OFFICE OF PREVENTION, PESTICIDES, AND

TOXIC SUBSTANCES

MEMORANDUM February 4, 2010

DP barcodes: 370457

Decisions: 390180

SUBJECT: Science Assessment of Pioneer ’s modeling addenda (MRIDs 478710-01, 478879-01, and 479108-01) in support of th eir application of registration for Optimum AcreMax1 Insect Protection

TO: Mike Mendelsohn, Senior Regulatory Specialist

Microbial Pesticide Branch Biopesticides and Pollution Prevention Division (7511P)

FROM: Jeannette Martinez, Ecologist

Microbial Pesticide Branch Biopesticides and Pollution Prevention Division (7511P)

SECONDARY Alan Reynolds, Entomologist

REVIEW: Microbial Pesticide Branch Biopesticides and Pollution Prevention Division (7511P)

ACTION REQUESTED:

The BPPD 1 has been asked to review three addenda by Pioneer (MRIDs 478710-01, 478879-01, and 479108-01) to complement their original modeling chapter (MRID 473567-08) submitted to support the Sec(3) registration request for Op timum® AcreMaxTM 1 Insect Protection (OAM1). OAM1 is a Bt corn seed blend consisting of 1) seeds that express a stack of Cry1F to target Lepidoptera corn pests and Cry34/35 to target corn rootworm (CRW ) and 2) seeds that express Cry1F only (refuge seed for CRW). The registrant requests a 5% seed blend registration with a separate 20% block refuge (containing 100% Cry34/35) for Lepidoptera planted immediately adjacent to the Bt field.

1 The use of BPPD in this review refers to the BPPD IR M team consisting of Jeannette Martinez and Alan Reynolds. UNITED STATES ENVIRONMENTAL PROTECTION AGENCY WASHINGTON, D.C. 20460- 2 - CONCLUSIONS AND RECOMMENDATIONS

1) Overall Conclusions

BPPD has reviewed Pioneer’s submissions fo r Optimum AcreMax1 (OAM1) including revised modeling with new movement data. In additi on, BPPD conducted an independent modeling analysis of Pioneer’s proposal using a model (deterministic and stochastic version) developed by

EPA/ORD. After careful review of Pioneer’s modeling (including parameter assumptions) and based on the analyses conducted by ORD (modeling efforts and probability analysis), BPPD concludes that the registrant’s a ssertion that a 5% seed blend is always superior to the current 20% block refuge deployment st rategies for OAM1 and corn root worm” is not scientifically supported.

Pioneer’s model made a number of assump tions on CRW movement, refuge location, and percent non-compliance. In their model, when these assumptions were included, a 5% seed blend

was more durable than a 20% bloc k refuge. However, BPPD has conc erns that some of Pioneer’s assumptions may not be realistic or conservati ve enough to support a reduced refuge for OAM1.

The following are Pioneer’s modeling parameters and assumptions which were not supported by BPPD’s and ORD’s analyses and were of con cern: random mating, neon ate dispersal, adult movement, planting location of block refuge s, 0% non-compliance, Lepidopteran refuge.

Considering all of the identifie d uncertainties and concerns discussed throughout this risk assessment, BPPD concludes that the modeling conditions/parameters used in Pioneer’s model (compliance, CRW adult dispersal, neonate moveme nt rather than late-instar movement, refuge placement to maximize distance between refuges) are unfavorable to blocks while being more

favorable to blends. This had the effect of bi asing Pioneer’s modeling analysis towards more

durable seed blends and le ss durable block refuges.

2) ORD and Pioneer models: a side-by-side comparison of modeling output using Pioneer’s benchmark parameter values

A side-by-side comparison (using Pioneer’s be nchmark parameter values)between the ORD’s

and Pioneer’s deterministic models showed that the ORD model predicte d greater durability for blocks than for seed blends (see Table below) . Predicted durability estimates were similar between the two models except for the 20% block refuges. A likely explanation for the predicted greater durability for 20% block re fuge strategy is that ORD chos e an effective refuge of 15% (due to non-random oviposition behavior and choice for refuge placement by growers). In order to duplicate Pioneer’s block ref uge result (7yrs), the effective refuge size would need to be decreased to 1%. This paramete r value (1%) was felt to be unreal istically low for a universal assumption (for further discussion see Section IV in this review).- 3 - Side-by-side comparison between predicted durability of ORD and Pion eer model with Pioneer’s benchmark
parameters Durability (time to resistance) Refuge Scenario Pioneer EPA/ORD 1

No Refuge 5 years 3 years 20% Block Refuge 7 years 14 years 5% Seed Blend Refuge 10 years 9 years

(Table 17 in Section IV )

1 15% effective refuge was chosen in ORD’s model as most likely value and fixed 30% non-compliance

3) ORD’s risk profile based on likely more realistic or conservative assumptions

The objective was to use BPPD’s and ORD’s a ssumptions for ‘likely more realistic and conservative’ parameter values and assumptions and to predict durability for 20% block and 5% seed blend under these conditions. In addition, a probability distribution was generated using a PERT distribution (setting a mini mum, most likely, and maximum value for each parameter of concern) to associate probabil ities with predicted outcomes.

a) Deterministic Model Considering other movement data and recalculat ing Pioneer’s movement rate by adjusting for errors, ORD’s deterministic model predicted that the mean time to resistance for the block refuge strategy was 11.95 years (56.55% of the simulations exceeded 10 years). For the seed mixture simulations, the mean time to resistance was 8.1 years (13.5% of the simulations exceeded 10 years) (see Table below).

ORD's risk profile for Cry34/35 deployed in a 20 % block and 5% seed blend based on more realistic assumptions and probability analysis
20% Block 5% Blend Mean Durability 11.95 yrs 8.1 yrs

% of simulations exceeding 10 years 56.6% 13.1%

(Table 18 in Section IV )

See Section C.2.b and Table 11 and Table 12 for assumptions and parameter values

b) Stochastic Model

The stochastic simulations with more conservati ve assumptions (i.e. square field size and no long-distance dispersal) predic ted that durability for Cry34/35 de ployed with a 20% block refuge would last approximately 13.9 year s for fields of typical size ( 40 ha); for large 144 ha fields (which are not average size but outliers), durability is predicted to be around 8.4 years.- 4 - 4) Options

i) Additional Data : Pioneer’s conclusions that a 5% s eed blend is superior to the 20% block refuge were the result of a number of uncertain (possibly unrealistic) assumptions that w ould have to hold true for the

entire Corn Belt. These include:

• Refuges would have to be plan ted consistently along the shor ter side of the Bt fields. This is against grower practice.

• Growers would have to move their refuge locations yearly. This is believed to be unlikely.

• Growers would have to plant refuges consistently over areas of lowest egg densities. This is probably not a realistic assumption.

• Adult male movement would have to be limited to Pioneer’s estimate of 15 m/d. ORD’s analysis demonstrated that Pion eer underestimated di spersal; after having corrected for errors, adult movement was similar to what was reported by Spencer (2009).

EPA/ORD’s interpretation of the data s uggest that non-random oviposition and refuge placement are most important rather than non- random mating being the threat to a block refuge system. This suggests a potentially plausible method that Pioneer might use to

bolster their claims that failu re of the block refuge system is imminent. If the relative density of eggs could be estimat ed in transects across fields of different sizes, and if more accurate estimates of how growers plant and move their refuges over years were available, then major differences between the ORD and Pioneer models could be resolved. Such a study might also provide an estimate of the contribution of beetles dispersing long distance.

ii) Different blend percentages: ORD explored the durability for different per centages of seed blends (2.5%, 5%, 7.5%, and 10%) and reported that predicted durab ility for the 10% seed blend was 11.3 yrs (with 51.6% of the simulations exceeding 11 year s). While this is still lower than the predicted durability for a 20% block refuge, the 10% seed blend had greatest durability among the percentages modeled and came closes t to the predicted 20% block durability.

iii) Temporal and/or geographi cally limited registration: BPPD has considered tempora lly/geographically-limited regist rations in the past because they may reduce the risk for resistance evolution. These registrations are typically limited by total U.S. acreage (usually <250,000) and have county-level acreage caps (usually <20,000). However, the modeling analyses conducted by Pioneer and ORD were not designed to address the risk of a time lim ited registration for OAM1. Therefore, BPPD cannot specifically quantify the risk of resistance evolution for such an option.

iv) Seed blend with a pyramid PIP: BPPD notes that models predict that pyram ids of two PIP toxins can increase the durability of the traits and allow for a reduction of the percentage of refuge. ORD’s- 5 - modeling showed that if two traits with the characterist ics of Cry34/35 were pyramided and deployed as a 5% seed blend that durabili ty would increase by more than three fold (52 years) over the sequential use of the tw o single toxins. Pioneer could consider the

addition of a second CRW-active toxin to OAM to improve the durability and potentially justify a 5% seed blend refuge.- 6 - BACKGROUND

In February, 2008, Pioneer Hi-Bred International, Inc. submitted the orig inal application to register Optimum® AcreMaxTM 1 Insect Protection, a new corn seed blend product that contains two different kinds of seeds from the already re gistered products Hercul ex XTRA and Herculex I. The new product would incorporate the CRW refuge into the seed bag. In addition to the seed mix request, Pioneer also requested a reduction in refuge for CRW from the standard 20% to 3.5% (±1.5).

A seed blend strategy and reduction in refuge from 20% to >2% represente d a paradigm shift for BPPD’s Insect Resistance Management (IRM) pr ogram, and based on the recommendations of a prior SAP (2000), the Agency was discouraged from permitting seed mixtures as a refuge option for Lepidoptera due to potentially high movement of larvae between Bt a nd non-Bt plants. BPPD conducted a risk assessment (BPPD 2008a and 2008b) and presented preliminary conclusions to the Scientific Advisory Panel for pe er review on February 23-24, 2009.

The SAP (2009) expressed concern about the proposal to greatly reduce refuge for a single toxin, non-high dose PIP. The Panel concluded that there was no compelling evidence to support a reduction in refuge from 20% to >2% from the data provided by Pioneer or found in the public literature. However, the Panel agreed that a seed mix strategy would lend itself to corn rootworm biology. The final recommendation by the Panel was the use of a 20% seed blend for OAM1 due to the non-high dose charac teristics of Herculex Cry34/35 corn. Among other things, the Panel commented on the study by Lefko et al. (2008), upon which Pioneer based its assumptions for the value of the major resistance gene fr equency (0.00035) in the simulation model. The Panel concluded that there was no rigorous ev idence in Pioneer’s re ported data that the frequency of major resistance genes was very low. Using the formulae by Andow and Alstad (1998), the Panel calculated the initial frequency of the resistance allele to be 0.0015 but stated

that Lefko et al. (2008) could not be used reliably to esta blish an initial freque ncy or a reasonable upper bound estimate for the frequency of the ma jor resistance allele due to a number of problems with the study. The SAP further stated that consider ing the limitations of the study, even this estimated value (0.0015) could not be considered a generous upper bound estimate. With respect to Pioneer’s domina nce value chosen for the major resistance allele (0.05), the

Panel stated that such a valu e was a reasonable upper bound estimat e for genes that confer more than 300 fold resistance (fitness of RR genot ype/fitness of SS genotype). Evidence suggests, however, that resistance in CRW may be based on genes of relatively minor effects that confer less than 50-fold resistance. Caprio et al. (2000) suggested that for gene s of this level of effect, no upper bound of less than 1.0 could be assumed.

In October 2009, Pioneer submitted an amended re quest for a 5% OAM1 s eed blend registration. To support this request, the registrant pr ovided three new addenda (MRIDs 478710-01, 478879- 01, and 479108-01) to their original modeling subm ission, which are subject of this review. BPPD received permission from Pioneer to cons ult with academia and other CRW experts on the latest data submission. Their comments and expert opinions are cited in this document. BPPD will discuss its independent simulation analys es of the seed mix proposal using “USEPA population genetics two locus model – POPGEN” and a variation of Caprio et al.’s stochastic- 7 - model (2006). The results of BPPD’s independent modeling are discussed in Section III of this

review.

I. CRW BIOLOGY

a. Additional Data on CRW Biology

Nowatzki et al. (2003a and 2003b) conducted a field study (mark-recapture) with western corn rootworm ( Diabrotica virgifera virgifera , WCR) using a rubidium la beling techniqu e to quantify short range, in-field movement of adult beetles from the time of their emergence. The experiment was conducted in two corn fields near Mead (NE) during 2002 and 2003.

Corn plants were treated with RbCl at the center of a non-transgenic corn field and then infested with 200 WCR eggs/plant. Larvae feeding on the treated plants were labeled with RbCl nondisruptively as were newly emerged adults. Be etle movement was then tracked with traps positioned in a circular pattern at fixed distan ces (50 ft, 100 ft, 200 ft, 300 ft, 400 ft, and 600 ft) from the center infested area to the field edge . Beetles were collected throughout the emergence time at regular intervals.

Newly emerged beetles were collected concurrently from the center infested area of the fields in 2003, fed a non-Rb treated diet of corn in the lab, and sub-sampled da ily for five days in order to determine the Rb-decay rate after emergence. Fi rst order exponential decay model was fit to natural log transformed data (Rb concentrations). According to th e model, labeled beetles were distinguishable from unlabeled beetles for approx imately four days (no difference between male and female beetles).

The Rb-decay curve allowed 1) estimating the time from when beetles le ft the treated area to when they were captured in the traps but 2) required an assumption regarding unknown Rb

concentration of individuals af ter they had stopped feeding on Rb-treated plants (= t 0 ). Nowatzki

et al. assumed that a beetle’s t 0 -Rb concentration was equal to th e average Rb concentration of beetles captured in traps within the Rb-treated region of the corn field. The rate of movement was calculated using 1) the distance traveled from the center of the fiel d to the trap and 2) estimated time elapsed since feeding had ceased.

Estimated beetle movement results sepa rated by timing and sex are listed in Table 2 . In summary, males moved an average of 13.9 m/da y prior to female emergence and 15.2 m/day during peak female emergence when the data were pooled across both years. Females moved on average 1.9 m/day during peak female emergen ce and 13.1 m/day during post-female emergence (data pooled across years).

Pioneer concluded from this study that male WC R beetles movement was much less than what was previously assumed (45 m/day). The regist rant proposes that 15 m/day for males and 13 m/day for females should be a more realistic estimate of WCR beetle in-field movement.- 8 - Table 1. Time periods capturing beetle mo vement based on biological significance Relevant time periods Female presence Estimated movement rate Pre-female emergence > 10% Male Peak-female emergence 40-70% Male and female Post-female emergence > 90% Female

Table 2. Male and Female Beetle Movement Results from Rb Study (data from 2002 and 2003)

Time

Period Sex N Mean

Movement (m/day) St Dev

(m/day) Min.

(m/day) 25 th

Percentile

(m/day) Median

(m/day) 75 th

Percentile

(m/day) Max.

(m/day)

2002

Prefemaleemergence M 305 13.2 23.1 0 0 4.9 15.9 220.8

F 59 0.06 0.47 0 0 0 0 3.6 Peak-

femaleemergence M 125 16.0 26.2 0 0.7 7.0 23 188.5

Postfemaleemergence F 26 12.8 17.7 0 0 0 30.4 41.6

2003

Prefemaleemergence M 47 18.6 30.2 0 0 6.3 25.7 152.7

F 16 8.8 13.8 0 0 0 17.9 38.9 Peak-

femaleemergence M 13 7.6 15.2 0 0 0 10.2 40.0

Postfemaleemergence F 41 13.3 13.6 0 0 7.9 25.5 41.4

Combined Across Years

Prefemaleemergence M 352 13.9 24.2 0 0 4.9 18.6 220.8

F 75 1.9 7.2 0 0 0 0 38.9 Peak-

femaleemergence M 138 15.2 25.4 0 0 6.7 22.6 188.5

Postfemaleemergence F 67 13.1 15.2 0 0 6.4 26.0 41.6

Modified from MRID 478879-01- 9 - II. PIONEER MODELING (MRIDs 478710-01, 478879-01, and 479108-01)

A. Purpose

With the support of three further modeling anal yses, Pioneer seeks to demonstrate that the

addition of a refined and better characterized mode ling parameter - an explicit in-field dispersal component for adult corn rootworm - adds to the durability of a seed mixture such as OAM1 and makes the 5% seed blend superior to block refuge strategies for 59122-7 corn.

B. Simulation Modeling: Assumptions and Scenarios

a) Assumptions: Pioneer’s original model (MRID 473567-08) was de terministic and spatially implicit but has since been modified to be spatia lly explicit between and within Bt and refuge fields in order to accommodate the incorporation of adult CRW in-field movement. No previously published or publicly available CRW model has incorporated and simulated the effects of such a parameter on the durability of 59122 maize; rather, previous models assumed an instantaneous and even distribution of refuge beetles in the Bt corn once they reached the edge of the field. Pioneer reported that such previously assumed CR W mixing/movement characteristics become increasingly unrealistic as Bt field size increases.

Pioneer modified and varied several input va lues for main modeling parameters (see value ranges in Table 3 ) based on the feedback received from the SAP 2009 and EPA to more conservatively assess the durability of 59122-7 mai ze for different refuge deployment scenarios. In Pioneer’s revised modeling, the effect of a ma jor resistance gene on the trait durability in absence of a minor gene was inve stigated as to avoi d uncertainties around th e interaction of the two genes. In the original model, Pioneer ha d assumed, based on interpretations of results by Lefko et al. (2007) that the pr esence of a minor gene allowe d for sufficient survival of susceptible genotypes which helped delay the onset of major resist ance evolution. In the latest modeling submission, the registra nt provided 1) worst-case, 2) benchmark, and 3) best-case scenarios for main parameters in the model. Most other assumptions of the original model remained in the modified version (e.g., de nsity-dependent mortality, overwintering egg mortality) with the exception of, for ex ample, pre-dispersal tasting mortality.

The model consists of a grid representing 100 m x100 m (1 ha) subunits (cel ls) within a field or set of fields. The program has a limit of 50 x 50 cells for a total evalua tion of 2500 ha. For the simulations reported in Pioneer ’s addenda, a maximum size of 4x10 cells (400 m x 1000 m) or 40 ha was assumed. Within each cell, the WCR simulation model by Onstad (2006) was implemented to capture larval population dynami cs. Movement of CRW adults between cells was based on the data generated by Nowatzki et al. (2003b). A cell in the model either represented a refuge block, a 59122 maize block, or a seed mix block (5% seed blend) depending on the scenario modeled. Once male dispersa l had occurred, mating was initiated, and oviposition was modeled uniform ly within each cells.- 10 - Overwintering mortality occurred during the egg stage and was se t at 50% (Godfrey et al. 1995). Larvae were represented by two life- stages (early and late-stage la rva), and larval movement and mortality occurred during the early stage only (f irst instars). Density dependent mortality for later-instars was introduced by using Onstad’s density dependent surv ival algorithm (Onstad et
al. , 2006). Emergence of adults was based upon the emergence curves by Nowatzki et al. (2008). Males emerged on average 7 days before females; emergence in Bt fields was further delayed by an additional 7 days for both sexes.

The model code was verified to confirm the expected behavior of the model for extreme scenarios (Onstad communi cation with Pioneer).

Table 3. Modeling Parameters and Input Values

Parameter Range values Worst-case - BPPD

defined* Worst-case - Pioneer

defined Benchmark

value Best-case value

Initial adult density/ha N/A 80,000 80,000 80,000 80,000

Proportion of males N/A 0.5 0.5 0.5 0.5 Plants/ha N/A 79,027 79,027 79,027 79,027 Initial frequency of resistance gene 0.0005-0.01 0.01 0.01 0.001 0.0005

Dominance 0.025-0.2 0.2 0.1 0.05 0.025 Early SS-survival 0.0125-0.05 0.05 0.025 0.025 0.0125 Early RR-survival N/A 1 1 1 1 % off-type seed N/A 0.75 0.75 0.75 0.75 Fecundity N/A 356 356 356 356 SS-oviposition rate N/A 0.32 0.32 0.32 0.32 RR-oviposition rate 0.32-1 1 1 1 0.32 Probability of leaving Bt N/A 0.625 0.625 0.625 0.625

Probability of leaving non-Bt N/A 0.5 0.5 0.5 0.5

Early larval survival during movement N/A 0.5 0.5 0.5 0.5

Overwintering

survival N/A 0.5 0.5 0.5 0.5

♂ movement pre- ♀ emergence (m/d) N/A 15 15 15 15

♂ movement peak- ♀ emergence (m/d) N/A 15 15 15 15

♀ movement postmating (m/d) N/A 12 12 12 12

% block compliance 70-100 70-100 70-100 70-100 70-100

BPPD extracted this information from MRID 478710-01 and MRID 478879-01 * BPPD asked Pioneer to explore more cons ervative parameter values than in thei r worst-case situation. These values are reflected in the assumptions name d ‘worst-case - BPPD defined’. Shaded fields indicate parameters whos e values were changed for output runs- 11 - b) Refuge Deployment Strategies Modeled:

Several scenarios were modeled with different refuge deployme nt approaches and assumptions about grower compliance with refuge require ments (MRID 479108-01). BPPD lists some of

these in Table 4 . Pioneer then varied input values of uncertain paramete rs (Table 3) to explore a possible range of durability outco mes for each scenario and also to compare durability results between different deployment options.

Scenario #1 involved modeling of 100% 59122 in conti nuous corn fields w ithout any designated refuge portion.

Scenario #2 involved modeling of 59122 maize in a continuous landscape with a 20% block refuge. The refuge was moved to different lo cations twice during th e time modeled. Pioneer stated that, based on communications with growers and consultants, moving refuges from year to year was a realistic assumption, Grower comp liance with refuge was assumed to be 70%.

Scenario #3 simulated the infield strip refuge opti on with a 20% refuge and 80% compliance with refuge requirements.

Scenario #4 was a variation of scenario 2) and incorporated the planting of 59122 corn by growers for four years followed by a 5% seed bl end starting the fifth gr owing season. 59122 corn was modeled with 20% block refuges (location moved once) and a 75% gr ower compliance with refuge requirements.

Scenario #5 modeled the 20% infield strip refuge with 75% compliance with refuge requirements. After four initial years with infiel d strip refuges, a 5% seed blend was introduced.

Scenario #6 involved the modeling of a 5% seed blend without a prior deployment of a block refuge.

Table 4. Different Refuge Deployment Scenarios Modeled by Pioneer

Scenario

Number Refuge Option

#1 No refuge #2 20% Block (moved location twice) with 70% compliance #3 20% strip refuge with compliance ranging from 50-83% #4 20% Block only (moved location once) for 4 yrs with 75% compliance; introduction of 5%

seed blend after 4 th year #5 20% strip refuge only for 4 yrs with 75% compliance; introduction of 5% seed blend after 4 th

year #6 5% Seed blend only; no inclusion of prior use-history

C. Sensitivity Analyses

Pioneer conducted numerous sensitivity analyses for parameters with uncertainty by varying the input values in block, strip, and seed blends to assess effects on estimated durability of DAS59122-7. Those parameters that had an effect on th e estimated durability are labeled ‘sensitive’ in Table 5. However, in their or iginal submission Pioneer used the descriptions ‘relatively- 12 - sensitive’ and ‘relatively insens itive’. Since Pioneer did not define what was meant by ‘relative’ as compared to potentially ‘very’ sensitive, B PPD simplified the descriptions by using the terms

‘sensitive’ and insensi tive’ only. The parameters investigated were:

• % seed blend: range of 2.5% - 10%

• Strip width (number of refuge rows): range of 4 rows – 20 rows

• % strip compliance: 50% - 100%

• % block refuge compliance: 50% - 100%

• Block refuge position: fixed or variable

• Initial adult density ; range of 20,000-160,000

• Overwintering survival: 0.2, 0.5, and 0.8

• Male movement (during pre- and peak-female emergence): range of 5m/d- 45m/d

• Female movement (post-mating): range of 5m/d - 45m/d

• Dominance of resistance gene: range of 0.05 – 0.2

• Survival rate of SS-genotype at ear ly life-stage: range of 0.0125 – 0.05

• Initial resistance allele fr equency: range of 0.0005 – 0.01

Whether parameters were or were not sensitive is displayed in Table 5 . In Pioneer’s model, dose, initial R-allele frequency, dominance and overwintering survival were sensitive parameters under all three deployment strategies. Compliance rate was a sensitive parameter for the 20% strip and block refuge. Movements of male and female adults dur ing different time periods were sensitive parameters and varying the distance dispersed per day aff ected the durability of the trait when deployed as a 20% block refuge. Varying adu lt movement in the 5% seed blend and infield strip option did not affect the durability of the trait.

Table 6 shows the durability outcomes (in years) for se nsitivity analyses of various seed blends implemented after four years of only 1) 20% bl ock refuge (refuge in same location and 100% compliance), 2) 20% strip refuge with 75% comp liance, and 3) 20% block refuge (refuge moved once in four years) with 75% comp liance. The last scenario in Ta ble 6 looks at only a seed blend option without considering prior use history. Each deployment st rategy was modeled with worst case, benchmark, and best-case parameter va lues/assumptions. Among all scenarios and combinations of assumptions and seed blends , the 20% strip refuge assuming 100% compliance had the greatest durability among all refuge deployment options. Among the remaining refuge options, the following order of decreasing durability was observed.

For Pioneer’s defined best-case parameter values , durability measured in years was second highest for the 20% block refuge with 100% comp liance with seed blends ranging from 2.5% to 10% (19 to >20 years). The seed blend deployment without considering prio r use history resulted in a durability ranging from 17 to > 20 years. There were small differences in years predicted to resistance between the blend percentages, and the higher blends had be tter durability. The 20% strip refuge with 75% compliance and various seed blends resulted in a durability ranging from 16 to >20 years. The 20% block refuge deploy ment assuming 75% compliance followed by seed blends resulted in a durability ranging from 15 to19 years.- 13 - For Pioneer defined worst-case parameter values , a 20% block refuge with 100% compliance as well as the 20% strip refuge with 75% complian ce both followed by seed blends after four years had the greatest durability (5 years for seed blends ranging from 2.5-10%). Seed blends ranging from 2.5% to 10% resulted in a durability of 4 years (2.5%-3.5%) or 5 years (5%, 7.5%, and 10%). There was essentially no difference in pr edicted durability betw een seed blends of different percentages for the worst-case a ssumptions. The 20% block refuge with 75% compliance followed by seed blends of different per centages resulted in four years of durability.

For Pioneer’s benchmark parameter values , durability for seed blends ranged from 8 to12 years; higher blend percentages predicted higher durabil ity. For 20% block refuge with 100% and 75% compliance followed by seed blends after four ye ars, durability ranged from 8 to 10 years and from 7 to 9 years, respectively.

Table 7 shows the sensitivity results for trait durabili ty measured in years for the block refuge when compliance was varied for the 20% block re fuge. With an assumption of 50% compliance, the trait was predicted to last 8 years; with 70% and 80% compliance, the trait was predicted to last 10 years; and with 90% compliance, the tr ait was predicted to la st 13 years; with 100% compliance, the trait was predicted to last more than 20 years.

Table 8 shows the sensitivity results for trait durabili ty measured in years for the strip refuge when compliance was varied for the 20% strip re fuge strategy. When farmers were compliant 50% of the time with planting refuges, then Pionee r’s model predicted that resistance to the trait evolved in 8 years. With 70%, 80%, and 90% compliance, resistance did not evolve until 9 years, 10 years, and 12 years, respectively.

Table 9 shows the sensitivity results for three de ployment strategies (20% block with 100% compliance, 20% infield strip with 100% comp liance, and 5% seed bl end without prior usehistory) when main parameters, such as domin ance and initial allele frequency of the major resistance gene, and dose assumptions were varied.

When dominance for the major resistance allele was increased from 0.025 to 0.2, the durability decreased from 17 to 6 years for the 20% block re fuge, 20 to 7 years for the 20% strip refuge, and 15 to 5 years for the 5% seed blend. The re lationship between deployment options and their estimated trait durability was generally: 20% Strip > 20% Block > 5% Blend

.

When initial resistance allele frequency was increased from 0.0005-0.01, the durability decreased from 11 to 6 years for the 20% block refuge, 16 to 8 years for the 20% strip refuge, and 12 to 5 years for the 5% seed blend. The relationship be tween deployment options and their estimated trait durability was generally: 20% Strip >5% Blend ≈ 20% Block

.

When survival of the susceptible genotype was increased from 0.0125 to 0.05, the durability increased from 9 to 13 years for the 20% block re fuge, 14 to 18 years for the 20% strip refuge, and10 to 12 years for the 5% seed blend. The rela tionship between deployment options and their estimated trait durability was generally: 20% Strip > 20% Block ≈ 5% Blend

.- 14 - Table 5. Sensitivity analyses for th ree different refuge deployment scenarios

Parameters 20% strip Refuge 20% block 5% seed blend Comments

Dose Sensitive Sensitive Sensitive Mortality for SSgenotypes was
varied from 95-

98.75%

R-allele frequency Sensitive Sensitive Sensitive Initial frequency was

varied from 0.0005-

0.1

Dominance Sensitive Sensitive Sensitive Value was varied

from 0.025-0.2.

Compliance Sensitive Sensitive N/a 100% was assumed when the parameter

value was held constant. Sensitivity values were explored

ranging from 50-

90%.

Initial density Insensitive Insensitive Insensitive Sensitivity values were explored ranging from 20,000-160,000.

Movement 1 Insensitive Sensitive Insensitive

Movement 2 Insensitive Sensitive Insensitive

Movement 3 Insensitive Sensitive Insensitive Sensitivity values were explored ranging from 5-45 m/day.

Refuge

location N/a Sensitive N/a Unclear from

submissions what

was modeled.

Overwinter.

Survival Sensitive Sensitive Sensitive Value was varied

from 0.2-0.8. In the sensitivity analyses, Pioneer changed the value of one parameter at the time

to determine the effect such a change had on the durability of the Bt trait under two refuge deployment scenarios. For the remaining parameters that were held constant, benchmark parameter values were assigned for most

cases.

Compliance was varied

from 50%-90%. Hence noncompliance ranged from 10%-50% and represented the percentage of growers who planted 0% refuge.

1 male movement prior to female emergence

2 male movement during peak female emergence

3 post-mating female

movement

Table extracted and modified from MRID 478879-01 and MRID 479108-01- 15 - Table 6. Sensitivity Analysis for Seed Blend P ercentage and Effects on Du rability of 5 9122 Corn

Worst Case

–Pioneer defined

Initial Resistance Allele Frequency=0.01

Dominance Value =0.1

surv-ss= 0.025 ovip-ss=0.32

ovip-rr =1 Benchmark

Initial Resistance Allele Frequency=0.001

Dominance Value =0.05

surv-ss= 0.0125

ovip-ss=0.32

ovip-rr =1 Best-case

Initial Resistance Allele

Frequency =0.0005 Dominance value=0.025,

surv-ss= 0.0125

ovip-ss=0.32 ovip-rr =0.32

Various % Seed Blends Following Block Refuge after 4 Years Refuge Deployment Strategy 2.5 3.5 5 7.5 10 2.5 3.5 5 7.5 10 2.5 3.5 5 7.5 10

20%+Fixed+

100% compliance followed by

various % blended refuge 5 5 5 5 5 8 9 9 10 10 19 >20 >20 >20 >20

20% strip with

75% and

100% compliance ; followed by

various % blended refuge 5

6 5

6 5

6 5

6 5

6 9

10 9

10 10

11 10

12 11

12 16

18 18

19 19

>20 20

>20 >20

>20

20% block

with variable

locations (moved once in four years)

with 75% compliance

rate ; followed by various % blended refuge 4 4 4 4 4 7 8 8 9 9 15 17 18 19 19

Various %

blended refuge 1 4 4 5 5 5 8 9 10 11 12 17 19 >20 >20 >20

Table extracted and modified from MRID 478879-01 and MRID 479108-01

1 These are seed blend options that do not take into consideration prior use-history of 59122 corn.- 16 - Table 7. Sensitivity Analysis for 20% Bloc k Refuge Compliance and Predicted Durability Block Compliance

Parameter Low Med High

Refuge Scenarios 20% block fixed

location 20% block fixed

location 20% block fixed

location Initial Adult Density/Hectare 80000 80000 80000 Proportion of Males 0.5 0.5 0.5

Plants/Hectare 79072 79072 79072 Initial Resistant Allele Frequency 0.001 0.001 0.001

Dominance Value of Resistant Allele 0.05 0.05 0.05 Survival Rate of Susceptible at Early Stage 0.0125 0.0125 0.0125

Survival Rate of Resistant at Early Stage 1 1 1

Offtype Seed Proportion 0.0075 0.0075 0.0075 Fecundity 356 356 356 Oviposition Rate of Susceptible in

Toxin 0.32 0.32 0.32

Oviposition Rate of Resistant in

Toxin 1 1 1

Probability of Leaving Bt 0.625 0.625 0.625 Probability of Leaving Normal 0.5 0.5 0.5 Early Larval Survival Rate During

Movement 0.5 0.5 0.5

Over-winter Survival Rate 0.5 0.5 0.5
Pre Female Emergence Male

Movement* 45 45 45

Peak Female Emergence Male

Movement 15 15 15

Post-Mating Female Movement 12 12 12 Block Refuge Position (Fixed or Years Changed) Fix Fix Fix

Block Compliance (% Compliance) 50% 70%/80%/90% 100%
Block Durability (Year when R allele > 50%) 8 10/10/13 >20

Table extracted and modified from MRID 478879-01- 17 - Table 8. Sensitivity Analysis for 20% Infield St rip Refuge Compliance and Predicted Durability
Strip Compliance Parameter Low Med High

Refuge Scenarios 20% Strip 20% Strip 20% Strip

Initial Adult Density/Hectare 80000 80000 80000

Proportion of Males 0.5 0.5 0.5

Plants/Hectare 79072 79072 79072

Initial Resistant Allele Frequency 0.001 0.001 0.001

Dominance Value of Resistant Allele 0.05 0.05 0.05

Survival Rate of Susceptible at Early Stage 0.0125 0.0125 0.0125

Survival Rate of Resistant at Early Stage 1 1 1

Offtype Seed Proportion 0.0075 0.0075 0.0075

Fecundity 356 356 356

Oviposition Rate of Susceptible in Toxin 0.32 0.32 0.32

Oviposition Rate of Resistant in Toxin 1 1 1

Probability of Leaving Bt 0.625 0.625 0.625

Probability of Leaving Normal 0.5 0.5 0.5

Early Larval Survival Rate During

Movement 0.5 0.5 0.5

Over-winter Survival Rate 0.5 0.5 0.5

Pre Female Emergence Male Movement 15 15 15

Peak Female Emergence Male Movement 15 15 15

Post-Mating Female Movement 12 12 12

Strip Refuge Position (Fixed or Years Changed) NA 1 NA NA

Strip Width (rows) 4 4 4

Strip Compliance (% Compliance) 50% 70%/80%/90% 100%

Strip Durability
(Year when R allele > 50%) 8 9/10/12 14

Table extracted and modified from MRID 478879-01- 18 - Table 9. Predicted Durability for DAS-59122-7 When Sens itive Parameters Were Varied for 5% Seed Blend, 20% Block, and 20% Infield Strip Refuge

Dominance Initial Resistance Allele Frequency Survival Refuge Deployment

0.025 0.05 0.1 0.2 0.0005 0.001 0.01 0.0125 0.025 0.05 20% block (fixed) + 100% compliance 17 9 7 6 11 9 6 9 11 13

20% strip, 100% compliance 20 14 10 7 16 14 8 14 15 18

5% seed blend 15 10 7 5 12 10 5 10 11 12

Table created from informa tion extracted from MRID 478879-01

D. Output Results

The “output results” section discusses Pion eer’s durability estimates for different 59122 deployment options for the different parameter sc enarios (i.e. worst-case, benchmark, and bestcase), while Pioneer’s sensitivity analysis sectio n explored the ranges of values for relevant parameters in order to determine how robust a pr edicted output result would be. Predicted output (durability) and sensitiv ity analyses need to be considered jointly.

Table 10 lists the results from simulation runs with different assumptions (worst-case, best-case, and benchmark assumptions) and various deployment strategies: 1) no refuge; 2) 5% seed blend following a i) 20% block refuge (location moved twice in four years) with 70% compliance; ii) 20% block refuge (location moved once in four years) with 75% compliance; iii) 20% strip refuge with 75% compliance; iv) 20% strip refuge with 85% compliance; and 3) 5% seed blend without prior-u se history and 100% compliance.

After a meeting with the BPPD IRM team in October 2009, Pioneer modeled a new worst-case scenario, which is referred to in the Table 10 as ‘worst cas e - BPPD defined’.

Assuming BPPD’s worst case defined parameters, the no-refuge scenario resulted in the ‘least amount of time to product failure’ (3 years), wh ile both the 20% block refuge scenarios and the 5% seed blend (with no prior use history) scenario predicted f our years to product failure. The 20% strip refuge scenarios with 75% and 80% comp liance resulted in the greatest durability (5 years).

Assuming Pioneer’s worst-case defined parameters, the 20% stri p refuge scenario with 83% compliance resulted in the greatest durability (6 years), while a) the 20% strip refuge with 75% and 50% compliance and b) the 5% seed ble nd predicted 5 years to product failure. The remaining scenarios predicted the same period to product failure as under BPPD’s worst case defined assumptions (3 years for no refuge and 4 years for the 20% block refuge scenarios).

For benchmark parameters , the trait durability was predicted to be highest for the 20% strip refuge with 75% compliance (80% compliance not evaluated) and the 5% seed blend (10 years- 19 - both) followed by the two block refuge deploymen t strategies (8 years both) and no refuge strategy (5 years).

For best-case parameter values, the trait durability was pr edicted to be more than 20 years for the 5% seed blend, 19 years for the 20% strip ref uge with 75% compliance (80% compliance not evaluated), 18 years for 20% block refuge with 75% compliance rate for four years followed by 5% seed blend thereafter, 13 years for 20% bloc k refuge with 70% compliance, and 9 years for the no-refuge deployment scenario.

Table 10. Durability for DAS-59122-7 und er different refuge deployment strate gies as expressed in ‘years to product failure’

Worst Case -

BPPD defined Worst Case –

Pioneer defined Benchmark

Parameters Best-case Scenario

Refuge Deployment Strategy R-allele freq =0.01

Dominance = 0.2

SS-survival = 0.05 Density = 160,000

Movement= 5m/d 1 R-allele freq = 0.01

Dominance = 0.1

SS-survival = 0.025 R-allele freq = 0.001

Dominance = 0.1

SS-survival = 0.025 R-allele freq = 0.0005

Dominance = 0.025

SS-survival = 0.0125

No refuge 3 years 3 years 5 years 9 years 20% Block (moved location twice), 70% compliance 4 years 4 years 8 years 13 years

20% strip refuge with compliance ranging

from 50-83% 4 years (50%) 5 years (80%) 5 years (60%) 6 years (83%) Not conducted Not conducted

20% Block (moved location once), 75% compliance followed by 5% seed blend after 4 years 4 years 4 years 8 years 18 years

20% strip refuge, 75% compliance followed by 5% seed blend 5 years 5 years 10 years 19 years

5% Seed blend 4 years 5 years 10 years >20 years

Table extracted and modified from MRID 478879-01 and MRID 479108-01 SS-oviposition rate was assumed to be 0.32 for wo rst case, benchmark, and best-case scenarios RR-oviposition rate was assumed to be 1 for wo rst-case and benchmark and 0.32 for best-case

1 Pre-female emergence (male movement), peak-female emergence (male movement), and post-female emergence (female movement) parameters were set to 5m/day.

E. Pioneer Conclusions

Pioneer concluded: “Depending upon the dimensi ons of the field, there can be areas of a 59122 field inaccessible to refuge b eetles. These distant ends of the 59122 field become zones of continuous selection that may have expected durabilities similar to the no-refuge scenario (five years durability). Thus, although the block refu ge can have good durability in a best-case scenario, it may lead to unintenti onal selection for resistance in th ese "hot spots." Alternatively, with a 5% blended refuge, there is never a risk of hot spots with uninten tional selection because susceptible CRW males emerging from refuge plants will be in close proximity (3.6 m) to any- 20 - female beetles that emerge from 59122 plants an ywhere in the field. Moreover, blending refuge ensures 100% compliance with CRW refuge requi rements without the gr ower having to incur any extra work, planning or expense.

In each scenario modeled, implementing a blen ded CRW refuge extends the durability of 59122 maize compared to a 20% block refuge, incl uding scenarios when 59122 maize has been in commercial use for four years.

The results of all the scenarios modeled here dem onstrate the superiority of a 5% blended refuge relative to block refuge deployment strategies for 59122 maize and corn rootworm. Based upon these results, a blended corn rootworm ref uge, which ensures 100% corn rootworm refuge compliance, provides the most eff ective tool that the grower can use to delay the development of

corn rootworm resistance to 59122 maize. Movi ng to a blended refuge as quickly as possible will extend the durability of existing 59122 products.”

III. BPPD REVIEW AND CONCLUSIONS

During the risk assessment process, the BPPD/I RM team routinely considers realistic case scenarios when evaluating simulatio n models. However, it can be very useful to look closely at scenarios with worst-case assumptions especially in cases when a degree of uncertainty is associated with important modeling parameters (e.g. dominance, ini tial resistan ce allele frequency, adult movement).

Risk assessment conclusions are reached based on th e best scientific information available to the Agency at the time of the assessment which is uni que to any particular pest-crop system. In the case of CRW, where the larval life-stage inhabits a subterranean environment, there is less IRM knowledge than for above-ground pests of corn. Fo r example, it has been more challenging to determine the dose of Bt toxins targeting immatu re CRW or to conduct larval behavioral studies than for Lepidopteran target pests. Such uncertain ties have required that BPPD “bracket” the risk of putative resistance evolution. This goal ha s been accomplished by estimating risk using realistic assumptions based on available data and “defensible” worst-case assumptions in the model. With this approach, the ‘actual’ risk of resistance evol ution may lie somewhere within this range of potential risk. If modeling results predict little to no-change in estimated risk of resistance with worst-case assumptions, then science conclusions will be more robust and

defensible.

For this current risk assessment, BPPD has b een presented with a new opportunity to estimate

the risk of resistance evolution with a recen tly developed simulation model. Deterministic

simulations will be undertaken with the “U SEPA population genetics two locus model – POPGEN”. A probability analysis will compleme nt the deterministic simulations (conducted by Dr. Caprio) and based on a PERT distribution (min. value, most lik ely value, and max. value) for parameters of uncertainties. The Agency is now in a position to analyze a registrant submitted model, compare and assess durability results of two independent deterministic models, and estimate the probability of occurrence for a predicted durability result.- 21 - In addition to the analysis of Pioneer’s m odel using POPGEN and c onducting the probability analyses, a variation of a stocha stic, spatially explicit model de veloped by Caprio et al. (2006)

was used to assess the resistance evolution for tw o geographically distinct regions with different CRW biotypes. Resistance evolution was estimated in the western Corn Belt region where data from Nowatzki et al. (2003) s uggest that male CRW may disper se on average 15m/d (field size average between 50-60 acres and max. 250-300 acr es). Second, resistance evolution was

estimated in the Red Zone where data indicate (Spencer 2009, Spencer -personal communication) that CRW adults disperse at mu ch greater distances (between 30-200m/d; field size average between 40-80 acres). Given the uncertainty regarding CRW movement, potential differences in behavior need to be explored when estimating risk of resistance evolution.

A. Review of Pioneer’s Modeling Assumptions

Pioneer reported durability of 59122 corn as the number of years to product failure for all scenarios modeled (meaning resistan ce evolution in the target pestpopulation). Durability of a trait can also be expressed as the ‘relative rate of adaption’ (RRA is t ypically used in public literature), which is the speed at which resistance evolves under certain conditions (i.e. % refuge) with respect to a baseline. One advantage of usi ng RRAs is that they provide rates (rather than absolute values) and remind users of such data that modeling estimates should not be taken as

absolute estimates. Pioneer stated that their re sults were presented in years because time to product failure was very short for all simu lations and RRA’s would not have been distinguishable between different scenarios. The years of durab ility estimated by Pioneer and reported in this review should not be viewed as absolute time estimates of durability (or onset of resistance).

1. Pioneer’s scenarios

With respect to modeling scenarios, BPPD conc ludes that while the ‘n o-refuge’ deployment strategy is informative, it represents an unrealistic and overly conservative scenario. The estimated years to product failure from this scen ario should not be used to establish a realistic lower bound estimate for durability; however, a ‘ no-refuge’ scenario does serve as a useful “negative” control.

Pioneer focused their main risk assessment c onclusions on results from the 5% seed blend modeling, not including prior use history of 59122 co rn. BPPD believes that the 5% seed blend scenario by itself cannot be used to establish a realistic esti mate for durability of 59122 corn.

While it is informative to have this scenario in cluded in the risk assessment, much like the “norefuge” scenario, it is more re alistic to consider the prio r use-history of 59122 corn.

The registration for Event DAS-59122-7, also kn own as Herculex Rootworm (HXRW), was granted by the Agency in August, 2005 for co mmercial plantings be ginning with the 2006 growing season. Growers have since had the opport unity to plant four years of corn expressing the Bt toxin Cry34/35 (refuge optio ns available to-date are 20% ad jacent, block refuge or 20%- 22 - infield strip refuge > 4 rows wide). CRW have been exposed to Cry34/35 for a period of time during which a putative major resi stance gene could have increased its initial frequency in the pest population. Therefore, Pionee r’s modeling output with scenar ios that considered prior-use history of DAS-59122-7 corn are likely to be mo re reflective of real world conditions. With respect to grower adoption of infield refuge option, BPPD has heard anecdotal accounts that approximately 30% of corn growers ma y be planting infield strip refuges.

Scenario

Number Refuge Option

#1 No refuge 1
#2 20% Block (moved location twice) with 70% compliance #3 20% strip refuge with compliance ranging from 50-83% #4 20% Block (moved location once), 75% compliance followed by 5% seed blend after 4 years #5 20% strip refuge, 75% compliance followed by 5% seed blend

#6 5% Seed blend 1

1 unrealistic scenario - not supported by use-history of DAS-59122-7

2. Pioneer’s assumptions and uncertainties

BPPD re-emphasizes that the numbers of year s estimated under Pioneer’s different scenarios modeled represent relative values of durability and should not be taken at face value or as absolute estimates to predict durability of 59122 corn. Rather, output results from modeled scenarios should be compared relative to each ot her. However, the two worst-case scenarios using the most conservative parameter values resu lted in relative short time estimates for trait durability for all scenarios. This may indicate that under worst-case assumptions, regardless of the scenario, (no refuge, struct ured refuge with low complia nce rates, a combination of structured refuges and seed blends, or seed blends only) resistance evolution may develop quickly.

Worst-case : Pioneer assumed for worst-case scenarios (as well as benchmark parameter values) that ss- genotypic survival was 2.5%. This genotypic survival value was not directly measured in the registrant’s experiment but rather represen t the total average survival (0.025) measured on 59122 corn in the field (MRID 473567-05). BPPD c ontends that ss-survival on Cry34/35 could be set higher for worst case assumptions because the average survival estimated by Pioneer may not be a good representation of wh at has been observed as maximu m survival at some of their

individual field sites. Storer et al. (2006) reported that Cry34/35 su rvival in the field could be as low as 4% (not adjusted for density-dependence) . BPPD proposes the use 5% as a worst-case survival rate instead (see ‘w orst-case - BPPD defined’, Table 10 ).

A major resistance gene frequency of 0.01 appears to be a conservative value for this parameter although recent studies with WCR s uggest that the resistance allele frequency for Bt toxins may be as high as 0.02 (Hibbard, 2010, personal communication). Because Cry34/35 is not a high

dose toxin and allows for CRW survival on 59122 corn, BPPD concludes that a dominance value for the major resistance gene needs to be set hi gher than what Pioneer assumed in their worstcase simulations (0.1). BPPD recommends that a more conservative value for dominance, such as 0.2, be explored as a maximum value.- 23 - Worst-Case – Pioneer defined

R-allele freq = 0.01 Dominance = 0.1 SS-survival = 0.025

Benchmark : The term ‘benchmark’ implies that values under this description may be the most

realistic case. Pioneer chose an initial frequency of 0.001 for the putative major resistance gene; however, this is the value historically used fo r Lepidoptera. Considering how quickly resistance was selected for in the lab (Meihls et al. 2008; Lefko et al. 2008), 0.001 may be too low. Furthermore, Dr. Tabashnik (2009) stated (pub lic comment submitted to SAP, 2009) that the data from the selection experiments reported by Lefko et al. (2008) demonstrated rapid evolution of resistance by WCR to 59122 cor n, but with incomplete resistan ce. This rapid evolution of resistance may imply that either the major resist ance allele frequency or dominance value in the natural populations sampled were greater than wh at was assumed for these benchmark parameter

values. Pioneer’s choice of a be nchmark value for dominance (0.1) may be adequate if resistance allele frequency was increased to greater th an 0.001. Both dominance and resistance allele frequency have similar effects on the rate of resist ance evolution. If either value is set high in the simulation model, resistance can be expected to evolve quicker th an when they are both set at a lower frequency. If they both are set to a high valu e, resistance will evolve even faster in the

model. Since field resistance has not been observed or reported yet for CRW, benchmark parameter values should only be increased for on e or the other parameter. BPPD states that a reasonable range for the initial resistance alle le frequency is proba bly between 0.005 and 0.001.

A benchmark value of 2.5% for ss-survival due to toxin exposure and other causes may be adequate. Overall, BPPD considers Pioneer’s benchmark values as realistic best-case assumptions.

Benchmark Parameters

R-allele freq = 0.001 Dominance = 0.1 SS-survival = 0.025

Best-case : The SAP (2009) concluded that in considerati on of the limitations of Lefko et al. (2008), an estimated value of 0.0015 for a major re sistance gene was not justifiable. BPPD concurs with the SAP and concludes that a majo r resistance allele frequency in CRW may lie somewhere in the range of 1 in a 1000 (0.001) to 1 in 100 (0.01). As stated earlier in Section III of this review, BPPD considers Pioneer’s parame ter values for “best-case” unrealistically high to

establish an estimate of actual durability.

Compliance : Pioneer assumed that a certain percentage of growers did not plant a refuge at all. In other words, when 70% compliance is modeled, it is implied that 30% of growers planted 0% refuge. After reviewing AB STC’s (Agricultural Biotec hnology Stewardship Technical Committee) data (2002-2008), BPPD reported that in 2007 and 2008 an apparent drop in grower compliance with refuge requirements for PIPs had occurred as compared to prior years (BPPD, Best-Case Parameters
R-allele freq = 0.0005 Dominance = 0.025 SS-survival = 0.0125- 24 - 2009). Those particular results from the anonymous, 3 rd party survey did not differentiate between different degrees of non-compliance, an d reported non-compliance results per farm. The survey also assessed what percen tage of growers planted at leas t a 15% refuge. BPPD specified that in 2007, approximately to 89 % and 95% of the growers surveyed attested to having planted at least a 15% and a 10% refuge , respectively, while in 2008, appr oximately to 86% of growers planted at least a 15% refuge . Considering ABSTC’s data, BPPD concludes that Pioneer’s assumption of 30% non-compliance is too conser vative since this would mean that 30% of farmers planted a 0% refuge. However, the Agen cy has received anecdotal evidence that certain counties/areas appear to have a greater amount of non-compliance than captured by the ABSTC report. This point will be in corporated and evaluated in BPPD’s independent analysis. Compliance is a sensitive parameter in Pioneer’s model; BPPD will carefully consider Pioneer’s outputs with overly conservative compliance percentages that aim to represent refuge planting

behavior across the entire Corn Belt.

Uniform oviposition : Pioneer structured its model in such a manner that oviposition of eggs occurred uniformly in each cell. Work by Br anson (1986) suggests that CRW egg masses in fields are best described by a clumped distribution. This could indicate that female CRW are capable of detecting different hos t qualities. It is unclear what impact Pioneer’s assumption of uniform distribution of eggs may ha ve on the estimated durability of 59122.

Larval dispersal : Pioneer modeled larval dispersal as a one-time event occurring during the neonate stage and adjusted for density-depende nt mortality. BPPD questions whether densitydependence is a factor for neonates leading to di spersal and whether the main focus of dispersal

in the model should be on neonates. In Hibbard et al. (2004) density-dependent dispersal for neonates did not occur during a three-year field study even under high-density pressure (with artificial infestation). And Hibba rd et al. (2005) found that late -instar movement occurred from sufficiently damaged non-Bt pl ants to surrounding transgenic plants. Work by Dr. Hibbard indicates that it is more realistic to assume high la rval movement from late instars and little to no movement for neonates. However, statistically significant neonate movement from Bt plants to non-Bt plants was observed in one year of their field study (Hibba rd et al. 2005). BPPD concludes that late-instar movement in response to resource limitations appears to be real and

should be modeled.

In addition, Pioneer demonstrated in a lab expe riment with CRW larvae and Cry34/35 corn that when exposure to 59122 seedlings occurred and survival was measured at the neonate (0.41), second-instar (0.66), and thir d-instar stage (0.77) that a reduction to 0.002, 0.17, and 0.50, respectively, was apparent (M RID 473567-01). Pioneer’s data s uggests that as CRW larvae mature, they become less susceptible to Cry34/35. Hi bbard et al. (2005) stated that initial larval development on non-Bt roots followed by later moveme nt to Bt roots could accelerate resistance evolution if heterozygot e larvae (RS- genotype) experienced greater survival on Bt. This is supported by results of Meihls et al. (2008), where exposure of neonate larvae to Cry3Bb1 followed by rearing on isoline corn did not result in the development of resistance in choice tests while transferring 2 nd instars from isoline to Cry3Bb1 di d eventually result in resistance, although the level of resistance was reduced and it s development was slower than with complete rearing on Cry3Bb1 corn.- 25 - Based on information provided by Dr. Hibbard (personal communication, 2009) and published as well as unpublished data (Hibba rd et al. 2004 and 2005; Meihls et al., 2008; MRID 473567-01),

BPPD concludes that larval movement at the late r-instar stage is more realistic and important to model than movement at the neonate stage.

Adult beetle movement : Pioneer simulated beetle movement using an exponential distribution (MRID 478879-01) relying on the data generated in a two-year, one-loc ation (very limited geographic scope) rubidium study (Nowatzki et al . 2003), which showed that some male beetles moved as far as 220 ft/day (sample size 305) du ring pre-female emergence and 188 ft/day in 2002 (sample size 125) and 152 ft/day in 2003 (sam ple size 47) during peak female emergence (see Table 2 ). Pioneer concluded that mean male movement should be around 15m/d while mean

female movement was closer to 13m/d. The medi an for male ‘pre-female emergence’ movement ranged from 4.9-6.3m/d, for male ‘peak female emergence’ movement from 0-7m/d, and for male ‘post-female emergence’ movement from 0-7.9m/d. BPPD questions the uniform applicability of the reported ‘average m/d traveled. Dr. Joseph Spencer (University of Illinois) measured average dispersal of adult CRW in Il linois over the course of two planting seasons (ESA 2009; personal communication, 2009) in Bt corn fields a nd non-Bt soybean fields and reported that dispersal could be as low as 30m/d (average 50m/d) but as high as 80 m/d (very low CRW year in 2009). The prior year, Dr. Spencer measured adult male dispersal of up to >200m/d; he stated that when corn rootworm presence was abundant that adult dispersal was high. Dr. Hibbard confirmed the Spencer and Nowatz ki dispersal data and agreed with the SAP (2002) that the adult movement behavior within the WCR was different in these two locations (distinct biotypes) (Hibbard, personal communi cation, 2009). BPPD conc ludes that these two CRW biotypes (NE region and Red Z one) appear to have real differe nces in dispersal behaviors. Short distance dispersal may be mo re appropriate for the Nebraska region while greater dispersal distances appear to be more appropriate fo r populations in the Red Zone. The stochastic modeling output in Section III.C.3 will address th ese differences in adult behavior and explore their effects on 59122 corn separately in these two regions of the Corn Belt.

Probability of leaving non-Bt and Bt roots : Pioneer assumed that 50 % and 62.5% of larvae leave a non-Bt and Bt host, respectively. However, Hibba rd et al. (2004) report ed that between 0.57% and 5.17% of larvae were recovered from nearby non- Bt expressing roots of artificially infested non-Bt plants. On the other hand, Hi bbard et al. (2005) stated that the percentage of CRW larvae leaving Bt roots may be about 25% higher than what is reported for non-Bt roots. This suggests that Pioneer’s probabilities for larvae leav ing non-Bt and Bt s ource may be too high.

SS-Survival : Pioneer derived an overall survival para meter from their field experiment which measured emergence in pure stands of 59122 mai ze and refuge fields (Nowatzki et al., 2008). The mean survival value (0.05) was calcul ated by dividing mean emergence (across locations)/59122 plant by mean emergence (acr oss locations)/refuge plant. Then Pioneer

assumed that half of the survival was due to individuals being homo zygous susceptible (0.025). There is some uncertainty with regard to the su rvival parameter chosen for the susceptible (ss) genotype. The experiment by Nowatzki was conducte d in five different locations across the Corn Belt and over the course of one growing season (2007). The range of mortality in this timelimited study (one year) was 96.0-99.8% for WCR ( 64-94% for NCR). Mortality in this study is

a function of dose of toxin as well as environmen tal factors. Hence, during any given year the- 26 - range of mortality (or survival) could vary even if experimental locations remained the same. Mortality could in fact be lower if environmen tal conditions were favor able to CRW survival (hot and dry). Second, BPPD considered it importa nt to include simulations with lower limits of observed survival in realistic worst-case scenarios rather than to rely on average survival/mortality rates alone and proposed to Pioneer to explore ss-genotype survival at 5%. Pioneer’s own sensitivity analyses showed that output results (durability of trait) were sensitive to changes in this parameter (see Table 5 ), which warrants exploring this parameter further.

Mixing Technology : Pioneer originally reported that bags containing OAM1 had a 1.5% standard error of the mean; a 5% seed mix c ould contain percent ref uge seeds ranging from 3.5- 6.5%. Pioneer has since analyzed data from a tota l of 32 commercial-scale seed blending runs to date (Rood, personal communicatio n, 2009) and reported that seed bags containing the OAM1 product have a blend rate ra nging from 4.910% to 5.075% (95% confidence interval). The amount of seed in each run was enough to f ill approximately 1000 bags of seed. BPPD will model the variation of % refuge seed from 4% - 6% because under high-production output, it is

conceivable that the standard er ror of the mean could increase.

Sensitivity Analyses : Sensitivity analyses are an important part of simulation exercises. They help determine the effects on output results when a range of possible values is considered for parameters of interest (i.e. re veal where uncertainty may be re levant in system). A modeling parameter is determined to be insensitive when changes in its value pr oduce robust results (i.e. cause “little to no change” in predicted durabi lity of the Bt trait). When outputs results are relatively insensitive to changes in values, then uncertainties may be less of a concern for risk

assessment purposes.

The sensitivity analyses conducted by Pioneer show ed that for infield strip refuge, block refuge, and seed blend simulations, the output results were sensitive to changes in the following parameters: dose (survival), initial frequency of the major resistance gene, dominance, and (egg) overwintering survival. For strip and block refuge, output results were sensitive to changes in compliance rate. For block refuge alone, output resu lts were sensitive to movement parameters.

Main parameters of concern that have uncertainty are 1) initial frequenc y of the R-allele, 2) dominance, 3) larval survival on Bt corn, 4) % non-compliance with refuge, and 5) the adult CRW movement parameters chosen by Pioneer. In light of the un certainty, BPPD believes it is better to incorporate more conservative, but still realistic, parameter estimates in model analyses for survival rate, R-allele fr equency, dominance, % non-complia nce, and adult movement for block refuges.

The work of Godfrey et al. (1995) was the ba sis for the assumption that a 50% overwintering survival is appropriate for CRW, and therefore, a sensitivity anal yses for this parameter may not be realistic to consider (Onsta d testimonial, 2009). For grower co mpliance with refuge plantings, BPPD will consider a reasonable range of non-comp liance derived directly from its review of ABSTC’s CAP reports (Bailey, 2008 & 2009; B PPD, 2009), which are also confirmed by a survey conducted in Illinois (G ray, 2009), and consider the per centages of growers who planted

zero % and at least a 15% and 10% refuge. As stated in secti on II. A. 2, ABSTC reported that according to their third party anonymous survey approximately 95% and 89% of growers planted- 27 - at least a 10% (between 10-20 %) and 15% refuge (between 15 -20%), respectively, in 2007; approximately 86% of growers planted at leas t a 15% refuge in 2008. BPPD concludes based on the 2007 and 2008 compliance data that a minimu m, most likely, and maximum value for noncompliance (zero % refuge planted) should be 5%, 10%, and 60% (addressing anecdotal evidence of no-refuge planting in certain counties), respectivel y. BPPD does not concur with Pioneer’s universal assumption of 30% no-refuge non-compliance in their model; however, this percentage is within BPPD’s suggested range. Pioneer’s own sensitivity analysis showed that higher compliance with block refuges resulted in greater durability. When zero % compliance was set at 50%, 70%, and 100%, durability results were 8 years, 10 years, and greater than 20

years.

The sensitivity analysis for male movement pr ior to female emergence showed that this parameter was a very important and sufficiently sensitive when durability was estimated for 20% block refuges. When beetle movement was low (5 m/d) or high (45m/d), Pioneer estimated that durability ranged from 9 years to >20 years for block refuges. Based on the different dispersal/movement data available from Nowatz ki et al. (2003) and Dr. Spencer, as discussed earlier in this section, it will be important to further investigate and discuss the male movement

parameter.

B. BPPD Review of Pioneer’s Modeling Results

The modeling outputs discussed in this section are cited in Table 10 of this review.

Time to resistance using BPPD defined worst-case assumptions : The ‘no-refuge’ simulation represented the upper bound for risk in Pioneer’s modeling and predicted three years of durabi lity for 59122 corn, which is less than the time the product has already been deployed. This resu lt emphasizes that the time to product failure estimated by the ‘no refuge’ scenario is likely unrealistically low and in addition emphasizes that years-toresistance should not be ta ken as absolute values.

The ‘20% block refuge with 70% compliance’ predicted an estimated 4 years of product durability; this predicted durability is the same as for the strip refuge with 50% compliance. Since the two deployment strategi es with either 50% and 70% of compliance resulted in the same amount of years estimated to resistance ev olution, it appears that the strip refuge, which allows for better mixing between refuge and non -refuge beetles can handle a lower compliance rate compared to the block refuge. BPPD conclude s that this may be the most likely explanation for the ‘no difference in durability’ between the two greatly differing compliance percentages of block and strip scenarios.

A 20% strip refuge with 83% compliance proved more durable relative to a 20% block refuge with 75% compliance followed by a 5% seed bl end. BPPD concludes that the strip refuge (allowing for better mixing between refuge and non-refuge beetles) a nd higher compliance rate

fare better in Pioneer’s model than the block refuge with lowe r compliance followed by the seed blend. Introducing the seed blend after having had a block refuge for 4 years did not appear to improve durability relative to 1) the 20% strip refuge with variable co mpliance (50-83%) or 2)- 28 - the 20% block refuge with 70% compliance. As previously stated, BPPD believes that the universal assumption of 30% no-refuge non-co mpliance is overly pessimistic and not representative of the actual numbers reported. Th erefore, 20% block might have proven more durable with assumptions of higher compliance as is suggested by Pioneer’s sensitivity analysis

in Table 7

.

The strip refuge with 75% compliance followed by a 5% seed blend was more durable, although just by one year (one CRW generation), relati ve to a 5% seed blend (assuming no prior deployment of 59122 corn as block or strip re fuge). There was no difference in years of durability between 1) the 20% block refuge with 70% compliance, 2) the strip refuge with 50% compliance, 3) the block refuge with 75% comp liance followed by a seed blend, and 4) the 5%

seed mix.

BPPD concludes that based on Pioneer’s modeling, the 20% strip refuge w ith or without a seed mix after four years appeared to be the most dur able deployment strategies using BPPD’s worstcase assumptions. There was no difference in es timated durability between 20% block refuge scenarios (with or without seed blend) and 5% seed blend strategy (no prior deployment of 59122 assumed). BPPD further concludes that the compliance percenta ges used (% growers planting no refuge) were too low.

None of the scenarios predicte d great durability for 59122 with BPPD’s worst-case assumptions

in Pioneer’s model.

No refuge 20% Block,

70% compliance 20% Strip,

50% -80% compliance 20% Block, 75% compliance 4yrs,

5% seed mix 20% Strip, 75% compliance, 4 yrs, 5% seed mix 5% Seed

mix

BPPD

worst case 3 yrs 4 yrs 4 yrs 5 yrs 4 yrs 5 yrs 4 yrs

Info extracted from Table 10 above

Estimated time to resistance using Pioneer’s worst-case assumptions : Compared to results generated w ith BPPD defined worst-case a ssumptions, results for Pioneer’s worst-case differed only for the strip refuge with variable co mpliance and the 5% seed blend

strategy.

The strip refuge with variable compliance (50%-83 %) resulted in 5 yrs and 6 yrs of durability for 59122 corn compared to 5 yrs for the 5% seed bl end (with no prior block or strip deployment). BPPD concludes that using Pioneer’s worst-case assumptions, a 20% strip refuge with more realistic compliance rates proved to be more durable relative to all other deployment options. There was no difference predicted in durability be tween either block refuge scenarios (with or without seed blends after 4 years). Using Pioneer’s model, the 5% seed blend fared better than both block refuge options and resu lted in 5 years of durab ility rather than 4 years (block refuge deployment).- 29 - No refuge 20% Block,

70% compliance 20% Strip,

50% -83% compliance 20% Block, 75% compliance 4yrs,

5% seed mix 20% Strip, 75% compliance, 4 yrs, 5% seed mix 5% Seed

mix

Pioneer

worst case 3 yrs 4 yrs 5 yrs 6 yrs 4 yrs 5 yrs 5 yrs

Info extracted from Table 10 above

BPPD notes that under worst-case assumptions (BPPD defined and Pioneer defined), infield strip with 80% and 83% compliance ( Table 10 ) was more durable (5-6 years) than any seed blend scenario modeled (4-5 years) when the same pa rameter values were used. While the universal assumption of ‘20% of growers planting no refuge ’ is not supported by real data (Bailey 2008 & 2009; BPPD, 2009), it could re present a worst-case, es pecially in counties w ith very low refuge adoption. The lowest percentage of growers poten tially not planting any refuge appears to be around 15%. BPPD considers 15% a most-likely estimate for zero-% non-compliance across the

Corn Belt.

Estimated time to resistance using Pioneer’s benchmark assumptions : The ‘no-refuge’ scenario resulted in the least amount of estimat ed time to product failure; the strip refuge followed by seed blend, as well as the 5% seed blend scen ario resulted in the greatest number of years to product failu re. Since the 5% seed mix (without prior use-history for blocks or infield strips) is not a real istic scenario, BPPD concludes that with benchmark parameter values, the 20% strip refuge (75% compliance rate s) followed by seed ble nd is the most durable scenario in Pioneer’s model compared with th e scenarios accounting for prior use-history. BPPD notes that a comparison to a 20% strip refuge with variable complian ce could not be made

because Pioneer did not model this for benchmar k parameters. BPPD speculates that the 20% strip refuge option might have pr ovided the greatest durability compared to Pioneer’s other

scenarios. Pioneer’s model did not predict a difference in dura bility between the two block refuge options with or without a seed blend after four years.

No refuge 20% Block,

70% compliance 20% Strip,

variable compliance 20% Block, 75% compliance 4yrs,

5% seed mix 20% Strip, 75% compliance, 4 yrs, 5% seed mix 5% Seed

mix

Benchmark

Parameters 5 yrs 8 yrs Not modeled 8 yrs 10 yrs 10 yrs

Info extracted from Table 10 above

Estimated time to resistance using Pioneer’s best-case assumptions : The results from different scenarios fall into di stinct time categories likely because best-case assumptions allow for greater m odeling runs and hence durability.

BPPD notes that Pioneer did not provide resu lts for the 20% strip re fuge with variable compliance when using its best-case assumptions. Th is scenario predicted the greatest durability among all scenarios when more conservative assumptions were used. From the scenarios modeled, the 5% seed mix was the most durable (> 20 yrs) followed by the 20% strip (19 yrs) and block refuge (18 yrs) with variable compliance and seed blend. BPPD speculates that the 20% strip refuge option, had it been modeled (including more rea listic non-compliance numbers), could have produced the greates t durability under Pioneer’s be st-case assumptions. The 20% block refuge with variable compliance was predicte d to be less durable (1 3 yrs) then when seed- 30 - blends were introduced after the first four y ears of 59122 deployment. Even for this scenario, unrealistic non-compliance numbers decreased the durability for 59122 compared to seed blends. The ‘no-refuge’ option resulted in the least amount of time to product failure (9 yrs).

BPPD is concerned with outputs generated wi th ‘best-case’ assumptions for the reasons

discussed earlier in this review . BPPD believes that the dominan ce value and initial resistance allele frequency chosen may not be justifiabl e based on what is known about Cry34/35 toxicity and history and that these valu es could represent unrealistic be st-case values for estimating durability. Rather, BPPD considers Pioneer’s benc hmark parameter values as defensible ‘bestcase’ assumptions.

No refuge 20% Block,

70% compliance 20% Strip,

variable compliance 20% Block, 75% compliance 4yrs,

5% seed mix 20% Strip, 75% compliance, 4 yrs, 5% seed mix 5% Seed

mix

Best-case 9 yrs 13 yrs Not modeled 18 yrs 19 yrs >20 yrs

Info extracted from Table 10 above

BPPD’s concluding remarks re garding Pioneer’s results : BPPD questions Pioneer’s conclusi ons that the use of a blended refuge following a block refuge extends the durability of 59122 maize relative to continuation of a block refuge, and that, therefore, moving to a blended refuge as quick ly as possible would ex tend the durability of existing 59122 products.

1. Pioneer’s conclusion that the addition of an in-field movement parameter makes the 5% seed blend (OAM1) always superior to the 20% block refuge is not supported in all model simulations. Pioneer’s modeling ou tput shows that the 20% block refuge alone and followed by a

5% seed blend resulted in the same amount of estimated durability for 59122 corn (8 yrs) when Pioneer’s benchmark parameters (and unrealis tic non-compliance rates) were used. Also, Pioneer’s model supports the conclusion that stri p-refuges are more durable than seed blends under worst-case (less conservative) assumptions.

2. Pioneer has modeled universal adult CRW mo vement based on data developed by Nowatzki et al. (2003), which indicate that adult movement is limited (average 15m/d for males). Spencer (2009) found that adult CRW movement may de pend on population density and reported that movement ranged from 30m/d to over 200m/d. Even with Spencer’s data (2009), there is still uncertainty regarding what constitutes reasonabl e adult CRW movement and whether different biotypes display different movement behavior.

Pioneer’s sensitivity analysis (using benchmark a ssumptions) showed that greater male dispersal prior to female emergence increased the durabil ity 59122 corn with block refuge deployment to >20 years. Hence, assessing the effects of adult dispersal as reported in the Illinois region is of great importance (see BPPD’s own analysis in Section IV.C.3).

3. The assumption that 30% of corn growers across the Corn Belt plant no refuge is not supported by ABSTC’s data and underestimates non-compliance (see BPPD’s own analysis in Section IV.C.2 of this review). Pioneer’s sensitivity analysis s howed that when the percent of- 31 - growers who complied 100 % with refuge require ments was increased from 70% to 100% that durability was increased from 10 years to >20 years (refuges not moved and benchmark parameters assumed otherwise). Hence, durabilit y for 20% block refuges using Pioneer’s model, should be between 10-20 years rather than 8 year s (refuges moved) as re ported. In this case, durability for blocks would be greater than for seed blends.

4. Field size is an important consideration that will affect the durability of 59122 corn in a block refuge configuration. The greate r the distance between the non-Btportion and the opposite end of the Bt field, the greater the probability that a refuge male, assuming limited dispersal, may not find the potentially resistant female emerging from the Bt corn to mate with, and vice versa. However, if dispersal is greater in some regions of the Corn Belt (Spencer, 2009) than reported by Nowatzki et al. (2003), then field size may not be of such great important anymore. Section IV.C.2.a in this review will explore the di fferent planting behaviors by growers, CRW

movements encountered in the Corn Belt as well as the effect of egg densities in refuge and Bt

fields.

5. If larval movement had been assumed for late r instars (rather than neonates) while including lower mortality and greater tolerance for Cr y34/35, estimated durability of 59122 in Pioneer’s model might have been reduced for seed bl ends. Late-instar dispersal could allow for heterozygous resistant su rvival, which might speed up evoluti on of resistance in seed blends using a single, non-high dose toxin. Mallet and Porter (1992) conc luded that when larvae were able to move from plant to plant within a Bt seed mixture that resistance could occur faster than compared to pure stands of Bt.

6. Pioneer has set model conditions/parameters that are unfavorable to blocks (compliance, CRW adult movement, refuge placement, and field si ze) and that are more favorable to blends.

7. BPPD concludes that with Pione er’s realistic and mo re conservative assumptions, seed blend and block scenarios predict abou t equal durability for 59122 corn. S eed blends are not superior; but Pioneer’s modeling shows they are not worse e ither. Seed blends fare better when prior-use history is ignored and best cas e assumptions are modeled. Seed blends do not fare well under most conservative (worst -case) assumptions.

C. EPA/ORD’s ANALYSES OF A 5% SEED BLEND AS PROPOSED IN

PIONEER’S SUBMISSIONS

1. Purpose

For the first time, BPPD has access to an EPA-gene rated model to simulate resistance evolution in a target pest in response to pesticide exposure (“USEPA popul ation genetics two locus model, POPGEN”). In this particular section of the risk assessment, the main objective was to determine

whether Pioneer’s conclusions ar e supported by using an independe nt model. This includes the important claim that the adult infield movement parameter in a 5% seed blend would lead to greater random mating between adult CRW emerging from Bt and non-Bt plants and consequently extend the durability of 59122 corn over the currently approved, 20% block refuge.- 32 - Further objectives of EPA’s modeling efforts were to 1) assess whether Pioneer results could be replicated using their benchmark assumptions, 2) use BPPD’s and ORD’s assumptions for likely more realistic and conservative parameter values and assumptions and incorporate a probability analysis to assess which generated results (estimat ed durability for block or seed blend) would be more likely to occur, and 3) test alternate hypotheses.

To understand the relative risks rela ted to the evolution of resist ance in western corn rootworm between a block refuge deployment (as curren tly mandated by EPA) and a 5% seed-mixture deployment as requested by Pi oneer, EPA/ORD developed two models. The first was a modification of a spatially-explic it stochastic model (Caprio et al. 2006), and the second was a simpler, frequency-based deterministic model. The latter could be r un in a single simulation mode with a graphical interface to enter parameters or in a risk-assessment mode capable of running thousands of simulations to estimate the e ffects of parameter uncertainty. The stochastic model was used to develop realistic estimates fo r several parameters used in the deterministic

model and to assess the effect of different dispersal rates and fi eld sizes on the durability of a 20% block refuge strategy.

2. Deterministic Model (POPGEN) and Probability Distributions (Taken out of Caprio & Glaser, 2010)

a. Model Description

The deterministic model is a frequency-based model that incorporates non-compliance, nonrandom mating in compliant fields, and larval move ment. It includes a switch to vary between a block-refuge design and a seed-mixture desi gn. Appendix A (Caprio & Glaser, 2010) includes descriptions of the different parameters. No refuge was planted for non-compliant fields. CRW in ‘non-random mating populations ’ came from fields with ref uges. Non-compliant populations increase the amount of non-random mating and re duce refuge size. As noted in the Pioneer submission, a critical component of their m odeling was that refuges were moved every generation. While EPA/ORD believes that this may overestimate grower placement of refuges, this was incorporated into the model by defining the refuge size not as the physical area devoted to the refuge, but rather the proportion of the e ggs in a field that started out underneath the new refuge. These small refuge sizes were independe nt of non-compliance and resulted from growers moving their refuge to areas of the field were few eggs were oviposited the previous year. To incorporate this important parameter, the stochas tic model was used to estimate egg densities at the start of a year across the mode l’s four different field sizes ( Figures 1a-e ). In each case, a best

and worst scenario was estimated. In the best case scenario, the grower moved refuges to the opposite side of the field next to where the ref uge would have been pl anted in the neighboring field the previous year (these si mulations were run with all grow ers planting refuges to the same sides of their fields and swapping them every year). The worst case scenario was based on planting the refuge in the area of the field where the egg density was the lowest. Because the egg density distribution across fields has never been measured, it is unlik ely that growers could accomplish this, but it does represent a theoreti cal lower bound for effective refuge size. The effective refuge sizes for the four different field sizes were (from smallest to largest field size): 15%, 11%, 3% and 0.055%. As noted by Pioneer, few insects were moving into the center of- 33 - large fields as reflected by the effective refuge size of 0.055% for 144 ha fields. Other estimates for dispersal were higher and might depend on the regi on of the Corn Belt. To test the effect of higher dispersal rates, the vector of daily move ment was doubled (to 29m/d ay) and the large field size simulations were repeated (Fig. 1e). In this case the density of eggs in the center of the field was fifty times greater under the worst case scenario and the effective refuge size was 1%. Most fields in the Corn Belt, however, are smaller, in the range of 30-50 ha, and EPA/ORD decided to use a lower limit of 1% for this parameter.

Figure 1a. Simulated egg densities in transects across Bt-corn and associated refuges: a small 2.25 hectare (approximately 5.56 acre) field size. There is a 30m ro w of refuge along the edge of a 120m wide field of transgenic corn. The refuge is always planted along an edge and is switched every year. The previous years, refuge was in strip 5, while the current on e is in strip 1. In the model, gr owers always plant to the same side of their fields, so the reason there is a larger egg popula tion under strip 1 than in strip two is because there was a refuge to the left in another fiel d. In this case, if growers planted in strip one, the new refuge would get 19.8% of the overwintering eggs. On the other hand, if they planted in the area with the lowest density, the refuge would have 15% of the overwintering eggs.

Figure 1b. Simulated egg densities in transects across B t-corn and associated refuges : a 9 hectare (22.24 acre) field. The highest density is under the previous ye ar’s refuge, but we continue to get “drift” from neighboring fields. In this case 18.6% of the eggs wo uld be under the refuge in the default planting pattern, 12345 0 1000 2000 3000 4000 5000 6000Egg density/ 900m2

Number of 30 m wide rows 12345 0 1000 2000 3000 4000 5000 6000Egg density/ 900m2

Number of 30 m wide rows

1 2 3 4 5 6 7 8 9 10 0 5000 10000 15000 20000 25000Egg Density/900m2

Number of 30 m wide rows 1 2 3 4 5 6 7 8 9 10 0 5000 10000 15000 20000 25000Egg Density/900m2

Number of 30 m wide rows- 34 - but the refuge would only get 11% if growers planted the refuge in the area with the lowest density of eggs. The refuge in this case was 2 strips (60m) wide.

Figure 1c. Simulated egg densities in transect across Bt-corn and associated refuges: A 36 hectare field (88.96 acres, about an average field for many parts of the U.S.). With the default planting pattern, the refuge would receive 20% of the eggs, but only 3% if growers planted ov er the area with the lowest density. The refuge in this case was 4 strips wide (120m).

Figure 1d. Simulated egg densities in transect across Bt-corn and associated refuges: 144 hectare (355.82 acre) field size. At this size, the default planting wo uld result in 23% of the eggs in the new refuge, but the worst case drops to 0.055% (if growers plant in the middle ). The refuge in this case was 8 strips wide (240m). 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 0 5000 10000 15000 20000 25000 30000 35000 40000Egg density/ 900m2

Number of 30 m wide rows 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 0 5000 10000 15000 20000 25000 30000 35000 40000Egg density/ 900m2

Number of 30 m wide rows

1 2 3 4 5 6 7 8 9 1011 121314151617 181920 212223242526272829 303132333435363738 39 40 0 5000 10000 15000 20000 25000Egg density/ 900m2

Number of 30 m wide rows 1 2 3 4 5 6 7 8 9 1011 121314151617 181920 212223242526272829 303132333435363738 39 40 0 5000 10000 15000 20000 25000Egg density/ 900m2

Number of 30 m wide rows- 35 - Figure 1e. Simulated egg densities in transect across Bt-corn and associated refuges: doubled dispersal distance in a 144 hectare field. This figure has a co lumn for each 30x30m block rather than an average of 3 transects as in the other four figures.

b. Model Assumptions

Asymmetrical movement : Asymmetrical movement is defined as the probability that larvae that have survived exposure on a Bt-plant will m ove. This movement was included in the ORD’s seed mixture model, though the amount movement of susceptible larvae off of transgenic corn is uncertain (Hibbard et al., 2005). When larvae moved, they moved onto plant genotypes according to the ratio of those genotypes in the fi eld. In this case, a larva leaving a transgenic plant had a 95% probability of m oving onto another transgenic plant. As with other rates in the model, this movement rate was converted into a daily rate (depending on the days of larval movement parameter).

Developmental delays : To replicate the developmental delay of larvae on transgenic plants in the Pioneer model, the ability to delay insects was added by holding back a portion of each age group by a fraction. Normally, all the individuals in an age group would age one day in the model per time step. In the ORD model, 5% we re held back from each age group (though they did experience mortality) and did not age. This resulted in a mean developmental delay of 2.3 days. 4 292 580 868 40

76 112

148

184

220

256 328

364

400

436

472

508

544 616

652

688

724

760

796

832 904

940

976

1012

1048

1084

1120

1156

1192

1228

1264

1300

1336

1372

1408

1444

1480

1516

1552

1588 0 5000 10000 15000 20000 25000 30000 35000 40000Egg density/ 900m2

Number of 30 m wide rows 4 292 580 868

40

76 112

148

184

220

256 328

364

400

436

472

508

544 616

652

688

724

760

796

832 904

940

976

1012

1048

1084

1120

1156

1192

1228

1264

1300

1336

1372

1408

1444

1480

1516

1552

1588 0 5000 10000 15000 20000 25000 30000 35000 40000Egg density/ 900m2

Number of 30 m wide rows- 36 - Prior use-history : the deterministic version of the ORD model was run for four years as a 20% block refuge deployment. After th e initial four years (simulating prior use- history of 59122 corn) had passed, simulations were continued with a 5% seed blend strategy. For the 20% block refuge, no changes occurred for further simulations.

Uncertainty (probability) distributions : To incorporate the uncertainty in the parameters used in the deterministic model, distributions were deve loped based on data for the lowest and maximum possible values, as well as a most likely values pa rameters might take (as determined jointly by BPPD and EPA/ORD). These values were us ed in PERT distributions based on a beta

distribution with the most lik ely value weighted by 4 (Vose 2001). This was done for both the block refuge model ( Table 12 ) and seed blend (larval movement) model ( Table 13 ). Each model was run 2000 times, each time drawing parameter values from the relevant PERT distributions and using the sampled parameters as inputs to the model. The time it took for the resistance allele to exceed 50% was recorded for each simulation. EPA/ORD measured the mean, 25 th , and 75 th
percentiles of the time to resistance and determ ined the proportion of simulations in which the time to resistance exceeded 10 years (i.e., 11 years or longer). Eleven years was chosen as a cutoff point because the gene has already been in use for 4 years, and previous EPA-SAPs have suggested 15 years as a reasonable minimal goa l for the lifetime of single gene products.

The uncertainty distribu tions are described in Table 11 and Table 12 for the block refuge and the

seed blend model.

Common parameters:

1. Fitness of susceptible homozygotes on transg enic plants (Wss): The Pioneer estimate for this parameter was 0.0125. In the past, however, as transgenic events have been exposed to a wide variety of environments, Wss has often been found to ex ceed initial estimates.

2. Dominance (h): In some cases, dominance associ ated with events thatare not a true highdose is considerably greater than the Pioneer benchmark estimate. BPPD/ORD’s estimate

was centered on the Pioneer estimate but included the possibility that it could be 5x less or 5x greater.

3. Initial resistance allele freque ncy (IRF): While many estimate s of the initial resistance allele frequency for loci provi ding resistance to Bt toxins are in the range of 0.001 (for Lepidoptera), recent studies with western co rn rootworm have suggested they may be more common, perhaps as high as 0.02 (Hi bbard 2010, personal communication). These

two values were used as our maximum and minimum values, with 0.005 used as the most likely value.

Block refuge parameters:

1. Effective refuge size: In th e absence of non-compliance, th e physical refuge size was always assumed to be 20% of the area. N on-random oviposition and refuge placement could, however, lead to that 20% of the ar ea having more or less than 20% of eggs initially (see Figures 1a-e ). This is a complex paramete r that depends on the size and shape of fields, how growers physically place the refuges in the fields, how they move the refuges between years, and on corn rootworm dispersal rates. It is likely that this parameter is responsible for ma ny of the differences between the results of this model and- 37 - the model used by Pioneer. The effective ref uge size varied from 1% to 20%, with 15% as the most likely value.

2. Non-compliance: Non-compliance must be measured on a field by field basis, and BPPD’s interpretation is that a non-comp liance rate of 30% (meaning that 30% of growers planted 0% refuge) is an overesti mate. There may be localized areas, however, where non-compliance may be greater than 30%, and to incorporate th e effects of these areas, the maximum value of non-compliance was set to 60%. The minimum value was 5% with a most likely value of 10%.

3. Non-compliance mixing: This parameter is re lated to the rate of mixing per generation between compliant and non-compliant fields and may be seen as a measure of persistent non-compliance versus growers that are accident ally non-compliant in one or more fields. Bailey (2008 & 2009) suggested 5-7% of grow ers were persistently non-compliant, so 93% was chosen as a most likely value, 95% as the maximum and 85% as a conservative

minimum value.

4. Non-random mating: This parameter is an estimate of the proportion of the population emerging in the transgenic fields that mate s among themselves with no contribution from refuge insects. Based on the simulated disper sal experiment, it appears as though as many

as 50% of these individuals would be unlikel y to mate with refuge individuals in the largest field sizes. Because refuges are moved each year, these non-random populations are merged with the random mating populat ions for reproduction. The maximum value used was 50%, with a minimum of 5% and a most likely value of 30%.

Seed blend model parameters:

1. Base larval movement: Data suggest that there is limited larval movement among nontransgenic plants in the absence on high larv al densities (unlikely to be found in a 5% seed-mixture). The range for this parameter was from 1% to 5%, with a most likely value

of 3%.

2. Days of larval movement: Based on Mallet and Porter (1992), larv al movement affects the evolution of resistance by shifting dominance. This sh ift occurs gr adually over a number of days. The range of time over wh ich larval movement occurred ranged from 1 to 18 days, with a most likely value of 10 da ys. The mortality rate was assumed to be constant over this period, with the overall mortality rate equal to 1 – w, the relative fitness of the genotype.

3. Asymmetrical movement: Food choice bioassays in many species suggest that larvae will tend to taste and then move off of transgenic tissue at a greater ra te than non-Bt tissue. Hibbard et al (2005) found, in a two year study, that this rate varied in those two seasons

between 0 and 30%. Because of the limited data on this parameter, the range of this parameter was 0 to 50% with a most likely va lue of 30% (30% of la rvae will leave initial Bt host plant).- 38 - Table 11. Uncertainty parameter values us ed for the block refuge simulations Min Most Likely Max

Wss 0.01250 0.04000 0.08000

Dominance 0.01000 0.05000 0.20000

IRF 0.00100 0.00500 0.02000 Refuge 0.01000 0.15000 0.20000
Non-compliance 0.05000 0.10000 0.60000 Non-compliance mixing 0.85000 0.93000 0.95000 Non-random mating 0.05000 0.30000 0.50000

Table 12. Uncertainty parameter values us ed for the seed mixture simulations

Min Most Likely Max

0.01250 0.04000 0.08000

Dominance 0.01000 0.05000 0.20000

IRF 0.00100 0.00500 0.02000 Refuge 5.00000 5.00000 5.00000

Base larval movement 0.01000 0.03000 0.05000 Days of larval movement 1.00000 10.00000 18.00000
Asymmetrical movement 0.00000 0.30000 0.50000 Wss

c. Sensitivity Analyses

ORD generated effects-plots for relevant parameters to determine the change in estimated time to resistance evolution when a range of values used in EPA’s model was explored. The graphs below (Fig. 2) allow side-by-side comparisons of which paramete rs created greater changes in durability (i.e. were more sensitive). The y-axis represents the estimated time-to-resistance in years; the x-axis represents the numerical value of the parameter of interest that was varied.

It becomes clear from viewing the plots in Figure 2 that dominance, % ref uge planted, and initial resistance frequency were the most sensitive pa rameters and predicted the greatest changes in estimated durability in the model. Non-compliance had an intermediate effect on durability, and non-random mating, fitness, and non -compliance mixing had very little effect on the predicted durability. Other parameters, such as random mati ng, were less sensitive and did not affect the estimated years to resistance evolutio n as much as predicted by Pioneer.- 39 - Figure 2. Effects of changes in parameter values on the time to resistance evolution

e. Modeling Results

The IRM scenarios that were comp ared with the deterministic vers ion of the ORD model were a) the 20% block refuge with non-compliance, non-ra ndom mating, and b) the 5% seed blend (also referred to as the larval movement model) in corporating asymmetrical larval movement. The

distributions of the time to resistance for the block refuge strategy and the larval movement strategy with and without asymmetrical movement are presented in Figure 3 . Because the bars each represent a year, the density of the bars is al so the proportion of simulations that resulted in resistance evolving in that gene ration. The mean time to resistan ce for the block refuge strategy was 11.95 years, and 56.55% of the simulations ex ceeded 10 years. For the seed mixture (larval movement) model, the mean time to resistan ce was 8.1 years and 13.5% of the simulations exceeded 10 years. Non.compliance.mixing effects plot Non.random.mating effects plot Refuge. effects plot Non.compliance.effects plot

Non.compliance.mixing effects plot Non.random.mating effects plot Refuge. effects plot Non.compliance.mixing effects plot Non.random.mating effects plot Refuge. effects Non.compliance.effects plot- 40 - Figure 3. Distribution of time to resistance for a) the block refuge model and b) seed mixture model

Table 13. Regression coefficients for the regressi on on the results from the block refuge model

Coefficients Estimate Std Error t-value Pr(>|t|) Intercept 11.6958 2.2321 5.240 1.78e-07***

Dominance -84.9810 1.3097 -64.887 <2e-16***

IRF -629.7121 14.7731 -42.626 <2e-16*** Non-compliance -8.8690 0.4426 -20.037 <2e-16*** Non-compliance mixing 1.5415 2.4028 0.642 0.521

Non-random mating -0.7173 0.5336 -1.344 0.179 Refuge 54.6024 1.2589 43.374 <2e-16***

Wss 61.5433 3.4862 17.653 <2e-16***
Signif.codes: 0 ‘***’, 0.001 ‘**’, 0.01 ‘*’

Residual standard error: 1.797 on 1992 degrees of freedom Multiple R-squared: 0.8095, Adjusted R-squared: 0.8088 F-statistic: 1209 on 7 and 1991 DF, p-value: <2.2e-16

3. Stochastic Model (Taken out of Caprio & Glaser, 2010)

a. Model description

The stochastic model was a modification of a prev ious western corn rootworm model (Caprio et al. 2006). Biological parameters were primarily derived from that model with the exception of mortality rates, block size and dispersal rates.

The stochastic model used the same mortality rates on transgenic corn as were applied in Pioneer's submission. Two different dominance values ( h ) were utilized, 0.05 and 0.2. If W

ss is the likelihood that a susceptible individual will emerge from transg enic corn relative to non-Bt corn, and W

rr is standardized as 1, then the fitness of heterozygotes (W

rs ) is estimated as (W

rr – W
ss )* h +W
ss . When h =0.05, heterozygotes were approximately 5x more likely to survive thanDensity of total simulations

Generations to Resistance for Block Refuge

Model Generations to Resistance for Seed Blend

ModelDensity of total simulationsDensity of total simulations

Generations to Resistance for Block Refuge

Model Generations to Resistance for Seed Blend

ModelDensity of total simulations- 41 - susceptible genotypes and approximately 20x more likely to survive when h =0.2 and when W

ss = 0.01. Those rates decreased as the fitness of su sceptible homozygotes incr eased (a reflection of the difficulty in using dominance rather than the W

rs :W
ss ratio).

The simulated universe consisted of a 40x40 matrix divided into 30m x 30m blocks, or a total of 144 ha. Four different types of simulations were run with different field sizes. Because the current refuge strategy requires th at refuges be associated with each field, the four different

scenarios were:

• Field size of 5x5 blocks (each block is 30m x 30m, for a total of 2.25 ha);

• Field size of 10x10 blocks (9 ha field size);

• Field size of 20x20 blocks ( 36 ha field size); and

• One single field of size 40x40 block field of 144 ha. Mean field sizes in the U.S. range from as low as 2.5 ha/field to as high as 150 ha/field, with most fields in the 30-50 ha/field range. In each case, a 20% refuge wa s planted along one edge of the field. In the case of the 5x5 block fields, this refuge consisted of a single row of 5 blocks only along one edge. In the 10x10 block fields, the refuge was 2 rows wide and 10 blocks long. The refuge widths for the remaining scenarios were , of course, 4 and 8 rows wide, maintaining in each case a 20% refuge (by area).

Pioneer maintained that a square field is a best-case scenario (F lexner, 2009), but EPA feels that the opposite is true. It is not co mpletely clear from the Pioneer submission, but it appears that the registrant simulated rectangular fiel ds with refuges planted at the s hort ends of these fields. It is EPA’s belief that most growers plant lengthwis e along such fields to minimize the number of turns that would have to be made with pl anters. Corn entomologists (Meinke 2010, Hibbard 2010, personal communication) confirmed EPA’s assumption and felt that most growers would plant refuge fields along one long ed ge of the field. If dispersal of adults is in question, then as fields become more rectangular (length:width ratio in creased), refuges become thinner, but the maximum distance between any Bt-pla nt and the refuge decreases.

Under these conditions a square field (as EP A/ORD used) maximized the distance between refuge and Bt plants and can be seen as a wors t case. Pioneer's model appeared to maximize the distance between refuge and Bt pl ants, but again, this did not seem consistent with typical grower practices.

b. Assumptions

Adult dispersal : This was modeled based on Pioneer's dispersal data. EPA/ORD combined the data from Nowatzki et al. (2003) ( Fig. 5 ) and built probability distributions based on the frequency of beetles moving different distances. In s hort, if the total height of the bars in Figure 5 summed to 1, then the individual bar heights w ould represent the likelih ood that a beetle would move that distance. However, Pioneer’s data incl uded beetles that did not disperse, and the zero

movement class consisted of 45% of the beetles. EPA/ORD made one change from how Pioneer defined ‘adult dispersal’. EPA’s st ochastic model assumed that 45% beetles did not disperse (on a daily basis), while the remainder moved accord ing to the Nowatzki data. The difference in terminology of ‘dispersal’ may be important. Pioneer 's interpretation of the data is that beetles move an average of 14.5m/day. Using the same da ta, but only looking at beetles that move, the- 42 - average movement rate is ca. 29m/day. This is purely a difference based on definition. In EPA/ORD’s stochastic model, at the beginni ng of each day a beetle was assigned a random position in the 30x30m block before it moved, upon which it chose a random direction. The

model determined the distance the beetle had mo ved (based on Nowatzki’s data) and placed the insect in a new block. However, if the dispersi ng beetle moved only a short distance, then it may not have changed the 30x30 m block it originated in.

To determine how well the stochastic model di spersal kernel matched the Nowatzki data, a

simulation was run in which one bl ock was populated with adults and the remaining blocks were empty. The number of beetles along a transect through the fiel ds was counted every 3 days, corrected for the number of cells at a distance, and multiplied by the distance they had dispersed to estimate the mean dispersal rate (per Nowatzki) at different times. The dispersal rate estimated after 3 days was 17.6 m/day, which closely matche d the dispersal estimate by Nowatzki et al.

Given that EPA’s model used Pioneer’s actual da ta to parameterize the dispersal, EPA/ORD felt

that this was a suitable match.

Interestingly, EPA/ORD noticed that the estimat ed mean dispersal rate dropped over time. This

occurred because beetles did not move in a strai ght line day after day. Brow nian diffusion (based on a random walk) is usually assumed for insect dispersal. By confounding measurements over 3 days and using that data in a daily time-step mo del, Pioneer may have underestimated the daily dispersal rate of western corn rootworm. To dem onstrate this process, a simple spreadsheet was generated (see Table 14 ), where 10 individuals were simula ted that all moved 14.5 m/day. At the start of each day, a new direction was randomly drawn, and the beetles moved 14.5 m in that direction. The right-hand column estimated the dispersal rate using the Nowatzki method. Only the estimates from the first day accurately es timated dispersal rate/day (because all the individuals were moving away fr om the release point). Estimate s based on beetles’ displacement on subsequent days tended to underestimate dail y movement. After three days of movement the estimated dispersal rate/day was less then ha lf the actual value (spr eadsheet used random numbers, hence values would differ from ‘run to run’). Unfortunately, because EPA/ORD did not have access to the original data and did not know the capture distributions for beetles of different ages, the proble m could not be corrected.

Table 14. Beetle Count over Time in a Simulated Dispersal Experiment Dispersal Distance from Initial Release Point (m) Days 0 30 60 90 120 150 180 210 240 270 300 330 360 Sum m/day

1 0 130080 114240 43200 7680 18000 0 0 0 0 0 0 0 313200 27.22

4 0 92640 174720 138240 72960 72000 34560 35280 46080 38880 0 0 0 705360 17.64

7 0 40560 95040 155520 142080 90000 77760 23520 0 0 96000 0 0 720480 13.66

10 0 24720 57600 123120 115200 102000 112320 82320 0 0 24000 0 34560 675840 11.09

13 0 14640 37440 75600 96000 54000 60480 58800 30720 38880 0 0 34560 501120 9.28

16 0 7440 29760 56160 107520 108000 43200 0 15360 77760 72000 29040 34560 580800 8.98

19 0 6240 19200 28080 49920 54000 34560 35280 15360 19440 24000 0 0 286080 6.81- 43 - In retrospect, analysis of the data using a di ffusion model may have been more appropriate.

EPA/ORD estimated that Nowatzki et al. underestim ated the daily dispersal rate by a factor of 0.68. Using a definition of estimating the dispersal as the distance a beetle moves when it moves (this is purely semantics but criti cal to define), and correct by the above factor, the resultant dispersal rate is ca. 41m/day. It should also be noted that, because a different definition of dispersal was used, this rate is not the same as the 45m/day dispersal rate used in the sensitivity analysis in the Pioneer submission.

Figure 4. Beetle Position by Day after Release (using Nowatzki et al. (2003) Data)

Refuge movement : Several scenarios were explored for refuge movement. One scenario, used for the transect experiments presen ted below, used refuges that were moved every generation. All growers planted their refuge on the west side of their fields in one generation and moved them to

the east side in the next. Because the east side of one field is adjacent to the west side of a neighboring field, this scenario resulted in ref uges always being planted adjacent to a refuge from the previous year. Ultimately, random placem ent of refuges each year was chosen as the most likely grower behavior. This means that in some cases refuges are not moved, while in other cases they are moved to relatively isolated areas.

c. Results

EPA/ORD’s stochastic model was run for four different field sizes and two different adult dispersal rates (dispersal (1) represents Nowatzki et al.’s (2003) data; di spersal rate (2) is double Nowatzki’s rate) ( Fig. 6 ). Because the largest field size took the entire simulated universe, it was 0 30 60 90 120 150 180 210 240 270 300 330 360 0.5 5 50 500 5000

1 4 7 10 13 16 19 Beetle Position By Day After Release

1

4

7

10

13

16

19

Distance (m)Number of beetles

D a y P o s t - R e l e a s e- 44 - not possible to incorporate non-compliance. To addr ess this issue, the model was also run in an 80x80 cell mode (6400 cells, each 30x30m) for this field size, allowing simulation of 4 144 ha fields. These larger simulations were us ed in Figure 6 and in the data analysis.

Analysis of variance showed no significant impact of dispersal rate, but field size was highly significant (p << 0.001). When run with a sing le 144 ha field and non-compliance excluded, the times to resistance were much longer than when run in a comparable fashion to all the other stochastic simulations (mean durability = 3292 d, a pprox. 23 yrs). It was not expected that doubling the dispersal rate would have so little im pact on the time to resistance, and it suggests that the model is relatively insensitive to this parameter.

Using Pioneer's 40 ha field size as a benchmark, the stochastic model predicted an increase in durability of 3.94 fold for 20% block refuges compared to simulations with no refuge (see discussion). This is much higher than the 1.4 fo ld reported for this field size in the Pioneer simulations. The simulations presen ted here predict that a block refuge with 40 ha fields would last 13.9 years, almost double the time predicte d by Pioneer. The 144 ha simulations showed much lower durability (8.4 yrs, or 2.4 fold longer th an without a refuge). It may be relevant that

the dimensions of these fields had a 240m wide refuge and a 960 meter wide strip of Bt-corn. Pioneer's simulations may have been run with a 200m wide refuge and an 800 m wide strip of Bt-corn (this occurs in a 40 ha fi eld size in the Pioneer model and in a 144 ha field in the current model).

To determine further the sensitivity of the model to the rate of dispersal, the 36 ha field size was run with 2x, 4x and 8x dispersal. Dispersal wa s again not significant in an ANOVA (p=0.058). The 36 ha field size had only 4 refuges in the default 144 ha system, and this increased the variance when in any given year each field had a 30% chance of being non-compliant. There is a

24% chance that all Bt fields would have a ref uge in any year and a 0.8% chance that all four fields would not have any refuge in any year. In a larger system, the mean number of noncompliant fields would remain more constant ove r time (law of larger nu mbers), and there might be a better opportunity to investigate how disper sal interacts with refuge size and placement.

It is possible that dispersal has less effect th an might have been expected because there are

several different situations that arise in the m odel. When a refuge is not moved, lower dispersal rates result in a larger effective refuge. Simulations suggest this frequently delays resistance evolution, although it reduces the probability that an individual beetle wi ll move and mate at random with beetles emerging in Bt-fields (Capri o 2001). Similarly, if the refuge is moved next to the location of where a refuge had been plante d in a neighboring field in the previous year, lower rates of dispersal might al so maximize effective refuge size. In contrast, when refuges are moved to areas relatively distant from the locations of the previous years refuges, higher rates of dispersal would maximize the effective refuge size. It is clear, however, that these results suggest that the model is less sensitive than expected w ith respect to dispersal rates. Discussions over

whether the Nowatzki data over or underestimated movement rate s for the entire Corn Belt may perhaps be less criti cal than expected.- 45 - Figure 5. Means of four block simulations at each fi eld size (ha). Simulations used for the dark line used Nowatzki's data (15m/d); simulations used in the red line doubled that dispersal rate (30m/d). 140 days/year are used; hence, 1400 da ys would represent 10 years to resistance.

4. Discussion for Stochastic and Deterministic Results (Taken out of Caprio & Glaser, 2010)

Pioneer presented in Table 1 of its submissi on (MRID 478710-01) the results from their model using the benchmark parameters listed in Table 3 of this review. Using the deterministic model presented here with similar parameter values, similar but not identical durabilities were found ( Table 15 ).

In the case with no refuge, Pioneer's model sugge sted resistance would evolve in 5 years, while ORD’s deterministic model suggested durability would be 3 years. The stochastic model predicted a durability of 3.5 yrs in the absence of refuges. For comparison's sake, other rates

were measured relative to these baseline numbers . For the block refuge model, Pioneer suggested resistance might evolve in 7 years, or 1.4x th e time without a refuge. Assuming EPA’s most likely value of a 15% effective refuge (and ot herwise using Pioneer's benchmark values), a predicted durability of 14 years was obtained w ith ORD’s deterministic model, 4.7 fold longer than the simulation without a refuge. One possi ble reason for the large discrepancy is theMean Time to Resistance (days)- 46 - effective refuge size. In order to duplicate th e 1.4x durability increase noted by Pioneer, the effective refuge size would need to be decr eased to 1%. This was the minimum value in EPA/ORD’s uncertainty distribution for this parameter. This parameter value (1%) was felt to be the minimum because it would require the following likely unrealistic assumptions:

• Large fields (on the order of 150 ha);

• Dispersal rates used by Pioneer were not underestimated;

• Movement rates were typical of rootworm dispersal rates throughout the Corn Belt (Data by Spencer (2009) suggests otherwise); and

• Growers consistently moved refuges to regions of their fields with the lowest egg density (would require that growers had a mechanism to detect areas of low egg density).

Table 15. Years of Durability Predicted by Pion eer's and EPA/ORD's Model using Benchmark Parameter

Values Durability (time to resistance) Refuge Scenario Pioneer* ORD deterministic model 1 ORD stochastic model 2
No Refuge 5 years 3 years 3.5 years 20% Refuge 7 years 14 years 13.9 years 5% Seed Blend 10 years 9 years Not modeled

* using Pioneer’s benchmark va lues (Table 1, MRID478710-01)

1 using Pioneer’s benchmark values but 15% effective refuge as most likel y value and fixed 30% non-compliance

2 Stochastic model used Nowatzki’s dispersa l data, Pioneer’s survival data, and otherw ise different assumptions: simulated typica l field size a) 40 ha and outlier field si ze b) 144 ha; refuge pl acement random; dominance va lues of 0.05 and 0.2.

The overall risk profile from the uncertainty si mulations for block refuges was also impacted by other parameters, which deviated from the Pione er benchmark values. Although relative fitness, initial frequency and dominance used the same uncertainty distributions for both the block and seed blend simulations and were likely to aff ect both models in a similar manner, these parameters could shift both distri butions in time. The in clusion of the possibility of higher levels of dominance, in particular, could lead to shorter predicted durability estimates. Other parameters specific to the block refuge model c ould lead to the longer durability predicted in these simulations compared to Pioneer's simu lations. ORD’s distributi on for non-compliance was weighted more heavily towards lower values (w ith a most likely value of 10% non-compliance) than reported by CSPI (2009); based on BPPD’s analysis, that report overestimated noncompliance. EPA also assumed that most non-compliant growers were “partially” noncompliant, perhaps due to miscalculations ormistakes during planting. Only 5% of noncompliant growers were assumed to be pe rsistently non-compliant year after year.

For ORD’s seed blend (or larval movement) model, the analysis pr edicted a durability of 9 years, or a 3-fold increase in durability compared to ‘no refuge’. This compared reasonably to the Pioneer estimate of 10 years, or a 2-fold benefit. The values both compared quite well to the mean of EPA’s risk profile, 8.1 years.

These data suggest that although there were minor differences be tween the seed blend (larval movement) results generated by the Pioneer m odel and the model pres ented here, the major differences lie in the block refuge model. Although Pioneer suggested that non-random mating- 47 - was important in their results, EP A/ORD did not find that to be the case in their simulations ( Table 13 ). The parameter most likely to be respons ible for the differences between the two models is the effective refuge size. EPA agrees w ith the Pioneer assertion that it is possible that areas of large fields could have very low egg de nsities, and should growers manage to locate and plant refuges over those areas, the block refuge strategy could be severely compromised.

A complete understanding of the differences in the results is limited by details missing in the Pioneer data submission. The registrant simu lated a size of 4x10 cells (each cell as 100m x100m), or 40 ha. It was unclear if this was a sing le field or represented multiple smaller fields, but given their results, a single field is likely. The exact placement of the refuge in the 4x10 cell universe is also important and not stated. Based on the Ento mological Society of America presentation by Flexner (2009), it appears as though the refuge was located to one “thin” edge of this system (perhaps two 4 cell rows to one e dge), which would maximize the furthest distance between transgenic plants and the refuge but would seem to be at odds with common grower behavior (this is also supporte d by the fact that one cannot get 20% of the cells by planting along the long edge in this size). Again, based on the same information by Flexner (2009), it appears as though the only way for insects to reach the plants to the right of the sy stem (when the refuge was planted on the left side) was to move acros s the intervening 800m. In actual field systems, however, there are other fields surrounding the simulated field with refuges. Those refuges would on average be adjacent to the current fi eld, and the mean distance between the most distant field and the refuge would be 400m. Most simulations were designed as toruses (an insect moving off the left side of the sy stem arrives at the ri ght side as if the system were a donut) to overcome this problem, but the animation presen ted at ESA was not designed in this manner. This type of model minimizes the effects of di spersal and would tend to lower Pioneer's estimate of the durability of a block refuge system.

In the current stochastic model (discussed in Section C.3), EPA/ORD simulated multiple fields arranged in a large torus. Refuges were placed randomly within fields, and non-compliant fields were also chosen at random on a yearly basis. In these simulations, in some cases refuges in adjacent fields would be side-by-side, while in ot her cases they would lie at opposite ends. In the latter case there would clearly be an area with low egg densities between the two refuges, but that was apparently offset by refuges that must logically be more closely spaced in other areas. Both the Pioneer model and EPA’s dispersal kern el, based on the Pioneer dispersal data, ignored the potential of longer distance dispersal (Storer 2003). Although the contribution from such beetles is likely to be small, in areas of fields were there are essentially no eggs (see Figure 1d),

the contribution of these few i ndividuals could be significant.

There are a number of explanati ons for the differences in the pr edicted longevity of the block refuge strategy between the Pioneer simulations and those presented here. In most cases important details of the Pioneer model were not presented, which made it difficult to determine how certain key elements were modeled. The Pioneer model used a single 40 ha block of 100x100m cells arranged in a rectangular fash ion (4x10). In discussions with corn entomologists, they felt that most growers woul d plant lengthwise in such a field, and the refuge would be planted along either one of the long ed ges (here termed the east and west edges). Simulations of refuges planted to the short ends of these fields (the north and south edges) would not seem to be the normal grower practice and w ould tend to increase the mean distance between- 48 - refuges and Bt plants. This choice, if made, would increase the likeli hood of the “holes” or “pockets” in random mating mentioned by Pioneer in their submission and reduce predicted durability of the block refuge design.

Pioneer also provided little detail in the spatial aspects of thei r simulation. Was a single field simulated with all the refuge gathered in one place? Or were smaller fields simulated with multiple refuges? When the refuge was move d, where was it moved to? Was it moved to a random location, or was it always moved to the opposite end of the field from its previous location? The latter choice might lead to a sign ificant decrease in the e ffective refuge size and more rapid resistance developm ent for the block refuge.

How were boundary conditions treated? When an inse ct moved off of the field to the west side, for example, what happened to it? Did it die, was it reflected back into the system, or was a torus simulated? The choice here may be important as it can again impact the mean distance between refuge and Bt plants and ultimately effective re fuge size. As an example, if a string of 4x10 fields is considered, each 1000m long, each with its own 200m wide refuge, then the maximum distance between any two refuges would be 1600m, and the maximum distance between a refuge and a Bt plant would be 800m. The average di stance would be 800m between refuges and 400m between Bt plant furthest from a refuge and the nearest refuge. If Pioneer chose not to use a torus design, and simulated a single fiel d with a refuge at either th e west or east end, the maximum distance between a Bt plant and th e nearest refuge would be doubled (800m). In a simulation of a torus, insects leaving the east edge of the field w ill end up in the west ed ge, in essence creating a long string of identical fields. This design woul d correctly model the mean distance but without any variance. EPA/ORD simulations included multip le fields with random placement of refuges to either the east or west edge of a field, wh ich seems to best mimic grower behavior. This design meant that in some cases the refuges would be at opposite ends of adjacent fields, while in other cases they might be side-by-side; simulati ons of this kind allowed for the correct mean distance between refuges but also for variance in th is distribution.

EPA/ORD’s interpretation of the data indicates that rather than non-ra ndom mating being the threat to a block refuge system, it appears that non-random oviposition and refuge placement are most important. This suggests a potentially plau sible method that Pioneer might use to bolster their claims that failure of the block refuge syst em is imminent. If the relative density of eggs

could be estimated in transects across fields of different sizes (similar to Fig. 3a-e), and if more accurate estimates of how growers plant and move their refuges over years were available, then major differences between the two models could be resolved. Such a study might also provide an

estimate of the contribution of beetles dispersing long distance.

5. Simulating Other Deployment Strategies (Taken out of Caprio & Glaser, 2010)

a. Different seed blend proportions : The effect of the proportion of non-Bt seeds in a seed-blend was determined by varying this proportion from 0.025 to 0.10 in increments of 0.025. At each non-Bt propor tion, 3 replicates of

2000 simulations were run. Each simulation used random draws from the uncertainty- 49 - distributions outlined in the sections above. From each replicate, the mean of the distribution was

obtained and the Standard Error of Mean (SEM) determined base d on the means from the three replicates. The percent of the simulations that exceeded 10 years for each non-Bt proportion was

estimated from the first run for a non-Bt class.

These results suggest that, at l east in the modeled range, the mo re non-Bt seeds included in a seed-blend, the longer the expected durability of that seed-blend will be. This relationship in this case was very close to a lin ear relationship (adjusted R 2 = 0.9996).

When compared with the durabil ity predicted by ORD for a 20% block refuge (11.95 yrs, 56.6% of the simulations exceeding 11 yrs), a 10% seed blend was still slightly inferior (11.3 yrs, 51.6% of simulations exceeding 11 yrs). However, a comparison between the blends modeled and the 20% block refuge show that the 10% seed mix resu lt is most similar to th e block refuge strategy risk profile discussed earlier.

Table 16. ORD's Risk profile for Cr y34/35 deployed in different seed blend percentages based on BPPD and ORD assumptions and probability analysis
2.5% Blend 5.0% Blend 7.5% Blend 10.0% Blend 20% Block Mean Durability 6.6 yrs 8.1 yrs 9.7 yrs 11.3 yrs 11.95

SEM 0.014 0.013 0.005 0.028 Not provided

% of simulations exceeding 11 years 3.4% 13.5% 30.5% 51.6% 56.6%*

*Exceeding 10 years

b. Resistance simulation for dual-gene (pyramided) product : A resistance simulation was run for a two locus seed-mixture model. In this case the fitness of the susceptible genotyped (SS) a nd the dominance of resistance at each locus were independently determined according to the PERT distributions from the single locus model. This distribution is informative because it estimates the relative long evity of two genes stacke d into the same plant versus using the same two genes sequentially in single gene plants . The PERT distribution is a

version of the Beta distributi on and requires the same three distributions as the Triangle distribution, the minimum, the most likely value (o r mode), and the maximum. This is relevant because introducing a single gene event into a lands cape with dual gene events may lead to rapid resistance at the first locus and essentially turn all dual gene events that share the toxin into single gene events.

The results from the two locus deterministic model showed that, even though the second toxin has identical uncertainty distributions as the fi rst toxin, the mean expect ed lifetime of the dual gene product is 52.6 generations for a 5% seed blend, a 3.25 fold increase from the sequential use of two genes (16.2 years). Single gene products that share toxins with dual gene events can lead to a pseudo sequential use because the single gene event selects rapidly for resistance to one gene, and once this occurs the dual gene events are effectively rendered single gene events- 50 - Figure 6. Distribution of time to resis tance from 2000 simulations of a 5% seed blend deployment with two

resistance loci. The mean time to resistance was 52.6 generations.

IV. BPPD Risk Assessment Conclusions

A. BPPD’s overall conclusions

Pioneer submitted new modeling information in whic h they determined that the addition of an adult CRW infield-movement parameter in Bt seed mixtures allowed for the reduction of refuge

from 20% block to 5% seed blend. Pioneer conc luded that a 5% seed blend would extend the durability of Bt corn expressing Cry34/35 over th e current refuge requirement of a 20% block refuge.

BPPD has reviewed Pioneer’s submissions for Optimum AcreMax1 and in addition conducted an independent modeling analysis of Pioneer’s propos al using a model (determi nistic and stochastic version) developed by EPA/ORD. After careful review of Pioneer’s modeling assumptions and based on the analyses conducted by ORD (modeling efforts and probability analysis), BPPD concludes that the registrant’s a ssertion that a 5% seed blend is always superior to the current block refuge deployment strategies for OAM1 and corn rootworm” is not scientifically supported.- 51 - B. BPPD conclusions regarding Pioneer’s modeling

Pioneer’s model made a number of assump tions on CRW movement, refuge location, and percent non-compliance. In their model, when these assumptions were included, a 5% seed blend

was more durable than a 20% bloc k refuge. However, BPPD has conc erns that some of Pioneer’s assumptions may not be realistic or conservati ve enough to support a reduced refuge for OAM1.

During the risk assessment process, BPPD makes an effort to consider realistic case scenarios and parameters when evaluating simulation mode ls. However, it can be very useful to look closely at scenarios with worst-case assump tions especially in cases when a degree of uncertainty is associated with important mode ling parameters (e.g. dominance, initial resistance allele frequency, adult movement). Risk assessm ent conclusions are reached based on the best scientific information available to the Agency at the time of the assessment which is unique to any particular pest-crop system. This often includes a degree of uncertainty. Such uncertainties have required that BPPD “bracket” the risk of putative resistance evolu tion. This goal has been accomplished by estimating risk using realistic assumptions based on available data and “defensible” worst-case assumptions in the model. With this approach, the ‘actual’ risk of resistance evolution may lie somewhere within th is range of potential ri sk. If modeling results predict little to no-change in estimated risk of resistance with worst- case assumptions, then

science conclusions will be more robust and defensible.

The following is a summary of Pioneer’s mode ling parameter values and assumptions which were not supported by BPPD’s and ORD’s analysis.

1. Random mating : Pioneer reported that the addition of an infield movement parameter for

adult CRW in the 5% seed blend increas ed the random mating between refuge and putative resistant beetles emerging from Bt pl ants. EPA’s analysis however showed that random mating is not as sensitive a parameter as stated by Pioneer, a nd that it has little

effect on the resistan ce evolution (see Fig. 2 ). In fact, EPA’s mode ling revealed that egg density/distribution in corn fields is a much more important (sensitive) parameter that

affects the rate of resistance evolution. EPA determined that in ≥ 150 ha fields (366 acres), ‘dead zones’ could occur without any re fuge beetles moving into those parts of the fields (as was pointed out by Pioneer) and withou t eggs present. It is important to recall, however, that the average fiel d size in the Western Corn Belt is around 32 ha (80 acres) and in the Red Zone (WI, IL, IN, and MS) betw een 16 ha to 32 ha (40-80 acres). For such fields of average size, these ‘zones of c ontinuous selection’ did not occur with EPA’s

model.

2. Neonate dispersal : If larval movement had been assu med for later instars (rather than neonates) while including lower mortality and greater tolerance for Cry34/35, estimateddurability of 59122 in Pioneer’s model might have been reduced for seed blends. Lateinstar dispersal could allow for heterozygous resistant survival, which might speed up

evolution of resistance in seed blends with a single, non-high dose toxin. Mallet and Porter (1992) concluded that when larvae were able to move from plant to plant within a

Bt seed mixture that resistance could occur fa ster than compared to pure stands of Bt.

3. Adult movement : Pioneer used data from Nowatzki et al. (2003) to calculate the mean adult movement and included 45% of non-moving individuals into this calculation, which- 52 - is not how dispersal is typically calculate d. For example, Storer (2003) only included individuals that actually di spersed into his movement calculations. Pioneer’s approach decreased the estimated mean dispersal to 15m /day, which is a much more conservative

value than what would have been obtained otherwise (28m/day). This approach of calculating dispersal puts bloc k refuges at a disadvantage over seed blends. EPA/ORD

estimated that Nowatzki et al . further underestimated the daily dispersal rate by a factor of 0.68. Using a definition of estimating the di spersal as the distan ce a beetle moves when it moves, and correcting by the above f actor, the resultant di spersal rate is ca. 41m/day, which is similar to what was reported by Spencer (2009).

4. Planting location of block refuges : Pioneer stated in their submission that farmers moved their refuges to a new location every year. A critical component of their modeling was that refuges were moved every generation. EP A/ORD believes that this may overestimate grower placement of refuges. This refuge planting practice would tend to decrease the number of beetles generated each year a nd making refuges less effective (Storer 2003). Pioneer’s assumption that growers move thei r refuges each year may be unrealistic and certainly made block refuge deployment less durable. Fixing refuge locations and aiming for uniform distribution of refuges should help delay resistance evolution (Sisterson et al., 2005). Second, Pioneer appeared to have planted refuges at the far and narrow end of

fields in their model rather than along the long side of th e Bt portion of the field. Most growers, however, plant lengt hwise along such fields to minimize the number of turns

tractors must make. Pioneer's model maximizes the distance between refuge and Bt plants

but is not consistent with typical grower practices.

5. 0% non-compliance : Pioneer explored a range of compliance from 50-100% and used 70% and 75% for block refuge simulations. Th is assumption translates into 25-30% of growers planting 0% refuges. This percent of non-compliance is an overestimate. Actual data indicates that up to 89% of growers planted more than 15% of the required refuge, and that only 5%-15% of growers plan ted less than 10% refuge in 2007 and 2008.

6. Lepidopteran refuge : Pioneer proposes that the refuges adjacent to the Bt fields are planted with Herculex RW seed (100% CRW control). BPPD contends that this would further reduce the proposed refuge from 5% to 4.1% and possibly further increase the rate

of resistance evolution.

In light of the concerns identified above, BPPD concludes that model conditions/parameters in Pioneer’s model (compliance, CRW adult, neonate movement rather than later instar movement, refuge placement to maximize distance between re fuges) are unfavorable to blocks while being

more favorable to blends. This has the effect of biasing Pioneer’s modeling analysis towards more durable seed blends and less durable block refuges.

C. EPA/ORD’s deterministic and stochastic analyses

BPPD has collaborated with ORD on independent modeling efforts to assess the 5% seed blend strategy as proposed by Pioneer. As part of this effort, BPPD 1) modeled Pioneer’s data and assumptions (i.e. side-by-side comparison) using ORD’s deterministic version of their model and 2) used two separate models (det erministic and stochastic version) with parameter estimates that BPPD believes are likely more realistic or conservative.- 53 - ORD and Pioneer models: a side-by-side comp arison of modeling output using Pioneer’s benchmark parameter values A side-by-side comparison (using Pioneer’s be nchmark parameter valu es) between the ORD’s

and Pioneer’s deterministic models showed that the ORD model predicte d greater durability for blocks than for seed blends (see Table 17). Pred icted durability estimates were similar between the two models except for the 20% block refuges.

Table 17. Side-by-side comparison between predicte d durability of ORD and Pioneer model with Pioneer’s benchmark parameters Durability (time to resistance) Refuge Scenario Pioneer EPA/ORD 1

No Refuge 5 years 3 years 20% Block Refuge 7 years 14 years 5% Seed Blend Refuge 10 years 9 years

1 15% effective refuge was chosen in ORD’s model as most likely value and fixed 30% non-compliance

The following is a likely explanation as to w hy ORD’s deterministic model predicted greater durability for 20% block refuge strategy than Pioneer:

BPPD and ORD chose the effective refuge to be 15%, meaning it was assumed that 15% of the eggs were laid in the refuge due to non -random oviposition and refuge placement by growers (otherwise Pioneer’s benchmark parameters were used). In order to duplicate Pioneer’s block refuge result (7yrs), th e effective refuge size would need to be decreased to 1%. This parameter value (1%) was felt to be unrealistic ally low for a universal assump tion because it would require:

• Large corn fields of 150 ha (but average fiel d sizes are on the order of 30-50 ha); and that

• Pioneer’s dispersal rates are not underesti mated (however, non-moving insect were included in the dispersal calculation lowe ring the mean movement rate); and

• Pioneer’s assumed movement rates are typi cal of corn rootworm throughout the Corn Belt (Data by Spencer (2 009) suggests otherwise);

• Growers consistently move their refuges to re gions of their fields with the lowest egg density (would require that grow ers had a mechanism to detect areas of low egg density).

ORD’s risk profile based on more rea listic or conservative assumptions:

a) Deterministic Model Considering average movement data as repor ted by Spencer (2009) and as calculated from Nowatzki’s data aver correcting for errors, ORD’ s deterministic model predicted that the mean time to resistance for the block refuge stra tegy was 11.95 years (56.55% of the simulations exceeded 10 years). For the seed mixture simulations, the mean time to resistance was 8.1 years (13.5% of the simulations exceeded 10 years) (see Table 18).- 54 - Table 18. ORD's risk profile for Cry34/35 deployed in a 20% block and 5% seed blend based on more realistic assumptions and probability analysis
20% Block 5% Blend Mean Durability 11.95 yrs 8.1 yrs

% of simulations exceeding 10 years 56.6% 13.1%

See Section C.2.b and Table 11 and Table 12 for assumptions and parameter values

BPPD concludes that the 20% block refuge ex tends the durability of Cry34/35 with greater probability than Pioneer’s proposed 5% seed bl end when more realistic assumptions for the following are made:

• Greater rates of adult CRW movement as suggested by Spencer (2009) and (after ORD

corrected error in movement calculat ions) by Nowatzki et al. (2003) data;

• Refuge placement by growers (at the long side of Bt fields); and

• Compliance percentages (betw een 1-15% of no refuge plan ting but with maximum of

60% to address anecdotal communication of non -compliant counties in some regions of the Corn Belt);

b) Stochastic Model ORD’s stochastic model predicted greater durabil ity (like ORD’s deterministic version) for the 20% block when Pioneer’s benchmark parameters were used (see Tabl e 19). ORD modeled Bt fields of square shape rather than rectangular (as Pioneer), which is less favorable for block refuges.

Table 19. Risk Profile of ORD's st ochastic model for 20% block refuges Durability (time to resistance) Refuge Scenario Pioneer deterministic model ORD stochastic model No Refuge 5 years 3.5 years 20% Block Refuge (40 ha fields) 7 years 13.9 years

20% Block Refuge (144 ha fields – atypical field size) Not modeled 8.4 years

The stochastic results predicted that durability for Cry34/35 deployed with a 20% block refuge would last approximately 13.9 years for fields of typical size; for large 144 ha fields (which are not average size but outliers), durab ility is predicted to be around 8.4 years. The stochastic model also analyzed the effect of two different di spersal rates on the durability of Cry34/35 when deployed in a 20% block refuge. These two differe nt dispersal scenarios were explored because there are reported differences in adult CRW movement from Ne braska (Nowatzki et al. (2003)) and Illinois (Spencer 2009) implicating that CRW movement may be different throughout the

Corn Belt. ORD did not find that doubling the dispersal rate us ed by Nowatzki et al. (2003) increased the durability significan tly. However, long-distance dispersal was not modeled (i.e. >200 m/d) and may be an important factor for larg er, atypical field sizes with areas of low egg- 55 - density. Discussions over whether the Nowatzki data over or undere stimated movement rates for the entire Corn Belt may perhaps be less critical than expected. An analysis of the stochastic results suggests that there may be an inter action between dispersal and effective refuge.

Summary of ORD’s modeling (det erministic and stochastic)

ORD’s predicted durability for block refuges rang es from 12 to 14 years in both the deterministic and stochastic simulations. Three different appr oaches were used to in the process of the analysis: 1) Pioneer’s benchmar k assumptions were incorporated (side-by-side comparison, see Table 17) and 2) BPPD’s and ORD’s more realis tic parameter assumptions were considered for the deterministic simulations, and 3) the stocha stic model was used to predict block refuge durability for two different dispersal rates a nd different field sizes (typical and outlier). EPA/ORD’s analysis also included a probability di stribution for the deterministic part of the assessment to establish which durability results we re more likely to occur. BPPD believes that these factors have allowed ORD to conduct a more comprehensive analysis than was submitted by Pioneer.

At the present time, based on the available data and independently genera ted evidence discussed in this risk assessment, BPPD cannot recomm end a registration for OAM1, a 5% seed blend expressing Cry34/35 to control CRW, because OR D’s modeling supports that such an action

would increase the risk of unreasonable advers e effects (i.e. resist ance evolution).

D. Strip refuge configuration

In Pioneer’s assessment, predicted durability for infield strip refuges was assessed and compared to blocks and seed blends. For the most part , infield strip deployment was superior to the predicted durability of 20% block refuges and 5% seed blends. ORD’s preliminary exploration suggest that predicted durability fo r infield strips is not as strai ght forward and may be affected by effective refuge size which coul d make the strip refuge deploymen t either inferior or superior

to blocks. ORD determined that evaluation of durability for strips needed further analyses.

E. Options

1. Pioneer’s conclusions that a 5% seed blend is superior to the 20% block refuge were the result of a number of possibly unrealistic assu mptions that would have to hold true for the

entire Corn Belt. These include:

• Refuges would have to be plan ted consistently along the shor ter side of the Bt fields. This is against grower practice.

• Growers would have to move their refuge locations yearly. This is believed to be unlikely.

• Growers would have to plant refuges consistently over areas of lowest egg densities, which meant that they would have to be able to somehow determine where egg

densities were lowest. This is probably not a realistic assumption.- 56 - • Adult male movement would have to be limited to Pioneer’s estimate of 15 m/d. ORD’s analysis demonstrated that Pion eer underestimated di spersal; after having corrected for errors, adult movement was similar to what was reported by Spencer (2009). Data by Spencer suggested that a dult CRW movement occured on the order of 30 m/d to greater than 200 m/d (average 50 m/d) and further suggested that adult movement may be positively affect ed by greater population density.

EPA/ORD’s interpretation of the data s uggest that non-random oviposition and refuge placement are most important rather than non- random mating being the threat to a block refuge system. This suggests a potentially plausible method that Pioneer might use to

bolster their claims that failu re of the block refuge system is imminent. If the relative density of eggs could be estimat ed in transects across fields of different sizes, and if more accurate estimates of how growers plant and move their refuges over years were available, then major differences between the ORD and Pioneer models could be resolved. Such a study might also provide an estimate of the contribution of beetles dispersing long distance.

2. ORD explored the durability for different proportions of seed blends (0.025, 0.05, 0.075, and 0.10) and reported that predicted durab ility for the 10% seed blend was 11.3 yrs (with 51.6% of the simulations exceeding 11 year s). While this is still lower than the predicted durability for a 20% block refuge , the 10% seed blend came closest to the predicted block durability.

3. BPPD has considered tempora lly/geographically-limited regist rations in the past because they may reduce the risk for resistance evolution. These registrations are typically limited by total U.S. acreage (usually <250,000) and have county-level acreage caps (usually <20,000). However, the modeling analyses conducted by Pioneer and ORD were not designed to address the risk of a time lim ited registration for OAM1. Therefore, BPPD cannot specifically quantify the risk of resistance evolution for such an option.

4. BPPD notes that models predict that pyram ids of two PIP toxins can increase the durability of the traits and allow for a reduction of the percentage of refuge. ORD’s modeling showed that if two traits with the characterist ics of Cry34/35 were pyramided and deployed as a 5% seed blend that durabili ty would increase by more than three fold (52 years) over the sequential use of the tw o single toxins. Pioneer could consider the

addition of a second CRW-active toxin to OAM to improve the durability and potentially justify a 5% seed blend refuge.- 57 - 0 REFERENCES

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Other:

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