e of honey bee s to planter exhaust . N o recommendations exist for disposal of used talc. In short, growers planting treated seeds using currently recommended practices for planting and cleaning of equipment will result in large volumes of exhaust talc being distribut ed to the outside environment.

“ In their field experiment, low exposure levels and no adverse effects were observed for bee colonies placed “in harm’s way” around the perimeter of a field as it was planted with treated corn seeds. Overall, the publication represents an interesting case study, but it does not provide any significant new insights into exposure of honeybe e s to neonicotinoid insecticides. ”

Th e first part of th is statement is misleading , as it implies that we designe d and conducted this field study to document a dverse effects of planting upon colonies. T his was never an intended part of the study. There was no measure of colony health at any time (before, during or after the study).

Simply put, w e used bee colonies placed near planted fields as pollen collectors. We placed pollen traps in each hive and analyzed the bee

- collected pollen to determine 1) what proportion of pollen collected by foraging bees was corn pollen, and 2) whether pollen collected by honey bees in these environments was

co ntaminated with pesticides , and whether that level of contamination increased following planting of treated seed and associated planter exhaust

. This portion of our study demonstrated that foraging bees collected large amounts of corn pollen while it was present (experiment

- wide mean of ca. 44%), and that neonicotinoids were found in 10 of 20 samples analyzed (ranging from 1.2

- 7.4 ppb thiamethoxam and from 1.1

- 88 ppb clothianidin). These levels of neonicotinoids are up to 10

- fold higher than those reporte d by previous studies of bees foraging near treated crops (4).

Our study and others have shown that seed treatment pesticides are found in the pollen of corn and other plants grown from treated seeds ( 7,8 ). Corn pollen is abundant across large areas of t he continental US during late June and early July each year , so it is important to determine whether bees use this food4 resource . The collection of this pollen by foraging honey bees reflects that dietary exposure to pesticides occurs later in summer as we ll , and not solely during the spring planting period.

These link age s between honey bees and seed treatment insecticides were not known to our fellow research ers and , more importa ntly, by the agricultural stakeholders who either use seed treatment insecticides or live and work in areas where they a re used. The response from growers and beekeepers alike has overwhelmingly demonstrated that they fe el this work identifies critical

, previously unexplored links between planting of treated see d and honey bees

.

T he primary contribution of our studies is two

- fold: 1) we document that bee

- kills were associated with the presence of seed treatment insecticides in each sample tested ; and 2) evaluate and provide an initial “ ranking ” ( using concentratio n s of neonicotinoids as a guideline ) for several honey bee exposure routes. These types of data are a key step in determining when and where risk s to pollinators occur and can be readily mitigat ed

. For example, the extremely high levels of seed treatment pesticides found in t alc exhaust ed from planters stands out as a potential source of acute exposure and a previously uni dentified t arget for remedial action. The lower concentrations found in many other sample types are certainly worthy of further study as well, and may act as important route s for sublethal exposure.

Referenc e s 1. Krupk e, C. et al. (2012). Multiple Routes of Pesticide Exposure for Honey Bees Living near Agricultural Fi e lds, PloS ONE vol.7

.

2

. US Environmental Protection Agency, Office of Pesticide Programs. Fact s heet Clothianidin (2003) EPA Publication 7501C; www.epa.gov/opprd001/ facts h eets/ clothianidin.pdf.

3 . Laurino D . et al. (2011)

. T oxici t y of neonicotinoid insecticides to honey bees: laboratory tests. Bull Insectol 64: 107

– 113.

4

. A n drea Tapparo et.al. Assessment of the E nvironmental Exposure of Honeybees to Particulate Matter Conta i ning Neonicotinoid Insecticides Coming from Corn Coated Seeds, Environ. Sci. Technol., vol. 46, 2592

- 2599, 2012;

5 . US Environmental Protection Agency, Office of Pesticide Prog r ams. Fact s heet Clothianidin (2003) EPA Publication 7501C; www.epa.gov/opprd001/factsheets/clothianidin.pdf.

6

. Iwasa T . et al. (2004)

. Mechanism for the differential toxici t y of neonicotinoid insecticides in the honey bee, Apis mellifera . Crop Prot 23: 371

– 378

.

7

. Cutler G

. and Scott

- Dupree C

. (2007) Exposu r e to clothianidin seed

- treated canola has no long

- term impa c t on honey bees. J Econ Entomol 100: 765

– 772.

8

. Bonmatin JM et al. (2005)

. Quantificati o n of imidacloprid update in maize crops. J Agri Food Chem 53 : 5336

– 5341

.