OFFICE OF PREVENTION, PESTICIDES, AND

TOXIC SUBSTANCES

MEMORANDUM September 22, 2008

SUBJECT: Review of Pioneer’s Benefits Do cument in Support of a Sec(3) Product Registration for Optimum® AcreMaxTM 1 Insect Protection, also known as Stack Herculex XTRA (Cry34/35Ab1 +Cry1F) with Herculex I (Cry1F, refuge seed for CRW) [MRID 473567-10 and supplemental data]

TO: Mike Mendelsohn, Regulatory Action Leader

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

FROM: Jeannette Martinez, Biologist

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

SECONDARY Alan Reynolds, Entomologist

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

ACTION REQUESTED:

BPPD 1 has been asked to review the Benefits Document submitted by Pioneer Hi-Bred, International, Inc. in suppor t of the Optimum® AcreMaxTM 1 Ins ect Protection registration. The product, for which Pioneer Hi-Bred, International, Inc. seeks a regi stration, is a corn seed blend containing seeds that express Cry34Ab1, Cry35Ab1, and Cry1F in a Bt stack for corn rootworm and lepidopteran protection and seeds that expr ess the event Cry1F for lepidopteran protection and constitute an incorporated refuge for corn rootworm. In a ddition, Pioneer seeks to 1) reduce the non-Bt CRW refuge requirement from 20% to at least 2% seed mix, and 2) replace the 20% CRW refuge requirement for the common ref uge with the 100% HXRW (Cry34/35Ab1) option of up to 25% refuge.

1 The use of BPPD in this review refers to BPPD IRM team. UNITED STATES ENVIRONMENTAL PROTECTION AGENCY WASHINGTON, D.C. 20460- 2 - I. CONCLUSIONS AND RECOMMENDATIONS

1. BPPD notes that many of the uncertainties noted below are also germane to Insect Resistance Management (IRM). BPPD will be conducting a separate assessment of IRM for Optimum® AcreMaxTM1 (OAM1) and will also include a disc ussion of these issues in that document. Pioneer may address these uncertainties (and a ny deficiencies) as a part of the IRM plan development for OAM1.

2. Provided that Pioneer addresse s the uncertainties described below, BPPD concludes that OAM1 should have the following benefits: 1) redu ced pesticide use (in refuge), 2) increased grower compliance with IRM requirements, a nd 3) grower benefits (including ease of planting).

3. Restricted use pesticides are still used today in combination with Bt products to combat CRW infestations. Pioneer states that Optimum AcreMax1 would contribut e to a reduction of such conventional pesticides because of incr eased rootworm protection (reduced refuge) and thus result in benefits to field workers, by-st anders, and environment. BPPD notes that while this assumption is probable, Pioneer did not provide field efficacy studies comparing rootworm control between blocked refuge (c urrent IRM option) and seed blend refuge (proposed option) but rather between different percentages of seed blends. Therefore, the above claimed ‘direct’ benefits, while possible, are not based on hard data but assumptions.

4. BPPD agrees that a seed blend product containing the refuge al ready in the bag would lead to 100% grower compliance for planting a refuge. However, whethe r refuge plants will be randomly distributed and the correct % of ref uge is planted depends on the distribution of refuge seed in the bag at time of planting and non-biased ‘dropping’ of seeds, i.e. properly functioning planting equipment should refuge se ed and Bt seed not have same phenotype. Furthermore, BPPD does not have data to a ssess the accuracy of Pioneer’s new seed mixing technology (see discussion under 6).

5. Pioneer states that Optimum AcreMax 1 would reduce economic damage to corn growers by providing maximized CRW protection on all acres w ith a reduced in-field seed blend refuge

of ≥ 2% and eliminating the non-Bt corn for CRW protection in common refuge for lepidopterans and CRW. Pioneer did not provide fi eld efficacy studies comparing rootworm control between blocked refuge a nd seed blend refuge, and so ther e is no data to support this claim. In addition, whether Optimum AcreMax 1 would reduce economic damage through reducing the in-field refuge to ≥ 2 % rather than any other percen tage tested is also not as obvious as the registrant repor ts (see discussion under 9).

6. Pioneer states that HXX and HX1 refuge s eeds were uniformly mixed by hand 10 times before being packed into bags. After having reviewed the ‘Trait Durability Plan’, BPPD learned that Pioneer plans on bl ending seeds with modern te chnology (no further description provided) to accurately control th e blend ratio in a uniform pro cess. Pioneer states that the resulting blended products will be tested to en sure acceptable plantabil ity for growers. BPPD notes that the accuracy of this new technology should have been field tested prior to the submission of the registration request for Op timum AcreMax 1. In addition, the registrant- 3 - does not provide any description of this new technology. BPPD requests that Pioneer provide a description of their new mixi ng technology and data to demonstr ate accuracy (repeat of the dropper experiment, including reporting of standard error).

7. Pioneer assessed the distribution of refuge in bags after having been shipped long-distance and states that approximately 33% of total ref uge seed were distributed into each third of a bag and that the counts of refuge seed were consistent across differe nt blended seed even when the refuge seed had a different shape (i .e. flat vs. round). B PPD would like to have more information on how many replicates/bags there were for each seed phenotype

combination tested.

8. Pioneer assessed the uniform release of seeds from planters by collecting twelve samples during the simulated dispensing of a bag. The regist rant states that when refuge seed content was 20% in the bag, the percentage of refuge seeds in the samp les collected from the planter ranged from 15% to 20% and that there was no trend towards greater or less refuge seeds being planted at any time. Pioneer proposes to use ≥ 2% refuge seed content in Optimum AcreMax1. Since Pioneer proposes to use ≥ 2% refuge seed blend for Optimum AcreMax1, Pioneer should provide BPPD with estimated error for the 20% seed blend experiment. The standard error becomes very important when co nsidering the implementation of a small seed blend refuge as proposed by the registrant. In addition, BPPD would also like to know how large the sample sizes were each time a sample was taken from the planter (sample consists of how many kernels). BPPD recommends that Pioneer provide this information to resolve

some of the uncertainties.

9. Pioneer also assessed the distribut ion of refuge plants in a fi ve acre field using a simulated approach with glyphosate tolerant and non-glyph osate tolerant hybrid seed and concluded that in the ten plots surveye d, an average of 21.8% (±2.5) plan ts were injured. BPPD agrees that this matches the expected 20% “refuge” seed planted. The registrant ran a linear regression analysis to show that the distribution of refuge plants in the ten plots did not differ (R 2 =0.02). However, nothing was said in Pi oneer’s report about testing for random distribution of plants in the field. Since Pioneer relies on the random distribution of refuge plants, the random distribution needs to be stat istically assessed. Furt hermore, BPPD would like to know how many plants were assessed in th e sub-plots surveyed. It is not apparent to the reviewers how many plan ts are represented in a 4 row x 50 feet plot.

10. BPPD is unclear how Pioneer could conclude that only ≤ 10% refuge seed provided similar protection as 100% HXX with no st atistically significant reducti on in yield. BPPD looked at the 2007 efficacy results and did not see suppor ting evidence that woul d lead to such a conclusion. Rather, when three effects where measured at four treatment levels in eight different locations, there was no statistically significant difference between treatments (0%, 5%, 10%, 20% refuge) with the ex ception of one effect measured in two treatments at one location).- 4 - II. BACKGROUND

The registration for Cry1F Event TC1507 (Event DAS -01507-1), also known as Herculex I, was first granted by the Agency on May 18, 2001 for commercial plantings beginning with the 2002 growing season. The registration was extende d from September 30, 2001 to October 15, 2008 (US EPA 2004). Herculex I expresse s the Cry1F insecticidal crystal protein as well as a pat gene expressing the PAT (phosphinothrin acetyl transfer ase) protein which confers tolerance to the herbicidal active ingredient gluf osinate-ammonium. Its primary targ et pest is the European corn borer (ECB, Ostrinia nubilalis ), but other target pest s are corn earworm (CEW, Helicoverpa

zea ), southwestern cornborer (SWCB, Diatrea grandiosella ), fall armyworm (FAW, Spodoptera frugiperda ), western bean cutworm (WBCW, Richia albicosta), and black cutworm (BCW, Agrotis ipsilon ). The main target pests for which IR M requirements apply are ECB, CEW, and

SWCB.

The registration for Event DAS-59122-7, also know n as Herculex Rootworm, was granted by the Agency in August, 2005 for commercial plan tings beginning with th e 2006 growing season. Herculex Rootworm expresses Bt toxins Cr y34Ab1 and Cry35Ab1, which are efficacious against the main target pests western corn root worm and northern corn rootworm (CRW, Diabrotica species). Maize containing Event 59122-7 has been modified to express the PAT protein which confers tolerance to gluphosinate-aluminum herbicide.

In June, 2005 Pioneer and Mycogen Seeds submitte d an application to re gister Herculex XTRA (Event TC1507 x DAS59122-7); the product was conditionally regist ered in October 2005 (EPA Reg. No. 68467-6, 299964-5) and released to growers in 2006. Herculex XTRA is the result of a conventional breeding cross between Herculex I and Herculex R ootworm and is considered a stacked PIP because it targets two different ki nds of pests (lepidopter a and coleoptera) and contains two separate PIP active ingredients.

In February, 2008, Pioneer Hi-Bred International, Inc. submitted an application to register Optimum® AcreMaxTM 1 Insect Protection, a new blended seed product that contains two different kinds of seeds of the already register ed products Herculex XTRA and Herculex I. The new product incorporates the CRW refuge into the seed bag. In this review, BPPD evaluates and comments on the registrant’s benefits claims associated with this new seed blend.

III. REVIEW OF PIONEER’S PU BLIC INTEREST DOCUMENT

General benefits as they apply to Herculex XTRA (HXX) and Herculex I (HX1) have already been addressed in BPPD’s past reviews, and be nefits summaries are available in the respective BRADs (US EPA, 2005, US EPA 2004). The benefits and uncertainties discussed in this review are those that arise from joining the two pr oducts, HXX and HX1, in a corn seed blend as Optimum AcreMax 1 and from a proposed reduction in refuge.- 5 - a. Environmental benefits

Reduced pesticide use

Pioneer provides data showing that since th e introduction of Bt-CRW products in 2003, corn acres treated with chemical pe sticides (pyrethroids, carbamate s, and organophosphates), some of which are restricted use insecticides, have been reduced by 43% from 2004 to 2006. Then Pioneer states that such pesticide use can be further reduced with Optimum AcreMax 1 because there is more CRW protection packed into the seed blend (proposed reduction from 20% block refuge to ≥ 2% seed mix refuge) eliminating the need to spray corn with conventional pesticides and resulting in direct benefits to field workers, by-standers, and the environment. BPPD notes that while this assumption is probable, Pioneer did not provide field effi cacy studies comparing

rootworm control between blocke d refuge (currently required op tions) and seed blend refuge. Rather different percentages of seed blends were tested. Therefore, these direct benefits, while possible, are not based on hard data, and BPPD cannot further comment on them.

BPPD notes that the following may be indirect benefits of introducing Optimum AcreMax 1 into the market as stated by Pioneer: reduced ener gy consumption for manufacture, transport, and application of chemical insecticides; reduced wa ste streams arising from pesticide manufacture; reduced disposal of pesticide waste containers and residues from pes ticide applications.

b. Growers benefits

Pioneer states that Optimum AcreMax 1 would reduce economic damage to corn growers by providing maximized CRW protection on all acres with 1) a reduced in-field seed blend refuge

of ≥ 2% and 2) eliminating non-Bt corn for CRW pr otection in common refuge for lepidopterans and CRW. As mentioned above, part of this cl aim may be probable; how ever, Pioneer did not provide field efficacy studies comparing rootwo rm control between blocked refuge and seed blend refuge but rather between different percen tages of seed blends. In addition, there is no overwhelming evidence, as discussed in the effi cacy section of this review, that Optimum

AcreMax 1 would reduce economic da mage by reducing the refuge to ≥ 2% rather than 5% or 10% (see Tables 2 and 3).

With Optimum AcreMax 1, Pioneer has incorp orated the CRW refuge (seed containing nonCRW Bt trait) in the seed bag, which would tran slate into 100% grower compliance with respect to fulfilling refuge requirements. In addition, it would make the already complex CRW corn refuge requirements and corn management less complex for corn growers. ABSTC’s compliance assurance statistics (phone su rvey, on-farm visits) show th at compliance with refuge requirements for individual trait Bt corn (i n 2007, 80 % compliance with refuge distance and size) is higher than for the stacked Bt corn products (in 2007, compliance is 66% for refuge distance and 70% for refuge size). Pioneer attr ibutes this to the more complicated refuge deployment requirements. To summarize, Pione er states that Optimum AcreMax1 would 1) eliminate the challenges with respect to IRM re quirements for ECB/CRW stacked Bt corn since the refuge is already contained in the bag and 2) protect the durability of the product. BPPD agrees that compliance with refuge planting would be 100%. However, whether refuge plants- 6 - will be randomly distributed and the correct percent of refuge will be planted depends on the distribution of refuge seed in the bag at time of planting and non-biased ‘dropping’ of seeds, i.e. properly functioning planting equipment of grower s should the phenotype of refuge seed and Bt

seed differ.

It appears that Optimum AcreMax 1 may have ad ditional benefits to growers with respect to weed management (i.e. planting of similar maturity and herbicide tolerant corn to control weed, handling multiple seed sources in same field cha llenging) as Pioneer states because the product

contains an herbicide tolerant (HT) trait. However, BPPD notes that the widespread introduction of all HT traits into the environment may also increase the speed of evolution of resistance in weedy plants and brings into question the durab ility of any HT trait. In addition, herbicide

tolerant traits in PIPs are not pesticidal and, thus, are not di rectly regulated by EPA/BPPD. The reviewers will not comment further on the pr oposed benefits of herbicide tolerance.

c. Uniformity, Plantability, and Efficacy of Product

BPPD would like to discuss the mi xing of the various seed types (d ifferent size and shape) as reported in ‘uniformity during seed shipping’ section of the benefits chapter. Pioneer states that “seed [refuge seed and non-refuge seed] was uniformly blended by hand mixing the seed ten times before packaging 80,000 kernels per bag.” B PPD notes that this type of mixing lacks a methodology that ensures consistent mixing of 1) large quantities of seed per se and 2) when different people are employed as handlers. The mi xing of the refuge seed with the non-refuge seed is the first crucial step in a sequence of steps upon which a random distribution of refuge plants in the field hinges. After having revi ewed the ‘Trait Durability Plan’ (MRID473567-09), BPPD learned that Pioneer plans on blending seeds with modern technology (no further description provided) to accurately control the bl end ratio in a uniform process. Pioneer states that the resulting blended products will be tested to ensure accepta ble plantability for growers. BPPD notes that the accuracy of this new technol ogy should have been field tested prior to the submission of the registration request for Optim um AcreMax 1. In addition, the registrant does not provide any description of this new technology. BPPD would like to get additional information from Pioneer regarding their large- scale mixing technology and data to demonstrate accuracy (repeat of the dropper experiment, includ ing reporting of standard error). Most of the IRM benefits are contingent upon a viable seed mixing process.

Uniformity of seed in seed bags during shipping

Pioneer assessed the impact of seed sizes and seed shapes on uniformity within in a bag after it had been shipped. The phenotypes chosen for this experiment were 1) mid-sized flat, 2) midsized round, 3) large flat 4) la rge round, and 5) small flat. Each package or bag contained 80%

HXRW seed treated with Poncho 250 (insecticide treatment hencefor th referred to as P250) and 20% non-Bt hybrid treated with Poncho 1250 (insectic idal treatment henceforth referred to as P1250). The registrant packed five different s eed phenotypes in six pair -wise (80% Bt and 20% refuge seed) combinations, shipped them via UPS from Iowa to Michigan, then to Indiana and on to Illinois, and finally back to Iowa. Upon re turn of the shipment, uniformity within each- 7 - shipping box was determined by examining the dist ribution within each th ird of a box. Pioneer states that approximately 33% of total refuge s eed was distributed into each third of a bag and that the counts of refuge seed were consistent across different blended seed even when the refuge seed had a different shape (i.e. flat vs. round) . BPPD would like to know how many bags were tested per seed phenotype combina tion; replication would instill more confidence into the results.

Uniform release of seed from planter

To test whether refuge seed would be differen tially segregated by a planter machine, Pioneer tested an entire bag of 80% simulated Bt seed with P250 and 20% simulated refuge seed with P1250 (same phenotype as simulated Bt seed), which contained enough kernels to plant 2.5 acres. Twelve times during the dropping of see d, samples were collected to determine the percentage of refuge and non-refuge seed. Pioneer states that % refuge seed ranged from 15% to 20% of the seed dropped through th e planter and that there was no trend towards gr eater or less refuge seeds being planted at any time. Since Pioneer proposes to use ≥ 2% refuge seed blend for Optimum AcreMax1, Pioneer should provide BPPD w ith estimated error fo r the 20% seed blend experiment. The standard error becomes very im portant when considering the implementation of a small seed blend refuge as proposed by the registrant. In addition, BPPD would also like to know how large the sample sizes were each time a sample was taken from the planter (sample consists of how many kernels). Since such info rmation is important for IRM, BPPD recommends that Pioneer provide this information to resolve some of the uncertainties.

Uniformity in the field

Pioneer planted 80% glyphosat e tolerant hybrids (medium-si zed flat seed) and 20% nonglyphosate tolerant hybrids (medium-sized flat seed) to simulate the Bt treatment refuge plants, respectively. At the V4-V6 stage, the five acre pl ot was sprayed with glyphosate; fourteen days later, ten sub-plots (4 rows x 50 ft each) were a ssessed within the sprayed area to determine the number and distribution of inju red ‘refuge’ plants. Pioneer repor ts that an average of 21.8% plants per plot (±2.5%) were injured thus matc hing the 20% simulated refuge seeds in the bag.

No difference in distribution of glyphosate injured plants was detect ed in the ten plots surveyed when a linear regression analysis was conducted (R 2 =0.02); no significant relationship was detected from the first to the last seed planted in these subplots. BPPD notes that the number of plants injured in the subplots matches the 20% “refuge” seed in the bag closely. However, the linear regression di d not test the distribution of refuge plants per se but whether there was a difference in the relative percenta ge of refuge plants between s ubplots. Since Pioneer appears to rely on the random distribution of refuge plants, the distribution of refuge plants needs to be

determined. BPPD would also like to know how many plants were assessed in the sub-plots surveyed. It is not apparent how many plants are represented in a 4 row x 50 feet plot.

In the benefits chapter submitted, Pioneer appears to use the terms “uniform” and “random” (referring to position of refuge plants in the fi eld) synonymously: “Structured refuges as blends require that the blended seed be uniformly distributed across the field so that susceptible insects are nearby any resistant insects that are emerging” and “.. seed bags show uniform distribution of- 8 - CRW refuge seed [in container] during ha ndling and shipping...the random distribution of refuge plants throughout the field maximizes proxim ity of susceptible corn rootworm insects to any potential resistant insects, thereby preserving th e long-term trait durability”. It is not clear to BPPD how “uniformity of seeds in the bag” a nd “uniform planting” tr anslates into “random distribution of refuge plants in the field”. The two terms are not synonymous, and “random

distribution” cannot be achieved with “uniform planting”. At this point BPPD assu mes that this is purely a matter of semantics. However, BPPD aw aits an analysis to test the data from the glyphosate experiment for random distribution of refuge plants.

Efficacy and yield benefits

Pioneer conducted a two year study (2006-2007) to assess efficacy and yield benefits of a blended seed refuge. The purpose of the study was to determine the effect s of a blended refuge approach on field standability, root ratings, and yield (three effects measured at each treatment level). The 2006 experiment was replicated at eight different locations in a randomized block design with four replicates for all five treatments . The treatments were 1) untreated control, 2)

conventional seed treated with P1250, 3) 100% HXRW seed treate d with P250, 4) 10% seed mix

of conventional seed treated with P250 and 90% HXRW seed treated with P250, and 5) 20%

seed mix of conventional seed treated with P1250 and 80% HXRW seed treated with P250. During July 2006, five roots per plot were rando mly selected, root damaged scored using the Iowa State 0-3 node injury scoring, and yield de termined at end of the growing season. Under low insect pressure, Pioneer repor ts little difference in node injury or yields between treatments (no data provided). Under high insect pressu re, all HXRW treatments had approximately equal grain yield and node injury (see Table 1 belo w) and the 10% and 20% seed blends were equivalent to the control of 100% HXRW (59122 maize).

Table 1 Pooled Plant Damage and Yield Results Under High Insect Pressure by Treatments for 2006 Season (derived from Table 3 in MRID 473567-10)

Treatment HXRW Hybrid Refuge Hybrid IST on Refuge Hybrid Node Injury

Score Grain Yield (Bu/a) % Root Lodging

Control 9 P250 1.56 155 27

Poncho 1250 9 P1250 0.65 179 5

HXRW 9 None P250 0.10 192 4

10% seed blend 9 9 P250 0.32 188 6

20% seed blend 9 9 P1250 0.17 193 5 LSD (p=0.5) 0.22 9 10

In 2007, the study was repeated with the addition of HX1 as refuge seed (treated with P250 and P1250). Otherwise testing conditi ons and locations were the sa me as in 2006. Pioneer reports that insect pressure was moderate in six out of eight locations; insect pressure was high in the remaining two locations. According to Pioneer’s report, the 5% and 10% seed mix treatments had similar root lodging and node injury as th e 100% HXX treatment, and both root damage ratings and lodgings were higher for the 20% re fuge treatments than the 100% HXX treatment.

Pioneer concludes that HXX/ HX1 seed blends with ≤ 10% refuge seed provided similar- 9 - protection with no statistically significant reduction in yield as compared to 100% HXX. Based on the results provided in Table 2, BPPD does not see statistically significant differences between node injury, % root lodging, and grain yields betw een any of the HXX treatments except with respect to node fo r the 20% refuge with P250. So BPPD is unclear how Pioneer could conclude that only ≤ 10% refuge seed provided simila r CRW protection compared to 100% HXX. Furthermore, the data indicate that the 20 % refuge seed produced the same grain yield as the 100% HXX (no refuge).

Table 2 Pooled Plant Damage and Yield Results by Treatments for the 2007 Season

Treatments Node Injury

Score Grain Yield (Bu/a) % Root Lodging

% HXX % HX1 (Poncho rate) 0 100 (P1250) 0.60 c 182 b 19 b 0 100 (P250) 0.89 d 178 b 16 b

100 0 0.10 a 192 a 4 a 95 5 (P1250) 0.10 a 190 a 5 a 95 5 (P250) 0.19 ab 194 a 6 a 90 10 (P1250) 0.14 ab 194 a 5 a 90 10 (P250) 0.15 ab 194 a 6 a 80 20 (P1250) 0.17 ab 192 a 4 a 80 20 (P250) 0.25 b 192 a 9 a LSD (p=0.05) 0.12 7 6 HXX contains Cry34/35 and Cry1F; HX1 contains Cry1F

Upon request, Pioneer supplied BPPD with supplem ental data (no MRID assigned) for all eight locations which is presented in Table 3 and discussed below by the Agency. There was no statistically significant differe nce between mean node injuries and grain yields of HXX treatments at seven out of eight locations (exception at Readly n, Iowa). In Readlyn, mean node injury of the 10% and 20% refuge treatments with P250 differed from the other HXX treatments at that location. No yield data could be collect ed at Readlyn due to st orm damage. There was no statistically significant difference between the me an root lodgings of any of the HXX treatments at all eight locations tested. BPPD recalls from Pi oneer’s report that six of the eight locations had moderate insect pressure, while at two locations, insect pressure was considered high. In order for Pioneer’s conclusion to hold “that only the ≤ 10% refuge seed provides similar protection and yield”, there would have had to be more substa ntial and supporting evidence especially from the high infestation locations. Based on the results provided by Pioneer, BPPD does not see a strong case for concluding that only the ≤ 10% refuge seed provides sim ilar protection and yield as compared to 100% HXX.- 10 - Table 3 Plant Damage and Yield Results by Location and by Treatments for the 2007 Season

Treatments Node Injury

Score Grain Yield (Bu/a) % Root Lodging

% HXX % HX1 (Poncho rate) Orange City, IA 0 100 (P1250) 0.54 b 115 b 0 a 0 100 (P250) 0.81 c 89 c 3a

100 0 0.18 a 125 ab 0 a 95 5 (P1250) 0.16 a 130 ab 1 a 95 5 (P250) 0.19 a 140 a 1 a 90 10 (P1250) 0.21 a 122 ab 0 a 90 10 (P250) 0.18 a 141 a 0 a 80 20 (P1250) 0.31 a 120 ab 0 a 80 20 (P250) 0.34 a 130 ab 0 a LSD (p=0.05) 0.20 25 NS Mankato, MN 0 100 (P1250) 0.60 b 134 b 53 c 0 100 (P250) 0.60 b 140 b 42 bc

100 0 0.06 a 152 a 20 a 95 5 (P1250) 0.11 a 152 a 22 ab 95 5 (P250) 0.15a 154 a 25 ab 90 10 (P1250) 0.17 a 156 a 22 ab 90 10 (P250) 0.15 a 154 a 34 abc 80 20 (P1250) 0.20 a 161 a 15 a 80 20 (P250) 0.22 a 156 a 31 ab LSD (p=0.05) 0.19 12 21 Readlyn, IA 0 100 (P1250) 2.47 d --- 11 b 0 100 (P250) 2.90 d --- 37 c

100 0 0.53 a

--- 0 a 95 5 (P1250) 0.81 ab --- 0 a 95 5 (P250) 0.79 ab --- 0 a 90 10 (P1250) 0.80 ab --- 0 a 90 10 (P250) 1.15 bc --- 0 a 80 20 (P1250) 1.01 ab --- 0 a 80 20 (P250) 1.56 c --- 1 a LSD (p=0.05) 0.52 --- 7 Henderson, NE 0 100 (P1250) 0.44 b 223 a 3 a 0 100 (P250) 0.66 b 221 a 2 a

100 0 0.07 a 223 a 2 a 95 5 (P1250) 0.07 a 211 a 0 a 95 5 (P250) 0.10 a 219 a 3 a 90 10 (P1250) 0.06 a 222 a 0 a 90 10 (P250) 0.07 a 212 a 3 a 80 20 (P1250) 0.14 a 222 a 0 a 80 20 (P250) 0.16 a 214 a 2 a LSD (p=0.05) 0.26 NS NS Johnston, IA 0 100 (P1250) 0.54 b 197 a 4 a 0 100 (P250) 0.99 c 206 a 5 a

100 0 0.22 a 207 a 3 a 95 5 (P1250) 0.18 a 202 a 4 a- 11 - Treatments Node Injury

Score Grain Yield (Bu/a) % Root Lodging

% HXX % HX1 (Poncho rate) 95 5 (P250) 0.15 a 203 a 2 a 90 10 (P1250) 0.22 a 213 a 4 a 90 10 (P250) 0.25 ab 213 a 2 a 80 20 (P1250) 0.27 ab 208 a 5 a 80 20 (P250) 0.43 ab 211 a 5 a LSD (p=0.05) 0.31 NS NS Rochelle, IL 0 100 (P1250) 0.89 b 197 a 48 b 0 100 (P250) 0.71 b 185 a 45 b

100 0 0.07 a 209 a 2 a 95 5 (P1250) 0.09 a 204 a 6 a 95 5 (P250) 0.07 a 210 a 4 a 90 10 (P1250) 0.08 a 211 a 2 a 90 10 (P250) 0.06 a 201 a 0 a 80 20 (P1250) 0.11 a 214 a 2 a 80 20 (P250) 0.19 a 206 a 15 a LSD (p=0.05) 0.29 NS 21 Princeton, IL 0 100 (P1250) 0.33 b 214 a 0 a 0 100 (P250) 0.34 b 222 a 0 a

100 0 0.05 a 220 a 0 a 95 5 (P1250) 0.07 a 212 a 0 a 95 5 (P250) 0.06 a 214 a 0 a 90 10 (P1250) 0.07 a 223 a 0 a 90 10 (P250) 0.08 a 226 a 0 a 80 20 (P1250) 0.07 a 214 a 0 a 80 20 (P250) 0.10 a 221a 0 a LSD (p=0.05) 0.19 NS NS Windfall, IN 0 100 (P1250) 0.76 b 193 b 0 a 0 100 (P250) 2.02 c 186 b 0 a

100 0 0.12 a 210 a 0 a 95 5 (P1250) 0.12 a 215 a 0 a 95 5 (P250) 0.16 a 215 a 0 a 90 10 (P1250) 0.24 a 211 a 0 a 90 10 (P250) 0.26 a 210 a 0 a 80 20 (P1250) 0.22 a 206 a 0 a 80 20 (P250) 0.43 a 207 a 0 a LSD (p=0.05) 0.32 10 NS NS = no significant differences- 12 - IV. REFERENCES

Bailey, L., R. Binning, L. Higgins, D. Langer, R. Layton, S. Lefko, T. Nowatzki, E. Owens, and T. Rood. 2008. Trait Durability Plan for Op timum® AcreMaxTM1 Insect Protection Following Commercialization. Unpublished report submitted to the US EPA (MRID 473567-09).

Davis, P. and S. MacIntosh. 2008. Benefits Document of Optimum® AcreMaxTM 1 Insect Protection. Unpublished report submitt ed by Pioneer Hi-Breed Internat ional, Inc. to the US EPA (MID 473567-10).

Others:

US EPA. 2005. Bacillus thuringiensis Cry34Ab1 and Cry35Ab1 Protei ns and the Genetic Material Necessary for Their Production (P lasmid Insert PHP 17662) in Event DAS-59122-7 Corn. Biopesticide Registration Action Document

US EPA. 2004. Biopesticide Re gistration Action Document Bacillus thuringiensis Cry1F Corn. Updated in 2005