CONCLUSIONS AND UNCERTAINTIES

1. PRELIMINARY RISK ASSE SSMENT CONCLUSIONS

BPPD has reviewed Monsanto’s and Dow AgroSc iences’ submissions for a 5% SmartStax seed mixture including biological and efficacy data as well as modeli ng of the rate of resistance evolution. In addition, EPA’s Office of Re search and Development (ORD) conducted a independent analysis of the applicants’ proposal using a deterministic and probabilistic model. After careful review of the applicants’ mode ling and the analyses conducted by ORD, BPPD reached the following conclusions:

• For corn rootworm (CRW), a 5% seed mi xture and 5% structured refuge have comparable durabilities predicted by both the applicants’ and EPA’s models;

• For European corn borer (ECB), worst-case re sults estimated that a 5% seed mixture was less durable than a 5% structured refuge but did not develop resist ance within 52 years (conservatively assuming three generations pe r year for ECB). BPPD notes that a 5%

seed blend is still an over all durable Insect Resistance Management (IRM) strategy for ECB (considering the results from M onsanto/Dow and EPA/ORD modeling);

• For Southwestern corn borer (SWCB), worst- case results estimated that a 5% seed mixture modeled by EPA/ORD was also less dura ble than a 5% struct ured refuge strategy although resistance did not evolve within 24 years (conservatively assuming three generations per year for SWCB). As with ECB, BPPD notes that a 5% seed blend is still an overall durable Insect Resistance Manage ment (IRM) strategy for SWCB (considering both the results of Monsanto and EPA/ORD).

Monsanto Company and Dow AgroSciences separately submitted efficacy, adult emergence, and larval movement data, which served to support some of their assumptions made in their simulation models. Monsanto conducted modeling for lepidopteran pests, while Dow performed modeling on corn rootworm. The mo deling analyses focused on the time to resistance or ‘rate of adaptation’ for European corn borer, Southwestern corn borer, and corn rootworm from the use of a 5% SmartStax seed mixture (95% Bt and 5% non-Bt seed). Dow’s model predicted similar

increases in corn rootworm resistance allele frequencies for both re fuge strategies, while Monsanto’s model suggested that lepidoptera woul d not evolve resistance to SmartStax within 100 generations (33 yrs or 50 yrs, depending on the number of generations per year) when deployed in a 5% seed blend.

EPA/ORD’s simulation analyses were conducte d separately for CRW and ECB/SWCB using a two-locus deterministic and probabilistic model

, which is an extension of the model used in Caprio and Glaser (2010a). The analysis was conducted by running 1000 simulations, each time re-sampling a group of uncertain parameters using a PERT- Beta distribution (setting a minimum, most likely, and maximum value for each parameter of concern). For each pest, the model output generated a probability distribution fo r durability (time to resistance) based on the

1000 simulations that were conducted. To anal yze these distributions , BPPD established durability estimates for various levels of risk ( i.e., the likelihood that a resistance event would occur within a particular timeframe ). For example, a 5% level of ri sk would mean that in 95% of the simulations, resistance evolved in greater time than the durability estimate predicted.

- 2 -Conversely, in the other 5% of the simulations resistance developed with in the predicted time

frame.

In the CRW model simulations (see Table i), the m ean time to resistance was 82 years for a 5% seed blend and 53 years for a 5% structured refuge, a 1.5-fold diffe rence. As described above, a probability distribution was also created from the simulations for the estimated time to resistance with a seed blend and structured refuge. With a 5% level of risk, the durab ility of SmartStax seed for CRW was 33 years, while the bl ock refuge was 22 years. This means that in only 5% of the 1000 simulations conducted, resistance evolved within 33 years (seed blend) and 22 years (structured refuge).

In the EPA/ORD analysis that was conducted for ECB and SWCB, the same deterministic, probabilistic model was employed as for CRW, a lthough three-loci were us ed to represent the three lepidopteran-active toxins in SmartStax. This modeling estimated that a 5% SmartStax seed mixture was approximately 85% less durable fo r the European corn borer and approximately

38% less durable for the Southweste rn corn borer than a 5% stru ctured refuge. As with CRW, however, seed blends were still an overall dur able IRM strategy for bot h lepidopteran species. From the probability distribu tion for the estimated time to resistance, EPA/ORD’s modeling estimated that with a 0% level of risk (meaning that all simulations resulted in a time to resistance that was at least as high as the dur ability estimate) resistance would not evolve in European corn borer within 52 years (seed blen d) and 375 years (structu red refuge) (see Table ii). For Southwestern corn borer , resistance did not develop with in 24 years (seed blend) and 39 years (structured refuge) (see Table iii). Assuming a 5% level of risk, resistance was estimated to evolve in ECB within 150 years with a seed bl end and 1018 years with a structured refuge. For SWCB, durability was 46 years with a seed blend and 69 years with a structured refuge with a

5% level of risk.

A general qualitative (non-quant itative) comparison can be made between the results of the different models listed in Table i-iii, yet cau tion is warranted because assumptions, parameter values, and model structures differ. Despite the different assumptions made by different modelers, the applicants and EPA/ORD predicte d similar outcomes for a 5% SmartStax seed mixture and structured refuge target ing lepidoptera and corn rootworm.

Table i. Durability estimates by EPA/ORD and Dow fo r 5% SmartStax seed mixture and 5% structured refuge targeting Corn rootworm

1 For benchmark and worst-case assumptions Dow CRW Model EPA/ORD CRW model Refuge Scenario Increase in Resistance Allele Frequency 1 5% Level of Risk Mean Time to

Resistance

5% Seed Blend 33 years 81.9 years 5% Structured Refuge Similar rates of increase 2 for the 20 years modeled 22 years 52.9 years

2 Dow’s model predicted similar durability for SmartStax under both refuge scenar ios but no actual values were provided

Table 47 in this risk assessment

- 3 -Table ii. Durability estimates by EPA/ORD and Mo nsanto for 5% SmartStax seed mixture and 5% structured refuge target ing European corn borer

1 Simulations were run with the assumptions of three and two generations per year Monsanto ECB Model EPA/ORD ECB model Refuge Scenario Time to Resistance Level of Risk Time to Resistance 1

0% 158 gen. (52 yrs or 79 yrs) 5% Seed Blend > 100 gen (>33 or >50 yrs) 5% 451 gen (150 yrs or 225 yrs)

0% 1125 gen (375 yrs or 562 yrs) 5% Structured Refuge >100 gen (>33 or >50 yrs) 5% 3055 gen (1018 yrs or 1527 yrs)

Table 48 in this risk assessment

Table iii. Durability estimates by EPA/ORD and Mo nsanto for 5% SmartStax seed mixture and 5% structured refuge targetin g Southwestern corn borer

1 Simulations were run with the assumption of three and two generations per year Monsanto SWCB Model EPA/ORD SWCB model Refuge Scenario Time to Resistance Level of Risk Time to Resistance 1

0% 73 gen (24 yrs or 36 yrs) 5% Seed Blend > 100 gen (>33 or >50 yrs) 5% 138 gen (46 yrs or 69 yrs)

0% 118 gen (39 yrs or 58 yrs) 5% Structured Refuge >100 gen (>33 or >50 yrs) 5% 207 gen (69 yrs or 103 yrs)

Table 49 in this review

2. UNCERTAINTIES

Although BPPD identified several uncertainties in this risk assessment, the registrants and EPA/ORD addressed most of them by making c onservative assumptions in their simulation

models.

a) There may be regions in the Corn Belt wh ere corn earworm coul d overwinter. Such potential areas need to be identified b ecause they could contribute to increased selection in the target pest. No analyses have been conducted by the applicants or

EPA/ORD to assess the risk of re sistance evolution in such areas.

b) Under high infestation levels, some corn ear worm larvae were found in the ears of pure SmartStax corn and in seed mixture plots suggesting that some survival should be expected for this main target pest. Hence, SmartStax may not be as efficacious against CEW under high infest ation pressure.

c) Data show that some fraction of the adu lt ECB population takes pa rt in long-distance dispersal and that this behavior may be a nor mal part of ECB life-history. These insects

can cover more than 12 km in one flight attempt. Results by Dorhout et al. (2008) further suggest that a fraction of 1-d old females may engage in obligate migratory flights. Mark-recapture studies in aggregation sites have failed to collect more than 1%

- 4 -of the released individuals. In order to de termine whether a structured refuge some distance away from the Bt field or an integrat ed refuge via seed blend is the better IRM option for ECB, additional information will be helpful with respect to the proportion of the population engaging in obligate migratory behavior and timing of mating with respect to dispersal.

d) There is some uncertainty as to the increa se in the effective refuge in the EPA/ORD model for lepidopteran pests. While ECB a nd SWCB are mobile pests and both likely deposit their eggs in a uniform fashion (one assumption that increases the effective refuge in the model), the extent of their dispersal and the proportion of a population engaging in long distance movement (as di scussed above) is an uncertainty. BPPD

recommended to EPA/ORD to increase adult dispersal for lepidoptera compared to corn rootworm in the simulation model, which created greater mixing between compliant and non-compliant fields and should reduce th e rate of adaptation to SmartStax. As a result, the EPA/ORD simulations for lepidopterans may be too conservative and have

overestimated the time to resistance in E CB and SWCB in the block refuge simulations.

e) There is uncertainty with respect to ECB’s pr opensity for late instar larval dispersal off non-Bt plants and whether olde r larvae would still be highly susceptible to surrounding SmartStax plants in a seed mixture environment.

f) Typically the assumption has been made th at since ECB and SWCB are similar in many ways that ECB can serve as a surrogate for SWCB to address uncertainties about biology and genetics. The applicants’ efficacy data, however, suggest that SmartStax is

somewhat less toxic to older instars of SWCB. Results of a larval exposure study by Monsanto support the concern that SWCB survival is higher on SmartStax than for ECB. BPPD concluded that simulation models should incorporate su ch information in the analyses. There is currently a lack of data on the propensity of SWCB larval ‘plantto-plant’ movement and on how ECB and SWCB differ in this respect, if at all.

g) BPPD recommended that EPA/ORD use a mo st likely value of 0.05 (a conservative overestimate) for the SWCB initial resistance allele frequency of Cry1A.105 and Cry2Ab (PERT distribution). This value was c hosen because SmartStax is less toxic to

SWCB than ECB and because of uncertainti es with dose. Accordingly, the EPA/ORD model predicted a shorter time to resist ance for SWCB, which may underestimate the durability of SmartStax for both IRM strate gies. There is still uncertainty, however, with how to appropriately characterize initia l resistance allele frequency in simulation

models.

h) The applicants did not address (or report) the standard error of mixing for the new technology in their modeling analyses. Howe ver, Monsanto modeled a worst-case for

5% SmartStax seed blend scen arios of 1% effective refuge for ECB and SWCB, and it could be argued that the sta ndard error of mixing was alread y included in their analyses for lepidoptera. However, it is unclear whether Dow’s modeling results (rate of adaptation) for corn would have been affected by incorporating such a standard error of mixing.

- 5 -i) Monsanto and Dow concluded that although 3 rd instar CRW larvae were not affected by the two toxins expressed in SmartStax that “they will suffer little or no selection for Bt

resistance because of their tolerance to th e Bt proteins”. BPPD is concerned, however, that there is uncertainty with characteriz ing the tolerance of later instar CRW to Cry34/35Ab1 and Cry3Bb1.

j) For the SmartStax toxins, Dow/Monsanto made a density-dependent adjustment to their dose estimates based on density/survival re lationships developed by Onstad et al. (2006). The resulting dose mortality profile was: Cry34/35Ab1 (99.75%), Cry3Bb1 (99.75%), and Cry34/35Ab1/Cry3Bb1 pyramid (99.95%). On the other hand, BPPD has also considered separate work by Hibbard et al. (2010), which suggests that density-dependent mortality o ccurs at higher egg density le vels than those assumed by Dow/Monsanto. In light of this research, BPPD recommended in its 2009 risk

assessment of SmartStax that dose should also be evaluated without a densitydependent adjustment. The non-adjusted dose profile for the SmartStax toxin is: Cry34/35Ab1 (94.2%), Cry3Bb1 (97.5%), and Cry34/35Ab1/Cry3Bb1 pyramid (98.2%).

k) Male CRW emergence was reduced (up to 60 times) in 5% SmartStax seed mixtures when compared to male emergence in non-Bt control plots. BPPD is concerned with potentially negative effects this might ha ve on the product durability and questions whether additional modeling needs to be done including this information. The SAP (2009) concluded that a reduction in the numbe r of males from Bt seed blends could negatively affect the effective refuge.

l) Dow did not include density-dependent disper sal from non-Bt plants in seed mixtures. BPPD notes that under sufficiently high CRW infestation levels the number of larvae on refuge plants could build up to a point where density dependent movement could occur and some survival on SmartStax pl ants surrounding refuge plants should be expected. It is unclear what effect such a parameter for density-dependent dispersal would have on Dow’s predicted durability of seed mixtures.

m) Initial resistance allele frequencies in the target pests are unknown for the five toxins expressed in SmartStax and some reasonably co nservative estimates had to be made by all parties (applicants and EPA). EPA/ORD based its assumptions on a toxin’s date since first commercialization, the dose profile as supported by baseline susceptibility and/or the resistance monitoring data, as we ll as on results from lab selection studies. Initial resistance allele frequencies are likely to be sensitive parameters in all three

models and can overestimate time to resistan ce evolution if assumed to be low.

- 6 -I. BACKGROUND

A) Regulatory Background Information

Monsanto developed MON 89034 x MON 88017 (tra de name YieldGard Triple), which was registered in 2008 with MON 89034. Dow previ ously registered TC1507 x DAS 59122-7 (trade name Herculex Xtra) in 2005. Sin ce their original registration, Monsanto and Dow AgroSciences have collaborated to pyramid these single gene events into a multi-trait Bt corn product called SmartStax to control the main lepidopteran and coleopteran target pests of corn in the US: European corn borer, corn earworm, southweste rn corn borer, fall armyworm worm, and corn

rootworm.

SmartStax was conditionally registered by the Agen cy in July, 2009 with a 5% structured refuge for lepidoptera and corn rootworm . As a condition of registration BPPD requested that additional data be developed to address uncertainties with dose (Cry3Bb1 and Cry34/35) and initial resistance allele frequency to support a 5% re fuge for corn rootworm. BPPD also recommended revised modeling incorporating these additional data to assess the ri sk of resistance evolution in

corn rootworm.

In December 2009, Monsanto and Dow submitted a reque st to the Agency to register SmartStax

with a 5% seed blend in the Co rn Belt and cotton growing regions where corn earworm is not a significant pest. In this review, BPPD summ arized and discussed the studies submitted by Monsanto and Dow to support thei r registration request and indepe ndently assessed the risk of resistance evolution in Lepidopt eran and Coleopteran target pe sts using a model developed by

US EPA/ORD.

B) Additional Biological Backgrou nd Information of Target Pests

This section represents a summary of BPPD’s literature search regarding additional biological

information relevant to this risk assessment.

1. European Corn Borer (ECB)

Adult movement: European corn borers have a lo cal dispersal mechanism and long- distance dispersal ability. The proportions and frequency of individuals in a population enga ging in dispersal before or after mating are not available, but thes e are important parameters in simulation models that can impact modeling results relative to an increase or decrea se of resistance evolu tion. Some models suggest that limited dispersal can accelerated the evolut ion of resistance (Alstad and Andow 1995); other models show that this may not always hold true especially when female dispersal is limited, refuges remain in the same location year afte r year, and male dispersal is sufficient enough (Caprio 2001; Guse et al. 2002; Ives and Andow 2002; Heimpel et al. 2005).

In an early European corn borer study, Caffrey and Worthl ey (1927) documented that the introduced species travelled close to 45 km across Lake Erie, which implies that this distance

- 7 -could be covered by within one generation. Th e proportion of the population that undertakes such long-distance traveling is unclear.

Mason et al. (1996) established that many ECB take a dvantage of aggrega tion sites (usually clusters of weeds or grasses) near corn fiel ds for mating. Females appear to mate the second night after pupal eclo sion. Another study suggested that it may be possible to manipulate aggregation sites to increase the likelihood of random mating betw een susceptible and potentially resistant ECB (Hellmich et al. 1998). In a recapture expe riment aimed at studying local dispersal and mating behavior, Hunt et al. (2001) released 3000 to 7500 adults (reared on colored diet) in irrigated and non-irrigated fields in Nebraska. Adults we re allowed to disperse between 24 h -72 h before trapping began; the r ecapture period lasted three days. Hunt et al. reported that more adults were caught per trap 1) within and near irrigated than non-irrigated fields, 2) within as opposed to outside corn fields, and 3) near the release site than away from the release site. The recapture rate at <210 m from the release point was <0.8%. More males and mated females were

found within rather than outside the fields. Si gnificantly more males and mated females were caught near the release site. Hunt et al. concluded that these results supported the contention that females mostly mated in corn fields and remained near emergence sites.

Showers et al. (2001) conducted a mark-recapture study (conducted during 1986-1988) to determine the general movement pattern of ECB out to 64 km within 3 days after the release time and from the release point (f lush bar sampling near release sites and wing-type traps). In 1986, less than 1% of marked individuals were ca ught within 3.2 km from the release point. One male each was recaptured at 6.4 and 9.6 km (effe ctive ~11.52 km). In 1987, the overall recapture proportion still was less than 1% bu t significantly more males were recaptured at the 800 m trap distance than at the 9.6 km trap distance. Two ma les were collected at the 19.2 km trap distance. In 1988, they found two females and one male that had travelled 14.2 km within 100 minutes of

their release. One male was ca ught 25.6 km from the release point; two males and one female were caught at 38.4 km, one male at the 40.2 km tr ap site, and one female at 49.1 km from the release point. They found in their mark-releas e-recapture study that adult ECB males and females could disperse great distances within 1-3 days after their re lease (23-49 km). While airflow likely contributed to the long-distance movement, Showers et al. stated that the majority of ECB may be capable of flying such great dist ances but that this study did not resolve whether

such movement was rare or common.

Bailey et al. (2007) released marked ECB adults into herbaceous borders surrounding cultivated corn fields in the south of France during 2003 to estimate what proportion of the marked individuals would remain near the release po int over a 12-h period. Their recapture success (7.7%-44.8%) suggested that within the first 12 hours of emergence, ECB did not engage in long-distance dispersal if habita t conditions were favorable (dense herbaceous borders within close proximity of em ergence site). Bailey et al. further reported that th e typical distance covered by adults within the first 12 hours (5 m- 35 m) might be insufficient to ensure random mating between resistant ECB emerging from the Bt field and susceptible ECB emerging from the refuge field.

Dalecki et al. 2006 conducted a two-year pre-dispersal-ma ting study in France to estimate the probability that 1) young ECB (<24-h) were found in field borders two days after their release

- 8 -and 2) mating of these individua ls occurred amongst themselves rather than with immigrants from other fields. They found that when marked indi viduals (24 h old) were placed in fields that the proportion of recaptures averaged 4.3% and ranged from 0.2%- 26.9%. Approximately 97% of the recaptured females were mated (28% of al l the released virgin females). A carbon isotope analysis of the spermatophore revealed that released females mated with both wild type (immigrant) and marked males and that the pr oportion of marked males mating with marked females ranged from 0% to 67% among mated fe males. Their study results further support the hypothesis that “males mate irrespective of whethe r they are immigrants or residents of a corn field”. Some evidence from their study suggests, however, that immigrating females may have a lower mating success than resident females.

In contrast, Reardon et al. (2006) conducted a mark-release-recapture study to test whether newly released ECB could be retained in aggr egation sites near emergence locations. Their recapture results indicated that le ss than 1% of the released insect s remained in aggregation plots for one day. Reardon et al. concluded that this might imply a possibility of obligatory migration of young ECB adults.

Dorhout et al. (2008) tested this hypothesis in a flight mill experiment; their results suggested that 1-day old unmated female ECBs could enga ge in obligate migratory dispersal of >20 km.

More than 65% of 1-d old females flew for one hour continuously (n-tot al = 42), while only 45%

of 5-d old unmated females flew for the same time duration (n-total = 45). Approximately 5% of

the 1-d and 2-d old unmated females flew for 8 h ours continuously, and none of 3-d and 5-d old females flew for such a long and uninterrupted time period. Twenty-five km and 20 km were the greatest distances covered by unmated females a nd male, respectively, du ring the 8-hr flight period.

Dorhaut et al. (2010) investigated fli ght differences of unmated ECB both infected and uninfected by Nosema pyrausta in a flight mill study and found that moderately infected individuals had reduced flight cap acity compared to slightly in fected and non-infected adults. Flight duration and total flight distance were reduced. For exam ple, they found that 77% of uninfected or slightly infected 1-d old females flew at least 1⁄2 km comp ared to only 47% of moderately infected females. On e-day old, uninfected females flew distances up to 3-fold greater than 3-day old females (as measured by median distance); this result supports an earlier observation by Dorhout et al. (2008) that 1-d old un mated females engage in obligate migration. Their preliminary analysis of data for 3-d old ad ults suggested that flight distance and duration were not negatively impacted by Nosema infection. It was observed that females generally flew further than males, irrespective of whether they were parasitized.

Kim et al. (2009) investigated the genetic variability of ECB using eight microsatellite loci of ECB along two 720 km, perpendicular transects in Io wa. No genetic variability was detectable at this scale suggesting large dimens ions for ECB populations. Showers et al. (2001) and Kim et al. (2008) both provide empirical evid ence from the field supporting that long-distance dispersal in

ECB is not unusual.

Larval movement: ECB larvae are capable of significant, plant-to-p lant movement within corn fields. Research

- 9 -conducted in non-transgenic corn sh owed that the vast majority of larvae did not move more than two plants within a row (Ross & Ostlie 1990); di spersal occurred soon after hatching and was not density-dependent. Between 50-56% of the larvae remained on the infested plants and did not disperse. In transgenic cor n, unpublished data (used in mode ling work) from Dr. Fred Gould (cited in Onstad & Gould 1998), however, indicate d that approximately 98% of susceptible ECB neonates moved away from plants containing Bt. Hi s data showed that the probability of leaving a non-Bt plant was approximately 90%.

Multi-year studies by Hellmich (1996, 1997, and 1998) attempted to quantify th e extent of plantto-plant larval movement. It was observed that 4 th instar larvae were capable of moving up to six corn plants within a row and six corn plants acr oss rows from a release point. Movement within a row was much more likely than movement acro ss rows (not surprising, due to the fact that plants within are row are more likely to be “touch ing” as opposed to those across rows). In fact, the vast majority of a movement cross rows was limited to one plant. This type of information has obvious implications for optimal refuge de sign. Larvae moving across Bt and non-Bt corn rows may be exposed to sub-lethal doses of protein increasing the li kelihood of resistance (Mallet & Porter 1992). Given th e extent of ECB larval movement between plants, seed mixes were previously determined to be an infe rior refuge option (Mal let & Porter 1992, SAP 1998, SAP 2000, and Onstad & Gould 1998).

Prasifka et al. (2009) found that 51% and 36% of re sistant and susceptible ECB larvae, respectively, moved off Cry1Ab expr essing corn plants in their la b experiment (total of 87%). This is less than the 98% reported by Onstad and Gould (1998). Prasifka et al. observed lower levels of movement of suscepti ble individuals in seed blends than previously estimated and hypothesized that susceptible larvae may experi ence greater mortality in seed mixtures.

Egg density

Sorenson et al. (1993) reported that the di stribution of egg densities of second generation ECB was random and that oviposition was relatively un iform throughout corn fields in North Carolina (three year study) which suggests that in mature corn, female ECB may not differentiate between different types of host quality. These findings in North Carolina were consistent with the oviposition patterns observed by second generation ECB in Kansas corn fields and first generation ECB in Minnesota corn fields.

2. Southwestern Corn Borer (SWCB)

Adult movement Observations by Rolston (1955) found that adults were capable of dispersing and ovipositing 2.4 km from their source site. Post-mating dispersa l may be significant. Rolston (1955) and Langille and Keaster (1973) showed that between 66%-91% of females mated within 24-48 hours after emergence. In addition, Langille and Keaster (19 73) observed that males engaged in a preferred pre-copulatory flight behavior in field of emergence. Guse et al. (2002) concluded from this that random mating occurred in the natal field.

- 10 -In a mark/recapture experiment to study local movement (Buschman et al. 1999), the following observations were made regardi ng SWCB: 1) more males than fe males were captured at greater distances from the release point (similar to ECB) ; 2) most recaptures of SWCB were within 100 feet of the release site, although some were also noted at 1200 feet; and 3) the movement patterns for ECB and SWCB appeared to be similar in most regards. The 1999 results, however, were hampered by low SWCB numbers available for testing.

In a mark-release-recap ture study by Qureshi et al. (2006), a limited proportion of marked SWCB were recaptured (2.1-17.9% for males and 0-4.2% for females). The majority (90%) of recaptures occurred within a 300 m perimeter from the release point. Only 1% of marked males were caught outside the release field. A substantial number of immigrant adults were also captured at the center of the experimental fields and likely come from refuge fields nearby (587- 1387 m away from the perimeter of the study fiel ds) suggesting that male dispersal can be greater in SWCB than suggested by the mark-recapt ure results. Thirty-eig ht percent and 14% of the marked and wild females, respectively, were unmated.

3. Corn Rootworm (CRW)

Adult movement: Little empirical data have been published on eco logical and environmenta l factors that affect dispersal and oviposition behavior of adult CRW. Published literature indicates that phenology of maize, density-dependence, a nd environmental factors such as soil texture and elevation are some factors affecting adult CRW disper sal spatially or temporally (Meinke et al. , 2009).

Tethered flight studies conducted by Naranjo (1990) and Coats et al. (1986) provided evidence that young female western corn rootworm (WCRW) were more likely to engage in longer sustained flights than males. The maximum time duration measured in Coats’ study converted to about 24 km/day – 39.4 km/day. Approximately 25% of the individuals that dispersed long distance were primarily females in their pre-ovipositional phase. Grant and Seevers reported that the majority of dispersal occurred prior to ovi position and that the ti me from emergence to oviposition was between 12-23 days (Grant & Seevers, 1989). They also stated that female dispersal was the primary dispersa l mechanism for the WCRW species.

Scott et al. (2004) and others (Witkowski et al. 1975; Van Woerkom et al. (1983)) showed that some atmospheric conditions and light winds (pre dominantly occurring early in the morning and evening) contributed to long range dispersal of WCRW out of co rn fields. Typical summer cold fronts with wind speeds of greater than 1.5 m/s -4m/s provide another predictable venue for longdistance dispersal of WCRW of up to tens of kilometers (Grant & Seevers, 1989).

Nowatzki et al. (2003) conducted a field study (mark-recapture) with western corn rootworm ( Diabrotica virgifera virgifera , WCRW) using a rubidium labeling technique (Nowatzki et al. 2003b) to quantify short range, in -field movement of adult be etles from the time of their emergence. The experiment was conducted in Nebraska during 2002 a nd 2003. Beetle movement was tracked with traps positioned in a circular pattern at fixed distances (50 ft, 100 ft, 200 ft, 300 ft, 400 ft, and 600 ft) from the center of the infested area to the field edge. Beetles were collected throughout the emergence time at regular intervals. Estimated dispersal results suggested that

- 11 -males moved 605 ft within 1-2 days from the center of the field. Few females were captured outside the infested area; after peak female emerge nce, females were also trapped at 605 ft away

from the central infested area.

Spencer found evidence of long-ra nge dispersal of the variant CRW during his 2007 experiments in Bt corn and non-Bt soybean fields in Illinois. He measured adult dispersal of greater than 200 m/d. For 2009, he reported that adul t CRW of the variant type coul d move as little as 30 m/d (average 50 m/d) but as much as 80 m/d (very low CRW year in 2009) and stated that greater corn rootworm abundance appeared to result in greater adult dispersal (S pencer 2009; personal communication, 2009).

Larval movement: In their larval movement study, Hibbard et al. (2003) reported that 0.75% and 6% of larvae

moved across several rows. Under hi ghest infestation scenarios, larv ae moved as far as 3 plants

down the row and 0.76 meter across. Larval moveme nt was largely dependent on the density of plants within a row, food availability, soil porosity, and level of soil compaction. Hibbard et al. (2004) concluded that larval movement in experiments conducted from 2000-2002 was also dependent upon egg density per plant and that density-dependent dispersal for neonates did not occur during the three-year field study even under high-density pressure (with artificial infestation). Higher WCRW egg densities lead to greater larval movement and did not significantly reduce larval recovery; however, adu lt recovery was lower, hence, suggesting most density dependence for WCRW occurred afte r larvae had established themselves.

Hibbard et al. (2005) found that late-instar movement occurred from sufficiently damaged nonBt plants to surrounding transgenic plants and indicated that it wa s more realistic to assume high

larval movement from late instars and little to no movement for neonates. However, statistically significant neonate movement from Bt plants to n on-Bt plants was observed in one year of their field study (Hibbard et al. 2005). Hibbard et al. stated that in itial larval development on non-Bt roots followed by later movement to Bt root s could accelerate resistance evolution if heterozygote larvae (RS- genotype ) experienced greater survival on Bt. This is supported by

Meihls et al. (2008) where exposure of neonate larvae to Cry3Bb1 followed by rearing on isoline

corn did not result in the de velopment of resistance in choice tests, while transferring 2 nd instars from isoline to Cry3Bb1 did eventual ly lead to resistance in the selected strain, although the level of resistance was reduced and its developmen t was slower than with complete rearing on Cry3Bb1 corn.

Egg density: Work by Branson (1986) suggests that CRW egg masses in fields are best described by a clumped distribution. This may indicate that fema le CRW are capable of detecting different host qualities.

Density dependence:

Storer (2003) used an eq uation developed by Onstad et al. (2001),

7.0 42.21 1 1 d 

## S

d ×+ −≡ , to

model density-dependent mortality in CRW where S d is the probability that larvae survive density-dependence and d is the larval density in millions per hectare.

- 12 -Onstad et al. (2006) used published data from WCRW a nd NCRW to investigate the relationship between density and survivorship from eggs to adulthood in the absence of pesticide exposure. They found that larval survival in non-Bt corn was density-dependent and that NCRW had generally lower survival than WCRW. A series of equations was derived that fit the data well and which allowed estimating survival of CRW due to density-i ndependent factors.

Hibbard (2010) determined from a multi-year study that density dependent mortality in CRW was apparent at 851 eggs (95% CI = 678-1024) per 30.5 cm. An estimated 54.4% of larvae were killed that established on plants with highest egg densities (hi gh infestation year). When data were pooled across years, density-dependent eff ects began at approximate ly 800 viable eggs per 30.5 cm of row (approximately per 1.6 plants).

II. PROBLEM FORMULATION

The Federal Insecticide, Fungi cide and Rodenticide Act (FIF RA) requires that registered pesticide uses do not pose unreasonable adverse e ffects to the environment. The purpose of this risk assessment is to provide insight into the pot ential effects to the environment as measured by the risk of resistance evolution in Bt corn target pests (lepidoptera and corn rootworm) associated with the use of a 5% seed blend registra tion for SmartStax corn. Monsanto and Dow AgroSciences requested to amend their SmartSta x registration by replacing the 5% structured refuge with a 5% seed mixture for the Co rn Belt and some parts of the Cotton Belt.

This risk assessment focuses on assessing the risk of resistance developing to a 5% SmartStax seed mixture in three main target pests: Europe an corn borer, Southwestern corn borer, and corn rootworm. The registrant proposed to use the existing 5% structured refuge strategy for SmartStax in most parts of the Cotton Belt so as to not further contribute to the risk of resistance evolution in corn earworm. Although it is known that CEW migrate northward during the growing season to corn-growing regions (i.e., th e U.S. Corn Belt and Canada), CEW typically are not capable of overwintering in these regi ons. Rather, CEW are known to overwinter in the South, often in cotton fields. Some reverse migr ation from the Corn Belt to the Cotton Belt was observed by Gould et al. 2002, which sparked interest and the need to quantify the impact of south-north and north-south migration on the adap tation rates in CEW. Computer simulations by the Agricultural Biotechnology Stewardshi p Technical Committee (ABSTC) showed no significant interaction between th e percent of the late summer adult CEW population in the south that is made up of immigrants and the date at which migrants return and no effect of return migration on the resistance gene frequency.

In the case of fall armyworm (FAW), a secondary target pest of SmartStax in the continental US, the species distribution is currently limited to areas of southern Florida and southern Texas; hence FAW are not currently a corn pest in the regions proposed for the adoption of a 5%

SmartStax seed mixture. Should future climate change elicit a range expansion for FAW and expand its overwintering capacity into the Corn Belt, then a revised risk assessment will be needed for this target pest and the proposed 5% SmartStax seed mixture.

- 13 -A. Seed Mixture vs. Structured Refuge

Seed mixtures incorporating Bt and non Bt crop seeds have been a topic of discussion for almost

two decades. While such an Insect Resistance Management (IRM) strategy has obvious benefits ( i.e., no grower compliance component, facilitating the planting of Bt and refuge fields), a variety of researchers have indi cated that under some circumstances this approach could lead to more rapid evolution of resistance in some targ et pests (e.g., Mallet and Porter, 1992; Onstad and Gould, 1998; Davis and Onstad, 2000 and more).

Scientific Advisory Panels (1998 and 2000) discour aged the Agency from the use of Bt seed mixtures to control lepidopter a target pest because substan tial larval movement could be expected between Bt and non-Bt plants leading to more rapid selection of resistance. Conversely,

the 2009 SAP concluded that a 20% seed blend stra tegy for Bt corn with low- or medium-dose effects on corn rootworm (Coleoptera: Chryso melidae) was supported by the insect’s limited larval movement. But the SAP specifically no ted that this recomm endation should not set precedence for other Bt crops targeting other pests (SAP 2009).

Theoretical work by Mallet and Porter (1992) show ed that insect resistance was accelerated in seed mixtures compared to Bt stands where 10% of the population was no t exposed to selection, when the probability of larval movement ranged from intermediate to high ( ≥ 0.2), intensity of selection was great, and dominance of the resistance gene was low. They also reported that when insects selectively chose their food source (avoi dance of toxic substanc es), then effective dominance was increased, and seed mixtures could delay resistan ce evolution but that predicted outcomes depended also on other circumstances.

Using Mallet and Porter’s model, Tabashnik (19 94) reported that 10% Bt seed mixtures were more durable than pure Bt stands and that bloc k refuges of 10% were at least as durable (and more) as 10% seed mixtures when inheritance of resistance was recessive, partly recessive, or additive. As the percentage of refuge increase d from 0-50%, Tabashnik fu rther reported that a block refuge strategy performed equally well or better than seed mixtures. However, seed mixtures in conjunction with re fuges were the most durable of all deployment strategies

evaluated.

Onstad and Gould (1998) recommen ded 20% block refuges adjacent to the Bt fields over seed blend strategies because of un certainties surrounding expression of high-dose against the target pest. Their modeling results predicted that bloc k refuges would be more durable than seed

mixtures aimed at contro lling lepidopteran pests.

In a 2-yr field study with seed mixture expres sing Cry1Ab and non-Bt corn, Davis and Onstad (2000) obtained larval dispersal data, which were used to parameterize their simulation model

and assess the effect on the durabil ity of seed mixtures. Davis and Onstad observed that survival (after recovery) was lower for larvae that successfully dispersed from Bt to non-Bt plants compared to larvae that dispersed from non-Bt to non-Bt plants; this should favor heterozygote genotypes and make inheritance of resistance le ss recessive (Mallet and Porter 1992). In addition, increased mortality of susceptible larvae moving off refuge plants onto Bt plants would

result in a reduced effective re fuge. They reported that neonate dispersal away from Bt plants

- 14 -was greater than dispersal observed from non-Bt pl ants and noted that extensive use of seed mixtures could select for populations with impr oved dispersal capabilities . The empirical data was used to parameterize the simulation model (Onstad and Gould, 1998), and it was determined that while seed mixtures delay evolution of resi stance in ECB they are less effective at doing so than IRM strategies using a 20% block refuge.

Carierre et al. (2004) concluded that the differences in Bt trait durability predicted by block refuges and seed mixtures in Mallet and Porter (1992) and Taba shnik (1994) were caused by a reduction in refuge insects in seed mixtures that had moved from non-Bt pl ants to Bt plants and could be overcome if the percent of non-Bt plants in seed mixtur es was increased (compared to blocks).

B. Uncertainties

1. BPPD recognizes that to determine whether a 5% seed mixture ( both lepidoptera and coleopteran controlling agents) is a superior, equivalent, or inferior strategy to the currently approved 5% block refuge for SmartStax, a number of uncertainties and questions need to be considered.

2. There is evidence that some fraction of the adult ECB population may take part in longdistance dispersal (references described above) and that this behavior is a normal part of ECB life-history. Evidence s uggests that these insects are able to cover more than 12 km in one flight attempt. Results by Dorhout et al (2008) suggest that a fraction of 1-d old females appear to engage in obligate mi gratory flights. Mark-recapture studies in aggregation sites have failed to collect more than 1% of the released individuals. To determine whether a structured refuge some distance away from the Bt field or an integrated refuge via seed blend is the better IRM option for ECB, information with respect to the proporti on of the population engaging in obligate migratory behavior and timing of mating with respect to dispersal is crucial.

3. The proportion of the female ECB populati on that would have to engage in preovipositional long distance disper sal before durability of a 5% SmartStax seed mixture became compromised and whether that proportion would be a reasonable assumption is

uncertain.

4. When susceptible ECB larvae move from Bt pl ants to non-Bt plants and their survival is lowered compared to susceptible larvae that have not previously been exposed to Bt (Onstad & Gould 1998), then a se ed mixture can be expected to decrease the effective refuge. Likewise, if susceptib le larvae leave non-Bt plants, arrive on Bt plants, and are subsequently killed, then seed mixtures can be expected to redu ce the refuge population to less than what can be expected to emer ge from a block refuge. In either case, literature supports that such conditions would increase the risk of resistance evolution in the target pests. An assessment is needed of whether the reducti on in effective refuge is substantial and could reduce the overall durability of the Bt product.

- 15 -5. Based on the timing of mating for SWCB (within 24-48h) and the pre-copulatory flight behavior over natal fields by males, it has been suggested that random mating occurs in the field for this target pest. Results by Qureshi et al. (2006), however , suggest that SWCB are also capable of dispersing great er distances. Based on the limited studies available in the public literatu re, it appears that mating co uld primarily take place in natal fields but that greate r dispersal may also occur af ter the mating phase. Whether mating occurs before or after dispersal or whether only a fraction of females disperse during the pre-mating phase is important in formation and can shape the IRM approach

recommended.

6. There may be regions in the Corn Belt wh ere corn earworm coul d overwinter. Such potential areas need to be identified b ecause they could contribute to increased selection in corn earworm, which is a ma in target pest of both cotton and corn.

7. A reduction in male CRW in Bt mixtures wa s discussed by the SAP (2009) with respect to 5% Cry34/35 Bt corn mixtures. The SAP (2009) concluded that a reduction in number of males could negativel y affect refuge effectiveness. This concern also applies for the pyramided Bt corn mixture ta rgeting CRW and should be addressed.

8. Different types of events and behaviors have been documented to affect WCRW adults’ dispersal spatially and temporally in the US Corn Belt. Despite this additional evidence for long distance dispersal, some data gaps st ill exist and should be addressed so that dispersal can be best incorporated into si mulation models. For example, Spencer (2009) reported that individuals of the variant WCRW type disper sed more than 200 m/day; however, the proportion of individuals in a population undertaking this sort of long distance movement is unknown. Likewise, uncer tainties associated with proportion of populations dispersing during cold front events (wind speeds of > 1.5 m/s) and frequencies of such events make it challeng ing to include this mode of dispersal.

9. It is unclear what proportion of female CRW populations would have to engage in preovipositional long distance disp ersal before durability of a 5% SS seed mixture could potentially be compromised.

10. It is unclear how non-compliance will affect th e durability of a 5% block refuge relative

to a 5% seed blend and whether such non- compliance assumptions are reasonable for

the US.

- 16 -III. MONSANTO/DOW STUDY SUMMARIES

A. Corn Rootworm Movement, Dose, and Survival (Appendix 5 (RPN-08-518); Appendix 6 (RPN-09-429); Appendix 7 (RPN-09-159); Appendix 9 (NA09S2J005); Appendix 10 (NA09S2J007); and Appendix 11 (NA09S2J008))

1. RPN-09-159 - Movement and Survival of Western Corn Rootworm in Seed

Mixtures of MON 89034 × TC1507 × MON 88017 × DAS-59122- 7 in 2009 U.S. Field Trials (Appendix 7)

Monsanto conducted a field study in 2009 to evalua te the survival and movement of CRW in 100% SmartStax, its seed mixtures , and structured refuge plots as measured by root damage.

The treatments were:

• 100% SmartStax with Poncho 250 on Bt seeds;

• 5% seed blend with treated non-Bt seeds (Poncho 250);

• 10% seed blend with treated non-Bt seeds (Poncho 250); and

• 100% non-Bt treated with Poncho 250.

Three sites were planted with large non-replicated strip plots (at least 16 rows wide and 300 ft long) in areas with natural CRW infestations (Flanagan (IL), G ilbert (IA), and Livingston (OH). Control sites with root damage ratings > 0.5 were selected to evaluate the efficacy of Bt treatments. In each seed mix plot, roots from fi ve groups of plants were assessed using Oleson et
al. ’s (2005) nodal injury rating. A group of plants consisted of 10 Bt plants surrounding one nonBt plant. For the control plots and 100% Smar tStax plots, 15 plants were non-systematically

selected and their roots assessed.

Study results : The Flanagan (IL) site was not analyzed due to lo w pest pressure that year, and Monsanto did not provide a discussion of results in their submi ssion. Results from the Gilbert (IA) site were

removed from the data set because adverse weathe r conditions interfered with root collections. Hence results reported are for Livingston (OH) only.

Root damage was highest in contro l plots and non-Bt plants in seed mix plots verifying that corn rootworm presence was abundant in Bt and non- Bt treatments. There was no statistically significant difference in damage on non-Bt plants from differen t treatments although the root damage in the 5% seed blend was numerically lower when compared to the root damage

observed in the 10% seed blend and control plot ( Table 1 ). Monsanto concluded from these results of no-difference that non-Bt plants in al l three treatments fulfille d their refuge function.

The SmartStax plants had relatively little dama ge in the pure stand and seed blend plots. However, the damage on SmartStax plants in th e 10% plot was significantly greater than in the 5% seed blend and 100% SS plot. Monsanto conc luded that some CRW movement occurred in the seed mix plots from non-Bt to SS plants that lead to significant diffe rences in damage among treatments. When the damage to SS plants surro unding the non-Bt plant wa s assessed in the seed blends, no effect of spatial location on root damage was detected. Monsanto concluded that

- 17 -CRW movement off no n-Bt plants was low and di d not differentially affect SS plants adjacent to non-Bt plants.

Table 1. Root damage ratings for Sm artStax and Non-Bt plants of diff erent Treatments (Livingston, Ohio)

Treatment Plant Sampled Mean Rating Standard Error Significance (p<0.05)

100% SmartStax Bt 0.010 0 A

95% SmartStax Bt 0.028 0.01 B

90% SmartStax Bt 0.052 0 C

5% non-Bt non-Bt 0.736 0.36 A

10% non-Bt non-Bt 1.270 0.38 A

100% non-Bt non-Bt 1.252 0.24 A
Modified and extracted from Appendix 7, p. 604, MRID 479437-01

Registrant conclusions : Based on the level of root damage observed on non- Bt plants, 5% and 10% seed blends appeared to have supported comparable numbers of larvae on refuge plants as di d plants in the non-Bt plot. SmartStax plants had low levels of damage in the 5% and 10% seed blend suggesting that there was limited larval movement off refuge pl ants. Monsanto concluded that a 5% seed blend with SmartStax should be an effective refuge strategy for CRW while producing comparable amounts of refuge insects as an appropr iately placed 5% structured refuge.

BPPD Review BPPD notes that the differences in damage between the SmartStax treatments in this spatially and temporally limited study were statistically significant ( Table 1 ), although SmartStax-plants from the 95% seed blend suffered less damage than what would be expected if there was a linear relationship between damage incurred and percentage of Bt plants in plots. Damage to SmartStax plants in a 95% seed mixture was closer to damage of plants in pure SmartStax plots rather than being intermittent between 100% and 90% seed mixtures.

The damage observed on non-Bt plants in the 5% seed mixture was lower, yet not statistically significant, than damage incurred to non-Bt pl ants in 10% plot and controls. The relationship between damage levels and proportion of refuge s eed was again not linear. Refuge plants in the 10% mix incurred damage more similar to refuge plants in the contro l plots (100% non-Bt).

Monsanto’s conclusion that the le vel of root damage on non-Bt pl ants in 5% and 10% seed mix plots indicated that these plants supported co mparable numbers of la rvae as the corresponding structured refuge is plausible. But the additional root protection received by non-Bt plants in 5% seed blends suggests that the eff ective refuge in a 5% seed mixtur e could be lower than expected in a structured refuge. There is uncertainty whethe r such a reduction in effective refuge in a 5% seed mixture could significantly increase the rate of resistance evolution in CRW.

Unfortunately, Monsanto was unable to successfully repeat this experiment in multiple locations.

It would have been informative to see whether the observed trend of dama ge levels was present

at other locations.

- 18 -2. RPN-08-518 - Movement and Survival of Western Corn Rootworm on the

Combined Trait Product MON 89034 × TC1507 ×MON 88017 × DAS-59122-7 and a NonBt Control (Appendix 5)

Monsanto conducted a lab study and field study to investigate CRW survival and plant-to-plant movement on SmartStax and non-Bt plants. The growth chamber experiment was designed to assess 1) larval survival and 2) adult emergence of WCRW, while the field experiment explored the effects of CRW feeding on SmartStax and no n-Bt plants on i) la rval plant-to-plant movement, ii) survival, a nd iii) adult emergence.

a) Growth chamber/lab experiment : Two treatments were tested in this experiment: individual (caged) pots co ntaining either a) one SmartStax plant or b) one non-Bt plant. Each treatment received one of three levels of

inoculation with CRW larv ae: plants were either inoculated with i) 40 1 st instar, ii) 20 2 nd instar, or iii) 20 3 rd instar larvae. The study was set up as a randomized complete block design with a total of six plants and three replications in each (SmartStax and non-Bt) treatment making it a total of 18 plants per treatment; there were two plan ts per sublevel in each replicate (total of six for SmartStax and non-Bt each). The growth chamber was the blocking-factor, and three different chambers were used containing one of the replicates for each treatment.

Potted plants were inoculated with neonates at the V2/V3 stage and with second and third instar larvae at the V4/V5 stage. After the inoculation had occurred, plants were caged in preparation of emerging adults. Adult collections occurred three times per week and continued until no adults were collected for five consecutive sample dates.

ANOVA was used to compare head widths and weight between treatments for each sublevel (larval infestation types) and the percentage of emerged adults between treatments.

Study Results : SmartStax provided high level of control of 1 st instar (neonate) WCRW, somewhat less control

of 2 nd instar (yet still significan tly different from non-Bt), an d no significant control of 3 rd instar larvae (see Table 2 ). For neonate infestations, adult emergence was 93% lower on SmartStax plants than on non-Bt plants.

Adults that emerged from the 1 st instar inoculation on SmartStax were smaller and weighed less than their non-Bt counterparts (s tatistically sign ificant). Results at the remaining two sublevels were not significantly different betw een SmartStax and non-Bt plants (see Table 3 ).

- 19 -Table 2. Average % WCRW emergence from SmartStax and non-Bt plants in growth ch amber experiment

Treatment

sublevel Treatment Mean % Emergence Standard Error P –value (0.05)

adults from 1 st instar SmartStax 2.0 0.87

adults from 1 st instar non-Bt 30.5 1.60 <0.0001

adults from 2 nd instar SmartStax 17.2 1.69

adults from 2 nd instar non-Bt 46.3 5.32 0.0284

adults from 3 rd instar SmartStax 30.8 5.07

adults from 3 rd instar non-Bt 31.1 8.73 0.9684

Table extracted and modified from Appendix 5, p. 339

Table 3. Average weight of emerging WCRW in growth chamber experiment

Treatment

sublevel Treatment Mean Weight (mg) Standard Error P –value (<0.05)

adults from 1 st instar SmartStax 1.34 0.13

adults from 1 st instar non-Bt 1.69 0.10 0.0022

adults from 2 nd instar SmartStax 1.17 0.06

adults from 2 nd instar non-Bt 1.88 0.42 0.2016

adults from 3 rd instar SmartStax 1.39 0.05

adults from 3 rd instar non-Bt 1.46 0.24 0.8136

Table extracted and modified from Appendix 5, p. 339

Registrant conclusions: SmartStax was highly toxic to first instar larv ae but somewhat less to second instars and not

toxic to third instar larvae. Th e registrant concluded that 3 rd instars would be able to move and survive in 5% SmartStax seed blends, but they would suffer little or no selection for Bt resistance because of their tolerance to the Bt proteins in SmartStax. Growth chamber conditions favored survival of WCRW, and consequent emergence in both treatments was higher than what should be expected under field conditions. Growth cham ber conditions were expected to increase the survival to adulthood by decreasing density depend ent and independent mort ality expected in the field. The relative impact of SmartStax in the la b setting on different life-stages should reflect qualitatively what would occur under field conditions.

b) Field experiment : Two treatments were tested in this experimen t: 1) one artificially infested non-Bt plant surrounded by 12 non-infested SS plants , and 2) one artificially in fested non-Bt plant surrounded by 12 non-infested non-Bt plants. The central pl ant was inoculated with 2400 WCRW eggs. The experiment was arranged in a split-plot design with four replicates; the treatments were arranged non-systematically within each replication. The sub-plot factor was the distance of a plant from the inoculated center plant. Five distances were included (see Figure 1 below): one, two, and three plants distant from cen ter plant within same row ( 17.8 cm, 35.6 cm, 53.4 cm), and across and diagonal from inoculated plant in neighborin g row (76.2 cm and 78.3 cm). In addition, three single-row plots of non-artificially infested non-Bt plants (7) were planted to obtain baseline information on natural rootworm infestation pressure.

- 20 -Figure 1. Planting arrangement within each field plot used to examine WCRW larval movement and emergence (reproduced from Monsanto’s submission, Appendix 5, p. 334)

Five weeks after the inoculation, plants in the three rows of each plot (treatment and control) were individually caged. Collection of adults occurred once or twice a week until no beetles had emerged for 14 days. Nodal injury was determined to the plants. In addition, thorax width, and weight for WCRW adults emerging in each trea tment were measured. Monsanto recorded sex and number of emerged adults, thorax widths, and weights. Root damage was assessed using the nodal injury score by Oleson et al. (2005).

For the statistical analysis, a dult WCRW were grouped based on th e distance of inoculated plant and plant from which they emerged. ANOVA was used to compare percentage of beetles between each distance class. Seize and weights of adults were compared between each distance class while pooling across distance classes bu t excluding the central infested plant.

Study results : A smaller proportion of beetles was found in plot s where the central plants were surrounded by non-Bt plants (24.9%) than in pl ots where central plants were surrounded by SmartStax plants (47.6%); this difference was stat istically significant (p=0.0472). The difference in proportion of beetles obtained from immediately adjacent SmartS tax or non-Bt plants was not statistically significant (p=0.3455). But a greate r proportion of beetles was found on non-Bt plants that were two plants (p = 0.0023) or three plants re moved (p=0.0059) from the central plant (see Table 4 ) than the proportion of beetles re ported on SmartStax pl ants of the same distance from center.

There was no statistically significant difference in weights and thorax wi dths reported between adults having emerged from SmartStax or non-Bt treatments (results not shown).

Root damage was uniformly low in the experiment with greater damage to non-Bt roots than SmartStax roots (actual data not provided by Monsanto).

- 21 -Table 4. Percent cumulative adult emergence at different distances from infested center plant in SmartStax

and non-Bt treatments
Distance from

center Treatment % Cumulative

mean emergence SE p-value

0 SmartStax 47.6 10.1

0 non-Bt 24.9 6.6 0.0472

17.8 SmartStax 20.6 4.6

17.8 Non-Bt 31.3 9.6 0.3455

35.6 SmartStax 0 0

35.6 Non-Bt 18.1 9.2 0.0023

53.4 SmartStax 0 0

53.4 Non-Bt 12.6 3.2 0.0059

76.2 SmartStax 11.7 6.0

76.2 Non-Bt 8.7 6.0 0.6502

78.3 SmartStax 20.0 8.3

78.3 Non-Bt 4.1 1.0 0.0503

Table extracted and modified from Appendix 5, p.341

Registrant conclusions : Monsanto concluded from the emer gence results that a larger perc entage of insects successfully dispersed off the central, heavily infested plan t onto surrounding plants when the surrounding plants were non-Bt plants than wh en they were SmartStax plants.

Monsanto proposed that the results of “no-di fference” for body weight and thorax width for emerged beetles between different treatments i ndicated that a) larval movement occurred at similar times in non-Bt and SmartStax plots, and b) larvae establishing on SmartStax plants had comparable fitness to those moving to a refuge plant. Larval movement off central infested, nonBt plants appeared to be dr iven by “condition of the plant an d density-dependent effects”. Ultimate survival to adult emergence was imp acted by the toxicity of SmartStax plants surrounding the artificially infested, non-Bt plant.

Root damage results were consistent with the low adult emergence observed. Monsanto

concluded that this was an i ndication of low larval establ ishment across the experiment.

BPPD Review In Monsanto’s lab emergence study, the average 1 st instar emergence on non-Bt was 30.56% and 2.08%; hence average emergence for 1 st instar larvae on Bt compar ed to the control was 6.8%. Average emergence for 2 nd instar larvae on Bt compared to the control was 37.1%. There was no difference in emergence among 3 rd instar lave from Bt and non- Bt plants. Therefore, overall larval mortality in this experiment was 95.7%. B PPD concludes that in this lab study SmartStax was only highly toxic to first instar larvae, to a lesser extent to second instar larvae, and not toxic to third instar larvae (p<0.968). As Monsanto st ated this implies that if older CRW larvae disperse from non-Bt plants in a SmartStax s eed mixture (in response to density-dependence) and arrive on a SmartStax plant that they will no t be affected by the toxin. Whether they are not exposed to selection for resistan ce, however, is an uncertainty.

- 22 -Monsanto’s conclusion from the field experiment makes sense: more CRW disperse successfully from central infested plants when surrounded by non-Bt plants. BPPD not es that there was no statistically significant difference in the cumula tive % of emerged beetles from the SmartStax and non-Bt plants immedi ately surrounding the central infested plants.

SmartStax plants in the second a nd third distance classes had zero adults on them suggesting that movement and/or survival beyond the surrounding SmartStax plant was not likely. However, cumulative percent emergence was greater on Smar tStax plants in distance class 4 (72.6 cm – directly opposite of central plant) and 5 (78.3 cm - diagonal from central infested plant) than on the corresponding non-Bt plants. One of those tw o comparisons was statistically significant. Monsanto did not provide an explan ation regarding this observation.

Monsanto did not provide root damage result s from this experiment, although data were

collected. It is unclear whethe r there were any statistically significant differences between

distance classes of different treatments.

3. NA09S2J007 - Movement and Survival of West ern Corn Rootworm Larvae on Pairs

of MON 89034 × TC1507 × MON 88017 × DAS-59122- 7 Corn Plants and Non-Bt Corn Plants under Field Conditions (Appendix 10)

Dow AgroSciences conducted a one-year field e xperiment in 2009 using pairs of SmartStax and non-Bt plants to determine movement of W CRW larvae and surviva l/emergence to adulthood. The trial was set up in a field in Mount Vernon (I N) that was planted to soybeans the previous year (assuring that no corn rootworm eggs were in the ground); the ar ea has no history of

rotation-resistant CRW.

The experiment was set up in a randomized comple te block design with f our replicates. Blocks were four rows wide (30 inch row spacing) and 20 feet long.

Three pairs of plants were randomly selected in the two center rows of each treatment plot. The

treatments were:

• 2 non-Bt plants under one emergence cage. Only one of the two plants was artificially infested with WCRW eggs (12 cages total/experiment); the other plant was not infested.

• 2 SmartStax plants under one emergence cage. Only one of the two plants was artificially infested with WCRW eggs (12 cages total/experiment).

• 1 SmartStax plant/1 non-Bt plant under one emergence cage. The SmartStax plant was infested with WCRW eggs (12 cages tota l/experiment), the n on-Bt plant was not.

• 1 SmartStax plant/1 non-Bt plant under one emergence cage. The non-Bt plant was infested with WCRW eggs (12 cages total/experiment), the SmartStax plant was not.

The designated plant in each plot (as descri bed above) was infested with 500 WCRW eggs. Following the day of artificial infestation, em ergence cages were set up over each plant pair. Nineteen days after the infestation, emergence cages were sealed and checked every three to seven days for adult CRW. When emergence had stopped, the cages were checked for adults for

an additional two weeks.

- 23 -Beetles were counted from each plot, and their sex was determined. Chi-square analysis was applied to assess whether there was a significan t difference in sex ratio between treatments.

Study results: No adults emerged from the pure SmartStax treatmen t cages (total of 12); a total of forty-five adults emerged from the pure non-Bt treatment cages (total of 12); a total of 32 adults emerged from the mixed treatment cages where the non-Bt plants were i noculated with eggs; and four beetles emerged from the mixed treatment cages where the SmartStax plants were inoculated with eggs ( Figure 2 ).

Sex ratios in only one of the treatments deviated significantly from 50:50 (p>0.1).

Figure. 2. Total WCRW emergence by sex from dual-plant emergence experiment with SmartStax and non-Bt plants (Figure reproduced from Dow’s submission, Appendix 10, p. 685); * symbol indicates the plant type that was inoculated with 500 eggs.

Registrant conclusions: Results from the plant pair emergence study provi de evidence that WCRW larval survival is expected to be low among larvae that move between Bt and non-Bt plants. Survival was reduced by half in SmartStax /non-Bt plant pair cages wh en the refuge plant was inoculated with WCRW eggs as compared to emergence from the non-B t/non-Bt plant pairs. When the SmartStax plant was infested in the combination plant pair, emer gence was very low. Dow concluded that very few larvae survive to adulthood when they moved from SmartStax to non-Bt plants.

BPPD Review Dow’s inoculation with 500 eggs was below the threshold where density dependent mortality was detected by Hibbard et al. (2010); hence, BPPD notes that it is unlikely that a lot of densitydependent movement occurred in this experi ment. The majority of the reported 45 and 31 emerged adults in the non-Bt*/non-Bt and non- Bt*/Bt treatment likely emerged from the

inoculated non-Bt. Dow did not report whether the difference be tween these two treatments was statistically significant. In the case of these two treatments, it w ould have been informative to compare ranges of emergence as well. Survival in the control treatment was 9% which seems

- 24 -reasonable considering that 90-95% of corn rootworm mortality is caused by densityindependent factors. Surviv al (emergence) in the Bt 2 /non-Bt treatment was 0.8%, which

demonstrates that SmartStax is very toxic to neonates. Apparen tly some larvae where able to leave the toxic plant and recover on the non-Bt plant. Survival in the non-Bt 2 /Bt treatment was

6.4% and 0% in the Bt/Bt treatment. The reduc tion in adult emergence between control and

treatments is listed in BPPD’s Table 5 below. One correction should be mentioned; Dow stated

that the reduction in adults was more than 50% in non-Bt 2 /Bt treatments compared to the control. However, that reduction was only 29%.

Table 5. Total and relative percent surviv al of WCRW in di fferent treatments

Treatment Non-Bt*/non-Bt (control) Bt*/non-Bt Non-Bt*/Bt Bt*/Bt

Total emergence 45 4 32 0

Total survival 1 9% 0.8% 6.4 0%

Reduction in survival with respect to total emergence in control 91.2% 28.9% 100%

* indicates which plant pair was inoculated with 500 WCRW eggs

1 This includes larvae that have survived toxin exposure and density-independent fact ors causing mortality (500 eggs/plant) BPPD generated table from data provided in Figure 2

4. RPN-08-429 - Evaluation of Corn Rootworm Efficacy and Adult Emergence from

the Combined Trait Corn Product MON 89304 × TC1507 × MON 88017 × DAS 59122-7 When Used in Seed Mixtures in 2007 and 2008 Field Trials (Appendix 6)

Monsanto conducted a two-year efficacy and emer gence study in severa l Illinois sites using

artificial or natural CRW infestations in SmartStax seed mixture and control plots.

Treatments for WCRW in tw o total sites during 2007:

• 100% SmartStax;

• 100% non-Bt corn;

• 95% SmartStax with 5% non-Bt corn; and

• 90% SmartStax with 10% non-Bt corn.

In 2007, treatment plots (12 ft x 12 ft, and caged ) were planted and re plicated (randomized complete block design, 4 reps) at two sites (Hoope ston and Flatville, Illin ois). Adult NCRW and WCRW were collected weekly w ith an aspirator, and root da mage was assessed using the 0-3 rating scale (Oleson et al. 2005). ANOVA was used for emergence and root damage data to

assess differences across and within sites. The pair-wise test (with a Tukey multiple comparison p-value adjustment) was used to as sess differences between treatments.

2 Plant type receiving inoculation of 500 WCRW eggs

- 25 -The treatments for WCRW and NCR W in three and two total sites, respec tively, during 2008:

• 100% SmartStax with Poncho 250;

• 100% Non-Bt corn with Poncho 250;

• 100% MON 88017 with Poncho 250 (expressing Cry3Bb1)

• TC1507 x DAS-59122-7 with Poncho 250 (aka Herculex XTRA (HXX), expressing Cry34/35);

• 95% SmartStax with 5% non-Bt corn treated with the seed treatment Poncho 250; and

• 95% SmartStax with 5% non-Bt corn treated with Poncho 625.

In 2008, these treatment plots (12 ft x 12 ft, cag ed) were planted and replicated (randomized complete block design with 4 reps) at three sites, Hoopston (IL), Waterman 3 (IL), and

Monmouth 3 (IL). Emerged beetles were collected with an aspirator and counted weekly; root damage was assessed and statistically analy zed. ANOVA was used for emergence and root damage data to assess differences across and w ithin sites. Weights for female and male CRW were recorded. The pair-wise test (with a T ukey multiple comparison p-value adjustment) was

used to assess differences between treatments.

a. Study Results for Western Corn Rootworm

i. Observed WCRW emergence (2007 and 2008) : Non-Bt plots had the highest number of males a nd females emerge (statistically significant) relative to the other treatments ( Tables 6

, 7 , and 8 ). The total number of adults varied between the two different locations, Flattville and H oopeston, where the field experiments were held. Flatville had a greater number of WCRW; however, the pattern of emergence was consistent in both sites with highest numbers of beetles from non-Bt sites, intermediate numbers from seed mixtures, and lowest numbers of beetles from SmartStax sites. These differences were statistically significan t at both locations.

• non-Bt > 10% seed mix > 5% seed mix > 100% SmartStax.

In 2008, the pattern of emergence was similar:

• non-Bt > single events (Cry34/35Ab1; Cry3Bb1) > 5% seed mix > 100%

SmartStax.

In both years, the observed emergence from seed mix plots was either less (51% with both sexes pooled in 5% blends; 54% in 10% blends) or sl ightly in excess of what would have been expected based on a weighted average of th e SmartStax and non-Bt plot mean emergence numbers. Monsanto concluded that the seed mix pl ots were acting as effici ent (or somewhat less efficient) in terms of WCRW b eetle production compared to pure stands of non-Bt plants. Where the seed mix plots were less efficient in produc ing adults, Monsanto inferred that additional mortality occurred in the blends by larvae movi ng from non-Bt plants to adjacent SmartStax plants. At all sites, significantly more female and male beetles emerged from the non-Bt control plots than from either single ev ents or SmartStax mixtures.

3 Artificial infestations of plots at these sites

- 26 -Table 6. Adult (male and fe male) WCRW emergence (2007 data) Flatville, IL Hoopeston, IL

Treatment Total

emergence Mean

emergence Tukeys (p<0.05) Total

emergence Mean

emergence Tukeys (p<0.05)

100% SmartStax 66 16.5 C 43 10.8 D

95% SmartStax

5% non-Bt 84 21.0 C 199 49.8 C

90% SmartStax

10% non-Bt 194 48.5 B 344 86.0 B

100% Non-Bt 1282 320.5 A 7652 1913.0 A

Modified from MRID479437-01, volume 2

Table 7. Female WCRW emergence (2008) Hoopeston Monmouth 1 Waterman 1

Treatment Mean

emergence Tukeys (p<0.05) Mean

emergence Tukeys (p<0.05) Mean

emergence Tukeys (p<0.05)

100%

SmartStax 11.8 C 5.0 B 25.7 C

100% Cry3Bb1 230.8 B 24.7 B 110.0 B

100% HXX 105.2 B 16.0 B 28.7 C

95%

SmartStax 5%

non-Bt

w/P250 130.8 B 7.3 B 35.3 C

95%

SmartStax 5%

non-Bt

w/P625 77.8 B 20.7 B 33.4 C

100% Non-Bt 1015.6 A 680.7 A 333.7 A

Modified from MRID479437-01, volume 2

1 artificially infested plots

- 27 -Table 8. Male WCRW emergence (2008) Hoopeston Monmouth 1 Waterman 1

Treatment Mean

emergence Tukeys (p<0.05) Mean

emergence Tukeys (p<0.05) Mean

emergence Tukeys (p<0.05)

100%

SmartStax 6.6 C 1 B 4.1 C

100% Cry3Bb1 51.0 B 11.7 B 25.0 B

100% HXX 48.4 B 17.7 B 13.3 BC

95%

SmartStax

5% non-Bt

w/P250 29.6 BC 3.0 B 12.4 BC

95%

SmartStax

5% non-Bt

w/P625 33.6 BC 30.0 B 7.9 BC

100% NonBt 488.2 A 190.3 A 240.3 A

Modified from MRID479437-01, volume 2

1 artificially infested plots

ii) Emergence of WCRW over time : In general, beetle emergence from the non-Bt plots tended to occur ea rlier than for other treatments as evidenced by greater percentage emergence in the first sampling period for both

males and females of WCRW. This result is cons istent with previous observations in the CRW literature and was thoroughly di scussed during the SAP (2009). Of the Bt treatments in the current study, the greatest delays in beetle em ergence were consistently associated with the SmartStax plots, as evidenced by relative lo w emergence in the first sampling period and relatively high emergence in the later sampling periods. Figures 3-6 show % female and male emergence over time at Waterman (IL) and Manmouth (IL) (Hoopeston not shown here).

- 28 -Waterman Illinois Site

0 10 20 30 40 50 60

1234567

Collection times% female emergence (WCR)

SS

95/5 w/P250

95/5 w/P625

non-Bt

Figure. 3. Timing of mean female WCRW emergence at Waterman, Illinois, with seven collection dates (BPPD generated graph; data used from Table 16 in Appendix 6).

Waterman Illinois Site

0 10 20 30 40 50 60 70 80

1234567

Collection times% male emergence (WCR)

SS

95/5 w/P250

95/5 w/P625

non-Bt

Figure. 4. Timing of mean male WCRW emergence at Waterm an, Illinois, with seven collection dates (BPPD generated graph; data used from Table 17, Appendix 6).

- 29 -Monmouth Illinois Site

0 10 20 30 40 50 60 70

123

Collection times% female emergence (WCR)

SS

95/5 w/P250

95/5 w/P625

non-Bt

Figure. 5. Timing of percent female emergence at Monmouth, Illinois, with only three collection dates (BPPD generated graph; data used from Table 18, Appendix 6).

Monmouth Illinois Site

0 10 20 30 40 50 60 70 80 90

123

Collection times% male emergence (WCR)

SS

95/5 w/P250

95/5 w/P625

non-Bt

Figure. 6. Timing of mean male WCRW emergence at Monmouth, Illinois, with only three collection dates (BPPD generated graph; data used from Table 19, Appendix 6).

iv. Root damage : Root damage found in 2007 at Hoopeston and Flatvi lle locations was consistent with emergence of adults. Damage was greatest in non-Bt plot s followed by seed mix plots and finally by pure

- 30 -stand SmartStax plots. At Hoopeston, the ins ect pressure was greater during 2007, and the differences in root damage rati ng for non-Bt plots compared to a ll other Bt plots was significant ( Table 9 ). Refuge plants within the seed mix plots had greater dama ge than SmartStax plants but less damage than plants in non-Bt plots ( Table 10 ). This observation corresponded with the decreased emergence observed in seed mix plots ( Table 6 ).

Table 9. WCRW root damage ra tings for refuge and different seed bl end Bt treatments (2007 growing season) Hoopeston, IL Flatville, IL

Treatment Mean Std. Error Tukeys (p <0.05) Mean Std. Error Tukey’s (p <0.05)

100% SmartStax 0.02 0.008 0.4 0.008 B

95% SmartStax 0.02 0.006 0.8 0.023 B

90% SmartStax 0.03 0.005 0.9 0.018 B

100% Non-Bt 0.11 0.052 No difference

1.75 0.395 A
Modified from MRID 479437-01, volume 2

Table 10. WCRW root damage by individual components (2007) Flatville, IL Hoopston, IL

Treatment Plant Mean Std. Error Tukeys (p <0.05) Mean Std. Error Tukey’s (p <0.05)

100%

SmartStax Bt 0.02 0.008 0.04 0.008 BC

95% SmartStax Bt 0.01 0 0.03 0.007 C

90% SmartStax Bt 0.01 0 0.05 0.004 BC

10% non-Bt non-Bt 0.04 0.010 0.14 0.038 B

5% non-Bt non-Bt 0.03 0.012 0.13 0.039 B

100% non-Bt non-Bt 0.11 0.052 No

difference

1.75 0.395 A

Root damage rating results in 2008 resembled those of 2007 with the exception of treatment

“95% SmartStax and 5% non-Bt with Poncho 625” which incurred significantly greater damage

than other Bt treatments.

v. Weight of adult beetles : In 2008, Monsanto collected information on adult beetle weight in each treatment. Females tended to be larger than males at all three locat ions. While there were small differences between locations (adults smaller at Monmouth), there were no consistent trends among treatments.

Monsanto concluded that this was an indication that size and fitness of adults from different treatments had to be comparable.

b. Study Results for Northern Corn Rootworm

i. Observed NCRW emergence : Similar patterns of emergence were reported fo r NCRW in 2008 (as for WCRW in 2007-2008) at the two sites where significant na tural infestations were found ( Table 11 and 12 ). The observed emergence from seed mix plots was slightly less ( 75% for females; 80% for males) compared to what would have been expected based on a weight ed average of the SmartStax and non-Bt plot

- 31 -means. Monsanto inferred that the seed mix was marginally less efficient at producing NCRW beetles than pure stan ds of non-Bt plants.

Table 11. Female NCRW emergence (2008) Monmouth, IL Waterman, IL

Treatment Mean

emergence Tukeys (p<0.05) Mean

emergence Tukeys (p<0.05)

100% SmartStax 1.0 B 7.0 B

100% Mon 88017 5.0 B 14.9 B

TC x DAS x non-Bt * 21.3 B 9.4 B

95% SmartStax/5%

non-Bt w/P250 4.0 B 5.1 B

95% SmartStax/5%

non-Bt w/P625 6.7 B 5.3 B

100% non-Bt 72.0 A 54.4 A
* Unclear what percentage DAS-59122 was te sted. Details not provided by Monsanto.

Table 12. Male NC RW emergence (2008) Monmouth, IL Waterman, IL

Treatment Mean

emergence Tukeys (p<0.05) Mean

emergence Tukeys (p<0.05)

100% SmartStax 2.0 B 0.7 C

100% Mon 88017 4.3 B 7.0 B

TC x DAS x non-Bt * 14.7 B 2.9 BC

95% SmartStax/5%

non-Bt w/P250 3.3 B 2.4 BC

95% SmartStax/5%

non-Bt w/P625 5.0 B 1.6 BC

100% non-Bt 70.3 A 32.6 A
* Unclear what percentage DAS-59122 was te sted. Details not provided by Monsanto.

ii. Emergence of NCRW over time : In general, beetle emergence from the non-Bt plots tended to occur ea rlier than for other treatments as evidenced by greater percentage emergence in the first sampling period for both

males and females of NCRW. This result is cons istent with previous observations of delayed CRW emergence from the single CRW-pro tected events, MON 88017 and DAS-59122-7, compared with non-Bt controls (EPA, 2009). Of the Bt treatments in this study, the greatest delays in beetle emergence were consistently as sociated with the SmartStax plots, as evidenced by relative low emergence in the first sampling pe riod and relatively high emergence in the later sampling periods. Figures 7 and 8 display the % emergence per sex for different collections times at Waterman. (Manmouth data are not shown).

- 32 -Waterman Illinois Site

0 10 20 30 40 50 60

1234567

Collection times% female emergence (NCR)

SS

95/5 w/P250

95/5 w/P625

non-Bt

Figure. 7. Timing of female NCRW emergence at Waterman, Illin ois, with seven collection times (BPPD generated; data used from Table 20, Appendix 6).

Waterman Illinois Site

0 10 20 30 40 50 60 70 80

1234567

Collection times% male emergence (NCR)

SS

95/5 w/P250

95/5 w/P625

non-Bt

Figure. 8. Timing of male NCRW emergence at Waterman Illinois with seven collection dates (BPPD generated; data used from Table 21, Appendix 6).

iii. Root damage and adult beetle weight The pattern observed for damage caused by NCRW in 2008 was comparable to the observations

made for WCRW in 2008.

The observations made with respect to weight of adult NCRW beetles were comparable to the observations made for WCRW (2008).

- 33 -Monsanto conclusions:

Monsanto concluded that the resu lts of the two-year study demons trated that non-Bt plants in seed mix plots acted as productive sources of beet les. Most beetles emerged at a similar time as beetles from non-Bt plots (which should be when resistant insects would emerge from a seed mix plots). The production of beetles was comparable to (at worst 50% of) what would be expected from a separate non-Bt refuge of the same percen tage. Monsanto concluded that, therefore, seed mix can be an efficient refuge strategy for SmartStax for both WCRW and NCRW.

BPPD Review

BPPD concurs with Monsanto th at WCRW and NCRW em ergence data show that non-Bt plots had the largest number of beetles emerge with seed blends having been intermittent, and pure SmartStax plots having had the fewest numbers. The study results support the conclusion that

95% and 90% SmartStax seed mixtures were effi cacious against CRW (no st atistical significance between emergence numbers from SmartSta x and SmartStax seed mixture plots).

Sex specific emergence data by sampling date prov ided a greater degree of information when the registrant incorporated more than three sampling dates. With more collection times, actual bimodal or trimodal distributions for emergen ce became apparent for SmartStax treatments, which otherwise would have remained undetected (see Figures 3 through 6 ). Both these factors (seed mixes and multi-modal emergence patterns) should facilitate mixing (temporally and spatially) between adult CRW emerging from Bt and non-Bt plants in seed mixtures. Most individuals from non-Bt plots emerged early during the sampling interval.

Male emergence in corn rootworm was reduced up to 60 times (data not shown above) in 5%

seed mixtures with MON 88017 x DAS-59122-7 as co mpared to the non-Bt controls (Tables 14- 23, pages 392-397, Appendix 6). The SAP (2009) conc luded that delays in emergence between

the two sexes observed in 5% seed mixtures wi th DAS-59122-7 corn were less of a concern for refuge effectiveness, however, that more im portantly a reduction in number of males could negatively affect how effec tive a refuge would be.

A reduction in beetle production from SmartStax s eed mixtures (at worst 50%) compared to what would be expected based on emergence from non- Bt refuge fields sugge sts that the effective refuge may at times be lower. The effects on resistance evolution need to be investigated, preferably in a simulation model.

The root damage ratings suggest that refuge plan ts in seed mixtures r eceive additional protection from the surrounding SmartStax plants hence le ading to a reduction in effective refuge.

- 34 -5. NA09S2J005 - Efficacy of MO N 89034 × TC1507 × MON 88017 × DAS-59122-7 Corn Mixed with Non-Bt Refuge Corn (Appendix 9)

Study Methods: Dow conducted two efficacy trials at Fowler (IN) and Sidney (IL) in 2008 and Arlington (WI) and Fowler (IN) in 2009. All treatments were re plicated 4 times within each trial using a randomized complete block design. Plots were eight rows wide (30" row spacing) and 20 ft long. At each location all plants within a plot were artificially infested with WCRW eggs (500 eggs/plant).

In late July/early August, a sample of fifteen plants was collected from each seed blend plot, and root damage was evaluated. A sample consisted of five non-Bt plants, one Bt plant adjacent to the non-Bt expressing plant, and one Bt plant distant from the non-Bt plant (total of 15 plants). Percentage of roots that did not exceed economic threshold was determined (i.e. consistency) at 0.75 (low stress environment) and 0.25 (threshol d under high environmental stress). Corn rootworm injury levels and consis tency were assessed using ANOVA.

The following treatments were planted in 2008:

• 100% SmartStax (SSX)

• 95% SmartStax (SSX) seed blend w/5% non-Bt seeds

• 90% SmartStax (SSX) seed blend w/10% non-Bt seeds

• 90% TC1507 x DAS-59122 (HXT) seed blend w/10% non-Bt seed

• 100% non-Bt seed

The following treatments were planted in 2009:

• 100% SmartStax

• 95% SmartStax w/5% non-Bt seeds

• 90% SmartStax w/10% non-Bt seeds

• 95% TC1507 x DAS-59122 w/5% non-Bt seed

• 95% MON 89034 x MON88017 w/ 5% non-Bt seed

• 100% non-Bt seed

Study Results:

All locations with artific ial infestations had high CRW pressure in both years; node injuries in non-Bt plots were >2.16 and > 2.8 in 2008 and 2009, respectively. Overall there was a trend towards less injury on non-Bt pl ants in seed mixtures; however , not all observed differences were statistically significant when compared to the non-Bt contro l plots. There was a trend of increased injury for Bt plants adjacent to non-Bt plants compared to Bt plants some distant away from non-Bt plants. This trend however was not st atistically significant in both years. All seed mixture plots had root damage below 0.75, and most seed mixture plots had root damage ratings below 0.25 ( Figures 9-10 ; only some data shown here).

Consistency ratings for SS plants (in the 5% seed mixtures) some distance from non-Bt plants remained high at the 0.75 root damage level (9 5%-100%). The consistency ratings for the same SS plants ranged from 85%-100% at the 0.25 root damage for 2008 and 2009.

- 35 -Consistency ratings for SS plants adjacent to non-Bt plants were 70%-100% and 87%-100% at the 0.25 and 0.75 root damage level, respectively in 2008 and 2009 ( Figures 11-12 ; only some data shown for the consistency comparisons at the 0.75 level).

Figure 9. Mean root injury by WCRW on null plants (non-Bt), Bt plants adjacent to null plants, and Bt plants distant from null plants in Sidney (IL) 2008 (Tukey’s, p<0.05). Figure reproduced from Fig. 1in MRID479437-01, Appendix 9, p. 660)

Figure. 10. Mean root injury by WCRW on null plants (non-Bt), Bt plants adjacent to null plants, and Bt plants distant from null plants in Fowler (IN) 2008 (Tukey’s, p<0.05). Figure reproduced from Fig. 2 in MRID479437-01, Appendix 9, p. 661)

- 36 -Figure 11. Consistency at the 0.75 root damage level for null plan ts, Bt plants adjacent to null plants, and Bt plants some distance away from null plants at Sidney (IL) 2008 (Tukey’s, p<0.05). (Figure reproduced from Fig. 5 in MRID479437-01, Appendix 9, p. 664)

Figure 12. Consistency at the 0.75 root damage level for null plan ts, Bt plants adjacent to null plants, and Bt plants some distance away from null plants at Fowler (IN) 2008 (Tukey’s, p<0.05). (Figure reproduced from Fig. 7 in MRID479437-01, Appendix 9, p. 666)

- 37 -Dow’s conclusions about CRW larval move ment and efficacy were as follows:

• CRW efficacy of a 95% SS seed mixture is very similar compared to a 100% SS pure stand;

• CRW damage to non-Bt plants in such a 95% SS with 5% non-Bt seed mixture may be

reduced because of less larval movement ont o refuge plants from surrounding Bt plants; however, surrounding Bt plants incurred additi onal feeding damage from movement of larvae off heavily infested non-Bt plants.

• Efficacy benefits of seed mixtures dec line with increasing % non-Bt seeds. Less protection of non-Bt roots is expected since more refuge plants will have refuge plants as neighbors. Damage to surrounding SS-plants be comes more prevalent in seed mixtures

with 10 % of non-Bt seeds.

BPPD Review BPPD notes that Dow observed density-dependent dispersal off heavily infested non-Bt plants onto SmarStax plants. This supports Monsanto’s la b study findings that ol der instars are likely not affected by the CRW toxins expressed in SmartStax and that SmartStax is mainly highly toxic against younger instars. BPPD notes that the root injury damage (> 2) incurred on non-Bt corn supports that CRW infestations were great during th e two years; hence the root damage ratings below the economic threshold (0.75) indicate that seed mixtures were efficacious against CRW. Dow’s 2 year, multilocation, root damage study appears to support the c onclusion that SmartStax plants more distant from non-Bt plants incur less root damage than immediately adjacent SmartStax plants; in fact, there was no difference in damage of distant SS plants in 100% SS plot s and 95% and 90% seed mixtures. The slightly greater damage incurred to SS plants adjacent to non-Bt plants in seed mixtures supports the notion that older CRW la rvae dispersed but few survived (damage less than 0.50) ( Figures 9 and 10 ). The reduced root damage obser ved in non-Bt plants immediately adjacent to SS-plants in this study seems to sugges t that the effective refuge in seed blends may be lower than in a compar able structured refuge.

BPPD finds that the consistenc y ratings (0.75 root damage level) did not differ significantly between SmartStax plants adjacent to non-Bt plants in 95% and 90% seed mixtures (see Figures

11 and 12 ). Interestingly, the same comparison between percent consistency ratings of SS plants (adjacent) in 90% SmartStax blends (approxi mately 85%) and 90% He rculex XTRA (Cry34/35 only- approximately 50%)) showed no st atistically significant difference.

6. NA09S2J008 - Survival of Wester n Corn Rootworm Larvae in

Mixed Stands of MON 89034 × TC 1507 × MON 88017 × DAS-59122-7 Corn

Plants and Non-Bt Corn Plants under Field Conditions (Appendix 11)

Dow AgroSciences conducted an experiment wi th different percentages of SmartStax seed blends targeting CRW to assess emergence of a dults and larval movement under field conditions.

- 38 -Study Methods: The trial was conducted at Fowler (IN) and Sidne y (IL) in 2009. All treatme nts were replicated 4x within each trial using a randomized complete block design. Plots were eight rows wide and 20 ft long. At each location all plants within a pl ot were artificially infested with WCRW eggs (500 eggs per plant). Some level of natural infestation was present, and the artifici al infestation was to ensure high CRW pressure.

At approximately the third instar stage of CRW larvae, plants were cut back to 1⁄2 meter above ground, and emergence cages (4.1 m x 2.9 m x 2.2 m spanning across three rows) were put in place. Once emergence began, collection of adults occurred every 3-7 days and continued either through August or September until emergence had ceased.

Statistical analyses were carried out for each location separately. The total number of beetles emerged per plot were calculated. The number of days to first emergence and days to 50% emergence were determined and analyzed usi ng ANOVA with treatment, replicate, and their interaction as dependant variab les. Post-ANOVA means separation (least-squared estimates) was conducted using Tukey’s Honest Sign ificant Difference (HSD) test (P ≤ 0.05).

The following treatments were planted in 2009:

• 100% SmartStax

• 95% SmartStax seed blend w/5% non-Bt seeds

• 90% SmartStax seed blend w/10% non-Bt seeds

• 95% TC1507 x DAS-59122 w/5% non-Bt seed

• 95% MON 88017, 5% non-Bt seed

• 100% non-Bt seed

Study Results: In the 100% non-Bt plots, an average of 1626 a nd 785 adults were collected from emergence cages at the Fowler (IN) and Sidne y (IL) location, respectively. In 100% SS plots, an average of

4.75 and 8 adults were collected at Fowler and Sidney, respectively. Based on the emergence data from 100% treatment plot s and ignoring larval movement from plant-to-plant, it was expected that approximately 167 and 86 beetles would emerge from 10% blend, and 86 and 47 would emerge from 5% seed blends at Fowler and Sidney, respectively. The actual means of beetles in the 10% blends were 139 and 65, some what below the expected number. The actual

means of beetles in the 5% blends were 86 and 75 and either matched or exceeded the expected

number for 5% seed blends. The actual number of beetles emerging from the 5% mixtures with single traits (Cry34/35 or Cry3Bb1) was higher than for the 5% mi xtures using both traits (data

not shown in Table 13 ).

- 39 -Table 13. Observed average adult emergence from pur e stands and expected a nd observed emergence from different percentages of SmartStax for seed blend

Location Treatment Average # of

adults observed # of adults expected based on pure stand

emergence
100% SmartStax 8

100% refuge 785

90% SmartStax 65 86 Sidney

95% SmartStax 75 47

100% SmartStax 4.75

100% refuge 1626

90% SmartStax 139 167* Fowler

95% SmartStax 86 86 BPPD generated Table. Single gene s eed mixture results are not shown.

* 10% adults from 1626 observed in non-Bt and 90% adults from 4.75 adults observed in Sm artStax = 167 adults total

There was temporal overlap in mean emergence between adults from non-Bt plots and SmartStax plots. Significant differences we re apparent in mean number of days to first emergence between

100% non-Bt and 100% SmartStax tr eatments as well as seed mixt ure treatments and pure stand

SmartStax treatments at both locations. There wa s no statistically significant difference between results from SmartStax seed mixtures and non-Bt plots.

With respect to median emergence the results we re not completely synchronous at Sidney; there were significant differences in median emergence between 100% SmartStax and 100% non-Bt plots as well as 100% SmartStax and 95% Sm artStax seed mixture plots. At Fowler, no differences between all treatments were apparent.

Registrant conclusions: Emergence results from the study showed that SmartStax seed blends and non-Bt plots had greater numbers of adult emergence than pure stands of SmartStax. Th e numbers of adults emerging from different seed mixture configura tions were lower than expected (with larval movement) and in one case higher than expect ed. Lower numbers suggested a larval (net) movement from non-Bt plants to SmartStax pl ants, while the higher number of emergence suggested that larval movement occurred from SmartStax plants to non-Bt plants. But these reported effects on survival via movement were weak, and overall study re sults supported that

SmartStax seed mixtures could be as eff ective for WCRW as structured refuges.

BPPD Review

Dow concluded that the observed reduction in adult emergence on non- Bt plants in seed blends stems from larval dispersal onto surrounding Bt plants. Hibbard et al. (2010) reported that the density-dependent dispersal threshold for CRW was somewhere between 800 and 850 eggs/ 30cm- row. Hence, BPPD suspects that dispersal from non-Bt plants in SmartStax seed mixtures was unlikely. Instead, BPPD hypothesi zes that some larvae, after an initial taste of Bt roots, initially recovered, dispersed (a ssumption of asymmetrical disper sal off Bt roots) onto non-Bt plants, yet incurred additional mortality due to prior Bt exposure.

- 40 -With respect to the “mean days to median emergence” at Fowler and Sidney, BPPD notes that reported results between non-Bt and 100% SmartS tax were significant at Fowler but not at Sidney although the difference in median emergen ce between the two treatments was 6 days at both locations. Conversely, a differe nce of two days in “mean days to median emergence” was significant between 95% SmartStax and 100% Smar tStax at Sidney, although biologically this is unlikely to represent a significant difference; howe ver, the difference of four days between 95%

SmartStax and 100% non-Bt was not found to be st atistically significant. BPPD concludes from the “days to median emergence” results that mixi ng of susceptible and poten tially resistant adults is facilitated in seed mixtures compared to pure Bt stands with structured refuges (difference in mean days to median emergence greater).

B. Lepidoptera Movement, Dose, and Survial (Appendix 1 (RPN-09-075); Appendix 2 (RPN-09-137); Appendix 3 (RPN-08-088); Appendix 4 (RPN-08-549); Appendix 9 (NA09S2J005))

1. RPN-09-075- Survival of European Corn Borer and Southwestern Corn Borer

Larvae on the Combined Trait Product MON 89034 × TC1507 × MON 88017 × DAS59122-7 and a Non-Bt Control (Appendix 1)

Monsanto carried out a laboratory study to assess European corn borer and Southwestern corn borer larval survival and development on the triple gene component of SmartStax during different developmental stages. In addition, an artificial infestation e xperiment was set up at Jerseyville (IL) to examine plant to plant movement of ECB and corn earworm (CEW) on SmartStax and non-Bt corn plants. Larval survival was very low for both species and data were confounded by a natural CEW infestation; hence data were not presented in Monsanto’s report.

For ECB and SWCB each, a total of 350 rearing c ontainers were set up; larvae at different developmental stages were fed SmartStax and non- Bt corn tissue. Each of the seven treatments (Population 1- Population 7) c onsisted of 50 containers; one 24 hour-old neonate larva was contained per container. Fresh Bt or non-Bt plant tissue were pr ovided every two days. At that point, mortality was scored and head capsule width of dead larvae measured. Every fourth day, larvae were transferred to a next exposure tr eatment. The duration for the experiment was 29 days and 26 days for ECB and SWCB, respectively.

Study Methods: SmartStax and non-Bt corn seeds (same hybrid genetic background as positive treatment) were planted in 10” and 12” pots; plants were maintained in a growth chamber to provide fresh feeding material for the larval experiment. Plants used as food source were tested for presence or absence of Bt proteins befo re being fed to larvae.

Seven larval populations were set up consisti ng of 50 larvae each. Seven exposure scenarios were applied to each population (see Table 14 below). All larval populations began their development on non-Bt tissue and then were transf erred to SmartStax tissue at 5 different time points (4, 8, 12, 16, or 20 days). ECB larvae in population 2 were at the second instar, in

- 41 -population 3 mostly at the third instar, in population 4 at the th ird and fourth instars, in population 5 at the fourth and fift h instar, and in population 6 at th e fifth instar when transfers

occurred from non-Bt to SmartStax tissue. P opulation 1 (negative c ontrol) and population 7 (positive control) consisted of 50 neonate larvae each that were kept on an exclusive non-Bt or SmartStax diet, respectively, for the entire study duration.

SWCB larvae in population 2 were at the s econd instar, population 3 at the third instar, population 4 at the fourth instar, Population 5 mos tly at the fifth instar, and population 6 were at

the fifth instar when the larvae were transfer red from the non-Bt control tissue to SmartStax tissue. Population 1 and population 7 consisted of 50 neonate larvae each that were kept on an exclusive non-Bt diet and SmartStax diet, respectively, for the entire study duration.

Table 14. Exposure Scenarios and Different Population Treatments Food Source Exposure (Intervals of 4 days) Population S 0 S 1 S 2 S 3 S 4 S 5 S 6 P 1 NB NB NB NB NB NB NB

P 2 NB SSX SSX SSX SSX SSX SSX

P 3 NB NB SSX SSX SSX SSX SSX

P 4 NB NB NB SSX SSX SSX SSX

P 5 NB NB NB NB SSX SSX SSX

P 6 NB NB NB NB NB SSX SSX

P 7 SSX SSX SSX SSX SSX SSX SSX Table reproduced from Table 1 in MRID 479437-01 of Appendix 1, p. 54

SSX = SmartStax leaf tissue

NB = Non-Bt leaf tissue Px = Population treatments

S X = Food source exposure of 4 day interval

Study Results:

European corn borer: Highest survival (42.55%) was obs erved at day 29 of the experi ment in population 1 (negative control) where larvae exclusively fed on non-Bt le af tissue. Most surviving insects reached the pupation stage, which indicated that the test syst em was suitable for growth and development of

ECB larvae.

Mortality in population P7 was 100% by day 8 of the experiment; larvae were exclusively exposed to Bt corn (SmartStax) tissue in this treatment. Populations P2 (SmartStax exposure at day 4), P3 (SmartStax exposure at day 8), and P4 (SmartStax exposure at day 12) incurred 100% mortality by Day 8, Day 14, and Day 22, respectivel y. Second to fourth instar larvae did not survive exposure to SmartStax and died soon af ter exposure. Larvae in Populations P5 and P6 did not get exposed to Bt corn tissue until Day 16 and Day 20 respectively; a small proportion of

individuals in these two treatme nts were able to survive SmartStax exposure unt il the end of experiment (P5 = 6.38% surviv al and P6 = 9.38% survival). Table 15 below shows the cumulative survivorship for differe nt larval treatments/populations.

These survival results for ECB were graphically summarized by Monsanto in Figure 13 below.

- 42 -Figure 13. Cumulative % Survival of ECB Larvae in Seven Experimental Treatments (Figure reproduced from Figure 1 in MRID 479437-01, Appendix 1, p. 59)

Table 15. Cumulative survival of seven ECB populations reared on SmartStax and non-Bt corn tissue

Assay days %

survival

P1 %

survival

P2 %

survival

P3 %

survival

P4 %

survival

P5 %

survival

P6 %

survival

P7

2 100 85.4

4 100 100 27.7

6 91.8 54 4.3

8 83.3 0 100 0

10 81.3 0 52.4 0

12 75 0 2.4 100 0

14 75 0 0 90 0

16 75 0 0 65 100 0

18 72.9 0 0 43.6 97.2 0

20 70.8 0 0 15.4 97.2 100 0

22 68.8 0 0 0 66.7 97.2 0

24 64.5 0 0 0 48.7 88.6 0

26 55.3 0 0 0 23.1 58.3 0

29 42.6 0 0 0 6.4 9.4 0 Table reproduced and modifi ed from Table 2 in MRID 479437-01, Appendix 1, p. 58

Southwestern corn borer: Highest survival (85.71%) was obs erved at day 26 of the expe riment in population 1 where larvae exclusively fed on non-Bt leaf tissue (contr ol treatment). Monsanto inferred that the test system was suitable for growth and development of SWCB larvae.

- 43 -Mortality in population P7 was 100% by day 4 of the experiment; larvae were exclusively exposed to Bt corn (SmartStax) tissue in this treatment. Populations P2 (SmartStax exposure at day 4) and P3 (SmartStax exposure at day 8) incurred 100% mortalit y by Day 10 and Day 18, respectively. Larvae in populations P4, P5, and P6 did not get exposed to Bt corn tissue until Day 12, 16, and 20 respectively, and some individuals were able to survive toxic exposure from SmartStax until Day 29 when the experiment was terminated (P4 = 4.17%, P5 = 80.43% survival and P6 = 93.33% survival). Table 16 below shows the cumulative survivorship for different larval treatments/populations.

These survival results for SWCB were gr aphically summarized by Monsanto in Figure 14 below.

Figure 14. Cumulative % Survival of SWCB Larvae in Seven Experimental Treatments (reproduced from Figure 2 in MRID 479437-01, Appendix 1, p. 61)

- 44 -Table 16. Cumulative survival of seven SWCB popula tions reared on SmartSt ax and non-Bt corn tissue

Assay days %

survival

P1 %

survival

P2 %

survival

P3 %

survival

P4 %

survival

P5 %

survival

P6 %

survival

P7

2 100 58

4 100 100 0

6 95.9 76 0

8 91.8 8 100 0

10 89.8 0 88 0

12 87.8 0 34 100 0

14 85.7 0 18 100 0

16 85.7 0 2 85.4 100 0

18 85.7 0 0 57.5 100 0

20 85.7 0 0 29.2 97.8 100 0

22 85.7 0 0 14.6 93.5 100 0

24 85.7 0 0 12 89.1 100 0

26 85.7 0 0 4.2 80.4 93.3 0 Table reproduced and modifi ed from Table 3 in MRID 479437-01, Appendix 1, p. 60

Registrant conclusions:

ECB larvae from first to fifth in star were highly susceptible to SmartStax corn; larvae from first to fourth instar died soon after being placed on SmartStax, and even fifth instars had low survival and probably would not have survived to pupation. These results were consistent with those of field studies that found no ECB larvae or pupae on SmartStax corn at mu ltiple sites over two years, and laboratory studies showing high susc eptibility of ECB larvae to various Cry1 and Cry2 proteins in artificial diet. This means that ECB larvae moving off non-Bt plants onto SmartStax plants within a seed mix would not survive, regardless of the age at which they

moved.

The results demonstrated that SWCB larvae from firs t to fourth instar were highly susceptible to SmartStax corn; all larvae from first to third inst ar, and almost all fourth instars, died soon after being placed on SmartStax. These results were consistent with those of field studies that found few SWCB larvae or pupae on SmartStax corn at mu ltiple sites over two years. This means that SWCB larvae moving off non-Bt plants onto Smar tStax plants within a seed mix are highly unlikely to survive, regardless of the age at which they moved.

BPPD Review

The cumulative survival numbers in Table 15 show that European corn borer larvae were highly susceptible to the triple gene product SmartStax es pecially at the first f our instar levels. BPPD noticed that control mortality in the negative control was rather high (approximately 58%), and, therefore, recommends that Monsanto correct ob served mortalities (using Abbott’s correction) based on the high control mortality.

It was unclear whether those la rvae that survived exposure at a later instar stage underwent successful pupation and emergence to adulthood. Based on this study alone and the uncertainty with results from P5 and P6, BPPD states that if a fourth instar larva left a non-Bt corn plant (5 th

- 45 -instar larvae likely will not move or eat much) and arrived on a corn plant expressing the triple gene product of SmartStax, it might have a six pe rcent probability of su rvival (pupation/adult emergence). Considering, however, that Monsan to and Dow have not found any ECB pupae or larvae in other field studies with corn plants expressing SmartStax, it may be unlikely that such a large proportion of 4 th instars survived SmartStax expos ure in actuality. An alternative explanation for no adult survival on SmartSta x surrounding non-Bt plants could be that ECB dispersal occurs during the early larval life-stage (Ross and Ostlie 1990) and not for older instars. If, however, there was a small fraction of 4 th instars (with RS-genotype) that dispersed and that were more tolerant to SmartStax exposure, then such genotypes could be selected for in a seed

blend environment and contribute to an increase in the resistance allele frequency. In such a case,

Bt could also select fo r different behavior ( i.e. , dispersal during later lif e-stage as discussed by Davis and Onstad 2000) in the targ et pest. More information on late instar movement for ECB is needed to determine whether the presen t uncertainties are reasons for concern.

The control mortality in Sout hwestern corn borer populations was much more reasonable (approx. 15%), and so BPPD has more confiden ce in the actual cumulative survivorship percentages reported by Monsanto. But Monsanto should also correct SWCB mortalities using Abbott’s correction. For comparison purposes , survival in SWCB was higher for 3 rd , 4 th , and 5 th

instars than for ECB; however, the duration of the experiment was three days shorter than the one for ECB, which could explain some of incr eased survival observed for SWCB. The results suggest, however, that SmartStax is somewhat less toxic for SWCB than ECB. The same

uncertainties and concerns with late instar movement discussed for ECB apply to SWCB.

2. RPN-09-137 Movement and Survival of Lepidoptera in Seed Mixtures of MON

89034 × TC1507 × MON 88017 × DAS-59122-7 in 2009 U.S. Field Trials (Appendix 2)

In 2009, Monsanto carried out a one-year field study in three US locations to determine survival and movement of key Lepidoptera target pests (ECB, SWCB, CEW, and sugar cane borer, SCB) in various SmartStax seed mixture and SmartStax and non-Bt corn pure stands.

Study Methods: The experiment was designed as a complete randomized design with 2 replicates for 100% SmartStax, 95% SS, 90% SmartStax, and 100% non-Bt corn and carried out in Loxley (AL), Leland (MS), and Winnsboro (LA) during the 2009 growing season. Each plot was three sets of four rows wide and 100 ft long. At Leland and Winns boro, one set of rows in a plot was used to track damage from SWCB and SCB, the second and third set of rows were used to track SWCB and CEW, and CEW and SCB, respec tively. At Loxley, two sets of ro ws in a plot were used to track ECB damage, and the third set of rows was used to track CEW damage. Refuge plants in

seed mixtures were detected af ter the plots had been sprayed w ith one or two applications of glufosinate at Winnsboro, Loxley, and Leland; SmartS tax plants were tolerant to the herbicide, while refuge plants were not. In cases where he rbicide damage did not co incide with expected pest damage, a lateral flow strip test was used to discern between Bt and non-Bt plants in seed

mixtures.

- 46 -Refuge plants in seed mixture plots were non-sy stematically chosen and artificially infested where natural pest densities were low. Damage ra tings in seed mixture plots were obtained from selected non-Bt plants and surrounding SmartStax plants to evaluate larval movement and survival. In pure stand plots (SmartStax and cont rol), 30 non-systematically chosen and infested plants were evaluated for pest damage.

For CEW , the percentage of damaged kernels and number of live larvae found on the primary and/or secondary ear were evaluated.

For ECB , the artificial infestation failed at L oxley, and these data were not reported.

For the first generation SWCB , leaf damage was assessed using a modified Guthrie rating scale (Guthrie et al. 1960). For the first and second generati on of SWCB, the number of live larvae and pupae was taken in addition to the number and length of primary and secondary ear galleries as well as stalk galleries:

1 meaning that no visible leaf injury had occurred or small amount of shot -hole type injury on a few leaves;

2 meaning that mall amounts of shot-holes type lesions were detectable on a few leaves;

3 meaning that shot-hole injury was common on several leaves;

4 meaning that several leaves ha d shot holes and elongated lesions;

5 meaning that several leaves contained elongated lesions;

6

meaning that several leaves contai ned elongated lesions (ca. 1 inch);

7

meaning that long lesions were common on one-half of the leaves;

8 meaning that long lesions were co mmon on about 2/3 of the leaves; and

9 meaning that most leaves contained long lesions.

For first and second generation SCB , the number of entry holes in stalk, live larvae, pupae, and number and total length of combin ed stalk galleries were recorded.

Treatments planted at each location were:

• 100% non-Bt (NK603)

• 100% SmartStax seed blends (M ON 89034 X TC1507 X MON 88017 X DAS-59122-7 with Poncho 250)

• 95% SmartStax seed blends with 5% non-Bt (NK603 with Poncho 250)

• 90% SmartStax seed blends with 10% non-Bt (NK603 with Poncho 250)

Statistical Analysis: Damage data per site were summarized by treatment. Pair-wise comparisons were performed using SAS to evaluate differe nces among SmartStax plants from different treatments. Analysis also focused on SmartStax plan ts in seed mixtures and their relative position to the nearest non-Bt plant (1 plant away, 2 plants away, or > than 2 plants away). Differences between non-Bt plants in pure stand and mixed plots were evaluated.

- 47 -Study Results: Loxley (AL): insufficient numbers of ECB established fr om the artificial infestations. CEW were

able to establish themselves from the artificial infestations, and those data were evaluated. FAW was present due to natural infest ations, and hence, damage ratings were collected for this pest.

Corn earworm : larval establishment was apparent in non-Bt plants in 5% and 10% seed mixtures. However, CEW numbers were approximately 3-fold less than the number of CEW that established themselves on plants in non-Bt pl ots. Almost no surviving adults were found on SmartStax plants in seed mix or pure stand plots (see Table 17).

Table 17. Number of CEW found in each treatment (Loxley, AL)

Substance Plant type Mean number of larvae Standard error Comparison (P-value < 0.05)

100% SmartStax Bt 0.00 0 *

95% SmartStax Bt 0.00 0 *

90% SmartStax Bt 0.00 0 *

5% non-Bt non-Bt 0.16 0.05 B

10% non-Bt non-Bt 0.20 0.04 B

100% non-Bt non-Bt 0.52 0.07 A

Table modified from Table 1 in MRID479437-01, Appendix 2, p. 85 * Insufficient variation to conduct comparisons

Fall armyworm : No statistically significant differences were detectable in the degree of foliar damage caused by FAW on non-Bt plants in seed mixtures (5% and 10%) and pure stand plots. Almost no damage was present on SmartStax plan ts in pure stand or seed mixture plots.

Table 18. Foliar damage by FAW to SS and non-Bt plants in different treatments

Substance Plant type Mean leaf damage Standard error Comparison (P-value < 0.05)

100% SmartStax Bt 0 0 *

95% SmartStax Bt 0.01 0.01 *

90% SmartStax Bt 0.01 0 *

5% non-Bt non-Bt 3.45 0.17 A

10% non-Bt non-Bt 3.55 0.12 A

100% non-Bt non-Bt 3.88 0.18 A

Table modified from Table 2 in MRID479437-01, Appendix 2, p. 85

Leland (MS): An analysis was conducted for SW CB only. Insufficient numbers of CEW

established themselves on Bt and non-Bt co rn to gain meaningful information.

For the first generation of SWCB, the number of galleries per pl ant was significantly greater on non-Bt plants in seed mixtures compared to non -Bt plants in pure sta nd plots; the range for number of galleries on non-Bt plants in control, 5% a nd 10% plots were 0.30, 1.00, and 0.77, respectively (see Table 19 ). The second generation caused less damage than the first generation, and the numbers of galleries per non-Bt plants in control, 5%, and 10% treatment plots were 1.29, 0.46, and 0.63, respectively. There was no stat istically significant difference between different seed blend percentages. SmartStax plan ts had very few galleries regardless of insect

- 48 -generation and seed mixture or pure stand plot s; the observed mean damage ranged from 0-0.03 galleries per stalk.

Table 19. Number of first and second generation galleries from SWCB feeding per stalk (Leland, MS)

Substance Plant type Mean number for

1 st

/2 nd generation Standard error for

1 st

/2 nd generation Comparison (Pvalue < 0.05) for

1 st

/2 nd generation

100% SmartStax Bt 0.00/0.02 0/ 0.02 */ *

95% SmartStax Bt 0.03/ 0.02 0.01/ 0.01 */ *

90% SmartStax Bt 0.02/ 0.01 0.01/ 0.01 */ *

5% non-Bt non-Bt 1.00/ 0.46 0.30/ 0.14 B/ B

10% non-Bt non-Bt 0.77/ 0.63 0.13/ 0.11 B/ B

100% non-Bt non-Bt 0.30/ 1.29 0.08/ 0.16 A/ A
Table modified from Appendix 2, p. 86 * Insufficient variation to conduct comparisons

Winnsboro (LA): CEW and SCB infestations were heavy in this location, and damage to plants was exacerbated by a late planting date of treatment plots.

Corn earworm: densities were greater on plants in non-Bt plots than seed mixture plots, and mean numbers ranged from 1.2-1.9 per ear of refuge plant (see Table 20 ); the observed mean number of larvae found on non-Bt plants in mixt ure and non-Bt plots di ffered significantly. The

difference between the mean numb er of larvae found on refuge plants in 5% and 10% seed mixtures was not significant.

The mean number of larvae found on SmartStax plants in pure stand plots and seed mixture plots was significantly lower than on non-Bt plants in pure stands. Some larvae were found on SmartStax plants under these heavy infestati on conditions; those mean numbers ranged from 0.32-0.61 per ear of SS plant. When the level of CE W survival on SmartStax plants in seed mix plots was analyzed based on the pos ition of these plants relative to the nearest non-Bt plants, no significant effect was detectable for CEW.

Table 20. Number of CEW larvae per primary ear (Winnsboro, LA)

Substance Plant type Mean number of

larvae Standard

error Comparison (Pvalue < 0.05)

100% SmartStax Bt 0.32 0.07 C

95% SmartStax Bt 0.61 0.04 D

90% SmartStax Bt 0.41 0.02 C

5% non-Bt non-Bt 1.24 0.12 B

10% non-Bt non-Bt 1.19 0.09 B

100% non-Bt non-Bt 1.88 0.16 A

Table modified from Appendix 2, p. 86

Sugarcane borer: The mean number of first generation gall eries per stalk in non-Bt plants did not differ among treatments, and the mean number ranged from 1.07-1.57 per stalk. The mean

- 49 -numbers of 2 nd generation galleries were higher in non-Bt plants and ranged from 2.61-4.55 per stalk. There was a statistically significant difference between th e galleries per stalk (non-Bt plant) in 5% and 10% seed (see Table 21 ). Little damage was found on SS plants from first generation SCB regardless of what seed mixture percentage (0%, 5%, or 10%). Higher damage was apparent from second generation SCB feedi ng. The range of mean galleries per stalk for 2 nd
generations was 0.28-0.60. The damage to SmartS tax plants in the 10% seed blend was significantly greater than the damage observed in 100% SmartStax and 5% SmartStax blends. When the levels of survival on SmartStax plants in seed mix plots were analyzed based on the position of these plants relative to the nearest no n-Bt plants, a significant effect was observed; SmartStax plants next to refuge plants in both mixtures had greater number of galleries per stalk than more distant SmartStax plants. The SCB galleri es in the SmartStax plants revealed very few live pupae (see Table 22 ).

Table 21. Number of first/seco nd generation SCB galleries per stalk (Winnsboro, LA)

Substance Plant type Mean number for

1 st

/2 nd generation Standard error for

1 st

/2 nd generation Comparison (P-value < 0.05) for 1 st /2 nd
generation

100% SmartStax Bt 0.00/0.28 0/ 0.09 */ C

95% SmartStax Bt 0.08/ 0.41 0.03/ 0.05 */ C

90% SmartStax Bt 0.05/ 0.60 0.01/ 0.06 */ D

5% non-Bt non-Bt 1.28/ 2.61 0.48/ 0.33 A/ B

10% non-Bt non-Bt 1.07/ 3.71 0.22/ 0.49 A/ A

100% non-Bt non-Bt 1.57/ 4.55 0.19/ 0.27 A/ A

Table modified fr om Tables 5 and 6 in MRId479437-01, Appendix 2, p.86

Table 22. Mean number of second generati on SCB pupae per stalk, Winnsboro (LA)

Substance Plant type Mean number

pupae Standard error Comparison (Pvalue < 0.05)

100% SmartStax Bt 0 0 *

95% SmartStax Bt 0.01 0 *

90% SmartStax Bt 0.01 0 *

5% non-Bt non-Bt 0.78 0.13 B

10% non-Bt non-Bt 0.58 0.18 B

100% non-Bt non-Bt 0.33 0.24 A

Table modified from Table 8 in MRID 479437-01, Appendix 2, p.87

Registrant conclusions: “In conclusion, the non-Bt plants in the 5% a nd 10% seed mix plots c onsistently supported significant numbers of CEW, SCWB , and SCB larvae. The densities of these pests on the non-Bt plants within the seed mix plots could be comparable to ≤ three-fold than on the plants in the non-Bt plots. The lower larval densities in th e seed mix plots presumably reflected larval movement off non-Bt plants onto surrounding SmartS tax plants. In addition, SmartStax plants in the seed mix plots had very few surviving larvae or pupae, meaning that there was little ability of

larvae that moved off non-Bt plants to establish on Smar tStax plants regardless of instar. Therefore, a 5% seed mix should be an effectiv e refuge strategy for SmartStax for the primary lepidopteran target pests.” (Appendix 2, p.84)

- 50 -BPPD Review

Corn earworm: BPPD notes that corn earworm were present on SmartStax plants in different seed mixture percentages. Monsanto stated th at infestation was high that year (1.9 larvae/non-Bt plant) and, hence, lead to some survival on SmartStax pl ants. For example at Winnsboro, for every 10 Bt plants, there were three life larvae in 100% Smar tStax plots; for every 10 Bt plants, there were approximately six and four life larvae in 5% and 10% SmartStax seed blends. At Loxley, the mean number of CEW in the control plot was 5 for every 10 plants; those numbers reflected larvae that successfully established after the artif icial infestation had occurred. This is much less than what was seen at Winnsboro (LA). Mons anto reported that survival on surrounding SmartStax plants in the seed mixtures and Smar tStax pure plot was zero percent. BPPD cautions

that this number needs to be considered rela tive to the number of larvae/control plant. In summary, the experiment at the two locations appear to show that if CEW infestation is low then SmartStax may be efficacious against CEW; however , if infestation and pest pressure are high, then some CEW survival should be expected on SmartStax plants.

While these reported survival numbers probably do not represent a great proportion of the CEW population densities during that ye ar, it is still substantial if one considers that SmartStax expresses three different Bt toxi ns against Lepidoptera. As st ated in BPPD (2009), however, since TC1507 has shown reduced efficacy against CEW with damage and in festation frequently not statistically different than the non-Bt controls, SmartStax ma y not express a functional highdose against CEW. For MON 89034 (BPPD 2007), dose tests (rather than field efficacy tests) have shown variable survival for CEW ranging fr om 0% (leaf disk test - yet great control mortality was noted) to 9% (kernel test).

Monsanto did not request a seed blend registra tion for cotton growing re gions where CEW are a major pest. BPPD recommends that Monsanto de termine where in the Corn Belt corn earwom

could overwinter because a 5% SmartStax seed bl end in such areas could increase the risk of resistance evolution in the target pest.

Sugarcane borer: The mean number of sugarcane borer pupae on non- Bt plants in seed mixtures (5% and 10%) was greater than in 100% non-Bt plots. This difference was statistically significant. No explanation for this observation was provided by Monsanto. Sugarcane borer infestations were heavy and natural at the Winnsboro (LA) lo cation. Hence BPPD suspects that there was increased dispersal off surrounding Bt plants onto non-Bt plants. The results of this time limited and one-location study suggest that non-Bt plants in SS mixtures gi ve refuge to a greater number of larvae/pupae than non-B t plants in the control plots. This could be viewed as initial support for potential asymmetrical dispersal in this target pest. It appears that SmartStax was efficacious against controlling SCB in both 5% and 10% s eed mixtures; although the difference in damage was statistically significant. There was no difference in damage on SS plants from 5% and 100%

SS mixtures.

Fall armyworm: The fall armyworm damage found on non-Bt plants in seed mixtures and control plots did not differ statistically, which implies that the non-Bt plants in all treatments supported a similar

- 51 -amount of individuals irrespective of the percentage of SS plants. SmartStax plants selected for sampling in pure stands incurred no FAW damage, while SS plants in the seed mixture treatments incurred minimal damage; the damage results reported did not differ statistically. Since SS plants were likely non-systematically sel ected (this part of the report was not clear), the results suggest that FAW survival on SmartSta x is unlikely. These results do not provide information about larval movement and subsequent survival as an older in star since the SS plants selected for damage evaluation did not a ppear to be neighboring non-Bt plants.

Southwestern corn borer: The SWCB results from Leland (MS) showed that minimal damage occurred to SS plants in all treatments. Damage to non-Bt plan ts was greatest in th e seed blends compared to the control suggesting that there may have been increased move ment off Bt plants onto refuge plants. These preliminary results together imply, based on dama ge reports as opposed to reported numbers of live larvae, that non-Bt plants in SS seed mixtures could provide as effective a refuge as non-Bt plants in the control. However, more informa tion is needed before such a conclusion can be

made.

3. RPN-08-088 - Assessment of the Efficacy of Lepidopteran and ColeopteranProtected Corn MON 89034 × TC1507 × MON 88017 × DAS-59122-7 Grown as a Seed Mix in 2007 U.S. Field Trials (Appendix 3)

In 2007, Monsanto assessed the efficacy of SmartStax and different percent seed mixtures against major lepidopteran pests. The target pest s of concern were European corn borer (ECB),

Ostrinia nubilalis (Hübner), corn earworm (CEW), Helicoverpa zea (Boddie), Southwestern corn borer (SWCB), Diatraea grandiosella Dyar, sugar cane borer (SCB), Diatraea saccharalis (Fabricius), and fall armyworm (FAW), Spodoptera frugiperda (J.E. Smith).

Study Methods:

Monsanto established field trials evaluati ng the efficacy of Smar tStax and MON 89034 x TC1507 against five target pest in 23 different locations of ma jor US corn growing areas;

Monsanto relied on a combination of natural and ar tificial infestation. Expe rimental plots were 8 rows wide by 20-100 ft long. Treatments were as signed to plots using a randomized complete block design with 2 or 4 replicates. The efficacy of SmartStax in pure stands and seed mixtures were evaluated using damage criteria appropriate for each insect pest and compared with non-Bt

controls. The treatments were:

• 100% SmartStax

• 95% SmartStax seed ble nd with 5% non-Bt seed

• 90% SmartStax seed blend with 10% non-Bt seed

• 100% MON 89034 x TC1507 (lepidopteran act ive toxins of SmartStax only)

• 95% MON 89034 x TC1507 (lepidopteran act ive toxins of SmartStax only)

• 90% MON 89034 x TC1507 (lepidopteran act ive toxins of SmartStax only)

• 100% non-Bt

- 52 -Statistical analyses for each trial were performed across and w ithin sites at the 0.05 level of significance. Results of statistical analyses for e ach trial are detailed in the Monsanto’s Appendix

3.

European corn borer : Artificial infestations were acco mplished by applying neonates using a bazooka applicator tool. Foliar da mage was assessed using a modifi ed 0-9 Guthrie scale, where 0 indicates ‘no damage’ and 9 indicates a majority of leaves with long feeding lesions. Stalk damage was evaluated by vertical dissection an d measuring tunnel length in centimeters or

inches.

Corn earworm : Artificial infestations were accomplishe d by applying neonates using a bazooka applicator tool. Ear damage was assessed by counting the number of damaged kernels or by using the Widstrom scale where 1 is damage to th e silks only, 2 is feeding to 1cm beyond the ear tip, and further values measure additional feeding in 1 cm increments beyond the ear tip. Total larvae per ear and percentage of plan ts infested were recorded as well.

Southwestern corn borer : Artificial infestations were accomp lished by applying neonates using a bazooka. Stalk damage was assessed by vertical dissection, and tunnel lengths were measured in

centimeters or inches. Total number of cav ities in stalks and ear were recorded.

Sugarcane borer : Damage was assessed by evaluating vert ically dissecting st alks and recording

the total number of cavities in stalks and ears as well as total number of larvae per ear.

Fall armyworm : Artificial infestations we re accomplished by applying neonates using a bazooka applicator tool. Foliar damage was assessed using the 0-9 Da vis scale where 0 indicates no damage and 9 indicates that the whorl is destroyed.

Study Results: European corn borer results : Foliar damage: recorded damage at Seymour (IL) and Flatville (IL) was low. Pooled foliar damage results across lo cations are shown in Table 23 . Foliar damage results at individual locations were: 1.1 and 1.0 for SmartStax; 1.6 and 1.5 for 95% SS; 1.5 and 2.0 for 90% SS; and 4.0 and 3.4 for 100% non-Bt corn. No significant di fferences in damage were detected between SS pure stand and SS seed mixtures within locati ons and across sites. Significant differences were detected, however, between means of all Bt treatments and means of non-Bt treatments. For example at Seymore, SS pure stands incurred a mean damage of <1.4, 10% and 5% seed blends had a mean damage of 1.8, and non-Bt controls averaged 4.0.

Table 23. Foliar damage for ECB by in dividual components (across sites)

Treatment Plant Type Mean Foliar Damage (Guthrie scale) Standard Error Tukeys (p<0.05)

100% SS Bt 1.1 0.06 C

95% SS Bt 1.3 0.23 BC

90% SS Bt 1.4 0.09 BC

5% non-Bt non-Bt 1.8 0.14 BC

10% non-Bt non-Bt 2.2 0.27 B

100% non-Bt non-Bt 3.7 0.31 A

Table modified from Appendix 3, p. 176

- 53 -Mean stalk damage (as measured by inches of tunnelin g): at Blair (NE) , Cambridge (IA), Lebanon (IL), and Waterman (IL) no stalk tunneling was observed for SS plants in any of the Bt treatments; only stalk damage was observed fo r non-Bt plants. The di fference between non-Bt and SS plant damage was statistically significant ( Table 24 )

Table 24. Stalk tunneling (inches) for ECB by indivi dual components of treatments (across sites)

Treatment Plant Type Mean Stalk Tunneling (inches) Standard Error Tukeys (p<0.05)

100% SS Bt 0 0 B

95% SS Bt 0 0 B

90% SS Bt 0 0 B

5% non-Bt non-Bt 0.8 0.25 AB

10% non-Bt non-Bt 1.3 0.45 AB

100% non-Bt non-Bt 1.8 0.23 A

Table modified from Appendix 3, p. 177

“Monsanto summarized that SmartStax provided a high level of control of foliar and stalk feeding by ECB larvae. No ECB stalk damage was observed on SmartStax alone or in the SmartStax component of the seed mixtures at any site. Across all lo cations, ECB damage was consistently lower for SmartStax and the seed mi xtures than the non-Bt c ontrols, with a general trend across sites of more damage on non-Bt c ontrols, followed by slightly less damage to nonBt plants in 10% seed mix and 5% seed mixture, and no damage to SS. The data indicated that the non-Bt component of the seed mixtures wa s consistently supporti ng ECB populations, with the level of damage and insect su rvival proportional to the percentage of non- Bt plants present.” (Appendix 3)

Corn earworm results : Across-site summary results (21 sites in 11 different states) were as follows: Using the Widstrom scale for foliar damage, assessing the number of da maged kernels, percentage of plants infested, and recording the number of larvae per ear, Monsanto found significantly less damage to SS plants than non-Bt contro l plants. However, the difference in ke rnel damage in seed mixtures and non-Bt treatments was not significant. No difference in the number of larvae found per ear was detected among any treatments. Difference in foliar damage was non-significant between treatments containing SS plants.

Monsanto summarized an evaluation of damage metrics across all locations for SmartStax and seed mixtures (90% and 95%) that found signifi cantly less damage compared to the non-Bt control. Similarly, all damage evaluations for the lepidopte ran active component of SmartStax (MON 89034 × TC1507) and its seed mixtures were found to be significantly lower than non-Bt controls, with a general trend ac ross sites of slightly more folia r damage and damaged kernels on the 90% (10% non-Bt) seed mixture than the 95% (5% non-Bt) seed mixture.

Southwestern corn borer results : Across-site summary results: When average leaf damage , number of cavities, larvae, and inches of tunneling were compared by treatments across s ites, significantly less damage was found in all SmartStax treatments than in non-Bt treatments. No differences in damage between the

SmartStax seed mixtures were detected.

- 54 -Except for foliar damage rating results, no di fference was found between SmartStax and MON 89034 x TC1507 treatments (there is no difference be tween the lepidoptera ta rgeting Bt proteins of the two products). However, damage ratings for 90% MON 89034 x TC1507 treatments were significantly higher than for 100% SmartSta x and 100% MON 89034 x TC1507 treatments (see Table 25).

Table 25. Mean foliar damage across sites due to SWCB

Treatment Mean damage Standard Error Tukeys (p< 0.05)

100% SS 0.46 0.16 C

100% MON 89034 x TC1507 0.56 0.17 C

95% MON 89034 x TC1507 0.93 0.20 BC

90% MON 89034 x TC1507 1.42 0.35 B

Non-Bt 8.21 0.32 A

Table extracted from MRID 479437-01, Appendix 3, p. 192

Monsanto concluded that the evaluations fo r SWCB found significantly less damage and fewer larvae in SmartStax and MON 89034 × TC1507 plots than non-Bt controls and that comparable

levels of control were observed in th e 95% and 90% MON 89034 × TC1507 seed mixtures. There was a general trend across sites of slightly more damage and larvae on the 90% (10% nonBt) seed mixture than the 95% (5% non-Bt) seed mixture.

Sugarcane borer results : The experiment was conducted at Needville (TX); hence, no across-site results were available. The numbers of cavities and number of larvae pe r ear were significantly lower in SS, MON 89034 x TC1507, and seed mixture treatments than non-Bt controls (see Table 26 and 27).

Table 26. Mean number of cavi ties and SCB larvae per ear

Treatment Mean # of

Cavities SE Tukeys (p< 0.05) Mean # of

Larvae SE Tukeys (p< 0.05)

100% SS 0.08 0.08 B 1.14 0.26 B

100% MON 89 x TC 0.18 0.15 B 1.35 0.48 B

95% MON 89 x TC 0.54 0.09 B 1.83 0.25 B

90% MON 89 x TC 0.68 0.35 B 2.04 0.46 B

Non-Bt 18.46 2.96 A 13.09 1.86 A

Table modified fr om MRID 479437-01, Appendix 3, p. 196

Table 27. Mean number of SCB larvae per ear

Treatment Mean # of Larvae SE Tukeys (p< 0.05)

100% SS 1.14 0.26 B

100% MON 89 x TC 1.35 0.48 B

95% MON 89 x TC 1.83 0.25 B

90% MON 89 x TC 2.04 0.46 B

Non-Bt 13.09 1.86 A

Table modified fr om MRID 479437-01, Appendix 3, p. 196

Monsanto stated that under high natura l pressure, SmartStax and MON 89034 × TC1507 materials provided excellent contro l of SCB. The number of cavitie s and larvae in Bt treatments were significantly lower than in non-Bt contro ls, and the damage and number of larvae were

- 55 -similar in Bt treatments. However, there was a ge neral trend of slightly more damage and larvae on the 90% (10% non-Bt) than the 95% (5% non-Bt) seed mixture.

Fall armyworm results : Across-site results: The experiment was conducted at th ree US sites, Leland (MS), Loxley (AL), and Union City (TN). Average foliar damage was significantly lower for SS and MON 89034 × TC1507 treatments than non-Bt controls. No differences in damage were evident between SS and MON 89034 × TC1507 plots. Table 28 summarizes m ean foliar damage results across all sites and range of values for each treatment.

Table 28. Mean and range of foliar dam age for Fall armyworm (across sites)

Treatment Mean Foliar Damage (Range) Standard error Tukeys (p< 0.05)

100% SS 0.14 (0-0.30) 0.06 B

100% MON 89034 x TC1507 0.26 (0.01-0.50) 0.11 B

95% MON 89034 x TC1507 0.72 (0.06-1.06) 0.20 B

90% MON 89034 x TC1507 0.90 (0.38-1.64) 0.22 B

Non-Bt 6.81 (3.88-9.00) 0.66 A

Table modified fr om MRID 479437-01, Appendix 3, p. 197

In summary, Monsanto stated that damage in the non-Bt control averaged > 4 on the Davis scale

across sites and was 9 at Uni on City indicating severe FAW damage. Achieved control was excellent for all Bt treatments with average ratings ranging from 0 to 1.64. A general trend was observed where slightly more damage in the 90 % (10% non-Bt) seed mixtures occurred than the 95% (5% non-Bt) seed mixture.

Registrant conclusions:

The results of these trials with SmartSta x and MON89034 × TC1507, alone or in 95% and 90% seed mixtures, against major lepido pteran pests of corn indicated th at the Bt treatment tested had significantly less foliar, stalk, and ear damage from these ma jor pests across all locations compared to non-Bt controls.

The general trend for damage across sites was as follows: non-Bt control > 10% seed mix > 5% seed mix > SmartStax. However, with generally lo w pest pressure, analysis of individual refuge components (5% non-Bt, 95% SmartStax, 10% non-Bt and 90% SmartStax) found few statistically significant differe nces in damage among 100%, 95% and 90% lepidopteran-resistant material, or between 5% and 10% non-Bt refuge (when the anal ysis was conducted).

BPPD Review

BPPD concurs with Monsanto th at SmartStax seed mixtures (5% and 10%) were efficacious against controlling ECB as is visible from the results for foliar and stalk damage.

Monsanto was able to demonstrat e that the lepidopteran active t oxins in SmartStax and its seed mixtures were efficacious against CEW. They reported no difference in number of larvae between 100% SS, 95% SS, 90% SS, and non-Bt c ontrol. BPPD understands that this was an efficacy experiment and not a st udy designed to look at movement of larvae; hence the larval numbers were not reported for individual componen ts (SS and non-Bt plants) of the treatments.

- 56 -This information would have been informative to have in addition to the mean number of larvae (per ear) per treatments. Also, it was not clear what the ratio of Bt to refuge plants was in seed mixture samples. This information could have pr ovided additional weight to Monsanto’s efficacy

results and conclusions.

BPPD concurs with Monsanto that SmartStax and its seed mixtures (95% and 90%) were efficacious in controlling SWCB. This conclusion was supported by results for stalk damage, tunnel length, total number of cavities, and total number of la rvae found per treatments. While Monsanto provided the mean number of SWCB la rvae found on plants and showed that there was no statistically significant difference between any of the treatments tested, it would have

been informative to have data for the individu al components (Bt and non-Bt plants) of SmartStax (as described in the CEW part of the study. Monsanto could have bolstered their conclusions by providing in their report a br eakdown of the mean number of larvae/ear found on individual SmartStax components or at a minimum, by repor ting the proportion of non- Bt plants contained in seed mixture samples.

BPPD concludes that SmartStax and its lepidop teran active components were efficacious in controlling the sugarcane borer. B PPD’s comments made above with respect to additional data reporting apply for this data set as well. More in formation about sample composition (ratio of Bt plants to non-Bt plants) and num bers of larvae found on SmartStax and refuge plants would have been illuminating.

BPPD concurs with Monsanto that across-site fo liar damage results show that SmartStax and its Lepidopteran active components were efficacious against fall armyworm.

4. RPN-08-549 - Assessment of the Efficacy of Lepidopteran and ColeopteranProtected Combined Trait Corn Product MON 89034 × TC1507 ×MON 88017 × DAS59122-7 and Combinations of MON 89034 × TC1507 × MON 88017 × DAS-59122-7 Grown as a Seed Mix in 2008 U.S. Field Trials (Appendix 4)

In 2008, Monsanto conducted field trials to asse ss the efficacy of SmartStax alone and seed mixtures containing SmartStax a nd non-Bt seed against several lepidopteran target pests in different locations of the US Corn Belt. The targ et pests of concern were European corn borer

and corn earworm.

Study Methods:

Monsanto established field trials evaluati ng the efficacy of Smar tStax and MON 89034 x TC1507 against three target pest in different loca tions of major US corn growing areas. Trial

sites were located in Blue Eart h (MN), Lincoln (MN), Clay (IL), Warren (IL), Tippecanoe (IN), and Shelby (OH). Experimental plots were 24 rows wide by 150-200 ft long. Treatments were assigned to plots using a randomi zed complete block design with no replicates. Damage ratings were taken from eight internal rows within plot s. Statistical analyses were performed across all sites at the 0.05 level of significan ce. Each site was a replicate.

- 57 -The treatments tested were:

• 100% SmartStax • 95% SmartStax seed blends and 5% non-Bt (NK603) • 90% SmartStax seed blend and 10% non-Bt (NK603) • 90% SmartStax seed blend and 10% non-Bt (NK603) • 100% non-Bt control (NK603)

European corn borer : plant damage was assessed by counting the number of cavities per plant.

Corn earworm : plant damage was assessed by collecting data on the number of larvae found per ear (20 plants/plot).

Study Results:

European corn borer results : No statistically significant differences were found among treatments in the number of cavities per plant (see Table 29). However, since ECB population density was low, the results cannot be further interpreted.

Table 29. ECB damage (cavities) across all sites (2008 data)

Treatment Mean number of cavities/plant

with SE Tukeys (p <0.05)

100% SmartStax 0 (0) A

95% SmartStax 0.01 (0.01) A

90% SmartStax 0.01 (0.01) A

90% MON89 X TC 0 (0) A

100% non-Bt 0.23 (0.19) A

Corn earworm results : All Bt treatments had significantly less CEW per ear compared to the control treatments; no larvae were found on Smar tStax ears. The plants examined in 100% SmartStax plots did not show any evidence of CEW larval presence. No statistically significant differences were reported betw een larvae found on 100% SmartStax and SmartStax seed blend treatments (see Table 30).

Table 30. Corn ear worm damage across all sites (2008 data)

Treatment Mean number of larvae/ear

with SE Tukeys (p <0.05)

100% SmartStax 0 (0) B

95% SmartStax 0 (0) B

90% SmartStax 0.01 (0.01) B

90% MON89 X TC 0.01 (0.01) B

100% non-Bt 0.30 (0.10) A

- 58 -Registrant conclusions: In summary, Monsanto stated th at evaluations of SmartStax and its seed mixtures demonstrated that the SS corn had significantl y less plant and ear damage than non-Bt controls acr oss all of the sites. Natural population density for Lepidopteran pests targeted was low, especially for ECB. There was a general trend of more damage to corn in 10% than 5% seed mixture, with the least and most damage found in SmartSta x and control plots, respectively.

BPPD Review Based on the very low number of CEW larvae found per ear and mean numbers of ECB cavities found on plants in non-Bt treatments, it appears th at Monsanto did not have great infestation pressure for both Lepidoptera pests. But the B PPD IRM team noted that the results in the CEW portion of the experiment were stat istically significant. Greater pest pressure might have revealed possible differences in efficacy between the Bt treatments. Monsanto did not describe how many plants per treatment plot were examined in the European corn borer experiment. It is unclear whether the study could have been improved by increasing the number of plants sampled. Nonetheless, these field trials supplement the efficacy trials described by Monsanto in Appendix

3.

5. NA09S2J005 - Efficacy of MO N 89034 × TC1507 × MON 88017 × DAS-59122-7 Corn Mixed with Non-Bt Refuge Corn (Appendix 9)

Dow conducted a two-year efficacy field study to evaluate protection from ECB damage to Bt and non-Bt plants in different SmartStax seed mixtures. All four locations were artificially

infested with ECB larvae.

Study Methods: Field trials were set up at Fr ankfort (IN), Fowler (IN), a nd Sidney (IL) in 2008 and Arlington (WI) and Fowler (IN) in 2009. All treatments were replicated 4 times within each trial using a randomized complete block design. Plots were eight rows wide (30" row spacing) and 20 ft long. At each location all non-Bt plants in row 3 of each seed mixture pl ot were artificially infested with ECB neonates at the early w horl stage. In pure stand plots, three randomly selected plants

were selected in row 3 and infested with 50 larv ae per whorl. Subsequent injury was assessed to non-Bt plants and adjacent plants 21 days after infestations had occurred. The 1-9 leaf injury scale was used to measure ECB damage.

The following treatments were planted in 2008:

• 100% SmartStax

• 95% SmartStax seed mixt ure and 5% non-Bt seeds

• 90% SmartStax seed mixture and 10% non-Bt seeds

• 90% TC1507 x DAS-59122 and 10% non-Bt seed

• 100% non-Bt seed

The following treatments were planted in 2009:

• 100% SmartStax

- 59 -• 95% SmartStax seed mixt ure and 5% non-Bt seeds

• 90% SmartStax seed mixture and 10% non-Bt seeds

• 95% TC1507 x DAS-59122 and 5% non-Bt seed

• 95% MON 89034 x MON88017 and 5% non-Bt seed

• 100% non-Bt seed

ECB injury scores were analyzed using analyses of variance (ANOVA) with treatment, replicate, and their interaction as depe ndant variables. Post-ANOVA m eans separation (least-squared estimates) was conducted using Tukey’s Honest Significant Difference (HSD) test (P < 0.05).

Registrant results:

Infestations were successful at al l four locations as measured by m ean injury ratings in controls ranging from 3-5.

In 100% non-Bt plots (controls), movement of larvae occurred within the row and across the row. In seed mixture plots at three locations, there was no evidence that larval movement had occurred across the row except for one location in a 90% seed mixture plot (mean damage rating of 1.25). At two locations there was evidence th at larval movement occurred within the row (mean damage rating of 1.18). No surviving ECB la rvae were found, however. In two of the four locations, the infested non-Bt plants in 95% seed mixtures had significantl y less damage than the non-Bt plants in 100% non-Bt plot s. Figures 15 and 16 show two of the four locations with their respective leaf damage rati ngs/different treatments.

Figure 15. European corn borer injury (leaf injury scal e 1–9) as an indicator of movement and survival at Frankfort (IN) in 2009. “Null plant” refers to a non-Bt plant infested in a seed mixture plot. (Figure 13 in Appendix 9, p. 672)

- 60 -Figure 16. European corn borer injury (leaf injury scale 1–9) as an indi cator of movement and survival at Fowler (IN) in 2009. “Null plant” ref ers to a non-Bt plant infested in a seed mixture plot (Figure 14 in Appendix 9, p. 673).

Registrant conclusions: In summary, Dow concluded that SmartStax plants exhibited minimal leaf feeding whether they were planted in a pure stand or mixed with non-Bt plants. SS plants adjacent to or distant from non-Bt plants were not signifi cantly affected by ECB feeding; the only damage observed was consistent with initial feeding scars by older la rvae. Dow proposed that ECB might have first fed on a non-Bt plant and then moved to a neighboring plant but were unable to overcome the three highly effective Bt proteins in SmartStax target ing Lepidoptera. Dow furthe r stated that insects

resistant to one toxin would still be controlled by the other two.

Across the four locations there was a trend (signi ficant at one location) for reduced damage to the non-Bt plants in a seed mix compared with a pure stand of non-Bt pl ants. This effect was greater for non-Bt plants in a 95% seed mix than for non-Bt plants in a 90% seed mix. This observed trend of reduced damage was presumably due to the lack of ECB larval survival on SS plant and subsequent migration to a neighboring non-Bt plant.

BPPD REVIEW BPPD notes that Dow demonstrated that 1) non-Bt plants in SS seed mixtures ( Figure 15 ) may occasionally obtain additional protection from su rrounding Bt plants (hence reducing effective refuge as compared to structured refuge) and 2) larval survival is unlikel y if larvae manage to move across a row or onto a neighboring plant (as im plied by very little feeding damage to those

- 61 -SmartStax plants). The results of this experiment appear to su pport that the thre e lepidopteran active Bt proteins in SmartS tax together are highly toxic to European corn borers.

C. Modeling

1. MSL0022311 - Modeling the Impact of a Five-Percent Seed Mixture on the Evolution of Lepidopteran Resi stance to MON 89034 × TC1507 × MON 88017 × DAS-59122-7 (Appendix 8)

In this section, BPPD lists Monsanto’s objectives , model structure, assumptions, results and their conclusions followed by BPPD’s analysis, incl uding uncertainties and conclusions. BPPD did not discuss the results for single and dual toxin s imulations that were prov ided by the registrant, rather, BPPD extracted ECB and SWCB results for the triple to xin simulations only. Monsanto provided single gene (20% stru ctured refuge) and dual gene (5 % structured refuge) simulation results as a comparison to the triple gene (5 % seed mix and structured refuge) results for lepidopteran pests. Given that Sm artStax (a triple gene pyramid ed product for lepidopteran) is currently registered with a 5% structured refuge, BPPD did no t see the need for comparisons with those additional analyses conducted by Monsanto. Tables 31 and 32 list Monsanto’s choice for values of certain relevant parameters. Tabl es 33 and 34 list ECB simulation results, while

Tables 35 and 36 list SWCB results for benchmark and worst-case assumptions as well as

different scenarios.

A 95% SmartStax [aka 5% Refuge in a Bag (RIB)] was proposed as a potential insect resistance management (IRM) strategy for SmartStax corn. Monsanto used a three locus deterministic model to evaluate the durability of a 5% seed mi xture (95% SS and 5% refuge seed) compared to the durability of a 5% structur ed refuge using SmartStax to control ECB and SWCB. The model was parameterized using lab and field derived data from survival of the target pests.

Assumptions

• Model structure The model used is a three toxin deterministic model consisting of three compartments (Java SE runtime environment, v1.6.0.16) incorporating non-random oviposition and random mating (occurring distinctly in compliant and non-comp liant compartments). The model was run as a single, dual, and triple toxin m odel to compare 1) current single Bt protein products (Cry1A.105) with 20% structured refuge to 2) dual gene Bt products (Cry1A.105 x Cry2Ab2) with 5% structured refuge and 3) triple gene Bt pr oducts (Cry1A.105 x Cry2Ab2 x Cry1F) with a 5% seed blend refuge. The model was run for either 100 generations or until the resistan ce allele frequency exceeded 0.5. No natural refuge was considered for ECB and SWCB.

• Resistance Resistance was modeled with a single diallelic lo cus for each of the thre e toxins targeting ECB and SWCB. Resistance was assumed to be complete , which is rarely the case. No fitness cost

- 62 -was incorporated despite evidence that resistance to Bt usually has associated fitness costs.

Resistance alleles were assumed to be partially recessi ve/dominant. Heterozygous (RS)

individuals had two or five-times the survivor ship/fitness of suscepti ble (SS) genotypes.

Initial r-allele frequency was 0.005 for all loci and both Lepidopt era species, which was a higher frequency than indicated by resistance monito ring studies. The number of generations were recorded when the frequency of the r-alle le exceeded 0.5 (less than 100 generations).

• Dose of toxins

Dose scenarios that were modeled are shown in Table 31 . Generally, the dose benchmark values for SWCB were assumed to be slightly lower th an for ECB. Two scenarios were run benchmark

and worst case. Monsanto stated that benchmark va lues were conservative re presentations of best available data derived from efficacy studies.

• Seed mix refuge A seed mixture was implicitly modeled. In fields with a 5% refuge of SmartStax, Monsanto reduced the production of refuge ECB and SWCB to a fraction of the refuge beetles produced in a 5% structured refuge. A 5% seed mixture was conservatively assumed to reduce the effective refuge to 1% and 2.5% of the structured refuge (up to 5 times reductio n; field and lab data support an adult reduction of up to 3 times).

• Non-compliance with structured refuge Modeling compliance and non-compliance was achie ved by creating separate compartments for each. Grower non-compliance with the 5% structur ed refuge was modeled as random (accidental non-compliance) and non random (intentional n on-compliance) and varied between 0%, 20%, and 40%. Oviposition was varied from random across compartments (complete mixing between compliant and non-compliant fields) to being on ly within a compartment (no mixing between compliant and non-compliant fields).

Table 31. Dose scenarios and assumptions modeled for single, dual, and triple toxins targeting ECB and

SWCB Dose Assumptions Toxin 1 Toxin 2 Toxin 3

ECB benchmark 99.9 99.9 99.9

ECB worst-case 99 99 99

SWCB benchmark 99 90 99

SWCB worst-case 99 85 99

Modified from Monsanto submission Appendix 8 (Table 1), MRID 479437-01

- 63 -Table 32. Parameter values and scenario assumptions modeled to assess the risk of resistance evolution in

ECB and SWCB to SmartStax

Parameter Values Comment

Initial R-allele frequency 0.005 Initial frequency assumed for all loci and both Lepidopteran species.

Heterozygous survival 2x, 5x Heterozygous genotypes for all resistance loci had greater survival than SS-genotypes; resistance was assumed partially recessive.

Effective refuge for seed blends 1%-2.5% The percentage of refuge insects was

reduced to a fraction of what would be expected to emerge in a comparable percentage structured refuge. Grower non-compliance with structured refuge 0%, 20% and 40% Non-compliance was modeled as

random and non-random.

Dispersal or mixing between
compartments 0%, 50%, and 100% Mixing between compliant and noncompliant compartments was varied. Oviposition was assumed to occur in a random fashion during mixing

events. BPPD generated table

Results

Simulation results for ECB modeling scenarios: 1) Triple toxin (Cry1A.105 x Cry2Ab2 x Cry1F) with a 5% seed blend, 5% structured refuge, and different genetic and mixing assumptions : For benchmark simulations, resistan ce did not evolve in 5% seed mi xtures and structured refuges (compliant) within the 100 genera tions modeled; resistance allele frequencies did not increase. Without mixing (proportion of ovipositing females from one compartment that occurs in the other compartment) and with a 20% non-complian ce, resistance evolved in 2 generations for structured refuges and resistan ce allele frequencies exceeded 0.5; compliant fields remained unaffected. When Monsanto allowed for mixing to occur between compliant and non-compliant fields, then non-compliance could in crease to 40% without having an effect on time to resistance (>100 generations) for block refuges; resistan ce allele frequencies at that point were approximately 0.05 in non-compliant fields.

Resistance did not evolve within 100 generati ons when deployed in a 5% SmartStax seed mixture (effective refuge of 1%) and 5% structured refuge with worst-case assumptions such as, for example, high heterozygote larv al survival on Bt plants ( Table 34 ). The resistance allele frequencies for Cry1A.105, Cry2Ab2, and Cry1F re mained unchanged after 100 generations with a 1%, 2.5%, or 5% effective refuge indicating that a 5% seed mix refuge would be a very durable strategy for SmartStax. With 20% and 40% non-comp liance, resistance a llele frequencies did increase in the non-compliant fiel ds (in 3 generations), but complia nt fields remained unaffected regardless of the degree of mi xing between the fields. If the non-compliant fields were completely isolated (no mixing with compliant fields), then resistan ce evolved very rapidly (three generations). When mixing between th e compliant and non-compliant compartments was incorporated, then resistance did not evolve in 100 generations ev en in the non-compliant fields.

- 64 -Table 33. Number of generations to ECB res istance and final R-allele frequency with benchmark assumptions and different scenarios for 5% seed mix (implicit modeling) and 5% structured refuge Final Frequencies 1 and Generations

to Resistance Compliant fields Final Frequencies 1 and Generations

to Resistance Non-Compliant fields5% Percent refuge

%

Noncompl. % Mixing Cry1A.105 Cry2Ab2

Cry1F Cry1A.105 Cry2Ab2

Cry1F

seed

mix/

block 0 0 0.005, >100

20 0 0.005, >100 0.547, 2

20 50 0.005, >100 0.034, >100

20 100 0.005, >100 0.034, >100

40 0 0.005, >100 0.547, 2

40 50 0.005, >100 0.034, >100 block

40 100 0.005, >100 0.034, >100 Final Frequencies 2 and Generations

to Resistance in Compliant fields Final Frequencies 2 and Generations

to Resistance in Non-Compliant fields5% Percent refuge

%

Noncompl. % Mixing Cry1A.105 Cry2Ab2

Cry1F Cry1A.105 Cry2Ab2

Cry1F

seed

mix/

block 0 0 0.005, >100

20 0 0.005, >100 0.68, 2

20 50 0.005, >100 0.047, >100

20 100 0.005, >100 0.047, >100

40 0 0.005, >100 0.68, 2

40 50 0.005, >100 0.047, >100 block

40 100 0.005, >100 0.047, >100

Table modified fr om Appendix 8, p. 643, MRID 479437-01

1 Heterozygote fitness is 2x that of the susceptible genotype.

2 Heterozygote fitness is 5x that of the susceptible genotype.

Bolded lines show worst-case scenarios and results or seed mixtures and results

- 65 -Table 34. Number of generations to ECB resis tance and final R-allele frequency with worst-case assumptions and different scenarios for 5% seed mixtures and structured refuge Final Frequencies 1 and Generations

to Resistance in Compliant fields Final Frequencies 1 and Generations to

Resistance in Non-Compliant fields%Percentrefuge %

Noncomp

l. % Mixing Cry1A.105 Cry2Ab2

Cry1F Cry1A.105 Cry2Ab2

Cry1F

1 0 0 0.005, >100

2.5 0 0 0.005, >100

5 0 0 0.005, >100

20 0 0.005, >100 0.69, 3

20 50 0.005, >100 0.03, >100

20 100 0.005, >100 0.03, >100

40 0 0.005, >100 0.69, 3

40 50 0.005, >100 0.03, >100 5

40 100 0.005, >100 0.03, >100

Table modified fr om Appendix 8, p.644, MRID 479437-01

1 Heterozygote fitness is 5x that of the susceptible genotype. 1%, 2.5%, and 5% refuges with 0% non comp liance represent seed mixture scenarios

Bolded lines show worst-case scenarios and results or seed mixtures and results

Simulation results for SWCB modeling scenarios: 1) Triple toxin (Cry1A.105 x Cry2Ab2 x Cry1F) with a 5% seed blend, 5% structured refuge, and different genetic and mixing assumptions : For benchmark simulations, resistan ce did not evolve in 5% seed mi xtures and structured refuges (compliant) within the 100 genera tions modeled; resistance allele frequencies did not increase. Without mixing and with a 20% non-compliance, resistance evolved in four and seven generations for structured refuges and resistance allele frequencies exceeded 0.5; compliant

fields remained unaffected. When Monsanto allo wed for mixing to occur between compliant and non-compliant fields, then non-compliance could increase to 40% without having an effect on time to resistance (>100 generations) for block refuges; resistance alle le frequencies at that point were approximately 0.03 in non-compliant fields.

Resistance did not evolve within 100 generati ons when deployed as a 5% SmartStax seed mixture (effective refuge of 1%) and 5% struct ured refuge under the worst case scenarios; however, allele frequencies increased up to 0.01 (see Table 36 ). Monsanto therefore concluded that a 5% seed mix refuge represents a ve ry durable strategy for SWCB. With 20% noncompliance and 0% mixing, resistant allele frequenc ies increased to greater than 0.5 in three and four generations; however, in th e non-compliant fields resist ance did not evolve within 100 generations in the compliant fields. As soon as Monsanto allowed for some mixing to occur,

resistance did not evolve in SWCB even with a 40% non-compliance with structured refuge requirements; resistance allele frequencies at that point were approximately 0.03 in noncompliant fields.

- 66 -Table 35. Number of generations to SWCB res istance and final R-allele frequency with benchmark assumptions and different scenarios for 5% seed mix and 5% structured refuge Final Frequencies 1 and

Generations to Resistance Compliant fields Final Frequencies 1 and Generations to

Resistance Non-Compliant fields5% Percentrefuge %

Noncompl. % Mixing Cry1A.105 1
Cry2Ab2 1

Cry1F 1 Cry1A.105 Cry2Ab2 Cry1F

seed

mix/

block 0 0 0.005, >100

20 0 0.005, >100 0.782, 4 0.741, 7 0.782, 4

20 50 0.005, >100 0.013, >100 0.010, >100 0.013, >100

20 100 0.005, >100 0.013, >100 0.010, >100 0.013, >100

40 0 0.005, >100 0.782, 4 0.741, 7 0.782, 4

40 50 0.005, >100 0.013, >100 0.010, >100 0.013, >100 block

40 100 0.005, >100 0.013, >100 0.010, >100 0.013, >100 Final Frequencies 2 and

Generations to Resistance in Compliant fields Final Frequencies 2 and Generations to

Resistance in Non-Compliant fields5% Percentrefuge %

Noncompl. % Mixing Cry1A.105 2
Cry2Ab2 2

Cry1F 2 Cry1A.105 2
Cry2Ab2 2

Cry1F 2

seed

mix/

block 0 0 0.005, >100

20 0 0.005, >100 0.690, 3 0.569, 4 0.690, 3

20 50 0.006, >100 0.029, >100 0.026, >100 0.029, >100

20 100 0.006, >100 0.030 >100 0.027, >100 0.030, >100

40 0 0.005, >100 0.690, 3 0.569, 4 0.690, 3

40 50 0.006, >100 0.029, >100 0.026, >100 0.029, >100 block

40 100 0.006, >100 0.030 >100 0.027, >100 0.030, >100

Table modified fr om Appendix 8, p.641, MRID 479437-01

1 Heterozygote fitness is 2x that of the susceptible genotype.

2 Heterozygote fitness is 5x that of the susceptible genotype.

Bolded lines show worst-case scenarios and results or seed mixtures and results

- 67 -Table 36. Number of generations to SWCB res istance and final R-allele frequency with worst

- case assumptions and different scenarios for 5% seed mixtures and structured refuge Final Frequencies 1 and

Generations to Resistance Compliant fields Final Frequencies 1 and Generations to

Resistance Non-Compliant fields%Percentrefuge %

Noncompl. % Mixing Cry1A.105 1
Cry2Ab2 1

Cry1F 1 Cry1A.105 1 Cry2Ab2 1 Cry1F 1

1 0 0

2.5 0 0 0.011, >100 0.007, >100

5 0 0 0.006, >100

20 0 0.006, >100 0.69, 3 0.512, 4 0.69, 3

20 50 0.006, >100 0.031, >100 0.027, >100 0.031, >100

20 100 0.006, >100 0.032, >100 0.028, >100 0.032, >100

40 0 0.006, >100 0.69, 3 0.512, 4 0.69, 3

40 50 0.006, >100 0.031, >100 0.027, >100 0.031, >100 5

40 100 0.006, >100 0.034, >100 0.029, >100 0.034, >100

Table modified fr om Appendix 8, p.640, MRID 479437-01

1 Heterozygote fitness is 5x that of the susceptible genotype 1%, 2.5%, and 5% refuges with 0% non comp liance represent seed mixture scenarios

Bolded lines show worst-case scenarios and results or seed mixtures and results

BPPD REVIEW

Uncertainties with assumptions

• Structure of representing seed blends Monsanto chose an implicit approach to model seed blends. The deterministic model for a 5% SmartStax structured refuge was employed but with a reduced effective re fuge of 1% and 2.5% ; in addition, conservative assumptions about hete rozygote survival were included. The reduction in effective refuge was based on Monsanto’s field data that showed that at times the number of refuge insects produced in seed blends coul d be lower than in 100% non-Bt (simulating structured refuge counterparts). In seed blends, there is a concern that when larvae are mobile, that dispersal from a refuge plant to a Bt pl ant can convey increased survival because older

instars can tolerate the toxins better than neonates. Monsanto addressed the increased survival potential of older ECB and SWCB by adjusting heterozygote survival (see discussion below). BPPD concludes that this appear s to be a reasonable approach to modeling seed blends for a spatially implicit model.

• Dose of toxins Monsanto presented their base case and worst-case dose assumptions for each of the three toxins expressed in SmartStax targeti ng ECB and SWCB. Monsanto failed to state in their Table 1 of Appendix 8 ( Table 31 in this review) which toxin repr esented which Bt protein (Cry1A.105, Cry2Ab2, and Cry1F). This part of the submission was not clear. Monsanto also did not cite the specific studies that where used to support their efficacy claims.

- 68 -• Resistance allele frequencies Initial resistance allele frequency for Cry1A .105 and Cry2Ab2 in ECB and SWCB of 0.005 is a

conservative choice. The toxins were first commercialized in 2009 as MON 89304; hence the target pests have had two growing season of exposure to these Cry proteins. Cry1F in corn has been commercially available in the US Corn Belt since five growing season, and no shifts in susceptibility have been detected in the main land U.S. through resistance monitoring programs (in Puerto Rico there was fall army resistan ce to the Cry1Fa protein expressed in TC1507). Hence, BPPD concludes that even for Cry1F, an initial resistance allele frequency of 0.005 seems reasonable (in mainland US), although ma ybe less conservative than for Cry1A.105 and Cry2Ab2. Monsanto could have considered a frequency of 0.01 in light of the resistance monitoring results for ECB.

BPPD notes that Monsanto and Dow indicate th ere is an apparent close to high-dose for the lepidopteran active toxins in SmartStax. Their assumption to model the resistance alleles as partially recessive/dominant contra dicts that claim to some extent . BPPD assumes that this may be another conservative assumption built into th e model which then could be used to bolster Monsanto’s modeling conclusions.

• Heterozygote survival

As discussed in Mallet & Porter (1992), effective dominance is i nversely related to the dose of toxin. If heterozygote larvae are expected or known to move between refuge and Bt plants (with some survival occurring), then the effective dominance should be increased. Monsanto addressed this in their implicit modeling of seed blends by increasing survival of heterozygote genotypes two and five times that of susceptible genotypes. This appears to be a fairly conservative assumption that should increase the rate of re sistance evolution in M onsanto’s seed blend modeling.

• Mixing between compliant and non-compliant fields Estimated durabilities of SmartStax in compliant fields with structured refuges showed no difference to varying levels of gene flow (0 %, 50%, and 100% mixing). This parameter was sensitive and affected outcomes in non-compliant fields. BPPD questions whether the levels of sensitivity predicted by Monsanto’s model are re alistic. Furthermore, it was unclear whether the assumption of 0% mixing meant “no dispersal ou t of any compartment” or whether, adult dispersal was allowed to occur separately be tween compliant compartments and separately between non-compliant compartments while othe rwise maintaining a barrier to gene flow. Furthermore, when Monsanto modeled dispersal of a fraction of the lepidopteran population from one compartment to another, it was not cl ear how the dispersers were selected. BPPD assumes that some probability of dispersal (0, 0.5, and 1) was a ssigned for each adult, but this was not discussed by the registrant. In additi on, Monsanto modeled 0%, 50%, and 100% mixing between populations of different compartments . Currently, it is unknown what proportions of ECB and SWCB populations might engage in l ong-distance dispersal. Recapture studies studying local dispersal have genera lly recaptured a small percentage of the marked ECB adults, which could imply that dispersal is fa r and frequent. Furthermore, Showers et al. (2001) found that ECB adults are capable of dispersing great distances. In Monsanto’s model, when mixing (or dispersal) between compartments increased, resist ance evolution was greatly delayed in terms of

- 69 -years and allele frequency in both ECB and SWCB ( Tables 33-36 ). BPPD concludes that dispersal is an important and sensitive parameter.

• Standard error of mixing for the seed mixture technology Monsanto did not incorporate into the model or report to BPPD wh at the expected standard error of mixing would be for a SmartStax 5% seed blend technology. This information should have been provided to the Agency in support of the Section 3 registra tion application. Although Monsanto did not incorporate th e standard error of mixing into the modeling simulations, one could argue that since Monsanto modeled a conser vatively low effective re fuge of 1% that the standard error of mixing could be included in this number. Nonetheless, Monsanto should provide this information to BPPD.

• Uncertainty regarding SWCB biology Less is known about SWCB larval and adult disp ersal than for ECB; typically the assumption has been made that since the two species are clos ely related and their beha vior is similar that ECB can serve as a surrogate for SWCB to address uncertainties about biol ogy and genetics. It is unclear what percentage of females, if any, coul d potentially engage in long-distance pre-mating dispersal and what the spatial scale of random mating is. Such uncertainties could affect the durability estimates for SmartStax. There is some evidence that there may be greater (natal) field fidelity for SWCB. If SWCB primarily mate in th e natal field, then seed mixtures could be a preferred IRM strategy.

• Non-random oviposition by adults Monsanto chose to model oviposition as non-rand om. Data suggest, however, that at least for second generation instars of ECB, oviposition o ccurs in a uniform fashion where females do not discriminate different types of pl ants in a corn field (Sorenson et al. 1993). Whether non-random oviposition or the uniform oviposition would have a ffected the rate of adaptation differently is unclear. On the other hand, Monsanto wrote that they varied oviposit ion from random across compartments to being only w ithin a compartment. BPPD is not clear whether oviposition was modeled as random for dispersers but not for re sident adults. For future submissions, BPPD recommends that Monsanto provide a more thor ough description and rational for some of their parameter values chosen as to facilitate the Agency’s review of the modeling submission.

BPPD is unaware of oviposition data for SWCB; however, since ECB and SWCB are closely related species, it may be acceptable to assume uniform oviposition behavior for SWCB as well. BPPD questions whether Monsanto ran explor atory simulations with random and non-random oviposition and determined that non-random ovi position was more conservative choice.

ECB results For worst-case assumptions (refuge beetle output 1% and 5x greater survivorship for heterozygote resistant genotypes) , resistance allele frequenc y for all three toxins in seed mixture compartments did not exceed 0.011 after 100 generati ons, and resistance in ECB had not evolved at that point.

- 70 -For worst-case assumptions (20% and 40% non-comp liance with planting a st ructured refuge and no mixing between compliant and non-compliant co mpartments), resistance allele frequency for

all three toxins in structured re fuge compartments was 0.69 and resi stance in ECB evolved in just 3 and 4 generations. BPPD notes that no mixing may be an unrealistic assumption for European corn borers since it has been shown that they ar e capable of dispersing approximately 14 km in 100 minutes (Showers et al. 2001). Yet, localized movement for ECB has been documented by many sources, and some of this information was discussed in the backgr ound section of this

review.

When non-compliance was 20% an d 40% but mixing was increase d to 50% and 100%, then

resistance evolution did not occur within the time frame modeled (100 generations or approximately 50 years) and r-allele frequency did not exceed 0.034.

SWCB results

Resistance in SWCB did not evolve within 100 generations under worst-case and benchmark dose assumptions for the 5% seed mixture (inclu ding a reduction in adult emergence to 1% and 2% of adult produced in 5% bloc k refuges). Similarly, resistance did not evolve for structured refuges within the 100 generation time fr ame modeled when compliance was 100%.

For worst-case assumptions (20% and 40% non- compliance and no mixing between compliant and non-compliant compartments), for all three toxins in structured refuge compartments resistance allele frequency increased from 0.005 to 0.69 in just three generations for SWCB. BPPD notes that no mixing appears to be an unrealistic assumption fo r Southwestern corn borers. Percentages of local recaptures in mark-r ecapture studies have been low (yet higher than for ECB), and while females appear to be mating within the 48 hrs of em ergence, there may be substantial post mating dispersal (discusse d in background section of this review).

When non-compliance was 20% and 40% and mixi ng was increased to 50% and 100%, then resistance did not evolve during the time-frame modeled (100 generations or approximately 50 yrs) and final resistance alle le frequency increased to 0.031.

Overall BPPD conclusions: Monsanto’s modeling output s uggests that a 5% SmartStax s eed mixture targeting ECB and SWCB may be a more durable IR M strategy than the 5% structur ed refuge approach because non-compliance with refuge planting is elimin ated. BPPD notes, however, that it is unclear whether explicit modeling of a seed blend envi ronment and, for example, asymmetric larval dispersal (from refuge and Bt pl ant) would reveal significantly di fferent results in a spatially explicit model.

2. 091173 - Simulation Modeling of Corn Root worm Adaptation to Pyramided Bt Corn Plant Incorporated Protectants MON 88017 and DAS-59122-7 with Block and Seed Mix Refuges (Appendix 12)

- 71 -In this section, BPPD has summa rized Dow’s objectives, model structure, assumptions, results and their conclusions followed by uncertainties and conclusions identified by BPPD. Tables 37 through 39 list Dow’s choices for values of certain relevant parameters (and BPPD recommendations) and different scenarios; Fi gures 17 through 19 display output results for

different CRW scenarios.

Dow employed the stochastic, spatially explicit model developed by Storer (2008) and adapted it to include a seed mixture component. This mode l was the basis to evaluate the durability of MON88017 x DAS-59122-7 deployed with two differe nt refuge strategies (seed mixture and structured refuge). The rate of adaptation was es timated for a 5% seed blend and compared to the rate of adaptation estimated for a 5% block refu ge. The refuge strategies were modeled in the following scenarios: 1) 5% block refuges were as sumed to be present in all SmartStax fields simulating 100% grower compliance with refuge requirements; 2) 5% block refuges were assumed to be present in 80% SmartStax simu lating a 20% grower non-compliance with refuge requirements; and 3) 5% seed mixtures were assu med to be present in all SmartStax fields.

Methods: The modeling landscape consisted of 10 x 10 grid fields, each 25 ha in size; 100% adoption of SmartStax was incorporated. Cross-resistance was assumed to be negligible between Cry3Bb1 and Cry34/35. Dose estimates for the single trai ts (default value = 99.75%) and pyramided event (default value = 99.95%) were based on the fi eld study by Huckaba and Storer (2008). Individuals that were heterozygous (RS) at one locus or both lo ci had a fitne ss intermediate between homozygous susceptible a nd resistant genotypes; the fitness was estimated based on the functional dominance for the resistance allele (S torer 2008). Complete resi stance and no cost of

resistance were assumed in the model. Two additional dose scenarios were modeled and shown

in Table 38

.

A 7 day developmental delay for susceptible inse cts emerging in Bt fields was incorporated. Heterozygotes were not ascribed a delay because they were assumed to be unaffected by the PIP trait (complete resistance).

1. Block refuge modeling : The 5% structured refuge was modeled varying non-compliance with refuge plantings from 0-20%. The results were co mpared to modeling runs for the 5% seed blend with various assumptions for larval movement and survivorship (see Table 39 ).

Fecundity of females was increased from 23 e ggs/female to 100 eggs/female because modeling 100% SmartStax in the landscape lead to fre quent population extinction. While this change enabled comparing the resistance evolution in seed mixtures and structured refuges, it also had the side-effect of increasing dens ity-dependent mortality in the re fuges as well as the rate of adaptation.

Occasional non-compliance was assessed by randomiz ing (20%) non-compliant fields each year. Willful non-compliance (16%) was modeled by keepi ng non-compliant fields fixed each year (a 4 x 4 block in the center of a 10 x 10 matrix).

- 72 -Adult movement: female premating dispersal was assumed to be zero. Adult males moved by default 6 rows/day; a 25 ha field was assigned 625 rows. The probability of a male moving from a refuge row into a Bt row was given by 6/(0.05 x 625)= 0.192; the probability of a male moving from a Bt row into a refuge row was give n by 6/(0.95 x 625) = 0.010. Each day, there was a probability of 0.025 that an adult would leave a flowering field.

2. Seed mixture modeling : adult dispersal was assumed to be irrelevant because random mating

was assured in the seed mixture environment. Re sistance evolution was assumed to be driven by

larval movement and survival.

Movement : Larval movement ( ‘rmove’ in Table 39 ) could occur from Bt Æ Bt, Bt Æ non-Bt,

non-Bt Æ non-Bt, or non-Bt Æ Bt. Movement off an initial Bt plant was genotype specific and determined by the susceptibility to the Bt. Resi stant larvae for both Bt traits would not be expected to leave a suitable host. Parameter ‘rmove’ is the proba bility of successful larval movement from a Bt plant onto another plant. Table 37 provides the probabiliti es of leaving a Bt plant based on the larval genot ypes. For example, Cry34/35 and Cry3Bb1 may each lead to 10% dispersal of a particular genotype. Summed togeth er, this would lead to 19% successful dispersal (calculated as 10% of 100% of larvae and added to 10% of the remaining 90% of larvae). An insect that was fully resistant to one protein would not disperse in response to that particular toxin and have a 10% probability of successful dispersal.

Density dependence : This parameter was incorporated into the seed mixture model yet turned out to be of no significance because SmartSta x suppressed population densities sufficiently.

Recovery : The parameter for recovery ( ‘recover’ in Table 39 ) is the probability of survival for larvae that originated from Bt plants, dispersed, and then arrive d on a refuge (non-Bt expressing) plant. Five percent of the larvae that left Bt plants in a 5% s eed mixture arrived on non-Bt plants and were assigned a probability to recover ba sed on their susceptibility and genotype. The parameter for recovery was calculated in the same manner as the probability for leaving Bt plants for different genotypes. Values for this paramete r were calculated in the same manner as for

‘rmove’

.

Sensitivity analysis and results: The increase in resistance allele frequency (initial = 0.005) was explored after 20 generations for each of the three dose profiles (benchmark, low dose, and BPPD recommended dose – referred to as ‘unrealistic worst case’ by Dow) and various adult and larval (seed mix) dispersal as well as block refuge compliance parameter values. Each simulation was run 5 times to determine the role of stochastic processes on resistance evolution.

Dow investigated the effect of male pre-mati ng dispersal and assigned a probability of 0.192 and 0.010 to refuge males moving into the Bt field and Bt males moving in to the refuge field, respectively. No effect on resistance allele fr equency was observed. Dow did not explore female pre-mating dispersal because females are beli eved to mate shortly after emergence.

- 73 -1. Seed mixture simulations

When default and low dose estimates were used for SmartStax in conjunction with default heterozygote movement, the rate of increase of resistan ce allele frequency was similar for seed mixtures and block refuges of 5% (i.e., le ss than a 2-fold incr ease over 20 years) ( Figure 17

, scenarios 1, 2, 7, and 8).

The various sets of larval movement and recove ry parameter values that were explored are summarized in Table 39. Based on reported results by Hibbard et al. (2005) and Miehls et al. (2008), assumptions were made in the model that larval movement from Bt to non-Bt roots

occurred at moderate to low levels and that recovery/survival was between 5% and 15% for Bt/non-Bt pairs of corn plants. For worst-case heterozygote dispersal (RS-genotypes as likely to be repelled off Bt as SS-genotype s), the rate of adaptation was greater but followed the same pattern for respective scenarios shown in Figure 19 . Dow concluded that seed mixes provided a clear resistance management benefit even for unr ealistic, worst-case heterozygote dispersal.

2. Block refuge simulations

Little effect was observed on the rate of adaptation of the dual ge ne product with 20% accidental non-compliance when the dose was simu lated at benchmark or low dose ( Figure 17 ); however,

with BPPD’s recommended dose levels and willf ul non-compliance (16%), resistance allele frequency doubled in 7 generations ( Figure 18 ). Dow concluded that th e 5% block refuge was durable under default and low dose assumptions for non-compliance witnessed over the years (referred to as complete and incomplete compliance by Dow).

Table 37. Genotype specific probability of leaving a Bt plant Genotype Probability of leaving Bt plant

SSa/SSb 19% RSa/SSb, SSa/RSb 14.5%

RSa/RSb 9.5% RRa/SSb, SSa/RRb 5% RRa/RSb, RSa/RRb 5%

RRa/RRb 0%

Table 38. Different percent mortality a ssumptions for modeling corn rootwo rm resistance to SmartStax in a seed blend and block refuge

Toxin Dow- default assumption 1 Dow - low dose assumption 2 BPPD recommended low dose assumption 3

Cry34/35 99.75% 99% 94.2% Cry3Bb1 99.75% 99% 97.5% Pyramided toxins 99.95% 99.9% 98.2% BPPD generated table

1 Based on estimates of la rval mortality obtained in Huckaba and Storer (2008)

2 Based on lowest estimates of larval mortality in Huckaba and Storer (2008)

3 Based on reduction in adult emergence - these estimates were the basis for EPA’s acceptance of a 5% refuge for

SmartStax.

- 74 -Table 39. Input parameter value sets for la rval movement and recovery in a seed mix
Seed mix

scenarios Larval

movement

( rmove ) Overall recover Predicted survival on Bt/nBt pair Comments

1 0.0975 100% 9.8% Consistent with em pirical observations

2 0.0975 56% 5.53% Consistent with em pirical observations

3 0.0975 25% 2.49% Overall survival low

4 0.0975 6% 0.66% Overall survival very low

5 0.0975 0% 0.05% Overall survival very low

6 0.19 100% 19.04% Overall survival high

7 0.19 56% 10.73% Consistent with em pirical observations

8 0.19 25% 4.80% Consistent with em pirical observations

9 0.19 6% 1.24% Overall survival very low

10 0.19 100% 0.10% Movement high: survival very low

11 0.51 81% 41.34% Movement high: survival very high

12 0.51 25% 12.79% Movement high: surviv al consistent with empirical observations

13 0.75 100% 75.01% Movement very high: survival very high

14 0.75 81% 60.77% Movement very high: survival very high

15 0.75 25% 18.79% Movement very high: survival high

16 0.75 6% 4.74% Movement high: surviv al consistent with empirical observations

17 0.75 0% 0.05% Movement very high: survival very low Table modified from Appendix 12, p. 721 (MRID 479437-01) Shaded fields indicate parameter values consistent with em pirical observations

Figure 97. Increase in r-allele frequency in 20 yrs for differen t compliance scenarios for bl ocks and different larval movement and recovery parameter assumptions for seed mixtures when using default and low dose estimates with default heterozygote dispersal. Seed mixture scenarios with an asterisk are consistent with empirical observations (Appendix 12, p. 722, MRID 479437-01).

- 75 -Figure 18. Increase in resistance allele frequency in 7 years fo r different compliance scenarios for block refuges, different larval movement, and recovery parameter settings for seed mixes. Seed mixture scenarios with an asterisk are consistent with empirical observations (Appendix 12, p. 724, MRID 479437-01).

Figure 19. Increase in r-allele frequency in 20 yrs for differen t compliance scenarios for bl ocks and different larval movement and recovery parameter assumptions for seed mixtures when using default and low dose estimates with

worst-case heterozygote dispersal. Seed mixture scenarios w ith an asterisk are consistent with empirical observations (Appendix 12, p. 723, MRID 479437-01).

- 76 -Dow Conclusions : Dow’s model was conservative in many respects and should over-estimate the rate of resistance evolution in the target pest to SmartStax as reported in this revi ew. 1) The landscape was

modeled with continuous SmartSta x (with the exception of refuges/non-Bt corn); 2) resistance to SmartStax was modeled as complete resistance ( however, lab and field isolated r-alleles do not allow for 100% survival on Bt corn); 3) no fitness cost was associat ed with resistance (most field

and lab isolated r-alleles have a cost of resistance for the pest ); and 4) female fecundity was increased from 23 eggs to 100 eggs/female to avoid population extinctio n, which increased the rate of population adaptation.

Dow compared rates of adaptati on (not time to resistance) of rootworm populations from seed mixtures and structured refuge simulations. In all realistic and most unrealistic modeling scenarios, the durability for 5% seed mixtures us ing SmartStax was comparable to 5% structured refuge scenarios incorporating willful or accidental grower non-compliance. Dow stated that a 5% seed blend for the pyramid MON 88017 X DAS -59122-7 was supporte d by the modeling and was shown to be as durable as the 5% block refuge.

BPPD REVIEW

Uncertainties with assumptions

• Modeling of dispersal in fields at the “edge” of the landscape BPPD is unclear how Dow treated the boundary c onditions in their simulations model. Dow did not provide any information regarding the precise ‘fate’ of insects that moved off of the field along the boundary of the landscape. This information should have been provided in the modeling submission because the approach chosen for dealin g with dispersers along the boundary could possibly impact ( i.e., reduce) the effective refuge.

• Initial resistance allele frequency Dow set initial resistance allele frequency for both genes in the model at 0.005 and assumed that double homozygous resistant individuals occurred at a frequency of 6.25 x 10 -10 . BPPD notes that this initial frequency may be a like ly value for Cry34/35 and Cry3Bb1. Recent studies (Miehls et al. 2008 and Lefko et al. 2008) suggested that resistance allele frequencies could be as high as 0.02 in WCRW (Hibbard (2010), pe rsonal communication). To incorporate the uncertainties of this parameter into the mode ling analysis, Dow could have incorporated a probability analysis for this parameter using, for example, a PERT-Beta distribution with a probability distribution based on data for the lowest ( e.g., 0.001) and maximum ( e.g., 0.02) possible values as well as most likely value ( e.g., 0.005) or conducted a sensitivity analysis for

different initial resistance allele frequencies to demonstrate their effects on the time to resistance.

• Dominance
Dow stated that functional dominance of resist ance was assumed to be related to dose and consistent with assumptions of 25-fold resist ance in heterozygotes based on concentrationresponse results. Dow incorporated the dominan ce into the genotypic fi tness calculations and

- 77 -listed parameter h

a as the functional dominance (Appe ndix 12, p. 708). BPPD was unable to

determine what dominance value was used in those calculations.

• Pre-mating dispersal Dow assumed that females mated immediately af ter emergence and that there was no pre-mating dispersal mechanism. This assumption is likel y based on the ‘closed system’ study conducted by Ball (1957). However, Marquardt and Krupke (2 009) did not observe that newly emerged females mated immediately in the field. Their da ta suggested that some pre-mating dispersal

occurred in female WCRW. Tethered f light studies by Nara njo (1990) and Coats et al. (1986) provided evidence that female pre-mating disper sal is probable. BPPD notes that pre-mating dispersal for female western corn rootworm was a sensitive parameter in EPA’s modeling (Caprio and Glaser 2010b) and sign ificantly extended the durability of Bt corn in block refuges compared to seed blends. BPPD considers fema le pre-mating dispersal as a possibility and effects of such a parameter on the durability of the product should have been explored in the

model.

• Developmental delay for susceptible genotypes Based on the work published on developmental delays of susceptible corn rootworm in Bt corn, BPPD concurs with Dow on the 7-day assumption for susceptible genotypes.

• Dose of toxins and dens ity-dependent adjustment

BPPD has concerns with Dow’s dose claim for Cry34/35 (based on Huckaba and Storer, 2008) and Cry3Bb1. In Huckaba and Storer, Dow artificiall y infested Bt and control fields with a 3300 WCRW eggs/meter; dose (essentially larval mort ality) was determined using the methods of

Storer et al. (2006) by dividing the observed number of emerging adults by the expected number and correcting for density dependent mortal ity (based on Onstad 2006). In a personal communication with BPPD, Dr. Hi bbard stated that assuming a pproximately 75% viable eggs, that at 3300 eggs/meter, it was li kely that little, if any dens ity-dependent mo rtality occurred (based on work published by Hibbard et al. 2010). Hence, BPPD concludes that the lowest mortalities reported by Huckaba and Storer (2008), unadjusted for density-dependence, should be used to estimate the dose of Cry34/35 and Cr y3Bb1. Those dose estimates were referred to as

BPPD recommended in this review and as ‘unr ealistic’ dose assumptions in Dow’s submission (Appendix 12). BPPD disagrees with the characteriz ation of these dose levels as “unrealistic”. In addition, BPPD will closely evaluate Dow’s si mulation results derived with these more conservative (lower) dose values.

Toxin Dow- default assumption 1 Dow - low dose assumption 2 BPPD recommended low dose assumption 3

Cry34/35 99.75% 99% 94.2% Cry3Bb1 99.75% 99% 97.5% Pyramided toxins 99.95% 99.9% 98.2% BPPD generated table

• Heterozygote larval movement

Hibbard et al. (2005) reported that typi cal larval CRW movement of f a Bt plant was less than 10%. BBPD understands that Dow estimated prob ability of dispersal of heterozygote and susceptible genotypes as 5% and 10% for a sing le gene Bt plant based on this study. Dow

- 78 -assigned the same probability of leaving (5% and 10% of the remaining 95% and 90%) to the second gene of the SmartStax CRW product. In the absence of any actual data on genotypic CRW dispersal of SmartStax corn, Dow’s probab ility assumptions appear reasonable to BPPD (see Table 37 ).

• Movement off refuge plants

Hibbard et al. (2005) found late-instar movement occurring from si gnificantly damaged non-Bt plants to surrounding transgenic plants. Work by Dr. Hibbard indicates that it is more realistic to assume high larval movement from late instar s and little to no movement for neonates. Dow incorporated this process into the model yet f ound that density dependence did not become an issue with 5% refuge plants. BPPD disagrees th at density-dependence should not be considered on refuge plants in a 5% seed mixture. First, Hill (1975) observed that the number of eggs oviposited per female ranged from 595-1559 (sample size 11). Assuming that egg viability is 75%, that range could be 446 – 1169 eggs/female. Hibbard reported that the density-dependence threshold occurred at around 854 eggs/30.5 cm; he nce, the neonates from one clutch could potentially be enough to cause de nsity-dependent dispersal off a refuge plant. In addition, Dow observed in their efficacy study (Appendix 9) that movement off Bt plants onto surrounding SmartStax plants incurred additional damage to Bt plants (NA09S2J005 - Efficacy of MON 89034 × TC1507 × MON 88017 × DAS-59122-7 Corn Mixed with Non-Bt Refuge Corn). Hence, BPPD notes that under sufficient high CRW pressure, the number of larvae on refuge plants could build up to a point where density dependence occurs.

• Seed mix scenarios Thirteen seed mix scenarios were explored in Dow’s sensitivity analysis. Dow stated that movement, recovery, and survival assumptions in scenarios 1, 2, 7, and 8 were consistent with empirical observations. BPPD concurs with th ese assumptions and will not discuss unrealistic seed blend scenarios. When BPPD’s dose assump tions were considered for the four seed mix scenarios mentioned, Dow’s modeling predicted that seed mixes for the dual gene product were more durable than block refuges with 100% comp liance or accidental (1 8%) and intentional noncompliance (20%). The dose values recommende d by BPPD resulted in greater rates of adaptation for block refuge scenarios and at least seed blend scenar ios 1 and 2 (visually determined from graphs).

• Block refuge and non-compliance Dow reported that non-compliance of 20% (field) or 16% (by farm) had very little impact on the durability of the dual gene product. Conversel y, EPA/ORD’s sensitivity analysis of noncompliance in their CRW model (Caprio and Glaser 2010a) revealed that this parameter had the ability to significantly affect the durability of Cry34/35 when deployed as a single gene product with a structured refuge.

• Standard error of mixing for the seed blend technology

Dow did not address whether the st andard error of mixing seed ble nds was incorporated into the modeling scenarios. It is unclear whether such an error would ha ve a significant effect on the durability of the pyramided corn rootworm product.

- 79 -Using Dow’s model outputs and considering uncerta inties and assumptions, it appears that seed mixes for Cry34/35 and Cry3Bb1 are a viable (i.e ., durable) alternative CRW IRM strategy to block refuges for SmartStax protected corn.

IV. U.S. REGIONS WITH SMARTSTAX DEPLOYMENT AND REFUGE

STRATEGIES

Below, Dow listed the US corn and cotton growi ng regions where SmartStax is proposed to be marketed either with a 20% structured refuge or 5% seed mixture for Lepidoptera and a 5%

structured or 5% seed mixture for CRW.

Table 40. Registrant proposed refuge schemes for Smar tStax corn targeting CRW and Lepidoptera pest in different regions of the cotton growing region

Proposed refuge

scenarios Region Refuge size Structured refuge – infield

or adjacent Structured refuge – up to 1⁄2 mile away

from Bt field Refuge as seed mix proposed by Dow and

Monsanto

#1 Cotton growing

where CEW is a significant pest and WCRW, NCRW

and MCR are not significant: NC, SC, GA, FL, TN (only

the counties of Carroll, Chester, Crockett, Dyer, Fayette, Franklin, Gibson, Hardeman, Hardin, Haywood, Lake, Lauderdale, Lincoln, Madison, Obion, Rutherford, Shelby, and Tipton), AL, MS, LA, AR, VA (only the

counties of Dinwiddie, Franklin City, Greensville, Isle of Wight, Northampton, Southampton, Suffolk City, Surrey, and Sussex) 20% nonBt corn Yes Yes No

#2 Cotton growing 20% non- Yes No No

- 80 -Proposed refuge

scenarios Region Refuge size Structured refuge – infield

or adjacent Structured Refuge as seed refuge – up to mix proposed 1⁄2 mile away by Dow and

from Bt field Monsanto
where CEW is a significant pest and WCRW, NCRW,

and/or MCR are significant: TX (except the counties of Carson, Dallam, Hansford, Hartley, Hutchinson, Lipscomb, Moore, Ochiltree, Roberts, and Sherman), OK (only the counties of Beckham, Caddo, Comanche, Custer, Greer, Harmon, Jackson, Kay, Kiowa, Tillman, and Washita), MO (only the counties of Dunklin, New Madrid, Pemiscot, Scott, and Stoddard) Bt corn

#3 Cotton growing

where CEW is not a significant pest and WCRW, NCRW

and MCR are not significant: NM, AZ, CA, NV 5% non-Bt

corn Yes Yes Yes

#4 Non-cotton growing where WCRW,

NCRW and MCR are not significant: OR, WA, ID, MT, WY, UT, VA (except the counties of Dinwiddie, Franklin City, Greensville, Isle of Wight, Northampton, 5% non-Bt

corn Yes Yes Yes

- 81 -Proposed refuge

scenarios Region Refuge size Structured refuge – infield

or adjacent Structured Refuge as seed refuge – up to mix proposed 1⁄2 mile away by Dow and

from Bt field Monsanto Southampton, Suffolk City, Surrey, and Sussex), WV, PA, MD, DE, CT, RI, NJ, NY, ME, MA, NH, VT, HI, AK, TN (except

the counties of Carroll, Chester, Crockett, Dyer, Fayette, Franklin, Gibson, Hardeman, Hardin, Haywood, Lake, Lauderdale, Lincoln, Madison, Obion, Rutherford, Shelby, and Tipton)

Table extracted and modified from MRID 479437-01

BPPD Review

General comments: BPPD questions whether a clear line of biologica l significance can be drawn between the Cotton

and Corn Belt. It is recommended that Monsanto determine where overlap exists between the

Corn and Cotton Belt and where the rate of ad aptation in corn earworm could be compromised by using a seed mixture IRM strategy. In such re gions a more conservative approach should be chosen - 5% structured refuge - proposed refuge scenarios #2 or #3 should be applied.

- 82 -Table 41. BPPD response to applicants proposed refuge strategies in four different regions of the continental

U.S. Proposed refuge scenarios for US regions BPPD comments

#1 • BPPD understands this to mean that a 20% refuge is planted for each pest – one adjacent or within the Bt field, and one 1⁄2 mile away.

#2 • The registrants stated ‘n o’ under “refuge 1⁄2 mile away” heading. BPPD interpret this as meaning that only one refuge will be planted for CRW and Lepidoptera pest and that ther efore, the refuge will be adjacent or within the Bt field.

#3 • No corn rootworm refuge will be planted in these regions.

#4 • No corn rootworm refuge will be planted in these regions.

See Table 40 for more information

V. MIXING TECHNOLOGY FOR SMARTSTAX (taken out of MRID 479437-01)

“A seed mix as an IRM refuge strategy for SmartStax corn requires that an appropriate percentage of refuge seed can be reliably in corporated into each un it of seed distributed. Consistently producing a seed mix of a given percentage is a manuf acturing challenge. As proposed, the manufacturing process will target at l east 5% refuge seed per unit. The key issue in manufacturing will be the varia tion among units in the percentage of refuge seed. If that variability is low, and the percentage of ref uge can be accurately delivered, then the target percentage of refuge seed can be closer to the minimum set by the IRM-related scientific factors. If variability among units is greater, then some margin for error will need to be built into the manufacturing process to ensure that the percentage of units with re fuge seed less than the target is at an acceptable level. Th ese considerations mean that the manufacturing process for producing the seed mix needs:

1. Adequate characterization in terms of accuracy (relative to the target percentage) and unit-tounit variability. 2. A procedure that will record the percentage of refuge seed per unit during production in a statistically appropriate percentage of units 3. A process for capturing appropria te verification data from thir d parties involved in seed mix production such as licensees

An additional factor for consideration is the patt ern of refuge seeds in planted fields, with a preference for a more uniform distribution of these seed/plants, versus a more clumped distribution, because of possible impacts on insect damage and yield. However, the distribution is unlikely to affect resistance management: bot h block (i.e. extremely clumped) and uniform seed mix approaches provide similar and robust resistance management. The spatial distribution of plants derived from a seed mix will be determined by the way in which refuge and Bt seed are initially combined in preparing each unit, a nd subsequent mixing and/ or separating that may occur during the distribution and planting processes. Manufacturing processes will ensure

- 83 -relatively homogeneous mixing of Bt and refuge seed in each field as needed for optimal efficacy.”

BPPD Review Monsanto and Dow’s language suggests that th e companies have not yet finalized the mixing technology for a seed blend. No standard error of mixing was reported to the Agency with the application but should be communi cated so that BPPD is aware of any potential reduction in effective refuge from the proposed 5% SmartStax seed blend.

Based on the registrants’ modeling it appears jus tifiable that a uniform or clumped (more like block refuge) distri bution of refuge plants should not affect insect resistance management and increase the risk of resistance evolution, alt hough the registrants propos e to produce a relatively homogenous mixture for SmartStax. In addition, BPPD notes that the phenotypes of Bt and nonBt kernels need to be comparable in order to reduce planting problems. The phenotype of refuge seed should be as close as possible to the phenot ype of the Bt seed. In addition, Bt seeds should be visibly distinguishable from non-Bt seeds ( i.e., different color) as to, among other things, alert the farmer to potential planting problems ( i.e., retention of colored se ed in the hoper). Coloring the refuge seed may also be necessary to assure 1) proper germination of re fuge seed if seeds are stored and 2) the appropriate re fuge percentage for the followi ng growing season of a SmartStax seed mixture product.

VI. EPA/ORD MODELING - Deterministic Model (POPGEN) and Probability

Distributions

For this current risk assessment, EPA (jointly BPPD and ORD) conducted independent modeling analyses with a recently developed simulation mode l for European corn borer, southwestern corn borer, and corn rootworm to estimate the risk of resistance evolution in these target pests.

Deterministic simulations were undertaken with the “USEPA population genetics two locus and three locus model – POPGEN” developed by EP A’s Office of Research and Development (ORD), National Risk Management Research Laborat ory in Cincinnati, Ohio (Dr. John Glaser in cooperation with Dr. Michael Capr io of Mississippi State Univer sity). A probability analysis complemented the deterministic simulations (conducted by Dr. Caprio) based on a PERT distribution (min. value, most lik ely value, and max. value) for pa rameters of uncertainties. This modeling allows the Agency to compare and a ssess durability results of three independent models (Monsanto, Dow, and US EPA/ORD).

EPA/ORD conducted an independent modeling analysis for a 5% seed blend targeting corn rootworm (described in this section A and Capr io and Glaser 2010c) and le pidoptera target pests (Section B, Caprio and Glaser 2010d). The main objectives were to dete rmine 1) whether a 5%

SmartStax seed mixture was a durable IRM strate gy for Lepidoptera target pests compared to the current structured refuge paradigm in place a nd estimate 2) the probability of occurrence for a predicted durability result using POPGEN.

- 84 -A. EPA/ORD’s analysis of a 5% seed ble nd with a dual gene pyramid targeting CRW

1. Model Description

The risk assessments utilized a tw o locus deterministic model, an extension of the model used in Caprio and Glaser (2010a and 2010b). This was a two compartment model, one compartment for persistently non-compliant fields (continuously non-compliant) and a second for compliant and unintentionally non-compliant fields. The latter compartment was further divided into two compartments, one for the Bt-plants and the second for refuge (non-Bt) plants. This version of

the model extended the model used in Caprio and Glaser (2010b) to two independent resistance

loci. The model could be run as either a seed-mix ture model or a structured refuge model. The risk assessment was conducted by running each version 1000 times, each time re-sampling the parameters listed in Table 43 using PERT-Beta distributions 4 (Vose 2000). The value of each parameter and the time until resistance evolve d was recorded for each simulation. Several options exist for sensitivity analysis of this data, including Bayesian model analysis (BMA), stepwise regression, and standard ized regression coefficients. Although the use of standardized regression coefficients is somewhat controversial, they are appropriate in this case because the variance in each variable is determined by th e PERT-Beta parameters and is related to the uncertainty in the parameter.

2. Model Assumptions

Fitness in this non-additive intera ction was calculated as follows. For this example the mortality figures presented by Dow were used. The dominance of locus 1 was h 1 and the dominance at

locus 2 was h 2 . When locus 1 is homozygous resistant ( RR), then the fitness only depends on the locus 2 genotype (and vice versa when the locu s 2 genotype is RR). The exterior row and

column of Table 42 can therefore be filled out directly from the submitted data because one of the loci is fully resistant and only one locus is responsible for the mortality. The double homozygous susceptible genotype fitness (SSSS) can be estimated by the overa ll survivorship to the dual toxin product (also provided). If we look at each locus separately, the survivorship of a susceptible genotype SS is 0.0025 and RR is 1.0. Assuming a dominance of 0.2, the heterozygote fitness is 0.202; the SSSS fitness is 0.0005 and RRRR is 1.0. Making these assumptions, the following locus specific f itnesses can be determined (Table 42).

Table 42. Locus specific fitness

Locus 1

SS RS RR

SS 0.0005 A 0.0025

RS B C (RRRRRRSS)*h 2 +RRSS Locus 2

RR 0.0025 (RRRRSSRR)*h 1 +SSRR 1.0000

The remaining difficulty is filli ng out the cells marked A & B (s ingle heterozygotes) and C (the double heterozygote class). Making the assumpti on that the dominance properties would hold

4 Assumptions about min, max, and mode values of each parameter in Table 43 were made by BPPD based on an array of information such as, observed mortality in the fiel d, results from selection experiments for CRW toxins, and personal communications with academia.

- 85 -for these genotypes as well, th e fitness of the SSRS genotype (B) would be calculated as

h 2 *(SSRR-SSSS)+SSSS, while the fitness of the R SSS genotype (A) would be calculated as

h 1 *(RRSS-SSSS) + SSSS. Finally, the double heterozygote fitne ss (C) was calculated by using an average dominance: RSRS = (h 1 +h 2 )/2 * (RSRR – RSSS) + RSSS.

Table 43. PERT-Beta distribution for certain modelin g parameters in seed mix and structured refuge modeling

Variable Min Value Mode Max Value Comment Cry3Bb1fitness 0.008 0.058 0.1 Cry34/35 fitness 0.0008 0.038 0.08

Overall survival 0.0003 0.018 0.03 Always as low as lowest of the

two loci

Dominance 0.01 0.05 0.2 Drawn independently for both

loci Initial R-allele freq. 0.001 0.005 0.02 For each of the 2`` loci Variables specific to structured refuges Refuge size 0 0.03 0.06 Always set to 0.05 for seed-mix Non-compliance 0 0.3 0.6 Always set to 0 for seed-mix Non-compliance mixing 0.8 0.9 1

Non-random mating 0.2 0.5 0.7 Always set to 0 for seed-mix Variables specific to seed mixtures

Base larval movement 0.05 0.2 0.4 Always set to 0 for structured refuges Days of larval

selection 5 10 15

Asymmetrical larval

movement 0 0.3 0.5 Always set to 0 for structured refuges

3. Results

The mean time to resistance for the seed-mix ture model was 81.9 generations, while it was 52.9 generations for the structured refuge simulations. Histogram s of the results from the 1000

simulations of each version of the model were si milar in shape (Figures not shown here), though bootstrapping revealed significant differences (p <<0.01, R statistical package, sm.density function). A comparative de nsity plot for the two distributions is shown in Figure 20 . In the case of seed-mixtures, 2.5% of the simulations resulted in times until re sistance evolved of less than 30 generations, while 17.0% of the structured refuge simulations fell below this limit. To assist in assessing different levels of risk, different quantiles of the two distributions are shown in

Table 44 . The quantiles can be interpreted as levels of acceptable risk. If one accepted a 5% chance of not meeting a target as an acceptabl e level of risk, the seed-mixture would be acceptable if the target were 33 generations or less, while a structured refuge would only meet a target of 22 generations or less. The sensitiv ity analysis (Appendix 1 of Caprio and Glaser (2010c)) indicated that all variables except non-random mating and non- compliant mixing had statistically significant impacts on the time it took for resistance to evolve. The Bayesian model averaging suggested that non-co mpliant mixing should be included in the model. For seedmixtures, all variables except base larval m ovement, asymmetrical movement, and the time period over which larval movement occurred, had statistically si gnificant impacts on the time it

- 86 -took for resistance to evolve. The Bayesian m odel averaging suggested that the base larval

movement rate should be included as a factor . The difference can be seen in the BMA output (See Appendix 1 of Caprio and Glaser 2010c). The best fit model, th e one returned by the stepwise regression, did not include base larval movement, but the second best fit model did

include that variable. In both of the BMA runs only the two t op models were used as other models had significantly higher abso lute Bayesian information contents (i.e., they did not fit the data nearly as well).

Table 44. Quantiles for the distributi on of time (generations) until resistance evolve d for 1000 simulations

each of the seed mixture a nd structured refuge models Quantiles IRM strategy 0 5% 10% 25% 50%

seed mixture 23 33 39 51 70

structured refuge 12 22 26 34 45

Figure20. Direction comparison of the densities of th e distribution of the time until resistance evolved for 5% seed-mixtures and 5% structured refuges for the critical first 200 generations

- 87 -4. Discussion The results provide support for the use of a seed -mixture refuge strategy in a two locus corn rootworm system. There are several factors incl uded in the model that made the structured refuge less effective than one might have expected. As noted in Caprio and Glaser (2010a), corn rootworm movement and refuge placement, comb ined with source-sink dynamics, can alter the effective refuge size. If growers always plant refuges in the same place year after year, it is likely that that refuge will have more than 5% of the total egg lay within the 5% of the total area it contains. Alternatively, if the refuge is move d every year, it is possible that very few eggs would be laid under the new refuge site (as disc ussed in Caprio and Gl aser 2010). In addition, the proportion of the growers were assumed to be non-compliant year after year was increased to

a maximum of 60% to account for local areas where non-compliance might be higher than average. Some non-random mating (independent of effective refuge size) was also included in structured refuge simulations. Finally, asymmetrical movement of CRW larvae off of transgenic plants was included in the seed-mixture model. This can, at least in some cases, lead to an increased density of larvae on th e non-Bt plants in a seed-mixture, somewhat increasing the efficacy of that strategy. The latter two s uggestions, however, are not supported by EPA/ORD’s sensitivity analysis. It should be noted that several of these fact ors are dependent on specifics of CRW biology (low vagility, asymmetrical move ment), and caution should be used when extrapolating these results to other species.

It is relevant to note that thes e data present more risk for dual gene products than might have been expected from the response in Caprio and Glas er (2010a). This is due primarily to a change in our perception of the initial resistance allele frequency for the dual gene product for western corn rootworm that was changed to a mini mum of 0.001, a most likely value of 0.005 and a

maximum value of 0.02.

B. EPA/ORD’s Analysis of a 5% seed blend with a triple gene pyramid targeting European Corn Borer and Southwestern Corn Borer

1. Model Description

The same deterministic risk assessment model was used for these simulations as was used for the western corn rootworm simulations (though all three loci were used). An excerpt from Caprio and Glaser (2010a) is also included at the end of this report that more fully presents details of the

deterministic model.

All simulations were run for at least 1000 genera tions. Assuming that each gene should ideally last at least 15 years, the target time horizon for a three gene product was 45 years. Conservatively assuming that ther e are 3 generations per year for both species, this gives a target time goal of 135 generations. For both species, at least 1000 simulations were run for both the structured refuge and seed-blend options.

- 88 -2. Model Assumptions Parameters were adjusted for the unique biology of these insects and th e impacts of the three toxins on each species (see Tables 45 and 46 ). Some specific adjustments included an increase in the effective refuge size because these insect s are more mobile and a more even deposition of eggs across the field would be expected (see Capr io and Glaser 2010). Both the base larval and asymmetrical movement rates were increased. The proportion of consistent non-compliance was

maintained at the same level as the corn rootwo rm simulations, reflecting a concern over areas of concentrated non-compliance, but the amount of mixing between these fields and other fields was increased to reflect the greater overwinter ing dispersal of the two corn borer species.

Table 45. PERT parameters for ECB variables in the structured and seed blend simulations
Variable Min Value Mode Max Value Comment Cry1A.105 fitness 0.00001 0.001 0.005 Cry2Ab2 fitness 0.00001 0.001 0.01 Cry1F fitness 0.001 0.005 0.01

Overall survival 0.00001 0.001 0.005 Always as low as lowest of the

three loci

Dominance 0.01 0.05 0.2 Drawn independently for all

loci Initial R-allele freq. 0.001 0.005 0.02 For each of the 3 loci Variables specific to structured refuges Refuge size 0.025 0.04 0.06 Always set to 0.05 for seed-mix Non-compliance 0 0.3 0.6 Always set to 0 for seed-mix Non-compliance mixing 0.8 0.9 1

Non-random mating 0.2 0.5 0.7 Always set to 0 for seed-mix Variables specific to seed mixtures

Base larval movement 0.3 0.5 0.7 Always set to 0 for structured refuges Days of larval

selection 5 10 15

Asymmetrical larval

movement 0.8 0.9 0.95 Always set to 0 for structured refuges

- 89 -Table 46. PERT parameters for SWCB variables in the structured and seed blend simulations

Variable Min Value Mode Max Value Comment Cry1A.105 fitness 0.001 0.05 0.08 Cry2Ab2 fitness 0.001 0.05 0.1 Cry1F fitness 0.001 0.005 0.01

Overall survival 0.00001 0.005 0.008 Always as low as lowest of the

three loci

Dominance 0.01 0.05 0.2 Drawn independently for all

loci Initial R-allele freq. 0.0005 0.001 0.002 For each of the 3 loci Variables specific to structured refuges Refuge size 0.025 0.04 0.06 Always set to 0.05 for seed-mix Non-compliance 0 0.3 0.6 Always set to 0 for seed-mix Non-compliance mixing 0.8 0.9 1

Non-random mating 0.05 0.3 0.5 Always set to 0 for seed-mix Variables specific to seed mixtures

Base larval movement 0.3 0.5 0.7 Always set to 0 for structured refuges Days of larval

selection 5 10 15

Asymmetrical larval

movement 0.8 0.9 0.95 Always set to 0 for structured refuges

The greatest difficulty in simulating the time to resistance based on the submitted data is partitioning the non-multiplicative inte ractions between the loci. If the mortality caused by each toxin were independent of the mortality caused by the other toxins, one would expect the overall mortality to be the product of the survivorships to each toxin. In other words, if two toxins each kill 95% of the population, then one would expect th e second toxin to kill 95% of the 5% of the population that survived the first toxin and the joint survivorship should be 0.25%. In these

simulations EPA/ORD could estimate this non -multiplicative interact ion by comparing the overall survivorship drawn from our PERT di stribution with product of the individual toxin mortalities based on the draws from their resp ective PERT distributi ons. In a two locus simulation, there is only one interact ion, and all of the interaction can be attributed to that pair of loci. In these three locus simulations, th ere are three interac tions (Cry1Ac<->Cry2Ab2, Cry1Ac<->Cry1F, Cry2Ab2<->Cry1F), and it is impossible to accurately distribute the

interactions without additional da ta on the two locus interactions . In the absence of data the mortality effects of pairs of toxi ns, the attempt was made to distribute the interactions evenly between the three pairs of loci. Note that if an insect is resistant to two loci, then the mortality rates for the remaining locus are the same as if that toxin were used alone. The mortality of

W [SS][RR][RR] is therefore estimated as W [SS] . The overall survivorship is given by W [SS][SS][SS]

. The fitnesses that are difficult to determ ine based on the submitted data are: W [SS][SS][RR] , W [RR][SS][SS ] and W [SS][RR][SS] . EPA/ORD’s approach was to es timate the individual interactions by taking the cube root of the total non-multiplic ative interaction. There were several additional

constraints on the new fitness. The fitness of the double susceptible genot ype had to be less than or equal to either of the compone nt single susceptible genotypes. This ensured that the fitness of a genotype did not decrease as additional resistance alleles were added. Finally, the new fitness of the double susceptible genotype could not be le ss than the overall fitness. Note that the overall survivorship was already constrained to be at least as low as the lowest single toxin

- 90 -fitness. This process was repeated for all thr ee double susceptible genotypes. The remaining fitnesses involving heterozygous genotypes could then be calculated based on dominance values and fitness values already estimated (Fitness ca lculations contained in Caprio and Glaser, 2010c).

As noted in the CRW modeling report, ther e can be multiple ways to estimate double heterozygotes. For example, the fitness W [RS][RS][RR] could be calculated as (W [RR][RS][RR] – W [SS][RS][RR] ) * hl1 + W [SS][RS][RR] , or it could be calculated as (W [RS][RR][RR] – W [RS][SS][RR] ) * hl2 +

W [RS][SS][RR] . Another alternative is to use a mean dominance for double heterozygotes. Other options certainly exist. The options became clearer as the modeling progressed, and ORD was not entirely consistent in their use. Tes ting the two options sugge sted the model was not sensitive to the option chosen, and ORD chose to proceed with the calculations as shown.

3. Results The European corn borer simulations did not resu lt in any cases of resistance prior to the time goal of 135 generations. The seed -blend option was less durable, but the shortest time taken for resistance to evolve in any single simulation was 158 generations . The shortest time for the structured refuge option was l onger than 1000 generations (see A ppendix 1 in Caprio and Glaser, 2010d). The risk of resistance was somewhat great er with Southwestern corn borer than with European corn borer. The s eed-blend option was less durable and 4.5% of the seed blend

simulations resulted in resistan ce evolving in less than 135 genera tions (ca. 45 years). For the structured refuge option, the s hortest time to resistance was 118 generations and 0.2% of the

simulations resulted in the evol ution of resistance in less than 135 generations (see appendix 2 in Caprio and Glaser, 2010d).

4. Discussion The results presented here suggest that there is a low risk of resistance evolving within 45 years to SmartStax corn in either species (with <5% pr obability). These may be considered somewhat conservative estimates as potential fitness costs we re not included in the simulations. Given the long time frames observed for the evolution of resist ance, it is likely that fitness costs could have a significant impact in further de laying resistance. The genetics and interactions between fitness costs to different toxins are, however, unknown and would add consid erably to the uncertainty of the results. Considering the time frames alrea dy observed, the inclusion of fitness costs seemed superfluous. The inclusion of three loci significantly increased the complexity of these simulations. The non-multiplicative interactions between loci were impossible to accurately partition given the mortality/dose information. To the degr ee possible, inclusion of data on two locus interactions in future submissions would help to more accurately ev aluate the data. The solutions to the problems of three locus simulati ons presented here are merely a first attempt and will hopefully provide a basis for further improveme nt in methods and data needs to understand how resistance evolves in these complex systems.

- 91 -VII. BPPD PRELIMINARY RISK ASSESSMENT CONCLUSIONS

A. BPPD’s Overall Conclusions

Dow AgroSciences and Monsanto Company submitted new efficacy, emergence, and larval movement data and conducted simulation modeling for European corn borer, Southwestern corn borer, and corn rootworm to assess the risk of resistance evolution from the use of a 5% SmartStax seed mixture (95% Bt and 5% non- Bt seed). Dow AgroSciences and Monsanto concluded that a 5% seed blend would be an eq ually durable IRM strate gy for both lepidopteran and corn rootworm pest systems as the current refuge requirement of a 5% block refuge. Dow’s model predicted similar increases in CRW resist ance allele frequency for both refuge strategies; Monsanto’s model proposed that lepidoptera would not evolve re sistance to SmartStax within 100 generations (33 yrs or 50 yrs) wh en deployed in a 5% seed blend.

EPA/ORD’s simulation analysis using a two-locu s model predicts that a 5% SmartStax seed

blend is more durable for corn rootworm than a 5% structured refuges. The mean time to

resistance for seed blends and structured re fuge is approximately 82 years and 53 years, respectively. There is a low risk (5% probability) that resistance could evolve in 33 years with seed blends and 22 years with structured refuge s. A separate ORD analys is using a three-locus

model estimates that a 5% SmartStax seed mixtur e is approximately 15 times less durable than a 5% structured refuge for the European corn bor er and 1.6 times less durable for the southwestern

corn borer. The ORD simulations estimated that resistance would not ev olve in the European corn borer within 52 years with a seed mixture and 375 years with a structure refuge and in the Southwestern corn borer within 24 years (seed mixture) and 39 years (structured refuge).

After careful review of Dow’s and Monsant o’s data and modeling submission, EPA/ORD’s modeling analyses for a 5% SmartStax seed mixt ure, BPPD concludes that under assumptions of normal and/or high pest densities:

1) A 5% seed blend strategy at best exceeds the du rability of and at worst is equally durable as a 5% structured refuge for Smar tStax targeting corn rootworm.

2) A 5% SmartStax seed blend is less durable th an a 5% structured refuge when targeting ECB

and SWCB but an overa ll durable IRM strategy.

B. BPPD Conclusions Regardin g Monsanto’s Modeling

Monsanto’s three-locus deterministic modeling si mulations predicted that resistance would not evolve in ECB and SWCB within 100 generations when SmartStax was deployed as a 5% seed mix and 5% structured refuge; resistance allele frequencies did not incr ease during the time the model ran. Seed mixtures were predicted to be more durable when worst-case assumptions were made for structured refuges (40% non-comp liance and 0% mixing between non-compliant and compliant fields). Monsanto concluded that a 5% SmartStax seed mixture was a durable IRM strategy for Lepidoptera. BPPD notes that Mons anto’s simulations conservatively predicted a durability of approximately 33-50 years, dependi ng on whether the Lepidopteran target pest has

- 92 -two or three generations per ye ar. EPA/ORD’s independent analys is predicted that there was a small probability that resistance evolved in E CB and SWCB for this timeframe, which supports

Monsanto’s conclusion that a 5% seed mixture is a durable IRM st rategy for these target pests.

BPPD identified some uncertainties with some of Monsanto’s modeling assumptions that could potentially affect the rate of resistance evolution (listed below). However, Monsanto made

different conservative choices for some modeling parameters and assumptions that likely increased the time to resistance ( i.e. , initial resistance allele fr equency, 1% and 2.5% effective refuge, heterozygote genotype fitness, 0% mixi ng between compliant and non-compliant fields).

• Mixing between compliant and non-compliant fields Estimated durabilities of SmartStax in compliant fields with structured refuges showed no difference to varying levels of gene flow (0 %, 50%, and 100% mixing). This parameter was sensitive and affected outcomes in non-compliant fields. BPPD questions whether the levels of sensitivity predicted by Monsanto’s model are re alistic. Furthermore, it was unclear whether 0% mixing meant an overall restriction for dispersa l out of any compartment or whether dispersal was restricted between compartments with different compliance histories.

• Non-random oviposition by adults On one occasion Monsanto stated that they modeled oviposition in a non-random fashion, although there is evidence in the lite rature to the contrary (Sorenson et al. 1993). It is unclear whether oviposition is a sensitive parameter in Monsanto’s model and whether uniform or

random would affect the time to resistance evolution.

• Uncertainty regarding SWCB Much is presently unknown about SWCB with re spect to larval movement but also adult movement, although there are limited studies in the pub lic literature for the latter. It is unclear what percentage of females, if any, could potentially engage in long-distance premating dispersal, what the spatial scale of random mati ng is. These types of behavior could affect the durability estimates for SmartStax. There is some evidence, however, that SWCB have greater (natal) field fidelity than for E CB. If SWCB primarily mate in their natal field, then seed mixtures could potentially be a preferred IRM strategy.

• Standard error of mixing for the seed mixture technology Monsanto did not incorporate the expected stan dard error of mixing for a SmartStax 5% seed blend technology. However, one could argue that since Monsanto modeled a conservatively low effective refuge of 1% that the standard erro r of mixing might already be included in this number. Nonetheless, Monsanto should provide this information to BPPD.

C. BPPD Conclusions Regarding Dow’s Modeling

Dow’s modeling results showed that a 5% SmartSta x seed mixture, even with more conservative dose assumptions, was at least as durable as a 5% structured refuge strategy. BPPD’s dose

- 93 -assumptions increased the rate of adaptation for both seed blends and structured refuges. Dow did not provide “years to product failure” and instead focused on the ra te of adaptation for comparison purposes. Dow’s modeling results were supported by EPA/ORD’s independent modeling analysis. The two modeling approaches (Dow’s and EPA’s) support the conclusion that a 5% SmartStax seed mixture approach is a durable IRM strategy fo r corn rootworm. BPPD concludes that Dow’s simulations may represent a conservative approach to assess the durability of SmartStax targeting CRW.

BPPD identified some uncertainties with some of Dow’s modeling assumptions that could potentially affect the rate of resistance evolution (listed below). Dow also made conservative choices for other modeling parameters and model stru cture that likely contributed to an increase in the rate of adaptation obs erved in their simulations ( i.e. , landscape of continuous SmartStax; complete resistance; no fitness co st of resistance; and female f ecundity of 100 eggs per female).

• Boundary conditions for adult dispersal Dow did not provide details about how boundary conditions were treated in the model. It was unclear what the fate was of adults that disperse d off the field at the edge of the landscape. As stated in Caprio and Glaser (2010), the choi ce may be important because it can impact the distance between Bt and non-Bt plants and the ultimate effective refuge size.

• Initial resistance allele frequen cy and lack of sensitivity analysis The initial resistance allele frequency for both CRW genes was set at 0.005. Recent studies (Miehls et al. 2008 and Lefko et al. 2008) suggest that resistance allele frequencies could be as high as 0.02 in WCRW. Initial resistance allele frequency is a sensitive parameter in most resistance management models.

• Lack of female pre-mating dispersal Dow did not model female pre-mating dispersal. The literature provides s upport for the inclusion of such a parameter in CRW models. Pre-mating dispersal for female we stern corn rootworm was a sensitive parameter in EPA’s modeling (Caprio and Glaser 2010c) and significantly extended the durability of Bt corn in block refuges compared to seed blends.

• Benchmark assumptions for dose of toxi ns and density-dependent adjustment

BPPD evaluated the dose for corn rootworm in its SmartStax review (BPPD 2009b) and recommended that Dow use lower percent mo rtality assumptions (94.2% for Cry34/35 single trait, 97.5% for Cry3Bb1 single trait, and 98.2% for the pyram ided product) unadjusted for density-dependence when parameterizing their simulation model. This recommendation is supported by the findings in Hibbard et al. (2010).

• Lack of density-dependent di spersal off refuge plants BPPD is unclear why density-dep endence should not occur on refuge plants in a 5% SS mixture and notes that under sufficiently high CRW pressure the number of larvae on refuge plants could build up to a point where density dependent movement occurred (supported by additional damage to SmartStax plants in se ed mixtures, Appendix 9, MRID 479437-01).

• Standard error of mixing for the seed blend technology

- 94 -Dow did not address whether the st andard error of mixing seed ble nds was incorporated into the modeling scenarios. It is unclear whether such an error would ha ve a significant effect on the durability of the pyramided corn rootworm product.

D. BPPD’s Conclusions Regarding Efficacy and Larval Movement for Lepidoptera and Coleoptera target pests

• Efficacy of 5% SmartStax seed mixture against Lepidoptera; larval survival and

movement Based on the efficacy studies submitted, BPPD conc ludes that a 5% SmartStax seed mixture is efficacious against main lepidoptera target pest s, ECB, SWCB, and CEW. Lepidopteran larval movement from non-Bt plants onto surrounding Bt pl ants occurred as was visible by the incurred stalk feeding damage; survival to adulthood for ECB and SWCB was generally not observed. The submitted data also suggest that Sm artStax is somewhat less toxic to 3 rd , 4 th , and 5 th instars of SWCB than ECB (Appendix 1, MRID 479437- 01). Additionally, under high infestation levels, some corn earworm (CEW) larvae were fo und in the ears of SmartStax corn in 100% Bt plots and in seed mixture plot s suggesting that some survival should be expected (Appendix 2, MRID 479437-01).

Less is known about SWCB larval and adult disp ersal than for ECB; typically the assumption has been made that since the two species are closely related that their behavior is also similar. It will be beneficial for resistance management purposes to understand the propensity for SWCB (as well as ECB) to disperse at different life-st ages and to high-light were ECB and SWCB differ in this respect.

There is further support that refuge plants in seed mixtures may receive additional protection from the surrounding SmartStax plants, but this coul d lead to a reduction in effective refuge and lowers overall trait durability.

• Efficacy of 5% SmartStax seed mixture against CRW; larval survival and movement Based on all the efficacy studies submitted for CRW, BPPD concludes that SmartStax has the potential to be very efficacious against CRW esp ecially if natural infestation levels are low and no density-dependent dispersal occurs off non-Bt plants. However, should density-dependent

movement occur for 3 rd instar larvae of refuge plants in a SmartStax seed mixture, Monsanto’s emergence results from a greenhouse experime nt (Appendix 5, MRID 479437-01) suggest that those individuals may not be affected by the two expressed toxins. Overall larval mortality (for

1 st

and 2 nd instars) in this experiment was 95.7%. To summarize, BPPD does not concur with Dow’s and Monsanto’s close to high-dos e claim for SmartStax against CRW.

A reduction in male emergence was apparent for WCRW and NCRW in 5% SmartStax seed mixtures (up to 60 times). BPPD is concerned with potential effects this might have on product durability. The SAP (2009) concluded that delays in emergence between the two sexes observed in 5% seed mixtures with Cry34/35Ab1 corn were less of a concern for re fuge effectiveness yet

- 95 -cautioned that more importantly a reduction in number of males could negatively affect how effective a refuge would be.

E. BPPD’s Conclusions Regarding EP A/ORD’s Deterministic Analyses

BPPD has collaborated with EPA/ORD on independe nt modeling efforts to assess the 5% seed mixture strategy for SmartStax corn. As part of this effort, BPPD modeled the seed mixture strategy for lepidoptera and corn rootworm using ORD’s deterministic three locus and two locus models (deterministic and probabi listic) with parameter estimates that BPPD believes are most realistic and/or conservative (in absence of data or due to uncertainties). A general qualitative (non-quantitative) comparison can be made between the results of different models, yet caution is warranted because assumptions, parameter values, and model structures differ. Tables 47-49 show that despite the different assumptions made by different modelers , the applicants and EPA/ORD predicted similar outcomes for a 5% Sm artStax seed mixture and structured refuge targeting lepidoptera and corn rootworm.

a. Modeling of CRW

Considering average movement data and corrections as reported in Caprio and Glaser (2010 and 2010b), EPA/ORD’s deterministic model predicted th at the mean time to resistance for the 5% block refuge strategy was 52.9 year s (5% probability that resistance evolves within 22 yrs) and for the 5% seed mixture simulations was 81.9 y ears (with a 5% probabi lity that resistance evolved within 81.9 yrs) (see Table 47 ). ORD predicted that the 5% seed blend strategy was more durable than the block refuge strategy and the difference was statis tically significant (p << 0.01). EPA/ORD conservatively assumed that the re sistance allele freque ncy had a most likely value of 0.005 (min. value =0.001 and max. value = 0.02); hence the predicted average time to resistance was likely an underestimate of durability for both IRM strategies.

Table 47. Durability estimates by EPA/ORD and Dow fo r 5% SmartStax seed mixture and 5% structured refuge targeting corn rootworm

1 For benchmark and worst-case assumptions Dow Model CRW EPA/ORD CRW model Refuge Scenario Increase in Resistance Allele Frequency 1 5% Probability for

Resistance Event Average Years

5% Seed Blend 33 years 81.9 years 5% Structured Refuge Similar rates of increase 2 for the 20 years modeled 22 years 52.9 years

2 The Dow model predicted similar durability for SmartStax under both refuge scenario s; no actual values were provided

however.

• Initial resistance allele frequency is unknown for obvious reasons and had to be conservatively chosen based on a gene’s da te since first commercialization, the dose profile as supported by baseline susceptibility and/ or the resistance monitoring data, as

well as on results from lab selection studies. This parameter is sensitive to change and will likely affect the durability of the product. However, BP PD believes that the initial frequencies chosen for the dual gene were conservative.

- 96 -b. Modeling of ECB and SWCB

Using the same model structur e as for corn rootworm ( i.e., assumptions regarding compliance, refuge placement), yet incorpor ating corn borer’s propensity for greater dispersal, ORD’s deterministic, probabilistic model predicted that:

• Resistance in ECB would not evolve within 158 ge nerations (52 yrs or 79 yrs) in a 5% seed mixture and 1125 generations (375 yrs or 562 yrs) in a 5% structured refuge. There was a 5% probability that resistance could evolve within 451 generations (150 yrs or 225 yrs) in a 5% seed mixture and 3055 generations (1018 yrs or 1527 yrs) in a 5% structured refuge (see

Table 48 ).

• Resistance in SWCB would not evolve within 73 generations (24 yrs or 36 yrs) in a 5% seed mixture and 118 generations (39 yrs or 58 yrs) in a 5% structured re fuge. There is a 5% probability that resistance could evolve within 138 generations (46 yrs or 69 yrs) with a 5% seed mixtures and 207 generations (69 yrs or 103 yrs) with a 5% st ructured refuge (see Table

49 ).

Table 48. Durability estimates by EPA/ORD and Mo nsanto for 5% SmartStax seed mixture and 5% structured refuge target ing European corn borer

1 Simulations were run with assumptions of three and two generations per year Monsanto ECB Model EPA/ORD ECB model Refuge Scenario Time to Resistance Level of

Risk Time of Resistance 1

0% 158 gen. (52 yrs or 79 yrs) 5% Seed Blend > 100 gen (>33 or >50 yrs) 5% 451 gen (150 yrs or 225 yrs)

0% 1125 gen (375 yrs or 562 yrs) 5% Structured Refuge >100 gen (>33 or >50 yrs) 5% 3055 gen (1018 yrs or 1527 yrs)

- 97 -Table 49. Durability estimates by EPA/ORD and Mo nsanto for 5% SmartStax seed mixture and 5% structured refuge targetin g Southwestern corn borer

1 Simulations were run with assumptions of three and two generations per year Monsanto SWCB Model EPA/ORD SWCB model Refuge Scenario Time to Resistance level of

Risk Time of Resistance 1

0% 73 gen (24 yrs or 36 yrs) 5% Seed Blend > 100 generations (>33 or >50 yrs) 5% 138 gen (46 yrs or 69 yrs)

0% 118 gen (39 yrs or 58 yrs) 5% Structured Refuge >100 generations (>33 or >50 yrs) 5% 207 gen (69 yrs or 103 yrs)

• BPPD recommended that ORD use a most likely value of 0.05 for the initial resistance allele frequency of Cry1A.105 and Cry2Ab fo r SWCB, which may be a conservative

overestimate. This value was chosen because SmartStax is less toxic to SWCB than ECB

and because of uncertainties with dose. Hen ce, the ORD model predicted a shorter time to resistance for SWCB, which may underest imate the durability of SmartStax for both IRM strategies.

• There is some uncertainty as to the increase in the effective refuge chosen by EPA/ORD. ECB and SWCB are more mobile and are likely to deposit their eggs in a more uniform fashion than CRW; however, wh at the extent is of their mo vement (pre-mating as well as post-mating) is an uncertainty. BPPD recommended that EPA/ORD increase adult dispersal for lepidoptera compared to corn rootworm in the simulation model, which should create greater mixing between compliant and non-compliant fields and reduce the rate of adaptation. In EPA/ORD’s analysis, effective refuge significantly affected the durability of SmartStax. It will, therefore, be helpful to have more precise estimates on long-distance dispersal and propor tion of population that engages in this behavior so that simulation models can be more accurately pa rameterized and provide better estimates for product durability.

• The proportion of consistent non-compliance wa s conservatively maintained at the same

level as the corn rootworm simulations alt hough data support that co mpliance with refuge plantings for Bt products targ eting lepidoptera is higher th an for CRW. Non-compliance was determined to significantly affect estimates of product durability.

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Other:

EPA Scientific Advisory Panel. 1998. Transmittal of the Final Report of the FIFRA Scientific Advisory Panel Subpanel on Bacillus thuringiensis (Bt) plant-pesticides and resistance management, Meeting held on February 9 and 10, 1998

EPA Scientific Advisory Panel. 2000. Transmittal of meeting minutes of the FIFRA Scientific Advisory Panel meeting held October 18-20, 200 0 on Bt plant-pesticid es risk and benefit assessments: insect resistance management. Re port from P. Lewis (DFO) dated March 12, 2001.

EPA Scientific Advisory Panel. 2009. Transmittal of meeting minutes of the FIFRA Scientific Advisory Panel meeting held February 23-24, 2009 on the evaluation of the resistance risks from using a seed mix refuge with Pioneer’s Optim um AcreMax1 corn rootworm-protected corn. Memorandum from J.E. Bailey (DFO) to Dr. D. Edwards (Director of OPP) on May 6, 2009.

BPPD, 2007. Technical review of Monsanto Comp any’s submission regarding insect resistance management for MON 89034 corn. Memo from S. Matten and A. Reynolds to S. Cerelli on November 6, 2007.

BPPD, 2009a. Review of the Insect Resistan ce Management (IRM) plan for SmartStax (MON 89034 x TC1507 x MON 88017 x DAS 59122-7) corn. Memo from A. Reynolds to M. Mendelson on March 16, 2009.

BPPD, 2009b. Review of additional insect resi stance management information for SmartStax (MON 89034 x TC1507 x MON 88017 x DAS 59122-7) Bt corn. Memo from A. Reynolds to M. Mendelsohn on July 1, 2009.