- 2

- EXECUTIVE SUMMARY

T he BPPD IRM T eam concludes that Cry3Bb1 resistance is suspected in at least som e portions of four states in which “unexpected damage” reports originated (IA, IL, MN, and NE) and recommends that the Cry3Bb1

r emedial action plan be implemented for “suspected resistance”

. T he BPPD IRM Team’s conclusions are

based on multiple documented cases of unexpected “severe” corn rootworm damage to Cry3Bb1 fields (Gassmann et al. 2011a; Gray 2011a, c) and other undocumented reports from corn entomologists

.

The 2009 resistance monitoring data for Cry3Bb1 showed that field

- collected populations (co llected from Illinois, Iowa, and Nebraska) were less susceptible to the toxin than the laboratory reference colony. In 2009, every field

- collected population had a higher EC 50 and LC 50 than the laboratory control, in some cases by an order of magnitude. Sampling was conducted in three states (Nebraska, Illinois, and Iowa); the BPPD IRM Team recommends that Colorado, South Dakota, Minnesota, and western Wisconsin be included in future monitoring based on the registrant ’s reported Cry3Bb1 performance inqui ries

.

After reviewing Monsanto’s overall resistance monitoring strategy for Cry3Bb1 (including responses to previous Agency reviews), the BPPD IRM Team concludes that the registrant ’s current resistance monitoring program (as proposed) is inadequate an d likely to miss early resistance events. This is due to a sampling trigger (> 1.5 NIS) in fields with unexpected damage that is too high (and may miss potentially resistant populations) and a sampling regime that collects beetles too far (1

- 2 miles away) from problem fields.

CONCLUSIONS AND RECOMMENDATIONS

1. THE BPPD IRM TEAM REVIEW OF MONSANTO’S MONITORING PLAN AND RESPONSE TO BPPD (2010)

1 a) After review ing Monsanto’s monitoring plan, the BPPD IRM Team concludes that the registr ant’s

current resist ance monitoring program (as proposed) is in adequate and likely to miss early resistance events. This conclusion was based on the following observations:

**•** The registrant proposed to collect insects from problem fields within 1

- 2 miles from neighboring sites of

failed fields

. The BPPD IRM Team believes that this proposal is insufficient if the goal is to determine

whether Bt failure is due to resistance of CRW population. Since the majority of adult corn rootworm

may not disperse long distances , the greatest probability of capture of resistant genotypes should be in

the problem fields, possibly in adjacent fields, but less likely in fields 2 miles away during that particular year. To collect in neighboring sites is reasonable after infield collections have occ urred or during the following year because of possible adult movement

. It is imperative that t he language for this crucial

pr ocedure is unambiguous and clear and that Monsanto and EPA come to a timely agreement on the method of collecting adult CRW in prob lem fields. If the resistance m onitoring program is to be meaningful

, a more proactive, effective approach must be adopted

.

**•** The BPPD IRM Team concludes that Monsanto’s Greater Than Expected Damage ( GTED ) threshold of 1.5 ( Node Injury Scale, NIS ) for a sin gle toxin PIP with non

- high dose expression is generally too high.

The BPPD IRM Team stated in the 2010 review that injury ratings of 1.0 should trigger investigations. Ideally, a comparison should occur between performance of Bt and nearby refuge corn; ho wever, this may not be an option if grower s were non

- compliant with refuge requirements. In such non

- compliant- 3

- cases, the probability that severe root damage is due to resistance likely is greater and, therefore, the

BPPD IRM Team considers a threshold lev el of 1.0 for a single toxin PIP a more defensible (conservative) trigger to initiate adult collections. During years with great pest pressure (not associated with agronomic practices such as growing continuous corn for years), it may be reasonable to devi ate from the 1.0 threshold and use 1.5 (based on a graded scale where Bt damage is related to adult density and damage to refuge corn). The BPPD IRM Team concludes that f or SmartStax products ( i.e. SSX

RIB ), a threshold of 1.0 (as proposed by Monsanto) is not conservative enough; this was already discussed in BPPD (2010). The pyramid has greater activity against CRW and should, therefore, have a lower threshold (such as 0.5) to trigger beetle collections . Additionally, in a seed blend it will be inherently more difficult to detect Bt performance issues, t herefore, it may not be realistic to use the following threshold “50% of the plants must show greater than 0.5 (NIS) damage” before an investigation occurs. T he BPPD IRM T eam proposes that any clearly visibl e sign of damage exceeding

the threshold level (0.5) in a seed blend ( after trait expression has been confirmed and other causes ruled

out

, but irrespective of the percentage of Bt plant damage in that field ) trigger adult collections

.

**•** If Monsanto was un able to collect adults from problem fields because they were contacted too late by seed dealers, then it is important that adult sampling occur s in the problem areas the following season irrespective of pest pressure and damage in the old problem fields . I f growers sprayed their fields after they observed high densities of adults during a problem year or used other methods of control , then the

pest pressure and subsequent Bt damage should be less severe the following year. If Monsanto does not

collect in su ch problem areas during the following season because of “ below threshold damage ” in Bt fields (1.0 for single PIPs and 0.5 for SSX), th en they will miss early resistance events in those areas. If there is resistance but low pest pressure, damage can be exp ected to be lower than in cases where there

is high pest pressure coupled with resistant individuals in the population.

1 b) Monsanto reported that h igh rootworm pressure was typically the result of continuous

- corn plantings that built up CRW population d ensities over time and used this reasoning as a justification to use a

GTED threshold level of ≥ 1.5. T he BPPD IRM T eam notes that continuous planting of corn with Cry3Bb1 without rotation also leads to greater selection pressure. Hence, unexpected pest dam age in such Cry3Bb1 fields with agronomic practices as described by Monsanto should be reason for concern and has a greater probability to be due to resistance evolution in that population. T herefore, the BPPD

IRM T eam maintains that a threshold level of 1 .0 for a single toxin PIP is a more appropriate trigger to

initiate adult collections.

1 c ) Monsanto stated that they had been working with Dr. Ostlie in Minnesota to understand the greater than expected root damage observed in Cry3Bb1 corn during the late corn growing season in 2009. T he

BPPD IRM T eam interprets this to mean that Monsanto is unsur e of the reasons for Bt failure and notes

that this is an instance where collections should have occurred the following year ( despite low er damage

levels or popul ation numbers ) . If growers ‘bombed’ adults in fall of 2009 or used IPM approaches to manage their fields , it sh ould be expected that population densiti es and subsequent root damage were

low er in 2010. However, resistance genes could still be in the se popul ation s (as discussed above)

, which

is the reason why adult collections should be made once unexplained performance failure s have

occurred.

1 d ) Monsanto determined additional problem areas in South Dakota that were included into the sampling

scheme for ann ual resistance monitoring in 2010 and proposes to use these to obtain baseline susceptibility data. These sites should not serve to obtain baseline data (as proposed by Monsanto) but rather measure shifts in CRW susceptibility to Cry3Bb1.- 4

- 1 e ) Monsanto pro vided EPA with the number of performance inquiries (PI) for their Cry3Bb1 products in the Corn Belt from 2003 through 2010. The number of PIs ranged from 0 to over 100 per year d uring this time period. Monsanto reported that none of their follow

- up investi gations resulted, however, in finding resistant populations. With the exception of samples collected in 2007 and 2008, it is unclear to

t he BPPD IRM T eam how many of the PI’s reported in Table 4 resulted in adult collections and bioassays. This information should be provided for each state (each year) in addition to reporting the number of PIs. Furthermore, the BPPD IRM T eam recommends that Monsanto include information about the degree of pest pressure and environmental conditions in those states with PIs ( i.e. wet spring,

etc ).

2. THE BPPD IRM TEAM REVIEW OF 200 9 CRW MONITORING REPORT

2 a) After reviewing Monsanto’s 2009 monitoring data f or Cry3Bb1, t he BPPD IRM T eam concludes that the highest bioassay concentration tested (170 μg of Cry3Bb1 /cm 2 of diet) doe s not appear to be adequate as a diagnostic assay for detecting resistance in WCRW populations. The EC/LC 50 appears to detect shifts in WCRW susceptibility, but the bioassay results do not appear to be sensitive enough for discerning between populations wi th decreased susceptibility from those with actual resistance to the

toxin. Monsanto should discuss with EPA whether the ‘comparative weight’ approach between Bt and

non

- Bt treatments for field populations could be used to develop a viable resistance detec tion level.

2 b ) Most mean LC 50 values from 2009 population samples had to be extrapolated for the highest

concentration because populations were no longer sensitive at this level of exposure. Monsanto and the

BPPD IRM T eam agree that LC 50 results can be s omewhat misleading when correlating larval survival to level of toxin susceptibility because larvae can survive on Bt for some time without feeding. A 3

- day long assay may, therefore, capture less than the actual percent mortality due to Cry3Bb1 exposure. All field populations collected in 2009 had greater (mean) EC 50 and LC 50 values (measured and extrapolated) than those for the lab populations, in some instances by an order of magnitude.

2 c ) The only count y from which population samples were obtained bo th in 2009 and 2008 was McLean (IL). In 2008, the EC 50 and LC 50 mean values were14.6 μg/cm 2 and 38.7 μg/cm 2 ; in 2009, the EC 50 and

LC 50 means were 37.75 μg /cm 2 and 585.06 μg/cm 2 (extrapolated value) . Th is represents a two

- fold and

100

- fold increase for mea n EC and LC

- values, respectively in that location. S usceptibility in McLean

C ounty (IL) may have decreased during this time period, though the BPPD IRM team is not aware of any documented cases of field failures from this county

.

2 d ) Monsanto tested W CRW populations from three states: Nebraska, Illinois, and Iowa. T he BPPD IRM

T eam notes that these three states alone may not be sufficient and that Colorado, South Dakota (as proposed by Monsanto), Minnesota, and western Wisconsin may need to be included bas ed on the registra nt’s reported Cry3Bb1 performance inquiries and discussions with corn entomologists (Gassmann et al., 2011b).

2 e ) Regarding the identified deficiency with respect to the terms and conditions of the registration (identified in BPPD 2010)

, Monsanto stated that Mexican and western corn rootworm were genetically similar and that therefore the WCRW IRM strategy should be appropriate for the MCRW. T he BPPD

IRM T eam generally agrees that the assumptions may be appropriate ; however, we note that there could still be differences in the specific dose profile that should be addressed.- 5

- 3. THE BPPD IRM TEAM REVIEW OF UNEXPECTED CORN ROOTWORM DAMAGE IN

CRY3Bb1 FIELDS AND CONCLUSIONS REGARDING SUSPECTED RESISTANCE TO CRY3Bb1

T he BPPD IRM T eam has been m ade aware of a number of cases of unexpected corn rootworm (CRW) damage to Cry3Bb1 fields. Some of these reports have been documented in the literature, while others are anecdotal. This section summarizes these reports and provides T he BPPD IRM T eam ’s revi ew of them.

3a) T he BPPD IRM T eam reviewed Gassmann et al.’s (2011) report concluding that western corn rootworm (WCRW) had evolved resistance to Cry3Bb1 maize in certain parts of eastern Iowa. T he BPPD IRM

T eam notes that populations tested in the assay originated from within problem fields where a certain proportion of adults can be assumed to have undergone development (egg hatching, immature development and emergence to adult hood ). Some of the collected individuals may have also immigrated

into these B t fields from neighboring fields. If this was the case, then the resistance observed and described by Gassmann could be “diluted”. More important ly, those native adults fed on and finished their development in Cry3Bb1 maize, were viable, and produced offsp ring that w ere also resistant . The subsequent progeny tested in greenhouses had significant and much greater survival on Cry3Bb1 maize

than larvae that were fed on non

- Bt maize and whose parental lines origi nated from control fields.

3 b) In two published reports, Dr. Gray from the University of Illinois described severe performance issues with Monsanto’s Cry3Bb1 corn in Henry and Whiteside County of northwestern Illinois (Gray 2011a) and LaSalle County of north

- central Illinois (Gray 2011c) . The problem f ields had been planted to continuous corn for many years, Cry3Bb1 maize was planted to protect against WCRW damage, and similarities to nearby Iowa problem fields identified by Gassmann et al. (2011 a ) were noted. Between two to three nodes were missing on damaged root systems ( Gray, 2011b ); adults were numerous in these fields; lodging was prevalent; insect collection s were made to determine whether resistance was the cause of Cry3Bb1 field failures. Dr. Gray proposed that growers who encountered field perf ormance issues with CRW protected traits should consider rotating to a non

- WCRW host, using CRW soil

insecticide a t planting and different CRW Plant

- Incorporated Protectant (PIP) , and/or planting a pyramided PIP expressing multiple Cry toxins in 2012, but that a long term IPM approach was necessary for successful CRW management.

3c) Over the course of 2011, T he BPPD IRM T eam has had multiple conference calls with scientists from

academia and USDA

- ARS to discuss performance of Bt corn in the Corn Belt. Dur ing a recent call on August 25, 2011, performance issues regarding Monsanto’s Cry3Bb1 maize in Illinois were discussed (Gassman n et al. 2011b)

. T he BPPD IRM T eam was also informed that there were other severe efficacy issues for Monsanto’s Cry3Bb1 trait i n Nebraska (though spotty throughout the state) , Minnesota (north

central and southern locations) , and Iowa in 2011 (same areas as discussed in Gassmann et al. 2011a)

. Specifically in Minnesota, Dr. Ostlie reported that 2011 marked the third year that mode rate to severe corn rootworm damage in Cry3Bb1 corn was observed and that the problem was becoming more widespread. In Nebraska, Drs. Siegfried, Meinke, and Hunt reported that the 2011 growing season marked the fourth year where moderate to severe rootworm damage in Monsanto’s Cry3Bb1 corn was apparent. As in Illinois and Iowa, these problem fields were planted to continuous corn with Cry3Bb1 for several years in a row; no rotation with other crops occurred. It was unlikely that refuges were planted.- 6

- T

he BPPD IRM T eam concludes that Cry3Bb1 resistance is suspected in at least some portions of Iowa,
Illinois, Nebraska, and Minnesota

, where “ unexpected pest damage ” reports originated. This conclusion is based on the following considerations:

**•** Western corn r ootworm field populations in eastern Iowa have been documented with significant and increased survival on Cry3Bb1 ma ize compared to populations collected from non

- problem fields and assayed on non

- Bt maize (Gassmann et al. 2011a)

. T he BPPD IRM TEAM recomme nds additional work be conducted on these populations to assess survival to adulthood, fecundity, and any other reproductive effects that could impact the populations’ ability to survive in the field.

**•** Two recent reports published by the University of Illi nois discussed severe Cry3Bb1 corn damage in

northwestern and north

- central Illinois during the 2011 corn growing season (Gray 2011a, 2011c). According to Dr. Gray’s report, Henry County was among those that had severe Cry3Bb1 damage. Monitoring data suppl ied by Monsanto (see Table 6) from this county showed a six

- fold increase in mean

LC 50 from 2007 to 2008 (the last year data were tabulated specifically from this county), though this result can not be directly correlated with any incidents of field damage

.

**•** There are other reports of multiple and increased Cry3Bb1 performance failures in Nebraska and Minnesota (Gassmann et al. 2011b). Although Cry3Bb1 field failures throughout these four states (IA, IL, MN, and NE) of the Corn Belt are spotty, the frequenc y of such reports has apparently increased (through 2011).

**•** Resistance monitoring data for Cry3Bb1 have shown that field

- collected populations (collected from Illinois, Iowa, and Nebraska) are generally less susceptible to the toxin than the laboratory ref erence colony. In 2009, every field

- collected population had a higher EC 50 and LC 50 than the laboratory control, in some cases by an order of magnitude. This was also the case for populations collected in 2007 (see BPPD 2009). Monitoring data collected from 2005 to 2009 appear to show a large decrease in susceptibility over the time period (see Tables 5 and 6), however, much of the decrease in susceptibility is likely due to a change in toxin source in 2007.

Based on the conclusion of “suspected resist ance”, the BPPD IRM T eam recommends that the appropriate remedial action plan be implemented for Cry3Bb1 corn in areas experiencing unexpected field damage. The remedial action plan includes the following steps for “suspected resistance” (BPPD 2010b):

**•** In cases of suspected resistance, the registrant will instruct growers to do one or more of the following:

o During the present season, use conventional insecticides to control the adult stage of the suspected pest;

o During the following season, use an alternati ve pest control method to deter establishment of potentially resistant insects.

The remedial action plan requires the use of alternate control measures to mitigate suspected resistance.
However, the BPPD IRM T eam notes that if Cry3Bb1 resistance has indee d developed (i.e. “ confirmed

resistance ” ), a 5% refuge for pyramids (i.e. SmartStax expressing Cry3Bb1 and Cry34/35 targeting corn rootworm) will be substantially less durable and could ultimately compromise the second unrelated toxin used to control the p est (i.e. in this case Cry34/35).- 7

- I. B ACKGROUND

Corn rootworm ( CRW ) is among the most serious economic insect pests of corn in the United States (Levine &

Oloumi

- Sadeghi, 1991)

. Western corn rootworm ( Diabrotica virgifera virgifera

, WCRW ) is found from

M exico to the U.S

. Corn Belt and Canada and is widely distributed ; Northern corn rootworm ( D. barberi

, NCRW ) is found in the Midwest of the U.S and ha s a localized distribution . Together, they are the most prevalent Diabrotica pests in the U

. S . The Mexican corn rootworm ( D. virgifera zea , MCRW ) has a distribution

from Central America to the southern U.S. and is a sporadic problem in central Texas and southern Oklahoma

. In 2003, the first t ransgenic ( Bt ) corn was registered to control CRW. Today, there are cu rrently three registered

Bt toxins available from different registrants to control target pest damage, all of which do not express a high

dose against CRW . Simulation modeling predicts that pests are at greater risk of evolving resistance to Bt crops when toxins are less than high dose (Tabashnik et al.

, 2004).

In February 2003

, the Agency approved Monsanto’s registration of Bacillus thuringiensis ( Bt ) Cry3Bb1 protein and the genetic material (Vector ZMIR13L) necessary for its production. Corn expressing t he Cry 3Bb1 protein, designated event MON 863 (YieldGard Rootworm, EPA Reg. No. 525

- 528) by Monsanto, was the first Bt corn product registered to protect crops against corn rootworm (CRW) species.

In December 2005, MON 88017 and MON 88017xMON810 were appr oved for registration. MON 88017xMON 810 expresses Cry3Bb1 and Cry1Ab and is targeted against corn rootworm (CRW) larvae (Cry3Bb) and European corn borer (ECB)/stalk boring lepidopteran larvae (Cry1Ab). The product was created by conventional breeding in w hich MON 88017 (EPA Reg. No. 524

- LLR) was crossed with MON 810 (Yield G ard, EPA Reg. No. 524

- 489). The Cry3Bb1 toxin expressed in MON 88017 is equivalent to that in

MON 863

. The Cry3Bb1 protein produced in MON 88017 and MON 863 is a variant of the wild

- type Cry3Bb1 protein from Bt subsp ecies kumamotoensis , whereas the Cry1Ab toxin originated from Bt subsp ecies kurstaki

. When compared by amino acid sequencing, the Cry3Bb1 protein expressed in MON 88017 has been reported to be 99.8% similar to the Cry3Bb1 pro tein expressed in MON 863. The primary difference between the hybrids is that MON 88017 also expresses a gene for resistance to glyphosa te (Roundup) based herbicides.

MON 89034 x MON 88017 is a plan t

- incorporated protectant (PIP) that was registered for commercial use on June 10, 2008. MON 89034 expresses the Bt

- derived insecticidal proteins Cry1A.105 and Cry2Ab2. The Cry1A.105 toxin is a “chimeric” protein containing domains I and II and the C

- terminal from Cry1Ac and domain III from Cry1Fa (domain III ). The Cry2Ab2 protein is exactly the same as that currently expressed in Monsanto’s Bollgard II cotton.

SmartStax was conditionally registered by the Agency in July, 2009 with a 5% structured refuge for lepidoptera and corn rootworm. However, as a con dition of registration

, EPA requested further data be developed to address the uncertainties with dose (Cry3Bb1 and Cry34/35) and initial resistance allele frequency to support a 5% refuge for corn rootworm. EPA further recommended revised modeling to asse ss the risk of resistance evolution in corn rootworm incorporating these additional data.

This memorandum summarizes (Section I and II) and reviews (Section III) Monsanto’s 2009 CRW resistance monitoring report for Cry3Bb1 corn and response to the Agency’ s recommendations for the proposed monitoring plan and questions regarding unexpected pest damage reports (BPPD 20 10 ).- 8

- I. MONSANTO 200 9 MONITORING REPORT (MRID 482080

- 01 )

Objective:

To c ontinue ongoing monitoring of susceptibility to Bt Cry3Bb1 protein in geographically distinct populations

of WCRW by contrasting susceptibility of 2009 collections to a lab strain as well as data from previous years.

Methodology: DM Crop Research Group (independent party collecting corn pest samples ) collected adult CRW samples from

13 geographically distinct field populations in Nebraska, Iowa, and Illinois. Those population samples were sent to Custom BioProducts (an independent laboratory conducting bioassays for Monsanto since 2006), which

maintained the insect colle ctions and provided eggs and subsequent neonates for testing . Twelve samples of the thirteen samples yielded enough insects to perform bioassays with six replicates; one sample yielded enough individuals for four replicates. In addition, a (Monsanto) non

- diapausing lab strain produced enough eggs for six control replicates.

The E. coli produced Cry3Bb1 used was a solution of 4.1 mg/ml supplied by Monsanto Company. This solution

was diluted with 0.1% Titron

- X 100 to obtain a series of concentrations of the Cry3Bb1 protein for bioassay.

B ased on the concentration of protein in the stock solution, the maximum concentration that could be used in

the concentration

- response assays was 170.8 μg/cm 2 of diet.

Neonate larvae were used in diet overlay bioassays and exposed to different Cry3Bb1 concentrations (10.7 μg/cm 2 , 21.4 μg/cm 2 , 42.7 μg/cm 2 , 85.4 μg/cm 2 , and 170.8 μg/cm 2 ). Thirteen μL of each dilution was applied to 12 individual wells and allowed to dry prior to larval introduction. Larvae were non

- systematic ally selected and placed into wells of the tissue culture tray. After three days, mortality and survival were recorded.

The University of Nebraska performed statistical analyses using SAS and Probit Analysis to determine EC 50 , EC 95 , LC 50 , and LC 90 values as well as goodness of fit ( Chi

- Square probability) for each population tested

.

Study Results and Discussion: Monsanto reported that 2009 bioassay results indicated that WCRW populations throughout the Corn Belt remained susceptible to Cry3Bb1 as express ed in MON 88017 and MON 863

.

Observed EC 50 values ranged from 13.9 μg/cm 2 (Hamilton Co, NE) to 63.61 μg/cm 2 (Franklin Co, IA)

, representing a 4 .5

- fold difference in susceptibil ity. The Franklin County population sample had the greatest

EC 50 values of al l 13 populations. The second highest EC 50 value (41.33 μg/cm 2 ) was found in Sherman County (NE). With results from Franklin County excluded , the EC 50 values of the field populations ranged from 13.90

- 41.11 μg/cm 2 and fell within the 95% confidence interval of the 2008 field populations (2.07

- 43.14 μg/cm 2 ). Monsanto concluded that although the EC 50 value for the Franklin population was relatively high, that the bioassay results showed that the population was still susceptible to Cry3Bb1.

LC 50 values were m ore variable than EC 50 values. Higher LC

- values however are an artifact of the assay system because larvae are able to survive without feeding. LC 50 values of field derived populations ranged from 60.29

- 726.40 μg of Cry3Bb1/cm 2 (excluding populations where the statistical program failed to produce probit results).- 9

- Table 1 . Susceptibility of Western Corn Rootworm Populations to Cry3Bb1 measured by EC /LC 50 s (2009 Data)

Population # of Reps EC 50 (95% C.I.) (μg of Cry3Bb1/cm 2 ) LC 50 (95 % C.I.) (μg of Cry3Bb1/cm 2 )

Franklin Co, NE 6 63.61 (40.75

- 113.01) *

McLean Co, IL 6 37.75 (30.19

- 46.89) 585.06 ( 216.57

- 30312 )

Clay Co, I L 6 14.12 (8.97

- 19.12) *

Champaign Co, IL 6 39.88 (37.16

- 42.77) 579.18 ( 246.06

- 7377 )

Sherman Co, NE 6 41.33 (30.19

- 56.38) 314.32

9169.59

- 15850

Story Co, IA 6 28.88 (22.32

- 36.20) 726.40 (231.61

- 265519)

Scott Co, IA 6 16.70 (11.52

- 21.82) *

Bureau Co, IL 6 18.29 (11.17

- 25.59) 277.53 (146.01

- 1495)

Howard Co, NE 6 40.61 (25.85

- 62.82) *

Hamilton Co, NE 4 13.90 (6.79

- 20 .76) 60.29 (36.65

- 119.73)

Iroquois Co, NE 6 26.14 (17.26

- 36.57) *

Ford Co, NE 6 18.55 (13.04

- 24.07) 188.14 (108.04

- 651.35)

Peoria Co, IL 6 21.71 (15.47

- 28.24) 194.32 (113.44

- 627.48)

Laboratory Control 6 9.25 (3.22

- 15.22) 51.59 (33.98

- 82.79)

*POLO fail ed to produce probit results for these collections

Monsanto tested the diagnostic concentration of 170.8 μg/cm 2 for the second consecutive field season and

determined that a lthough 100% mortality was not obtain ed, survivors at this concentra t i on exhibite d severe stunting ( as measured by average body mass). The reduction in average mass ranged from 1⁄8 to 1⁄4 (0.03

- 0.06 mg) of the control groups average mass (0.25 mg)

. For example, survivors of the Franklin County population had an average body mass of 0.06 m g
, which was comparable to other field populations and significantly less than the mass of all field populations at the control dose.- 10

- Table 2 . Mean mortality and average mass of survivors from field populations at the highest Cry3B b1

concentration

Population % Mortality Across Replicates Average Mass of Survivors ( mg ) Across Replicates Maximum Mass Across Replicates ( mg )

Franklin Co, NE 50.00 0.06 0.09
McLean Co, IL 43.06 0.05 0.06 Clay Co, I L 52.78 0.03 0.05 Champaign Co, IL 3 4.72 0.05 0.08
Sherman Co, NE 45.83 0.05 0.08 Story Co, IA 40.28 0.05 0.08
Scott Co, IA 48.61 0.04 0.06
Bureau Co, IL 48.61 0.03 0.05
Howard Co, NE 33.33 0.05 0.08
Hamilton Co, NE 66.67 0.03 0.05 Iroquois Co, NE 58.33 0.04 0.06
Ford Co, NE 48.61 0. 06 0.08
Peoria Co, IL 52.78 0.04 0.07
Ranges 33.33

- 66.67 0.03

- 0.06 0.05

- 0.09 Laboratory Colony 68.06 0.03 0.04

Table modified from Monsanto’s original submission (MRID 482080

- 01)

Table 3 . Control mean mortality and mass of surv ivors of field populations on non

- Bt containing diet

Control

- Field

Population s Mean % Mortality

Across R eplicates Average mass of

S urvivors ( mg ) Across

R eplicates Maximum Mass Across

R eplicates ( mg )

All sites 9.24 0.25 0.30

Table extracted from Monsan to’s original submission (MRID 482080

- 01)

II. MONSANTO’S RESPONSE TO THE BPPD IRM TEAM ’S REVIEW OF THE APPLICANT’S 2008

MONITORING PLAN

Monsanto stated that it utilize d the root damage threshold level of 1.5 (on the 0

- 3 Node Injury Scale, NIS) in

Bt field s with greater than expected damage (GTED) and 50% of the sampled plants with >1.5 to determine if additional actions were needed. High rootworm pressure was typically the result of continuous

- corn plantings that built up CRW population densities over time or presence of diapausing variant of the NCRW or soybean

variant of WCR W (in corn

- soy

- corn crop rotations). Additionally, Monsanto proposed that a threshold of at
least 50% of the sampled plants with >1.0 node injury would be better for a pyramided produc t such as

SmartStax.

Monsanto investigated all product performance incidents using a standardized process for collection of agronomic and field information. Afte r the grower s informed local sales seed representative s (LSSR) , the sale

rep s investigate d the claim and collect ed information as well as representative root samples from the field (and adjacent refuge field if present). At this point, if the node injury damage was less than 1.5, Monsanto

stated that the LSSR work ed to understand the damage and res olve d the incident with the grower s . If the node injury damage was greater than 1.5, seed representative s work ed with the technology development- 11

- representative to assess the situation and verif ied if average root damage was greater than 1.5 and 50% of the

roots were damaged in the field of concern. After this assessment, they decide d whether the results met the criteria to trigger CRW beetle sampling. Sampling would occur at this time or

, if adults were no longer present, the field would be monitored the fo llowing year for root damage. If the root damage rating exceed ed 1.5 at that time, adults would be collected in the problem fields.

Monsanto reported that i n 2007 and 2008, five and eight samples of adult beetles, respectively were collected

from fields w ith excess damage (>1.5) and sent to Custom Bio

- Products for bioassays. No populations were collected from fields with performance issues in 2009 due to late notifications. None of populations that

stemmed from individuals collected in 2007 and 2008 showed a significant decrease in susceptibility relative

to histor ical results and routine monitoring conducted in those years (no actual bioassay data were submitted in this report) . For example, in 2008 Monsanto responded to 32 individual field performance iss ues (see Table

4 ). Of those only three had node injury scores >1.5, and in only two fields beetles could be collected. Additionally, Monsanto collected CRW samples in Colorado, Illinois, Kansas, and Nebraska where historical

pest pr essure and root damage were high. Eight populations were reared in the lab

, and standard diet bioassays

were conducted with neonates. The conclusions from the assays were that all eight populations remained susceptible to Cry3Bb1 when compared to populatio ns assa yed in the routine monitoring. Monsanto reported

that t he reasons for the ‘greater than expected damage’ in Cry3Bb1 corn were uncertain.

Table 4 . Number of performance inquiries by state and year for MON 88017 and MON 863 products

Table extracted from Monsanto’s submission (MRID 484368

- 01) *calculations based on 2.6 acres per unit of seed sold; SmartStax is not included in the 2010 statistics

1 PIs = performance inquiries

The BPPD IRM Team ( BPPD 2010) raised concern s with Mon santo’s protracted response to unexpected damage which could take up to two seasons before the registrant responds to a potential resistance event (according to t he BPPD IRM Team ’s internal analysis of Monsanto’s reports). Monsanto responded that their pol icy as good stewards of MON88017 and MON 863 was to respond to every case of GTED (greater than expected damage) and collect adult insects whenever possible. When Monsanto is contacted to o late in the State 2003 2004 2005 2006 2007 2008 2009 2010

CO 0 0 0 9 3 12 23 13

IL 0 13 4 13 15 0 3 0

IN 0 0 0 0 0 0 0 0

IA 0 0 2 11 21 3 23 13

KS 0 3 3 2 13 4 8 7

MN 0 0 2 0 0 1 15 3

NE 0 5 12 58 46 11 30 47

OH 0 0 0 0 1 0 0 0

SD 0 0 0 0 3 0 0 9

W I 0 0 4 0 0 0 4 1

Other 0 0 5 1 1 1 1 1 5

Total CRW

related P Is 1 0 21 32 94 103 32 107 98

MON CRW Bt

Acres * 354 1586 3271 9227 19869 28949 30631 28747

MON 863

Acres * 354 1586 3271 9227 18385 5899 2018 800

MON 88017

Acres * 0 0 0 0 1484 23050 28613 27 947- 12

- season to collect adults, the area of concern is monito red the following growing season

, and if GTED occurs again, adults will be collected

.

The BPPD IRM Team ( BPPD 2010) also disagreed with Monsanto on their plan to collect (potentially resistant) insects >1/2 mile away from problem fields. Monsanto clarifie d that once the GTED had been established in a

field t hat Monsanto representatives would collect 500

- 1000 individuals from the neighboring area within a 1 to 2 mile radius of the problem site.

In 2009, Dr. Ostlie reported Cry3Bb1 field failures in norther n Minnesota and both NCRW and WCRW adults were present in abundant numbers. The BPPD IRM Team ( BPPD 2010) requested that Monsanto provide a

detailed description about follow up activities in those areas of Minnesota. Monsanto reported that in most cases

th e root damage rating did not exceed 1.5 in 50% of the Cry3Bb1 plants , hence no further action was pursued

. In the Cry3Bb1 fields with GTED, no adult collections were made in 2009 because the reports were received

too late, and the same damage was not obser ved the following year. Monsanto has been working with Dr. Ostlie

in Minnesota to better understand the reports of GTED. However, Monsanto responded that in 2010 they followed up on performance inquiries in two affected southern Minnesota fields that were near a site that had reported problems in 2009. No adults were collected because no GTED was observed. Additionally, two populations in an affected area were sampled in South Dakota. Those were near 2009 Minnesota problem sites

and were included to initiat e additional monitoring in the area and to establish a baseline in the area.

The BPPD IRM Team ( BPPD 2010 ) recommended that Monsanto include MN and WI into their ABSTC sampling region 2 beginning with the 2009 CRW monitoring collections . In addition, we also recommend that

Monsanto obtain samples from the eastern IA region where Gassmann et al. (2011) reported Cry3Bb1 durability

issues. Monsanto stated that beginning in 2010

, ABSTC included additional population samples from areas in

Iowa and Minnesota , t hough none were collected in 2009 (b eetle densities were, however, low making it impossible to successfully collect in most regions )

. Additionally, Monsanto included three new locations in

northeastern IA and two new sites in SD from where beetles were col lected during the 2010 growing season (results to be submitted to Agency in 2011).

The BPPD IRM Team ( BPPD 2010 ) further requested that Monsanto report on the progress of developing assays for northern corn rootworm before March 31, 2011

. The BPPD IRM Te am also recommended that Monsanto take up monitoring and the development of baseline susceptibility data for NCRW as outlined in the

terms and conditions of the registration; join with other registrants and develop an ABSTC style unified sampling strategy for CRW based on high risk and adoption of PIPs. Monsanto re sponded that efforts were underway through ABSTC to review the available knowledge on NCRW rearing and bioassay, and to investigate any promising ne w developments but that presently no labs were successfully rearing and testing th e northern corn rootworm; companies joined and established a unified sampling strategy for WCRW (through ABSTC) and had established sampling regions.

The BPPD IRM Team ( BPPD 2010) had identified a deficiency in Monsanto’ s response to t he t erms and conditions of registration in that Monsanto had not reported whether IRM strategies designed for WCRW and

NCRW were appropriate for the Mexican corn rootworm. Monsanto reported (MRID 484368

- 01) that WCRW

and MCR W appear to be ge netically similar (Szalanski et al. 1999)

.- 13

- III. BPPD IRM T EAM REVIEW AND CONCLUSIONS

1. 200 9 MONITORING DATA

The BPPD IRM Team reviewed Monsanto’s LC 50 values for the 2009 CRW monitoring season (highest

concentration of 1 70 μg /cm 2 was used ) and note s that m ost mean values were extrapolations from lower

concentrations because populations were no longer sensitive at this level of exposure. Monsanto and the BPPD

IRM Team agree that LC 50 results can be somewhat misleading when correlating larval survival to leve l of toxin susceptibility because larvae can survive on Bt for some time without feeding. A 3

- day long assay may, therefore, capture less than the actual percent mortality due to Cry3Bb1 exposure.

W hen looking at the historical EC 50 and LC 50 ranges for f ield populations

, it appears that there is a visible downward trend in susceptibility since 2005 ( Table 5 ). Much of the apparent reduced susceptibility occurred between 2006 and 2007 and is likely due to a change in toxin source to less active version of Cry3Bb1 (see BPPD 2009)

. Because of this

, data collected prior to 2007 cannot be directly compared to the more recent

results. Nevertheless, the results show that field

- collected populations have been generally less susceptible to Cry3Bb1 than the laboratory control colonies. For example, all of the field populations collected in 2009 had greater (mean) EC 50 and LC 50 values (measure d and extrapolated) than those for the lab populations, in some

instances by an order of magnitude ( see Table 1)

. This was also the case in the 2007 collections (though not in 2008).

One population from Franklin County , Nebraska had a mean EC 50 value that fell outside of the 95% confidence interval of the 2008 field populations (2.07

- 43.14 μg/cm 2 ). M onsanto reported that there was no reason for concern because the mean weight of the sample was approximate ly one third that of the mean weight of the control population (see Tables 2 and 3 )

. It is unclear from the submitted report whether these individuals were

tracked to potential adulthood after the assay s were terminated or whether Monsanto decided that the decrease in weight meant that individuals were essentially unfit to survive. The BPPD IRM Team recommends that

Monsanto clar ify this point and concludes that the highest concentration tested (170 μg of Cry3Bb1 /cm 2 of diet) does not appear to be adequate for monitoring as a diagnostic con resistant in WCRW populations. The

EC/LC 50 appears to detect shifts in WCRW susceptibility, but the bioassay results do not appear to be sensitive enough for discerning between populations with decreased susceptibility from those with actual resistance to
the toxin

. It is recommended that Monsanto discuss with the Agency whether the ‘comparativ e weight’ approach between Bt and non

- Bt treatments for field populations could be used to develop a “ viable ” resistance

detection level.

The only county from which population samples were obtained both in 2009 and 2008 was McLean (IL). In 2008, the EC 50 and LC 50 means were14.6 μ g/cm 2 and 38.7 μ g/cm 2 , and their minimum and maximum values ranged from 7.5 to 21.4 μg /cm 2 and 19.1 to 63.6 μg /cm 2 , respectively; in 2009, the EC 50 and LC 50 mean values

were 37.75 μg /cm 2 and 585.06 μg /cm 2 (extrapolated value), a nd their minimum and maximum values ranged

from 30.19 μg /cm 2 to 46.89 μg /cm 2 and 216 to 39312 μg /cm 2

, respectively. T he 2009 values were a two

- fold

and > 100

- fold increase for mean EC and LC

- values, respectively , compared to 2008 values . The maximum

values of the ranges of 2008 increased by two

- and >60

- fold for estimated EC 50 and extrapolated LC 50 - values respectively. M ost m ean LC values in 2009 could not be measured at the highest concentration and had to be extrapolat ed based on treatments with lower conc entrations.

Monsanto tested CRW po pulations from three states : Nebraska, Illinois, and Iowa. The BPPD IRM Team notes that these three states alone may not be sufficient and that Colorado, Minnesota, and western Wisconsin may- 14

- need to be included based on Monsanto’s information (Table 4) regarding Cry3Bb1 performance issues and

other discussions with corn entomologists (Gassmann et al., 2011b).

Regarding the identified deficiency with respect to the terms and conditions of the registration (BPPD 2010)

, Mo nsanto stated that Mexican and western corn rootworm were genetically similar and that therefore the WCRW IRM strategy should be appropriate for the MCRW. The BPPD IRM Team generally agrees that the assumptions may be appropriate . We note, however, that t here could still be differences in the specific dose profile that should be addressed.

Table 5

. Susceptibility results across years for Cry3Bb1

Year EC 50 ( μg/cm 2 ) LC 50 ( μg/cm 2 )

field collected laboratory field collected laboratory

2005 0.2 8

- 2.64 N/A 0 .31

- 4.59 N/A

2006 0.64

- 1.88 N/A 1.43

- 22.22 N/A

2007 14.20

- 33.46 12.91 50.18

- 289.25 22.29

2008 7.3

- 30.4 21.8 24.5

- 335 87.9

2009 13.9

- 63.61 9.25 60.29

- 726.40 51.59

T able generated from data submitted by Monsanto.

2. MONSANTO’S MONITORING PLAN AND FU RTHER RESPONSES TO BPPD (2010)

After review ing Monsanto’s monitoring plan, the BPPD IRM Team concludes that the applicant’s current resistance monitoring program is ineffective and likely to miss early resistance events. This conclusion was

based on the f ollowing observations:

a. The registrant proposed to collect insects from problem fields within 1

- 2 miles from neighboring sites of

failed fields

. The BPPD IRM Team believes that this proposal is insufficient if the goal is to determine

whether Bt failure is due to resistance of CRW population. Since the majority of adult corn rootworm

may not disperse long distances , the greatest probability of capture of resistant genotypes should be in

the problem fields, possibly in adjacent fields, but less likely in fie lds 2 miles away during that particular year. To collect in neighboring sites is reasonable after infield collections have occurred or during the following year because of possible adult movement

. It is imperative that t he language for this crucial

pr ocedu re is unambiguous and clear and that Monsanto and EPA come to a timely agreement on the method of collecting adult CRW in problem fields. If the resistance m onitoring program is to be meaningful

, a more proactive, effective approach needs to be adopted

.

b. T he BPPD IRM Team concludes that Monsanto’s GTED threshold of 1.5 ( on the NIS) for a single toxin

PIP with non

- high dose expression is generally too high. The BPPD IRM Team stated in the 2010 review that injury ratings of 1.0 should trigger investigations

. Ideally, a comparison should occur between performance of Bt and nearby refuge corn; however, this may not be an option if grower s have

not compl ied with refuge requirements

. In such non

- compliant cases, the probability that severe root damage is due to re sistance should be greater and, therefore, the BPPD IRM Team considers threshold

level of 1.0 for a single toxin PIP a more defensible (conservative) trigger to initiate adult collections. During years with great pest pressure (not associated with agronomi c practices such as growing- 15

- continuous corn) , EPA notes that it may be reasonable to deviate from the 1.0 threshold and use 1.5 ( based on a graded scale where Bt damage is related to adult density and damage to refuge corn )

. The

BPPD IRM Team concludes tha t f or SmartStax products ( i.e. SSX RIB ) , a threshold of 1.0 (as proposed by Monsanto) is not conservative enough; this was already discussed in BPPD (2010). The pyramid has greater activity against CRW and should, therefore, have a lower threshold (such as 0.5 ) to trigger beetle

collections

. Additionally, in a seed blend it will be inherently more difficult to detect Bt performance issues, t herefore, i t may not be realistic to use the following threshold “50% of the plants must show greater than 0.5 (NIS) d amage” before an investigation occurs. The BPPD IRM Team proposes that any clearly visible sign of damage exceeding the threshold level (0.5) in a seed blend (irrespective of the percentage of Bt plant damage in that field ) trigger adult collections

.

c. If Monsanto is unable to collect adults from problem fields because they were contacted too late by seed

dealers , the n it is important that adult sampling occur s in the problem areas the following season irrespective of pest pressure and damage in the old pro blem fields . If growers sprayed their fields after they observed high densities of adults during a problem year or used other methods of control , then the

pest pressure and subsequent Bt damage should be less severe the following year. If Monsanto does not collect in such problem areas during the following season because of “ below threshold damage ” in Bt

fields (1.0 for single PIPs and 0.5 for SSX) , th en they will miss early resistance events in those areas. If there is resistance but low pest pressure, dam age can be expected to be lower than in cases where there

is high pest pressure coupled with resistant individuals in the population

.

Monsanto reported that h igh rootworm pressure was typically the result of continuous

- corn plantings that built up CRW po pulation densities over time and used this reasoning as a justification to use a GTED threshold level

of 1.5. The BPPD IRM Team notes that continuous planting of corn with Cry3Bb1 without rotation also leads to greater selection pressure. Hence, unexpected pest damage in such Cry3Bb1 fields with agronomic practices as described by Monsanto should be reason for concern and has a greater probability to be due to resistance evolution in that population. T herefore, the BPPD IRM Team maintains that a threshold l evel of 1.0 for a single

toxin PIP is a more appropriate trigger to initiate adult collections.

Monsanto stated that they had been working with Dr. Ostlie in MN to understand the greater than expected root damage observed in Cry3Bb1 corn during the late c orn growing season in 2009. The BPPD IRM Team interprets this to mean that Monsanto is unsur e of the reasons for Bt failure and notes that this is an example of instance where collections should have occurred the following year ( despite low er damage levels or population

numbers ) . If growers ‘bombed’ adults in fall 2009 or used IPM approaches to manage their fields , it sh ould be expected that population densiti es and subsequent root damage were low er in 2010. However, resistance genes

could still be in the se population s
, which is the reason why adult collections should be made once unexplained performance failure s have occurred.

Monsanto determined additional problem areas in South Dakota that were included into the sampling scheme

for annual resistance moni toring in 2010 and proposes to use these to obtain baseline susceptibility data. These sites should not serve to obtain baseline data but rather to measure shifts in CRW susceptibility to Cry3Bb1

.

Monsanto provided EPA with the number of performance inqui ries for their Cry3Bb1 products in the Corn Belt from 2003 through 2010. The number of PIs ranged from 0 to over 100 per year d uring this time period. Monsanto reported that none of their follow up investigations resulted , however, in finding resistant

pop ulations. With the exception of samples collected in 2007 and 2008, it is unclear to the BPPD IRM Team how many of the PI’s reported in Table 4 resulted in adult collections and bioassays

. This information should be provided for each state (each year) in a ddition to reporting the number of PIs. Furthermore, the BPPD IRM

Team recommends that Monsanto include information about the degree of pest pressure and environmental- 16

- conditions in those states with PIs ( i.e. wet spring, etc.).

The BPPD IRM Team notes t hat Dr. Gray provided information that supports Monsanto ’s reporting of having

had no Cry3Bb1 performance inquiries during the 2010 corn growing season in Illinois . Dr. Gray stated that damage to Cry3Bb1 maize was mild and that generally adult population l evels were low throughout the state. A wet spring was likely responsible for these conditions (Gr ay (2011b), personal communication ).

The BPPD IRM Team recommends that Monsanto provide an update on the development assays, resistance monitoring, and the d evelopment of baseline susceptibility data for northern corn rootworm with the next monitoring report.

3. THE BPPD IRM T EAM’S REVIEW OF UNEXPECTED CORN ROOTWORM DAMAGE IN

CRY3Bb1 FIELDS AND CONCLUSIONS REGARDING SUSPECTED RESISTANCE TO

CRY3Bb1

The BPPD IRM Team has been made aware of a number of cases of unexpected corn rootworm damage to Cry3Bb1 fields. Some of these reports have been documented in the literature, while others are anecdotal. This section summarizes these reports and contains the BPPD IRM T eam ’s analysis regarding “suspected resistance” to Cry3Bb1 corn.

During a presentation, Dr. Gassman n alerted the scientific community at the Entomological Society of America (ESA) meeting in San Diego (2010) that w estern corn rootworm had evolved resistan ce to Monsanto’s Cry3Bb1

corn in certain Iowa locations. T he official report w as subsequently published by Gassmann et al. (2011 a ). The following is the BPPD IRM Team ’s summary of the publication.

In 2009, Dr. Gassmann was alerted to severe feeding damage in four Cry3Bb1 fields in eastern Iowa. At that time, adults were collected in reported problem fields as well as in five non

- Bt control fields throughout the

state

. Rootworm damage was assessed in Bt fields but not the control fields. Interviews with far mers revealed

that growers had planted Cry3Bb1 maize for at least three continuous seasons in problem fields. No information about compliance was gathered although the authors speculated that non

- compliance may have contributed to

the observed effects

. The adults collected during 2009 were held in separate cages and allowed to mate at the Iowa State University . The resulting eggs/neonates were examined using a 17

- day long seedling assay on plants that were grown in the greenhouse. The assay used two differe nt Bt expressing seedlings (DeKalb Cry3Bb1 and Mycogen Cry34/35) and DeKalb and Mycogen non

- Bt varieties as controls. Survival of larvae from Cry3Bb1 problem fields was three times higher and significantly greater than survival of larvae from control fiel ds reared

on non

- Bt expressing plants. Gassmann et al.’s preliminary analysis suggested that WCRW resistance was incomplete, that problem fields contained a mixture of resistant and susceptible genotypes, or that a

combination of these conditions was prese nt. Survival of control larvae

, however, was significantly lowe r on Cry3Bb1 maize compared to non

- Bt expressing plants. Offspring of adults from problem field s had significantly

lower survival on Cr y34/35 compared to non

- Bt maize, and no difference was det ected between survival on Cry34/35 of offspring from problem and control fields.

A significant positive correlation was detected between the number of years growers chose to plant Cry3Bb1

maize and the survival on Cry3Bb1 maize of insects from problem fie lds

. The researchers concluded that

WCRW was evolving resistance in some areas of Iowa and that the following factors likely supported the

evolution of resistance: non

- high dose of Cry3Bb1 c orn resistance is non

- recessive and insufficient insects from

refu ge populations. Gassmann et al. (2011 a ) suggested that there was a direct corollary between their findings in the problem fields ( Cry3Bb1 grown for at least three consecutive years ) to the lab experiment by Meihls et al. (2008) where WCRW resistance evolve d after just three generations of selection on Cry3B maize.- 17

- During a conversation with the BPPD IRM Team , Dr. Gassmann explained that damage in the same 2009 Iowa problem fields did not occur in 2010 and 2011 because growers typically change their practi ce to avoid further field failures the following year (i.e. adult bombing, planting other PIPs, other IPM methods) . He noted though that similar Cry3Bb1 field damage occurred just a few miles from where the original field failure s were reported (Gassmann 2 011 , personal communication)

.

Additionally, the BPPD IRM Team has been made aware of severe Cry3Bb1 field damage in western Illinois

near the sites of the field failures reported by Gassmann (Gray 2011 b , personal communication ). A report published by the University of Illinois described severe performance issues with Monsanto’s Cry3Bb1 corn in Henry and Whiteside County (Gray 2011 a )

. The problem fields had been planted to continuous corn for many years, Cry3Bb1 maize was planted to protect against WCRW dam age , and similarities to nearby Iowa problem fields identified in Gassmann et al. (2011 a ) were noted. Between two to three nodes were missing on damaged root systems ( G assmann et al. 2011 b ); adults were numerous in thes e fields; lodging was prevalent; inse ct
collection s were made to determine whether resistance was the cause of Cry3Bb1 field failures. In the report it

was proposed that growers who encountered field

- performance issues with CRW protected traits should consider rotating to a non

- WCRW host, us ing CRW soil insecticide at planting and different CRW PIP, and/or planting a pyramided PIP expressing multiple Cry toxins in 2012 but that a long term IPM approach was

necessar y for successful CRW management (Gray 2011 a )

. On September 23, 2011, Dr. Gray published another report describing more Cry3Bb1 field failures (several nodes were completely missing from the roots) in north

central Illinois ( i.e. LaSalle C ounty ). The affected fields shared that they had been grown to Monsanto’s Cry3Bb1 corn for many successive years without crop rotation (Gray 2011 c ).

Over the course of 2011, the BPPD IRM Team has had multiple conference calls with scientists from academia

and USDA

- ARS to discuss performance of Bt corn in the Corn Belt. During a recent call on Augus t 25, 2011,

the BPPD IRM Team discussed with these corn entomologists the performance issues regarding Monsanto’s Cry3Bb1 maize in Illinois ( Gassman n et al. 2011b ). At that time, the BPPD IRM Team was informed that there

were other severe performance issue s for Monsanto’s Cry3B b1 trait in Nebraska (spotty though throughout the state) , Minnesota ( north

- central and southern locations ) , and Iowa in 2011 (some areas as discussed in Gassmann et al. 2011a)

. Specifically in Minnesota, Dr. Ostlie reported that 2011 marked the third year that moderate to severe corn rootworm damage in Cry3Bb1 corn was observed and that the problem was becoming more widespread. In Nebraska, Drs. Siegfried, Meinke

, and Hunt reported that the 2011 growing season marked the fourth year w here moderate to severe rootworm damage in Monsanto’s Cry3Bb1 corn was apparent . As in Illinois and Iowa, these problem fields were planted to continuous corn with Cry3Bb1 for several years in a row (cattle farmers who need corn for feed or growers who pla nted corn for ethanol production); no rotation with other crops occurred. It was unlikely that refuges were planted. Gravid females were collected from these sites and their offspring will be analyzed. According to these researchers

, many growers still pla nt this trait despite the incidences of rootworm damage . When growers encounter great numbers of adult corn rootworm, they

convert back to adult beetle “bombing” so that Cry3Bb1 can be planted again the following season.

The BPPD IRM Team reviewed Gassman n et al.’s report (2011 a ) and notes that population samples tested in the assay originated from within problem fields where a certain proportion can be assumed to have undergone development (egg hatching, immature development and emergence to adult hood ). S ome of the collected individuals may have also immigrated into these Bt fields from neighboring fields. If this was the case, then the resistance observed and described by Gassmann could be “diluted”. More important ly though, th ose native

adults fed on and finished their development in Cry3Bb1 maize, were viable, and produced offspring that were also resistant . The progeny tested in greenhouses had significant and much greater survival on Cry3Bb1 maize

than larvae fed on non

- Bt maize and whose parental line s origi nated from control fields. The proximity of

western Illinois fields with failed Cry3Bb1 maize is disconcerting

. The BPPD IRM Team concludes that- 18

- Gassmann et al. demonstrated that there is increased WCRW tolerance to Cry3Bb1 in some eastern Iowa fiel d populations, and that the observed response had a heritable component. The researchers did not analyze or report if the progeny survived to adulthood. To fully characterize these pop ulations the BPPD IRM Team

recommends additional work be conducted on th ese populations to assess survival to adulthood, fecundity, and any other reproductive effects that could impact the populations ’ ability to survive in the field.

Overall, t he BPPD IRM Team concludes that Cry3Bb1 resistance is suspected in at least some portions of the four states in which these reports originated (IA, IL, MN, and NE). This conclusion is based on the following weight of evidence:

**•** Gassmann et al. (2011a) showed that WCRW field populations in eastern Iowa had significant and

increased sur vival on Cry3Bb1 ma ize compared to populations collected from non

- problem fields and assayed on non

- Bt maize. The researchers did not report , however, whether the progeny

survived to adulthood.

**•** Two recent reports published by the University of Illinois di scussed severe Cry3Bb1 corn damage in

northwestern and north

- central Illinois during the 2011 corn growing season (Gray 2011 a, 2011 c ). According to Dr. Gray’s report, Henry County was among those that had severe Cry3Bb1 damage.

M onitoring data supplied by Monsanto (see Table 6 ) from this county showed a six

- fold increase in

mean LC 50 from 2007 to 2008 (the last year data were tabulated specifically from this county)

, though this result can not be directly correlated with any incidents of field damage

.

**•** Ther e are other reports of multiple and increased Cry3Bb1 performance failures in Nebraska and Minnesota (Gassmann et al. 2011b). Although Cry3Bb1 field failures throughout four states of the Corn Belt are spotty (IA, IL, MN and NE,) , the frequency of such rep orts ha s apparently increas ed (through the 2011 season)

.

**•** Resistance monitoring data for Cry3Bb1 have shown that field

- collected populations (collected from Illinois, Iowa, and Nebraska) are generally less susceptible to the toxin the laboratory reference colony. In 2009, every field

- collected population had a higher EC 50 and LC 50 than the laboratory control, in some cases by an order of magnitude. This was also the case for populations collected in 2007 (see BPPD 2009). Monitoring data collected from 20 05 to 2009 appear ed to show a large decrease in susceptibility over the time period (see Table s 5 and 6 ), however, the great drop off in susceptibility between 2006 and 2007 wa s likely due to a change in toxin source.- 1 9

- Table 6 . Susceptibility in Three Co unties across the Corn Belt as Measured by EC 50 and LC 50 Values (2005

- 2008 Data)

Population Mean EC 50 ( μg/cm 2 ) Mean LC 50 ( μg/cm 2 ) Scott County, IA (2005) 0.3 0.5 Scott County, IA (2006) 1.7 6.6 Scott County, IA (2007) 15.5 63.8 Scott County, IA (2008) 25.8 40.0
Henry County, IL (2005) 1.9 3.2 Henry County, IL (2006) 1.9 5.6 Henry County, IL (2007) 16.2 50.2 Henry County, IL (2008) 14.8 300.9
Seward County, NE (2005) 2.6 3.3 Seward County, NE (2006) 1.3 9.3 Seward County, NE (2007) 14.2 64.2

Sew ard County, NE (2008) 9.4 335.0
Monsanto Reference Strain (2007) 12.9 22.3 Monsanto Reference Strain (2008) 21.8 87.9

T able generated from data submitted by Monsanto.

Based on the conclusion of “suspected resistance”, the BPPD IRM Team recommends that the appropriate remedial action plan be implemented for Cry3Bb1 corn in areas experiencing unexpected field damage. The remedial action plan includes the following steps for “suspected resistance”:

**•** In cases of suspected resistance, the registrant will ins truct growers to do one or more of the following:

o During the present season, use conventional insecticides to control the adult stage of the suspected pest;

o During the following season, use an alternative pest control method to deter establishment of pot entially resistant insects.

The remedial action plan requires the use of alternate control measures to mitigate suspected resistance.
However, the BPPD IRM Team notes that if Cry3Bb1 resistance has indeed developed (i.e. “confirmed resistance”), a 5% refu ge for pyramids (i.e. SmartStax expressing Cry3Bb1 and Cry34/35 targeting corn rootworm) will be substantially less durable and could ultimately compromise the second unrelated toxin used to control the pest (i.e. in this case Cry34/35).- 20

- I V

. REFERENCES

BPPD. 20 09 . Review of Monsanto’s 200 5

- 2007 Corn Rootworm Monitoring Report and Revised CRW Resistance Monitoring Plan for MON 88017, MON 88017 x MON 810, and MON 89034 x MON 88017 (EPA Reg. Nos. 524

- 528, 524

- 545, 524

- 551, 524

- 552, and 524

- 576) . Memo from J C Martinez to Mike Mendelsohn

dated August 24, 2009

.

BPPD. 2010. Review of Monsanto’s 2008 Corn Rootworm Monitoring Report and Revised CRW Resistance Monitoring Plan for MON 88017, Mon 88017 x MON 810, MON 863, MON 863 x MON810, MON 89034 c

TC1507 x MON 8 8017 x DAS

- 59122

- 7, and MON 89034 x MON 88017. Memo from JC Martinez to Mike Mendelsohn dated June 30, 2010.

Gassmann AJ, JL Petzold

- Maxwell, RS Keweshan, and MW Dunbar. 2010. Screening field

- populations of western corn rootworm for susceptibility to Bt c orn. Presentation at the Entomological Society of America in San Diego, December 2010.

Gassmann AJ, JL Petzold

- Maxwell, RS Keweshan, and MW Dunbar. 2011 a . Field

- evolved resistance to Bt maize by western corn rootworm. PLOS one, Vol. 6 (7): 1

- 7

Gassmann A , M Gray, R Hellmich , T Hunt, L Meinke, B Siegfried, and K Ostlie. Personal communication with JC Martinez and A Reynolds on August 25 , 2011 b

.

A Gassmann, personal communication with JC Martinez on September 14 th , 2011.

M Gray. Severe root damage to Bt c orn observed in northwestern Illinois. The Bulletin, No. 20, August 26,

2011 a

. http://bulletin.ipm.illinois.edu/article.php?id=1555

M Gray, email communication with JC Martinez on August 1 8, 2011 b

.

M Gray. Additional reports of severe rootworm damage to Bt corn received: questions and answers. http://bulletin.ipm.illinois.edu/article.php?id=1569 . The Bulletin, No. 22, Sept ember 23, 2011 c

.

G Head. 2011. Conditions of registration for MON 88017 and MON 89034 x MON 88017, and response to EPA’s request for additional information on Monsanto’s resistance corn rootworm monitoring program. Monsanto unpublished report to the Agenc y (MRID 484368

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