UNITED STATESENVIRONMENTALPROTECTIONAGENCY WASHINGTON, D
.C
.20460



## DEC 082014 OFFICE OF CHEMICAL SAFETY AND POLLUTION PREVENTION

MEMORANDUM SUBJECT:



### TO:

FROM:

PEER 

### REVIEW: Review of2012 Com Rootworm Resistance Monitoring Data for Bt comproducts containing Cry34/35Abl. EPAReg Nos. 68467-5, -6; 29964-4, -5,-6,-10. MRID#: 492072-01,492418-01. PC Code: 006490. Decision#: 483490, 485700. DP Barcodes: 415358, 417219. Ann Sibold, Regulatory Action Leader Microbial Pesticides Branch Biopesticides and Pollution Prevention Division (7511P) AlanReynolds, Entomologist Microbial PesticidesBranch Biopesticides and PollutionPrevention
Jeannette Martinez, Ph.D., Ecologist/Entomologist

## ~~ Microbial Pesticides Branch

### ~ Biopesticides and PollutionPrevention Division (7511P)


### Action Requested The BPPD IRMTeam1 has been asked to review comrootworm resistance monitoring datafor the Cry34/35Ab1 Bt comtoxin. Submissions were made by DowAgroSciences and PioneerHiBredto address terms ofregistration for Bt comproducts that express Cry34/35, including the Herculex Rootworm and Optimum AcreMax product lines. The data covercomrootworm collectionsmade during the 2012 growing seasons. Two separate submissions were made for the diet bioassay (Dow) and Subletha l Seedling Assay (Pioneer) monitoring approaches.

1The BPPD IRM Team consis ts ofAlan Reynolds and Jeannette MartinezConclusions and Recommendations 1) Results from diet bioassays and Sublethal Seedling Assays (SSA) showed that the com rootworm (CRW) populations samp l ed during 2012 season were susceptible to the Cry34/35 toxin. The responses of these populations were similar to the susceptib le control colony and we re consistent with the results obtained in previous years. Despite this conclusion, the BPPD IRM Team is concerned that the diet bioassay approach using ECso/LCsos cannot reliably identify resistant populations, nor is there a functional ''diagnostic" die t bioassay. For these reasons, the BPPD IRM Team supports Pioneer's development and use of the SSA as a diagnostic on-plant assay for resistance detection. 2) Though the SSA can serve as an effective resistance monitoring too l , BPPD remains concerned by the potential for false negatives wi th the SSA, particularly among popu la t ion s · sampled from Cry34/35 fields with unexpected CRW damage. For th e past several years, Pioneer has tested a Cry34/35 selected co lon y ("Rochelle") as a positive control with the SSA. In the test in g, the Rochelle colony was clearly distinguishable f r om susceptible control and field populations, as the colony deve lop ed much better on Cry34/35 than the other tested populations

(evidenced by higher instar and body area r atios). But in only one ofthe four runs were the results statistically significant (p-value 0.05). Since Pioneer's resistance determination criter i a specify statistical significance, BPPD questions whether the Rochelle colony would be deemed resistant had it been a field-collected population. Populations that stand out as clearly less sens i t i ve to Cry34/35 (as Rochelle did in the 2012 testing) should be identified for further investigation regardless of whet h er they meet the strict stat i stica l criteria for resistance. 3) BPPD recommends that Pioneer consider the December, 2013 FIFRA Scientific Adv i sory Panel's (SAP) recommendations to improve the SSA for future testing. The SAP addressed the SSA as part of EPA's charge to eva l uate resistance monitoring for CRW. Improvements to the assay could be made by collecting live larvae from the funnels (instead of recovery with alcohol vials) for further study or using an agar based medium to observe developing l arvae. Some co n cerns were raised by the panel regarding possible decline in Bt expression over time in the plant and potential effects on the assay results. 4) Over the course of the 2012 grow i ng season, Dow received a "limited number" of unexpected damage reports from their customers related to Cry34/35 corn. Pioneer reported that no damage

claims were received by the company in 2012. One damage investigation (from Chickasaw Cou n ty, I owa) was described in the s u bmi tt ed report. ELISA testing verified the damaged plants as Cry34/35-expressing, but the site visit (early September) occurred too late in the season to sample adu l t CRW. At the recommendation ofDow, the field was rotated to soybean the . following season (20 13 ). It was unclear from the report whether there were other damage investigations during 2012. For future reporting, it is r ecommended that Dow/Pioneer provide an accounting of all damage reports from the season and a brief summary of their disposition. - 2-

### Background Btcornproducts expressingthe Cry34/35Abl toxin were first registered in 2005 by Dow AgroSciences and PioneerHi-Bred ("Herculex Rootworm" product lines). Subsequentto the initial registration, Cry34/35 has beenregistered in a number ofotherBtcornplant-incorpora ted protectants (PIPs), including stacked varieties (e.g., SmartStax) and seed blends (e.g., Optimum AcreMax). The Cry34/35 toxin is active against the corn rootworm (CRW) complex, including western corn rootworm (WCRW, Diabrotica virgifera virgifera) and northern corn rootworm (NCRW, Diabrotica barberi). As part ofthe terms ofregistration, all registrants ofCRW -protected Bt corn products (including Cry34/35) must monitor CRW populations for potential resistance. The monitoring effortshave included two components: 1) annual sampling andbioassays of CRWpopulations, and 2) investigations ofunexpected CRW damage in Cry34/35 fields. Past monitoring efforts and data (through the 2011 growing season) with Cry34/35 have been reviewed and summarized by EPA(see BPPD 2010a, 2013a). As documented in those reviews, CRW susceptibility has been assessed using diet

-based bioassays buthas been complicated by relatively low susceptibility to Cry34/35 (the toxin is not "high dose" for CRW) and lack ofa functional diagnostic assay to detect resistance. Since 2009, Pioneer has beenemployingthe Sublethal Seedling Assay (Nowatzki et al. 2008) as a second bioassay approach for resistance detection. This assay incorporates on-plant exposure (to seedlingmats) and can function more lik e a diagnostic tool for resistance detection (BPPD 2013a, b; SAP 2014). 

### Summary ofCornRootwormResistance Monitoring Submissions for Cry34/35 Two submissions were madeby Dowand/or Pioneerto address resistance monitoring activities during the 2012 growing season. A submission from Pioneer covered testing conducted withthe Sublethal Seedling Assay, while Dowand Pioneerjointly submitted a report documenting assays conducted with artificial diet. MRID# 492072-01: Monitoring Corn Rootworm Susceptibilityto EventDAS-59122-7

_ Usingthe Sublethal Seedling Assay (SSA): 2012 Collections (study submittedby Pioneer) MRID# 492418-01: Monitoring Corn Rootworm Susceptibilit y to Cry34/35Ab1 Event DAS 59122-7: 2012 Growing Season (Study submittedby Dow and Pioneer)


### A. Cry34/35 Diet Bioassays In accordance with the approved monitoring plan for Cry34/35 (MRID# 473342

-01), Dow and Pioneer
.are required to annually sample CR W populations and screen populations (with artificial diet bioassays orthe Sublethal Seedling Assay) for changes in susceptibilityto the toxin. Sampling has been coordinatedby ABSTC for all Btcorn registrants from three defined regions covering different WCRW variants. The regions also represent areas ofhigh Bt corn adoption



### - 3-and CRW pressure. Region 1 (soybean ro t ation resistant variety) includes Indiana and eastern Illinois; Region 2 (wild type) consists ofwestem Illinois , Iowa and Missouri; Region 3 (organophosphate resistant variant) encompasses Nebraska and Kansas. A minimum of2,000 adults are collected to establish each population for susceptibility testing.

2012 Sampling and Assays (MRID# 4 9 2 418-01) CRW sampling was conducted in late July / early August, 2012. Ten popula ti ons were collected

from Illinois (Region 1; 1 population), Iowa (Region 2; 4 populations) , Minnesota (Region 2; 1 population) and Nebraska (Region 3; 4 populations). Co ll ections were attempted at an additional 18 locations (in Iowa, Illinois, Indiana, Neb raska , and Wisconsin) but were not successfu l. Specific county locat ions of the collections are documented in Table 1 below. A Cry34/35- susceptible, non-diapausing control population (obtained from Crop Characteristics I nc., Farmington, MN) was also included in the assays. Eggs from the sampled adults were kept in diapause until testing (initiated the following March). Once removed from diapause, eggs were s t erilized and incubated until neonate hatching. Te sting was conducted by Custom Bio - Products (Maxwell, IA) using 96 well artificial diet trays supplied by Dow AgroSciences. The Cry34 /3 5 toxin was also provided by Dow and consisted of lyophilized powder preparations ofCry34Abl (lot TSN102172 ; 54% active ingredient) and

Cry35Ab1 (lot TSN102171; 37% active ingredient) mixed together in a 3:1 ratio by weight. Serial dilutions of Cry34/35 were prepared and overlaid on the diet trays to ach ie ve the following toxin levels: 0, 0.7 , 2.2, 6.7, 20.0, and 60.0 J.lg Cry34/35 / cm 2 diet surface. Ne onate WCRW were exposed to the serial dilutions (one larvae per well) and allowed to feed for five days after which mortality and (survivor) weight was recorded. Four to six replicates were run per population (each tray was considered a replicate); replicates were considered inva lid if the control mortality exceeded 25%. Mortality data were used to determine a LC va l ue for each population by probit

analysis. Lar va l weigh t data were grouped and converted to percent growth inhib it ion (relative to the control) to determine EC values. Results from the bioassays are detailed in Table 1 below. ECso susceptib ility to Cry34/35 was 0.70 -2 .94 J.lg/cm 2 for t he field-collected populations and 1.1 3 - 1.85 J.lglcm 2 for the unselected control colony. These results were reported to be within the historical range of Cry34/35 ECso values observed for WCRW (Table 2) . Simi la rly, the LCso results (range: 3.53 - 8.41 J.lg/cm 2 )

were comparable to (or lower than) previous years; however, LC so's could not be estab lished for 6 of the 10 populations because the data failed to converge in prohibit analyses. Dow and Pioneer noted that th e LCso values for the laboratory control (2.25 and 5.24 J..lg/cm 2 ) were actually an order of magnitude les s than the previous year (25 .86 J.lg / cm 2 ), suggesting that

control data from multiple sources would be of value for bioassay interpretation. To further analyze the data, the registrants compared the variation between the least and most sensitive populations (using the technique of Siegfried et al. 2007). Hi gher variation could be an indicator of local selection pressure causing decreased suscep tibili ty to Cry34 / 35. The variation of the 2012 data (ECso and LCso ratios) was 2 -5 fold, which was consistent with that from previous collections (3-6 fold since 2004), suggesting that natural variability could be responsible for the observed results rather than potential resistance. - 4-Overall, the report concluded that the diet bioassay data provided no indicationthat WCRW susceptibilityto Cry34/35 had declined during the 2012 growing season. Field-collected populations responded similarlytothe susceptible control colony and the variat ion betweenthe least and most sensitive populations remained low (similarto previousyears).
Table 1. Western comrootworm susceptibility to Cry34/35 using overlay diet bioassays: 2012 season (Table reproduced from Table 1in Appendix A, MRID# 492418-01) Population Replicates MeaoECso MeanLCso (95% Cl) · (95% Cl) (11glcm2 ) (!lg/cm2 ) Cerro Gordo County, IA 4 2: 15 6.41 (1.0 1

- 3.87) (1.30

- 18.12) Clay County, lA 5 2.94 (1.16-6.48) 

### ---- I Floyd County, IA 5 2.30 6.37 (I.39

- 3.57) (1.57

- 18.18) Hamilton County, IA 4 0.70 (0.60

- 0.80) 

### ---- I Lee County, IL 4 2.04 ( 1.07

- 3.48) 

## -·-- I Mower County, MN 6 2.66 8.41 (2
.32

- 3.02) (5.67 -12
. 16) Howard County,NE 4 1.64 3.53 (1.52

- 1.76) (2.46

- 4.86) Platte County, NE 6 2.20 I ( 1.39

- 3
.30) 

### ---- Stanton County,NE 6 2.30 I (1.72

- 3
.02) ........ York County, NE 5 1.41 I (0.98

- 1.90) 

## ---- Range 0
.70

- 2.94 3.53

- 8.41 Laboratory Control (Js' Run) 6 l.l3 2.25 (0.60

- 1.74) (0.39

- 5.05) Laboratory Control (rdRun) 6 1.85 5.24 (I.41

- 2.39) (2.06

- 8.60)
1LC50s could not be calculated for these populations because data failed toconverge in probit analysis
.



### - 5

-Table 2. Susceptibi li ty bioassay results 1 for Cry34/35

- 2004 to 2012 (Table genera t ed from data provided by Dow AgroSciences and Pioneer) Year ECso (J..Lg/cm 2 ) EC99 (J..Lg/cm 2 ) LCso (J..Lg/cm 2 ) 3 LC90 (J..Lg/cm 2 ) 2004-2005 0.8- 1.1 2.0-2.4 2006 0.8-2 . 3 21.0-56.6 1.2-7.3 9.2-78.1 2007 1.0- 3.7 51.9-213.6 3 .0 - 11.5 23.9-463 2008 2.0-7.9 83.8- 596.6 6.2-15.4 58.4-613.6 2009 1.9- 5.7 45.9-93.3 2010 2.4- 7.6 6.7-33.7

2011 2 2.6- 5 . 3 11.7- 39.9 2012 0 . 7-2 . 9 3.5 - 8.4 1 Results are for field populations collected by ABSTC; does not include results from fields with unexpected damage or other CR W populations.

2 Testing in 2011 was conducted with new toxin lots (activity was similar to the previous lots). 3 Numerous field populations are not included in the LCso results because data failed t o converge in probit analysis. BP P D IRM Team Review - 20 I 2 Diet Bioassays Growth inhibition (ECso and LCso) testing showed that the 2012 populations were sens itive to

Cry34 / 35 and responded simi l arly to CRW collected from previous seasons (Table 2) . LCso values could not be calculated in pro bit analysis for a number of populations in 2012, which has a l so been an issue for past bioassays. Though the field-collected populations were within historical susceptibility ranges, the control populations were significantly more sensitive (LCso) to the toxin than in 2011: 225 and 5.24 11g/cm 2 in 2012 compared to 25.86 p..tg/cm 2 in 2011. Since Dow/Pioneer noted that the laboratory methods and conditions were the same in previous years, the result is likely reflective of natural variation in CR W response to diet bioassays. BPPD agrees, with Dow / Pioneer's suggestion that using multiple sources for contro l population would be of value for Cry34/35 suscept i bility monitoring. Many of the counties sampled in 2012 had also been sampled in previous seasons (2009

- 201 1 ).

The Cry34 / 35 susceptibility was comparable (in many cases lower) in the se count i es during 2012 than in the previous collection years (see data summarized in BPPD 2013a) . BPPD cau t ions, however, that it is unclear (and probably unlikely) that the same locale s (i.e. , CRW populations) were sampled within these counties from year -to- year. Therefore, these results do not necessarily track changes in s us ceptibility to discrete CRW populations ove r the samp lin g period. As described in previous rev iew s (BPPD 20 13a, b) , BPPD has expressed concerns with the diet bioassay approach as an effective tool to detect potentially resistant field populations. The 2013 SAP evaluated diet bioassays for CR W and recommended that the approach has value for - 6-monitoring susceptibility (SAP 2014). Thepanel highlighted their standard use in the scientific community,the precise nature ofthe assays, the breadth ofdata that can be collected (lethal and sublethal responses), and logistical advantages for researchers. Onthe otherhand, the panel noted a number ofshortcomings ofdiet bioassays (as currently designed) that limit their ability to detect field resistance. Specifically, dietbioassays may not reflect survival oncomplants, LC measures have beenhighly variable, EC values may notbe sensitive enoughto identify early resistance, a "diagnostic" concentration capable ofkilling all susceptible andheterozygous resistant genotypes has not beendeveloped, and the assays may not be able to distinguish resistant populationsfrom susceptible ones. BPPD recognizes the scientific value ofdiet bioassays, but believes the limitations (as identifiedby the SAP) restrict their usefulness as a regulatory tool for resistance detection. A keycomponent ofIRM has beenthe effective identification ofresistant populations as early as possible so that mitigatory measures can be applied with a greater likelihood ofsuccess (BPPD 2013b). Onplant assays, which can effectively distinguish resistant populations from susceptible ones based on response to Btplants (Gassmann et.al. 2011), offer morepotential as a deterministic tool for resistance monitoring. Along these lines, Pioneerhas developed an on-plantassay (Sublethal Seedling Assay; Nowatzki et al. 2008) for Cry34/35 (see discussion in thefollowing section). B. Sublethal Seedling

### Assay Since 2009, Pioneer has beenutilizing the Sublethal Seedling Assayinconjunction with diet bioassays as a diagnostic monitoring tool for detecting resistance. The Sublethal Seedling Assay (SSA) was developedby Pioneer scientists withthe Cry34/35 toxin and entails exposure ofCRW larvae to root mats ofBt and non-Btplants (an "on-plant" assay). Methodology for the assays is described in Nowatzki etal. (2008), though Pioneerhas modified theprocedure based ontheir experiences with the test (see discussion in BPPD 2013a). SSA- 2012 Testing (MR!Ds 492072-01) As part ofthe 2012 testing, Pioneerincluded populations collected as part ofthe ABSTC annual sampling (see Table 1 above); however, due to limited egg availability orviability onlytwo of these populations could ultimately be tested. To buttress the sampling, Pioneer collected eight additional populations from the ComBelt, six ofwhich produced sufficient eggs for testing. The tested populations are listed in Table 3 below. Two control populations were also included in the assays: a Cry34/35 susceptiblepopulation (supplied by USDA-ARS, Brookings, SD) and a Cry34/35 resistant strain (supplied by Pioneer, termed "Rochelle-RR"). Testing methodologyfor the SSA involves exposing neonate CRW to seedling mats ofBtor non-Bt com (150 seedkernels permat). As impliedbythe name, the SSA does not measure mortality; rather, the test is designed to assess potential developmental effects ofBtexposure including growth (instar) and body size (larval body area). Forthe 2012 testing, each ofthe seedling mats in the test was infested with approximately 500 CRWeggs onthe soil surface and held in an environmental chamber (25 °C, 65% RH, 14:10 light/dark). For some populations with low egg availability, a lower infestationrate was used. Threeto four replicates (each mat 

### was considered areplicate) were established for each treatment (Bt and non-com). A separate hatch test using a subsample ofeggs in Petri dishes held in the same environmental chambers



### - 7-was conducted to determine the starting point of the test. After hatching was initiated, larvae were exposed for approximately 154 degree days to allow for growth and development. Larvae were then collected from the root mats using Burlese funnels positioned over containers with 70% ethanol. A sample of 30 larvae was obtained from each replicate; any population that did .

not produce at least 30 larvae in a majority of the rep l icates was considered non-viable. The samp l ed l arvae were then measured (head capsu l e width) to determine the developmental instar (1 5 \ 2nd, or 3rd). A second processing procedure was also used to assess body size. Samples were poured into Petri dishes , which were scanned and analyzed by imaging software to calculate the total body area (mm 2 ) of each larva. These procedures were the same as those used in the 2011 testing (described in MRID# 490085-01; reviewed in BPPD 20 1 3a). To interpre t the developmental data, Pioneer analyzed the d i stribution of larvae among all three instars. A mean instar was calcu l ated for each treatment group as follows: [1 *( # 1 st instars) + 2*( # 2nd instars) + 3*( # 3rd ins t ars)] I total # larvae. Bt and non - Bt trea t ments wer e compared to g e nerate a means ' ratio (mean instar of Bt I mean instar of non-Bt). If larval development on Cry34 1 35 equa l s or exceeds that on non-Bt, the ratio will be 2: 1.0 (a similar output as the third ins t ar odds ratio). The instar data were further analyzed by generating a Kolmogorov-Smirnov statistic (KS D) t hat accounts for the magnitude of difference between treatments (ranging from1 to 1, with 0 indicating no differences between distributions). Fisher ' s exact test wa s used to determine sta t istical s i gnificance. Imaging data were analyzed by calculating are a mean ratios (body area of Bt I body area of non - Bt) for each population. Similar to the instar data , a KS D statistics and Fisher's exact tests were employed to assess differences between the treatment

groups. Though not addressed in this submission , Pioneer has previously indicated (MRID # 490085-01) that its decision process to determine suspected resistance (triggering follow - up investigation) will include two criteria. F urther action will be taken if a population: • Shows sim il ar response when exposed to both 59122 (Cry34 1 35) and isoline at a significance l evel of p>0.05 for both methods (instar and image data); • Shows mean ratios (both instar and size) outside the 95% confidence inter v al established from data collected (in) previous years. SSA Results ( 2012) The susceptibility of each field population as measured by th e SSA is documented in Table 3 below. WCRW field populations collected in 2012 had a mean ins tar ratio range of 0. 710 to

0.883 , indicating t hat larvae developed more slowly on Bt than isoline com (KS D 2:. 0.256; p < 0.0001 ). By compar i son, the instar ratio for the two runs with the susceptible laboratory colony

were 0.733 and 0.902. Body area analysis of the field populations followed a sim i lar pattern, as larvae on Bt plants were smaller than those on non-B t. Mean body area ratios ranged from 0 .2 24 to 0.388 (control colony = 0.236 and 0.292), with all populations having significantly different distributions on Bt and non-B t (KS D 2:. 0.466; p < 0 .0 001). Two test runs were conducted with the "Rochelle" Cry34 / 35 - selected colon y . This population fared better on Bt com t han the field or laboratory colonies , with mean instar ratios of 0.972 (KS - 8 -D = 0.083; p = 0.0249) and 0.994 (KS D = 0.017; p = 0.6218). Similarly, the colony had higher body arearatios (0.712 and 0.764) than the field populations, though the Bt and non-Bt distributions were statistically different (KS D

## =0.517 and 0.443; p 

### < 0.0001). Despite the statistical ambiguity, Pioneernoted that the Rochelle colony had the lowest susceptibility to Cry34/35 ofthe populations tested. SSAdata from the 2012 season were comparableto those from field populations tested in previous years (see Table4). Since none ofthe 20 12 populations exhibited similardevelopment onBt and non-Bt, Pioneer concluded that "no field-collected populations [were] identified as resistant to 59122."
Table 3. Western corn rootworm susceptibility to Cry34/35 using the Sublethal Seedling Assay: 2012 season (Tablereproduced from Table 1 in Appendix A, MRID# 492418-0I)
Instar Ratio Body Area Population Mean Mean Instarmean Non-Bt Area mean instar: Bt instar: ratio Bt (mm2) (mm2) ratio non-Bt (Bt :non-Bt) (Bt: non Bt ) Cerro Gordo, IA (ABSTC) 2.22 2.68 0.830 2.49 6.40 0.388

Hamilton, lA (ABSTC) 2.40 2.80 0.857 3.64 10.02 0.364 Blue Earth, MN 2.36 2.67 0.883 2.89 9.04 0.319 Champaign, IL 2.08 2.84 0.733 2.54 8.64 0.293 Columbia, Wl 2.27 2.93 0.773 3.99 10.87 0.367 Delaware, I A 2.62 2.87 0.9 15 4.46 11 .62 0.384 Hutchinson, SO 2.27 2.92 0.776 3.06 10.81 0
.283 York, NE 2
.01 2.83 0.710 2.26 10.09 0.224

Range 0.710-0.915 0.224- 0.388 Laboratory Control (, .,Run) 2.16 2.39 0.902 1.93 6
.62 0.292 Laboratory Control (2ndRun) 2.10 2.87 0.733 2.43 10.30 0.236 Rochelle RR (1 11 Run ) 2.88 2.97 0.972 8.26 11.59 0.712 RochelleRR (2"d Run) 2.98 2.99 0.994 8.90 11.65 0.764

- 9-Table 4 . Sublethal Seedling Assay Results for Western Corn Rootworm, 2009- 2012 (Table created from data submitted by Pioneer) # l nstar (mean ratio Bt : non-Bt) Body Area (mean ratio Bt: non-Bt) Year Field Susceptible Selected Susceptible Pop. Field Control' Colony 2 Field Control' 2009 15 0.766- 0.996 0.781 --- 0.393 - 0.894 0.508 2010 7 0.734- 0.960 0.787 0.915 0.387 - 0.511 0.512 2011 11 3 0.687 - 0.883 1) 0.727 0.986 0.214-0.421 I ) 0 . 19 8 2) 0.830 2) 0.274 2012 8 0.710 - 0.883 I) 0.902 1) 0.972 0 . 224- 0 . 388 I ) 0.292 2) 0.733 2) 0.994 2) 0.236 1 Cry34/35-susceptibile control colony obtained from USDA-ARS (Brookings, SD). 2 York-RR (2010-2011) and Rochelle-RR (2012) have been selected on 59122 (Cry34 / 35) com. 3 Includes three populations collected from fields with unexpected CR W damage. The resu l ts from t h ese populations were within the ranges of the field-collected populations. BPPD IRMTeam Review- SSA Procedure and Results

Selected Colony 2 --- 0.692 I) 0.624 2) 0 . 633 I) 0.712 2) 0.764 Pioneer 's 2012 report documented the fourth year of testing with the SSA. Results from the 2012 assays were co nsis tent with the field colonies tested iri previous years and, coupled with the general lack of field damage reports , demons trated no discernable reduction in susceptibility to Cry34/35. In the 2012 testing, BPPD noted some variation in the instar ratios between the two control colony tests (0.902 and 0.733), though both runs were within the range observed for the field populations. Overall, thes e results support BPPD's previous conclusion (BPPD 2013b) that

the SSA can serve as a functional "diagnostic" test for CR W resistance monitoring and may b e more valuable in detecting potentially resistant populations than diet bioassays. Some of the populations were collected from counties in which CR W had been sampled and tested in previous years. However, unless the same fields or surrounding areas were sampled, it i s unlikely that discrete populations have been sampled over time. Two of the counties sampled during 2012 (Delaware, lA and Hutchinson, SD) had Cry34/35 fields with CRW damage in 2011 (see BPPD 2013a). Pioneer's report did no t specify where and how thes e populations were collected within the counties and if they were taken from locations proximate to the 2011 field

damage sites. The popula t ion collected from Delaware , lA had the highest instar ratio and second highest body area of th e tested field populations (see Table 3), though the larvae on Cry34/45 were still significantly less dev e loped than those on non-Bt corn. On the other hand, the Hutchinson, SD population appeared to have similar Cry34/35 susceptibility as the other tested field populations. In a previous review of the SSA, BPPD (2013a) expressed concerns regardi ng the potential se nsitivity of the assay in distinguishing resistant populations . In addition to te st ing a s usceptible control, Pioneer has a l so tested a Cry34/35-se l ected co l ony, "Rochelle -RR " (a separate colony, . - 10 -"York-RR," was tested in 2010 and 2011). This colony (at the time oftesting) had been selected on Cry34/35 for 28 of53 generations. 

### In testing with the SSA, the Rochelle colony was clearly distinguishable from susceptible control and field populations (see Table 3). The colony developed much betteron Cry34/35 than the other tested populations, as evidenced byhigher instar andbody arearatios. But in only one ofthe four runs were the results statistically significant (p-value 0.05). Since Pioneer's resistance determination criteria specify statistical significance, BPPD has questioned whether the Rochelle colony would be deemed· resistant had itbeen a field-collected population (BPPD 2013a, b). Pioneerhas noted that the selected colony is nonetheless readily identifiable as a population withless susceptibilityto Cry34/35 and evidence that the SSA functions as a diagnostic tool (Pioneer 2013). BPPD remains concerned by thepotential for false negatives with the SSA, particularly among field-collected populations or those sampled from Cry34/35 fields withunexpected CRW damage· . Further testing with selectedcolonies is recommended to improve the interpretation of the assay in the contextofresistance determinations. Populations that stand out as clearly less sensitive to Cry34/35 (as Rochelle did in the 2012 testing) should be identified for further investigationregardless ofwhether they meet the strict statistical criteria for resistance. EPA convened a Scientific Advisory Panel (SAP) meeting inDecember, 2013 to consider scientificuncertainties associated with CR W resistance monitoring. As part ofthe charge questions, thepanel reviewed the methodology ofon-plant assays (includingthe SSA) and providedrecommendations to improve theiruse. The panel concluded that on-plant assays have value by measuring "actual response ofthe western comrootworm larvae to cornplantsthat express Btprotein(s)" (SAP 2014). Regarding the SSA, the panel noted Pioneer's progress in optimizingthe assay by adjusting eggdensities and shortening the exposure period (methodology changes described in BPPD 2013a). Improvements to the assay could be made by collecting live larvae from thefunnels (instead ofrecovery with alcohol vials) for further study orusing an agar based medium to observe developing larvae. Some concerns were raised regarding possible decline in Btexpression overtime inthe plant and potential effects on the assay results (SAP 2014). BPPD recommends that Pioneerconsider the panel's comments to further develop and improve the SSA for future testing. C. Cry34/35Field DamageReports Overthe course ofthe 2012 growing season, Dow received a "limited number" ofunexpected damage reports from their customers related to Cry34/35 com. Pioneerreported that no damage claims were received by the company in 2012. One damage investigation was described in the submitted report. A grower in Chickasaw County, Iowa observed unexpected damage to a field plantedto Herculex Xtra (CrylF x Cry34/35). ELISA testing verified the damaged plants as Cry34/35-expressing, butthe site visit (early September) occurredtoo late in the season to sample adult CRW. Atthe recommendation ofDow, the field wasrotated to soybean the following season (2013). The site was revisited in 2013 to collect CRW though too few adults were found to establish a populationfor testing. Dow also noted that no other reports ofunexpected damage werereceived from the county during 2013.



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-It was unclear from the report whether the Chickasaw field was the only Cry34 / 35 damage investigation during 2012. For future reporting, it is recommended tha t Dow / Pioneer provide an

accounting of all damage reports from th e season and a brief summary of their disposition. Dow ' s response to the Chickasaw case appears to have been successfully mitigated by rotation to soybean: few CRW were reported the following year and no additional damage reports were received in the v icinity. BPPD believes that the use of IPM-based Bes t Management Practices (BMPs), particularly rotation to soybean, can be an eff e ct i ve means to suppress (or extirpate) a potentially**~esistant** population (BPPD 2013b). References BPPD, 2010a. Review of Dow's 2008 corn rootworm monitoring report and revised resistance monitoring plan for DAS-59122-7. J.C. Martinez memorandum toM. Mendelsohn, June 30 , 2010. BPPD, 2010b. Bacillus thuringiensis Cry34Abl and Cry35Ab1 Proteins and the Genetic Material Necessary for t heir Production in Event DAS-59122-7 Corn Biopesticide Registration

Action Document (BRAD). Revised September , 2010. Available at http: / / www.epa.gov / oppbppd 1/biopesticides/ingredients / tech docs/brad 006490.pdf. BPPD , 20 I Oc. Terms and Conditions for Bt Corn Registra t ions, September 30, 2010. Available at: http: /( www.epa.gov / pesticides / biopesticides / pipslbt-corn-terms-conditions.pdf . BPPD, 2011. U pdated BPPD IRM Review of Reports of Unexpected Cry3Bb1 Damage, Monsanto ' s 2009 Corn Rootworm Monitoring Report, and Revised Corn Rootworm Resistance Monitoring Plan for MON 88017, MON 88017 x MON 810, MON 863, MON 863 x MON810, MO N 89034 x TC1507 x MON 88017 x DAS-59122-7 , and MON 89034 x MON 88017. J. Martinez memorandum toM. Mendelsohn, November 22 , 2011. BPPD , 2012. BPPD IRM Team review of Monsanto's 2010 corn rootworm moni t oring data, unexpected _ damage reports for Cry3Bb1 e x pressing Bt Corn and academic report s ofCry3Bb1 field failures as well as corn rootworm resistance. J. Martinez and A. Reynolds memorandum t o A. Sibold, October 11, 2012. BPPD, 20 13a. Review of Corn Rootworm Resistance Monitoring Submissions and Data (2009
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2011) for Bt corn products containing Cry3'4 / 35Abl. A. Reynolds memorandum to A. Sibold, March 20 , 2013. BPPD, 2013b. White paper on corn rootworm resistance monitoring for Bt plant-i n corporated protectants. Ava i lable at: http: / / www.epa.gov / oppfead 1 / cb / csb _page / updates / 20 13 / cornrootworm-wp.h t ml. Gassmann, A.J. , J.L. Petzold-Maxwell, R.S. Keweshan, and M.W. Dunbar, 2011. Field-evolved

resistance to Bt maize by western com rootworm. PLoS ONE. 6(7): 1-7. - 12-Nowatzki, T.M
., S.A. Lefko, R.R. Binning, S.D
. Thompson, T.A. Spencer, and B.D. Siegfried, 2008
. Validationofa novel resistance monitoringtechnique for cornrootworm (Coleoptera: Chrysomelida) and eventDAS-59122-7 maize. J Appl. Entomol. 132: 177

- 188. Pioneer, 2013. Comments to public docket EPA-HQ-OPP-2013-0490, dated November 26, 2013. http://www.regulations.gov/ #!documentDeta il;D=EPA-HQ

-OPP

-2013

-0490-0025
. SAP, 2014. SAP Minutes No. 2014-01: A SetofScientific Issues Being Consideredby the Environmental ProtectionAgency Regarding Scientific Uncertainties Associated with Com Rootworm Resistance Monitoringfor Bt Corn Plant Incorporated Protectants (PIPs) December4-5, 2013 FIFRA Scientific Advisory Panel Meeting
. Available at: http://www.epa.gov /scipoly/sap/meetings/2013/december /120413minutes
.pdf.

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