UNITED STATES ENVIRONMENTAL PROTECTION AGENCY 

WASIIINGTO . D.C. 04 0 

APR 2 7 2016 

OFFICE Of' Cl IEMICAL SAFE I Y 

,\ND POLl l l I ION PREVENT lOt\ 

MEMORANDUM 

SUBJECT: 

T O: 

FROM: 

PEER 

REVIEW: 

Review of2013 and 2014 Com Rootworm Resistance Monitoring Data for 

Bt com products containing Cry34/35Abl. 

EPA Reg Nos. 68467-5, -6; 29964-4, -5, -6,-10. 

MRJD#: 492072-01, 4924 18-01, 497109-01, 497 11 0-0 1. 

PC Code: 006490. 

Decision#: 497811 , 497816. 

DP Barcodes: 424334, 425985. 

Ann Sibold, Regulatory Action Leader 

Microbial Pesticides Branch 

Biopesticides and Pollution Prevention Division (7511P) 

Alan Reynolds, Entomologist ~ (} 

Microbial Pesticides Branch V (;<------

Biopesticides and Pollution Prevention Division (7511 P) 

Jeannette Martinez, Ph.D., Ecologist/Entomologist · Q /\f}J 

Biopesticides 

Microbial Pesticides 

and Pollution 

Branch 

Prevention Division (75llP 

~ 

Action Requested 

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

the Cry34/35Ab I 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 corn rootworm 

collections made during the 2013 and 2014 growing seasons. Separate submissions were made 

for the diet bioassay (Dow and Pioneer) and Sublethal Seedling Assay (Pioneer) monitoring 

approaches. 

1 The BPPD IRM Team consists of Alan Reynolds and Jeannene Maninez 
Conclusions and Recommendations 

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

corn rootworm (CRW) populations sampled during 2013 and 2014 seasons 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. However, the 2013 SSA 

testing was confounded by methodological problems that greatly reduced CR W development 

(even in the non-Bt treatments). Pioneer appeared to have addressed these issues for the 2014 •I ; 

season. ' 

2) BPPD has previously expressed concerns with the diet bioassay approach as an effective tool 

to detect potentially resistant field populations (BPPD 2013b). In particular, diet bioassays may 

not predict 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. Despite these concerns, 

BPPD is open to improved strategies for diet bioassays if they can function reliably and 

consistently to detect resistant populations. To aid in the interpretation of these assays, BPPD 

recommends the use of a positive (Cry34/35-resistant) control in the assays. Pioneer has a 

resistant colony (Rochelle-RR) that is included with the SSA, though it is not known if this 

colony would be well-suited for artificial diet. 

3) For the SSA testing in 2014, Pioneer opted to discontinue use of body area analysis, stating in 

its report that the measure was highly correlated with the other measured variable (3rd instar 

ratios) and thus presumably redundant. BPPD has advocated for assessing two separate 

measures in CRW susceptibility bioassays to minimize the potential for false positives or 

negatives in the results (BPPD 2013b). However, in this case, BPPD agrees with Pioneer that 

the body area data do not appear to provide additional analytical value over the 3rd instar results. 

4) Consistent with a new framework for CRW insect resistance management 

(http://www.epa.gov/pesticides/new-reguirements-address-corn-rootworrn-resistance-bt-com), 

BPPD expects that the 20 14 report will be the last to include CRW populations sampled from 

random locations in the Com Belt. Future reports will focus on investigations of unexpected 

damage (UXD) fields, including sampling from these sites and susceptibility testing, as the 

primary resistance monitoring approach. 

5) Pioneer investigated 22 cases of unexpected damage (UXD) to Cry34/35 com in 2013 and 

three cases in 2014. Ofthe 2013 cases, Pioneer was able to collect and test insects from 12 UXD 

sites. The following season, Pioneer attempted to resample the remaining UXD sites and was 

able to collect and test an additional four sites. One population was collected from the 20 14 

UXD locations; the other two sites were rotated to soybean. Bioassays (diet and SSA) of the 

UXD populations revealed slightly elevated susceptibility (ECso and LCso) relative to other field 

population and controls, although the populations appeared to still be susceptible to Cry34/35. 

Mixed results were observed for two populations collected from UXD fields in Delaware 

County, lA. The first population, collected in 2014 from a 2013 UXD site, showed statistically 

- 2 -
significant growth as measured by SSA testing. The second, a 2014 UXD site, had relatively 

high ECso and LCso values, although the SSA results were comparable to the other field 

populations collected that year. It is unlikely that either population is completely resistant to 

Cry34/35, particularly when compared to Pioneer's Cry34/35-resistant "Rochelle" colony. 

Nonetheless, given the possibility of incomplete resistance and that both of these populations 

were collected from the same county BPPD recommends additional monitoring (i.e., UXD 

scouting) within Delaware County. 

Background 

Bt com products expressing the Cry34/35Ab I 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 

Acre Max). 

The Cry34/35 toxin is active against the corn rootwonn (CR W) 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 ofCRW-protected Bt 

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

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

CR W populations, and 2) investigations of unexpected CR W damage in Cry34/35 fields. Past 

monitoring efforts and data (through the 2012 growing season) with Cry34/35 have been 

reviewed and summarized by EPA (see BPPD 2010a, 2013a, 2014). 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 CR W) 

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 I ike a diagnostic tool for resistance detection (BPPD 20 13a, b; SAP 2014 ). 

In response to cases of field resistance to some Bt PIPs (Gassmann et a!. 20 11 ), EPA developed a 

new framework for CR W resistance management in early 20 16. The new framework includes 

new requirements for resistance monitoring that focus on cases of unexpected pest damage and 

standardize approaches for determining and responding to resistance. Details of the new 

framework can be found at: (http://www.epa.gov/pesticides/new-rcquirements-address-cornroot worm-resi stance-bt -corn). 

Summarv of Corn Rootworm Resistance Monitoring Submissions for Cry34/35 

Four total studies were submitted by Dow and/or Pioneer to address resistance monitoring 

activities during the 2013 and 2014 growing seasons. Submissions from Pioneer covered testing 

conducted with the Sublethal Seedling Assay, while Dow and Pioneer jointly submitted reports 

documenting assays conducted with artificial diet. The submitted reports include the following: 

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MRID# 495609-01: Monitoring Com Rootworrn Susceptibility to Event DAS-59122-7 

Using the Sublethal Seedling Assay (SSA): 2013 Collections 

(study submitted by Pioneer) 

MRJD# 495009-01: Monitoring Com Rootworm Susceptibility to Cry34/35Ab I Event 

DAS 59122-7: 2013 Growing Season (Study submitted by Dow 

and Pioneer) 

MRID# 497109-01: Monitoring Corn Rootworm Susceptibility to Cry34/35Ab I Event 

DAS 59122-7: 2014 Growing Season (Study submitted by Dow 

and Pioneer) 

MRID# 497110-01: Monitoring Susceptibility ofthe Insect Pest Diabrotica virgifera 

virgifera, LeConte to Maize Containing Event DAS-59122-7 

Using the Sublethal Seedling Assay: 2014 Collections (study 

submitted by Pioneer) 

A. Cry34/35 Diet Bioassays 

ln accordance with the approved monitoring plan for Cry34/35 (MRJD# 473342-0 I), Dow and 

Pioneer are required to annually sample CRW 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 com registrants from three defmed regions 

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

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 fourth sampling 

region (Colorado, South Dakota, and Wisconsin) was added for the 2014 testing to include 

"fringe" portions of the Com Belt. Collections included a minimum of2,000 adults to establish 

each population for susceptibility testing. 

Eggs from sampled adults are kept in diapause until testing (initiated the following March). 

Once removed from diapause, eggs are sterilized and incubated until neonate hatching. Testing is 

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

Dow AgroSciences. The Cry34/35 toxin is also provided by Dow and consists of lyophilized 

powder preparations ofCry34Ab1 (lot TSN1 02172; 54% active ingredient in 2013 and 2014) 

and Cry35Ab1 (lot TSN102171; 37% active ingredient in 2013 and 2014) mixed together in a 

3:1 ratio by weight. Serial dilutions of Cry34/35 are 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 11g Cry34/35/cm2 diet surface. 

Neonate WCRW are exposed to the serial dilutions (one larvae per well) and allowed to feed for 

five days after which mortality and (survivor) weight is recorded. Four to six replicates are run 

per population (each tray is considered a replicate); replicates are considered invalid if the 

control mortality exceeds 25%. Mortality data are used to determine a LC value for each 

population by probit analysis. Larval weight data are grouped and converted to percent growth 

inhibition (relative to the control) to determine EC values. 

-4-
2013 Sampling and Assays (MRJD# 492418-01) 

CRW sampling was conducted in late July through early September in 2013. Nine populations 

were collected from Illinois (Region 1; 1 population), Iowa (Region 2; 3 populations), Minnesota 

(Region 2; I population) and Nebraska (Region 3; 4 populations). Collections were attempted at 

an additional 18 locations (in Iowa, Illinois, and Nebraska) but were not successful. Specific 

county locations of the collections are documented in Table I below. Other populations were 

collected from fields with unexpected pest damage; these sites are documented in section C of 

this review. Two Cry34/35-susceptible, non-diapausing control populations were also included in 

the assays. These populations were obtained from Crop Characteristics Inc. (Farmington, MN) 

and French Agricultural Research Inc. (Lamberton, MN) was also included in the assays. 

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

1.61 - 3.97 flg/cm2 for the field-collected populations and 1.33 and 1.62 flg/Cm2 for the two 

unselected control colonies. 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: 4.37 -

11.70 flg/cm2) were comparable to (or lower than) previous years; however, LCso's could not be 

established for 3 of the 9 populations because the data failed to converge in prohibit analyses. 

Dow and Pioneer reported that the LCso values for the laboratory controls were I. 78 and 3.16 

flg/cm2. One population (York, NE) was noted as having non-overlapping ECso and LCso 

confidence intervals with the control colonies. However, the authors indicated that the results 

were still within the historical ranges for populations collected from this county (2007- 2012 

data submitted to EPA). 

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 2013 data (ECso and LCso ratios) was 2-3 fold, which was consistent with that from 

previous collections (2-6 fold since 2004), suggesting that natural variability could be 

responsible for the observed results rather than potential resistance. 

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

susceptibility to Cry34/35 had declined during the 2013 growing season. Field-collected 

populations responded similarly to the susceptible control colonies, and the variation between the 

least and most sensitive populations remained low. 

Table 1. Western com rootworm susceptibility to Cry34/35 using overlay diet bioassays: 2013 

season (Table reproduced from Table I in Appendix A, MRlD# 495009-01) 

Mean ECso Mean LCso 

Population Replicates (95% Cl) (95% CI) 

(~Jg/Cm ) (~Jg/cm ) 

Floyd County, lA (Charles City) 6 2.39 5.26 

( 1.62 - 3.44) (1.30 - 18.12) 

Floyd County, lA (Rudd) 6 2.19 4.37 

( 1.29 - 3.52) (2.61 - 6.41) 

Scon County, lA 6 2.89 4.93 . (2.23 - 3.69) (2.52 - 7.97) 

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Mean ECso Mean LCso 

Population Replicates (95% CI) (95%Cl) 

(Jlg/Cm2

) (Jlg/Cm2

) 

McLean County, IL 4 1.61 I 

(0.77 - 2.71) ----

Fillmore County, MN 5 2.68 I 

(1.10 - 5.51) ----

Clay County, NE 6 

3.38 6.80 

(2.66 - 4.28) (4.06- 10.14) 

Hamilton County, NE 5 2.36 5.81 

(1.69 - 3.23) (2.58- 9.43) 

Platte County, NE 6 

3.5 1 I 

(1.70 - 6.77) ----

York County, NE 5 3.97 11.70 

(3 .66 - 4.30) (5.69 - 17.67) 

Range (Field Populations) 1.61 - 3.97 4.37- 11.70 

Laboratory Control (Crop 6 

1.62 1.78 

Characteristics, Inc.) ( 1.28 - 2.0 I) (0.67 - 3.3 1) 

Laboratory Control (French Ag. 6 

1.33 3.16 

Research Services, Inc.) (0. 74 - 2.09) ( 1.66 - 4.77) 

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

2014 Sampling and Assays (MRID# 497109-01) 

Populations for the 2014 bioassays were sampled from each of three established collection 

regions from the following states: Illinois (Region 1; 3 populations), Iowa (Region 2; 3 

populations), Minnesota (Region 2; 1 population), and Nebraska (Region 3; 3 populations). In 

addition, ABSTC included a fourth region for 2014 that incorporates three " fringe" Com Belt 

states (Colorado, South Dakota, and Wisconsin). One population from South Dakota was 

collected and tested from this region. As with the 2013 monitoring, two Cry34/35-susceptible 

control populations (Crop Characteristics, Inc. and French Agricultural Research Inc.) were also 

included in the assays. Custom Bio-Products conducted the testing using the procedures 

described for the 20 13 assays above. 

Susceptibility of the collected populations is detailed in Table 2 below. WCR W populations 

collected by ABSTC had an ECso range of 1.31-5.73 J..lg/cm2 and an LCso range of2.47 - 16.85 

J..lg/cm2

. For the control colonies, the ECso of the Crop Characteristics colony was 1.01 - 1.68 

J..lg/cm2 and 7.12 J..lg/cm2 for the French Agricultural Research colony. LCso values for these 

colonies were 1.68 - 4.81 J..lg/cm2 (Crop Characteristics) and 44.72 !lg/cm2 (French Agricultural 

Research). The French Agricultural Research colony was markedly less sensitive to Cry34/35 

than in the 2013 testing (Table 2), though this differential was not addressed in the study report. 

The variation (ECso and LCso ratios of least and most sensitive populations) of the 2014 data was 

4.4 (ECso) and 6.8 (LCso) fold, which was slightly higher than in previous years. Much of the 

variation was due to one population (Clinton, IN) with lower susceptibility (ECso == 5.73 !lg/cm2

; 

LCso = 16.85 J..lg/cm2

) than the other field populations and the more sensitive control colony 

(Crop Characteristics). 

- 6 -
Similar to 2013, Dow/Pioneer concluded that the 2014 ABSTC monitoring demonstrated 

continued susceptibility to Cry34/35. In addition to the ABSTC populations, a number of 

collections were made in commercial Cry34/35 fields with unexpected damage. Bioassay results 

from these populations are described in section C of this review. 

Table 2. Western corn rootworm susceptibility to Cry34/35 using overlay diet bioassays: 2014 

season (Table reproduced from Table 2 in Appendix A, MRID# 497109-01) 

Mean ECso Mean LCso 

Population Replicates (95% Cl) (95% CI) 

()lg/cm2) {!t1icm2

) 

McLean County , IL 5 2.09 4.08 

( 1.17 - 3.40) ( 1.76 - 7.4 1) 

Shelby County, IL 6 2.72 6.39 

( 1.81 - 3.92) (3.46 - 10.52) 

Clinton County. IN 5 5.73 16.85 

(1.77 - 17.56) (no Cl reported) 

Floyd County, lA 6 1.56 2.47 

( 1.30 - 1.84) ( 1.60 - 3.50) 

Scott, lA 6 1.94 I 

(1.15 - 2.98) ----

Fillmore County, MN 6 3.6 1 I 

(3.03 - 4.26) ----

Clay County, E 6 3.44 11 .82 

(2.77 - 4.24) (7 .26 - 17. 16) 

Hamilton County, NE 6 3.49 10.47 

(2.98 - 4.06) (6.06 - 15.84) 

York County, NE 6 1.47 3.74 

(0.99 - 2.06) (2.12 - 5.78) 

Brookings County , SD 6 1.31 I 

(0.93 - 1.72) ----

Range (Field Populations) 1.31- 5.73 2.47 - 16.85 

Laboratory Control (Crop 5 1.0 I 1.68 

Characteristics, Inc.) - # I (0.8 1 - 1.22) (0.72 - 2.93) 

Laboratory Control (Crop 5 1.68 4.81 

Characteristics, Inc.) - #2 ( 1.39 - 2.00) ( 1.86 - 9.26) 

Laboratory Control (French Ag. 6 7.12 44.72 

Research Services. Inc.) (5.15 - 9.87) ( 17.71 - 75.80) 

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

Table 3. Susceptibility bioassay results 1 for field-collected CRW to Cry34/35- 2004 to 2014 

(Table generated from data provided by Dow AgroSciences and Pioneer) 

Year ECso (J..lg/cm2

) EC99 (J..lg/cm2

) LCso (J..lg/cm2

)

3 LC9o (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 

- 7-
Year ECso g/cm ) EC99 (!lg/cm2

) LCso (!lg/cm2

)

3 LC9o (~glcm ) 

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 

20 11 2 2.6 - 5.3 11.7- 39.9 

2012 0.7- 2.9 3.5- 8.4 

201 3 1.6 - 4.0 4.4 - I 1.7 

2014 1.3 - 5.7 2.5- 16.9 

1 Results are for field populations collected by ABSTC; does not include results from CRW sampled from fields 

with unexpected damage or other populations. 2 Testing in 20 II 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 

BPPD !RM Team Review - 2013 and 201-1 Diet Bioassays 

Growth inhibition (ECso and LCso) testing showed that most populations collected in 201 3 and 

2014 responded similarly to the susceptible control populations. LCso values could not be 

calculated in probit analysis for a number of populations in both years, which has also been an 

issue for past bioassays. BPPD agrees with Dow/Pioneer that the results were comparable to 

previous seasons (Table 3), though it is questionable how meaningful the diet bioassay results 

are in the context of making resistance determinations (see discussion below). 

In 20 13, three populations (Scott, Clay and York counties) had non-overlapping ECso confidence 

intervals (CI) with the controls; one population (York) also had a non-overlapping LCso CI with 

both controls. However, the magnitude of these difference was not great and the 20 13 

populations responded similarly to CRW collected from previous seasons (Table 2). Dow and 

Pioneer indicated that the results from York were similar to other CR W populations collected 

from the county in previous years. BPPD notes that York county was sampled in the two 

preceding seasons with the following results: 2011 (ECso = 3.38J..lg/cm2, CI = 2.81- 4.04; LCso 

= 14.90 11g/cm

2

, CI = 9.51- 20.96) and 2012 (ECso = 1.41J..lg/cm2

, CI = 0.98- 1.90; no LCso was 

calculated). The population was also more susceptible in the 2014 testing (ECso = 1.4 7 11g/cm2, 

CI = 0.99 - 2.06; LCso = 3.47 11g/cm2

, CI = 2.12 - 5.78). In addition, no cases of unexpected 

field damage to Cry34/3 5 corn were reported in the three counties during 2013 or 20 14. 

One of the control colonies included for the 2014 testing (French Agricultural Research) was 

much less susceptible to Cry34/35 than in 2013 (ECso = 1.33 J..lg/cm2, LCso = 3.16 11g/cm2 in 

2013 compared with ECso = 7.1 2 11g/cm2

, LCso = 44.72 ~t cm2 in 2014). This result was not 

specifically discussed in the study report; BPPD expects that difference may be due to natural 

variation in response to the toxin. When compared to the more sensitive control colony (Crop 

Characteristics), three populations (Fillmore, Clay, and Hamilton counties) had non-overlapping 

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ECso Cis. None of these populations had an overlapping LCso Cl, however, an unexpected 

damage site was investigated in Clay county during 2014 (see section C). 

Besides York County, many of the counties sampled in 2013 and 2014 had also been sampled in 

previous seasons (2009 - 20 12). The Cry34/35 susceptibility was comparable (in some cases 

lower) in these counties during 2013 than in the previous collection years (see data summari zed 

in BPPD 2013a, 2014). BPPD cautions, however, that it is unclear (and probably unlikely) that 

the same locales (i.e., CR W populations) were sampled within these counties from year-to-year. 

Therefore, these results do not necessarily track changes in susceptibility to discrete CR W 

populations over the sampling period. 

Though the populations collected for the annual (ABSTC) monitoring program were susceptible 

to Cry34/35, Pioneer reported 22 investigations of unexpected field damage in 2013. These 

cases are described in section C of this review. Dow received no unexpected damage reports 

during 2013. 

Dow/Pioneer responded to past issues of variability in the control population (see BPPD 2014) 

by including two susceptible colonies in the testing. Control populations from Crop 

Characteristics Inc. (Farmington, MN) and French Agricultural Research Inc. (Lamberton, MN) 

were included for 2013; in previous years testing was done with one or the other of those 

sources. Both of the control populations performed similarly in the 2013 bioassays. 

As described in previous reviews (BPPO 20 l3a, 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 CRW and concluded that the approach has value for 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 

proactively detect field resistance. Specifically, diet bioassays may not predict survival on corn 

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) have to date restricted their 

usefulness as a regulatory tool for resistance monitoring. One aspect that could improve the 

interpretations of diet bioassays would be the use of a positive control group (i.e., a resistant 

population). Along these lines, the new framework for CR W resistance management requires 

companies to include resistant colonies as part of resistance determination assays (additional 

details can be found at: (http://www.epa.gov/pcsticides/new-reguirements-address-comrootworrn-resistance-bt-corn). Pioneer has maintained a Cry34/35-resistant population for use in 

the Sublethal Seedling Assay (see section B), though it is unclear if this colony has been adapted 

for artificial diet. 

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 

- 9 -
20 13b ). On plant assays, which can effectively distinguish resistant populations from susceptible 

ones based on response to Bt plants (Gassmann et al. 20 11 ), offer potential as deterministic tool 

for resistance monitoring. Along these lines, Pioneer has developed an on-plant assay (Sublethal 

Seedling Assay; Nowatzki et al. 2008) for Cry34135 (see discussion in the following section). 

Despite the concerns with the current diet bioassay approach, BPPD is open to improved 

strategies for their use if they can function reliably and consistently to detect resistant 

populations. In particular, BPPD recommends the use of a positive (Cry34135-resistant) control 

in the assays to aid with interpretation of the results. Pioneer has a resistant colony (Roche II eRR) that is included with the Sublethal Seedling Assay, though it is not known if this colony 

would be well-suited for artificial diet. 

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 Cry34135 toxin and entails exposure of CRW 

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

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

their experiences with the test (see discussion in BPPD 20 13a). 

The current testing methodology for the SSA has been used since 20 11 and involves exposing 

neonate CRW to seedling mats ofBt or non-Bt corn (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). 

Each of the seedling mats in the test is infested with approximately 500 CR W eggs on the soil 

surface and held in an environmental chamber (25 °C, 65% RH, 14: I 0 light/dark). For some 

populations with low egg availability, a lower infestation rate may be used. Three to four 

replicates (each mat is considered a replicate) are established for each treatment (Bt and noncorn). A separate hatch test using a subsample of eggs in Petri dishes held in the same 

environmental chambers is conducted to determine the starting point of the test. After hatching 

is initiated, larvae are exposed for approximately 154 degree days to allow for growth and 

development. Larvae are then collected from the root mats using Burlese funnels positioned 

over containers with 70% ethanol. A sample of 30 larvae is obtained from each replicate; any 

population that does not produce at least 30 larvae in a majority of the replicates is considered 

non-viable. The sampled larvae are then measured (head capsule width) to determine the 

developmental instar (JS1

, 2"d, or 3rd). A second processing procedure is also used to assess body 

size. Samples are poured into Petri dishes, which were scanned and analyzed by imaging 

software to calculate the total body area (mm2

) of each larva. 

Both instar and body size data have been analyzed for the SSA, though Pioneer has adjusted how 

these data have been interpreted since the assay was first developed (see BPPD 20 13a, 2014 for 

discussion). For instar development, larvae are grouped into two categories, 151 + 2nd instar and 

3rd instar, so that percent achieving the 3rd instar can be determined. Bt and non-Bt treatments 

are compared to generate a third instar ratio (% 3rd instar on Bt I % 3rd instar on non-Bt). An 

odds ratio ([% 3rd instar Bt/1 - % 3rd instar Bt] I ([% 3rd instar non-Bt/1 - % 3rd instar non-Bt]) is 

- 10-
also determined. If larval development on Cry34/35 equals or exceeds that on non-Bt, both the 

3rct instar· and odds ratio will approach or exceed 1.0. Statistical analyses of the ratio data are 

conducted with a statistical significance of p ~ 0.05. Body size data have historically been 

analyzed in a similar manner; however, with the 2014 testing analysis of these data was 

discontinued. Pioneer indicated that the body size data was highly correlated with the instar data 

and therefore, presumably redundant. 

Though not addressed in the 2013 and 2014 submissions, 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 (Cry34/35) and isoline at a 

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

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

from data collected (in) previous years. 

SSA - 2013 Sampling and Testing (MR!Ds -195609-01) 

Pioneer included CRW populations from a number of sources in the 20 13 testing (listed in Table 

4 below). Nine CRW populations collected as part of the ABSTC annual sampling (see Table I 

above) were tested, though the study report indicated that two populations from McLean, IL 

were collected (instead of two from Floyd, lA as reported for the diet bioassays). In addition to 

these populations, Pioneer collected CR W from six other locations in Iowa and Illinois. The 

SSA assays also included I I populations (plus two control populations) sampled from fields with 

unexpected damage, although one of these colonies could not be tested due to limited egg 

viability. 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'} 

The susceptibility of each field population as measured by the SSA is documented in Table 4 

below. Pioneer indicated that the results did not align well with previous data in that the 3rd instar 

ratios were much lower than in previous years. A number of challenges were observed in the 

2013 testing, including irregular egg hatch and new 59122 seed lots that produced low root mass. 

For these reasons, Pioneer did not analyze the instar or body size data to calculate comparative 

rations and provided only raw summaries of the results. BPPD calculated the ratios from the 

data summaries (reported in Table 4 below), though no statistical analysis was conducted on the 

results. 

WCR W field populations collected in 2013 had a 3rd ins tar ratio range of 0.018 - 0.213, 

indicating that larvae developed more slowly on Bt than isoline com. However, development to 

3rd instar varied widely among the tested populations on non-Bt com, ranging from 23.4% to 

88.0%. The instar ratio for the two runs with the susceptible laboratory colony were 0.057 and 

0.039, though development to the 3rct instar was also low in both test runs (14. 1% and 38.6%). 

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 of field populations ranged from 0.138 to 

0.236; control colony body area ratios were 0.186 and 0.155. Despite the ambiguous data (due to 

- I I -
the problems encountered during testing), Pioneer identified one of the ABSTC-collected sites 

(Fillmore, MN: 3rd instar ratio = 0.213; body area ratio= 0.203) as a location for follow-up 

activities in subsequent years. 

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

generally fared better on Bt corn than the field or laboratory colonies, with 3rd instar ratios of 

0.210 and 0.447. In addition, development to the 3rd instar was higher on non-Bt corn (91.7% 

and 92.7%) than the control and field populations. On the other hand, Pioneer noted that 

development to the 3rd instar on Bt was lower (19.5% and 41.0%) than historically observed 

(>88.3%). Pioneer hypothesized that lower root masses produced by new 59122 seed lots could 

have been responsible for the reduced development. Similarly, the Rochelle colony had higher 

body area ratios (0.332 and 0.372) than the field populations, but lower than in previous years 

(Table 6). 

Pioneer's testing of populations collected from fields with unexpected damage is discussed in 

section C ofthis review. 

Table 4. Western com rootworm susceptibility to Cry34/35 using the Sublethal Seedling Assay: 

2013 season (Table reproduced from Table I in Appendix A, MRID# 495609-01) 

yd Insta r Ratio Body Ar ea 

Population %3rd %3rd 3'd lnstar ratio on-Bt Area mean 

instar: 81 inslar: (131 : non-131) 81 (mm2) (mm2) 

ratio 

non-Bt (81: non 81) 

ABSTC and Pioneer Collections 

Clay, NE (ABSTC) 2.2 48.4 0.045 0.99 6.50 0.152 

Fillmore, MN (ABSTC) 17.8 83.4 0.213 1.64 8.09 0.203 

Floyd, lA (ABSTC) 5.8 48.3 0.120 1.34 6.54 0.205 

Hamilton, NE (ABSTC) 1.5 23.4 0.064 0.69 3.90 0.177 

McLean, IL (Carlock- ABSTC) 4.4 43.2 0.102 1.07 6.44 0.166 

McLean, IL (Chenoa- ABSTC) 9.4 64.3 0.146 1.21 6.58 0. 184 

Platte, NE (ABSTC) 4.4 63.9 0.069 1.04 6.43 0.162 

Scott, lA (ABSTC) 6.8 31.9 0.213 0.99 4.19 0.236 

York, NE (ABSTC) 3.4 44.9 0.076 0.87 4.60 0.189 

Jefferson, lA (Pioneer) 4.8 67.3 0.071 1.26 6.24 0.202 

Hardin, lA (Pioneer) 1.6 88.0 0.018 1.21 8.78 0.138 

Marshall, lA (Pioneer) 5.5 83.7 0.066 1.32 7.49 0.176 

Ogle, IL (Pioneer) 2.4 66.7 0.036 1.1 3 6.60 0. 171 

- 12-
3rd lnsta r Ratio Body Area 

Population %3rd %3rd 3'd lnstar ratio Non-Bt Area mean 

instar: Bt instar: (Bt : non-Bt) Bt (mm2) (mm2) 

ratio 

non-llt (Bt: non Bt) 

Clayton, lA (Pioneer) 3.2 84.7 0.038 1.41 9.15 0.154 

Hamilton, lA (Pioneer) 5.5 80.4 0.068 1.34 8.40 0.160 

Range 0.018 - 0.213 0.138 - 0.236 

UXD Collections 

Lafayene, WI 3.2 16.2 0.198 1.02 4.24 0.241 

Linn, lA 3.2 56.7 0.056 1.10 6.92 0.159 

Benton, lA 18.5 97.9 0.189 2.08 10.92 0.190 

Franklin, lA (Hampton) 2.7 41.0 0.066 1.21 6.41 0.189 

Franklin, lA (Hansell) 5.6 52.6 0.106 1.22 5.71 0.214 

Jackson, lA 9.8 40.6 0.241 1.13 4.72 0.239 

Grant, SO 3.1 57.5 0.054 1.21 5.50 0.220 

Grundy, lA 7.2 40.8 0.176 1.07 5.18 0.207 

Grundy, lA -control 5.2 70.9 0.073 1.25 6.30 0.198 

Sioux, lA 9.8 51.7 0.190 1.17 4.95 0.236 

Sioux, lA -control 1.5 74.5 0.020 1.05 6.03 0.174 

Cherokee, lA 0.7 84.9 0.001 1.10 5.89 0.187 

Range 0.00 I - 0.24 1 0. 159 - 0.24 1 

Control Groups 

Laboratory Control ( f$' Run) 0.8 14. 1 0.057 0.56 3.01 0.186 

Laborato1y Control (2"J Run) 1.5 38.6 0.039 0.65 4.20 0.155 

Rochelle RR (!-''Run) 19.5 92.7 0.210 2.50 7.52 0.332 

Rochelle RR (2"4 Run) 41.0 91.7 0.447 2.98 8.02 0.372 

SSA- 2014 Sampling and Testing (MR!Ds -1971 10-01) 

As in the 2013 monitoring, Pioneer tested CR W populations collected by ABSTC and from 

fields with unexpected damage (Table 5 below). Nine of the ten ABSTC populations (Table 2 

above) tested in the diet bioassays were al so tested with SSA. An additional five populations 

- 13 -
collected from Bt fields with unexpected damage were included (reviewed in Section C below). 

Control groups included two Cry34/35 susceptible populations: the Brookings USDA-ARS 

reference colony and a Pioneer-supplied population designated "CRW SS" (a cross between the 

Brookings strain and field populations). The "Rochelle-RR" population resistant to Cry34/35 

was used as a positive control. 

Results from the 2014 SSA testing are detailed in Table 5 below. Pioneer opted to discontinue 

with the body area measurements with the 2014 testing, indicating that they were highly 

correlated with the instar data. Therefore, only the instar measurements and analyses were 

reported. As in 2013, Pioneer used a third instar ratio (i.e., percent of individuals reaching the 

third instar) instead of the mean instar ratio that was previously used. For statistical analysis, 

Pioneer also calculated an odds ratio - i.e., a quantification of the association between the 

probability of reaching the third instar and exposure to Bt corn. 

Third instar ratios of the field populations ranged from 0.059 to 0.493, similar to the values 

observed in 20 13 (3 rd in star ratio also measured) but lower than the ranges observed in 2009-

20 12 when a mean instar approach was used (Table 6). The three susceptible control groups had 

ratios of 0.226 (Brookings), 0.348 (Brookings), and 0.436 ("CRW SS"). Analysis of odds ratios 

showed that none of these populations had statistically significant development on Bt corn 

relative to non-Bt com. On the other hand, the Rochelle-RR colony had a statistically significant 

ratio of 1.119, indicating that the colony's development on Bt com exceeded that on non-Bt corn. 

Table 5. Western com rootworm susceptibi li ty to Cry34/35 using the Sublethal Seedling Assay: 

2014 season (Table reproduced from Table I in Appendix A, MRJD# 497110-01 ) 

3rd lnsta r Ratio 

Population %3rd % 3rd 3n1 lnstar ratio 

instar: Bt in star: (Bt : non-Bt) Odds ratio' 

non-Bt 

ABSTC Collections 

Shelby, NE 23.0 57.6 0.399 0.220 

Clinton, IN 28.9 58.7 0.493 0.286 

Floyd, lA 14.7 44.9 0.327 0.211 

Scott, lA 12.2 42.4 0.287 0.189 

Clay, NE 11.3 64.4 0. 176 0.071 

Hamilton. NE 12.2 65.3 0.186 0.074 

Howard, NE 4.0 67.8 0.059 0.020 

York, NE 7.3 70.1 0. 105 0.034 

Fillmore, MN 13.0 40.7 0.318 0.2 17 

Range 0.059 - 0.493 

- 14-
Jrd Jnstar Ratio 

Population %3rd %3rd J'd lnstar ratio iostar: Odds ratio1 

instar: Bt 

non-Bt (Ot : non-Bt) 

UXD Collecrions 

Delaware, lA (# I) 20.5 50.8 0.403 0.249 

Delaware, lA (#2) 33.0 44. 1 0.749 0.625* 

Fayette, lA 13.7 42.4 0.324 0.216 

Hamilton, lA 28.1 57.6 0.487 0.287 

Boone, NE 20.4 60.2 0.340 0. 170 

Range 0.324 - 0.749 

Comrol Groups 

Brookings Conrrol ( !" Run) 10.5 46.6 0.226 0.135 

Brookings Control (2"'1 Run) 18.0 5 1.7 0.348 0.205 

CRW SS Lab 25.5 58.5 0.436 0.243 

Rochelle RR 92.7 82.9 1.119 2.639* 

1 Values destgnated wtth a·'*" are stattsttcally stgo tficant at p :S 0.05. 

Table 6. Sublethal Seedling Assay Results for Western Corn Rootworm, 2009 - 2014 (Table 

created from data submitted by Pioneer) 

# lnsta r (mean ratio Bt : non-Bt)1 Body Area (mea n ratio Bt : non-Bt) 

Year Field Susceptible Selected Susceptible Selected Pop. Fie ld Control2 Colonyl Field Control 1 Colony2 

2009 15 0.766 - 0.996 0.781 --- 0.393 - 0.894 0.508 ---

2010 7 0.734-0.960 0.787 0.9 15 0.387- 0.511 0.5 12 0.692 

20 11 I I 0.687 - 0.883 1)0.727 0.986 0.2 14- 0.421 I) 0. 198 I) 0.624 

2) 0.830 2) 0.274 2) 0.633 

20 12 8 0.710 - 0.883 I) 0.902 I) 0.972 0.224 - 0.388 I) 0.292 I) 0.71 2 

2) 0.733 2) 0.994 2) 0.236 2) 0.764 

2013 15 0.0 18 - 0.2 13 I) 0.057 I} 0.210 0.138 - 0.236 I) 0.186 I) 0.332 

2) 0.039 2) 0.447 2) 0.155 2) 0.372 

I) 0.226 

20 14 9 0.059 - 0.493 2) 0.348 1.119 NT NT NT 

3) 0.436 

- 15-
1 Mean instar ratio was used 2009-20 II , third instar ratio was used 20 13-20 14. For e ither ratio, values approaching 

or exceeding I indicate little developmental effects due to Bt exposure. 2 Cry34/35-susceptibile control colony obtained from USDA-ARS (Brookings, SO). 

3 York-RR (20 I 0-20 II) and Rochelle-RR (20 12) have been selected on 59 122 (Cry34/35) corn. 

BPPD IRM Team Review - 2013 and 2014 SSA Testing 

BPPD agrees with Pioneer that the 2013 instar ratio results were aberrational compared with 

previous testing and were likely confounded by methodological issues in the testing. All test 

groups showed less development on both Bt and non-Bt treatments than in prior years, including 

the Cry34/35 selected colony. Body area data from 2013 were somewhat more consistent with 

the historical data than the instar ratios, but larvae still showed less development than would 

have been expected. 

Pioneer's 2013 report indicated that irregular egg hatch and new 59122 seed lots that produced 

low root mass may have affected the tests, resulting in reduced larval development. The 2014 

report did not indicate if these issues were resolved or were still a factor in the 2014 testing. The 

data from 2014 generally followed the pattern of the 2013 testing, although the instar ratios for 

field and control populations were somewhat higher and the selected colony had robust 

development on both Bt and non-Bt com. 

The 2013 and 2014 instar ratio results may also partially be explained by a change in how the 

test data were analyzed. Prior to 2013, Pioneer calculated a mean instar ratio in which the 

average instar development of the population is calculated for Bt and non-Bt com treatments. In 

2013 and 2014, Pioneer moved to a 3rd instar ratio by detennining the percentage of each 

population that reaches the 3rd instar on Bt and non-Bt treatments during the test period. This 

new approach could have the effect of creating more "separation" between a susceptible 

population (with low 3rd instar ratios) and a potentially resistant one (with a 3rd instar ratio 

approaching or exceeding l.O). In the 2009 - 2012 assays (Table 6), susceptible populations 

(field or control) often had mean instar ratios exceeding 0.9, not much lower than the positive 

control (resistant) colony. By contrast, in 2014 the field and susceptible control population ratios 

were< 0.5, compared to 1.11 9 for the resistant colony. 

Pioneer opted to discontinue use of body area analysis for 2014, stating in its report that the 

measure was highly correlated with the instar ratios and thus presumably redundant. BPPD has 

advocated for assessing two separate measures in CRW susceptibility bioassays to minimize the 

potential for false positives or negatives in the results (BPPD 201 3b). However, in this case, 

BPPD agrees with Pioneer that the body area data do not appear to provide additional analytical 

value over the 3rd instar results. In general, the body area mass analysis produced lower ratios 

than with the instar approach, even with the Cry34/35 selected colony (Table 6). None of the test 

runs with selected colony had a body area ratio approaching 1.0, the putative indication of 

resistance. Without the second analytical measure, it will be important to continue use of the 

selected colony as a positive control and additional point of comparison for the field populations. 

BPPD also presumes that Pioneer's resistance determination criteria (summarized above) will be 

updated to reflect that only one measure (3rd instar ratio) will be assessed for a resistance 

confirmation. 

- 16 -
A number of populations collected from damaged Cry34/35 were tested in 2013 and 2014 using 

the SSA. These populations are discussed in the next section. 

C. Cry34/35 Field Damage Reports 

2013 Reports 

During the 20 13 growing season, Pioneer reported a total of 22 unexpected damage (UXD) 

reports from their customers related to Cry34/35 corn. These fields were investigated using the 

protocols described in Pioneer's monitoring program (MRID# 480455-0 1). Pioneer was able to 

collect CR W adults from 16 of the fields, with 12 populations producing enough eggs for testing 

(see list in Table 7 below). Most ofthe UXD populations were from Iowa, with several others 

from Nebraska, South Dakota, and Wisconsin. Dow reported no cases of UXD among its 

customers during 2013. 

Populations collected from UXD fields were tested by diet bioassays and the Sublethal Seedling 

Assay (three populations were tested with diet assays only). Results for the diet assays are 

summarized in Table 7 below; SSA results for these populations are detailed in Table 4. In the 

diet assays, the ECso range of the UXD populations was 2.47 - 6.45 11g/cm2, compared to a range 

of 1.61 - 3.97 ~tg cm2 for the field populations collected by ABSTC. The ECsos of the two 

control populations were 1.33 and 1.62 11g/cm2. LCso values for the UXD populations were 5.56 

- 12.16 11g/cm2 (ABSTC population LCso = 4.3 7 - 11.70 11g/cm2

; control population LCsos = 

1.78 and 3.16 11g/cm2

) . The SSA results (instar and body area ratios) for the UXD populations 

were comparable to those from the ABSTC and control popul ations, though methodological 

issues likely confounded the 20 13 SSA testing (see discussion in section B of this review). 

Table 7. Cry34/35 Susceptibility of CR W populations collected from fields with unexpected 

damage: 2013 season (Table reproduced from Table 1 in Appendix B, MRID# 495009-01) 

Mean ECso Mean LCso 

Population Replicates (95% Cl) (95% CI) 

(11g/crn2) (11g/cm2) 

Benton County, lA 5 4.53 7.36 

(2.16 - 9.33) (3. 75 - 11.63) 

Cedar County, lA .., 2.81 9.73 .) 

( 1.82 - 4.27) (4.52 - 16.43) 

Cherokee County, lA 6 5.16 10.93 

(3.44 - 7.67) (7.70 - 14.27) 

Franklin County, lA (# I) 4 3.27 6.62 

(2.67 - 4.0 I) (3.53 - 10.08) 

Franklin County, lA (#2) 6 3.46 8.00 

( 1.95 - 6.03) (5.27 - 10.36) 

Jackson County, lA 3 3.46 8.38 

(2.41 - 4.90) (2.6 1 - 17.56) 

Grundy County, lA 5 4.49 10.92 

(3.1 0 - 6.43) (5.54 - 15.66) 

Grundy County, lA (control) 6 5.40 12.16 

(4.05 - 7.17) (8.40 - 16.46) 

- 17-
MeanECso Mean LCso 

Population Replicates (95% CI) (95% Cl) 

(~t g/cm ) (~g/cm ) 

Linn County , lA 6 

6.45 9.25 

(2.86 - 14.35) (5.69 - 13.23) 

Muscatine County, lA 3 

4.57 10.94 

(3.08 - 6.71) (5.25 - 17 .35) 

Sioux County, lA 4 3.20 8.07 

( 1.0 I - 8.97) (3.00 - 12.42) 

Sioux County, lA (control) 6 

3.29 5.81 

( 1.93 - 5.44) (2.94 - 8.57) 

Grant County, SD 6 

3.49 5.56 

(2.27 - 5.29) (3.24 - 8.15) 

Lafayette County, WI 6 

2.47 I 

(1.42 - 4.13) ............ 

Range (UXD Populations) 2.47 - 6.45 5.56 - 12.16 

Laborat01y Control (Crop 6 

1.62 1.78 

Characteristics, Inc.) (1.28 - 2.01 ) (0.67 - 3.3 1) 

Laborato1y Control (French A g. 6 

1.33 3.16 

Research Services, Inc.) (0.74 - 2.09) (1.66 - 4.77) 

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

2014 Reports 

Pioneer investigated three "AcreMax" fields with UXD in 2014 in accordance with established 

protocols (MRlD# 480455-01). CRW sampling was successful at one ofthe sites (Delaware 

County, lA # 1); the two other sites were rotated to soybean in 2015 and additional collections 

will not be attempted. Pioneer also followed up with 12 UXD sites from the 2013 season that 

could not be sampled that year. Six populations were collected from these 2013 sites, four of 

which produced sufficient progeny for testing. The populations tested are detailed in Table 8 

below. As in 20 13, Dow reported no cases ofUXD among their customers during 2014. 

UXD populations were tested with diet bioassays (Table 8) and the Sublethal Seedling Assay 

(Table 5). The UXD population collected in 20 14 (Delaware County, IA # 1) showed an e levated 

LCso (114.48 f.lg/cm2

) relative to the other field populations (ABSTC collections; LCso range= 

2.47- 16.85 f.lg/cm2) and control populations (LCso = 1.68 - 44.72 f.lg/cm2

) from that season. 

The ECso (7.05 f.lg/cm2

) of this population was also elevated relative to ABSTC populations 

(1.31- 5.73 f.lg/cm2

) and the Crop Characteristics control colony (ECso = 1.01 and 1.68 f.lg/cm2), 

but was comparable to the second control colony (French A g.; ECso = 7.12 f.lg/cm2

). On the 

other hand, SSA testing of this colony revealed 3rd instar odds ratio (0.249) that was similar to 

the other field and control populations. 

A second population from Delaware County. lA (#2) that was taken from a 2013 UXD site was 

also tested in 20 14. The ECso (5.78 f.lg/cm2

) and LCso (32.5 1 f.lg/cm2

) of this population were 

somewhat higher than the ABSTC populations and the Crop Characteristic control colonies, but 

were less than the French Ag. colony. SSA testing of this population, however, resulted in a 3rd 

instar odds ratio (0.625) that was statistically relevant and greater than any of the ABSTC or 

- 18 -
control populations. Pioneer's report concluded the biological relevance of the population was 

unclear but noted that the result could "be indicative of a shift in susceptibility for this 

population." 

For the other tested UXD populations, ECso and SSA testing results were comparable to the 

control and field populations. The LCso results for these populations were elevated, though 

similar to the French Ag control colony. But as noted in section A of this review, the French Ag 

colony was generally less susceptible to Cry34/35 than the other control colony and many of the 

field collected populations. 

Table 8. Cry34/35 Susceptibility of CR W populations collected from fields with unexpected 

damage: 2014 season (Table reproduced from Table 2 in Appendix B, MRID# 497109-01) 

Mean ECso Mean LCso 

Population Replicates (95% Cl) (95% Cl) 

{~Jg/cm ) (~Jg/cm ) 

Delaware County, lA (# I) 6 7.05 114.48 

(3.37 - 14.72) (55.08 - I 0 18.00) 

Delaware County, lA {#2) 1 2 5.78 32.51 

(2.72 - 11.83) (14.87 - 55.69) 

Fayene County, IA1 5 4.34 24.80 

(3.43 - 5.45) (10.90 - 48.14) 

Hamilton County, lA 1 6 5.02 39.64 

(3.3 1- 7.51) (16.89 - 109.96) 

Boone County, NE1 6 4.13 24.31 

(3.17-5.33) (14.5 1 - 40.19) 

Range (UXD Populations) 4.13-7.05 24.31 - 114.48 

Laborato1y Control (Crop 5 1.01 1.68 

Characteristics, Inc.) (0.81 - 1.22) (0.72 - 2.93) 

LaboratO!J' Control (Crop 5 1.68 4.81 

Characteristics. Inc.) ( 1.39 - 2.00) ( 1.86 - 9.26) 

Laborato1y Control (French Ag. 6 7.12 44.72 

Research Services. Inc.) (5.15 - 9.87) ( 17.71 - 75.80) 

1 2013 UXD site that was resampled and tested during 2014. 

BPPD IRM Team Review - UXD Reports 

In general, the UXD populations tested had slightly elevated susceptibility (ECso and LCso) 

relative to other field population and controls (Tables 7 and 8), but still appeared to be 

susceptible to Cry34/35. However, mixed results were observed for two populations collected 

from UXD fields in Delaware County, lA. The first population, collected in 2014 from a 2013 

UXD site, showed statistically significant growth as measured by SSA testing (Table 5). The 

second, a 2014 UXD site, had relatively high ECso and LCso values, although the SSA results 

were comparable to the other field populations collected that year. lt is unlikely that either 

population is completely resistant to Cry34/35, particularly when compared to the selected 

resistant "Rochelle" colony (SSA odds ratio of"Rochelle" was 1.11 9 compared to 0.625 for the 

Delaware population). Nonetheless, given the possibility of incomplete resistance and that both 

- 19-
of these populations were collected from the same county, BPPD recommends additional 

monitoring (UXD scouting) within Delaware County. 

BPPD is encouraged that two ofthe three UXD sites in 2014 were rotated to soybean in 20 15. 

BPPD believes that the use of JPM-based Best Management Practices (BMPs), particularly 

rotation to soybean, can be an effective means to mitigate (or extirpate) a potentially resistant 

population (BPPD 2013b). 

References 

BPPD, 2010a. Review ofDow's 2008 com 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 Cry34Ab1 and Cry35Abl Proteins and the Genetic 

Material Necessary for their Production in Event DAS-59 122-7 Com Biopesticide Registration 

Action Document (BRAD). Revised September, 2010. Available at 

http://www.epa.gov/oppbppd I /biopesticides/ingredients/tech docs/brad 006490.pdf. 

BPPD, 201 Oc. Terms and Conditions for Bt Com Registrations, September 30, 20 I 0. Available 

at: http://www.epa.gov/pesticides/biopesticides/pips/bt-com-tenns-conditions.pdf. 

BPPD, 20 11. Updated BPPD lRM Review of Reports ofUnexpected Cry3Bbl Damage, 

Monsanto's 2009 Com Rootworm Monitoring Report. and Revised Com Rootworm Resistance 

Monitoring Plan for MON 88017, MON 88017 x MON 810, MON 863, MON 863 x MON81 0, 

MON 89034 x TC1507 x MON 88017 x DAS-59122-7, and MON 89034 x MON 88017. J. 

Martinez memorandum to M. Mendelsohn, November 22, 2011 . 

BPPD,'2012. BPPD IRM Team review of Monsanto's 2010 com rootworm monitoring data, 

unexpected damage reports for Cry3Bbl expressing Bt Corn and academic reports ofCry3Bb1 

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

A. Sibold, October 11 , 2012. 

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

2011) for Bt corn products containing Cry34/35Ab I . A. Reynolds memorandum to A. Sibold, 

March 20, 2013. 

BPPD, 2013b. White paper on com rootworm resistance monitoring for Bt plant-incorporated 

protectants. A vai Jable at: http://www.cpa.gov/oppfcad I /cb/csb pagc/updatcs/20 13/comroot worm-wp.html. 

BPPD, 2014. Review of2013 Corn Rootworm Resistance Monitoring Data for Bt com products 

containing Cry34/35Abl. A. Reynolds memorandum to A. Sibold, December 8, 20 14. 

- 20-
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. ?LoS ONE. 6(7): 1-7. 

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: 

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