UN I TED STATES ENV IR ONMENTAL PROTECTION AGENCY W AS III N GTO . D .C .

**04 0** 



## APR 2 7 2016 OFF I CE Of' Cl I E MI CAL SAFE I Y
, \ND POLl l l I ION PR EVENT l Ot\

ME MORAND UM SU BJ E C T :

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EE R RE V IEW: Review of2013 and 2014 Com Rootworm Resistance Monitoring Data for Bt com products containing Cry34/ 35Abl. EPA R eg Nos. 68467-5 , -6; 29964-4 , -5 , - 6,-10. MRJ D# : 492072
- 01 , 4924 1 8
- 0 1 , 497 109-0 1 , 497 11 0-0 1. PC Code: 006490. Decision # : 497811 , 497816. DP Barcodes: 424334 , 425985. Ann S ibo ld , Regulatory Action Leader Microbial Pesticides Branch B iopesticides and Pollution Prevention Division (7511P) Alan Reyno lds, Entomo logist



# ~ (} Microbial Pesticides Branch V 

# (;<------ Biopesticides and Pollution Prevention Division ( 7511 P) J eannette Martinez , Ph.D. , Ecologist/Entomologist



# · Q /\f}J Microbial Pesticides B ranch



## ~ Biopesticides and Pollution Prevention Division (75ll P A cti o n Reque s ted 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 HiB red to address te r ms of reg istr atio n fo r B t com produc ts that express Cry34/35, i nclu di ng the Herculex Rootworm and Optimum AcreMax product lines. The data cover corn rootworm collections made during the 2013 and 2014 growing sea s ons. Separate submissions were made for the diet bioassay ( D ow and Pioneer) and Sublethal Seedling Assay (Pioneer) monitoring approac hes.

1 Th e BPPD IRM Te am consi sts o f Alan Rey nold s and Jeann en e M aninez

### Conclusions and Recommendations 1) Results from diet bioassays and Sublethal Seedling Assays (SSA) generally s howed that the corn rootworm (CRW) populations sampled duri ng 2013 and 2014 seasons were susceptible to the Cry34/ 35 toxin. The responses of these populations were simi lar to the susceptible control colony and were con s iste nt with the results obtained in previous years. However, the 2013 SSA testing was confounded by methodological problems that greatly reduced CRW development (even in the non-Bt treatments). Pioneer appeared to have addressed these issues for the 2014 •I ; season. ' 2) BPPD has previously exp re ssed concern s with the diet bioassay approach as an effective tool to detect pote ntially resi s tant field populations (BPPD 20 1 3b). In particular, diet bioassays may not predict survival on com plants, LC measures have been highly variable , EC va lue s may not be sens itive enough to identify early resistance , a " dia gnostic " concentration capab le of killing all susceptibl e and heterozygous resistant genotypes has not been developed , and the assays may not be able to distinguish res i stant populations from susceptible ones. Despite these concerns, BPPD i s ope n to improved strategies fo r diet bioassays if they can function reliably and consistently to detect resistant populations. To aid in th e interp retation of these assays, BPPD recommends the use of a positive (C ry34/ 35-resis tant) cont ro l in the assays. Pioneer has a re s is tant colony (Rochelle-RR) that is included with the SSA , though it is not known if thi s colony would be we ll-suited for artificial diet. 3) For the SSA testing in 2014 , Pioneer opted to discontinue use of body area analysis , stat in g in its r e port that the measure was highly correlated with the other meas ured va riabl e (3rd instar ratios) and thus presumably redundant. BPPD has advocated for assessing two separate m eas ures in CRW s usceptibility bioa ssays to minimize the potential for false positives or negatives in the results (BPPD 2013b). Ho weve r , in this case , BPPD agrees w ith Pioneer that th e body area data do not appear to provide additional analytical value over the 3rd instar results. 4) Co n s i s tent with a new framework fo r CRW in sect res istance m anag e ment (http: //www . epa .gov /pesticides/ new-reguirements-address-corn-rootworrn-resistance
- bt
- com) , BPPD expects that the 20 14 report will be the la st to include CRW populations sampled from random locations in the Com Belt. Future reports will focus o n investigations of unexpected damage (UXD) fi e ld s , includin g samp lin g from th ese sites and susceptibility testing , as the primary resistance monitoring approach. 5) Pion eer investigated 22 cases of unexpected damage (UX D) 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 s ites. Th e foll owing seaso n , Pioneer attempted to resample the remai nin g UXD sites and was abl e to co llect and t es t an addit ional four s ites . 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 reveale d s li ghtly elevated susceptibility (ECso and LCso) relative to other field population and controls , although the populations appeared to still be susceptib le to Cry34 / 35. Mixed r es ults were observed for two population s collected from UX D fields in Delaw are County , lA. The first population , collected in 20 14 from a 2013 UXD site , showed stat isticall y



### - 2
-significant growth as measured by SSA testing. The seco nd, a 2014 UX D site , had relativel y high ECso and LC so values , a lthough the SSA results were comparable to the other field populations collected that year. It is unlikely that ei th er population is completely resistant t o Cry34/ 35, particu larly when compared to Pi o neer's C ry 34/35- resi s tant " Roch e ll e" colony . Nonethe less , given the pos s ibility of incomplete resistanc e and that both of thes e population s were collected from the same co unty BPPD recommend s additional monitoring (i.e., UXD scout in g) within Delaware Co unty. 

### B a ckground Bt com product s ex pressing the Cry34/ 35Ab I toxin were first regi s tered in 2005 by Dow Ag roSciences and Pioneer Hi-Bred (" He rcul ex Rootworm·' product lin es) . Subsequent to th e initial registrat ion, Cry34/ 35 has been registered in a number of other Bt corn plant-incorporate d protectants (P IP s), including stacked varieties (e.g. , SmartStax) and see d blend s (e.g. , Optimum AcreMax). The Cry34/ 35 toxin is active against the corn rootwonn (C R 

### W) complex , including western corn rootworm (WCRW , 

### Diabroti ca v irgifera virgifera) and northern corn rootworm (NCRW, 

### Diabrotica barb e ri ) . As part of the term s of reg istrati on, a ll regi stran ts ofCRW-protected Bt corn products ( including Cry34/ 35) mu st monitor CRW populations fo r potential resistance. The monitoring e fforts ha ve included two component s: I) annual sam pling and bioassays of CRW popu l ations , and 2) inve s tigations of unexpected CR W damage in Cry34/ 35 fields . P ast monitoring efforts and data (through the 2012 growing seaso n) with Cry34/ 35 have been reviewed and s ummarized by E PA (see BPPD 20 1 0a , 2013a, 2014). As documented in those re v iews, CRW s u sc eptibilit y ha s been assessed using diet-based bioa ss ays but has been complicated by r e lati ve ly low s usceptibility to Cry34/ 35 (the toxin is not " high dose " for CR W) and lack of a functional diagno stic assay to detect resi s tance. S ince 2009, P ioneer has been e mpl oy ing the S ublethal Seedling Assay (Nowatzki et a l. 200 8) as a second bioassay approach fo r r es istance detection . Thi s assay incorporates on-plant exposure (to see dlin g mats) and can function more I ike a diagnostic tool for r es is tance detection ( BPPD 20 13a , b ; SAP 2014). In re s ponse to cases of field re s is tance to so me Bt PIP s (Gass mann e t a!. 20 1 1 ) , E PA developed a new framework for CR W re sis tanc e man ageme nt in early 20 16. T he new fram ework include s new requirement s for re s i s tanc e monitorin g that focus o n cas es of unexpected pest damage and sta ndardize approaches for determining and responding to resistance . Deta il s of the new framework can be found at: ( http ://www.epa . gov/p es ti c ides/ new- rcq uire ment s - address -c o rn rootwo rm-res is ta nce - bt - co rn ). 

### S umm a rv of Co rn Ro otw o rm R es is t a n ce Mo nitoring S ubmi ss i on s f or C ry 3 4/35 Four total studies were submitted by Dow and / or P ion ee r to address re s istance monitoring acti v iti es durin g the 20 13 and 20 14 growing seaso n s . Sub mission s f rom Pione er covered testing conducted with the S ubl et hal See dling A ssay, while Dow and Pioneerjointly s ubmitted report s documenting assays conducted w ith artificial diet. The s ubmitted reports include the following:

- 3
-MRID # 495609-01: Monitoring Com Rootworrn Susceptibility to Eve nt DAS-59122-7 Using the Sublethal Seedling Assay (SSA): 2013 Collections (study submitt ed by Pioneer) MRJD# 495009-01: Monitoring Com Rootworm Susceptibility to Cry34 / 35Ab I Event DAS 59122-7: 2013 Growing Season (Study subm itted 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 v ir gifera , Le Conte to Maize Containing Eve nt DAS- 59122-7 Us ing the Sublethal Seedli ng Assay: 2014 Collections (study subm itted by Pioneer) A. Cry3 4 /35 Diet Bioa ssays ln accordance with the approved monitoring p lan 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 Sub lethal Seedling Assay) for changes in susceptibility to the toxin. Sampling has been coordinated by ABSTC for all Bt com registrants from three defmed reg ion s cover in g different WCR W variants. The re gio ns also represent areas of high Bt corn adoption and C RW pressure. Re gion 1 (soybean rotation r esis tant variety) includes Indiana and eastern Illinoi s; Re gio n 2 (wi ld type) consists ofwestem Illinoi s , Iowa and Missouri ; Region 3 (o rganopho sphate resistant variant) enco mpasses Nebraska and Kansa s . A fourth sa mpling region (Co l orado , South Dakota , and Wisconsin) was added for the 201 4 testing to include "fri nge" portions of the Com Belt. Collections included a minimum of2,000 adu lts to establ ish eac h population for sus ceptib ilit y 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. Testi n g is conducted by Custom Bio-Products (Maxwe ll , IA ) using 96 we ll artificial diet trays s upplied by Dow Ag roScienc es. The Cry34/ 35 toxin i s 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 20 1 3 and 2014) mixed together in a 3: 1 ratio by weight. Serial di lutions of Cry34/35 are prepared and overlaid on the diet tray s to achieve the follow ing toxin le ve ls: 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 dilution s (one larvae per well) and allowed to feed for five days after wh ich mortality and (survivor) weight is recorded. Four to six replicate s are run per population (eac h 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 (relativ e to the contro l) to determine EC values.

-4-

### 2013 Sampling andAssays (M RJD# 492418-01) CRW samp ling was conducted in late July through early September in 2013. Nine populations were collected from Illinoi s (Region 1 ; 1 population) , Iowa (Region 2; 3 populations) , Minnesota (Region 2 ; I population) and Nebraska (Region 3; 4 populations). Collection s 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 fro m fie lds with un expected pest damage; these s it es a re documented in section C of this review. Two Cry34/ 35-suscept ible , non-diapausing control populations were a lso included in the assays. These populations were obtained from Crop Characteristics Inc. (Farm ington , MN) and French Agricultural Research Inc. (Lamberton , MN ) was also included in th e assays. Results fro m the bioassays are detailed in Table I below . ECso suscep tibility to Cry34/35 was 1.61
- 3.97 flg/cm 2 for the field-co ll ected populations and 1.33 and 1.62 fl g/C m 2 for the two unselected control colonies. These results were reported to be within the historical range of Cry34/ 35 EC so value s observed for WCRW (Table 2). Similarl y, the LCs o results (ran ge: 4.37
- 11.70 flg/ cm 2) were comparable to (or lower than) previous years; however , LC so' s could not be established for 3 of t he 9 populations because the data failed to converge in prohibit analyses. Dow and Pioneer report ed th at th e LCso values for the laborato ry controls were I . 78 and 3.16 fl g/cm 2

. One population (Yo rk , NE) was noted as havin g non-overlapping ECs o and LCso confidence intervals with the contro l colonie s . H owever, the authors indicated that the results were still with in the historical ranges for populations collected from this county (2007- 2012 data submitted to EPA) . To further a nal yze the data , the reg istrant s compared the variation between the least and most sensitive populations (us in g the technique of S iegfried et al. 2007). Higher variation could be an indicator of lo cal selection pressure causing decreased susceptib ilit y to Cry34/ 35. The variation of the 2013 data (ECs o and LC so ratios) was 2-3 fold , which was consistent w ith 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 susceptib ili ty to Cry34/ 35 had declined during the 2013 g rowing season. Fi eld-collected populations responded similarly to the susceptib le control colon ies , and the variation between the least and most sensitive populations remained low. Tab l e 1. Western com rootworm susceptibility to Cry34/ 35 u s i ng overlay d iet bioassays: 2013 season (Tab le reproduced from Table I in Appendix A , MRlD# 495009-01) Mea n EC so Mea n L Cso Popu lation Rep li cates ( 95 % C l) (95 % CI)

**(~J g/**  **Cm )**  **(~Jg/cm**

**)** Floyd Co unty , l A (Charl es 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)



### - 5
-Mea n ECso Mean LCso Pop ul a ti on Rep li cates (95% C I) (95%Cl) (Jlg/Cm2 ) (Jlg/Cm2 ) Mc Lean 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, In c.) ( 1.28
- 2.0 I) (0.67
- 3.3 1) Laborat ory Control (Fr en ch Ag. 6 1.33 3.16 R esearch Services, In c.) (0. 74
- 2.09) ( 1.66
- 4.77)
1 LC 50 s could not be calculated for these populations because data failed to converge in probit analy s is. 

### 2014 Sampling andAssays (M RID# 49 7 109-01) Populations for the 2014 bioas says were sampled from each of thre e established collection regio ns 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 inc luded a fourth region for 2014 that incorporates three " fringe" Com B e lt states (Colo rado , South Dakota , and Wisconsin). One population from South Dako ta was collected and tested from this region. As with the 2013 monitoring , two Cry34/35-susceptible co ntrol populations (Crop Characteristics , Inc. and French Agricultural Research Inc. ) we re also included in the assays. Custom Bio-Products conducted the t est in g using the proc edure s described for th e 20 13 assays above. Susceptibi lity of the collected populations is deta i led in Table 2 below. WCR W populations co ll ected by ABSTC had an ECso range of 1. 31-5.73 J..lg/cm 2 and an LCs o range of2.47
- 16.85 J..lg / cm2

. For th e control colonies, the ECso of the Crop Characteristics colony was 1.01
- 1.68 J..lg/ cm 2 and 7 . 12 J..lg/cm 2 for the French Agricultural Research colony. LC so values for these coloni es we re 1.68
- 4.81 J..lg/cm 2 (C rop Characteristics) and 44.72 !lg/cm2 (Frenc h Agricultural Research). The French Agricu ltural Research colony was markedly less sensitive to Cry34 / 35 than in the 2013 testing (Tab le 2) , though this differential was not addressed in the stu d y report. The variation (ECso and LCso ratios of least and most sensitive population s) of the 2014 data was 4.4 (ECso) and 6.8 (LCso) fold , which was s li ghtly higher than in previous years. Much of the variation was due to one population (Clinton, IN) with lower s usceptibility (ECso == 5.73 !lg / cm 2 ; LCso = 16.85 J..lg/cm2 ) than the other field populations and the more sensitive control colony (Crop Characteri stics ).



### - 6
-Similar to 20 1 3, Dow/ Pi o nee r concluded that the 2014 ABSTC monitoring d e monstrated continued s u sceptibilit y to Cry34/35. In addition to the ABSTC populations , a number of collection s were mad e in commerc ial Cry 3 4 / 35 fields with un ex pected damag e . Bioassay result s from th ese populations are described in sec tion C of thi s r eview. 

### Table 2. W es tern corn rootworm susceptibi lity to Cry34/ 35 u s in g overlay diet bioassays: 2014 season (Tab le re produc ed f rom Table 2 in Appendix A , MRID # 497109
- 01) Mean EC so Mean LC so Population Rep licates (95% C l ) (95% C I) ()lg/c m 2 ) {!t1ic m 2 ) McLean Co unt y , 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) Cl inton County. I N 5 5.73 16 .85 (1.77
- 1 7.56) (no C l reported ) Floyd County , lA 6 1. 56 2 .4 7 ( 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 Co unty , 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 Co unt y , SD 6 1.31 I (0.93
- 1.72) 

### ---- Range ( Field Populations ) 1. 3 1
- 5.73 2.47
- 16.85 Laboratory Contr ol ( Crop 5 1.0 I 1. 68 Charac teristics, In c.)
- # I (0.8 1
- 1 .22) (0.72
- 2.93) Laboratory Co ntr o l (Crop 5 1.68 4.81 Characteristics, In c.)
- # 2 ( 1 .39
- 2.00) ( 1.86
- 9.26) Lab o ra tory Contr o l ( F r e nch Ag. 6 7.12 44.72 Re search S ervices. In c.) (5.15
- 9.87) ( 17.71
- 75.80) 1 LC sos could not be calcu lated for th ese population s beca use data fai led to converge in probit analysis . 

### Tab le 3. S u s ceptibility bioa ss ay results 1 for fi e ld-coll ecte d C RW to Cry34/ 35- 2004 to 2014 (Table ge ne rated from data pro v ided by D ow AgroScience s and Pioneer) Year ECso ( J..l g/c m 2 ) EC99 (J..lg/cm 2 ) LCso (J..l g/c m 2 ) 3 LC9o ( J..l g/c m 2 ) 2004-2005 0.8
- 1.1 2.0-2.4 2 006 0.8-2.3 2 1. 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 EC so

**g/cm )**  EC 99 (!lg/cm 2 ) LC so (!l g/ c m 2 ) 3 LC9o

**(~ gl**  **cm )**  

### 20 08 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- 3 9.9 

### 2012 0.7- 2.9 3.5- 8.4 

### 201 3 1.6
- 4.0 4 .4
- I 1.7 

### 20 14 1.3
- 5 .7 2 . 5- 16.9

1 Result s are for field populations collected by ABSTC ; do es not include re s ults from CRW s ampl e d from fields w ith unexpected dama ge or other population s . 2 Testin g in 20 II was conducted with new toxin lots (activ it y was s imilar to th e previous lot s). 3 Numerous field population s are not incl ud ed in th e L Cso r es ults because data fail ed to con ve rge in probit anal ysis 

### BPPD !RM Team Review
- 2013 and 20 1-1 Di et Bi oassa ys Growth inhibition (E C so and LCs o) te sting s howed that most populati o n s coll ec ted in 201 3 and 20 14 r esp o nd e d s imilarl y to the sus c ep tible control populati o n s . LC so va lu es cou ld not be calcu lated in probit anal ys is for a number of population s in both years, whi c h has al so been an i ss u e for pa s t bio assays. BPPD agrees with Dow / Pione er that the res ult s were comparabl e to previ o us seaso n s (Tabl e 3), th ough it i s que st ionable how me an in gful the diet bi oassay r es ults are in the context of makin g re s i stanc e determinati o ns (see discussion below). In 20 1 3, three p opulation s (Scott , Clay and York counties) had non-o verlapping EC so co nfid ence intervals (C I) with the co ntrol s; o ne population (York) a lso had a non- overlappin g LCs o C I w ith both co ntrol s . H owever , the m agn itude of these difference was not g reat and the 20 13 popul atio n s responded s imilarl y to C RW collected from previous s ea so ns (Tab le 2). D ow and Pione e r indi cate d that th e result s from York were s imilar to other CRW populations co ll ec t ed from the county in previou s yea rs . BPPD no t es th at York county was s ampl e d in th e two prec ed ing seaso n s w ith the foll owin g result s: 2011 (EC so 

### = 3.38J..lg/cm 2 , C I 

## = 2.81- 4.04 ; LC so 

## = 14 .9 0 11 g/ c m 2 , C I 

## = 9 . 51- 20 . 96) and 20 12 (EC so 

### = 1.41J..lg/cm2 , CI 

## = 0 . 9 8- 1.90 ; no LC so was calculated). The popul atio n was also more s u sceptib le in the 20 14 te s tin g (EC so 

### = 1.47 11 g/ c m 2 , C I 

## = 0.99
- 2 .06; LCso 

### = 3 .47 11 g / cm2 , C I 

## = 2.12
- 5. 78 ). In addition , no cases of unexp ec ted field dam age to Cry34 / 3 5 corn we re r epo rted in th e thre e co unti es durin g 2013 or 20 14 . On e of the control co loni es included for th e 2 014 t es tin g (Fre nc h Agricultura l Researc h ) was much less s usceptible to Cry34 / 35 than in 20 1 3 (ECso 

## = 1.33 J..l g/ cm 2 , LCso 

## = 3.16 11 g/cm 2 in 20 1 3 co mp ared with EC so 

### = 7.1 2 11 g/ c m 2 , LCso 

### = 44 .7 2

**~t cm**  **2** in 2014). Thi s re s ult wa s not specificall y discu sse d in the study report; BPPD ex pect s that differen ce may be du e to n atural variation in re spon se to the toxin. Wh e n compared to th e more sens iti ve control co l o ny (Crop Characteri s tics ), three populations (Fi llmore , Cla y, and Ham ilt on counties) had non-overlappin g


### - 8 -ECso Cis. No ne of these populations had an ove rlapping LCso C l , however, a n un ex pected dama ge s ite wa s investi gated in Clay county during 2014 (see secti on C). B esides Yo rk County, man y of the counties sam pled in 2013 and 2014 had a l so been samp l ed in previous seaso ns (2009
- 20 12) . The Cry34/ 35 susceptibility was comparab le (in some cases lower) in these co unti es during 2013 than in th e previous co ll ectio n years (see data s ummari zed in BPPD 20 1 3a, 2014). BPPD cautions , however , that it is un c l ea r (and probabl y unlikely) th at the same loca les (i. e . , CR W population s) were sa mpled within th ese counties from year- to - yea r. Th erefore, these results do not necessarily track changes in s u sce ptibility to di sc re te CRW populations over the sampling period. T hou g h the pop ul ations co ll ected for the annual (A B STC) monitoring program were sus ceptibl e to Cry34/ 35, Pioneer repo rte d 22 investigations of unexp ected fi e ld damage in 20 1 3 . Th ese cases a re described in sect io n C of this r ev iew. D ow rec e i ved no un ex pecte d damage reports duri ng 2013. Dow/Pioneer respo nd ed to pas t issues of variab il ity in the co ntrol population (see BPPD 2014) by in cl udin g two s u sceptib le co lonies in th e testin g. Contro l populations from C rop Charac teri s tic s In c. (Farmin gton , MN) and Fr e nch Agricu ltural Resea rch Inc. (La mberton , MN) we re includ e d for 2 013 ; in previous years tes tin g was done w ith o ne or the other of tho se sources. Both of the control pop ul ati ons performed similarly in the 2013 bioa ssays . As described in previous re v iew s ( BPP O 20 l3a , b) , BPPD ha s expressed concerns w ith the diet bioassay app roac h as an effective tool to detect potentially res i s t a nt field populations. Th e 2013 SAP eva lu ate d diet bioa ssays for CRW and co n c luded that the a pproach ha s valu e for monitorin g s u sceptibilit y (SAP 20 14). The panel highlighted their s tand ard use in the sc ie ntifi c community , the precise nature of the assays , the br eadth of data that can be collecte d ( le th al an d s ublethal responses) , and logistical advantages for re searchers. On the other hand , the p ane l noted a number of s hortcomings of di et b ioassays (as currently design e d) that limit the ir ab ili ty to proactively detect field resi s tance. Specifically , diet bioassays may not predic t s urvi val on co rn plants , L C meas ures have been hi g hly v ariabl e, EC value s may not be s en s iti ve e nough to id e nti fy early res istanc e, a " diagnostic " concentration capable of killing all s u sce ptible and heterozygou s res istant genotypes has not been deve loped , and the assays may not be able to distingu i s h resistant population s from susceptible ones. BPPD recognizes the sc ientific val ue of diet bioa ssays, but believe s the limitations (as iden tifi ed by the SAP ) ha ve to date restricted their usefulne ss as a regulatory too l fo r r es is tance monitoring. On e aspect that could improve the interpretati o n s of diet bio assays wou ld be the u se of a po s itiv e control group (i. e . , a resistant population). Along th ese lin es, the new framework for C RW r es i stance man age m e nt requires companies to include res istant co lo ni es as part of resistanc e d etermination assays (add iti o nal details can be found at: ( http: //www . epa.gov/ pcsticides/ new-reguirements
- address-com
rootworrn-res i stance - bt
- corn ). Pioneer has maintained a Cry34/ 35 -res i stant popu lation for use in the Sub lethal Seed ling Assay (see section B ), though it is unc lear if this colony has been adapted for art ifi cial diet. A key compo nent of IRM ha s been the effective id entification of resistant populations as early as possible so that mi tigatory mea s ures can be applied with a g reat e r likelihood of s uccess (BPPD



### - 9
-20 13b). On plant assays , which can effective ly distinguish resistant populations from susceptibl e o nes ba se d on resp on se to Bt plants (Gass mann et al. 20 11 ), offer potential as dete rmini s ti c tool for resistance monitoring. Along thes e lin es, Pion eer has developed an on-plant a ssay (Sublethal Seedlin g Assay; Nowatzk i et a l. 20 08 ) for Cry34 135 (see di sc u ssio n in the f o llowin g section). Des pite the conc erns with the current diet bioassay approac h , BPPD is open to improved st r a tegie s for th e ir use i f they can fu nction reli ab ly and consistentl y to dete ct resi stant populations. In particular, BPPD recommend s the u se of a positive (Cry34 13 5-res is tant) control in the assays to aid with interpr eta tion of the results. Pione er has a res istant co lony ( RocheII eRR) that is includ ed with th e Sublethal Seedling Assay, though it is not kn o wn i f thi s colony would b e well-suited for artificial diet. 

### B. Sublethal Seedlin g Assay Since 2009, Pioneer ha s been utilizin g the Sublethal See dlin g Assay in conjunction w ith diet b ioassays as a diagno s tic monitoring tool for detecting resistance. The Sub lethal Seedlin g Assay (SSA) was developed by Pion eer scientists w ith th e Cry3 4 135 toxin and e ntails ex p os ure of CRW larvae to root mats ofBt and non-Bt plants (an " on-plant" a ss ay). Methodology for the assays i s desc rib ed in Nowatzki et al. (200 8 ), though Pi oneer ha s since modified the procedure bas e d on their experiences with the test (see discussion in BPPD 20 1 3a) . The current testing m et hodolo gy for the SSA has been used si nce 20 11 and invo lves exposing neo nate C RW to seed lin g mat s ofBt or non-Bt corn (150 seed kernel s per mat). As impli e d b y the nam e, the SSA doe s not meas ure mortalit y; rath er , the test is des igned to assess potential developmental effects ofBt exposure including growth ( in star) and body size (larv a l bod y area). Eac h of the seed lin g mats in th e test i s infested with approximately 500 CR W eggs on the s oil surface and he ld in an env ironm e ntal c hamb er (25 °C, 65% 

### RH , 14: 

### I0 ligh t/ dark). For some populations with low egg availability , a low er infestation rate may be used. Three to four rep licat es (each mat is considered a replicate) are established for eac h treatment (Bt and non corn). A separate hatch tes t u s in g a sub s ampl e of eggs in Petri di s he s held in the same environmental chambers is conducted to determine the startin g point of the tes t. After hatching is initi ated , larvae are exposed for approximately 1 54 degree days to allow fo r growth and d eve lopment. Larvae are then collected from the roo t mat s u s ing Burl ese funnels positioned over containers with 70 % ethanol. A s ample of 30 larvae is obtained from ea ch replicate ; any p op ulati o n that does not produce at least 30 larvae in a maj o ri ty of the replicate s is consid e red non-viable. The s ampled larvae are th en meas ured (he ad cap s ule width) to determin e th e developmental instar (JS 1 , 2"d , o r 3rd). A secon d proc ess ing procedure is a lso used to assess bod y size. Samp les are pour e d into P e tri dishes, w hich were scanned and analyzed by imaging softw are to calculate th e total body area (mm 2) of eac h larva. Both instar and body size data have b ee n analy ze d f o r the SSA, thou g h Pi oneer ha s adjusted how th ese d ata ha ve bee n interpreted since the assay was first deve lop ed (see BPPD 20 1 3a, 2 014 for di sc u ss io n). For instar development , larv ae are grouped into t wo categories , 151 + 2nd in s tar and 3rd instar , so t h at percent achi ev ing th e 3rd in s tar can be dete rmined. Bt and non-Bt treatm e nts are compared to generate a third inst ar ratio (% 3rd instar on Bt I % 3rd in s tar on no n- Bt). A n odds ratio ([% 3rd in star B t/ 1
- % 3rd in star Bt] I ( [% 3rd in star non-B t/ 1 - % 3rd in s tar 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 w ill approach or exceed 1.0 . Statistical ana lyses of the ratio data are conducted with a statistical s ignificance of p

**~** 0.05 . Body si z e 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 submiss i ons , Pioneer has previous ly indi cated (MRID # 490085
- 0 1) that its decision process to determine suspected resistance (triggering follow
- up investigation) w i ll include two criteria. Further action will be taken i f 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 (instar and image data); • Shows mean ratios (both instar and s ize) outside the 95% confidence interva l established from data collected (in) previous years. SSA
- 2013 S ampling and T es ting ( MR!Ds -195609-01 ) Pi oneer included CRW pop ul ati ons from a number of so urces in the 20 13 testi ng (liste d in Table 4 below). Nine CRW populations co ll ected as part of the ABSTC annual samp lin g (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, P ioneer co ll ected CRW from six other locations in Iowa and Illinois. The SSA assays also i nc l uded I I populations (plus two control populations) sampled from fields with unexpected damage, a lthough one of these colonies could not be tested due to l imited egg viability. Two control populations were also included in the assays: a Cry34/ 35 suscepti b le 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 docume n ted in Table 4 below. P ioneer ind icated that the res ul ts did not a l ign well wi th previous data i n that the 3rd in star ratios were m u ch l ower than in prev i ous years. A number of challenges were observed in t he 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 s ize 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 instar 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 suscepti ble laboratory co lony we re 0 . 057 and 0.039 , th o ugh develop ment to the 3rct instar was a lso low in bot h test runs (14. 1 % a nd 38.6%). B ody area ana lysis of the field populations fo ll owed a similar pattern , as larvae on Bt p l ants were smaller than those on non-Bt. Mean body area ratios of field populations ranged f rom 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 prob lems encountered during testing), Pionee r identified one of the ABSTC-co ll ected s ites (Fillmore , MN: 3rd instar ratio = 0 . 213; body area ratio= 0.203) as a location for follow-up activ i ties i n s u bsequent years . Two test ru n s were conducted with the " Rochelle " Cry34 / 35-selected colony. Thi s pop u lation generally fared better on Bt corn than the fie ld or laboratory co lonies , with 3rd instar rat i os of 0.210 and 0.447. In addition, development to the 3rd instar was high er on non-Bt corn (91.7% and 92 .7%) t h a n th e control a nd field populations. On the other hand, P ioneer noted that d evelopment 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 lot s could have been responsible for the reduced deve lopment. Simi larly, t he Roc h e ll e co lony had hi gher body area ratio s (0.332 and 0.372) than the fi e ld populations , but lower than in previous years (Tab le 6). Pioneer ' s testing of population s collected from fields with unexpected damage is discussed in section C ofthis rev iew. 

### 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 In sta r R a ti o Body A r e a Popul at ion %3rd %3 rd 3'd ln star ratio on -Bt Area mean instar: 81 inslar: ( 131 : non-131) 81 (mm 2 ) (mm 2 ) ratio non-Bt (8 1: non 81) ABSTC and Pioneer Co llections C la y, NE (AB STC) 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 (A BSTC) 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 P latte , N E (ABSTC) 4.4 63.9 0.069 1.04 6.43 0.162 Scott, lA (A BSTC) 6.8 31.9 0.213 0.99 4.19 0.236 York, NE (A BSTC) 3.4 44.9 0.076 0.87 4.60 0.189 J efferson , l A (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. 1 71

- 12-3rd ln s t a r R a ti o B o d y Ar ea Po pul ati o n %3 rd %3rd 3'd ln star ratio Non-Bt Area mean instar: Bt in s tar : (Bt : non-Bt) Bt (mm2 ) (mm 2 ) ratio non-llt (Bt: non Bt) Clayton, lA ( Pioneer) 3.2 84.7 0.038 1.41 9.15 0.154 Hamilton , l A ( Pion eer) 5.5 80.4 0.068 1.34 8.40 0.160 Range 0.018
- 0.213 0.138
- 0.236 UX D Co llections 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 B enton , 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 ( Han sell) 5.6 52.6 0.106 1.22 5.71 0.214 Jack so n, l A 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 Sio u x, l A 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 C herokee , lA 0.7 84.9 0 . 001 1.10 5.89 0.187 Range 0.00 I
- 0.24 1 0. 1 59
- 0 . 24 1 Co ntr o l Groups Labo rato r y Contr o l ( f$' Run) 0.8 14 . 1 0.057 0.56 3.01 0 .186 Labo rato 1 y Control (2"J Run) 1.5 38.6 0 . 039 0.65 4.20 0.155 R och e ll e RR (! -' 'Run) 19.5 92.7 0.210 2.50 7.52 0.33 2 R oche lle RR (2" 4 Run ) 41.0 91.7 0.447 2.98 8.02 0.372 SSA- 20 14 Sa mpling and Testing (MR!Ds -19 7 1 10-01 ) As in the 2013 monitoring , Pioneer tested C RW populations collected by ABSTC and from fields wi th unexpected damage (Table 5 below). Ni ne of the t en ABSTC populations ( Table 2 above) te s ted in the diet bioassays were a l so tested with SSA. An additional five populations

- 13
-collected from Bt field s with unexpecte d dam age were included (rev iewed in Section C be low). Control g roup s included two C ry34/ 35 s usc ept ible p op ulation s: the Brooking s USDA
- A R S reference colony and a Pioneer- supplied population de s ignated "CRW SS " (a cross betw ee n the Br ookings strai n and field populations). T he " Ro chelle -RR " population res is tant to Cry34 / 35 w as used as a pos itive control. Res ult s fro m th e 20 14 SSA testing are detailed in T able 5 below. Pi oneer opted to discontinue w ith the body a re a mea s urement s w ith the 2014 testin g, indicating that th ey were highly corre lat ed w ith the in s tar data. Therefore , only the instar meas urem e nts and analyses were repo rted. As in 20 13 , Pione er u se d a third in star rati o (i.e. , percent of individuals r each in g the third in s tar) in stead of the mean instar ratio th a t was previou s ly us ed. For statistical analy s is , Pioneer a lso calculated an odds ratio
- i .e., a quantification of the assoc iati on between the pro bability of reac hin g the third instar and ex pos ure to Bt corn. Third in star rati os of the field populations ran ged from 0.059 to 0.493, simi lar to the valu es obs erv e d in 20 1 3 (3 rd in star ratio also measure d ) but lower than the ran ges observed in 2009- 20 12 whe n a mea n in star approach was used (T ab le 6). Th e three susceptible control groups had ratio s of 0.226 (Brookings) , 0. 3 48 (Brooking s), and 0.436 ( " C RW SS") . A nalysi s of odds ratios showed that no ne of these population s had stati s ticall y significant d eve lopm e nt on Bt corn relative to non-Bt com. On the other hand , the Roch e ll e -RR colony had a statis ti cally sig nifican t rat io of 1.119 , indicatin g that the colon y 's dev e lopment on Bt com excee d e d that on non-Bt corn. 

### Tab le 5. Weste rn com rootworm susceptibi li ty to Cry34 / 35 u s ing the S ubl e thal See dlin g Assay: 20 14 season (T ab le re pr o duc ed from Table I in Appendix A , MRJD # 497110-01 )
3rd ln sta r Rat io Popu latio n %3 rd % 3rd 3n1 ln star ratio in star: Bt in s tar: (B t : non-Bt) Odds ratio' no n-Bt ABST C Collections She lb y, NE 23.0 57 .6 0.399 0.220 Cl inton , IN 28.9 58 .7 0.493 0.286

Flo yd , 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 H

owa rd , 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 1 7 Range 0.059
- 0.493

- 14-Jrd Jn s tar Rat io Popu latio n %3 rd %3rd J'd ln s tar ratio iostar : Odds ratio 1 in st ar: Bt non -Bt (O t : non-Bt) UX D Co ll ec

rio n s 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 , l A 2 8.1 57.6 0.487 0.287 Boone , NE 20.4 60.2 0.340 0. 170

Ran ge 0.324
- 0.749 Comr ol Gro ups Br

ookings Conrrol ( ! " Run) 10.5 46.6 0.226 0.135 Brookings Co ntr o l (2"' 1 Run) 18 .0 5 1. 7 0.348 0.205 CRW SS Lab 25.5 58.5 0.436 0.243 R och e lle RR 92.7 82.9 1.119 2.639* 1 Values de s tgnated wtth a·'* " are s tatt sttcally s t go tfic an t at p :S 0.05. Tab l e 6. S ubl ethal Seedling Assay Result s for Western Corn Rootworm , 2009
- 20 14 (Tab le c reated from data submitted by Pioneer)
# ln s t a r ( mean ratio Bt : no n- Bt) 1 B o d y Area ( mea n rati o Bt : non-Bt) Year F ie ld Susceptib le Se l ec t ed Suscep tibl e Se lected Pop . Fie ld Co ntrol 2 Co lonyl Fie ld Co ntrol 1 Colon y2 20 09 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 1 4- 0.421 I ) 0. 1 98 I ) 0.624 2) 0.830 2) 0.274 2) 0.633 20 12 8 0.710
- 0.883 I ) 0 .90 2 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 .0 39 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

- 1 5-1 Mean instar ratio was used 2009-20 II , third instar rati o was u se d 20 1 3-20 1 4. For e ith er ratio, va lu es approaching or exceeding I indicate little developmenta l effects due to Bt exposure. 2 Cry34 / 35-suscep tibil e control colony obtained from USDA-ARS (B rookings, SO). 3 York-RR (20 I 0-20 II) and Rochelle-RR (20 12) have bee n se lected on 59 122 (Cry34/ 35) corn. 

### BPPD IR M Team Rev iew - 2013 and 20 1 4 SSA Testing B P PD agrees w ith Pi oneer that the 20 1 3 instar ratio results were aberrational compared with p rev iou s tes tin g and were like ly confounded by me th o dol og ica l iss ues in the tes tin g . All test groups showed less development on both Bt and non-Bt treatments than in pr ior years, including th e Cry 34/35 selec ted colony. Body area data from 2013 we re so m ew hat more consistent wit h the historical data than the in star ratios , but larvae st ill showed less development than would have been expected. Pioneer's 20 1 3 report indicated that irr egular egg hatch and new 59122 seed lots that produced l ow root mass may hav e affected the tests, resulting in reduced larval development. The 2014 report did no t indicate if these issues we re r eso lve d o r were still a facto r in the 2014 testin g. The data from 20 1 4 ge nerally fo ll owed the pattern of the 20 13 t esting, al though the instar ratio s for field and co ntrol popul ation s were somewhat high e r an d th e se lect ed co lon y had ro bu st deve lopment on bot h Bt and non-Bt com. The 20 1 3 and 20 14 instar ratio re sul ts may also partially be explai ned by a change in how the te st data were analyzed. Pri or to 2013, Pioneer calculated a mean instar ratio in which the ave rage in star development of the population is calculated for Bt and non -Bt com treatments . In 2013 and 2014 , Pi oneer moved to a 3rd instar ratio by detennining the percentage of each population that reaches the 3rd in star on Bt and non- Bt treatments during the test period. This new approach cou ld h ave t he effect of c reatin g more "separation " between a suscept ibl e po pul atio n (with low 3rd ins t ar ratios) an d a potentially resistant one (with a 3rd in star ratio ap proachin g or excee din g l.O ). In the 20 09
- 20 1 2 assays (Tab le 6) , s usc ep tible populations (field or contro l) often had mean instar ratios exceeding 0.9 , not much lower than the positive co ntro l (res ist an t) colony. B y cont r ast, in 20 14 the fie ld and su sceptible control population ratio s were< 0.5, compared to 1.11 9 for th e resistant colony. Pioneer o pte d to disc o ntinu e u se of body area anal ys is for 2014, stating in it s rep ort that the measure was hi ghly corre lated with th e in star ratio s and thus presumably r edunda nt. BPPD has advocated for assessing two separate measures in C RW suscep tibili ty b ioassays to minimi ze the potential for fal se po sit iv es or nega ti ves 

### in the res ult s (BPPD 201 3b) . However, in this case, BPPD agrees with Pioneer t hat the body area data do not appear t o provide additional anal y tical value over the 3rd instar results. In general , the body ar ea mass analysis produced lower ratios than with the instar approach , even w ith the Cry34/ 35 se lec t ed co lony (Tabl e 6). No ne of the te st runs with selected colony had a body area ratio approac hin g 1.0 , th e putative indication of r esistanc e. Without t he se cond analytical m eas ure, it w ill be important to continue use of the selected co lony as a positive contro l and addi ti o na l po int of comparison for the fie l d populations. BP PD a l so presumes that Pioneer's resistan ce determination criteria (summarized above) will be updated to reflect that on ly o ne m easu re (3rd in star ratio) w ill be assessed for a resistance confirmation.

- 16 -A numb e r of population s co ll ec t e d from dam aged Cry34/ 35 were te s ted in 2013 an d 20 1 4 u s in g the SSA . Th ese population s a r e di scussed in the nex t sec ti on. 

### C. Cry34/ 35 F i e ld Dam age Reports 

### 2013 Reports During the 2 0 1 3 g rowin g seaso n , Pion ee r re p o rt e d a total of 22 une xpected dama ge (UXD ) report s from their customers re late d to C ry34 / 35 corn. Th ese fi e ld s were investigated using th e protocols described in Pi oneer 's monitorin g program (MRI D # 480455-0 1 ). Pioneer was able to collect C R W ad ults fro m 16 of th e field s , wi th 12 populations producing enough eggs for testing (see list in Tab le 7 b e low). Most ofthe UX D populations we re fro m Iowa , w i th seve ral others f rom Neb ra s k a , So u th Dak o ta , and Wiscon si n . Dow report e d no cases of UXD a mon g its customers durin g 2013. Populations collec ted from UX D field s we re tested by diet bioassays and t h e S ubl ethal Seedling Assay (three populations were tested w ith diet assays o nl y). Resu lts for the diet assays are s ummari zed in Tab le 7 below; SSA re s ul ts fo r these populations a r e detail ed in Table 4 . In the diet assays, th e ECso ra nge of the UX D popul ations was 2 .4 7
- 6.45 11g / cm 2 , co mpared to a range of 1.61
- 3.9 7

**~tg cm**  **2** for th e field popul ation s co ll ected b y A BSTC. The EC so s of the two contro l p op ulations were 1 .33 and 1.6 2 11g / c m2

. LCs o va lue s fo r the UXD p o pulati o ns we re 5.56

- 1 2.16 11g / cm 2 (ABS T C p o pulati o n LC so = 4.3 7
- 11.7 0 11 g/ cm 2 ; cont rol p o p ulation LCs o s = 1.78 and 3.16 11g / cm 2 )
. The SSA re s ult s (i n star and body area ratios) for the UXD populations we re com p a rab le to tho se f rom the A B STC a nd co ntrol p op ul ation s, though m ethodological i ss ue s likel y confounded th e 20 13 SSA testing (see discu ss ion in sec tion B of thi s rev i ew). Table 7. C ry34/ 35 Suscept ibility of CR W populations co ll ec ted from fie ld s wit h une x pect ed damage: 2013 seaso n ( Tabl e rep roduc ed from Tab le 1 in Ap p end ix B , MRID # 495009
- 01) Mean EC so Mean LC so Popu lation Replicates (95% C l ) (95% C I) ( 11 g/c rn 2 ) ( 11 g/c m 2) Benton County , l A 5 4.53 7.36 (2.16
- 9 . 33) (3. 75
- 11.63) Cedar Count y, lA .., 2.81 9.73 .) ( 1.82
- 4.27) (4.52
- 16.43) Cherokee Coun t y, lA 6 5 . 16 10.93 (3.44
- 7 .67) (7.70
- 14 .27) Franklin County , l A (# 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 , l A 3 3.46 8.38 (2.41
- 4.90) (2.6 1
- 17.56) Gru ndy Co unty , l A 5 4.49 10.92 (3.1 0
- 6.43) (5.54
- 15.66) Grundy County , l A (control) 6 5.40 12.16 (4.05
- 7.17 ) ( 8.40
- 16.46 )

- 17-MeanEC so Mean L Cso Population Replicates (95 % CI) ( 95 % C l)

**(~t g/**  **cm )**  **(~g/cm )**  Linn Count y , lA 6 6.45 9.25 (2.86
- 14.3 5) (5 .69
- 1 3.2 3 ) Mu s catine County , l A 3 4.57 10.94 (3.08
- 6.71) (5.25
- 17 .35) Sioux County , lA 4 3 . 20 8.0 7 ( 1.0 I - 8.97) (3.00
- 1 2 .42) Siou x C ounty , lA (contr o l) 6 3.29 5.81 ( 1.93
- 5.44 ) (2. 94
- 8 . 5 7) Grant County, SD 6 3.49 5.56 ( 2. 2 7
- 5.29 ) (3 .24
- 8.15 ) Lafayette County , WI 6 2 .47 I (1.4 2
- 4.13) ............ Range (U XD Populations ) 2.47
- 6.45 5.56
- 12 .16 La borat01y Control (C ro

p 6 1.62 1.78 C

harac te ri s tics, In c.) ( 1. 2 8
- 2.01 ) ( 0 . 67
- 3 . 3 1) Laborato 1y Control (Fren ch A g. 6 1. 3 3 3.16 Research S e rvice s, In c.) (0.74
- 2.09) (1.66
- 4.77)
1 LCsos could not be calcu lated for th ese populations because data fai led to converge in probit analy s is. 

### 2 014 R e ports Pioneer investigated three " AcreMax " fields with UX D 

### in 2014 in accordance with established protocols (MRlD# 480455
- 01). CRW samp lin g was successful at one ofthe sites (Delaware County , lA # 1 ) ; the two other sites were rotated to soybean in 2015 and additional co llections will not be attempted. Pioneer also followed up with 12 UXD sites from the 2013 season that cou ld not be samp led that year. Six populations were co ll ected from the se 20 1 3 s ites , 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 cu s tomer s during 2014. UXD populations we re tested with diet bioa ss ays (Table 8) and the Subletha l Seedling A ss a y (Tab le 5). The UXD population collected in 20 14 (De lawa r e Co unty , IA # 1) sho wed an e levated LC so ( 114.48 f.l g/ cm 2) re lati ve to the other field populations (ABSTC collections ; LC so ra nge= 2.47- 1 6.85 f.lg/ cm 2 ) and control population s (LC so = 1.68
- 44.72 f.lg/ cm 2) from that sea s on. The EC so (7.05 f.lg/cm2 ) of thi s population was al so e levated relative to ABSTC popu lations (1.31- 5.73 f.lg/ cm 2 ) and the Crop Characteri s tics control colony (E C so = 1.01 and 1.68 f.l g/ cm 2 ), but was co mp arab le to the second control co lony (Fre nch A g.; EC so = 7.12 f.lg/cm 2 ). On the other hand , SSA testing of thi s co lony reveal e d 3rd instar odds ratio ( 0.249) that was simi lar to the other field and control populations. A second population fro m Delaware C o unty. lA (#2) that wa s taken from a 2013 UXD s ite was also tested in 20 14. The ECso (5.78 f.lg/cm 2 ) and LC so (32.5 1 f.lg / cm 2 ) of this population were s omewhat higher than the ABSTC population s and the Crop Characteristic contro l colonies , but were less than the French Ag. colony. SSA testing of this popu la tion , however, resulted in a 3r d instar odds ratio (0.625) that was s tati stica ll y relevant and g re ater than any of th e ABSTC or

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-control populations. Pioneer 's r eport concluded the biological r elevance of the population was unclear but noted that the result could "be indicative of a shift in suscept ibili ty for this population. " For the other tested UXD populations, ECso and SSA t est ing results were comparable to the control and field populations. The LCs o 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 a nd many of the field collected populations. 

### Tab le 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 EC so Mean LC so Popu lation Re plicat es (95% Cl) (95% Cl)

**{~Jg/cm**

**) (~Jg/cm** 

**)** Delaware County, l A ( # I) 6 7.05 114.48 (3.37
- 14. 72) (55.08
- I0 18.00) Delaware County, l A {#2) 1 2 5.78 32.51 (2.72
- 11.83) (14.87
- 55.69) Fayene County, IA 1 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, NE 1 6 4.13 24.31 (3.17-5.33) (14.5 1
- 40.19) Range (U XD Populations) 4.13-7.05 24.31
- 114.48 Laborato1y Con tr ol (Crop 5 1.01 1.68 Characte ristics, In c.) (0.81
- 1.22) (0.72
- 2.93) LaboratO !J' Control (C r op 5 1.68 4.81 Characteristics. In c.) ( 1.39
- 2.00) ( 1.86
- 9.26) Laborato1y Control (French Ag. 6 7.12 44.72 Res ea rch Services. In c.) (5.15
- 9.87) ( 1 7.71
- 75.80) 1 2013 UXD site that was resampled and tested during 2014. BPPD IRM Team Review
- UXD Reports In genera l, the UXD populations tested had s lightl y elevated susceptibility (ECs o and LC so) 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 Delawar e County, lA. T he first population, collected in 2014 from a 20 1 3 UXD site, s howed statisticall y s ig nificant g rowth as measured by SSA testing (Tab le 5). The second, a 2014 U XD site, had relatively high ECs o and LCso values, although the SSA results were comparab le to the other field populations collected that year. lt is unlikely that either population is completely resistant to Cry34/ 35, particularly w hen compared to the se lected r esistant " Roch e ll e" colony (SSA odds ratio of" Roch e ll e" was 1 .11 9 com pared 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 sa me county, BPPD r eco mm e nd s additional monitoring (UXD scou tin g) wit hin D elaware County. BPPD is e ncouraged that two ofthe three UXD s ites in 20 14 were rotated to soybean in 20 1 5. BPPD believes that th e u se of JP M -b ased B est Management Practices ( B MPs) , particularl y rotation to soybean, can be an effective mean s to miti gate (o r extirpate) a potentia ll y resi stant population (BPPD 2013b). 

### References BPPD , 20 1 0a. Review of Do w ' s 2008 com rootworm monitoring report and revised resistance monitorin g plan for DAS-591 22 -7. J. C. Martinez memo randum toM . Mendelsohn , June 30, 2010 . BPPD , 20 10b. Bacillus thurin g iens is Cry34Ab 1 and Cry35Abl Prote in s and th e Ge neti c Materi a l Necessary for their Production in Event D AS-59 1 22
- 7 Com Biopesticide R egistrat ion Action D oc um e nt (BRAD). Re v i se d Se ptemb e r , 2010. Available at http ://www .epa . go v/oppbppd I /biop es ti c ides/ ingr e di e nt s/ t ec h docs/brad 0 0 6 490 . pdf. BPPD , 2 01 Oc. Terms and Co ndition s for Bt C om R eg i s tration s, September 30, 20 I 0 . Availabl e at: http ://www . epa. g ov / pesti c ides/b io pes ti c id es / pip s /b t- co m - te nn s-co nd i tio n s . pd f. BPPD , 20 11. Upda te d BPPD lRM Review of Rep orts ofUnexp ected Cry3Bbl D amage , Monsanto ' s 2009 Co m Rootworm Monitor in g Report. and Revised Com Rootworm Resi s tance Monitorin g Plan for MON 88017 , MON 88017 x MON 810 , MON 863 , MON 86 3 x MON81 0, MON 89034 x TC1507 x MON 88017 x D AS-59122-7 , and MON 89034 x M O N 88017. J. Martinez me morandum to M . Mendelsohn, Nove mb er 22 , 2011 . BPPD ,' 2012. BPPD IRM T eam re view o f Mo n sant o's 20 1 0 co m rootworm monitoring data , unexpecte d dama ge rep o rts f o r Cry3Bbl express in g Bt Corn and academic report s ofCry3Bb1 field failures as well a s co m rootworm resistance. J. Martinez and A. Reyno ld s me morandum to A. S ib o ld , October 11 , 2012. BPP D , 20 13a. Review of Com Rootworm R esistance Monitoring Submissions and Data ( 2009
- 20 1 1) for Bt corn products containing C ry 34 / 35Ab I . A. Re ynold s memorandum to A. S ib o ld , Marc h 20 , 20 1 3. BPPD , 2013b. White p ape r on com rootworm res is tance monitorin g for Bt plant-incorporated protectants. A vai Jable at: http ://www . c p a . go v / o pp f c ad I /c b / cs b pagc / updatcs/20 1 3 /c omro o tworm -wp.htm l . BPPD , 2014 . Review of2013 Corn Rootworm Resistance Monitorin g D ata f or Bt com products conta inin g Cry34 / 35Abl. A. Rey nold s memorandum to A. S ibold , Dec e mb er 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 O N E. 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: Chrysomelida) and event DAS-59122-7 maize . J Appf. Entomol. 132 : 177-188. Pione e r , 2013. Comments to public docket EPA-HQ-OPP-2013-0490 , dated November 26 , 2013. http: // www.regulations . gov/# !documentDetail:D = EPA
- H Q
- OP P
- 20 13-0490-0025 . SAP , 2014. SAP Minutes No . 2014-01 : A Set of Scientific I ssues Being Considered by the Environmental Protect ion Agency Regarding Scientific Uncerta inties Associated with Corn Rootworm Resistance Mon itoring for Bt Corn Plant Incorporated Protectants (PIPs) December 4-5, 2013 FIFRA Scientific Advisory Panel Meeting . Available at: http: //www . epa . gov/ sc ip o ly / sap/ meetinus/ 20 13 /decemberll2 0413minutes.pdf

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