CONCLUSIONS AND UNCERTAINTIES 

1. PRELIMINARY RISK ASSESSMENT CONCLUSIONS

BPPD has reviewed Monsanto’s and Dow AgroSciences’ submissions for a 5% SmartStax seed 

mixture including biological and efficacy data as well as modeling of the rate of resistance 

evolution. In addition, EPA’s Office of Research and Development (ORD) conducted a 

independent analysis of the applicants’ proposal using a deterministic and probabilistic model. 

After careful review of the applicants’ modeling and the analyses conducted by ORD, BPPD 

reached the following conclusions: 

• For corn rootworm (CRW), a 5% seed mixture and 5% structured refuge have 

comparable durabilities predicted by both the applicants’ and EPA’s models; 

• For European corn borer (ECB), worst-case results estimated that a 5% seed mixture was 

less durable than a 5% structured refuge but did not develop resistance within 52 years 

(conservatively assuming three generations per year for ECB). BPPD notes that a 5% 

seed blend is still an overall durable Insect Resistance Management (IRM) strategy for 

ECB (considering the results from Monsanto/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 durable than a 5% structured 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 Management (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 modeling 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 refuge strategies, while 

Monsanto’s model suggested that lepidoptera would 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 conducted 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 for durability (time to resistance) based on the 

1000 simulations that were conducted. To analyze 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 risk 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 within the predicted time 

frame. 

In the CRW model simulations (see Table i), the mean time to resistance was 82 years for a 5% 

seed blend and 53 years for a 5% structured refuge, a 1.5-fold difference. 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 durability of SmartStax seed 

for CRW was 33 years, while the block 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, although three-loci were used to represent the 

three lepidopteran-active toxins in SmartStax. This modeling estimated that a 5% SmartStax seed 

mixture was approximately 85% less durable for the European corn borer and approximately 

38% less durable for the Southwestern corn borer than a 5% structured refuge. As with CRW, 

however, seed blends were still an overall durable IRM strategy for both lepidopteran species. 

From the probability distribution 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 durability estimate) resistance would not evolve in 

European corn borer within 52 years (seed blend) and 375 years (structured refuge) (see Table 

ii). For Southwestern corn borer, resistance did not develop within 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 blend 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-quantitative) comparison can be made between the results of the 

different models listed in Table i-iii, yet caution is warranted because assumptions, parameter 

values, and model structures differ. Despite the different assumptions made by different 

modelers, the applicants and EPA/ORD predicted similar outcomes for a 5% SmartStax seed 

mixture and structured refuge targeting lepidoptera and corn rootworm. 

Table i. Durability estimates by EPA/ORD and Dow for 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 

Frequency1 5% Level of Risk Mean Time to Resistance 

5% Seed Blend 33 years 81.9 years 

5% Structured Refuge 

Similar rates of increase2

 for the 

20 years modeled 22 years 52.9 years 

2

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

Table 47 in this risk assessment 

- 3 -
Table ii. Durability estimates by EPA/ORD and Monsanto for 5% SmartStax seed mixture and 5% 

structured refuge targeting 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 Resistance1

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

5% Structured Refuge 0% 1125 gen (375 yrs or 562 yrs) >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 Monsanto for 5% SmartStax seed mixture and 5% 

structured refuge targeting 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 Resistance1

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

5% Structured Refuge 0% 118 gen (39 yrs or 58 yrs) >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 conservative assumptions in their simulation 

models. 

a) There may be regions in the Corn Belt where corn earworm could overwinter. Such 

potential areas need to be identified because 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 resistance evolution in such areas. 

b) Under high infestation levels, some corn earworm 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 infestation pressure. 

c) Data show that some fraction of the adult ECB population takes part in long-distance 

dispersal and that this behavior may be a normal 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 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, 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 increase in the effective refuge in the EPA/ORD 

model for lepidopteran pests. While ECB and 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 discussed 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 the 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 ECB and SWCB in the block refuge simulations. 

e) There is uncertainty with respect to ECB’s propensity for late instar larval dispersal off 

non-Bt plants and whether older larvae would still be highly susceptible to surrounding 

SmartStax plants in a seed mixture environment. 

f) Typically the assumption has been made that 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 such 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 most 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 chosen because SmartStax is less toxic to 

SWCB than ECB and because of uncertainties with dose. Accordingly, the EPA/ORD 

model predicted a shorter time to resistance for SWCB, which may underestimate the 

durability of SmartStax for both IRM strategies. There is still uncertainty, however, 

with how to appropriately characterize initial 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. However, Monsanto modeled a worst-case for 

5% SmartStax seed blend scenarios of 1% effective refuge for ECB and SWCB, and it 

could be argued that the standard error of mixing was already 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 3rd 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 the Bt proteins”. BPPD is concerned, however, 

that there is uncertainty with characterizing 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 relationships 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 occurs at higher egg density levels 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 have 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 number of males from Bt seed blends could 

negatively affect the effective refuge. 

l) Dow did not include density-dependent dispersal 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 plants 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 conservative 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 well 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 resistance evolution if assumed to be low. 

- 6 -
I. BACKGROUND 

A) Regulatory Background Information 

Monsanto developed MON 89034 x MON 88017 (trade name YieldGard Triple), which was 

registered in 2008 with MON 89034. Dow previously registered TC1507 x DAS 59122-7 (trade 

name Herculex Xtra) in 2005. Since 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, southwestern corn borer, fall armyworm worm, and corn 

rootworm. 

SmartStax was conditionally registered by the Agency 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% refuge for corn rootworm. BPPD also recommended 

revised modeling incorporating these additional data to assess the risk of resistance evolution in 

corn rootworm. 

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

with a 5% seed blend in the Corn Belt and cotton growing regions where corn earworm is not a 

significant pest. In this review, BPPD summarized and discussed the studies submitted by 

Monsanto and Dow to support their registration request and independently assessed the risk of 

resistance evolution in Lepidopteran and Coleopteran target pests using a model developed by 

US EPA/ORD. 

B) Additional Biological Background 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 local dispersal mechanism and long-distance dispersal ability. The 

proportions and frequency of individuals in a population engaging in dispersal before or after 

mating are not available, but these are important parameters in simulation models that can impact 

modeling results relative to an increase or decrease of resistance evolution. Some models suggest 

that limited dispersal can accelerated the evolution 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 after 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 Worthley (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. The proportion of the population that undertakes 

such long-distance traveling is unclear. 

Mason et al. (1996) established that many ECB take advantage of aggregation sites (usually 

clusters of weeds or grasses) near corn fields for mating. Females appear to mate the second 

night after pupal eclosion. Another study suggested that it may be possible to manipulate 

aggregation sites to increase the likelihood of random mating between susceptible and potentially 

resistant ECB (Hellmich et al. 1998). In a recapture experiment 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 were allowed to disperse between 

24 h -72 h before trapping began; the recapture 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. Significantly 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 (flush bar sampling near release sites and wing-type traps). In 

1986, less than 1% of marked individuals were caught within 3.2 km from the release point. One 

male each was recaptured at 6.4 and 9.6 km (effective ~11.52 km). In 1987, the overall recapture 

proportion still was less than 1% but significantly more males were recaptured at the 800 m trap 

distance than at the 9.6 km trap distance. Two males 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 caught 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 trap site, and one female at 49.1 km from the 

release point. They found in their mark-release-recapture study that adult ECB males and 

females could disperse great distances within 1-3 days after their release (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 distances 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 point 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 habitat conditions were favorable (dense herbaceous borders within 

close proximity of emergence site). Bailey et al. further reported that the 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-mating 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 individuals occurred amongst themselves rather than with immigrants 

from other fields. They found that when marked individuals (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 all 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 proportion of marked males mating with marked 

females ranged from 0% to 67% among mated females. Their study results further support the 

hypothesis that “males mate irrespective of whether 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 aggregation sites near emergence locations. Their 

recapture results indicated that less than 1% of the released insects 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 engage in obligate migratory dispersal of >20 km. 

More than 65% of 1-d old females flew for one hour continuously (n-total = 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 hours 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 and male, respectively, during the 8-hr flight 

period. 

Dorhaut et al. (2010) investigated flight 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 capacity compared to slightly infected and non-infected adults. 

Flight duration and total flight distance were reduced. For example, they found that 77% of 

uninfected or slightly infected 1-d old females flew at least 1⁄2 km compared to only 47% of 

moderately infected females. One-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 unmated females engage in obligate migration. 

Their preliminary analysis of data for 3-d old adults 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 Iowa. No genetic variability was detectable at 

this scale suggesting large dimensions for ECB populations. Showers et al. (2001) and Kim et al.

(2008) both provide empirical evidence from the field supporting that long-distance dispersal in 

ECB is not unusual. 

Larval movement: 

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

- 9 -
conducted in non-transgenic corn showed that the vast majority of larvae did not move more than 

two plants within a row (Ross & Ostlie 1990); dispersal 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 corn, unpublished data (used in modeling work) from Dr. Fred Gould 

(cited in Onstad & Gould 1998), however, indicated that approximately 98% of susceptible ECB 

neonates moved away from plants containing Bt. His 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 the extent of plantto-plant larval movement. It was observed that 4th instar larvae were capable of moving up to six 

corn plants within a row and six corn plants across rows from a release point. Movement within 

a row was much more likely than movement across rows (not surprising, due to the fact that 

plants within are row are more likely to be “touching” 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 design. Larvae moving across Bt and non-Bt corn 

rows may be exposed to sub-lethal doses of protein increasing the likelihood of resistance 

(Mallet & Porter 1992). Given the extent of ECB larval movement between plants, seed mixes 

were previously determined to be an inferior refuge option (Mallet & Porter 1992, SAP 1998, 

SAP 2000, and Onstad & Gould 1998). 

Prasifka et al. (2009) found that 51% and 36% of resistant and susceptible ECB larvae, 

respectively, moved off Cry1Ab expressing corn plants in their lab 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 susceptible individuals in seed blends than previously estimated and 

hypothesized that susceptible larvae may experience greater mortality in seed mixtures. 

Egg density 

Sorenson et al. (1993) reported that the distribution of egg densities of second generation ECB 

was random and that oviposition was relatively uniform 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 dispersal 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 (1973) 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 regarding SWCB: 1) more males than females 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-recapture 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 fields) suggesting that male dispersal can be 

greater in SWCB than suggested by the mark-recapture results. Thirty-eight 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 ecological and environmental factors that affect 

dispersal and oviposition behavior of adult CRW. Published literature indicates that phenology 

of maize, density-dependence, and environmental factors such as soil texture and elevation are 

some factors affecting adult CRW dispersal 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 oviposition and that the time from emergence to 

oviposition was between 12-23 days (Grant & Seevers, 1989). They also stated that female 

dispersal was the primary dispersal 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 (predominantly occurring early in the morning and 

evening) contributed to long range dispersal of WCRW out of corn 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 beetles from the time of their 

emergence. The experiment was conducted in Nebraska during 2002 and 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 emergence, females were also trapped at 605 ft away 

from the central infested area. 

Spencer found evidence of long-range 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 adult CRW of the variant type could 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 (Spencer 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 highest infestation scenarios, larvae moved as far as 3 plants 

down the row and 0.76 meter across. Larval movement 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, adult recovery was lower, hence, suggesting most 

density dependence for WCRW occurred after 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 was 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 non-Bt plants was observed in one year of their 

field study (Hibbard et al. 2005). Hibbard et al. stated that initial larval development on non-Bt 

roots followed by later movement to Bt roots 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 development of resistance in choice tests, while transferring 2nd instars 

from isoline to Cry3Bb1 did eventually lead to resistance in the selected strain, although the level 

of resistance was reduced and its development 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 female CRW are capable of detecting different host 

qualities. 

Density dependence: 

Storer (2003) used an equation developed by Onstad et al. (2001), 7.0 42.21

1 1

d Sd ×+ −≡ , to 

model density-dependent mortality in CRW where Sd 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 and 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-independent 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 (high infestation year). When data 

were pooled across years, density-dependent effects began at approximately 800 viable eggs per 

30.5 cm of row (approximately per 1.6 plants). 

II. PROBLEM FORMULATION 

The Federal Insecticide, Fungicide and Rodenticide Act (FIFRA) requires that registered 

pesticide uses do not pose unreasonable adverse effects to the environment. The purpose of this 

risk assessment is to provide insight into the potential 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 registration for SmartStax corn. Monsanto and Dow 

AgroSciences requested to amend their SmartStax registration by replacing the 5% structured 

refuge with a 5% seed mixture for the Corn 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: European 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., the U.S. Corn Belt and Canada), CEW typically 

are not capable of overwintering in these regions. Rather, CEW are known to overwinter in the 

South, often in cotton fields. Some reverse migration 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 adaptation rates in CEW. Computer simulations by 

the Agricultural Biotechnology Stewardship Technical Committee (ABSTC) showed no 

significant interaction between the 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 indicated that under some circumstances this approach could lead to 

more rapid evolution of resistance in some target pests (e.g., Mallet and Porter, 1992; Onstad and 

Gould, 1998; Davis and Onstad, 2000 and more). 

Scientific Advisory Panels (1998 and 2000) discouraged the Agency from the use of Bt seed 

mixtures to control lepidoptera target pest because substantial 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 strategy for Bt corn with low- or medium-dose 

effects on corn rootworm (Coleoptera: Chrysomelidae) was supported by the insect’s limited 

larval movement. But the SAP specifically noted that this recommendation should not set 

precedence for other Bt crops targeting other pests (SAP 2009). 

Theoretical work by Mallet and Porter (1992) showed that insect resistance was accelerated in 

seed mixtures compared to Bt stands where 10% of the population was not 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 (avoidance of toxic substances), then effective 

dominance was increased, and seed mixtures could delay resistance evolution but that predicted 

outcomes depended also on other circumstances. 

Using Mallet and Porter’s model, Tabashnik (1994) reported that 10% Bt seed mixtures were 

more durable than pure Bt stands and that block 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 increased from 0-50%, Tabashnik further reported that a 

block refuge strategy performed equally well or better than seed mixtures. However, seed 

mixtures in conjunction with refuges were the most durable of all deployment strategies 

evaluated. 

Onstad and Gould (1998) recommended 20% block refuges adjacent to the Bt fields over seed 

blend strategies because of uncertainties surrounding expression of high-dose against the target 

pest. Their modeling results predicted that block refuges would be more durable than seed 

mixtures aimed at controlling lepidopteran pests. 

 

In a 2-yr field study with seed mixture expressing 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 durability 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 less 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 refuge. They reported that neonate dispersal away from Bt plants 

- 14 -
was greater than dispersal observed from non-Bt plants and noted that extensive use of seed 

mixtures could select for populations with improved 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 resistance 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 Tabashnik (1994) were caused by a 

reduction in refuge insects in seed mixtures that had moved from non-Bt plants to Bt plants and 

could be overcome if the percent of non-Bt plants in seed mixtures 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 suggests 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 migratory 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 proportion 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 population that would have to engage in preovipositional long distance dispersal 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 plants 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 seed mixture can be expected to decrease the effective 

refuge. Likewise, if susceptible larvae leave non-Bt plants, arrive on Bt plants, and are 

subsequently killed, then seed mixtures can be expected to reduce the refuge population 

to less than what can be expected to emerge 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 reduction 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 greater distances. Based on the limited studies 

available in the public literature, it appears that mating could primarily take place in 

natal fields but that greater dispersal may also occur after 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 information and can shape the IRM approach 

recommended. 

6. There may be regions in the Corn Belt where corn earworm could overwinter. Such 

potential areas need to be identified because they could contribute to increased 

selection in corn earworm, which is a main target pest of both cotton and corn. 

7. A reduction in male CRW in Bt mixtures was 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 negatively affect refuge effectiveness. This concern also applies 

for the pyramided Bt corn mixture targeting 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 still exist and should be addressed so that 

dispersal can be best incorporated into simulation models. For example, Spencer (2009) 

reported that individuals of the variant WCRW type dispersed more than 200 m/day; 

however, the proportion of individuals in a population undertaking this sort of long 

distance movement is unknown. Likewise, uncertainties associated with proportion of 

populations dispersing during cold front events (wind speeds of > 1.5 m/s) and 

frequencies of such events make it challenging to include this mode of dispersal. 

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

potentially be compromised. 

10. It is unclear how non-compliance will affect the 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 evaluate 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), Gilbert (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 five 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% SmartStax plots, 15 plants were non-systematically 

selected and their roots assessed. 

Study results: 

The Flanagan (IL) site was not analyzed due to low pest pressure that year, and Monsanto did not 

provide a discussion of results in their submission. Results from the Gilbert (IA) site were 

removed from the data set because adverse weather conditions interfered with root collections. 

Hence results reported are for Livingston (OH) only. 

Root damage was highest in control 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 different 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 all three treatments fulfilled their refuge function. 

The SmartStax plants had relatively little damage in the pure stand and seed blend plots. 

However, the damage on SmartStax plants in the 10% plot was significantly greater than in the 

5% seed blend and 100% SS plot. Monsanto concluded that some CRW movement occurred in 

the seed mix plots from non-Bt to SS plants that lead to significant differences in damage among 

treatments. When the damage to SS plants surrounding the non-Bt plant was assessed in the seed 

blends, no effect of spatial location on root damage was detected. Monsanto concluded that 

- 17 -
CRW movement off non-Bt plants was low and did not differentially affect SS plants adjacent to 

non-Bt plants. 

Table 1. Root damage ratings for SmartStax and Non-Bt plants of different 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 did 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 plants. 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 appropriately 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 plants in 10% plot and controls. The relationship 

between damage levels and proportion of refuge seed was again not linear. Refuge plants in the 

10% mix incurred damage more similar to refuge plants in the control plots (100% non-Bt). 

Monsanto’s conclusion that the level of root damage on non-Bt plants in 5% and 10% seed mix 

plots indicated that these plants supported comparable numbers of larvae as the corresponding 

structured refuge is plausible. But the additional root protection received by non-Bt plants in 5% 

seed blends suggests that the effective refuge in a 5% seed mixture could be lower than expected 

in a structured refuge. There is uncertainty whether 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 damage 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 non-Bt plants on i) larval plant-to-plant 

movement, ii) survival, and iii) adult emergence. 

a) Growth chamber/lab experiment: 

Two treatments were tested in this experiment: individual (caged) pots containing either a) one 

SmartStax plant or b) one non-Bt plant. Each treatment received one of three levels of 

inoculation with CRW larvae: plants were either inoculated with i) 40 1st instar, ii) 20 2nd instar, 

or iii) 20 3rd 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 plants 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 1st instar (neonate) WCRW, somewhat less control 

of 2nd instar (yet still significantly different from non-Bt), and no significant control of 3rd 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 1st instar inoculation on SmartStax were smaller and weighed less 

than their non-Bt counterparts (statistically significant). Results at the remaining two sublevels 

were not significantly different between SmartStax and non-Bt plants (see Table 3). 

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

Treatment 

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

adults from 1st instar SmartStax 2.0 0.87 

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

adults from 2nd instar SmartStax 17.2 1.69 

adults from 2nd instar non-Bt 46.3 5.32 0.0284 

adults from 3rd instar SmartStax 30.8 5.07 

adults from 3rd 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 1st instar SmartStax 1.34 0.13 

adults from 1st instar non-Bt 1.69 0.10 0.0022 

adults from 2nd instar SmartStax 1.17 0.06 

adults from 2nd instar non-Bt 1.88 0.42 0.2016 

adults from 3rd instar SmartStax 1.39 0.05 

adults from 3rd 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 larvae but somewhat less to second instars and not 

toxic to third instar larvae. The registrant concluded that 3rd 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 chamber conditions were expected to increase the 

survival to adulthood by decreasing density dependent and independent mortality expected in the 

field. The relative impact of SmartStax in the lab setting on different life-stages should reflect 

qualitatively what would occur under field conditions. 

b) Field experiment: 

Two treatments were tested in this experiment: 1) one artificially infested non-Bt plant 

surrounded by 12 non-infested SS plants, and 2) one artificially infested non-Bt plant surrounded 

by 12 non-infested non-Bt plants. The central plant 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 center plant within same row (17.8 cm, 35.6 cm, 53.4 cm), and across 

and diagonal from inoculated plant in neighboring 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 treatment 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, adult WCRW were grouped based on the 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 but excluding the central infested plant. 

Study results: 

A smaller proportion of beetles was found in plots where the central plants were surrounded by 

non-Bt plants (24.9%) than in plots where central plants were surrounded by SmartStax plants 

(47.6%); this difference was statistically significant (p=0.0472). The difference in proportion of 

beetles obtained from immediately adjacent SmartStax or non-Bt plants was not statistically 

significant (p=0.3455). But a greater proportion of beetles was found on non-Bt plants that were 

two plants (p = 0.0023) or three plants removed (p=0.0059) from the central plant (see Table 4) 

than the proportion of beetles reported on SmartStax plants of the same distance from center. 

There was no statistically significant difference in weights and thorax widths 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 emergence results that a larger percentage of insects successfully 

dispersed off the central, heavily infested plant onto surrounding plants when the surrounding 

plants were non-Bt plants than when they were SmartStax plants. 

Monsanto proposed that the results of “no-difference” for body weight and thorax width for 

emerged beetles between different treatments indicated 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 driven by “condition of the plant and density-dependent effects”. 

Ultimate survival to adult emergence was impacted 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 indication of low larval establishment across the experiment. 

BPPD Review 

In Monsanto’s lab emergence study, the average 1st instar emergence on non-Bt was 30.56% and 

2.08%; hence average emergence for 1st instar larvae on Bt compared to the control was 6.8%. 

Average emergence for 2nd instar larvae on Bt compared to the control was 37.1%. There was no 

difference in emergence among 3rd instar lave from Bt and non-Bt plants. Therefore, overall 

larval mortality in this experiment was 95.7%. BPPD 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 stated this implies that if older CRW larvae 

disperse from non-Bt plants in a SmartStax seed mixture (in response to density-dependence) 

and arrive on a SmartStax plant that they will not be affected by the toxin. Whether they are not 

exposed to selection for resistance, 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 notes that there was no 

statistically significant difference in the cumulative % of emerged beetles from the SmartStax 

and non-Bt plants immediately surrounding the central infested plants. 

SmartStax plants in the second and 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 SmartStax 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 two comparisons was statistically significant. 

Monsanto did not provide an explanation regarding this observation. 

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

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

distance classes of different treatments. 

3. NA09S2J007 - Movement and Survival of Western 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 experiment in 2009 using pairs of SmartStax and 

non-Bt plants to determine movement of WCRW larvae and survival/emergence to adulthood. 

The trial was set up in a field in Mount Vernon (IN) that was planted to soybeans the previous 

year (assuring that no corn rootworm eggs were in the ground); the area has no history of 

rotation-resistant CRW. 

The experiment was set up in a randomized complete block design with four 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 total/experiment), the non-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 described above) was infested with 500 WCRW eggs. 

Following the day of artificial infestation, emergence 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 significant difference in sex ratio between treatments. 

Study results:

No adults emerged from the pure SmartStax treatment 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 inoculated 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 provide 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 when the refuge plant was inoculated with WCRW 

eggs as compared to emergence from the non-Bt/non-Bt plant pairs. When the SmartStax plant 

was infested in the combination plant pair, emergence 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 experiment. 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 between these two treatments was 

statistically significant. In the case of these two treatments, it would 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. Survival (emergence) in the Bt2

/non-Bt treatment was 0.8%, which 

demonstrates that SmartStax is very toxic to neonates. Apparently some larvae where able to 

leave the toxic plant and recover on the non-Bt plant. Survival in the non-Bt2

/Bt treatment was 

6.4% and 0% in the Bt/Bt treatment. The reduction 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-Bt2

/Bt treatments compared to the control. 

However, that reduction was only 29%. 

Table 5. Total and relative percent survival of WCRW in different treatments 

Treatment Non-Bt*/non-Bt 

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

Total emergence 45 4 32 0 

Total survival1 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 factors 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 emergence study in several Illinois sites using 

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

Treatments for WCRW in two 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 replicated (randomized 

complete block design, 4 reps) at two sites (Hoopeston and Flatville, Illinois). Adult NCRW and 

WCRW were collected weekly with an aspirator, and root damage 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 assess differences between treatments. 

 

2

 Plant type receiving inoculation of 500 WCRW eggs 

- 25 -
The treatments for WCRW and NCRW in three and two total sites, respectively, 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, caged) were planted and replicated (randomized 

complete block design with 4 reps) at three sites, Hoopston (IL), Waterman3 (IL), and 

Monmouth3 (IL). Emerged beetles were collected with an aspirator and counted weekly; root 

damage was assessed and statistically analyzed. ANOVA was used for emergence and root 

damage data to assess differences across and within sites. Weights for female and male CRW 

were recorded. The pair-wise test (with a Tukey 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 and 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 Hoopeston, 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 significant 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 slightly in excess of what would have been 

expected based on a weighted average of the SmartStax and non-Bt plot mean emergence 

numbers. Monsanto concluded that the seed mix plots were acting as efficient (or somewhat less 

efficient) in terms of WCRW beetle production compared to pure stands of non-Bt plants. Where 

the seed mix plots were less efficient in producing adults, Monsanto inferred that additional 

mortality occurred in the blends by larvae moving 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 events or SmartStax mixtures. 

 

3

 Artificial infestations of plots at these sites 

- 26 -
Table 6. Adult (male and female) 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 Monmouth1 Waterman1

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 Monmouth1 Waterman1

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 earlier 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 consistent with previous observations in the CRW 

literature and was thoroughly discussed during the SAP (2009). Of the Bt treatments in the 

current study, the greatest delays in beetle emergence were consistently associated with the 

SmartStax plots, as evidenced by relative low 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 Waterman, 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 Flatville locations was consistent with emergence 

of adults. Damage was greatest in non-Bt plots followed by seed mix plots and finally by pure 

- 30 -
stand SmartStax plots. At Hoopeston, the insect pressure was greater during 2007, and the 

differences in root damage rating for non-Bt plots compared to all other Bt plots was significant 

(Table 9). Refuge plants within the seed mix plots had greater damage 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 ratings for refuge and different seed blend 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 locations. 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 for NCRW in 2008 (as for WCRW in 2007-2008) at 

the two sites where significant natural 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 weighted 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 stands 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 tested. Details not provided by Monsanto. 

Table 12. Male NCRW 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 tested. Details not provided by Monsanto. 

ii. Emergence of NCRW over time: 

In general, beetle emergence from the non-Bt plots tended to occur earlier 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 consistent with previous observations of delayed 

CRW emergence from the single CRW-protected 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 associated with the SmartStax plots, as evidenced 

by relative low emergence in the first sampling period 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, Illinois, 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 results of the two-year study demonstrated that non-Bt plants in 

seed mix plots acted as productive sources of beetles. 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 percentage. 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 that WCRW and NCRW emergence 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 efficacious against CRW (no statistical significance 

between emergence numbers from SmartStax and SmartStax seed mixture plots). 

Sex specific emergence data by sampling date provided a greater degree of information when the 

registrant incorporated more than three sampling dates. With more collection times, actual 

bimodal or trimodal distributions for emergence 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 compared to the non-Bt controls (Tables 14-

23, pages 392-397, Appendix 6). The SAP (2009) concluded that delays in emergence between 

the two sexes observed in 5% seed mixtures with DAS-59122-7 corn were less of a concern for 

refuge effectiveness, however, that more importantly a reduction in number of males could 

negatively affect how effective a refuge would be. 

A reduction in beetle production from SmartStax seed mixtures (at worst 50%) compared to what 

would be expected based on emergence from non-Bt refuge fields suggests 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 plants in seed mixtures receive additional protection 

from the surrounding SmartStax plants hence leading to a reduction in effective refuge. 

- 34 -
5. NA09S2J005 - Efficacy of MON 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 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 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 (threshold under high environmental stress). Corn 

rootworm injury levels and consistency 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 artificial 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 plants in seed mixtures; however, not all observed differences 

were statistically significant when compared to the non-Bt control 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 statistically 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 (95%-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 plants, 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 plants, 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 movement 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 onto refuge plants from surrounding Bt plants; 

however, surrounding Bt plants incurred additional feeding damage from movement of 

larvae off heavily infested non-Bt plants. 

• Efficacy benefits of seed mixtures decline 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 becomes 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 lab study findings that older 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 the 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 conclusion 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 plots 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 larvae dispersed but few survived (damage less 

than 0.50) (Figures 9 and 10). The reduced root damage observed in non-Bt plants immediately 

adjacent to SS-plants in this study seems to suggest that the effective refuge in seed blends may 

be lower than in a comparable structured refuge. 

BPPD finds that the consistency 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 (approximately 85%) and 90% Herculex XTRA (Cry34/35 

only- approximately 50%)) showed no statistically significant difference. 

6. NA09S2J008 - Survival of Western Corn Rootworm Larvae in 

Mixed Stands of MON 89034 × TC1507 × MON 88017 × DAS-59122-7 Corn 

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

Dow AgroSciences conducted an experiment with different percentages of SmartStax seed 

blends targeting CRW to assess emergence of adults and larval movement under field conditions. 

- 38 -
Study Methods: 

The trial was conducted at Fowler (IN) and Sidney (IL) in 2009. All treatments 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 plot were artificially infested with WCRW eggs 

(500 eggs per plant). Some level of natural infestation was present, and the artificial 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 using ANOVA with treatment, replicate, and their 

interaction as dependant variables. Post-ANOVA means separation (least-squared estimates) was 

conducted using Tukey’s Honest Significant 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 and 785 adults were collected from emergence 

cages at the Fowler (IN) and Sidney (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 plots 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, somewhat 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% mixtures using both traits (data 

not shown in Table 13). 

- 39 -
Table 13. Observed average adult emergence from pure stands and expected and 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 seed mixture results are not shown. 

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

There was temporal overlap in mean emergence between adults from non-Bt plots and SmartStax 

plots. Significant differences were apparent in mean number of days to first emergence between 

100% non-Bt and 100% SmartStax treatments as well as seed mixture treatments and pure stand 

SmartStax treatments at both locations. There was no statistically significant difference between 

results from SmartStax seed mixtures and non-Bt plots. 

With respect to median emergence the results were 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% SmartStax 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. The numbers of adults 

emerging from different seed mixture configurations were lower than expected (with larval 

movement) and in one case higher than expected. Lower numbers suggested a larval (net) 

movement from non-Bt plants to SmartStax plants, 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 results supported that 

SmartStax seed mixtures could be as effective 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 hypothesizes that some larvae, after an initial taste of Bt roots, 

initially recovered, dispersed (assumption of asymmetrical dispersal 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% SmartStax were significant at Fowler but not at 

Sidney although the difference in median emergence between the two treatments was 6 days at 

both locations. Conversely, a difference of two days in “mean days to median emergence” was 

significant between 95% SmartStax and 100% SmartStax at Sidney, although biologically this is 

unlikely to represent a significant difference; however, the difference of four days between 95% 

SmartStax and 100% non-Bt was not found to be statistically significant. BPPD concludes from 

the “days to median emergence” results that mixing of susceptible and potentially 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 experiment 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 containers 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) consisted of 50 containers; one 24 hour-old neonate larva was 

contained per container. Fresh Bt or non-Bt plant tissue were provided 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 treatment. 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 before being fed to larvae. 

Seven larval populations were set up consisting 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 transferred 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 third and fourth instars, in 

population 5 at the fourth and fifth instar, and in population 6 at the fifth instar when transfers 

occurred from non-Bt to SmartStax tissue. Population 1 (negative control) 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 second instar, population 3 at the third instar, 

population 4 at the fourth instar, Population 5 mostly at the fifth instar, and population 6 were at 

the fifth instar when the larvae were transferred 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 S0 S1 S2 S3 S4 S5 S6

P1 NB NB NB NB NB NB NB 

P2 NB SSX SSX SSX SSX SSX SSX 

P3 NB NB SSX SSX SSX SSX SSX 

P4 NB NB NB SSX SSX SSX SSX 

P5 NB NB NB NB SSX SSX SSX 

P6 NB NB NB NB NB SSX SSX 

P7 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 

SX = Food source exposure of 4 day interval 

Study Results: 

European corn borer:

Highest survival (42.55%) was observed at day 29 of the experiment in population 1 (negative 

control) where larvae exclusively fed on non-Bt leaf tissue. Most surviving insects reached the 

pupation stage, which indicated that the test system 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, respectively. Second to fourth instar larvae did not 

survive exposure to SmartStax and died soon after 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 treatments were able to survive SmartStax exposure until the end of 

experiment (P5 = 6.38% survival and P6 = 9.38% survival). Table 15 below shows the 

cumulative survivorship for different 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 modified from Table 2 in MRID 479437-01, Appendix 1, p. 58 

Southwestern corn borer:

Highest survival (85.71%) was observed at day 26 of the experiment in population 1 where 

larvae exclusively fed on non-Bt leaf tissue (control 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% mortality 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 graphically 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 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 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 modified from Table 3 in MRID 479437-01, Appendix 1, p. 60 

Registrant conclusions: 

ECB larvae from first to fifth instar 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 multiple sites over two 

years, and laboratory studies showing high susceptibility 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 first to fourth instar were highly susceptible to 

SmartStax corn; all larvae from first to third instar, 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 multiple sites over two years. This means that 

SWCB larvae moving off non-Bt plants onto SmartStax 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 especially at the first four instar levels. BPPD 

noticed that control mortality in the negative control was rather high (approximately 58%), and, 

therefore, recommends that Monsanto correct observed mortalities (using Abbott’s correction) 

based on the high control mortality. 

It was unclear whether those larvae 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 (5th

- 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 percent probability of survival (pupation/adult 

emergence). Considering, however, that Monsanto 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 4th instars survived SmartStax exposure in actuality. An alternative 

explanation for no adult survival on SmartStax 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 4th 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 for different behavior (i.e., dispersal during later life-stage as discussed by 

Davis and Onstad 2000) in the target pest. More information on late instar movement for ECB is 

needed to determine whether the present uncertainties are reasons for concern. 

 The control mortality in Southwestern corn borer populations was much more reasonable 

(approx. 15%), and so BPPD has more confidence 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 3rd, 4th, and 5th

instars than for ECB; however, the duration of the experiment was three days shorter than the 

one for ECB, which could explain some of increased 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 Winnsboro, 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, respectively. At Loxley, two sets of rows 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 after the plots had been sprayed with one or two applications of 

glufosinate at Winnsboro, Loxley, and Leland; SmartStax plants were tolerant to the herbicide, 

while refuge plants were not. In cases where herbicide damage did not coincide 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-systematically chosen and artificially infested 

where natural pest densities were low. Damage ratings 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 control), 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 Loxley, 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 generation 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 had shot holes and elongated lesions; 

5 meaning that several leaves contained elongated lesions; 

6 meaning that several leaves contained elongated lesions (ca. 1 inch); 

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

8 meaning that long lesions were common 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 combined stalk galleries were recorded. 

Treatments planted at each location were: 

• 100% non-Bt (NK603) 

• 100% SmartStax seed blends (MON 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 differences among SmartStax plants from different 

treatments. Analysis also focused on SmartStax plants 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 from the artificial infestations. CEW were 

able to establish themselves from the artificial infestations, and those data were evaluated. FAW 

was present due to natural infestations, 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 plots. 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 plants 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 SWCB only. Insufficient numbers of CEW 

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

For the first generation of SWCB, the number of galleries per plant was significantly greater on 

non-Bt plants in seed mixtures compared to non-Bt plants in pure stand plots; the range for 

number of galleries on non-Bt plants in control, 5% and 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 statistically significant difference between 

different seed blend percentages. SmartStax plants had very few galleries regardless of insect 

- 48 -
generation and seed mixture or pure stand plots; 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 

1st/2nd generation 

Standard error for 

1st/2nd generation 

Comparison (Pvalue < 0.05) for 

1st/2nd 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 mixture and non-Bt plots differed significantly. The 

difference between the mean number 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 infestation conditions; those mean numbers ranged from 

0.32-0.61 per ear of SS plant. When the level of CEW survival on SmartStax plants in seed mix 

plots was analyzed based on the position 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 galleries 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 2nd generation galleries were higher in non-Bt plants and ranged from 2.61-4.55 per 

stalk. There was a statistically significant difference between the 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 feeding. The range of mean galleries per stalk for 2nd

generations was 0.28-0.60. The damage to SmartStax 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 non-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 galleries in the SmartStax plants revealed very few 

live pupae (see Table 22). 

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

Substance Plant type Mean number for 

1st/2nd generation 

Standard error for 

1st/2nd generation 

Comparison (P-value 

< 0.05) for 1st/2nd

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 from Tables 5 and 6 in MRId479437-01, Appendix 2, p.86 

Table 22. Mean number of second generation 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% and 10% seed mix plots consistently 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 the seed mix plots presumably reflected larval 

movement off non-Bt plants onto surrounding SmartStax 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 SmartStax plants regardless of instar. 

Therefore, a 5% seed mix should be an effective 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 that infestation was high that year (1.9 larvae/non-Bt plant) and, 

hence, lead to some survival on SmartStax plants. For example at Winnsboro, for every 10 Bt 

plants, there were three life larvae in 100% SmartStax 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 artificial infestation had occurred. This is much less 

than what was seen at Winnsboro (LA). Monsanto reported that survival on surrounding 

SmartStax plants in the seed mixtures and SmartStax pure plot was zero percent. BPPD cautions 

that this number needs to be considered relative 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 year, it is still substantial if one considers that SmartStax 

expresses three different Bt toxins against Lepidoptera. As stated in BPPD (2009), however, 

since TC1507 has shown reduced efficacy against CEW with damage and infestation frequently 

not statistically different than the non-Bt controls, SmartStax may 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 from 0% (leaf disk test - yet great control 

mortality was noted) to 9% (kernel test). 

Monsanto did not request a seed blend registration for cotton growing regions where CEW are a 

major pest. BPPD recommends that Monsanto determine where in the Corn Belt corn earwom 

could overwinter because a 5% SmartStax seed blend 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) location. 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 give refuge to a greater number 

of larvae/pupae than non-Bt 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% seed 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 selected (this part of the report was not clear), the 

results suggest that FAW survival on SmartStax is unlikely. These results do not provide 

information about larval movement and subsequent survival as an older instar since the SS plants 

selected for damage evaluation did not appear 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 plants was greatest in the seed blends compared to the control 

suggesting that there may have been increased movement off Bt plants onto refuge plants. These 

preliminary results together imply, based on damage 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 information 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 pests 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 evaluating the efficacy of SmartStax and MON 89034 x 

TC1507 against five target pest in 23 different locations of major US corn growing areas; 

Monsanto relied on a combination of natural and artificial infestation. Experimental plots were 8 

rows wide by 20-100 ft long. Treatments were assigned 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 blend with 5% non-Bt seed 

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

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

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

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

• 100% non-Bt 

- 52 -
Statistical analyses for each trial were performed across and within sites at the 0.05 level of 

significance. Results of statistical analyses for each trial are detailed in the Monsanto’s Appendix 

3. 

European corn borer: Artificial infestations were accomplished by applying neonates using a 

bazooka applicator tool. Foliar damage was assessed using a modified 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 and measuring tunnel length in centimeters or 

inches. 

Corn earworm: Artificial infestations were accomplished 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 the 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 plants infested were recorded as well. 

Southwestern corn borer: Artificial infestations were accomplished 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 cavities in stalks and ear were recorded. 

Sugarcane borer: Damage was assessed by evaluating vertically dissecting stalks and recording 

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

Fall armyworm: Artificial infestations were accomplished by applying neonates using a bazooka 

applicator tool. Foliar damage was assessed using the 0-9 Davis 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 locations 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 differences in damage were detected between 

SS pure stand and SS seed mixtures within locations 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 individual 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 tunneling): 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 for non-Bt plants. The difference between non-Bt 

and SS plant damage was statistically significant (Table 24) 

Table 24. Stalk tunneling (inches) for ECB by individual 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 locations, ECB damage was 

consistently lower for SmartStax and the seed mixtures than the non-Bt controls, with a general 

trend across sites of more damage on non-Bt controls, 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 was consistently supporting ECB populations, with 

the level of damage and insect survival 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 damaged kernels, percentage of plants infested, 

and recording the number of larvae per ear, Monsanto found significantly less damage to SS 

plants than non-Bt control plants. However, the difference in kernel 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 significantly less damage compared to the non-Bt 

control. Similarly, all damage evaluations for the lepidopteran 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 across sites of slightly more foliar 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 sites, 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 difference was found between SmartStax and MON 

89034 x TC1507 treatments (there is no difference between the lepidoptera targeting Bt proteins 

of the two products). However, damage ratings for 90% MON 89034 x TC1507 treatments were 

significantly higher than for 100% SmartStax 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 for 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 the 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 per 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 cavities 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 from 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 from MRID 479437-01, Appendix 3, p. 196 

Monsanto stated that under high natural pressure, SmartStax and MON 89034 × TC1507 

materials provided excellent control of SCB. The number of cavities and larvae in Bt treatments 

were significantly lower than in non-Bt controls, and the damage and number of larvae were 

- 55 -
similar in Bt treatments. However, there was a general 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 three 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 mean foliar damage results across all sites 

and range of values for each treatment. 

Table 28. Mean and range of foliar damage 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 from 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 Union 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 SmartStax and MON89034 × TC1507, alone or in 95% and 90% 

seed mixtures, against major lepidopteran pests of corn indicated that the Bt treatment tested had 

significantly less foliar, stalk, and ear damage from these major 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 low pest pressure, analysis of individual refuge 

components (5% non-Bt, 95% SmartStax, 10% non-Bt and 90% SmartStax) found few 

statistically significant differences in damage among 100%, 95% and 90% lepidopteran-resistant 

material, or between 5% and 10% non-Bt refuge (when the analysis was conducted). 

BPPD Review 

BPPD concurs with Monsanto that 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 demonstrate that the lepidopteran active toxins 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 control. BPPD understands that this was an 

efficacy experiment and not a study designed to look at movement of larvae; hence the larval 

numbers were not reported for individual components (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 provided 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 larvae found per treatments. While 

Monsanto provided the mean number of SWCB larvae 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 individual 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 breakdown of the mean number of larvae/ear found on individual 

SmartStax components or at a minimum, by reporting the proportion of non-Bt plants contained 

in seed mixture samples. 

BPPD concludes that SmartStax and its lepidopteran active components were efficacious in 

controlling the sugarcane borer. BPPD’s comments made above with respect to additional data 

reporting apply for this data set as well. More information about sample composition (ratio of Bt 

plants to non-Bt plants) and numbers of larvae found on SmartStax and refuge plants would have 

been illuminating. 

BPPD concurs with Monsanto that across-site foliar 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 assess the efficacy of SmartStax alone and seed 

mixtures containing SmartStax and non-Bt seed against several lepidopteran target pests in 

different locations of the US Corn Belt. The target pests of concern were European corn borer 

and corn earworm. 

Study Methods: 

Monsanto established field trials evaluating the efficacy of SmartStax and MON 89034 x 

TC1507 against three target pest in different locations of major US corn growing areas. Trial 

sites were located in Blue Earth (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 randomized complete block design with no replicates. Damage ratings 

were taken from eight internal rows within plots. Statistical analyses were performed across all 

sites at the 0.05 level of significance. 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 SmartStax ears. The plants examined in 100% 

SmartStax plots did not show any evidence of CEW larval presence. No statistically significant 

differences were reported between 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 that evaluations of SmartStax and its seed mixtures demonstrated 

that the SS corn had significantly less plant and ear damage than non-Bt controls across 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 SmartStax 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 that Monsanto did not have great infestation 

pressure for both Lepidoptera pests. But the BPPD IRM team noted that the results in the CEW 

portion of the experiment were statistically 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 MON 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 Frankfort (IN), Fowler (IN), and 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 plot were artificially infested 

with ECB neonates at the early whorl stage. In pure stand plots, three randomly selected plants 

were selected in row 3 and infested with 50 larvae 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 mixture 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 mixture 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 dependant variables. Post-ANOVA means separation (least-squared 

estimates) was conducted using Tukey’s Honest Significant Difference (HSD) test (P < 0.05). 

Registrant results: 

Infestations were successful at all four locations as measured by mean 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 that larval movement occurred within the row 

(mean damage rating of 1.18). No surviving ECB larvae were found, however. In two of the four 

locations, the infested non-Bt plants in 95% seed mixtures had significantly less damage than the 

non-Bt plants in 100% non-Bt plots. Figures 15 and 16 show two of the four locations with their 

respective leaf damage ratings/different treatments. 

Figure 15. European corn borer injury (leaf injury scale 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 indicator of movement and survival at 

Fowler (IN) in 2009. “Null plant” refers 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 significantly affected by ECB feeding; the only damage observed was 

consistent with initial feeding scars by older larvae. 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 targeting Lepidoptera. Dow further stated that insects 

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

Across the four locations there was a trend (significant at one location) for reduced damage to 

the non-Bt plants in a seed mix compared with a pure stand of non-Bt plants. 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 surrounding Bt plants (hence reducing effective 

refuge as compared to structured refuge) and 2) larval survival is unlikely if larvae manage to 

move across a row or onto a neighboring plant (as implied by very little feeding damage to those 

- 61 -
SmartStax plants). The results of this experiment appear to support that the three lepidopteran 

active Bt proteins in SmartStax 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 Resistance 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, including uncertainties and conclusions. BPPD did 

not discuss the results for single and dual toxin simulations that were provided by the registrant, 

rather, BPPD extracted ECB and SWCB results for the triple toxin simulations only. Monsanto 

provided single gene (20% structured 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 SmartStax (a triple gene pyramided product for lepidopteran) is 

currently registered with a 5% structured refuge, BPPD did not 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. Tables 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 mixture (95% SS and 5% refuge seed) compared to 

the durability of a 5% structured 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-compliant compartments). The model was run as a 

single, dual, and triple toxin model 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 products (Cry1A.105 x Cry2Ab2 x Cry1F) with a 5% seed 

blend refuge. The model was run for either 100 generations or until the resistance allele 

frequency exceeded 0.5. No natural refuge was considered for ECB and SWCB. 

• Resistance 

Resistance was modeled with a single diallelic locus for each of the three 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 recessive/dominant. Heterozygous (RS) 

individuals had two or five-times the survivorship/fitness of susceptible (SS) genotypes. 

Initial r-allele frequency was 0.005 for all loci and both Lepidoptera species, which was a higher 

frequency than indicated by resistance monitoring studies. The number of generations were 

recorded when the frequency of the r-allele 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 than for ECB. Two scenarios were run benchmark 

and worst case. Monsanto stated that benchmark values were conservative representations 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 reduction; field and lab data 

support an adult reduction of up to 3 times). 

• Non-compliance with structured refuge 

Modeling compliance and non-compliance was achieved by creating separate compartments for 

each. Grower non-compliance with the 5% structured refuge was modeled as random (accidental 

non-compliance) and non random (intentional non-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 only 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, resistance did not evolve in 5% seed mixtures and structured refuges 

(compliant) within the 100 generations 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-compliance, resistance evolved in 2 generations for 

structured refuges and resistance 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 increase to 40% without having an effect on time to resistance 

(>100 generations) for block refuges; resistance allele frequencies at that point were 

approximately 0.05 in non-compliant fields. 

Resistance did not evolve within 100 generations 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 larval survival on Bt plants (Table 34). The resistance allele 

frequencies for Cry1A.105, Cry2Ab2, and Cry1F remained 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-compliance, resistance allele frequencies did 

increase in the non-compliant fields (in 3 generations), but compliant fields remained unaffected 

regardless of the degree of mixing between the fields. If the non-compliant fields were 

completely isolated (no mixing with compliant fields), then resistance evolved very rapidly 

(three generations). When mixing between the compliant and non-compliant compartments was 

incorporated, then resistance did not evolve in 100 generations even in the non-compliant fields. 

- 64 -
Table 33. Number of generations to ECB resistance 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 fields 

5% 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 fields 

5% 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 from 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 resistance 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 

%Percent 

refuge 

% 

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 from 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 compliance 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, resistance did not evolve in 5% seed mixtures and structured refuges 

(compliant) within the 100 generations 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 allowed 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 allele frequencies at that point 

were approximately 0.03 in non-compliant fields. 

Resistance did not evolve within 100 generations when deployed as a 5% SmartStax seed 

mixture (effective refuge of 1%) and 5% structured 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 very durable strategy for SWCB. With 20% noncompliance and 0% mixing, resistant allele frequencies increased to greater than 0.5 in three and 

four generations; however, in the non-compliant fields resistance 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 resistance 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 fields 

5% Percent 

refuge 

% 

Noncompl. 

% 

Mixing Cry1A.1051

Cry2Ab21

Cry1F1 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 fields 

5% Percent 

refuge 

% 

Noncompl. 

% 

 Mixing Cry1A.1052

Cry2Ab22

Cry1F2

Cry1A.1052

Cry2Ab22

Cry1F2

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 from 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 resistance 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 

%Percent 

refuge 

% 

Noncompl. 

% Mixing Cry1A.1051

Cry2Ab21

Cry1F1

Cry1A.1051 Cry2Ab21 Cry1F1

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 from 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 compliance 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 refuge of 1% and 2.5% ; 

in addition, conservative assumptions about heterozygote 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 could 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 plant 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 appears 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 targeting ECB and SWCB. Monsanto failed to state in their Table 1 of 

Appendix 8 (Table 31 in this review) which toxin represented 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 mainland U.S. through resistance monitoring programs 

(in Puerto Rico there was fall army resistance 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 maybe 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 there 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 contradicts that claim to some extent. BPPD assumes that this may 

be another conservative assumption built into the model which then could be used to bolster 

Monsanto’s modeling conclusions. 

• Heterozygote survival

As discussed in Mallet & Porter (1992), effective dominance is inversely 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 resistance evolution in Monsanto’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 realistic. Furthermore, it was unclear whether the 

assumption of 0% mixing meant “no dispersal out of any compartment” or whether, adult 

dispersal was allowed to occur separately between compliant compartments and separately 

between non-compliant compartments while otherwise 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 clear how the dispersers were selected. BPPD 

assumes that some probability of dispersal (0, 0.5, and 1) was assigned for each adult, but this 

was not discussed by the registrant. In addition, 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 long-distance dispersal. Recapture studies 

studying local dispersal have generally recaptured a small percentage of the marked ECB adults, 

which could imply that dispersal is far 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, resistance 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 what 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 registration application. Although 

Monsanto did not incorporate the standard error of mixing into the modeling simulations, one 

could argue that since Monsanto modeled a conservatively low effective refuge 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 dispersal than for ECB; typically the assumption 

has been made that since the two species are closely related and their behavior is similar that 

ECB can serve as a surrogate for SWCB to address uncertainties about biology and genetics. It is 

unclear what percentage of females, if any, could 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 the natal field, then seed mixtures could be a 

preferred IRM strategy. 

• Non-random oviposition by adults 

Monsanto chose to model oviposition as non-random. Data suggest, however, that at least for 

second generation instars of ECB, oviposition occurs in a uniform fashion where females do not 

discriminate different types of plants in a corn field (Sorenson et al. 1993). Whether non-random 

oviposition or the uniform oviposition would have affected the rate of adaptation differently is 

unclear. On the other hand, Monsanto wrote that they varied oviposition from random across 

compartments to being only within a compartment. BPPD is not clear whether oviposition was 

modeled as random for dispersers but not for resident adults. For future submissions, BPPD 

recommends that Monsanto provide a more thorough 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 exploratory simulations with random and non-random 

oviposition and determined that non-random oviposition was more conservative choice. 

ECB results 

For worst-case assumptions (refuge beetle output 1% and 5x greater survivorship for 

heterozygote resistant genotypes), resistance allele frequency for all three toxins in seed mixture

compartments did not exceed 0.011 after 100 generations, and resistance in ECB had not evolved 

at that point. 

- 70 -
For worst-case assumptions (20% and 40% non-compliance with planting a structured refuge and 

no mixing between compliant and non-compliant compartments), resistance allele frequency for 

all three toxins in structured refuge compartments was 0.69 and resistance 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 are 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 background section of this 

review. 

When non-compliance was 20% and 40% but mixing was increased 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 (including a reduction in adult emergence to 1% and 

2% of adult produced in 5% block refuges). Similarly, resistance did not evolve for structured 

refuges within the 100 generation time frame 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 for Southwestern corn 

borers. Percentages of local recaptures in mark-recapture studies have been low (yet higher than 

for ECB), and while females appear to be mating within the 48 hrs of emergence, there may be 

substantial post mating dispersal (discussed in background section of this review). 

 

When non-compliance was 20% and 40% and mixing 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 allele frequency increased to 0.031. 

Overall BPPD conclusions: 

Monsanto’s modeling output suggests that a 5% SmartStax seed mixture targeting ECB and 

SWCB may be a more durable IRM strategy than the 5% structured refuge approach because 

non-compliance with refuge planting is eliminated. BPPD notes, however, that it is unclear 

whether explicit modeling of a seed blend environment and, for example, asymmetric larval 

dispersal (from refuge and Bt plant) would reveal significantly different results in a spatially 

explicit model. 

2. 091173 - Simulation Modeling of Corn Rootworm 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 summarized 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; Figures 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 model was the basis to evaluate the durability of 

MON88017 x DAS-59122-7 deployed with two different refuge strategies (seed mixture and 

structured refuge). The rate of adaptation was estimated for a 5% seed blend and compared to the 

rate of adaptation estimated for a 5% block refuge. The refuge strategies were modeled in the 

following scenarios: 1) 5% block refuges were assumed 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 simulating a 20% grower non-compliance with refuge 

requirements; and 3) 5% seed mixtures were assumed 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 traits (default value = 99.75%) and pyramided event 

(default value = 99.95%) were based on the field study by Huckaba and Storer (2008). 

Individuals that were heterozygous (RS) at one locus or both loci had a fitness intermediate 

between homozygous susceptible and resistant genotypes; the fitness was estimated based on the 

functional dominance for the resistance allele (Storer 2008). Complete resistance 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 insects 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 compared 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 eggs/female to 100 eggs/female because modeling 

100% SmartStax in the landscape lead to frequent population extinction. While this change 

enabled comparing the resistance evolution in seed mixtures and structured refuges, it also had 

the side-effect of increasing density-dependent mortality in the refuges as well as the rate of 

adaptation. 

Occasional non-compliance was assessed by randomizing (20%) non-compliant fields each year. 

Willful non-compliance (16%) was modeled by keeping 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 given 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. Resistance 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. Resistant larvae for both Bt traits would not be 

expected to leave a suitable host. Parameter ‘rmove’ is the probability of successful larval 

movement from a Bt plant onto another plant. Table 37 provides the probabilities of leaving a Bt 

plant based on the larval genotypes. For example, Cry34/35 and Cry3Bb1 may each lead to 10% 

dispersal of a particular genotype. Summed together, 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 SmartStax 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 arrived on a refuge (non-Bt expressing) 

plant. Five percent of the larvae that left Bt plants in a 5% seed mixture arrived on non-Bt plants 

and were assigned a probability to recover based 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 parameter 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-mating dispersal and assigned a probability of 0.192 and 

0.010 to refuge males moving into the Bt field and Bt males moving into the refuge field, 

respectively. No effect on resistance allele frequency was observed. Dow did not explore female 

pre-mating dispersal because females are believed 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 resistance allele frequency was similar for seed 

mixtures and block refuges of 5% (i.e., less than a 2-fold increase over 20 years) (Figure 17, 

scenarios 1, 2, 7, and 8). 

The various sets of larval movement and recovery 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-genotypes), 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 unrealistic, worst-case heterozygote dispersal. 

2. Block refuge simulations

Little effect was observed on the rate of adaptation of the dual gene product with 20% accidental 

non-compliance when the dose was simulated at benchmark or low dose (Figure 17); however, 

with BPPD’s recommended dose levels and willful non-compliance (16%), resistance allele 

frequency doubled in 7 generations (Figure 18). Dow concluded that the 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 assumptions for modeling corn rootworm resistance to SmartStax in a 

seed blend and block refuge 

Toxin Dow- default 

assumption1

Dow - low dose 

assumption2

BPPD recommended 

low dose assumption3

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 larval 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 larval 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 empirical observations 

2 0.0975 56% 5.53% Consistent with empirical 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 empirical observations 

8 0.19 25% 4.80% Consistent with empirical 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: survival 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: survival 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 empirical observations 

Figure 97. Increase in r-allele frequency in 20 yrs for different compliance scenarios for blocks 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 for 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 different compliance scenarios for blocks 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 with 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 review. 1) The landscape was 

modeled with continuous SmartStax (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 associated 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 extinction, which increased the 

rate of population adaptation. 

Dow compared rates of adaptation (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 using 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 supported 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 conditions 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 dealing 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 likely 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), personal communication). To incorporate the 

uncertainties of this parameter into the modeling 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 resistance was assumed to be related to dose and 

consistent with assumptions of 25-fold resistance in heterozygotes based on concentrationresponse results. Dow incorporated the dominance into the genotypic fitness calculations and 

- 77 -
listed parameter ha as the functional dominance (Appendix 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 after emergence and that there was no pre-mating 

dispersal mechanism. This assumption is likely based on the ‘closed system’ study conducted by 

Ball (1957). However, Marquardt and Krupke (2009) did not observe that newly emerged 

females mated immediately in the field. Their data suggested that some pre-mating dispersal 

occurred in female WCRW. Tethered flight studies by Naranjo (1990) and Coats et al. (1986) 

provided evidence that female pre-mating dispersal 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 significantly extended the durability of Bt corn in block refuges 

compared to seed blends. BPPD considers female 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 density-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 artificially infested Bt and control fields with a 3300 

WCRW eggs/meter; dose (essentially larval mortality) 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 mortality (based on Onstad 2006). In a personal 

communication with BPPD, Dr. Hibbard stated that assuming approximately 75% viable eggs, 

that at 3300 eggs/meter, it was likely that little, if any density-dependent mortality 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 Cry3Bb1. Those dose estimates were referred to as 

BPPD recommended in this review and as ‘unrealistic’ dose assumptions in Dow’s submission 

(Appendix 12). BPPD disagrees with the characterization of these dose levels as “unrealistic”. In 

addition, BPPD will closely evaluate Dow’s simulation results derived with these more 

conservative (lower) dose values. 

Toxin Dow- default 

assumption1

Dow - low dose 

assumption2

BPPD recommended 

low dose assumption3

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 typical larval CRW movement off a Bt plant was less than 

10%. BBPD understands that Dow estimated probability of dispersal of heterozygote and 

susceptible genotypes as 5% and 10% for a single 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 probability assumptions appear reasonable to BPPD 

(see Table 37). 

• Movement off refuge plants 

Hibbard et al. (2005) found late-instar movement occurring from significantly 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 instars and little to no movement for neonates. Dow 

incorporated this process into the model yet found that density dependence did not become an 

issue with 5% refuge plants. BPPD disagrees that 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; hence, the neonates from one clutch could 

potentially be enough to cause density-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 these assumptions and will not discuss unrealistic 

seed blend scenarios. When BPPD’s dose assumptions 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% compliance or accidental (18%) and intentional noncompliance (20%). The dose values recommended by BPPD resulted in greater rates of 

adaptation for block refuge scenarios and at least seed blend scenarios 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. Conversely, 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 standard error of mixing seed blends was incorporated into the 

modeling scenarios. It is unclear whether such an error would have a significant effect on the 

durability of the pyramided corn rootworm product. 

- 79 -
Using Dow’s model outputs and considering uncertainties 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 growing 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 SmartStax 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 biological 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 adaptation in corn earworm could be compromised 

by using a seed mixture IRM strategy. In such regions 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 ‘no’ 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 therefore, 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 incorporated into each unit of seed distributed. 

Consistently producing a seed mix of a given percentage is a manufacturing challenge. As 

proposed, the manufacturing process will target at least 5% refuge seed per unit. The key issue in 

manufacturing will be the variation among units in the percentage of refuge seed. If that 

variability is low, and the percentage of refuge 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 refuge seed less than the target 

is at an acceptable level. These 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 appropriate verification data from third parties involved in seed mix 

production such as licensees 

An additional factor for consideration is the pattern 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: both 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, and 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 the 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 communicated 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 justifiable that a uniform or clumped (more like 

block refuge) distribution of refuge plants should not affect insect resistance management and 

increase the risk of resistance evolution, although the registrants propose 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 phenotype 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 seed in the hoper). Coloring 

the refuge seed may also be necessary to assure 1) proper germination of refuge seed if seeds are 

stored and 2) the appropriate refuge percentage for the following 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 model 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 EPA’s Office of Research and Development 

(ORD), National Risk Management Research Laboratory in Cincinnati, Ohio (Dr. John Glaser in 

cooperation with Dr. Michael Caprio of Mississippi State University). A probability analysis 

complemented the deterministic simulations (conducted by Dr. Caprio) based on a PERT 

distribution (min. value, most likely value, and max. value) for parameters of uncertainties. This 

modeling allows the Agency to compare and assess 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 Caprio and Glaser 2010c) and lepidoptera target pests 

(Section B, Caprio and Glaser 2010d). The main objectives were to determine 1) whether a 5% 

SmartStax seed mixture was a durable IRM strategy for Lepidoptera target pests compared to the 

current structured refuge paradigm in place and estimate 2) the probability of occurrence for a 

predicted durability result using POPGEN. 

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

1. Model Description

The risk assessments utilized a two 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-mixture 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 distributions4 (Vose 2000). The value of each 

parameter and the time until resistance evolved was recorded for each simulation. Several 

options exist for sensitivity analysis of this data, including Bayesian model analysis (BMA), 

stepwise regression, and standardized 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 the PERT-Beta parameters and is related to the 

uncertainty in the parameter. 

2. Model Assumptions

Fitness in this non-additive interaction was calculated as follows. For this example the mortality 

figures presented by Dow were used. The dominance of locus 1 was h1 and the dominance at 

locus 2 was h2. When locus 1 is homozygous resistant (RR), then the fitness only depends on the 

locus 2 genotype (and vice versa when the locus 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 overall 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 fitnesses 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)*h2+RRSS Locus 2 

RR 0.0025 (RRRRSSRR)*h1+SSRR 1.0000 

The remaining difficulty is filling out the cells marked A & B (single heterozygotes) and C (the 

double heterozygote class). Making the assumption 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 field, results from selection experiments for CRW toxins, and 

personal communications with academia. 

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

h2*(SSRR-SSSS)+SSSS, while the fitness of the RSSS genotype (A) would be calculated as 

h1*(RRSS-SSSS) + SSSS. Finally, the double heterozygote fitness (C) was calculated by using 

an average dominance: RSRS = (h1+h2)/2 * (RSRR – RSSS) + RSSS. 

Table 43. PERT-Beta distribution for certain modeling 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-mixture model was 81.9 generations, while it was 52.9 

generations for the structured refuge simulations. Histograms of the results from the 1000 

simulations of each version of the model were similar in shape (Figures not shown here), though 

bootstrapping revealed significant differences (p <<0.01, R statistical package, sm.density 

function). A comparative density 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 resistance 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 acceptable 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 sensitivity 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-compliant mixing should be included in the model. For seedmixtures, all variables except base larval movement, asymmetrical movement, and the time 

period over which larval movement occurred, had statistically significant impacts on the time it 

- 86 -
took for resistance to evolve. The Bayesian model 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, the 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 top models were used as other 

models had significantly higher absolute Bayesian information contents (i.e., they did not fit the 

data nearly as well). 

Table 44. Quantiles for the distribution of time (generations) until resistance evolved for 1000 simulations 

each of the seed mixture and 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 the 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 included 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, combined 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 moved every year, it is possible that very few eggs 

would be laid under the new refuge site (as discussed in Caprio and Glaser 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 the non-Bt plants in a seed-mixture, somewhat increasing the 

efficacy of that strategy. The latter two suggestions, however, are not supported by EPA/ORD’s 

sensitivity analysis. It should be noted that several of these factors are dependent on specifics of 

CRW biology (low vagility, asymmetrical movement), and caution should be used when 

extrapolating these results to other species. 

It is relevant to note that these data present more risk for dual gene products than might have 

been expected from the response in Caprio and Glaser (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 minimum 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 generations. 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 there 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 the 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 insects are more mobile and a more even deposition of 

eggs across the field would be expected (see Caprio 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 rootworm 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 overwintering 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 interactions 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 the 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 interaction by comparing the 

overall survivorship drawn from our PERT distribution with product of the individual toxin 

mortalities based on the draws from their respective PERT distributions. In a two locus 

simulation, there is only one interaction, and all of the interaction can be attributed to that pair of 

loci. In these three locus simulations, there are three interactions (Cry1Ac<->Cry2Ab2, 

Cry1Ac<->Cry1F, Cry2Ab2<->Cry1F), and it is impossible to accurately distribute the 

interactions without additional data on the two locus interactions. In the absence of data the 

mortality effects of pairs of toxins, 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 determine 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 estimate the individual interactions 

by taking the cube root of the total non-multiplicative interaction. There were several additional 

constraints on the new fitness. The fitness of the double susceptible genotype had to be less than 

or equal to either of the component 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 less 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 three double susceptible genotypes. The remaining 

fitnesses involving heterozygous genotypes could then be calculated based on dominance values 

and fitness values already estimated (Fitness calculations contained in Caprio and Glaser, 

2010c). 

As noted in the CRW modeling report, there 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. Testing the two options suggested 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 result 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 longer than 1000 generations (see Appendix 1 in Caprio and Glaser, 

2010d). The risk of resistance was somewhat greater with Southwestern corn borer than with 

European corn borer. The seed-blend option was less durable and 4.5% of the seed blend 

simulations resulted in resistance evolving in less than 135 generations (ca. 45 years). For the 

structured refuge option, the shortest time to resistance was 118 generations and 0.2% of the 

simulations resulted in the evolution 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% probability). These may be considered somewhat 

conservative estimates as potential fitness costs were not included in the simulations. Given the 

long time frames observed for the evolution of resistance, it is likely that fitness costs could have 

a significant impact in further delaying resistance. The genetics and interactions between fitness 

costs to different toxins are, however, unknown and would add considerably to the uncertainty of 

the results. Considering the time frames already 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 degree possible, inclusion of data on two locus 

interactions in future submissions would help to more accurately evaluate the data. The 

solutions to the problems of three locus simulations presented here are merely a first attempt and 

will hopefully provide a basis for further improvement 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 equally durable IRM strategy 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 resistance allele frequency for both refuge strategies; 

Monsanto’s model proposed that lepidoptera would not evolve resistance to SmartStax within 

100 generations (33 yrs or 50 yrs) when deployed in a 5% seed blend. 

EPA/ORD’s simulation analysis using a two-locus 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 refuge 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 refuges. A separate ORD analysis using a three-locus 

model estimates that a 5% SmartStax seed mixture is approximately 15 times less durable than a 

5% structured refuge for the European corn borer and 1.6 times less durable for the southwestern 

corn borer. The ORD simulations estimated that resistance would not evolve 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 Monsanto’s data and modeling submission, EPA/ORD’s 

modeling analyses for a 5% SmartStax seed mixture, BPPD concludes that under assumptions of 

normal and/or high pest densities: 

1) A 5% seed blend strategy at best exceeds the durability of and at worst is equally durable as a 

5% structured refuge for SmartStax targeting corn rootworm. 

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

and SWCB but an overall durable IRM strategy. 

B. BPPD Conclusions Regarding Monsanto’s Modeling 

Monsanto’s three-locus deterministic modeling simulations 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 increase 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-compliance 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 Monsanto’s simulations conservatively predicted a 

durability of approximately 33-50 years, depending on whether the Lepidopteran target pest has 

- 92 -
two or three generations per year. EPA/ORD’s independent analysis predicted that there was a 

small probability that resistance evolved in ECB and SWCB for this timeframe, which supports 

Monsanto’s conclusion that a 5% seed mixture is a durable IRM strategy 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 frequency, 1% and 2.5% effective 

refuge, heterozygote genotype fitness, 0% mixing 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 realistic. Furthermore, it was unclear whether 0% 

mixing meant an overall restriction for dispersal 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 literature 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 respect to larval movement but also adult 

movement, although there are limited studies in the public 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 mating 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 ECB. 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 standard 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 error 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% SmartStax 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 rate 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 for 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 structure that likely contributed to an increase 

in the rate of adaptation observed in their simulations (i.e., landscape of continuous SmartStax; 

complete resistance; no fitness cost of resistance; and female fecundity 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 dispersed off the field at the edge of the landscape. As 

stated in Caprio and Glaser (2010), the choice may be important because it can impact the 

distance between Bt and non-Bt plants and the ultimate effective refuge size. 

• Initial resistance allele frequency 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 support for the inclusion 

of such a parameter in CRW models. Pre-mating dispersal for female western 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 toxins and density-dependent adjustment 

BPPD evaluated the dose for corn rootworm in its SmartStax review (BPPD 2009b) and 

recommended that Dow use lower percent mortality assumptions (94.2% for Cry34/35 single 

trait, 97.5% for Cry3Bb1 single trait, and 98.2% for the pyramided 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 dispersal off refuge plants 

BPPD is unclear why density-dependence 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 seed mixtures, Appendix 9, MRID 479437-01). 

• Standard error of mixing for the seed blend technology

- 94 -
Dow did not address whether the standard error of mixing seed blends was incorporated into the 

modeling scenarios. It is unclear whether such an error would have 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 concludes that a 5% SmartStax seed mixture is 

efficacious against main lepidoptera target pests, ECB, SWCB, and CEW. Lepidopteran larval 

movement from non-Bt plants onto surrounding Bt plants 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 SmartStax is somewhat less toxic to 3rd, 4th, and 5th instars 

of SWCB than ECB (Appendix 1, MRID 479437-01). Additionally, under high infestation 

levels, some corn earworm (CEW) larvae were found in the ears of SmartStax corn in 100% Bt 

plots and in seed mixture plots suggesting that some survival should be expected (Appendix 2, 

MRID 479437-01). 

Less is known about SWCB larval and adult dispersal 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-stages 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 could 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 especially if natural infestation levels are low and 

no density-dependent dispersal occurs off non-Bt plants. However, should density-dependent 

movement occur for 3rd instar larvae of refuge plants in a SmartStax seed mixture, Monsanto’s 

emergence results from a greenhouse experiment (Appendix 5, MRID 479437-01) suggest that 

those individuals may not be affected by the two expressed toxins. Overall larval mortality (for 

1st and 2nd instars) in this experiment was 95.7%. To summarize, BPPD does not concur with 

Dow’s and Monsanto’s close to high-dose 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 refuge 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 EPA/ORD’s Deterministic Analyses 

BPPD has collaborated with EPA/ORD on independent 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 probabilistic) 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% SmartStax 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 that the mean time to resistance for the 5% 

block refuge strategy was 52.9 years (5% probability that resistance evolves within 22 yrs) and 

for the 5% seed mixture simulations was 81.9 years (with a 5% probability 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 statistically significant (p << 

0.01). EPA/ORD conservatively assumed that the resistance allele frequency 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 for 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 

Frequency1

5% Probability for 

Resistance Event Average Years 

5% Seed Blend 33 years 81.9 years 

5% Structured Refuge 

Similar rates of increase2

 for the 

20 years modeled 22 years 52.9 years 

2

 The Dow model predicted similar durability for SmartStax under both refuge scenarios; 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 date 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, BPPD believes that the initial 

frequencies chosen for the dual gene were conservative. 

- 96 -
b. Modeling of ECB and SWCB 

Using the same model structure as for corn rootworm (i.e., assumptions regarding compliance, 

refuge placement), yet incorporating corn borer’s propensity for greater dispersal, ORD’s 

deterministic, probabilistic model predicted that: 

• Resistance in ECB would not evolve within 158 generations (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 refuge. 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% structured refuge (see Table 

49). 

Table 48. Durability estimates by EPA/ORD and Monsanto for 5% SmartStax seed mixture and 5% 

structured refuge targeting 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 Resistance1

 

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

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

- 97 -
Table 49. Durability estimates by EPA/ORD and Monsanto for 5% SmartStax seed mixture and 5% 

structured refuge targeting 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 Resistance1

 

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

5% Structured Refuge 0% 118 gen (39 yrs or 58 yrs) >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 for 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. Hence, the ORD model predicted a shorter time 

to resistance for SWCB, which may underestimate 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, what the extent is of their movement (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 proportion of population that engages in this behavior so that 

simulation models can be more accurately parameterized and provide better estimates for 

product durability. 

• The proportion of consistent non-compliance was conservatively maintained at the same 

level as the corn rootworm simulations although data support that compliance with refuge 

plantings for Bt products targeting lepidoptera is higher than for CRW. Non-compliance 

was determined to significantly affect estimates of product durability. 

- 98 -
VIII. LITERATURE CITED 

Alstad, DN and DA Andow. 1995. Managing the evolution of insect resistance to transgenic 

plants. Science, 268: 1894-1896 

Bailey RI, D Bourget, A-H Le Pallec, and S Ponsard. 2007. Dispersal and settling preferences of 

European corn borers in maize field borders. J. Appl. Ecol., Vol. 44: 385-394 

HJ Ball. 1957. On the biology and egg-laying habits of the western corn rootworms. J. Econ. 

Ent., Vol. 50:126-128 

TF Branson. 1986. Larval feeding behavior and host-plant resistance in maize. In J.L. Krysan 

and A.T. Miller (eds), Methods for the study of pest Diabrotica. Springer, New York. 

Caffrey DJ and LH Worthley, L. 1927. A progress report on the investigations of the European 

corn borer (U.S. Department of Agriculture, Washington, DC), USDA Bull. 1476. 

MA Caprio. 2001. Source-sink dynamics between transgenic and non-transgenic habitats and 

their role in the evolution of resistance. J. Econ. Entomol., Vol.94: 698-705 

Caprio M and J Glaser. 2010a. Simulation models evaluation of pest resistance development to 

refuge in the bag concepts related to Pioneer submissions. EPA-ORD letter report to EPAOPP/BPPD dated January 22, 2010. 

Caprio M and J Glaser. 2010b. The use of simulation models to evaluate pest resistance 

development to refuge in the bag concepts related to Pioneer submission. EPA-ORD letter report 

to EPA-OPP/BPPD dated April 12, 2010. 

Caprio M and J Glaser. 2010c. Risk assessment of the evolution of resistance in western corn 

rootworm and lepidoptera target pests of SmartStax corn. Letter report to EPA-OPP/BPPD dated 

October 29, 2010. 

Caprio M and J Glaser. 2010d. Risk Assessments of the evolution of resistance in European Corn 

Borer and Southwestern Corn Borer to SmartStax corn. Letter report to EPA-OPP/BPPD dated 

October 29, 2010. 

Carriere, Y, MS Sisterson, and BE Tabashnik. 2004. Resistance management for sustainable use 

of Bacillus thuringiensis crops in integrated pest management, p. 65-95. Book chapter in “Insect 

Pest Management: Field and Protected Crops”, Springer, 2004. 

Coats, SA, JA Mutchmor, and JJ Tollefson. 1986. Study of migratory flight in the western corn 

rootworm (Coleoptera: Chrysomeldiae). Environmental Entomology, Vol. 15: 620-625 

Davis PM and DW Onstad. 2000. Seed mixtures as a resistance management strategy for 

European corn borers (Lepidoptera: Crambidae) infesting transgenic corn expressing Cry1Ab 

protein. J. Econ. Entomol., Vol. 93 (3): 937-948 

- 99 -
Dorhout DL, TW Sappington, and ME Rice. 2008. Evidence for obligate migratory flight 

behavior in young European corn borer (Lepdioptera: Crambidae) females. Env. Ent., Vol. 37: 

1280-1290 

Dorhout DL, TW Sappington, and ME Rice. 2010. Flight behaviour of European corn borer 

infected Nosema pyrausta. Journal of Applied Entomology 

Grant RH and KP Seevers. 1989. Local and long-range movement of adult western corn 

rootworm (Coleoptera: Chrysomelidae) as evidenced by washup along southern lake Michigan 

shores. Envir. Entomology, Vol. 18 (2): 266-272 

Gould F, N Blair, M Reid, TL Rennie, and S Micinski. 2002. Bacillus thuringiensis-toxin 

resistance management: stable isotope assessment of alternate host use by Helicoverpa zea. 

Proc. Natl. Acad. Sci. 99: 16581-16586. 

Guse CA, DW Onstad, LL Buschman, P Porter, RA Higgins, PE Sloderbeck, GB Cronholm, and 

FB Peairs. 2002. Modeling the development of resistance by stalk-boring Lepidoptera 

(Crambidae) in areas with irrigated transgenic corn. Env. Entomol., Vol.31: 512-526 

Heimpel GE, C Neuhauser, and DA Andow. 2005. Natural enemies and the evolution of 

resistance to transgenic insecticidal crops by pest insects: the role of egg mortality. Env. 

Entomol., Vol.34: 512-526 

Hellmich RL, RL Pingel, and WR Hansen. 1996. Influencing European corn borer (Lepidoptera: 

Crambidae) aggreagation sites in small grain crops. Env. Entomol., Vol. 27(2): 253-259 

RL Hellmich, 1996. 1996 Resistance management related field experiments: Ames, IA. 

Unpublished study submitted to EPA (part of Monsanto’s 1997 IRM research report). No MRID 

#. 

RL Hellmich. 1997 Resistance management related field experiments: Ames, IA. Unpublished 

study submitted to EPA (part of Novartis’ 1997 IRM research report), MRID # 444754-01. 

RL Hellmich. 1998 Resistance management related field experiments: Ames, IA. Unpublished 

study submitted to EPA (part of AgrEvo’s 1998 IRM research report), MRID # 447443-01. 

Hellmich RL, L.S. Higgins, J.F. Witkowski, J.E. Campbell, and L.C. Lewis, 1999. 

Oviposition by European corn borer (Lepidoptera: Crambidae) in response to various transgenic 

corn events. J. Econ. Entomol. 92 (5): 1014-1020. 

Hibbard BE, DP Duran, MR Ellersieck, and MM Ellsbury. 2003. Post-establishment movement 

of Western corn rootworm larvae (Coleoptera: Chrysomelidae) in central Missouri corn. Journal 

of Economic Entomology, Vol. 96(3): 599-608 

- 100 -
Hibbard BE, MI Higdon, DP Duran, YM Schweikert, and MR Ellersieck. 2004. Role of egg 

density on establishment and plant-to-plant movement by western corn rootworm larvae 

(Coleoptera: Chrysomelidae). Journal of Economic Entomology, Vol. 97(3): 871-882 

Hibbard BE, TT Vaughn, IO Oyediran, TL Clark, MR Ellersieck. 2005. Effect of Cry3Bb1 

expressing transgenic corn on plant-to-plant movement by western corn rootworm larvae 

(Coleoptera: Chrysomeldiae). Journal of Econonomic Entomology, Vol. 98(4): 1126-1138 

Hibbard BE, LN Meihls, MR Ellersieck, and DW Onstad. 2010. Density-dependent and densityindependent mortality of the Western corn rootworm: impact on dose calculations of rootwormresistant Bt corn. J. Econ. Entomol., Vol. 103 (1): 77-84 

RE Hill. 1975. Mating, oviposition patterns, fecundity and longevity of the western corn 

rootworm. Journal of Economic Entomology, Vol. 68 (3): 311-315 

Huckaba RM and N Storer. 2008. Field measures of western corn rootworm (Coleoptera: 

Chrysomelidae) mortality caused by Cry34/35 and Cry3Bb1 proteins in the combined trait corn 

product MON 89034 x TC1507 x MON 88017 x DAS-59122-7. Unpublished report (MRID 

474449-11) submitted to the Agency. 

Hunt TE, LG Higgley, JF Witkowski, LJ Young, and RL Hellmich. 2001. Dispersal of adult 

European corn borer (Lepidoptera: Crambidae) within and proximal to irrigated and nonirrigated corn. J. Econ. Entomol., Vol. 94 (6): 1369-1377 

Ives AR and DA Andow. 2002. Evolution of resistance to Bt crops: directional selection in 

structured environments. Ecology Letters, Vol. 5: 792-801 

Kim KS, JM Bagley, BS Coates, RL Hellmich, and TW Sappington. 2009. Spatial and temporal 

genetic analysis show high gene flow among European corn borer (Lepdioptera: Crambidae) 

populations across the central U.S. corn belt. Env. Ent., Vol. 38: 1312-1323 

Langille RH, and AJ Keaster. 1973. Mating behavior of the southwestern corn borer. (Diatraea 

grandiosella). Environ. Entomol. 2: 225Ð226. 

Lefko SA, TM Nowatzki, SD Thomposon, RR Binning,MA Pascual, ML Peters, EJ Simbro, and 

BF Stanley. 2008. Characterizing laboratory colonies of western corn rootworm (Coleoptera: 

Chrysomelidae) selected for survival on maize containing event DAS-59122. Journal of Applied 

Entomology, Vol. 132: 189-204 

Mallet J and P Porter. 1992. Preventing insect adaptation to insect-resistant crops: are seed 

mixtures or refugia the best strategy? Proc. Royal Soc. London B, 250: 165-169 

Marquardt, PT and CH Krupke. 2009. Dispersal and mating behavior of Diabrotica virgifera 

virgifera (Coleoptera: Chrysomelidae) in Bt corn fields. Environ. Entomol., Vol.38(1): 176-182 

- 101 -
Mason CE, ME Rice, DD Calvin, JW Van Duyn, WB Showers, WD Hutchinson, JF Witkowski, 

RA Higgins, DW Onstad, and GP Dively. 1996. European corn borer: ecology and management. 

North Central Region Ext. Publ. No. 327. Iowa State University, Ames, IA. 

Meihls LN, ML Higdon, BD Siegfried, TA Spencer, NJ Miller, TW Sappington, MR Ellersieck, 

and BE Hibbard. 2008. Increased survival of western corn rootworm on transgenic corn within 

three generations of on-plant greenhouse selection. Proceedings of the National Academy of 

Science 105: 19177-19182. 

Meinke LJ, TW Sappington, DW Onstad, T Gullemaud, NJ Miller, J Komaromi, N Levay, L 

Furian, J Kiss, and F Toth. 2009. Western corn rootworm (Diabrotica virgifera virgifera 

LeConte) population dynamics. Agricultural and Forest Entomology, Vol.11: 29-46 

SE Naranjo. 1990. Comparative flight behavior of Diabrotica virgifera virgifera and Diabrotica 

barberi in the laboratory. Ent. Experimantalis et Aplicata, Vol. 87: 383-394 

Nowatzki TJ, SD Blair, and LJ Meinke. 2003. Comparative movement and mating behavior of 

adult western corn rootworm (Coleoptera: Chrysomeldia) in YieldGard rootworm transgenic and 

a conventional cornfield. Poster presentation at the Entomological Society of America meeting in 

Cincinnati (Ohio). 

Nowatzki TJ, N Bradley, KK Warren, S Putnam, LJ Meinke, DC Gosselin, FE Harvey, TE Hunt, 

and B Siegfried. 2003b. In-field labeling of western corn rootworm adults (Coleoptera: 

Chrysomelidae) with Rubidium. Journal of Economic Entomology, Vol. 96(6): 1750-1759 

Onstad DW and F Gould. 1998. Modeling the dynamics of adaptation to transgenic maize by 

European corn borer (Lepidoptera: Pyralidae). J. Econ. Entomol., Vol. 91: 585-593 

Oleson JD, YL Park, TM Nowatzki, and JJ Tollefson. 2005. Node-injury scale to evaluate root 

injury by corn rootworms (Coleoptera: Chrysomelidae). J. Econ. Entomol., Vol. 98: 1-8 

Onstad DW, and F Gould. 1998. Modeling the dynamics of adaptation to transgenic maize by 

european corn borer (Lepidoptera: Pyralidae). J. Econ. Entomol. 91: 585-593. 

Onstad DW, CA Guse, J. L. Spencer, E. Levine, and M. E. Gray. 2001. Modeling the dynamics 

of adaptation to transgenic corn by western corn rootworm (Coleoptera: 

Chrysomelidae). J. Econ. Entomol. 94: 529-540. 

Onstad DW, BE Hibbard, TL Clark, DW Crowder, and KG Carter. 2006. Analysis of densitydependent survival of Diabrotica (Coleoptera: Chrysomelidae) in cornfields. Env. Entomol., 

Vol. (35(5): 1272-1278 

Prasifka JR, RL Hellmich, ALB Crespo, BD Siegfried, and DW Onstad. 2009. Video-tracking 

and on-plant tests show Cry1Ab resistance influences behavior and survival of neonate Ostrinia 

nubilalis following exposure to Bt maize. J. Insect Behavior, Vol. 23: 1-11 

- 102 -
Qureshi JA, LL Bushcman, JE Throne, and SB Ramaswami. 2006. Dispersal of adult Diatrea 

grandiosella (Lepdioptera: Crambidae) and its implications for corn borer resistance management 

in Bacillus thuringiensis Maize. Ann. Enomol. Soc. Am Vol. 99 (2): 279-291 

Reardon BJ, DV Sumerford, and TW Sappington. 2006. Dispersal of newly eclosed European 

corn borer adults (Lepidoptera: Crambidae) from corn into small-grain aggregation plots. J. 

Econ. Entomol., Vol. 99: 1641-1650 

JE Rolston. 1955. The southwestern corn borer in Arkansas. Ark. Agric. Exp. Stn. Bull. 

Fayetteville 553. 

Ross SE and KR Ostlie, 1990. Dispersal and survival of early instars of European corn 

borer (Lepidoptera: Pyralidae) in field corn. J. Econ. Entomol. 83 (3): 831-836. 

Scott IA, JL Spencer, TR Mabry, and E Levine. 2004. Influence of atmospheric conditions on 

high-elevation flight of Western corn rootworm (Choleoptera: Chrysomelidae). Environmental 

Entomology, Vol. 33(3):650-656 

Showers WB, RL Helmich, ME Derrick-Robinson, and WH Hendrix III. 2001.Aggregation and 

dispersal behavior of marked and released European corn borer (Lepidoptera: Crambidae) adults. 

Env. Entomol., Vol. 30(4): 700-710 

Sorenson CE, GG Kennedy, JW Van Duyn, and JR Bradley, Jr. 1993. Distribution of second 

generation European corn borer, Ostrinia nubilalis, egg masses in field corn and relationship to 

subsequent tunneling damage. Entomol. Exp. Appl. 68: 15-23 

Spencer J. 2009. Personal communication. E-mail response and summary of unpublished data 

submitted to J.C. Martinez (EPA), dated December 28, 2009. 

N Storer. 2003. A spatially explicit model simulating Western corn rootworm (Coleoptera: 

Chrysomelidae) adaptation to insect-resistant maize. J. Econ. Entomol., Vol. 96 (5): 1530-1547 

Storer NP, JM Babcock, and JM Edwards. 2006. Field measures of western corn rootworm 

(Coleoptera: Chrysomelidae) mortality caused by Cry34/35Ab1 proteins expressed in Maize 

event 59122 and implications for trait durability. Journal of Economic Entomology, Vol. (99 (4): 

1381-1387 

N Storer. 2008. Simulation modeling of corn rootworm adaptation to pyramided Bt corn plant 

incorporated protectants MON 88017 x DAS-59122-7. Unpublished report (March 27, 2008) 

submitted to the Agency. 

BE Tabashink. 1994. Delaying insect adaptation to transgenic plants: seed mixtures and refugia 

reconsidered. Proc. Royal Soc. London B, 255: 7-12 

Van Woerkom GJ, FT Turpin, & JR Barret Jr. 1983. Wind effect on western corn rootworm 

(Coleoptera: Chrysomelidae) flight behavior. Environmental Entomololgy, Vol 12: 196-200 

- 103 -
- 104 -

D Vose. 2000. Risk analysis: a quantitative guide. Wiley & Sons, New York. 

Witkowski JF, JC Owens, and JJ Tollefson. 1975. Diel activity and vertical flight distribution of 

adult western corn rootworms in Iowa cornfields. Journal of Economic Entomology, Vol. 68: 

351-352 

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, 2000 on Bt plant-pesticides risk and benefit 

assessments: insect resistance management. Report 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 Optimum 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 Company’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 Resistance 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 resistance 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.