UNITED STATES ENVIRONMENTAL PROTECTION AGENCY 

WASHINGTON, D.C. 20460 

DEC 0 8 2014 

OFFICE OF CHEMICAL SAFETY 

AND POLLUTION PREVENTION 

MEMORANDUM 

SUBJECT: 

TO: 

FROM: 

PEER 

REVIEW: 

Review of 2012 Com Rootworm Resistance Monitoring Data for 

Bt com products containing Cry34/35Abl. 

EPA Reg 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) 

Alan Reynolds, Entomologist 

Microbial Pesticides Branch 

Biopesticides and Pollution Prevention 

Jeannette 

Microbial 

Biopesticides 

Martinez, 

Pesticides 

and Pollution 

Ph.D.

Branch 

, Ecologist/Entomologist 

Prevention Division (7511 P) 

~~ 

~ 

Action Requested 

The BPPD IRM Team 1 has been asked to review com rootworm resistance monitoring data for 

the Cry34/35Ab1 Bt com toxin. Submissions were made by Dow AgroSciences and Pioneer HiBred to address terms of registration for Bt com products that express Cry34/35, including the 

Herculex Rootworm and Optimum AcreMax product lines. The data cover com rootworm 

collections made during the 2012 growing seasons. Two separate submissions were made for the 

diet bioassay (Dow) and Sublethal Seedling Assay (Pioneer) monitoring approaches. 

1 The BPPD IRM Team consists of Alan Reynolds and Jeannette Martinez 
Conclusions and Recommendations 

1) Results from diet bioassays and Sublethal Seedling Assays (SSA) showed that the com 

rootworm (CRW) populations sampled during 2012 season were susceptible to the Cry34/35 

toxin. The responses of these populations were similar to the susceptible control colony and 

were 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" diet 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 tool, BPPD remains 

concerned by the potential for false negatives with the SSA, particularly among populations 

· sampled from Cry34/35 fields with unexpected CRW damage. For the past several years, 

Pioneer has tested a Cry34/35 selected colony ("Rochelle") as a positive control with the SSA. 

In the testing, the Rochelle colony was clearly distinguishable from susceptible control and field 

populations, as the colony developed much better on Cry34/35 than the other tested populations 

(evidenced by higher instar and body area ratios). 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 questions whether the Rochelle colony would be deemed 

resistant had it been a field-collected population. Populations that stand out as clearly less 

sensitive to Cry34/35 (as Rochelle did in the 2012 testing) should be identified for further 

investigation regardless of whether they meet the strict statistical criteria for resistance. 

3) BPPD recommends that Pioneer consider the December, 2013 FIFRA Scientific Advisory 

Panel's (SAP) recommendations to improve the SSA for future testing. The SAP addressed the 

SSA as part of EPA's charge to evaluate 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 larvae. Some 

concerns 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 growing 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 

County, Iowa) was described in the submitted 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 adult 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 recommended that Dow/Pioneer provide 

an accounting of all damage reports from the season and a brief summary of their disposition. 

- 2-
Background 

Bt corn products expressing the Cry34/35Abl toxin were first registered in 2005 by Dow 

AgroSciences and Pioneer Hi-Bred ("Herculex Rootworm" product lines). Subsequent to the 

initial registration, Cry34/35 has been registered in a number of other Bt corn plant-incorporated 

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 of the terms of registration, all registrants of CRW -protected Bt 

corn products (including Cry34/35) must monitor CRW populations for potential resistance. 

The monitoring efforts have included two components: 1) annual sampling and bioassays of 

CRW populations, and 2) investigations of unexpected 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 but has been complicated by 

relatively low susceptibility to Cry34/35 (the toxin is not "high dose" for CRW) and lack of a 

functional diagnostic assay to detect resistance. Since 2009, Pioneer has been employing the 

Sublethal Seedling Assay (Nowatzki et al. 2008) as a second bioassay approach for resistance 

detection. This assay incorporates on-plant exposure (to seedling mats) and can function more 

like a diagnostic tool for resistance detection (BPPD 2013a, b; SAP 2014). 

Summary of Corn Rootworm Resistance Monitoring Submissions for Cry34/35 

Two submissions were made by Dow and/or Pioneer to address resistance monitoring activities 

during the 2012 growing season. A submission from Pioneer covered testing conducted with the 

Sublethal Seedling Assay, while Dow and Pioneer jointly submitted a report documenting assays 

conducted with artificial diet. 

MRID# 492072-01: Monitoring Corn Rootworm Susceptibility to Event DAS-59122-7 

_Using the Sublethal Seedling Assay (SSA): 2012 Collections 

(study submitted by Pioneer) 

MRID# 492418-01: Monitoring Corn Rootworm Susceptibility to Cry34/35Ab1 Event 

DAS 59122-7: 2012 Growing Season (Study submitted by 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 or the Sublethal Seedling Assay) for changes in susceptibility to the toxin. 

Sampling has been coordinated by ABSTC for all Bt corn registrants from three defined regions 

covering different WCRW variants. The regions also represent areas of high Bt corn adoption 

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and CRW pressure. Region 1 (soybean rotation 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 populations were collected 

from Illinois (Region 1; 1 population), Iowa (Region 2; 4 populations), Minnesota (Region 2; 1 

population) and Nebraska (Region 3; 4 populations). Collections were attempted at an additional 

18 locations (in Iowa, Illinois, Indiana, Nebraska, and Wisconsin) but were not successful. 

Specific county locations of the collections are documented in Table 1 below. A Cry34/35-

susceptible, non-diapausing control population (obtained from Crop Characteristics Inc., 

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 

sterilized and incubated until neonate hatching. 

Testing was conducted by Custom Bio-Products (Maxwell, IA) using 96 well artificial diet trays 

supplied by Dow AgroSciences. The Cry34/35 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 achieve the following 

toxin levels: 0, 0.7, 2.2, 6.7, 20.0, and 60.0 J.lg Cry34/35/cm2 diet surface. Neonate 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 invalid if the control mortality 

exceeded 25%. Mortality data were used to determine a LC value for each population by probit 

analysis. Larval weight data were grouped and converted to percent growth inhibition (relative 

to the control) to determine EC values. 

Results from the bioassays are detailed in Table 1 below. ECso susceptibility to Cry34/35 was 

0.70 -2.94 J.lg/cm2 for the field-collected populations and 1.1 3 - 1.85 J.lglcm2 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). Similarly, the LCso results (range: 3.53 - 8.41 J.lg/cm2

) 

were comparable to (or lower than) previous years; however, LCso's could not be established for 

6 of the 10 populations because the data failed to converge in prohibit analyses. Dow and 

Pioneer noted that the LCso values for the laboratory control (2.25 and 5.24 J..lg/cm2

) were 

actually an order of magnitude less than the previous year (25.86 J.lg/cm2

), 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). Higher variation could be an 

indicator of local selection pressure causing decreased susceptibility 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. 

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Overall, the report concluded that the diet bioassay data provided no indication that WCR W 

susceptibility to Cry34/35 had declined during the 2012 growing season. Field-collected 

populations responded similarly to the susceptible control colony and the variation between the 

least and most sensitive populations remained low (similarto previous years). 

Table 1. Western com rootworm susceptibility to Cry34/35 using overlay diet bioassays: 2012 

season (Table reproduced from Table 1 in 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 (rd Run) 6 1.85 5.24 

(I .41 - 2.39) (2.06 - 8.60) 

1 LC50s could not be calculated for these populations because data failed to converge in probit analysis. 

- 5 -
Table 2. Susceptibility bioassay results1 for Cry34/35 - 2004 to 2012 (Table generated from data 

provided by Dow AgroSciences and Pioneer) 

Year ECso (J..Lg/cm2

) EC99 (J..Lg/cm2) LCso (J..Lg/cm2)

3 LC90 (J..Lg/cm2

) 

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 to 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 sensitive to 

Cry34/35 and responded similarly 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 

also 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/cm2 in 2012 compared to 25.86 p..tg/cm2 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 control population 

would be of value for Cry34/35 susceptibility 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 these counties during 2012 

than in the previous collection years (see data summarized in BPPD 2013a). BPPD cautions, 

however, that it is unclear (and probably unlikely) that the same locales (i.e., CRW populations) 

were sampled within these counties from year-to-year. Therefore, these results do not 

necessarily track changes in susceptibility to discrete CRW populations over the sampling 

period. 

As described in previous reviews (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). The panel highlighted their standard use in the scientific 

community, the precise nature of the assays, the breadth of data that can be collected (lethal and 

sublethal responses), and logistical advantages for researchers. On the other hand, the panel 

noted a number of shortcomings of diet bioassays (as currently designed) that limit their ability 

to detect field resistance. Specifically, diet bioassays may not reflect survival on com plants, LC 

measures have been highly variable, EC values may not be sensitive enough to identify early 

resistance, a "diagnostic" concentration capable of killing all susceptible and heterozygous 

resistant genotypes has not been developed, and the assays may not be able to distinguish 

resistant populations from susceptible ones. BPPD recognizes the scientific value of diet 

bioassays, but believes the limitations (as identified by the SAP) restrict their usefulness as a 

regulatory tool for resistance detection. A key component of IRM has been the effective 

identification of resistant populations as early as possible so that mitigatory measures can be 

applied with a greater likelihood of success (BPPD 2013b). On plant assays, which can 

effectively distinguish resistant populations from susceptible ones based on response to Bt plants 

(Gassmann et. al. 2011 ), offer more potential as a deterministic tool for resistance monitoring. 

Along these lines, Pioneer has developed an on-plant assay (Sublethal Seedling Assay; Nowatzki 

et al. 2008) for Cry34/35 (see discussion in the following section). 

B. Sublethal Seedling Assay 

Since 2009, Pioneer has been utilizing the Sublethal Seedling Assay in conjunction with diet 

bioassays as a diagnostic monitoring tool for detecting resistance. The Sublethal Seedling Assay 

(SSA) was developed by Pioneer scientists with the Cry34/35 toxin and entails exposure ofCRW 

larvae to root mats of Bt and non-Bt plants (an "on-plant" assay). Methodology for the assays is 

described in Nowatzki et al. (2008), though Pioneer has modified the procedure based on their 

experiences with the test (see discussion in BPPD 2013a). 

SSA- 2012 Testing (MR!Ds 492072-01) 

As part ofthe 2012 testing, Pioneer included populations collected as part of the ABSTC annual 

sampling (see Table 1 above); however, due to limited egg availability or viability only two of 

these populations could ultimately be tested. To buttress the sampling, Pioneer collected eight 

additional populations from the Com Belt, six of which 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 susceptible population (supplied by USDA-ARS, Brookings, SD) and a 

Cry34/35 resistant strain (supplied by Pioneer, termed "Rochelle-RR"). 

Testing methodology for the SSA involves exposing neonate CRW to seedling mats ofBt or 

non-Bt com (150 seed kernels per mat). As implied by the name, the SSA does not measure 

mortality; rather, the test is designed to assess potential developmental effects ofBt exposure 

including growth (instar) and body size (larval body area). For the 2012 testing, each of the 

seedling mats in the test was infested with approximately 500 CRW eggs on the 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 infestation rate was used. Three to four replicates (each mat 

was considered a replicate) were established for each treatment (Bt and non-com). A separate 

hatch test using a subsample of eggs in Petri dishes held in the same environmental chambers 

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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 replicates was considered non-viable. The 

sampled larvae were then measured (head capsule 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 (mm2

) of each larva. These procedures were the same as those used 

in the 2011 testing (described in MRID# 490085-01; reviewed in BPPD 2013a). 

To interpret the developmental data, Pioneer analyzed the distribution of larvae among all three 

instars. A mean instar was calculated for each treatment group as follows: [1 *(# 1st instars) + 

2*(# 2nd instars) + 3*(# 3rd instars)] I total # larvae. Bt and non-Bt treatments were compared to 

generate a means 'ratio (mean instar of Bt I mean instar of non-Bt). If larval development on 

Cry34135 equals or exceeds that on non-Bt, the ratio will be 2: 1.0 (a similar output as the third 

ins tar odds ratio). The instar data were further analyzed by generating a Kolmogorov-Smirnov 

statistic (KS D) that accounts for the magnitude of difference between treatments (ranging from1 to 1, with 0 indicating no differences between distributions). Fisher's exact test was used to 

determine statistical significance. Imaging data were analyzed by calculating area 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. Further action will be taken if a population: 

• Shows similar response when exposed to both 59122 (Cry34135) and isoline at a 

significance level of p>0.05 for both methods (instar and image data); 

• Shows mean ratios (both instar and size) outside the 95% confidence interval established 

from data collected (in) previous years. 

SSA Results (2012) 

The susceptibility of each field population as measured by the 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 that larvae developed more slowly on Bt than isoline com (KS D 2:. 0.256; p < 

0.0001 ). By comparison, 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 similar pattern, as 

larvae on Bt plants were smaller than those on non-Bt. Mean body area ratios ranged from 0.224 

to 0.388 (control colony = 0.236 and 0.292), with all populations having significantly different 

distributions on Bt and non-Bt (KS D 2:. 0.466; p < 0.0001). 

Two test runs were conducted with the "Rochelle" Cry34/35-selected colony. This population 

fared better on Bt com than 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 area ratios (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, Pioneer noted that the Rochelle colony had the lowest susceptibility to 

Cry34/35 of the populations tested. 

SSA data from the 2012 season were comparable to those from field populations tested in 

previous years (see Table 4). Since none of the 2012 populations exhibited similar development 

on Bt 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 (Table reproduced from Table 1 in Appendix A, MRID# 492418-0 I) 

Instar Ratio Body Area 

Population Mean Mean Instar mean Non-Bt Area mean 

instar: Bt in star: 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 0.915 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 (2nd Run) 2.10 2.87 0.733 2.43 10.30 0.236 

Rochelle RR (111 Run) 2.88 2.97 0.972 8.26 11.59 0.712 

Rochelle RR (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) 

# lnstar (mean ratio Bt : non-Bt) Body Area (mean ratio Bt: non-Bt) 

Year Field Susceptible Selected Susceptible Pop. Field Control' Colony2 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) 0.198 

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 results from these 

populations were within the ranges of the field-collected populations. 

BPPD IRMTeam Review- SSA Procedure and Results 

Selected 

Colony2 

---

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 consistent with the field colonies tested iri previous years and, coupled with the 

general lack of field damage reports, demonstrated 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, these 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 be 

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 

is 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 not specify where and how these populations were 

collected within the counties and if they were taken from locations proximate to the 2011 field 

damage sites. The population collected from Delaware, lA had the highest instar ratio and second 

highest body area of the tested field populations (see Table 3), though the larvae on Cry34/45 

were still significantly less developed 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 regarding the potential 

sensitivity of the assay in distinguishing resistant populations. In addition to testing a susceptible 

control, Pioneer has also tested a Cry34/35-selected colony, "Rochelle-RR" (a separate colony, 

. - 10 -
"York-RR," was tested in 2010 and 2011). This colony (at the time of testing) had been selected 

on Cry34/35 for 28 of 53 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 better on Cry34/35 than the other tested populations, as evidenced by higher 

instar and body area ratios. But in only one of the 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 

it been a field-collected population (BPPD 20 13a, b). Pioneer has noted that the selected colony 

is nonetheless readily identifiable as a population with less susceptibility to Cry34/35 and 

evidence that the SSA functions as a diagnostic tool (Pioneer 2013). 

BPPD remains concerned by the potential for false negatives with the SSA, particularly among 

field-collected populations or those sampled from Cry34/35 fields with unexpected CRW 

damage·. Further testing with selected colonies is recommended to improve the interpretation of 

the assay in the context of resistance determinations. Populations that stand out as clearly less 

sensitive to Cry34/35 (as Rochelle did in the 2012 testing) should be identified for further 

investigation regardless of whether they meet the strict statistical criteria for resistance. 

EPA convened a Scientific Advisory Panel (SAP) meeting in December, 2013 to consider 

scientific uncertainties associated with CR W resistance monitoring. As part of the charge 

questions, the panel reviewed the methodology of on-plant assays (including the SSA) and 

provided recommendations to improve their use. The panel concluded that on-plant assays have 

value by measuring "actual response of the western com rootworm larvae to corn plants that 

express Bt protein(s)" (SAP 2014). Regarding the SSA, the panel noted Pioneer's progress in 

optimizing the assay by adjusting egg densities and shortening the exposure period (methodology 

changes described in BPPD 20 13a). 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 larvae. Some concerns were raised regarding possible 

decline in Bt expression over time in the plant and potential effects on the assay results (SAP 

2014). BPPD recommends that Pioneer consider the panel's comments to further develop and 

improve the SSA for future testing. 

C. Cry34/35 Field Damage Reports 

Over the course of the 2012 growing season, Dow received a "limited number" of unexpected 

damage reports from their customers related to Cry34/35 com. Pioneer reported 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 planted to Herculex Xtra (CrylF x 

Cry34/35). ELISA testing verified the damaged plants as Cry34/35-expressing, but the site visit 

(early September) occurred too late in the season to sample adult CRW. At the recommendation 

of Dow, the field was rotated to soybean the following season (2013). The site was revisited in 

2013 to collect CRW though too few adults were found to establish a population for testing. 

Dow also noted that no other reports of unexpected damage were received 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 that Dow/Pioneer provide an 

accounting of all damage reports from the 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 vicinity. BPPD believes that the use of IPM-based Best Management Practices 

(BMPs), particularly rotation to soybean, can be an effective 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 their 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 Registrations, September 30, 2010. Available 

at: http:/(www.epa.gov/pesticides/biopesticides/pipslbt-corn-terms-conditions.pdf. 

BPPD, 2011. Updated 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, 

MON 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 monitoring data, 

unexpected_damage reports for Cry3Bb1 expressing Bt Corn and academic reports ofCry3Bb1 

field failures as well as corn rootworm resistance. J. Martinez and A. Reynolds memorandum to 

A. Sibold, October 11, 2012. 

BPPD, 20 13a. Review of Corn Rootworm Resistance Monitoring Submissions and Data (2009 -

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-incorporated 

protectants. Available at: http:/ /www.epa.gov/oppfead 1/cb/csb _page/updates/20 13/cornrootworm-wp.html. 

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. 

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Nowatzki, T.M., S.A. Lefko, R.R. Binning, S.D. Thompson, T.A. Spencer, and B.D. Siegfried, 

2008. Validation of a novel resistance monitoring technique for corn rootworm (Coleoptera: 

Chrysomelida) and event DAS-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/#!documentDetail;D=EP A-HQ-OPP-20 13-0490-0025 . 

SAP, 2014. SAP Minutes No. 2014-01: A Set of Scientific Issues Being Considered by the 

Environmental Protection Agency Regarding Scientific Uncertainties Associated with Com 

Rootworm Resistance Monitoring for Bt Corn Plant Incorporated Protectants (PIPs) 

December 4-5, 2013 FIFRA Scientific Advisory Panel Meeting. Available at: 

http://www .epa.gov/scipoly/sap/meetings/20 13/december/120413minutes.pdf. 

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