Petition for the Determination of Non regulated Status for Glyphosate-Tolerant

Canola MON 88302

The undersigned submits this petition under 7 CFR § 340.6 to request that the

Administrator make a determination that the article should not be regulated under

7 CFR Part 340

June 27, 2011

OECD Unique Identifier: MON 883Ø2-9

Monsanto Petition Number: 11-CA-233U

Submitted by:

Margaret A. Wideman, M.S.

Monsanto Company 800 North Lindbergh Blvd. St. Louis, MO 63167 Phone: (314) 694-2680

Fax: (314) 694-3080 E-mail: margaret.a.wideman@monsanto.com

Prepared by:

A.E. Bernhard, B.S., K.S. Clark, B.S., K.S. Crowley, Ph.D., B. Hammond, Ph.D., D.R. Lundry, B.S., M.A. McPherson, Ph.D., K.A. Robinson, Ph.D., D.M. Ruezinsky, Ph.D., A. Silvanovich, Ph.D., Z. Song, M.S., J.L. Vicini, Ph.D., J.M. Ward, Ph.D., M.A. Wideman, M.S.

Contributors and/or Pr incipal Investigators:

S.A. Adams, B.S., S.M. Arackal, M.S., T. Bhakta, Ph.D., M.S. Bleeke, Ph.D., J.J. Finnessy, M.S., J.M. Harrison, M.S., M.A., J.L. Honegger, Ph.D., K.R. Lawry, M.S., A. Mehrsheikh, Ph.D., K.E. Niemeyer, B.S., D.A. Saltmiras, Ph.D., D.A.B.T., J.K. Soteres, Ph.D., Q. Tian, Ph.D., H. Tu, M.S.Monsanto Company 11-CA-233U 2 of 500 RELEASE OF INFORMATION

Monsanto is submitting the information in this petition for review by the USDA as part of the regulatory process. By submitting this information, Monsanto does not authorize its release to any third party. In the event the USDA receives a Freedom of Information Act request, pursuant to 5 U.S.C., § 552, and 7 CFR Part 1, covering all or some of this information, Monsanto expects that, in adva nce of the release of the document(s), USDA will provide Monsanto with a copy of the material proposed to be released and the opportunity to object to the release of a ny information based on appropriate legal grounds, e.g ., responsiveness, confiden tiality, and/or competitiv e concerns. Monsanto understands that a copy of this information may be made available to the public in a reading room and upon individual request as part of a public comment period. Except in accordance with the foregoing, Monsanto does not authorize the release, publication or other distribution of this information (incl uding website posting) without Monsanto's prior notice and consent.

©2011 Monsanto Company. All Rights Reserved.

This document is protected under copyright la w. This document is for use only by the regulatory authority to which it has been submitted by Monsanto Company and only in support of actions requested by Monsanto Co mpany. Any other use of this material, without prior written consent of Monsanto, is strictly prohibited. By submitting this document, Monsanto does not grant any party or entity any right to license or to use the information or intellectual property described in this document.CERTIFICATION

The undersigned certifies that, to the best knowledge and belief of the undersigned, this petition includes all information and views on which to base a determination, and that it

includes all relevant data and information known to the petitioner that are unfavorable to

the petition.

?

Margaret A. Wideman, M.S. Regulatory Affairs Manager

Address:

Monsanto Company

800 North Lindbergh Blvd., C3SD

St. Louis, MO 63167

Tel: (314)-694-2680

Fax: (314)-694-3080

Monsanto Company II-CA-233U 30f500Monsanto Company 11-CA-233U 4 of 500 EXECUTIVE SUMMARY

The Animal and Plant Health Inspection Serv ice (APHIS) of the United States (U.S.) Department of Agriculture (USDA) has resp onsibility under the Plant Protection Act (Title IV Pub. L. 106-224, 114 Stat. 438, 7 U.S.C. § 7701-7772) to prevent the introduction and dissemination of plant pe sts into the U.S. APHIS regulation 7 CFR § 340.6 provides that an applicant may petition APHIS to evaluate submitted data to determine that a particular regulated arti cle does not present a pl ant pest risk and no longer should be regulated. If APHIS dete rmines that the regulated article does not present a plant pest risk, the petition is gr anted, thereby allowing unr estricted introduction

of the article.

Monsanto Company is submitting this requ est to APHIS for a determination of nonregulated status for the new biotec hnology-derived canola pr oduct, MON 88302, any progeny derived from crosses between MON 88302 and conventional canola, and any progeny derived from crosses of MON 88302 with biotechnology-derived canola that have previously been granted nonregul ated status under 7 CFR Part 340.

Product Description

Weed competition can be a major limiting factor in canola pr oduction leading to significant yield reductions. Monsanto Company has de veloped a second-generation glyphosate-tolerant canola product, MON 883 02, designed to provide growers with improved weed control through greater flexibi lity for glyphosate herbicide application. MON 88302 produces the same 5-enolpyruvylshikimate-3

- phosphate synthase (CP4 EPSPS) protein that is produced in commercial Roundup Ready ® crop products, via the incorporation of a cp4 epsps coding sequence. The CP 4 EPSPS protein confers tolerance to the herbicide glyphosate, the ac tive ingredient in th e family of Roundup agricultural herbicides.

MON 88302 utilizes an improved promoter sequence to enhance CP4 EPSPS expression in male reproductive tissues ( i.e. , pollen), compared to the promoter used to drive CP4 EPSPS production in the first-generati on product, Roundup Ready canola (RT73). Enhanced CP4 EPSPS expression in the male reproductive tissues of MON 88302 allows the greater flexibility of gl yphosate herbicide applications as MON 88302 plants can be sprayed with higher rates of glyphosate and at later stages of development with no detectable impact to male fertility. Glyphosate is a systemic herbicid e and is translocated in the plant, generally from a strong source tissue ( e.g., leaf) to rapidly developing, or sink tissue. Sink tissues, such as pollen, th at accumulate glyphosate and lack sufficient CP4 EPSPS expression are considered to be at risk for glyphosate inju ry. By virtue of enhanced CP4 EPSPS expression in male reproductive tissues, MON 88302 provides tolerance to glyphosate during the sensitive reproductive stages of growth, and enables the application of glyphosate at higher rates and at later stag es of development than is possible with the current R oundup Ready canola product.

® Roundup and Roundup Ready are registered trademarks of Monsanto Technology, LLCMonsanto Company 11-CA-233U 5 of 500 Use of MON 88302 will enable growers to ap ply Roundup herbicide at higher rates and at later stages of developmen t than is possible with the cu rrent product facilitating: 1) better control of tough-to-kill weeds; 2) an increased opport unity to control weeds if glyphosate application is delayed due to weat her or equipment failure; 3) an enhanced ability to tailor labeled glyphos ate applications to the weed development stage instead of the canola developmental stage; and 4) enha nced protection of canola plants at more advanced development stages. Use of MON 88302 will provide growers with the opportunity to ensure weeds that may impact yields are removed at the optimal time while minimizing the potential for crop injury.

Data and Information Presented Confirms th e Lack of Plant Pest Potential and the Food and Feed Safety of MON 88302 Compared to Conventional Canola

The data and information presented in this petition demonstrate MON 88302 is agronomically, phenotypically, and compositiona lly comparable to conventional canola, with the exception of the intr oduced trait. Moreover, the data presented demonstrate MON 88302 is unlikely to pose an increased plant pest risk, including weediness or adverse environmental impact, compared to conventional canola. The food, feed and environmental safety of MON 88302 was c onfirmed based on multiple, well established

lines of evidence:

 Canola is a familiar crop that has a histor y of safe consumption, and serves as an appropriate basis of co mparison for MON 88302.

 A detailed molecular characterization of the introduced DNA demonstrated a single, intact copy of the transgenic inse rt in a single locu s within the canola

genome.

 The CP4 EPSPS protein in MON 88302 is id entical to the CP4 EPSPS protein produced in several other commercially av ailable crops that have been reviewed by USDA and previously deregulated ( e.g., Roundup Ready soybean, Roundup Ready 2 Yield soybean, Roundup Rea dy corn 2, Roundup Ready canola, Roundup Ready sugar beet, Roundup Ready cotton, Roundu p Ready Flex cotton and Roundup Ready alfalfa). The safety of CP4 EPSPS proteins present in biotechnology-derived crops ha s been thoroughly assessed, and is the subject of numerous publications. The mode of action of CP4 EPSPS protein and how it confers glyphosate tolerance has been exte nsively studied and is well documented in peer reviewed publications.

 A compositional assessment confirmed that MON 88302 seed is compositionally equivalent to seed of conventional canola.

 An extensive evaluation of MON 88302 phe notypic and agronomic characteristics and environmental interactions demonstrated MON 88302 has no increased plant pest potential compared to conventional canola.Monsanto Company 11-CA-233U 6 of 500  An assessment of potential impact to non-target organisms (NTOs) and endangered species indicated that, under normal agricultural conditions, MON 88302 is unlikely to have adverse e ffects on these organisms, similar to

conventional canola.

 Evaluation of MON 88302 using inte nded and current cultivation and management practices for canola concl uded that deregulatio n of MON 88302 will not significantly impact canola agro nomic practices or land use.

Canola is a Familiar Crop and Ebony is an Appropriate Comparator to MON 88302

Brassica napus is a versatile crop that provides both food and f eed to the global economy and whose biology is well understood and doc umented. There are numerous terms used to describe oil-producing B. napus varieties including oilseed rape, rapeseed, rape, low erucic acid rapeseed and canola. For purposes of this petition, B. napus will be referred to as oilseed rape and the term canola wi ll be used to denote B. napus varieties that produce low (< 2%) erucic acid oil and have levels of glucosinolates below the OECD standard of 30 μmoles/g in meal.

Most U.S. canola production is concentrated in the northern Great Plains where drier, shorter growing seasons make maize an d soybean production less attractive. Approximately 1.0 to 1.5M acres of canola are planted annually in eight states with the majority (>85%) being produced in North Dakot a. Spring and winter canola varieties have been developed which permit production in both the northern and southern Great Plains, respectively.

Brassica napus is not generally regarded as an environmentally hazardous, colonizing, or invasive species in undisturbe d natural ecosystems. Although B. napus has some characteristics typical of weedy species such as high reproductive capacity, rapid growth and multiple pollination mechanisms (self, wind, insect), it also has many characteristics typical of domesticated speci es including low genetic divers ity, lack of long-distance seed dispersal mechanisms, limited populatio n persistence, lack of primary seed dormancy and an inability to compete well with perennial species. Brassica napus is not listed as an invasive weed in the Catalog of Invasive Plant Species of the U.S., nor is it present on the lists of noxious weed species maintained by the federal government (7 CFR § 360).

The Brassica napus canola variety used as the recipi ent for the DNA insertion to create MON 88302 was Ebony, a non-transgenic conv entional spring canola variety. Ebony was used as the conventional canola comparator (referred to in this petition document as the conventional control) in the safety assessment of MON 88302. MON 88302 and the conventional control have similar geneti c backgrounds with the exception of the cp4 epsps expression cassette.Monsanto Company 11-CA-233U 7 of 500 Molecular Characterization Verified the In tegrity and Stability of the Inserted DNA

in MON 88302

MON 88302 was developed through Agrobacterium -mediated transformation of hypocotyls from canola variety Ebony u tilizing plasmid vector PV-BNHT2672.

PV-BNHT2672 contains one T-DNA that is delin eated by Left and Right Border regions.

The T-DNA contains the cp4 epsps coding sequence under the control of the FMV/Tsf1 chimeric promoter, the Tsf1 leader and intron sequences, and the E9 3 ′ untranslated region. After transformation a nd subsequent rounds of self- pollination, plants containing only a single T-DNA insertion were identified resulting in production of glyphosatetolerant canola MON 88302.

Molecular characterization by Southern bl ot analyses determined that MON 88302 contains one copy of the T-DNA at a singl e integration locus. These data also

demonstrated that MON 88302 does not contain detectable backbone sequences from the plasmid vector. The complete DNA sequence of the insert and adjacent genomic DNA sequences in MON 88302 confirmed the integrity of the inserted cp4 epsps expression cassette within the inserted sequences and identified the 5 ′ and 3 ′ insert-to-genomic DNA junctions. Southern blot analysis demonstr ated that the insert in MON 88302 has been maintained over multiple generations of breed ing, thereby confirming the stability of the insert. Further, results from segregation an alyses show inheritance and stability of the insert were as expected across multiple generations, which corroborates the molecular insert stability analysis and establishes the genetic behavior of the T-DNA in MON 88302 at a single chromosomal locus.

Data Confirms CP4 EPSPS Protein Safety

A multistep approach was used to characterize and assess the safety of the CP4 EPSPS protein expressed in MON 88302 resulting from the ge netic modification. The physicochemical characteristics of the CP4 EPSPS protein in MON 88302 were

determined and shown to be equivalent to those of an E. coli -produced CP4 EPSPS protein that has been used in CP4 EPSPS protei n safety studies. Th e expression levels of the CP4 EPSPS protein in selected tissues of MON 88302 were determined. An assessment of the allergenic potential of the CP4 EPSPS prot ein supports the conclusion that the CP4 EPSPS protein does not pose a signi ficant allergenic risk to humans or animals. In addition, the donor orga nism for the CP4 EPSPS coding sequence, Agrobacterium sp. strain CP4, is ubiquitous in th e environment and is not commonly known for human or animal pathogenicity or allergenicity. Bioinformatics analysis determined that CP4 EPSPS protein does not sh are amino acid sequence similarities with known allergens, gliadins, glutenins, or protei n toxins. The CP4 EPSPS protein is rapidly digested in simulated digestive fluids and dem onstrates no oral toxi city in mice at the level tested. Hence, the consumption of the CP4 EPSPS protein from MON 88302 or its progeny is considered safe fo r humans and animals.Monsanto Company 11-CA-233U 8 of 500 MON 88302 is Compositionally Equivalent to Conventional Canola

Previous Roundup Ready crops reviewed by the USDA, including the first-generation product Roundup Ready canola (RT73), have ha d no biologically relevant compositional changes identified, and there is no reason to expect expression of the CP4 EPSPS protein

in MON 88302 would affect nutritionally important nutr ients, toxicants, and antinutrients present in seed from this new product.

Safety assessments of biotechnology-derive d crops follow the comparative safety assessment process in which the composition of grain and/or other raw agricultural commodities of the biotechnology-derived crop are compared to the appropriate conventional counterpart that has a history of safe use. Compositional assessments were performed using the principles and analytes outlined in crop-specific OECD consensus documents, in this case for canola composition.

Compositional analysis comparing MON 883 02 to the conventional control variety (Ebony) and commercial reference varie ties demonstrated that MON 88302 is compositionally equivalent to conventional canola. The background genetics of the

conventional control were similar to th at of MON 88302, but did not contain the cp4 epsps expression cassette. The commercial refe rence varieties were used to define the natural variability of key nutrients, toxicants, and anti- nutrients in canola varieties that have a history of safe consumption. Th e samples utilized for compositional analysis

were obtained from two U.S. sites and three Canadian sites.

Nutrients assessed in this analysis in cluded proximates (ash, carbohydrates by calculation, moisture, protein, and total fat), fi bers (acid detergent fiber [ADF], neutral detergent fiber [NDF], and total dietary fibe r [TDF]), amino acids (18 components), fatty acids (FA; C8-C24,), vitamin E ( α -tocopherol), and minerals (calcium, copper, iron, magnesium, manganese, phosphorus, potassium , sodium, and zinc) in seed. The

toxicants assessed in seed included erucic aci d and glucosinolates (a lkyl glucosinolates [including 3-butenyl, 4-pe ntenyl, 2-hydroxy-3-butenyl , and 2-hydroxy-4-pentenyl glucosinolates], indolyl glucosinolates [including 3-indolylmethyl and 4-hydroxy-3- indoylmethyl], and total glucosi nolates). The anti-nutrients assessed in seed included phytic acid and sinapine (as sinapic acid).

Combined-site analyses were conducted to de termine statistically significant differences ( α = 0.05) between MON 88302 and the conventiona l control seed samp les. Statistical

results from the combined-site data were eval uated using considerations relevant to the safety and nutritional quality of MON 88302 when compared to the co nventional control.

Considerations used to assess the relevance of each combined-site statistically significant difference included: 1) the relative magnitude of the difference in the mean values of nutrient, toxicant, and anti-nutrient components between MON 88302 and the conventional control; 2) whether the MON 883 02 component mean value is within the range of natural variability of that component as represented by the 99% tolerance

interval of the commercial reference varietie s grown concurrently in the same trial; 3) evaluation of the reproducibility of the statistically significant ( α = 0.05) combined-site component differences at indivi dual sites, and 4) an assessmen t of the differences withinMonsanto Company 11-CA-233U 9 of 500 the context of natural variability of co mmercial canola compositi on published in the scientific literature. If statistically significant differences detected in the individual site analyses were not observed in the combined -site analysis, they were not considered further for the compositional assessment of safety.

The levels of assessed components in M ON 88302 were comparable to those in the

conventional control and within the range of variability of commercial reference varieties grown concurrently in the same field trial. Of the 51 components statistically analyzed, 42 were not significantly different from the conventional control. Where statistically significant differences ( α = 0.05) were observed between MON 88302 and the

conventional control in the co mbined-site analysis, the magnitudes of the differences

were small in relation to their natural vari ability as established by the 99% tolerance

interval established from the commercial re ference varieties grow n concurrently. The mean values for the significantly different components were within the 99% tolerance interval, differences were not observed across all individual sites, a nd with the exception of alkyl glucosinolates for which a range of values was not available, mean values were

within the natural vari ability of canola components as repor ted in the scientific literature for conventional canola that have a history of safe consumption. Based on these results, the observed differences were not meaningful to food and feed safety or nutritional value, and led to the conclusion that MON 88302 is compositionally equivalent to conventional canola that has a history of safe consumption.

MON 88302 Does Not Change Canola Plan t Pest Potential or Environmental

Interactions

Plant pest potential of a biotechnology-de rived crop is assessed from the basis of familiarity that the USDA recognizes as an important underlying concept in risk assessment. The concept of familiarity is based on the fact that the biotechnologyderived plant is developed from a conventiona l plant hybrid or vari ety whose biological properties and plant pest potential are well known. Familiarity considers the biology of the plant, the introduced trai t, the receiving environment, and the interactions among these factors. This provides a basis fo r comparative risk assessment between a biotechnology-derived plant and the convent ional control. Thus, the phenotypic, agronomic, and environmental interactio n assessment of MON 88302 included the genetically similar conventional control as a co mparator. This evalua tion used a weight of evidence approach and considered sta tistical differences between MON 88302 and the conventional control with respect to reproducibility, magnitude, and directionality. The observations were taken on plants not treate d with glyphosate in order to evaluate only the impact of the introduced trait in MON 88302. Comparison to a range of commercial references established the range of natural va riability for canola, and provided a context from which to further evaluate any statis tical differences. Characteristics assessed included: seed dormancy and germina tion, pollen morphology, and plant phenotypic

observations and environmental interacti on evaluations conducted in the field. Commercial references grown concurrently were used to establish a range of natural variability for each assessed characteri stic in canola. The phenotypic, agronomic, and

environmental interaction assessment dem onstrated that MON 88302 is comparable toMonsanto Company 11-CA-233U 10 of 500 the conventional control. Thus, MON 88302 is unlikely to have increased weediness or plant pest potential compared to conventional canola.

Seed dormancy and germination characteri zation indicated that MON 88302 seed had no changes in the dormancy or germination ch aracteristics that coul d be indicative of increased plant weediness or pest potential of MON 88302 compared to the conventional control. No statistically significant differences were detected ( α = 0.05) between

MON 88302 and the conventional control for percent viable pollen or pollen grain diameter. Furthermore, no visual diffe rences in general pollen morphology were

observed between MON 88302 and the conventi onal control, demons trating that the introduction of the glyphosate-tolerance tra it did not alter the overall morphology or pollen viability of MON 88302 compared to the conventional control.

The field evaluation of phenotypic, agronomic , and environmental characteristics of MON 88302 also supports the conclusion that MON 88302 is not likely to have increased weediness or plant pest potential or an altered environmental impact compared to

conventional canola. The evaluations were con ducted at eight field sites in the U.S. and

nine field sites in Canada. These 17 field s ites provided a diverse range of environmental and agronomic conditions representative of commercial canola production areas in North America. Assessments included 12 phenotypic a nd agronomic characteristics, as well as observations for plant responses to abiotic st ressors and plant-dis ease and plant-arthropod interactions. The observed phenotypic ch aracteristics were comparable between

MON 88302 and the conventional control. Across sites, da ta show no statistically significant differences between MON 88302 and the conventional cont rol for early stand count, seedling vigor, seed maturity, lodging, plant he ight, visual rating for pod shattering, quantitative pod shattering, seed quality, yield, and final stand count. Two statistically significant differences were detected between MON 88302 and the

conventional control in the combined-site analysis. MON 88302 reac hed first flowering

later than the convent ional control (61.1 vs. 56.2 days). However, the mean value of MON 88302 for days to first flowering was within the na tural variability of the commercial reference varieties (45.9 – 67.5 days). Therefore, the difference in days to first flower is unlikely to be biologically meaningful in terms of increased weediness potential. MON 88302 also ha d higher harvested seed moisture than the conventional control (13.2% vs. 11.7%). However, the mean value of MON 88302 for harvested seed moisture was within the natural variability of the commercial reference varieties (7.5% – 14.8%). Therefore, the differe nce in seed moisture is unlikely to be biologically meaningful in terms of incr eased weediness potential.

In a qualitative assessment of plant respons e to abiotic stress, no differences were

observed between MON 88302 and the conv entional control for 130 out of 131 comparisons involving 9 assessed abiotic st ressors. One differe nce was observed in abiotic stress response in Observation 1 fo r frost damage at the MBBR site (where

MON 88302 was severe and the conventional control was m oderate). However, the observed frost damage to MON 88302 was with in the range of the damage observed among the commercial reference varieties (slight – severe). In addition, the difference was not observed during any of the other 12 fr ost damage observations among the sites. Thus, the single difference for frost damage wa s not considered biologically meaningfulMonsanto Company 11-CA-233U 11 of 500 in terms of plant pest potential or an a dverse environmental impact from MON 88302 compared to conventional canola.

In a qualitative assessment of plant response to disease damage and arthropod damage, no

differences were observed between MON 88302 and the conventional control for any of the 141 comparisons involving 16 assessed diseas es or for any of the 165 comparisons for any of the 13 assessed arthropods among a ll observations at the sites.

In addition, damage by two common pests, flea beetles and seed pod weevil was evaluated quantitatively at four sites. No statistically significant differences were

detected between MON 88302 and the conventional control from the four sites evaluated.

In a quantitative assessment of pest and be neficial arthropod abundance, no statistically significant differences were detected be tween MON 88302 and the conventional control for any of the 51 comparisons, including 36 ar thropod pest comparisons and 15 beneficial arthropod comparisons, among the observa tions at the four sites.

In summary, the phenotypic, agronomic, and environmental interaction data were evaluated to characterize MON 88302, and to a ssess whether the introduction of the trait in MON 88302 alters the plant pest potential compared to conventional canola. The evaluation, using a weight of evidence ap proach, considered the reproducibility, magnitude, and direction of detected di fferences (trends) between MON 88302 and the conventional control and comparison to the ra nge of the commercial reference varieties. Results from the phenotypic, agronomic, an d environmental interactions assessment indicated that MON 88302 does not possess weedy characteristics, increased susceptibility or tolerance to specific ab iotic stress, diseases, or arthropods, or characteristics that would confer a plant pest risk or a significant environmental impact compared to conventional canola.

MON 88302 Will Not Adversely Affect NTOs or Threatened or Endangered Species

Evaluation of the impacts of MON 88302 on non-target organisms (NTOs) is a component of the plant pe st risk assessment. Si nce MON 88302 does not possess pesticidal activity, all organisms that inte ract with MON 88302 are considered to be

NTOs. The environmental assessment of MON 88302 indicated that it poses no adverse effect on NTOs or endangered species using current and intended agricultural practices.

The assessment indicates that the CP 4 EPSPS protein found in MON 88302 did not unexpectedly alter plant-arthr opod interactions, including bene ficial arthropods, or alter disease susceptibility compared to the conve ntional control.

The safety of the CP4 EPSPS protein presen t in biotechnology-derived crops has been extensively assessed. A mouse gavage study demonstrated no acute oral toxicity and consequently the low potential for impact to terrestrial vertebrate NTOs including threatened and endangered vertebrate species. In addition, the history of safe use of Roundup Ready crops and the ubiquitous pres ence of functionally identical EPSPS proteins in plants and microbes in the enviro nment make it unlikely that the presence of CP4 EPSPS protein in MON 88302 will have a significant impact on the receivingMonsanto Company 11-CA-233U 12 of 500 environment, including soil function and water quality. Data from the 2009 North American phenotypic and agronomic st udy including observational data on

environmental interactions su ch as plant-disease inter action, arthropod damage and arthropod abundance, were collected at se lect sites for MON 88302 and the conventional control. These results suppor t the conclusion of no adverse environmental impact from cultivation of MON 88302 to non-target organi sms and no increased incidence of disease in MON 88302. Taken together, these data support the conclusion that MON 88302 has no reasonable mechanism for harm to NTOs, or impact to threatened and endangered species compared to the cultivat ion of commercial canola.

Brassica napus is predominantly self-pollinating a lthough interplant (plants that are touching one another) cross pollination rates range from 12% to 55% with a mean of 30%. Most (98.8%) of pollen travels less than twelve meters from its source although dispersal due to pollinators may occur ove r greater distances at low frequency. Brassica

napus produces a large amount of po llen that can remain viable up to four to five days under field conditions. This, c oupled with the potential for B. napus pollen movement, suggests the possibility for hybridization between B. napus and related species. Species

with which B. napus can hybridize under field conditions are found throughout regions in the U.S. where canola is grown. There are re ports of hybridization under field conditions

with B. napus as the pollen donor with six species including Brassica rapa, Brassica juncea, Brassica oleracea, Hirschf eldia incana, Raphanus raphanistrum, and Sinapis

arvensis . In all cases th ese hybrids had decreased envir onmental fitness evidenced by a variety of characteristics including decreased pollen vi ability, seed production, and seedling survival when compared to parental varieties, making gene flow between species unlikely.

Gene flow from B. napus canola to B. napus vegetables ( e.g., Swedes or rutabaga, Siberian kale) is possible as they ar e members of the same species. Since B. napus has

chromosomes in common with B. rapa and B. oleracea

, B. napus gene flow to B. rapa vegetables ( e.g., turnip and Chinese cabbage) and B. oleracea vegetables ( e.g., cabbage, cauliflower, broccoli, collards, kale, Brussels sprouts) is less likely but may occur.
However, B. napus

, B. rapa and B. oleracea vegetables are not c onsidered weedy, and are generally harvested prior to flowering, preventing cross-pollina tion, hybridization and

seed formation. Thus the potential for B. napus gene flow and introgr ession into closely related vegetable species is low. In the unlikely event that trait introgression and persistence of hybrids between MON 88302 and related species occurred, these plants could be controlled by mechanical or chemical means.

Deregulation of MON 88302 Will Not Sign ificantly Impact Canola Agronomic

Practices or Land Use

An assessment of current canola agronomic practices was conducted to determine

whether the cultivation of MON 88302 has the potential to impact current canola and weed management practices. Canola fields are typically highly managed agricultural

areas that are dedicated to crop production. MON 88302 is li kely to be used in common rotations on land previously used for agri cultural purposes. Certified seed production will continue to use well-established industry practices to deliver high quality seed withMonsanto Company 11-CA-233U 13 of 500 MON 88302 to growers. Cultivation of MO N 88302 is not expected to differ from current canola cultivation using glyphosate-to lerant canola, with the exception of an opportunity to use glyphosate during an expanded window of application and at rates higher than those currently re commended and authorized. Du e to the expanded timing of in-crop applications to MON 88302 glyphosate treatments may be later in the growing season than current labeled uses. Monsanto submitted amended labeling to the U.S. Environmental Protection Agency (U.S. EPA) in February 2011 that proposes to modify the current use pattern of glyphosate in canola based on MON 88302.

MON 88302 is similar to conventional canola in its agronomic, phenotypic, ecological, and compositional characteristics and has levels of resistance to insects and diseases comparable to conventional canola. Ther efore, no significant impacts on current cultivation and management practices for ca nola are expected following the introduction of MON 88302. Based on this assessment, the introduction of MON 88302 will not impact current U.S. canola cultivation prac tices or weed management practices, other

than intended weed control benefits.

Conclusion

Based on the data and information presented in this petition, it is concluded that MON 88302 is not likely to be a plant pest. Therefore, M onsanto Company requests a

determination from APHIS that MON 88302 and any progeny derived from crosses

between MON 88302 and conventional Brassica species or deregul ated biotechnologyderived crop be grante d nonregulated status und er 7 CFR Part 340.Monsanto Company 11-CA-233U 14 of 500 TABLE OF CONTENTS

RELEASE OF INFORMATION......................................................................................2

CERTIFICATION .............................................................................................................3

EXECUTIVE SUMMARY ...............................................................................................4

TABLE OF CONTENTS ................................................................................................14

LIST OF TABLES ...........................................................................................................18

LIST OF FIGURES .........................................................................................................22

ABBREVIATIONS AND DEFINITIONS .....................................................................24

I. RATIONALE FOR THE DEVELOPMENT OF MON 88302 ...............................27

I.A. Basis for the Request for a Determ ination of Nonregulated Status under 7 CFR § 340.6 ...............................................................................................................27

I.B. Rationale for the Development of Glyphosate-Tolerant Canola

MON 88302 ...................................................................................................................27

I.C. Submissions to Other Regulatory Agencies ..........................................................28

I.C.1. Submission to the FDA ...............................................................................28

I.C.2. Submission to the EPA ...............................................................................28

I.C.3. Submissions to Fore ign Government Agencies ..........................................29

II. THE BIOLOGY OF CANOLA ................................................................................30

II.A. Canola as a Crop ..................................................................................................30

II.B. Characteristics of the Recipient Plant ..................................................................32

II.C. Canola as a Test System in Product Safety Assessment ......................................32

III. DESCRIPTION OF TH E GENETIC MODIFICATION ....................................33

III.A. The Plasmid Vector PV-BNHT2672 ..................................................................33

III.B. Description of the Transformation System .........................................................33

III.C. The cp4 epsps Coding Sequence and the CP4 EPSPS Protein ...........................34

III.D. Regulatory Sequences ........................................................................................37

III.E. T-DNA Borders ..................................................................................................37

III.F. Genetic Elements Outside of the T-DNA Borders ..............................................37

IV. CHARACTERIZATION OF THE GENETIC MODIFICATION .....................41

IV.A. Southern Blot Analysis to Determine Insert and Copy Number of

T-DNA I in MON 88302 ...............................................................................................47

IV.A.1. T-DNA Probes 1 and 3 ............................................................................47

IV.A.2. T-DNA Probe 2 ........................................................................................48

IV.B. Southern Blot Analysis to De termine the Presence or Absence of PV-BNHT2672 Backbone Sequences in MON 88302 .................................................51Monsanto Company 11-CA-233U 15 of 500 IV.B.1. Backbone Probe 4 ....................................................................................51

IV.B.2. Backbone Probe 5 ....................................................................................51

IV.B.3. Backbone Probe 6 ....................................................................................52

IV.C. Organization and Sequence of the Insert and Adjacent DNA in

MON 88302 ...................................................................................................................56

IV.D. PCR and DNA Sequence Analyses to Examine the MON 88302

Insertion Site ................................................................................................................ ..58

IV.E. Southern Blot Analysis to Examine Insert Stability in Multiple

Generations of MON 88302 ..........................................................................................60

IV.E.1. T-DNA Probes 1 and 3 .............................................................................60

IV.F. Inheritance of the Ge netic Insert in MON 88302 ...............................................63

IV.G. Genetic Modification Ch aracterization Conclusion ...........................................67

V. CHARACTERIZATION AND SAFETY ASSESSMENT OF THE

CP4 EPSPS PROTEIN PRODUCED IN MON 88302 .................................................68

V.A. Identity and Function of the CP4 EPSPS Protein from MON 88302 ..................68

V.B. Characterization and Equiva lence of CP4 EPSPS Protein from

MON 88302 ...................................................................................................................69

V.C. Expression Levels of CP 4 EPSPS Protein in MON 88302 .................................70

V.D. Assessment of Potential Alle rgenicity of the CP4 EPSPS Protein ......................72

V.E. Safety Assessment Summary of CP4 EPSPS Protei n in MON 88302 ................72

V.E.1. The Donor Organism Ha s a History of Safe Use ......................................72

V.E.2. CP4 EPSPS Proteins Are Common in Food and Feeds.............................73

V.E.3. CP4 EPSPS Catalyzes a Specific Enzyme Reaction .................................73

V.E.4. CP4 EPSPS Protein in MON 88302 is Not Homologous to Known Allergens or Toxins................................................................................................73

V.E.5. CP4 EPSPS Protein in MON 88302 Is Labile in in vitro Digestion Assays ....................................................................................................................73

V.E.6. CP4 EPSPS Protein in M ON 88302 is Not Acutely Toxic .......................74

V.F. CP4 EPSPS Protein Characteri zation and Safety Conclusion .............................74

VI. COMPOSITIONAL ASSESSMENT OF MON 88302 .........................................76

VI.A. Compositional Equivalence of MON 88302 Seed to Conventional

Canola ........................................................................................................................ ....77

VI.A.1. Nutrient Levels in Canola Seed ...............................................................79

VI.A.2. Anti-Nutrient Levels in Canola Seed .......................................................80

VI.B. Compositional Assessment of MON 88302 Conclusion ..................................104

VII. PHENOTYPIC, AGRONO MIC, AND ENVIRONMENTAL

INTERACTIONS ASSESSMENT ...............................................................................106Monsanto Company 11-CA-233U 16 of 500 VII.A. Characteristics Measured for Assessment ......................................................106

VII.B. Interpretation of Phenotypic and Environmental Interaction Data .................110

VII.B.1. Interpretation of Detected Differences Criteria ....................................110

VII.C. Comparative Assessments of the Phenotypic, Agronomic, and

Environmental Interaction Characteristics of MON 88302 ........................................113

VII.C.1. Seed Dormancy and Germination Characteristics ................................113

VII.C.2. Field Phenotypic, Agronomic , and Environmental Interactions

Characteristics ......................................................................................................116

VII.C.3. Pollen Characteristics ...........................................................................125

VII.D. Conclusions for Phenotypi c, Agronomic, and Environmental

Interactions Evaluation ................................................................................................126

VIII. U.S. AGRONOMIC PRACTICES.....................................................................127

VIII.A. Introduction ...................................................................................................127

VIII.B. Overview of U.S. Canola Production ............................................................128

VIII.B.1. Canola Production ...............................................................................128

VIII.B.2. Canola Seed Production ......................................................................134

VIII.C. Production Management Considerations .......................................................138

VIII.D. Management of Insects ..................................................................................146

VIII.E. Management of Diseases and Other Pests .....................................................149

VIII.F. Weed Management .........................................................................................150

VIII.G. Second Generation Glyphosat e-tolerant Canola – MON 88302 ...................157

VIII.H. Crop Rotation Practices in Canola .................................................................158

VIII.I. Canola Volunteer Management .......................................................................163

VIII.J. Weed Resistance to Glyphosate Herbicide .....................................................165

VIII.K. Stewardship of MON 88302 ..........................................................................165

VIII.L. Impact of the Introduction of MON 88302 on Agricultural Practices ...........166

IX. ENVIRONMENTA L CONSEQUENCES ...........................................................167

IX.A. Introduction ......................................................................................................167

IX.B. Plant Pest Assessment of MO N 88302 Insert and Expressed Protein ..............168

IX.B.1. Characteristics of the Gene tic Insert and Expressed Protein .................168

IX.B.2. Compositional Characteristics ...............................................................169

IX.B 3. Phenotypic and Agronomi c and Environmental Interaction

Characteristics ......................................................................................................170

IX.C. Weediness Potential of MON 88302 ................................................................173

IX.D. Potential for Pollen Mediated Gene Flow and Introgression ...........................180Monsanto Company 11-CA-233U 17 of 500 IX.D.1. Potential for Pollen Mediated Gene Flow and Introgression of B. napus ................................................................................................................180

IX.D.2. Potential for Pollen Mediated Gene Flow and Introgression of B. napus to Closely Related Brassica Species .....................................................182

IX.D.3. Potential for Pollen Mediated Gene Flow and Introgression of B. napus to Brassica Vegetables ..........................................................................185

IX.D.4. Potential for Pollen Mediated Gene Flow and Introgression of B. napus to Related Species in the Family Brassicaceae .....................................185

IX.D.5. Transfer of Genetic Info rmation to Species with Which Brassica

napus Cannot Interbreed (Horizontal Gene Flow) ...............................................194

IX.E. Potential Impact on Canola Agronomic Practices ............................................194

IX.F. Summary of Plant Pest Assessments ................................................................194

X. ADVERSE CONSEQUENC ES OF INTRODUCTION .......................................196

REFERENCES ...............................................................................................................198

APPENDICES ................................................................................................................217

Appendix A: USDA Notifications ................................................................................218

Appendix B: Materials, Methods, and Results for Molecular Analyses of

MON 88302 ..................................................................................................................... 219

Appendix C: Materials, Methods, a nd Results for Characterization of

CP4 EPSPS Protein Produced in MON 88302 ............................................................226

Appendix D: Materials and Methods Used for the Analysis of the Levels of

CP4 EPSPS Protein in MON 88302 .............................................................................245

Appendix E: Materials, Methods, and Individual-Site Results for Compositional Analysis of MON 88302 Canola Seed .................................................250

Appendix F: Materials, Methods and Results for Seed Dormancy and

Germination Assessment of MON 88302 .....................................................................307

Appendix G: Materials, Methods and Individual Site Results from Phenotypic, Agronomic and Environmental Interaction Assessment of

MON 88302 under Field Conditions ............................................................................312

Appendix H: Materials and Methods for Pollen Morphology and Viability

Assessment .................................................................................................................... ..339

Appendix I: Petitioner’s Environmental Report ........................................................343

Appendix J: Herbicide Resistance ...............................................................................451

Appendix K: Potential Impact of Glyphosate on Human Health and the

Environment ................................................................................................................... 469Monsanto Company 11-CA-233U 18 of 500 LIST OF TABLES

Table III-1. Summary of Gene tic Elements in PV-BNHT2672 ..................................38

Table IV-1. Summary Chart of th e Expected DNA Segments Based on Hybridizing Probes and Restriction Enzymes Used in MON 88302 Analysis ...........45

Table IV-2. Summary of Ge netic Elements in MON 88302 ........................................46

Table IV-3. Segregation of the cp4 epsps Gene During the Development of

MON 88302 ..................................................................................................................... ..66

Table V-1. Summary of CP4 EPSPS Prot ein Levels in Canola Tissues from

MON 88302 Grown in 2009 U.S. and Canadian Field Trials ......................................71

Table VI-1. Summary of Differences (p < 0.05) for the Comparison of Canola Seed Component Levels for MON 88302 vs. the Conventional

Control ....................................................................................................................... .......83

Table VI-2. Statistical Summary of Combined-site Canola Seed Nutrients

for MON 88302 vs. Conventional Control .....................................................................93

Table VI-3. Statistical Summary ofCombined-site Canola Seed Antinutrients for MON 88302 vs. Conventional Control ..................................................101

Table VI-4. Literature Ranges for Components in Canola Seed ..............................102

Table VII-1. Phenotypic, Agronomic and Environmental Interaction

Characteristics Evaluated in U.S. and Canadian Field Trials or Laboratory

Studies ....................................................................................................................... ......108

Table VII-2. Germination Characteri stics of MON 88302 and Conventional

Control ....................................................................................................................... .....115

Table VII-3. Field Phenotypic E valuation Sites for MON 88302 during 2009........118

Table VII-4. Combined-Site Comparison of MON 83302 to Conventional Control During 2009 for Phenotypic and Agronomic Characteristics .....................119

Table VII-5. Summary of Qualitative and Quantitative Environmental Interaction Assessments During 2009 ..........................................................................123

Table VII-6. Combined-Site Analysis : Quantitative Assessment of Flea Beetle and Seedpod Weevil Damage to MON 88302 Compared to the

Conventional Control in 2009 Field Trials ..................................................................124

Table VII-7. Pollen Characteristic s of MON 88302 Compared to the

Conventional Control ....................................................................................................126

Table VIII-1. Canola Production in the United States, 2001 – 2010 ........................131

Table VIII-2. U.S. Canola Production by State in 2010 ............................................132

Table VIII-3. North Dakota Canola Production Costs and Returns from

2005 – 2009.................................................................................................................... ..133

Table VIII-4. Insect Pests by Canola Development Stage in Spring and

Winter Canola in the U.S. .............................................................................................148Monsanto Company 11-CA-233U 19 of 500 Table VIII-5. Common Weeds in Spring Canola Production in North

Dakota ........................................................................................................................ .....152

Table VIII-6. Most Common Weeds in Winter Canola in the Great Plains ...........152

Table VIII-7. Most Common Weeds in Spring and Winter Canola in the

Pacific Northwest ...........................................................................................................15 2

Table VIII-8. Herbicide Usage in Canola in North Dakota in 2008 .........................153

Table VIII-9. Grass Weed Species Re sponses and Persistence in Soil to Herbicides Applied in Canola Production ...................................................................155

Table VIII-10. Broadleaf Weed Species Responses to Herbicides Applied in

Canola Production .........................................................................................................156

Table VIII-11. Rotational Practices in the U.S. Following Canola Production ......161

Table VIII-12. Ratings for Post-emergence Control of Volunteer Canola in Labeled Rotational Crops .............................................................................................164

Table IX-1. Summary of Literature on the Persistence of Canola Outside of

Cultivated Fields ............................................................................................................1 75

Table IX-2. Summary of Representative Studies of Brassica napus Cross

Pollination Rates at Various Distances ........................................................................181

Table IX-3. Summary of Published Lit erature on Unassisted Hybridization

under Field Conditions with B. napus as the Male Parent .........................................189

Table A-1. USDA Notifications A pproved for MON 88302 and Status of

Trials Conducted under These Notifications ...............................................................218

Table B-1. Hybridization Conditions of Utilized Probes ...........................................222

Table C-1. N-Terminal Sequence of the MON 88302-produced CP4 EPSPS .........229

Table C-2. Summary of the Tryptic Masses Identified for the MON 88302- produced CP4 EPSPS Using MALDI-TOF Mass Spectrometry ...............................231

Table C-3. Comparison of Immunoreactive Signal Between MON 88302-

and E. coli -produced EPSPS Proteins ..........................................................................236

Table C-4. Molecular Weight Comparison Between the MON 88302- and E.

coli -produced CP4 EPSPS Proteins .............................................................................237

Table C-5. CP4 EPSPS Functional Activity Assay ....................................................243

Table D-1. Protein Extraction Methods for Tissue Samples .....................................247

Table E-1. Commercial Reference Canola Varieties .................................................250

Table E-2. Re-expression Formulas for Statistical Analysis of Composition

Data .......................................................................................................................... .......258

Table E-3. Statistical Summary of Site MBNW Canola Seed Nutrient

Content for MON 88302 vs. the Conventional Control ..............................................260

Table E-4. Statistical Summary of Si te MBNW Canola Seed Anti-nutrient

Content for MON 88302 vs. the Conventional Control ..............................................268Monsanto Company 11-CA-233U 20 of 500 Table E-5. Statistical Summary of Site MBPL Canola Seed Nutrient Content

for MON 88302 vs. the Conventional Control .............................................................269

Table E-6. Statistical Summary of Si te MBPL Canola Seed Anti-nutrient

Content for MON 88302 vs. the Conventional Control ..............................................277

Table E-7. Statistical Summary of Site MNCA Canola Seed Nutrient

Content for MON 88302 vs. the Conventional Control ..............................................278

Table E-8. Statistical Summary of Si te MNCA Canola Seed Anti-nutrient

Content for MON 88302 vs. the Conventional Control ..............................................286

Table E-9. Statistical Summary of Si te NDVA Canola Seed Nutrient Content

for MON 88302 vs. the Conventional Control .............................................................287

Table E-10. Statistical Summary of Site NDVA Canola Seed Anti-nutrient

Content for MON 88302 vs. the Conventional Control ..............................................295

Table E-11. Statistical Summary of Site SKSA Canola Seed Nutrient

Content for MON 88302 vs. the Conventional Control ..............................................296

Table E-12. Statistical Summary of Si te SKSA Canola Seed Anti-nutrient

Content for MON 88302 vs. the Conventional Control ..............................................304

Table F-1. Starting Seed of MO N 88302, Control and Commercial Canola Reference Varieties Used in Dormancy Assessment ...................................................309

Table F-2. Dormancy and Germinatio n Characteristics of MON 88302 and

the Conventional Control Seed .....................................................................................310

Table G-1. Starting Seed for Phen otypic, Agronomic and Environmental

Interaction Assessment ..................................................................................................314

Table G-2. Field and Pl anting Information ................................................................315

Table G-3 Data Missing or Excluded from Analysis .................................................321

Table G-4. Individual Site Phenot ypic Comparison of MON 88302 to

Conventional Control ....................................................................................................323

Table G-5. Abiotic Stress Respon se Evaluations of MON 88302 and Conventional Control Using an Observational Severity Scale ..................................329

Table G-6. Disease Damage Evalua tions of MON 88302 and Conventional Control Using an Observational Severity Scale ..........................................................330

Table G-7. Arthropod-Related Da mage Evaluations of MON 88302 and Conventional Control Using an Observational Severity Scale ..................................331

Table G-8. Individual Site Analysis: Qu antitative Assessment of Flea Beetle and Seedpod Weevil Damage to MON 88302 Compared to the Conventional

Control in 2009 Field Trials ..........................................................................................332

Table G-9. Abundance of Pest Arthro pods in Cone Beat Sheet Samples Collected from MON 88302, Conventi onal Control and the Commercial

Reference Varieties ........................................................................................................333Monsanto Company 11-CA-233U 21 of 500 Table G-10. Abundance of Beneficial Arthropods in Cone Beat Sheet Samples Collected from MON 88302, Conventional Control and the

Commercial Reference Varieties ..................................................................................335

Table H-1. Starting Seed for Pollen Morphology and Viability Assessment ...........341

Table I-1. Summary of Potent ial Environmental Impacts ........................................348

Table I-2. Certified Canola Seed Standards ...............................................................363

Table I-3. Common Weeds in Spring Canola Production in North Dakota............379

Table I-4. Most Common Weeds in Wi nter Canola in the Great Plains .................380

Table I-5. Most Common Weeds in Spr ing and Winter Canola in the Pacific

Northwest ..................................................................................................................... ...380

Table I-6. Herbicide Usage in Canola in North Dakota in 2008 ...............................382

Table I-7. Weed Species Responses to Herbicides in Canola ....................................383

Table I-8. Herbicide Resistant Commo n Weeds in Top Five U.S. CanolaProducing States in 2010 ...............................................................................................386

Table I-9. Summary of Re presentative Studies of B. napus Cross Pollination

Rates at Various Distances ............................................................................................392

Table I-10. Cross Pollination Potentia l for Canola with Related Vegetable Seed Species .................................................................................................................. ..395

Table I-11. Glyphosate Use on Roundup Ready Crops .............................................426

Table J-1. U.S. Glyphosate Resistant Weeds through March 2011..........................457

Table J-2. Management Recommendations for Control of Glyphosate Resistant Weeds in Canola and Rotational Crops ......................................................461

Table K-1. Established Glyphosate Tol erances in Selected Crops Prior to Glyphosate-tolerant Crops (1993) ................................................................................476

Table K-2. Summary of EPA Approvals for Glyphosate Use in Glyphosatetolerant Crops................................................................................................................. 477

Table K-3. Aggregate Exposure Assessment for Glyphosate ....................................479Monsanto Company 11-CA-233U 22 of 500 LIST OF FIGURES

Figure III-1. Circular Map of PV -BNHT2672 Showing Probes 1-6 ..........................35

Figure III-2. Schematic of the Development of MON 88302 ......................................36

Figure III-3. Deduced Amino Acid Sequence of the CP4 EPSPS Precursor

Protein ....................................................................................................................... ........40

Figure IV-1. Schematic Representation of the Insert and Flanking DNA in

MON 88302 ..................................................................................................................... ..44

Figure IV-2. Southern Blot Analysis to Determine Insert and Copy Number

of T-DNA I in MON 88302: Probes 1 and 3 .................................................................49

Figure IV-3. Southern Blot Analysis to Determine Insert and Copy Number

of T-DNA in MON 88302: Probe 2 ................................................................................50

Figure IV-4. Southern Blot Analysis to Determine the Presence or Absence of the PV-BNHT2672 Backbone Sequences in MON 88302: Probe 4 ........................53

Figure IV-5. Southern Blot Analysis to Determine the Presence or Absence of the PV-BNHT2672 Backbone Sequences in MON 88302: Probe 5 ........................54

Figure IV-6. Southern Blot Analysis to Determine the Presence or Absence of the PV-BNHT2672 Backbone Sequences in MON 88302: Probe 6 ........................55

Figure IV-7. Overlapping PCR Analys is across the Insert in MON 88302 ...............57

Figure IV-8. PCR Amplification of the MON 88302 Insertion Site in

Conventional Canola .......................................................................................................59

Figure IV-9. Breeding History of MON 88302 .............................................................61

Figure IV-10. Southern Blot Analysis to Examine Insert Stability in Multiple

Generations of MON 88302: Probes 1 and 3 .................................................................62

Figure IV-11. Breeding Path for Ge nerating Segregation Data for

MON 88302 ..................................................................................................................... ..65

Figure VII-1. Schematic Diagram of Agronomic and Phenotypic Data Interpretation Methods .................................................................................................111

Figure VIII-1. Planted Canola Acres by County in the U.S. in 2009 ........................131

Figure C-1. MALDI-TOF MS Cover age Map of the MON 88302-produced

CP4 EPSPS Protein........................................................................................................232

Figure C-2. Western Blot Analysis of MON 88302- and E. coli -produced

CP4 EPSPS Proteins ......................................................................................................235

Figure C-3. Molecular Weight and Purity Analysis of the MON 88302- produced EPSPS Protein ...............................................................................................238

Figure C-4. Glycosylation Analysis of the MON 88302-produced CP4 EPSPS

Protein ....................................................................................................................... ......241

Figure I-1. Canola Growing Regions of Canada and the U.S. ..................................356

Figure I-2. U.S. Canola Oil Production and Demand ................................................358Monsanto Company 11-CA-233U 23 of 500 Figure I-3. U.S. Canola Harvested Seed Production 1991-2010 ...............................359

Figure I-4. U.S. 2010 Canola Harvested Seed Production by State (metric tons) ......................................................................................................................... ........360

Figure J-1. Weed Resistance to Vari ous Herbicide Chemical Families ...................454

Figure K-1. Tier I Endangered Species Analysis .......................................................486Monsanto Company 11-CA-233U 24 of 500 ABBREVIATIONS AND DEFINITIONS 1

~ Approximately a.e. Acid Equivalent a.i. Active Ingredient

AA Amino Acid ADF Acid Detergent Fiber

AOSA Association of Offi cial Seed Analysts Ave Average BBCH Scale Bayer, BASF, Ciba-Geigy and Hoechst Cereal Grain Growth

Scale

BLOCKS A database of amino acid motifs found in protein families

BLOSUM Blo cks Su bstitution Matrix, used to score similarities between pairs of distantly related prot ein or nucleotide sequences

BSA Bovine Serum Albumin bw Body Weight C8-C24 8-24 Carbon-Chain Fatty Acids CFIA Canadian Food Inspection Agency CFR Code of Federal Regulations

CI Confidence Interval cp4 epsps Codon optimized coding sequence of the aroA gene from Agrobacterium sp. strain CP4 encoding CP4 EPSPS CP4 EPSPS 5-Enolpyruvylshikimate-3-phos phate synthase protein from the Agrobacterium sp. strain CP4

CPI Canola Protein Isolate CSFII Continuing Surveys of Food Intakes by Individuals DAP Days After Planting DDI Daily Dietary Intake DEEM-FCID Dietary Exposure Evalua tion Model-Food Commodity Intake

Database

DTT Dithiothreitol dw Dry Weight

E. coli Escherichia coli EDTA Ethylenediaminetetraacetic Acid ELISA Enzyme-l inked Immunos orbent Assay EPA Environmental Protection Agency EPSP 5-enolpyruvylshikimate-3-phosphate EPSPS 5-enolpyruvylshikimate- 3-phosphate synthase enzyme FA Fatty Acid FAO/WHO Food and Agriculture Organi zation of the United Nations/World Health Organization FFDCA Federal Food, Drug and Cosmetic Act

1 Alred, G.J., C.T. Brusaw, and W.E. Oliu. 2003. Handbook of Technical Writing, 7th edn., pp. 2-7. Bedford/St. Martin's, Boston, MA.Monsanto Company 11-CA-233U 25 of 500 FIFRA Federal Insecticide, Fungicide, and Rodenticide Act fw Fresh Weight

g g-force GLP Good Laboratory Practice GRAS Generally Recognized As Safe HEPES N-[2-(hydroxyethyl)piperazine -N'-(2-ethanesul fonic acid)]

ILSI International Life Sciences Institute

kDa Kiloda lton

LB Laemmli buffer

LOD Limit of Detection LOQ Limit of Quantitation

MALDI-TOF-MS Matrix Assisted Laser De sorption Ionization - Time of Flight Mass Spectrometry

MMT Million metric tons MOE Margin of Exposure MW Molecular Weight MWCO Molecular Weight Cuto ff NDF Neutral Detergent Fiber NFDM Non-Fat Dry Milk NIST National Institute of Standards and Technology

NOAEL No Observable Adverse Effect Level NR Nitrogen Requirements NTOs Non-Target Organisms OD Optical Density OECD Organization for Economic Co-operation and Development ORF Open Reading Frame

OSL Over Season Leaf PBS Phosphate Buffered Saline PBST Phosphate Buffered Saline containing 0.05% (v/v) Tween-20 PCC Previous Crop Credit PCR Polymerase Chain Reaction PEP Phosphoe nolp yruvate

P i Inorganic phosphate

PPA Plant Protection Act ppm Parts Per Million

PRESS Predicted Re sidual Sum of Squares PVDF Polyv inylidene Dif luoride RBD Refined, Bleached, and Deodorized RED Re-registration Eligibility Decision

RfD Reference Dose RQ Risk Quotient S3P Shikimate-3-phosphate Sarkosyl N -lauroylsarcosine, sodium salt

SD Standard Deviation SDS-PAGE Sodium Dodecyl Sulfate-Polya crylamide Gel Electrophoresis

SE Standard ErrorMonsanto Company 11-CA-233U 26 of 500 SGF Simulated Gastric Fluid sp. Species STN Soil Test Nitrate-nitrogen index SNP Single Nucleotide Polymorphism TDF Total Dietary Fiber

T-DNA Transfer DNA Tm Melting Temperature

Tz Tetrazolium

YP Yield PotentialMonsanto Company 11-CA-233U 27 of 500 I. RATIONALE FOR THE DEVELOPMENT OF MON 88302

I.A. Basis for the Request for a Determ ination of Nonregulated Status under 7 CFR § 340.6

The Animal and Plant Health Inspection Serv ice (APHIS) of the United States (U.S.) Department of Agriculture (USDA) has res ponsibility, under the Plant Protection Act (Title IV Pub. L. 106-224, 114 Stat. 438, 7 U.S.C. § 7701-7772) to prevent the introduction and dissemination of plant pe sts into the U.S. APHIS regulation 7 CFR § 340.6 provides that an applicant may petition APHIS to evaluate submitted data to determine that a particular regulated arti cle does not present a pl ant pest risk and no longer should be regulated. If APHIS dete rmines that the regulated article does not present a plant pest risk, the petition is gr anted, thereby allowing unr estricted introduction

of the article.

Monsanto Company is submitting this requ est to APHIS for a determination of nonregulated status for the new biotec hnology-derived canola pr oduct, MON 88302, any progeny derived from crosses between MON 88302 and conventional canola, and any progeny derived from crosses of MON 88302 with biotechnology-derived canola that have previously been granted nonregul ated status under 7 CFR Part 340.

I.B. Rationale for the Development of Glyphosate-Tolerant Canola MON 88302

Monsanto Company has developed a sec ond-generation glyphosate-tolerant canola product, MON 88302, designed to provide growers with impr oved weed control through greater flexibility for glyphosate herbicide a pplication. Weed competition can be a major limiting factor in canola production leading to significant yield redu ctions (CCC, 2006). Certain perennial weeds, such as Canada thistl e, are known to be par ticularly important to control in canola production. For example, studies have demonstrated that only 10 Canada thistle plants per square meter have resulted in 10% yield loss while 40 plants per square meter have resulted in over 50% yield loss (CCC, 2006). Glyphosate is highly effective against the majority of annual and perennial grasses and broad-leaf weeds including Canada thistle ( NDSU, 2005; Padgette et al., 1996). Glyphosate has been shown to have a favorable safety profile by the U.S. EPA (1993a) which has concluded that use of glyphosate will not pose unreasonabl e risks to humans or the environment.

MON 88302 was produced by incorporation of the cp4 epsps coding sequence from the

common soil bacterium Agrobacterium sp. strain CP4. The cp4 epsps coding sequence directs the production of th e 5-enolpyruvylshikimate-3- phosphate synthase (termed CP4 EPSPS) that is less sensitive to inhibition by glyphosate compared to the endogenous plant EPSPS. Hence, the CP4 EPSPS renders MON 88302 tolerant to glyphosate, the active ingredient in the Roundup family of ag ricultural herbicides. The transformation cassette in MON 88302 employs sequences from the promoter of the TsfI gene from Arabidopsis thaliana and enhancer sequences from the 35S promoter from the figwort mosaic virus (FMV) to enhance CP4 EPSPS production in male reproductive tissues (Feng et al., 2010). Glyphosate is a syst emic herbicide and is translocated in the plant generally from a strong source tissue ( e.g., leaf) to rapidly deve loping, or sink tissueMonsanto Company 11-CA-233U 28 of 500 (Devine et al., 1993). Sink tissu es, such as pollen, that accu mulate glyphosate and lack sufficient CP4 EPSPS expression are considered to be at risk for gl yphosate injury. By virtue of enhanced CP4 EPSPS expressi on in male reproductive tissues, MON 88302 provides tolerance to glyphosate during the sens itive reproductive stages of growth (Feng et al., 2010). Use of MON 88302 will enable growers to apply Roundup herbicide at higher rates and at later stages of development than is possi ble with the current product facilitating: 1) better contro l of tough-to-kill weeds; 2) an increased opportunity to control weeds if glyphosate application is dela yed due to weather or equipment failure; 3) an enhanced ability to tailor labeled glyphosat e applications to weed development stage instead of the canola developmental stage; and 4) enhanced protection of canola plants at more advanced development stages. Use of MON 88302 will provide growers with the opportunity to ensure weeds that may impact yields are removed at the optimal time while minimizing the potential for crop injury.

I.C. Submissions to Other Regulatory Agencies

Under the Coordinated Framework for Regul ation of Biotechnology, the responsibility for regulatory oversight of biotechnology-derived crops fa lls primarily on three U.S. agencies: U.S. Food and Drug Administration (FDA), the United States Department of Agriculture (USDA), and in the case of plant incorporated protectants, the Environmental Protection Agency (EPA). Deregulati on of MON 88302 by USDA constitutes only one component of the overall regulato ry oversight and review of this product. As a practical matter, MON 88302 cannot be released and marketed until FDA and USDA have completed their reviews and assessments under their respective jurisdictions.

I.C.1. Submission to the FDA

MON 88302 falls within the scope of th e 1992 FDA policy statement concerning regulation of products derive d from new plant varieties, including those developed through biotechnology (U.S. FDA, 1992). In co mpliance with this policy, Monsanto has

initiated a consultation with the FDA (BNF No. 127) on th e food and feed safety and compositional assessment of MON 88302. Monsanto submitted a safety and nutritional assessment summary document to the FDA in March 2011.

I.C.2. Submission to the EPA

The EPA has authority over the use of pesticid e substances under the Federal Insecticide, Fungicide and Rodenticide Act (FIFRA), as amended (7 U.S.C § 136 et seq .). Monsanto submitted amended labeling to the U.S. Enviro nmental Protection Agency (U.S. EPA) in February 2011 for EPA Registratio n Numbers 524-537 (Roundup WeatherMAX ®
Herbicide) and 524-549 (Roundup PowerMAX ® Herbicide), that propose to modify the current use pattern of gl yphosate in canola based on MON 88302. The post emergence (in-crop) use of glyphosate in Roundup Ready canola was first approved by the U.S. EPA in March 1999. Although the amended labeling increases the rate of application and widens the application period relative to ca nola development, this use of glyphosate does not present any new environmental exposure s cenarios not previously evaluated for useMonsanto Company 11-CA-233U 29 of 500 on other Roundup Ready crops which have alre ady been deemed acceptable by the U.S.

EPA.

I.C.3. Submissions to Forei gn Government Agencies

To support commercial introduction of MON 88302 in the U.S., re gulatory submissions will be made to countries that import signif icant quantities of canola and canola products

from the U.S. This results in submissi ons to a number of additional governmental regulatory agencies including, but not limited to the Ministry of Agriculture, People’s Republic of China; Japan’s Ministry of Ag riculture, Forestry, and Fisheries and the Ministry of Health, Labor, and Welfare; the Canadian Food Inspection Agency and Health Canada; the Intersectoral Commissi on for Biosafety of Genetically Modified Organisms, Mexico; the European Food Safe ty Authority, Food Standards Australia New Zealand, the Korea Food and Drug Admini stration, and the Rural Development Administration of Korea, as well as to re gulatory authorities in other canola importing countries with functioning regulatory systems. As appropriate, notificat ions will be made to countries that import significant quantitie s of canola and canola products and do not have a formal regulatory review process for biot echnology-derived crops.Monsanto Company 11-CA-233U 30 of 500 II. THE BIOLOGY OF CANOLA

Brassica napus L. is a versatile crop that provid es both food and feed to the global economy, and whose biology is well underst ood and documented. There are numerous terms used to describe oil-producing B. napus varieties including oi lseed rape, rapeseed, rape, low erucic acid rapeseed and ca nola. For purposes of this document, B. napus will be referred to as oilseed rape and th e term canola will be used to denote B. napus varieties that produce lo w (< 2%) erucic acid oil and have levels of glucosinolates below the accepted standard of 30 μmol es/g in meal (OECD, 2001).

The Organisation for Economic Co-operation and Development Consensus Document on the Biology of Brassica napus (OECD, 1997) provides key information on:

- general description of B. napus biology, including taxonomy and morphology and use

of B. napus as a crop plant

- agronomic practices in B. napus cultivation

- geographic centers of origin

- reproductive biology

- inter-species/genus introgression into relati ves and interactions with other organisms

- summary of the ecology of B. napus

Additional information on the biology of B. napus can be found on the Canadian Food Inspection Agency website (CFIA, 2005), and the Australian Government Department of Health and Ageing (Office of the Gene T echnology Regulator) webs ite (OGTR, 2008). Information on the taxonomy of B. napus can be found in the U.S. Department of Agriculture Natural Resources Conservati on Service PLANTS database (USDA-NRCS, 2010b).

To support the evaluation of the plant pe st potential of MON 88302 relative to conventional canola, additional information regarding several aspects of canola biology can be found elsewhere in this petition. This includes: agronomic pr actices for canola in Section VIII; volunteer manage ment of canola in Section V III; and inter-species/genus introgression potential in Section IX.

II.A. Canola as a Crop

While canola oil can be derived from any one of three species: Brassica napus, Brassica rapa, and Brassica juncea (OGTR, 2008; U.S. FDA, 1988; 2000)

, most canola

oil is derived from Brassica napus . Oilseed rape is a member of the mustard (Brassicaceae) family, and has been cultivated by ancient civilizati ons in Asia and the Mediterranean primarily for its use as oil in lamps (Colton and Sykes, 1992). Later B. napus oil was used as an industrial lubricant, and today there is still demand for high erucic oil in a variety of industrial applications.Monsanto Company 11-CA-233U 31 of 500 Until recently, the presence of the naturally occurring toxicants, erucic acid in the oil fraction and glucosinolates in the meal has made rapeseed oil and meal derived from B. napus unattractive for human consumption a nd as an animal feed, respectively, particularly in western countries . High erucic acid rapeseed oil (as much as 50% of total fatty acids) has been shown to have cardiopathic potential resulting in a weakening of the heart muscle in experimental animals (Bo zcali et al., 2009; Chien et al., 1983) and high levels of glucosinolates made oilseed rape meal unsuitable for use in animal nutrition because of anti-nutritional, goitrogenic (s uppresses thyroid function), reproductive, and palatability problems (Fenwick et al., 1989). However, in the 1960s intensive breeding programs resulted in the deve lopment and introduction of lo w erucic acid or canola (Canadian oil, low acid) va rieties of oilseed rape (OECD, 2001; OGTR, 2008). At approximately the same time low erucic acid varieties of B. rapa were introduced (OECD, 2001). Slightly later, in the 1980s, low erucic acid varieties of B. juncea were developed (CCC, 1999). However, B. napus varieties are the most commonly grown canola oil-producing varieties in th e U.S. (Boyles et al., 2009).

Brassica napus , an amphidiploid (chromosome n=19, AA and CC genomes), is thought to be derived from a cross between two diploid Brassica species, B. rapa (chromosome n=10, AA genome) and B. oleracea (chromosome n=9, CC genome). Brassica napus has the greatest sexual compatibility with B. rapa and B. juncea under natural field conditions, but has also been known to outcr oss with some wild relatives including Raphanus raphanistrum (wild radish) and Hirschfeldia incana (shortpod mustard) (OECD, 1997; OGTR, 2008).

There are spring and winter biotypes of canol a varieties. Spring ca nola, a cool season crop, is grown in Canada, southern Australia, northern China, and in the northern Great Plains region of the U.S. Spring canola is slow growing and does not compete well with weeds in its early growth stages. Closely related weeds like wild mustard, stinkweed and shepherd’s purse are often problematic in commercial spring canola fields, and weeds must be controlled early in the spring canola life cycle to avoid yield loss due to competition (OECD, 1997). Winter canola is planted in the fall, requires vernalization (exposure to winter cold) to flower, and is grown in parts of Europe, Asia, the northwestern U.S. and in the central portions of the U.S. Great Plai ns. Winter canola, once established, suppresses and out-competes most annual weeds (Boyles et al., 2009). In addition to various pre-emergent weed control options, varieties of canola having tolerance to glyphosate, glufos inate and imazamox herbicides for weed control in canola fields are widely available.

Canola oil is currently the world’s third la rgest source of vege table oil (15%) after soybean oil (28%) and palm oil (32%) (A SA, 2010; USDA-ERS, 2010). Canola oil appeals to health conscious consumers because it has the lowest level of saturated fat of all edible oils, th e second-highest level of monounsaturated fat, and is free of artificial trans-fatty acids (Brown et al., 2008; USDA-ER S, 2010). Canola seed is also processed into canola meal which is used as high protei n animal feed. Canola meal is the second largest protein meal produced in the world. However, it is relatively small compared to soybean meal. Global production of canola m eal was 30.8 million metr ic tons (MMT) in 2008/2009 compared to 151.6 MMT for soybean m eal (USDA-ERS, 2010). Canola mealMonsanto Company 11-CA-233U 32 of 500 contains less protein than soybean meal (34-38% vs. 44-49%) and fewer key amino acids (USDA-ERS, 2010).

The United States produced only about 0.7 MMT (1.1%) of the world’s canola production (58 MMT) in the growing season 2008/2009 (USDA-FAS, 2010). The European Union, Canada, and China were the largest producers of canola with 19.0, 12.6, and 12.1 MMT, respectively.

II.B. Characteristics of the Recipient Plant

The B. napus canola variety used as the recipi ent for the DNA insertion to create MON 88302 was Ebony, a non-transgenic conven tional spring canola variety registered with the Canadian Food Inspection Agen cy in 1994 by Monsanto Company (CFIA, 2010). Ebony originated from a cross of varie ties (Bienvenu × Alto) × Cesar. Selection criteria for the non-transgenic variety incl uded yield, oil and pr otein content, and tolerance to the fungus Leptosphaeria maculans , commonly known as blackleg (CFIA, 1994). Ebony was used to produce the glyphos ate-tolerant canola MON 88302 because it responds well to Agrobacterium -mediated transformation and tissue regeneration.

II.C. Canola as a Test System in Product Safety Assessment

Ebony was used as the conventional canola comp arator (referred to in this document as the conventional control) in the safety assessment of MON 88302. The background genetics of the conventional control are similar to that of MON 88302, but do not contain

the cp4 epsps expression cassette. In addition, co mmercial conventional canola varieties (referred to in this consultation document as co mmercial reference varieties) were used to establish ranges of natural variability or re sponses representative of commercial canola

varieties. The commercial reference varietie s used at each location were selected based on their availability and agronomic fit for the geographic region.Monsanto Company 11-CA-233U 33 of 500 III. DESCRIPTION OF TH E GENETIC MODIFICATION

MON 88302 was developed through Agrobacterium -mediated transformation of hypocotyls from Ebony canola variety uti lizing plasmid vector PV-BNHT2672. This section describes the plasmid v ector, the donor gene, and the re gulatory elemen ts used in the development of MON 88302 as well as the deduced amino acid sequence of the CP4 EPSPS protein produced in MON 88302. In this section, tr ansfer DNA (T-DNA)

refers to DNA that is transferred to the plant during transformation. An expression cassette is comprised of sequences to be transcribed and the regulatory elements necessary for the expression of those sequences.

III.A. The Plasmid Vector PV-BNHT2672

PV-BNHT2672 was used in th e transformation of canola to produce MON 88302 and is shown in Figure III-1. The elements included in this plasmid vector are described in Table III-1. PV-BNHT2672 is approximately 9.7 kb and contains one T-DNA that is delineated by Left Border and Right Bo rder regions. The T-DNA contains the cp4 epsps coding sequence under the control of the FMV/Tsf1 chimeric promoter, the Tsf1 leader and intron sequences, and the E9 3 ′ untranslated region. The ch loroplast transit peptide CTP2 directs transport of the CP4 EPSPS protei n to the chloroplast and is derived from

CTP2 target sequence of the Arabidopsis thaliana shkG gene.

The backbone sequence of PV-BNHT2672, locat ed outside of the T-DNA, contains two origins of replication for maintenan ce of plasmid vector in bacteria ( ori V and ori-pBR322 ), a bacterial selectable marker gene ( aadA ), and a coding sequence for repressor of primer protein ( rop ) for maintenance of plasmid vector copy number in Escherichia coli (E. coli) . A description of the genetic elements and their prefixes ( e.g., B-, P-, L-, I-, TS-, CS-, T-, and OR-) in PV-BNHT2672 is provided in Table III-1.

III.B. Description of the Transformation System

MON 88302 was developed through Agrobacterium -mediated transformation of canola hypocotyls, based on the method describe d by Radke et al. (1992), utilizing PV-BNHT2672 (Figure III-1). In summary, hypocotyl segments were excised from Ebony seedlings grown in the dar k. After co-culturing with Agrobacterium carrying the vector, the hypocotyl segments were placed on callus growth medium that contained carbenicillin, ticarcillin dis odium and clavulanate potassium to inhibit the growth of

excess Agrobacterium . The hypocotyls were then placed in selection media containing glyphosate to inhibit the growth of untrans formed cells and plant growth regulators conducive to shoot regeneration. Rooted R 0 plants with normal phenotypic

characteristics were selected and transferred to soil for growth and further assessment.

The R 0 plants generated through the Agrobacterium -mediated transformation were transferred to soil for growth and then self pollinated to produce R 1 seed. R 0 and R 1 plants were evaluated for tolerance to glyphos ate and screened for the presence of the T-DNA ( cp4 epsps expression cassette) and absence of backbone sequence ( ori V ). Subsequently, the cp4 epsps homozygous R 1 plant was self-pollinate d to give rise to R 2Monsanto Company 11-CA-233U 34 of 500 plants. Homozygous R 2 plants containing one copy of the T-DNA inserted at a single locus, were identified by a combination of analytical techniques including glyphosate spray, polymerase chain reaction (PCR), and Southern blot analysis, resulting in production of glyphosate-tolerant canola MON 88302. MON 88302 was selected as the lead event based on superior phenotypic char acteristics and its comprehensive molecular profile. Regulatory studies on MON 88302 were initiated to furt her characterize the genetic insertion and the expressed protei n, and to establis h the food, feed, and environmental safety relative to conventiona l canola. The major steps involved in the development of MON 88302 are de picted in Figure III-2.

III.C. The cp4 epsps Coding Sequence and the CP4 EPSPS Protein

The cp4 epsps expression cassette, or T-DNA in this petition, encodes a 47.6 kDa CP4 EPSPS protein consisting of a single pol ypeptide of 455 amino acids (Figure III-3) (Padgette et al., 1996). The cp4 epsps coding sequence is th e codon optimized coding sequence of the aroA gene from Agrobacterium sp. strain CP4 encoding CP4 EPSPS (Barry et al., 2001; Padgette et al., 1996). The CP4 EPSPS protein is similar and functionally identical to e ndogenous plant EPSPS enzymes, but has a much reduced affinity for glyphosate, the ac tive ingredient in Roundup agri cultural herbicides, relative to endogenous plant EPSPS (Barry et al., 2001; Padgette et al., 1996).Monsanto Company 11-CA-233U 35 of 500 Probe DNA Probe Start Position (bp) End Position (bp) Total Length (kb)

1 T-DNA Probe 1 1 2287

~ 2.3

2 T-DNA Probe 2 2231 3618

~ 1.4

3 T-DNA Probe 3 3562 4910

~ 1.3

4 Backbone Probe 4 4911 6564

~ 1.7

5 Backbone Probe 5 6512 8383

~ 1.9

6 Backbone Probe 6 8329 9664

~ 1.3

Figure III-1. Circular Map of PV-BNHT2672 Showing Probes 1-6 A circular map of PV-BNHT2672 used to develop MON 88302 is shown. Genetic elements and restriction sites (in bold) used in Southern analyses (with positions relative to the first base pair of th e plasmid vector) are shown on th e exterior of the map. The probes used in the Southern analyses are show n on the interior of the map and listed in

the table. PV-BNHT2672 c ontains a single T-DNA. PV-BNHT2672 9664 bp

Ase I 4026 Ase I 8525

Bam HI 3768 Sca I 1307 B-Right Border Region

P-

FMV/Tsf1

L- Tsf1

I- Tsf1

TS- CTP2

CS- cp4 epsps

T-

E9 B-Left Border Region OR- oriV CS- rop OR- ori-pBR322 aadA

T-DNA

2Monsanto Company 11-CA-233U 36 of 500 Figure III-2. Schematic of the Development of MON 88302 Transformed Ebony, a conventional canola variety, hypocotyl segments with the vector PV-BNHT2672 in A grobacterium tumefaciens

Selected transformants and generated rooted shoots from the transformed hypocotyls segments

Identified MON 88302 as lead candi date and further evaluated its

p rogeny in the laboratory and fiel d for insert integrity, glyphosate tolerance and agronomic performance Assembled A grobacterium binary plasmid vector PV-BNHT2672 an d

transferred to A grobacterium tumefaciens strain ABI

Evaluated the transformed plan ts for tolerance to glyphosate and screened the transformed plan ts for the presence of T-DNA ( cp4 epsps expression cassette) and absence of the T-DNA backbone ( Ori V )

Selected homozygous plants usi ng a quantitative polymerase chain

reaction methodMonsanto Company 11-CA-233U 37 of 500 III.D. Regulatory Sequences

The cp4 epsps coding sequence in MON 88302 is under the regulation of the FMV/Tsf1 chimeric promoter, the Tsf1 leader and intron sequences, and the E9 3 ′ untranslated region. The FMV/Tsf1 chimeric promoter, which directs transcription in plant cells, contains enhancer sequences from the promot er of the FMV 35S RNA (Richins et al., 1987) combined with the promoter from the Tsf1 gene of Arabidopsis thaliana that encodes elongation factor EF-1 α (Axelos et al., 1989). The Tsf1 leader sequence is the 5' untranslated region from the Tsf1 gene of Arabidopsis thaliana (Axelos et al., 1989). The

E9 3' untranslated region is the 3' untranslated region of the pea ( Pisum sativum ) ribulose-1,5-bisphosphate car boxylase small subunit ( rbcS2 ) E9 gene (Coruzzi et al., 1984) and is present to dire ct polyadenylation of the cp4 epsps transcript. The chloroplast transit peptide CTP2 directs tr ansport of the CP4 EPSPS protein to the chloroplast and is derived from CTP2 target sequence of the Arabidopsis thaliana shkG gene (Herrmann, 1995; Klee et al., 1987).

III.E. T-DNA Borders

PV-BNHT2672 contains Right Border and Le ft Border regions (Figure III-1 and Table III-1) that were derived from Agrobacterium tumefaciens plasmids. The border regions each contain a 24-25 bp nick site th at is the site of DNA exchange during transformation (Barker et al., 1983; Depicker et al., 1982; Zambryski et al., 1982). The border regions separate the T-DNA from the pl asmid backbone region and are involved in the efficient transfer of the T-DNA into the canola genome.

III.F. Genetic Elements Outside of the T-DNA Borders

Genetic elements that exis t outside of the T-DNA border regions are those that are

essential for the maintenance or selection of PV-BNHT2672 in bacteria. The origin of replication, ori V, is required for the maintenance of the plasmid in Agrobacterium and is derived from the broad host plasmid RK2 (Stalker et al., 1981). The orig in of replication, ori-pBR322, is required for the maintenance of the plasmid in E. coli and is derived from the plasmid vector pBR322 (Sutcliffe, 1979). Coding sequence rop encodes the repressor of primer (ROP) protein which is necessary for the maintenance of plasmid copy number

in E. coli (Giza and Huang, 1989). The selectable marker aadA is a bacterial promoter and coding sequence for an enzyme from tran sposon Tn7 that confers spectinomycin and streptomycin resistance (Fling et al., 1985) in E. coli and Agrobacterium during molecular cloning. As these elements are ou tside the border regions, they are not expected to be transferre d into the canola genome. The absence of the backbone sequence in MON 88302 has been confirme d by Southern blot analyses (see Section IV.B).Monsanto Company 11-CA-233U 38 of 500 Table III-1. Summary of Gene tic Elements in PV-BNHT2672

Genetic Element Location

in Plasmid Function (Reference)

T-DNA

B 1 -Right Border Region 1-357 DNA region from Agrobacterium tumefaciens containing the Right Border sequence used for transfer of the T-DNA (Depicker et al., 1982; Zambryski et al., 1982) Intervening Sequence 358-427 Sequence used in DNA cloning

P 2

- FMV/Tsf1 428-1467 Chimeric promoter consisting of the promoter

of the Tsf1 gene from the Arabidopsis thaliana encoding elongation factor EF-1 α (Axelos et al., 1989) and enhancer sequences from the 35S promoter from the figwort mosaic virus (Richins et al., 1987)

L 3

- Tsf1 1468-1513 5' untranslated leader (exon 1) from the Arabidopsis thaliana Tsf1 gene encoding elongation factor EF-1 α (Axelos et al., 1989)

I 4

- Tsf1 1514-2135 Intron from the Arabidopsis thaliana Tsf1 gene encoding elongation factor EF-1 α (Axelos et al., 1989) Intervening Sequence 2136-2144 Sequence used in DNA cloning

TS 5

- CTP2 2145-2372 Targeting sequence from the shkG gene encoding the chloroplast transit peptide region

of Arabidopsis thaliana EPSPS (Herrmann, 1995; Klee et al., 1987) that directs transport of the CP4 EPSPS protein to the chloroplast

CS 6

- cp4 epsps 2373-3740 Codon optimized coding sequence of the aroA gene from the Agrobacterium sp

. strain CP4 encoding the CP4 EPSPS protein (Barry et al., 2001; Padgette et al., 1996) Intervening Sequence 3741-3782 Sequence used in DNA cloning

T 7

- E9 3783-4425 3' untranslated sequence from the rbcS2 gene of Pisum sativum (pea) encoding the Rubisco small subunit (Coruzzi et al., 1984) Intervening Sequence 4426-4468 Sequence used in DNA cloning

B-Left Border Region 4469-4910 DNA region from Agrobacterium tumefaciens containing the Left Border sequence used for transfer of the T-DNA (Barker et al., 1983; Zambryski et al., 1982)Monsanto Company 11-CA-233U 39 of 500 Table III-1. Summary of Genetic Elements in PV-BNHT2672 (continued)

Genetic Element Location

in Plasmid Function (Reference)

Vector Backbone Intervening Sequence 4911-4996 Sequence used in DNA cloning

OR 8

- oriV 4997-5393 Origin of replication fr om the broad host range plasmid RK2 for maintenance of plasmid in Agrobacterium (Stalker et al., 1981) Intervening Sequence 5394-6901 Sequence used in DNA cloning

CS- rop 6902-7093 Coding sequence for repressor of primer protein for maintenance of plasmid copy

number in E. coli (Giza and Huang, 1989) Intervening Sequence 7094-7520 Sequence used in DNA cloning

OR- ori-pBR322 7521-8109 Origin of replica tion from pBR322 for maintenance of plasmid in E. coli (Sutcliffe, 1979) Intervening Sequence 8110-8639 Sequence used in DNA cloning

aadA 8640-9528 Bacterial promoter, coding sequence, and 3 ′ untranslated region for an aminoglycoside-modifying enzyme, 3''(9)- O -nucleotidyl-transferase from the transposon Tn7 (Fling et al., 1985) that confers spectinomycin and streptomycin resistance Intervening Sequence 9529-9664 Sequence used in DNA cloning

1 B, Border

2 P, Promoter

3 L, Leader

4 I, Intron

5 TS, Targeting Sequence

6 CS, Coding Sequence

7 T, Transcription Termination Sequence

8 OR, Origin of ReplicationMonsanto Company 11-CA-233U 40 of 500 1 MAQVSRICNG VQNPSLISNL SKSSQRKSPL SVSLKTQQHP RAYPISSSWG

51 LKKSGMTLIG SELRPLKVMS SVSTAC MLHG ASSRPATARK SSGLSGTVRI
101 PGDKSISHRS FMFGGLASGE TRITGLLEGE DVINTGKAMQ AMGARIRKEG

151 DTWIIDGVGN GGLLAPEAPL DFGNAATGCR LTMGLVGVYD FDSTFIGDAS
201 LTKRPMGRVL NPLREMGVQV KSEDGDRLPV TLRGPKTPTP ITYRVPMASA
251 QVKSAVLLAG LNTPGITTVI EPIMTRDHTE KMLQGFGANL TVETDADGVR
301 TIRLEGRGKL TGQVIDVPGD PSSTAFPLVA ALLVPGSDVT ILNVLMNPTR
351 TGLILTLQEM GADIEVINPR LAGGEDVADL RVRSSTLKGV TVPEDRAPSM

401 IDEYPILAVA AAFAEGATVM NGLEELRVKE SDRLSAVANG LKLNGVDCDE

451 GETSLVVRGR PDGKGLGNAS GAAVATHLDH RIAMSFLVMG LVSENPVTVD

501 DATMIATSFP EFMDLMAGLG AKIELSDTKA A

Figure III-3. Deduced Amino Acid Sequence of the CP4 EPSPS Precursor ProteinThe amino acid sequence of the CP4 EPSPS precursor protein was deduced from the fulllength coding nucleotide sequence present in PV-BNHT2672. The 76 amino acid CTP2, the transit peptide of the Arabidopsis thaliana EPSPS protein, is underlined. CTP2 targets CP4 EPSPS protein to the chloroplasts. At the chloroplast the CTP2 is cleaved producing the mature 455 amino acid CP4 EPSPS protein that begins with the methionine at position 77.Monsanto Company 11-CA-233U 41 of 500 IV. CHARACTERIZATION OF THE GENETIC MODIFICATION

Characterization of the DNA insert in M ON 88302 was conducted by Southern blot, PCR and DNA sequence analyses. Th e results of this characterization demonstrate that MON 88302 contains a single copy of the cp4 epsps expression cassette, i.e. , the T-DNA, is stably integrated at a single locus and is inherited according to Mendelian principles over multiple generations. These conclusions were based on several lines of evidence: 1) Southern blot analyses assayed the en tire canola genome for the presence of T-DNA

and the absence of the backbone se quences derived from PV-BNHT2672, and demonstrated that only a single copy of the T-DNA was inserted at a single site; 2) DNA sequence analyses determined the exact sequence of the inserted DNA and the DNA sequences flanking the 5' and 3' ends of the insert, and allowed a comparison of the T-DNA sequence to the plasmid vector to conf irm that only the expe cted sequences were integrated; 3) DNA sequences flanking the 5' and 3' ends of the insert were compared to the sequence of the insertion site in conventional canola to identify any rearrangements that occurred at the insertion site during tr ansformation;. 4) Southern blot analysis

demonstrated that the insert in MON 88302 has been maintained over multiple generations of breeding, thereby confirming the stability of the insert; 5) segregation analyses show inheritance and stability of th e insert were as expected across multiple generations. Taken together, the charact erization of the genetic modification demonstrates that a single copy of the T-DNA wa s inserted at a single locus of the canola genome and that no plasmid vector backbone sequences are present in MON 88302.

Southern blot analyses were used to determine the copy numb er and insertion sites of the integrated DNA as well as the presence or absence of plas mid vector backbone sequences. The Southern blot strategy wa s designed to ensure that all potential transgenic segments would be identified. The entire canola genome was assayed with probes that spanned the complete plasmid vector to detect the presen ce of the insert as well as confirm the absence of any plasmi d vector backbone sequences. This was accomplished by using probes that were not more than 2.5 kb in length to ensure a high level of sensitivity. This high level of se nsitivity was demonstrated for each blot by detection of a positive control added at 0.1 genome equivalent . Two sets of restriction enzymes were specifically chosen to fu lly characterize the T-DNA and detect any potential fragments of the T-DNA and backbone sequences. The restriction enzyme sets were chosen such that each enzyme set cl eaves once within the inserted T-DNA and at

least once within the known DNA flanking the 5' or 3' end of the insert. As a consequence, at least one segment containing a portion of the insert with the adjacent 5' flanking DNA generated by one set of the enzyme (s) is of a predictable size and overlaps with another predictable size segment containi ng a portion of the insert with the adjacent 3' flanking DNA generated by another set of th e enzyme(s). This two-set-enzyme design

ensures that the entire insert is identified in a predictable hybridization pattern. This strategy also maximizes the possi bility of detecting an insert ion elsewhere in the genome that could be overlooked if that the fragment co-migrated on the gel with an expected fragment.

To determine the number of copies and insert ion sites of the T-DNA, and the presence or absence of the plasmid vector backbone seque nces, duplicated sample s that consisted ofMonsanto Company 11-CA-233U 42 of 500 equal amounts of digested DNA were run on th e agarose gel. One set of samples was run for a longer period of time (long run) than th e second set (short run). The long run allows for greater resolution of large molecular we ight DNA, whereas the short run allows for retaining the small molecular weight DNA on th e gel. The molecular weight markers on the left of the figures were used to estimate the sizes of the bands presen t in the long run lanes of the Southern blots, and the molecula r weight markers on the right of the figures were used to estimate the sizes of bands pres ent in the short run la nes of the Southern blots (Figure IV-2 through Figure IV-6). Sout hern blot analyses determined that a single copy of the T-DNA was inserted at a si ngle locus of the canola genome, and no additional genetic elements, including b ackbone sequences, from PV-BNHT2672 were

detected in MON 88302.

PCR and DNA sequence analyses complement the Southern analyses. PCR and DNA sequence analyses performed on MON 88302 determined the complete DNA sequence of the insert and flanking genomic DNA seque nces in MON 88302, confirmed the predicted organization of the genetic elements within the insert, and determined the sequences flanking the insert. In addition, DNA sequen ce analyses confirmed that each genetic

element in the insert is intact and the se quence of the insert is identical to the corresponding sequence in PV-BNHT2672. Furt hermore, genomic organization at the MON 88302 insertion site was determined by co mparing the 5' and 3' flanking sequences of the insert to the sequence of the in sertion site in conventional canola.

The stability of the T-DNA present in MON 88302 across multiple generations was demonstrated by Southern blot fingerprint analysis. Genomic DNA from multiple generations of MON 88302 (Figur e IV-9) was digested with one of the enzyme sets used for the insert and copy number analyses and was hybridized with two probes that detect restriction segments that encompass the entire insert. This fingerprint strategy consists of two insert segments each containing its adjacen t genomic DNA that assesses not only the stability of the insert, but also the stability of the DNA directly ad jacent to the insert.

Segregation analysis was conducted to determ ine the inheritance and stability of the

T-DNA insert in MON 88302. Results from this analysis demonstrated the inheritance and stability of the insert were as expected across multiple generations (Figure IV-11, Table IV-3), which corroborates the molecular in sert stability analysis and establishes the genetic behavior of the T-DNA at a single chromosomal locus.

The Southern blot analyses confirmed that the T-DNA reported in Figure IV-1 represents the only detectable insert in MON 88302. A circular map of PV-BNHT2672 annotated with the probes used in the Southern blot analysis is presented in Figure III-1 and the genetic elements within the MON 88302 insert are summarized in Table IV-2. A linear map depicting restriction sites within the insert as well as within the DNA immediately flanking the insert in MON 88302 is shown in Figure IV-1. Based on the plasmid map and the linear map of the in sert, a table summarizing the expected DNA segments for Southern analyses is presented in Table IV -1. The results from the Southern blot analyses are presented in Figure IV-2 thr ough Figure IV-6. PCR amplification of the

MON 88302 insert and the insertion site in the conventional control (Ebony) for DNA sequence analysis are shown in Figure IV-7 and Figure IV-8, respectively. TheMonsanto Company 11-CA-233U 43 of 500 generations used in the genera tional stability analysis are de picted in the breeding history shown in Figure IV-9 and the results from the generational stability analysis are presented in Figure IV-10. The breeding path for generating the segregation data is shown in Figure IV-11 and the results for th e segregation analysis are presented in

Table IV-3. Materials and methods used fo r the characterization of the insert in MON 88302 are found in Appendix B.Monsanto Company 11-CA-233U 44 of 500Figure IV-1. Schematic Representation ofthe Insert and Flanking DNA in MON 88302A linear map of the insert and DNA flanking the insert in MON88302 is shown. Right-angled arrows indicate the ends of theintegrated T-DNA and the beginning of the flanking DNA. Identified on the linear map are genetic elements within the insert, as wellas the sites of the restriction enzymes used in the Southern analyses with positions relative to the first base pair of the DNAsequencerepresented in this map. The relative sizes and locations of the T-DNA probes and theexpected sizes of restriction fragmentsareindicated in the lower portion of the scheme.This schematic diagram is not drawn to scale. Locations of genetic elements andT-DNA probes are approximate. Probesare also shown in Figure III-1.~1.4 kb~3.8 kbAseI~4.3 kb>1.8 kbSalI andScaIProbe 1Probe 2Probe 3

B-Right Border Region r1 P- FMV/Tsf1 L- Tsf1 I- Tsf1

TS- CTP2 CS- cp4 epsps T- E9 B-Left Border Region r1AseI 4552AseI 757SalI 6093ScaI 1833AseI 59755' Flanking DNA3' Flanking DNA16174Monsanto Company 11-CA-233U 45 of 500Table IV-1. Summary Chart of the Expected DNA Segments Based on Hybridizing Probes and Restriction Enzymes Used inMON 88302 Analysis1probe template spikes were used as positive hybridization controls in Southern blot analyses when multiple probes were hybridized to the Southern blotsimultaneously.2‘~~’ indicates that probe template spikes were not used.3‘--’ indicates that the combination of the restriction enzymes was not used in the analysis.Southern Blot Analysis T-DNA BackboneInsertStabilityFigure NumberIV-2IV-3IV-4IV-5IV-6IV-10Probe Used1, 324561, 3Probing Target Digestion Enzyme Expected Band Sizes on Each Southern BlotPlasmidPV-BNHT2672BamHI andScaI~2.5 kb~7.2 kb~2.5 kb~7.2 kb~7.2 kb~7.2 kb~2.5 kb~7.2 kbProbe Template Spikes1N/A~2.3 kb~1.3 kb~~2~~2~~2~~2~2.3 kb~1.3 kbMON 88302AseI~3.8 kb~1.4 kb~3.8 kbNo band No band No band~3.8 kb~1.4 kbSalI andScaI>1.8 kb~4.3 kb~4.3 kbNo band No band No band--3Monsanto Company 11-CA-233U 46 of 500 Table IV-2. Summary of Ge netic Elements in MON 88302

Genetic Element Location in Sequence Function (Reference)

5' Flanking Sequence 1-839 DNA sequence adjacent to the 5' end of the insertion

site

B 1 -Right Border Region r1 840-882 DNA region from Agrobacterium tumefaciens containing the Right Border sequence used for transfer of the T-DNA (Depicker et al ., 1982; Zambryski et al., 1982) Intervening Sequence 883-952 Sequence used in DNA cloning

P 2

- FMV/Tsf1 953-1992 Chimeric promoter consisting of the promoter of the Tsf1 gene from the Arabidopsis thaliana encoding elongation factor EF-1 α (Axelos et al., 1989) and enhancer sequences from the 35S promoter from the figwort mosaic virus (Richins et al., 1987)

L 3

- Tsf1 1993-2038 5' untranslated leader (exon 1) from the Arabidopsis

thaliana Tsf1 gene encoding elongation factor EF-1 α (Axelos et al., 1989)

I 4

- Tsf1 2039-2660 Intron from the Arabidopsis thaliana Tsf1 gene encoding elongation factor EF-1 α (Axelos et al., 1989) Intervening Sequence 2661-2669 Sequence used in DNA cloning

TS 5

- CTP2 2670-2897 Targeting sequence from the shkG gene encoding the chloroplast transit peptide region of Arabidopsis

thaliana EPSPS (Herrmann, 1995; Klee et al., 1987) that directs transport of the CP4 EPSPS protein to the chloroplast

CS 6

- cp4 epsps 2898-4265 Codon optimized coding sequence of the aroA gene from the Agrobacterium sp

. strain CP4 encoding the CP4 EPSPS protein (Barry et al., 2001; Padgette et al., 1996) Intervening Sequence 4266-4307 Sequence used in DNA cloning

T 7

- E9 4308-4950 3' untranslated sequence from the rbcS2 gene of Pisum

sativum encoding the Rubisco small subunit (Coruzzi et al., 1984) Intervening Sequence 4951-4993 Sequence used in DNA cloning B-Left Border Region r1

4994-5267 DNA region from Agrobacterium tumefaciens containing the Left Border sequence used for transfer of the T-DNA (Barker et al ., 1983; Zambryski et al., 1982) 3' Flanking Sequence 5268-6174 DNA sequence adjacent to the 3' end of the insertion

site

1 B, Border

2 P, Promoter

3 L, Leader

4 I, Intron

5 TS, Targeting Sequence

6 CS, Coding Sequence

7 T, Transcription Termination Sequence

r1 Superscripts in Left and Right Border Regions indi cate that the sequences in MON 88302 were truncated compared to the sequences in PV-BNHT2672.Monsanto Company 11-CA-233U 47 of 500 IV.A. Southern Blot Analysis to Determ ine Insert and Copy Number of T-DNA I in

MON 88302

The numbers of copies and insertion sites of the T-DNA sequences in the canola genome were evaluated by digesting MON 88302 and conventional control genomic DNA samples with the restriction enzyme Ase I or the combination of restriction enzymes Sal I

and Sca I and hybridizing Southern blots with probe s that span the T-DNA (Figure IV-1). Each restriction digest is expected to pr oduce a specific banding pattern on the Southern blots (Table IV-1). Any addi tional copies and/or integratio n sites would be detected as

additional bands on the blots.

The restriction enzyme Ase I cleaves once within the in serted T-DNA and within the known genomic DNA flanking the 5' and 3' ends of the insert (IV-1 ). Therefore, if T-DNA sequences were present as a single copy at a single integration site in MON 88302, the digestion with Ase I was expected to generate two border se gments with expected sizes of ~3.8 kb and ~1.4 kb (Figure IV -1 and Table IV-1). The combination of restriction enzymes Sal I and Sca I cleaves once within the inserted T-DNA and within the known genomic DNA flanking the 3' end of the insert (Figure IV-1). If T-DNA sequences were present as a single copy at a single integration s ite in MON 88302, the digestion with Sal I and Sca I was expected to generate two border segments with expected sizes of >1.8 kb and ~4.3 kb (Figure IV-1 and Table IV-1).

The Southern blots were hybridized with T-DN A probes that collectively span the entire inserted DNA sequence (Figures III-1 and IV-1, Probe 1, Probe 2, and Probe 3). Conventional control genomic DNA dige sted with the re striction enzyme Ase I and spiked with either probe templates and/or digested PV-BNHT2672 DNA served as positive hybridization controls. The positiv e hybridization control was spiked at approximately 0.1 and 1 genome equivalents to demonstrate sufficient sensitivity of the Southern blot. Conventional control genom ic DNA digested with the appropriate restriction enzymes was used as a negative co ntrol. The results of these analyses are shown in Figure IV-2 and Figure IV-3.

IV.A.1. T-DNA Probes 1 and 3

Conventional control genom ic DNA digested with Ase I (Figure IV-2, Lane 1 and Lane 5) or the combinati on of restriction enzymes Sal I and Sca I (Figure IV-2, Lane 3 and Lane 7) and simultaneously hybridized w ith Probe 1 and Probe 3 (Figures III-1 and IV-1) produced no detectable hybridization band s as expected for the negative control. Conventional control genomic DNA digested with Ase I and spiked with the PV-BNHT2672 DNA, previously digested with the combination of restriction enzymes

Bam HI and Sca I (Figure III-1), produced tw o bands at ~7.2 kb and ~2.5 kb (Figure IV-2, Lane 10), as expected. Conve ntional control genomic DNA digested with

Ase I and spiked with probe templates of Probe 1 and Probe 3 (Figure III-1) produced the expected bands at ~2.3 kb and ~1.3 kb (Figure IV-2, Lane 11 and Lane 12). Detection of the positive controls indicates that the probe s hybridized to their target sequences.Monsanto Company 11-CA-233U 48 of 500 MON 88302 DNA digested with Ase I and simultaneously hybridized with Probe 1 and Probe 3 (Figures III-1 and IV-1) produced the expected bands at ~3.8 kb and ~1.4 kb (Figure IV-2, Lane 2 and Lane 6). MON 88302 DNA digested with the combination of restriction enzymes Sal I and Sca I and hybridized with Probe 1 and Probe 3 (Figures III-1 and IV-1) produced two bands at ~2.7 kb and ~4.3 kb (Figure IV-2, Lane 4 and Lane 8), which is consistent with the expected >1.8 kb and ~4.3 kb bands (Figure IV-1 and Table IV-1), respectively.

The results presented in Figur e IV-2 indicate that the se quences covered by Probe 1 and Probe 3 reside at a single detectable locus of integration in MON 88302.

IV.A.2. T-DNA Probe 2

Conventional control DNA digested with Ase I (Figure IV-3, Lane 1 and Lane 5) or the combination of restriction enzymes Sal I and Sca I (Figure IV-3, Lane 3 and Lane 7) and hybridized with Probe 2 (Figures III-1 a nd IV-1) produced no detectable hybridization bands as expected for the negative control. Conventional control genomic DNA digested

with Ase I and spiked with the PV-BNHT2672 DNA, previously digested with the combination of restriction enzymes Bam HI and Sca I (Figure III-1), produced a unique band at ~2.5 kb (Figure IV-3, Lane 10 and La ne 11), as expected. Detection of the positive controls indicates that the probe hybridized to its target sequence.

MON 88302 DNA digested with Ase I and hybridized with Probe 2 (Figures III-1 and IV-1) produced the expected band at ~3.8 kb (Figure IV-3, Lane 2 and Lane 6). MON 88302 DNA digested with the co mbination of restriction enzymes Sal I and Sca I and hybridized with Probe 2 (Figures III-1 and IV-1) produced the expected band at ~4.3 kb (Figure IV-3, Lane 4 and Lane 8, Figure IV-1, and Table IV-1).

The results presented in Figure IV-3 indi cate that the sequence covered by Probe 2 resides at a single detectable locu s of integration in MON 88302.Monsanto Company 11-CA-233U 49 of 500 Figure IV-2. Southern Blot Analysis to Determine Insert and Copy Number of

T-DNA I in MON 88302: Probes 1 and 3

The blot was simultaneou sly hybridized with two 32 P-labeled probes that span a portion of the T-DNA sequence (Figure III-1, Probe 1 and Probe 3). Each lane contains approximately 10 μg of digested genomic DNA. Arrows denote th e size of the DNA, in kilobase pairs, obtained fr om 1 Kb DNA Extension Ladder on the ethidium bromide stained gel. Lane desi gnations are as follows:

Lane

1 Conventional control ( Ase I)

2 MON 88302 ( Ase I)

3 Conventional control ( Sal I/ Sca I)

4 MON 88302 ( Sal I/ Sca I)

5 Conventional control ( Ase I)

6 MON 88302 ( Ase I)

7 Conventional control ( Sal I/ Sca I)

8 MON 88302 ( Sal I/ Sca I)

9 Blank

10 Conventional control ( Ase I) spiked with PV-BNHT2672 ( Bam HI/ Sca I) [~1 genome equivalent]

11 Conventional control ( Ase I) spiked with Probe 1 and Probe 3 [~1 genome equivalent]

12 Conventional control ( Ase I) spiked with Probe 1 and Probe 3 [~0.1 genome equivalent] 1.0 1.6 2.0 3.1 4.1 5.1 6.1 7.1 8.1 10 15 20 40

1.0 1.6 2.0 3.1 4.1 5.1 6.1 7.1 8.1 10 15 20 40

0.5 1 2 3 4 5 6 7 8 9 10 11 12 LONG RUN SHORT RUNMonsanto Company 11-CA-233U 50 of 500 Figure IV-3. Southern Blot Analysis to Determine Insert and Copy Number of

T-DNA in MON 88302: Probe 2 The blot was hybridized with a 32 P-labeled probe that span s a portion of the T-DNA sequence (Figure III-1, Probe 2). Each lane contains approx imately 10 μg of digested genomic DNA. Arrows denote the size of th e DNA, in kilobase pa irs, obtained from

1 Kb DNA Extension Ladder on the ethidium bromide stained gel. Lane designations are

as follows:

Lane

1 Conventional control ( Ase I)

2 MON 88302 ( Ase I)

3 Conventional control ( Sal I/ Sca I)

4 MON 88302 ( Sal I/ Sca I)

5 Conventional control ( Ase I)

6 MON 88302 ( Ase I)

7 Conventional control ( Sal I/ Sca I)

8 MON 88302 ( Sal I/ Sca I)

9 Blank

10 Conventional control ( Ase I) spiked with PV-BNHT2672 ( Bam HI/ Sca I) [~1 genome equivalent]

11 Conventional control ( Ase I) spiked with PV-BNHT2672 ( Bam HI/ Sca I) [~0.1 genome equivalent] 1.0 1.6 2.0 3.1 4.1 5.1 6.1 7.1 8.1 10 15 20 40

0.5

1.0 1.6 2.0 3.1 4.1 5.1 6.1 7.1 8.1 10 15 20 40 1 2 3 4 5 6 7 8 9 10 11 LONG RUN SHORT RUNMonsanto Company 11-CA-233U 51 of 500 IV.B. Southern Blot Analysis to Determine the Presence or Absence of

PV-BNHT2672 Backbone Se quences in MON 88302

To determine the presence or absence of the PV-BNHT2672 backbone sequences, MON 88302 and conventional control genomic DNA were digested with the restriction

enzyme Ase I or the combination of restriction enzymes Sal I and Sca I, and hybridized with one of the three backbone probes that collectively span the entire backbone sequences (Figure III-1, Probe 4, Probe 5, a nd Probe 6). If backbone sequences are present in MON 88302, then probing with ba ckbone probes should re sult in hybridizing bands. Conventional control genomic DNA di gested with the restriction enzyme Ase I and spiked with digested PV-BNHT2672 DNA served as positive hybridization controls. The positive hybridization control was spik ed at approximately 0.1 and 1 genome equivalents to demonstrate sufficient sensit ivity of the Southern blot. Conventional control genomic DNA digested with the appropriate restriction enzymes was used as a negative control. The results of these anal yses are shown in Figures IV-4, IV-5, and

IV-6.

IV.B.1. Backbone Probe 4

Conventional control DNA digested with Ase I (Figure IV-4, Lane 1 and Lane 5) or the combination of restriction enzymes Sal I and Sca I (Figure IV-4, Lane 3 and Lane 7) and hybridized with Probe 4 (Figure III-1) produc ed no detectable hybridization bands as expected for the negative control. Conventional control DNA digested with Ase I and spiked with the PV-BNHT2672 DNA, previously digested with the combination of restriction enzymes Bam HI and Sca I (Figure III-1), produced a unique band at ~7.2 kb (Figure IV-4, Lane 10 and Lane 11), as expected. Detection of the positive controls indicates that the probe hybridi zed to its target sequence.

MON 88302 DNA digested with Ase I (Figure IV-4, Lane 2 and Lane 6) or the combination of restriction enzymes Sal I and Sca I (Figure IV-4, Lane 4 and Lane 8) and hybridized with Probe 4 produced no detectable bands.

The results presented in Figure IV-4 indi cate that MON 88302 cont ains no detectable backbone sequences covered by Probe 4.

IV.B.2. Backbone Probe 5

Conventional control DNA digested with Ase I (Figure IV-5, Lane 1 and Lane 5) or the combination of restriction enzymes Sal I and Sca I (Figure IV-5, Lane 3 and Lane 7) and hybridized with Probe 5 (Figure III-1) produc ed no detectable hybridization bands as expected for the negative control. Conventional control DNA digested with Ase I and spiked with the PV-BNHT2672 DNA, previously digested with the combination of restriction enzymes Bam HI and Sca I (Figure III-1), produced a unique band at ~7.2 kb (Figure IV-5, Lane 10 and Lane 11), as expected. Detection of the positive controls indicates that the probe hybridi zed to its target sequence.Monsanto Company 11-CA-233U 52 of 500 MON 88302 DNA digested with Ase I (Figure IV-5, Lane 2 and Lane 6) or the combination of restriction enzymes Sal I and Sca I (Figure IV-5, Lane 4 and Lane 8) and hybridized with Probe 5 produced no detectable bands.

The results presented in Figure IV-5 indi cate that MON 88302 cont ains no detectable backbone sequences covered by Probe 5.

IV.B.3. Backbone Probe 6

Conventional control DNA digested with Ase I (Figure IV-6, Lane 1 and Lane 5) or the combination of restriction enzymes Sal I and Sca I (Figure IV-6, Lane 3 and Lane 7) and hybridized with Probe 6 (Figure III-1) produc ed no detectable hybridization bands as expected for the negative control. Conventional control DNA digested with Ase I and spiked with the PV-BNHT2672 DNA, previously digested with the combination of restriction enzymes Bam HI and Sca I (Figure III-1), produced a unique band at ~7.2 kb (Figure IV-6, Lane 10 and Lane 11), as expected. Detection of the positive controls indicates that the probe hybridi zed to its target sequence.

MON 88302 DNA digested with Ase I (Figure IV-6, Lane 2 and Lane 6) or the combination of restriction enzymes Sal I and Sca I (Figure IV-6, Lane 4 and Lane 8) and hybridized with Probe 6 produced no detectable bands.

The results presented in Figure IV-6 indi cate that MON 88302 cont ains no detectable backbone sequences covered by Probe 6.Monsanto Company 11-CA-233U 53 of 500 Figure IV-4. Southern Blot Analysis to Determine the Presence or Absence of the PV-BNHT2672 Backbone Sequen ces in MON 88302: Probe 4 The blot was hybridized with a 32 P-labeled probe that spans a portion of the plasmid vector backbone sequences (Fi gure III-1, Probe 4). Each la ne contains approximately 10 μg of digested genomic DNA. Arrows denot e the size of the DNA, in kilobase pairs,

obtained from λ DNA/ Hind III fragments on the ethidium bromide stained gel. Lane designations are as follows:

Lane

1 Conventional control ( Ase I) 2 MON 88302 ( Ase I) 3 Conventional control ( Sal I/ Sca I) 4 MON 88302 ( Sal I/ Sca I) 5 Conventional control ( Ase I) 6 MON 88302 ( Ase I) 7 Conventional control ( Sal I/ Sca I) 8 MON 88302 ( Sal I/ Sca I)

9 Blank 10 Conventional control ( Ase I) spiked with PV-BNHT2672 ( Bam HI/ Sca I) [~1 genome equivalent] 11 Conventional control ( Ase I) spiked with PV-BNHT2672 ( Bam HI/ Sca I) [~0.1 genome equivalent] 23.1

9.4

6.6

4.4

2.3

2.0 23.1

9.4

6.6

4.4

2.3

0.6 2.0 1 2 3 4 5 6 7 8 9 10 11 LONG RUN SHORT RUNMonsanto Company 11-CA-233U 54 of 500 Figure IV-5. Southern Blot Analysis to Determine the Presence or Absence of the PV-BNHT2672 Backbone Sequen ces in MON 88302: Probe 5 The blot was hybridized with a 32 P-labeled probe that spans a portion of the plasmid vector backbone sequences (Fi gure III-1, Probe 5). Each la ne contains approximately 10 μg of digested genomic DNA. Arrows denot e the size of the DNA, in kilobase pairs,

obtained from λ DNA/ Hind III fragments the on the ethidium bromide stained gel. Lane designations are as follows:

Lane

1 Conventional control ( Ase I) 2 MON 88302 ( Ase I) 3 Conventional control ( Sal I/ Sca I) 4 MON 88302 ( Sal I/ Sca I) 5 Conventional control ( Ase I) 6 MON 88302 ( Ase I) 7 Conventional control ( Sal I/ Sca I) 8 MON 88302 ( Sal I/ Sca I)

9 Blank 10 Conventional control ( Ase I) spiked with PV-BNHT2672 ( Bam HI/ Sca I) [~1 genome equivalent] 11 Conventional control ( Ase I) spiked with PV-BNHT2672 ( Bam HI/ Sca I) [~0.1 genome equivalent] 23.1

9.4

6.6

4.4

2.3

2.0 23.1

9.4

6.6

4.4

2.3

0.6 2.0 1 2 3 4 5 6 7 8 9 10 11 LONG RUN SHORT RUNMonsanto Company 11-CA-233U 55 of 500

Figure IV-6. Southern Blot Analysis to Determine the Presence or Absence of the PV-BNHT2672 Backbone Sequences in MON 88302: Probe 6 The blot was hybridized with a 32 P-labeled probe that spans a portion of the plasmid vector backbone sequences (Figure III-1, Probe 6). Each lane contains approximately 10 μg of digested genomic DNA. Arrows denote the size of the DNA, in kilobase pairs,

obtained from λ

DNA/ H ind III fragments on the ethidium bromide stained gel. Lane designations are as follows:

Lane 1 Conventional control ( Ase I) 2 MON 88302 (

Ase I) 3 Conventional control ( Sal I/ Sca I) 4 MON 88302 ( Sal I/ Sca I) 5 Conventional control ( Ase I) 6 MON 88302 (

Ase I) 7 Conventional control ( Sal I/ Sca I) 8 MON 88302 ( Sal I/ Sca I)

9 Blank 10 Conventional control ( Ase I) spiked with PV-BNHT2672 ( Bam HI/ Sca I) [~1 genome equivalent] 11 Conventional control ( Ase I) spiked with PV-BNHT2672 ( Bam HI/ Sca I) [~0.1 genome equivalent] 23.1

9.4

6.6

4.4

2.3

2.0 23.1

9.4

6.6

4.4

2.3

0.6 2.0 1 2 3 4 5 6 7 8 9 10 11 LONG RUN SHORT RUNMonsanto Company 11-CA-233U 56 of 500 IV.C. Organization and Sequence of the Insert and Adjacent DNA in MON 88302

The organization and sequence of the el ements within the MON 88302 insert was confirmed by DNA sequence analysis. PCR primers were designed with the intent to amplify two overlapping DNA amp licons that span the entire length of the insert and the associated DNA flanking the 5' and 3' ends of the insert (Figure IV-7). The amplified PCR products were subjected to DNA sequence an alyses. This analysis determined that the DNA sequence of the MON 88302 insert is 4428 bp long (Table IV-2) and is identical to the corresponding T-DNA sequence of PV-B NHT2672 as described in Table III-1.Monsanto Company 11-CA-233U 57 of 500 Figure IV-7. Overlapping PCR Analys is across the Insert in MON 88302

PCR was performed on both conventiona l control genomic DNA and MON 88302 genomic DNA using two pairs of primers to generate overlapping PCR fragments from MON 88302 for sequence analysis. Five microliters of each of the PCR reactions was loaded on the gel. The expected product size for each amplicon is provided in the illustration of the insert in MON 88302 that app ears at the bottom of the figure. Arrows on the agarose gel photograph deno te the size of the DNA, in kilobase pairs, obtained

from 1 Kb DNA Ladder on the ethidium bromide stained gel. Lane designations are as

follows:

Lane

1 1 Kb DNA Ladder

2 Conventional control

3 MON 88302

4 No template DNA control

5 Conventional control

6 MON 88302

7 No template DNA control

8 1 Kb DNA Ladder 1.0 1.6 2.0 3.1 4.1

0.5 12

5.1

3.1

0.5 1.0 1.6 2.0 4.1 12

5.1 1 2 3 4 5 6 7 8 Product A Product B

5 ́ Flank 3 ́ Flank Product B: ~ 3.3 kb Product A: ~ 3.3 kbB-Right Border Regionr1P-FMV/Tsf1L-Tsf1I-Tsf1TS-CTP2CS-cp4 epspsT-E9B-Left Border Regionr1Monsanto Company 11-CA-233U 58 of 500 IV.D. PCR and DNA Sequence Analyses to Examine the MON 88302 Insertion Site

PCR and sequence analyses were perf ormed on genomic DNA extracted from

MON 88302 and the conventional control to examine the M ON 88302 insertion site. The PCR was performed with a forward primer specific to the genomic DNA sequence flanking the 5' end of the inse rt paired with a reverse prim er specific to the genomic DNA sequence flanking the 3' end of the insert (Figure IV-8). The amplified PCR product

from the conventional contro l was subjected to DNA sequen ce analysis. Alignments

between the conventional contro l sequence obtained from this analysis and the sequences immediately flanking the 5' and 3' end of the MON 88302 insert were separately performed to determine the integrity and geno mic organization of the insertion site in MON 88302. From these alignment analyses , a 9 base pair in sertion immediately adjacent to the 3' end of the MON 88302 insert and a 29 base pair deletion from the conventional genomic DNA were identified. Such changes are quite common during plant transformation; these changes presum ably resulted from double-stranded break repair mechanisms in the plant during the Agrobacterium -mediated transformation process (Salomon and Puchta, 1998). A si ngle nucleotide difference between the conventional control sequence and the genomic DNA sequence flanking the 3' end of the

MON 88302 insert was also identified. The di fference was most likely caused by a single nucleotide polymorphism (SNP) segregating in the canola population (Trick et al., 2009).Monsanto Company 11-CA-233U 59 of 500

Figure IV-8. PCR Amplification of the MON 88302 Insertion Site in Conventional

Canola PCR was performed on both conventional control genomic DNA and MON 88302 genomic DNA, using Primer A specific to the 5' flanking sequence and Primer B specific to the 3' flanking sequence of the insert in MON 88302, to generate DNA fragments for sequence analysis. The insertion site in conventional control (top) and MON 88302 (bottom) are illustrated at the bottom of the figure. Five microliters of each of the PCR reactions were loaded on the gel. Arrows on the agarose gel photograph denote the size of the DNA, in kilobase pairs, obtained from 1Kb DNA Ladder on the ethidium bromide stained gel. Lane designations are as follows:

Lane

1 1 Kb DNA Ladder

2 Conventional control

3 MON 88302 4 No template DNA control

5 1 Kb DNA Ladder 1 2 3 4 5

1.0 1.6 2.0 3.1 4.1

0.5 12

6.1

3.1

0.5 1.0 1.6 2.0 4.1 12

6.1

Primer A Primer B

Primer A Primer B

5 ́ Flank 3 ́ FlankB-Right Border Regionr1P-FMV/Tsf1L-Tsf1I-Tsf1TS-CTP2CS-cp4 epspsT-E9B-Left Border Regionr1Monsanto Company 11-CA-233U 60 of 500 IV.E. Southern Blot Analysis to Examine Insert Stability in Multiple Generations of

MON 88302

In order to demonstrate the st ability of the insert in M ON 88302, Southern blot analysis was performed using genomic DNA extracted from leaf tissues from four breeding generations of MON 88302. For reference, the breeding history of MON 88302 is presented in Figure IV-9. The specific genera tions tested are indicat ed in the legend of Figure IV-9. The R 3 generation was used for the molecular characterization analyses shown in Figure IV-2 through Figure IV-6. To analyze insert stability, additional samples from three generations of MON 88302 were evaluated by Southern blot analysis and compared to the R 3 generation. Genomic DNA, isolated from each of the selected generations of MON 88302, was digested with the rest riction enzyme Ase I and simultaneously hybridized with Probe 1 and Probe 3 (Figures III-1 and IV-1), which was designed to detect both fr agments generated by the Ase I digest. Any instability

associated with the insert would be detected as extra bands within the fingerprint on the

Southern blot. The Southern blot has the same controls as described in Section IV.A.1.

IV.E.1. T-DNA Probes 1 and 3

Conventional control genomic DNA di gested with restriction enzyme Ase I and simultaneously hybridized with Probe 1 and Pr obe 3 (Figures III-1 and IV-1) produced no hybridization signals (Figure IV-10, Lane 1) as expected for the negative control. Conventional control genomic DNA digested with Ase I and spiked with the PV-BNHT2672 DNA, previously digested with the combination of restriction enzymes

Bam HI and Sca I (Figure III-1 and Table IV-1), pr oduced the expected bands at ~2.5 kb and ~7.2 kb (Figure IV-10, Lane 8). Conven tional control genomic DNA digested with

Ase I and spiked with probe templates of Probe 1 and Probe 3 pr oduced the expected bands at ~2.3 kb and ~1.3 kb (F igure IV-10, Lane 9 and Lane 10). Detection of the positive controls indicates that the probes hybridized to their ta rget sequences.

MON 88302 genomic DNA digested with Ase I and hybridized with Probe 1 and Probe 3 (Figures III-1 and IV-1) is expected to produ ce a Southern fingerprint with two bands at ~3.8 kb and ~1.4 kb (Figure IV-1 and Table IV-1 ). Southern fingerprints produced from multiple generations (Figure IV-10, Lane 2, La ne 4, Lane 5, and Lane 6), of MON 88302 are consistent with the one produced fr om the fully characterized generation R 3 (Figure IV-2, Lane 2 and Lane 6, and Figur e IV-10, Lane 3), indicating that MON 88302 contains one copy of the T-DNA insert that is stable across multiple generations.Monsanto Company 11-CA-233U 61 of 500 Figure IV-9. Breeding History of MON 88302

R 0 corresponds to the transformed canola plant. All generations were self pollinated.

 designates self- pollination. The R 3 generation was used for the molecular

characterization and commercial development of MON 88302. The R 2 , R 3 , R 4 , R 5a , and

R 5b (bolded in the breeding tree) generations of MON 88302 were used for analyzing the stability of the insert across generations. R 5b was propagated independently of R 5a beginning with the R 3 generation. R 0 - Originally transformed plant ;  -self pollination R 0 (transformation)

R 1

R 2

R 3

R 4

R 5a R 5b R 4b  Commercial Development 







 Monsanto Company 11-CA-233U 62 of 500 Figure IV-10. Southern Blot Analysis to Examine Insert Stab ility in Multiple

Generations of MON 88302: Probes 1 and 3

The blot was simultaneou sly hybridized with two 32 P-labeled probes that span a portion of the T-DNA sequence (Figure III-1, Probe 1 an d Probe 3). Each lane contains ~10 μg of digested genomic DNA. Arrows denote th e size of the DNA, in kilobase pairs,

obtained from 1Kb DNA Extension Ladder on th e ethidium bromide stained gel. Lane designations are as follows:

Lane

1 Conventional control ( Ase I)

2 R 2 generation of MON 88302 ( Ase I)

3 R 3 generation of MON 88302 ( Ase I)

4 R 4 generation of MON 88302 ( Ase I)

5 R 5a generation of MON 88302 ( Ase I)

6 R 5b generation of MON 88302 ( Ase I)

7 Blank 8 Conventional control ( Ase I) spiked with PV-BNHT2672 ( Bam HI/ Sca I) [~1 genome equivalent] 9 Conventional control ( Ase I) spiked with probe templates Probe 1 and Probe 3 [~1 genome equivalent] 10 Conventional control ( Ase I) spiked with probe templates Probe 1 and Probe 3 [~0.1 genome equivalent] 1 2 3 4 5 6 7 8 9 10

1.0 1.6 2.0 3.1 4.1 5.1 6.1 7.1 8.1 10 15 20 40

0.5Monsanto Company 11-CA-233U 63 of 500 IV.F. Inheritance of the Genetic Insert in MON 88302

During development of MON 88302, segregati on data were recorded to assess the inheritance and stability of the coding se quence present in MON 88302. Chi-square ( χ 2 ) analysis was performed over se veral generations to confirm the segregation and stability

of the MON 88302 insert. The χ 2 analysis is based on testin g the observed segregation ratio to the expected segregation rati o according to Mendelian principles.

The MON 88302 breeding path for generating segregation data is described in Figure IV-11. The transformed R 0 plant was self-pollinated to generate R 1 seed. From

the R 1 segregating population, an indi vidual plant homozygous for the cp4 epsps coding sequence (subsequently designated MON 88302 ) was identified via TaqMan PCR copy number assay and Southern blot copy number analysis. The cp4 epsps homozygous R 1 plant was self-pollinated to give rise to R 2 plants that were self -pollinated to produce R 3 seed. At each generation, the homozygous plants were tested for the expected segregation pattern of 1:0 ( positive: negative) for the cp4 epsps gene using a glyphosate spray test and/or TaqMan PCR assay.

An individual cp4 epsps positive R 3 plant, which was confirmed by Endpoint TaqMan PCR assay, was crossed to a Monsanto proprie tary canola inbred, which does not contain the MON 88302 insert, via traditional breedi ng techniques to produce hemizygous F 1 seed. The resulting F 1 plant, was shown to contain a single copy of the cp4 epsps gene by real-time TaqMan PCR, and was th en self-pollinated to produce F 2 seed. A cp4 epsps hemizygous F 2 plant from the F 2 population was shown to contain a single copy of the cp4 epsps gene by real-time TaqMan PCR and was then self-pollinated to produce the F 3 population. A cp4 epsps hemizygous F 3 plant from the F 3 population was shown to contain a single copy of the cp4 epsps gene by real-time TaqMan PCR and was self-pollinated to produce the F 4 population. The copy number of the cp4 epsps gene in

the F 2, F 3, and F 4 populations was then assessed using a real-time TaqMan PCR assay.

A χ 2 analysis was performed on each of the F 2 , F 3 , and F 4 populations using the statistical program R (Version 2.10.1) to compare the observed segregation ratio of cp4 epsps coding sequence to the expected ratio according to Mendelian principles of inheritance. The Chi-square was calculated as:

χ 2 = ∑ [( o – e ) 2 / e]

where o = observed frequency of the genotype or phenotype and e = expected frequency of the genotype or phenotype. The level of st atistical significance was predetermined to be 5% ( α = 0.05).

The results of the χ 2 analysis of the MON 88302 segreg ating progeny are presented in

Table IV-3. The χ 2 value in the F 2 , F 3 , and F 4 populations indicated no statistically significant difference between the observed and expected 1:2:1 segregation ratioMonsanto Company 11-CA-233U 64 of 500 (homozygous positive: hemizygous: homozygous negative) of cp4 epsps coding sequence. These results suppor t the conclusion that the cp4 epsps expression cassette in MON 88302 resides at a single lo cus within the canola genome and is inherited according to Mendelian principles of i nheritance. These results ar e also consistent with the molecular characterization data indicating th at MON 88302 contains a single, intact copy

of the cp4 epsps expression cassette inserted at a si ngle locus in the canola genome.Monsanto Company 11-CA-233U 65 of 500Figure IV-11. Breeding Path for Generating Segregation Data for MON 88302An individual hemizygous plant from each of the F1, F2, and F3populations was self-pollinated toproduce the population of the nextgeneration. Chi-square analyses wereconducted on segregation data from the F2, F3, and F4populations.Transformed R0PlantR1R2R3(homozygous)cross with a Monsanto proprietary canola inbredwhich does not contain the MON 88302 insertF1(hemizygous)F2(expected segregation ratio 1:2:1)(homozygous positive : hemizygous : homozygous negative)F3(homozygous positive : hemizygous : homozygous negative)F4(homozygous positive : hemizygous : homozygous negative)(expected segregation ratio 1:2:1)(expected segregation ratio 1:2:1)Monsanto Company 11-CA-233U 66 of 500Table IV-3. Segregation of thecp4 epspsGene During the Development of MON 883021:2:1 SegregationGenerationTotalPlants*Observed #PlantsHomozygousPositiveObserved #PlantsHemizygousObserved #PlantsHomozygousNegativeExpected #PlantsHomozygousPositiveExpected #PlantsHemizygousExpected #PlantsHomozygousNegativeχ2ProbabilityF2220 51 122 47 55.00 110.00 55.00 2.76 0.2511F3166 39 94 33 41.50 83.00 41.50 3.35 0.1874F4198 53 97 48 49.50 99.00 49.50 0.33 0.8465*Plants were evaluated for the copy number of thecp4 epspsgene using a real-time TaqMan PCR assay.Monsanto Company 11-CA-233U 67 of 500 IV.G. Genetic Modification Characterization Conclusion

Molecular characterization of MON 88302 by Sout hern blot analyses demonstrated that the T-DNA was inserted into the canola genome at a single locus containing one copy of

the cp4 epsps expression cassette. No additional elements were detected other than those

associated with the insert. Moreover, no backbone sequence was detected in the genome

of MON 88302.

DNA sequence analyses performed on MON 88302 determined the complete DNA sequence of the insert in MON 88302, confir med the predicted organization of the genetic elements within the insert, determin ed the sequences flanking the insert, and examined the MON 88302 insertion site. Sequence analysis of the T-DNA insertion site indicated that a 9 base pair insertion immediately adjacent to the 3' end of the MON 88302 insert and a 29 base pair de letion from the conventional genomic DNA occurred during the insertion of the T-DNA into the conventional canola to form MON 88302. In addition, a single nucleo tide difference between the conventional control sequence and the know n DNA sequence flanking the 3' end of the MON 88302 insert was also identified. This single nuc leotide difference was mo st likely caused by single nucleotide polymorphism (SNP) segrega ting in the canola popula tion (Trick et al., 2009).

Southern blot analysis of multiple MON 88302 generations demonstrated that the inserted DNA has been stably maintained through multiple generations of breeding, thereby, confirming the stability of the insert . Results from segregation analyses show inheritance and stability of the insert were as expected across multiple generations, which corroborates the molecular insert stability anal ysis and establishes th e genetic behavior of the T-DNA in MON 88302 at a single chromosomal locus.Monsanto Company 11-CA-233U 68 of 500 V. CHARACTERIZATION AND SA FETY ASSESSMENT OF THE

CP4 EPSPS PROTEIN PR ODUCED IN MON 88302

Characterization of the introduced protein in a biotechnology-der ived crop is important to establishing food, feed, and environmental safety. As described in Section IV,

MON 88302 contains a cp4 epsps expression cassette that, when transcribed and translated, results in the expres sion of the CP4 EPSPS protein.

This section summarizes: 1) the iden tity and function of the CP4 EPSPS protein produced in MON 88302; 2) demonstration of the equivalence of the plant-produced and

E. coli -produced proteins; 3) the level of the CP4 EPSPS protein in MON 88302 plant tissues; 4) assessment of the potential allergenic ity of the CP4 EPSPS protein produced in MON 88302; and 5) the food and feed safety assessment of the CP4 EPSPS protein produced in MON 88302. The data support a co nclusion that MON 88302 is safe for the human or animal consumption based on severa l lines of evidence summarized below.

V.A. Identity and Function of th e CP4 EPSPS Protein from MON 88302

The enzyme, 5-enolpyruvylshikimate-3-phospha te synthase (EPSPS), catalyzes one of the enzymatic steps of the shikimic acid pathway, and is the target for the broad spectrum herbicide glyphosate (Haslam, 1993; Herrmann and Weaver, 1999; Kishore et al., 1988; Steinrücken and Amrhein, 1980). The shik imic acid pathway and EPSPS enzymes are ubiquitous to plants and microorganisms, but ab sent in mammals, fis h, birds, reptiles, and insects (Alibhai and Stallings , 2001). EPSPS proteins have be en isolated from both plant and microbial sources and their properties have been extensively stud ied (Harrison et al., 1996; Haslam, 1993; Klee et al., 1987; Sc hönbrunn et al., 2001; Steinrücken and Amrhein, 1984). The plant and microbial enzy mes are mono-functional with a molecular weight of 44-51 kDa (Franz et al., 1997; Kishore et al., 198 8). EPSPS enzymes catalyze the transfer of the enolpyruvyl group fr om phosphoenolpyruvate (PEP) to the 5-hydroxyl of shikimate-3-phosphate (S3P), thereby yielding in organic phosphate and 5- enolpyruvylshikimate-3-phosphate (EPSP) (Ali bhai and Stallings, 2001). Shikimic acid is a substrate for the biosynthesis of the arom atic amino acids (phenyl alanine, tryptophan and tyrosine) and other aromatic molecules necessary for plant growth.

The EPSPS transgene in M ON 88302 is derived from Agrobacterium sp. strain CP4 ( cp4

epsps ). The cp4 epsps coding sequence encodes a 47.6 kDa EPSPS protein consisting of a single polypeptide of 455 amino acids (Padge tte et al., 1996). The CP4 EPSPS protein is similar and functionally id entical to endogenous plant EP SPS enzymes, but has a much reduced affinity for glyphosate, the active ingredient in Roundup agricultural herbicides, relative to endogenous pl ant EPSPS (Padgette et al., 19 96). In conventional plants, glyphosate blocks the biosynthesis of EPSP, th ereby depriving plants of essential amino acids (Haslam, 1993; Steinrücken and Amrh ein, 1980). In Roundup Ready plants, which are tolerant to Roundup agricultural herbicid es, requirements for aromatic amino acids

and other metabolites are met by the continued action of th e CP4 EPSPS enzyme in the presence of glyphosate (Padgette et al., 1996). The CP4 EPSPS protein expressed in

MON 88302 is identical to the CP4 EPSPS proteins in other Roundup Ready crops including Roundup Ready soybeans and Roundup R eady 2 Yield soybeans, as well as theMonsanto Company 11-CA-233U 69 of 500 CP4 EPSPS in Roundup Ready corn 2, Roundup Ready canola, Roundup Ready sugar beet, Roundup Ready and Roundup Ready Flex cotton and Roundup Ready alfalfa.

V.B. Characterization and Equivale nce of CP4 EPSPS Protein from MON 88302

The safety assessment of crops derived th rough biotechnology incl udes characterization of the physicochemical and functional propertie s of the protein(s) produced from the inserted DNA, and confirmation of the safe ty of the protein(s). The safety of E. coli

produced CP4 EPSPS protein has been as sessed previously and the results are summarized by Harrison et al. (1996). For the existing CP4 EPSPS safety data set to be applied to CP4 EPSPS protein produced inMON 88302, the equivalence of the plantand E. coli -produced protein was established. The MON 88302-produced CP4 EPSPS protein was characterized and the equivalen ce of the physicochemical characteristics and functional activity between the MON 88302 -produced CP4 EPSPS protein and the

E. coli -produced CP4 EPSPS protein was assessed using a panel of an alytical tests, including: 1) N-terminal sequence analysis of th e MON 88302-produced CP4 EPSPS protein to establish identity; 2) matrix-assist ed laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis of peptides derived from tryptic digested MON 88302-produced CP4 EPSPS protein to estab lish identity; 3) west ern blot analysis using anti-CP4 EPSPS polyclonal antibodies to establish identity and immunoreactive equivalence between MON 88302 -produced protein and the E. coli -produced protein; 4) sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to establish equivalence of the apparent molecular we ight between MON 88302-produced protein and

the E. coli -produced protein; 5) glycosylatio n analysis of the MON 88302-produced CP4 EPSPS protein to establish the equi valence between the MON 88302-produced and

E. coli -produced CP4 EPSPS proteins; and 6) CP 4 EPSPS enzymatic activity analysis to demonstrate functional equivalence between MON 88302-produced and the E. coli

produced protein. The details of the material s, methods, and results are described in Appendix C while the conclusions are summarized as follows.

A comparison of the MON 88302-produced CP4 EPSPS protein to the E. coli -produced CP4 EPSPS protein confirmed the identi ty of the MON 88302-produced CP4 EPSPS protein and established the equivalence of the plant produced protein to the E. coli

produced CP4 EPSPS protein. The identity of the CP4 EPSPS protein isolated from the seed of MON 88302 was confirmed by N- terminal sequencing, MALDI-TOF MS analysis of peptides produced after trypsin digestion, and by western blot analysis using anti-CP4 EPSPS polyclonal antibodies. Th e N-terminus of the MON 88302-produced CP4 EPSPS protein matched the predicted amino acid sequence translated from the cp4 epsps coding sequence. The MALDI-TOF MS analysis yielded peptide masses

consistent with the e xpected peptide masses from the translated cp4 epsps coding sequence. The MON 88302-produced CP4 EPSPS protein was detected on a western blot probed with antibodies specific for CP 4 EPSPS protein. Furthermore, the immunoreactive properties and electrophoret ic mobility of the MON 88302-produced CP4 EPSPS protein were shown to be equivalent to those of the E. coli -produced CP4 EPSPS protein.. The apparent molecu lar weight, glycosylation status, and functional activity of the MON 8830 2-produced CP4 E PSPS protein and E. coli -produced CP4 EPSPS protein were also all found to be equivalent. Taken together, these dataMonsanto Company 11-CA-233U 70 of 500 provide a detailed characterization of the CP4 EPSPS protein isolated from MON 88302 and establish its equivalence to the E. coli -produced CP4 EPSPS protein. Furthermore, since CP4 EPSPS proteins isolated from other Roundup Ready crops have been previously demonstrated to be equivalent to the E. coli -produced CP4 EPSPS protein, by inference, the MON 88302-produced CP4 EPSPS protein is equivalent to the CP4 EPSPS proteins expressed in other Roundup Ready crops , all of which have been deregulated by

USDA-APHIS.

V.C. Expression Levels of CP4 EPSPS Protein in MON 88302

CP4 EPSPS protein levels in various tis sues of MON 88302 relevant to the risk assessment were determined by a validat ed enzyme-linked immunosorbent assay (ELISA). Tissues of MON 88302 were collected from four replicate plots planted in a randomized complete block field design during the 2009 growing season from the following three field sites in the U.S.: Po wer County, Idaho; Wilkin County, Minnesota; and McHenry County, North Dakota, and the following three field sites in Canada: Portage la Prairie, Manitoba; Newton, Manitoba; and Saskatoon, Saskatchewan. These field sites were representative of canola producing regions suitable for commercial production. Forage, seed, over-season leaf (O SL-1 through OSL-4), and root (Root-1 and Root-2) tissue samples were collected from each replicated plot at all field sites.

CP4 EPSPS protein levels were determined in all eight tissue types. The results obtained

from ELISA are summarized in Table V-1 a nd the details of the materials and methods are described in Appendix D. CP4 EPSPS protein levels in MON 88302 across tissue types ranged from 22 to 500 μg/g dw. Th e mean CP4 EPSPS protein levels were determined across six sites with the exception of seed (5 sites), OSL-1 (5 sites), OSL-2 (3 sites), and Root-2 (4 sites). Sample collect ions are detailed in Appendix D. The mean CP4 EPSPS protein levels were highest in le af (ranging from OSL-1 at 180 μg/g dw to OSL-3 at 230 μg/g dw), followed by forage (170 μg/g dw), root (ranging from Root-2 at 38 μg/g dw to Root-1 at 82 μg/g dw), and seed (27 μg/g dw).Monsanto Company 11-CA-233U 71 of 500 Table V-1. Summary of CP4 EPSPS Prot ein Levels in Canola Tissues from

MON 88302 Grown in 2009 U.S. a nd Canadian Field Trials

Tissue 1 Development Stage 2 Days

After Planting

(DAP) CP4 EPSPS Mean (SD) Range (μg/g fw) 3 CP4 EPSPS Mean (SD) Range (μg/g dw) 4 LOQ/LOD 5

(μg/g fw)

Forage 30 BBCH 37 – 57 18 (4.4) 170 (22) 0.91/0.28

14 - 28 120 - 210

Seed 99 BBCH 118 – 132 25 (5.2) 27 (5.6) 0.91/0.81

21 - 43 22 - 46

OSL-1 13-14 BBCH 23 - 40 23 (10) 180 (40) 0.91/0.098

10 - 45 110 - 250

OSL-2 17-19 BBCH 32 - 54 22 (5.9) 180 (41) 0.91/0.098

18 - 37 120 - 250

OSL-3 30 BBCH 37 - 57 31 (6.3) 230 (50) 0.91/0.098

20 - 41 130 - 300

OSL-4 60-62 BBCH 51 - 61 36 (14) 210 (80) 0.91/0.098

20 - 85 110 - 500

Root-1 30 BBCH 37 - 57 19 (4.1) 82 (17) 0.91/0.60

11 - 25 46 - 100

Root-2 71-73 BBCH 49 - 81 10 (3.3) 38 (14) 0.91/0.60

7.0 - 17 24 - 62

1 OSL = over-season leaf.

2 The development stage at which each tissue was collect ed. The canola growth stages are based on the

Bayer, BASF, Ciba-Geigy and Hoechst Cereal Grain Growth Scale (BBCH) (BBCH, 2001).

3 Protein levels are expressed as the arithmetic mean and standard deviation (SD) as microgram ( μ g) of

protein per gram (g) of tissue on a fresh weight basis (fw). The means, SD, and ranges (minimum and maximum values) were calculated for each tissue across a ll sites. The numbers of samples (n) figured into the calculations are as follows: forage n = 20, seed n = 16, OSL-1 n = 16, OSL-2 n = 9, OSL-3 n = 20, OSL-4 n = 20, Root-1 n = 19, and Root-2 n = 11. Sample collections are detailed in Appendix D.

4 Protein levels are expressed as the arithmetic mean and standard deviation (SD) as microgram ( μ g) of

protein per gram (g) of tissue on a dry weight basis (dw). The dry weight values were calculated by dividing the μ g/g fw by the dry weight conversion factor obtained from moisture analysis data.

5 LOQ=limit of quantitation; LOD = limit of detection.Monsanto Company 11-CA-233U 72 of 500 V.D. Assessment of Potential Alle rgenicity of the CP4 EPSPS Protein

The allergenic potential of an introduced protein is assesse d by comparing the

biochemical characteristics of the introduced protein to biochemical characteristics of known allergens (Codex Alimentarius, 2009). A pr otein is not likely to be associated with allergenicity if: 1) the protein is fr om a non-allergenic source; 2) the protein represents a very small portion of the total plant protein; 3) the protein does not share structural similarities to known allergens ba sed on the amino acid sequence; and 4) the protein is rapidly digested in mammalian gastrointestinal systems. The CP4 EPSPS protein has been assessed for its potential allergenicity according to these safety assessment guidelines.

1) The CP4 EPSPS protein originates from Agrobacterium sp. strain CP4 an organism that has not been reported to be a source of known allergens.

2) The CP4 EPSPS protein represents no more than 0.01% of the total protein in the

seed of MON 88302.

3) Bioinformatics analyses demonstrated that the CP4 EPSPS protein does not share amino acid sequence similarities with known allergens and, ther efore, is highly unlikely to contain immunol ogically cross-reactive al lergenic epitopes.

4) Finally, in vitro digestive fate experiments condu cted with the CP4 EPSPS protein demonstrate that the protein is rapidly digested in si mulated gastric fluid (SGF) and in simulated intestinal fluid (SIF).

Taken together, these data support the conc lusion that the CP4 EPSPS protein does not pose a significant allergenic ri sk to humans or animals.

V.E. Safety Assessment Summary of CP4 EPSPS Protein in MON 88302

Numerous factors have been considered in the safety assessment of the CP4 EPSPS protein and a comprehensive food and f eed assessment of CP4 EPSPS protein was conducted. The results are summarized below along with the conclusions reached from

the assessment.

V.E.1. The Donor Organism Has a History of Safe Use

The donor organism, Agrobacterium sp. , strain CP4 is not known for human or animal pathogenicity, and is not commonl y allergenic (FAO-WHO, 1991). Agrobacterium sp. strain CP4 has been previously reviewed as a part of the sa fety assessment of the donor organism during Monsanto consultations with the FDA regarding Roundup Ready soybean (1994), Roundup Ready canola (1995), Roundup Ready cotton (1995), Roundup Ready 2 corn (1996), Roundup Ready sugar b eet (1998), and Roundup Ready Flex cotton (2005). Further, the Environmental Protec tion Agency has established an exemption from the requirement of a tolerance for re sidues of CP4 EPSPS protein and the genetic material necessary for its production in all plants (U.S. EPA, 1996b).Monsanto Company 11-CA-233U 73 of 500 V.E.2. CP4 EPSPS Proteins Are Common in Food and Feeds

The CP4 EPSPS protein present in MON 88302 is similar to EPSPS proteins consumed in a variety of food and feed sources. CP4 EPSPS protein is homologous to EPSPS proteins naturally present in plants, including food crops ( e.g., soybean and maize) and fungal and microbial food sources such as baker’s yeast ( Saccharomyces cerevisiae ), all of which have a history of safe human consum ption (Harrison et al., 1996; Padgette et al., 1996). The similarity of the CP4 EPSPS protei n to EPSPS proteins in a variety of foods supports extensive human consumption of the fa mily of EPSPS proteins and the lack of health concerns. The ubiquitous presence of homologous EPSPS enzymes in food crops and common microorganisms establishes th at EPSPS proteins, and their enzymatic activity, pose no hazards for human and animal consumption.

V.E.3. CP4 EPSPS Catalyzes a Specific Enzyme Reaction

EPSPS exerts its functions in the shikimate pa thway that is integral to aromatic amino acid biosynthesis in plants and microorga nisms (Levin and Sprinson, 1964; Steinrücken and Amrhein, 1980). Therefore, this enzyme and its activity are f ound widely in food and feed derived from plant and microbial sources. Genes for numerous EPSPS proteins have been cloned (Pad gette et al., 1996) and the catalyt ic domains of this group of proteins are conserved. B acterial EPSPS proteins have b een well characterized with respect to their three dimensional X-ray crys tal structures (Stal lings et al., 1991) and

detailed kinetic and chemical mech anisms (Anderson and Johnson, 1990).

V.E.4. CP4 EPSPS Protein in MON 88302 is Not Homologous to Known Allergens

or Toxins

The CP4 EPSPS protein does not share amino acid sequence similarities with known allergens or protein toxins that have adverse effects to mammals. This has been demonstrated by extensive assessment with bioinformatic tools, such as the FASTA sequence alignment tool and eight-amino aci d sliding window search. An amino acid sequence is considered to have allergenic poten tial if it has an exact sequence identity of at least eight linearly contiguous amino acids with a potential allergen epitope (Hileman et al., 2002; Metcalfe et al., 1996). Using a sliding window of less than eight amino acids can produce matches containing significant uncertainty depending on the length of the query sequence (Silvanovich et al., 2006) and are not useful to the allergy assessment process (Thomas et al., 2005).

V.E.5. CP4 EPSPS Protein in MON 88302 Is Labile in in vitro Digestion Assays

The CP4 EPSPS protein is readily digestible in simulated gastric fluid (SGF) and simulated intestinal fluid (SIF ) (Harrison et al., 1996). Ra pid degradation of the fulllength CP4 EPSPS protein in SGF and SIF re duces the exposure of CP4 EPSPS protein

to cells of the small intestine in a biologically active form.Monsanto Company 11-CA-233U 74 of 500 V.E.6. CP4 EPSPS Protein in MON 88302 is Not Acutely Toxic

An acute oral toxicology study with mi ce was conducted with a CP4 EPSPS protein (Harrison et al., 1996) that was shown to be physicochemically and functionally equivalent to the CP4 EPSPS protein produced in MON 88302. Results indicate that the CP4 EPSPS protein did not cause any adverse effects in mice, therefore the No Observable Adverse Effect Level (NOAEL) for CP4 EPSPS is considered to be 572 mg/kg, the highest dose level tested.

V.E.6.1 Human and Animal Exposure to the CP4 EPSPS Protein

A common approach used to assess potential health risks from chemicals or other potentially toxic products is to calculate a Margin of Exposure (MOE) between the lowest NOAEL from an appropriate animal toxicity study and an estimate of human exposure. However, the primary human food cu rrently produced from canola is refined, bleached, and deodorized (RBD) oil. Because RBD oil contains negligible amounts of protein (Martín-Hernández et al., 2008), o il produced from MON 88302 will contain negligible levels of CP4 EPSPS protein. Th erefore an MOE was not calculated for the CP4 EPSPS protein since there is minimal, if any, dietary exposure to this protein from consumption of foods derived from MON 88302. Furthermore, the safety of CP4 EPSPS has been extensively assesse d (Harrison et al., 1996) a nd several Roundup Ready crops that produce CP4 EPSPS have been reviewed by FDA and other regulatory agencies. They concluded the Roundup Ready crops were safe for consumption.

The potential CP4 EPSPS protei n exposure to animals from consumption of MON 88302 in feeds was evaluated by calculating an estima te of daily dietary intake (DDI). The highest percentage of CP4 EPSPS protein (g/k g bw) per total protein consumed was in the dairy cow, 0.0141% (g/g) of the to tal dietary protein intake (0.00084 g CP4 EPSPS/kg bw divided by 6 g dietary protein which is the total dietary protein intake for the cow). The chicken and pig percenta ges of the CP4 EPSPS protein consumed as part of the daily protein intake are much less than for the dairy cow. At the most, poultry, swine and lactating dairy cattle would be consuming less than 0.015% (g/g) of their total protein intake as CP4 EPSPS protein from MON 88302. Therefore, there is minimal exposure to MON 88302 CP4 EPSPS in relati on to the total protein consumed.

V.F. CP4 EPSPS Protein Characte rization and Safety Conclusion

The data and information provided in this s ection address the questions important to the food and feed safety of the CP4 EPSPS pr otein in MON 88302 including its potential allergenicity and toxicity. To summarize, the physicochemical characteristics of the CP4 EPSPS protein were determined and s hown to be equivalent to those of an E. coli

produced CP4 EPSPS protein. The expressi on levels of the CP4 EPSPS protein in

selected tissues of MON 88302 were determin ed. An assessment of the allergenic potential of the CP4 EPSPS protein supports the conclusion that the CP4 EPSPS protein does not pose a significant allergenic risk to humans or animals. In addition, donor organism for the CP4 EPSPS coding sequence, Agrobacterium sp. strain CP4, is ubiquitous in the environment and is not commonly known for human or animalMonsanto Company 11-CA-233U 75 of 500 pathogenicity, or allergenicit y. The CP4 EPSPS protein lack s structural similarity to allergens, toxins or other proteins known to have adve rse effects on mammals. The CP4 EPSPS protein is rapidly di gested in simulated digestiv e fluids and demonstrates no oral toxicity in mice at the level tested. Based on the above information, the consumption of the CP4 EPSPS protein from MON 88302 or its progeny are considered safe for

humans and animals.

The protein safety data presented herein support the conclusion that food and feed products containing MON 88302 or derived from MON 88302 are as safe as canola currently on the market for human and animal consumption.Monsanto Company 11-CA-233U 76 of 500 VI. COMPOSITIONAL AS SESSMENT OF MON 88302

Several Roundup Ready crops that produce the CP4 EPSPS protein have been reviewed by the USDA. The CP4 EPSPS protein expressed in MON 88302 is identical to the CP4 EPSPS protein in other Roundup Ready crops and the mode of action of CP4 EPSPS protein is well understood. Previous Roundup R eady crops reviewed by the USDA have had no biologically relevant compositional changes identified, and there is no reason to expect the CP4 EPSPS protein in MON 883 02 would affect nutritionally important nutrients, toxicants, or anti- nutrients present in seed from this new product.

Safety assessments of biotechnology-derive d crops follow the comparative safety assessment process (Codex Alimentarius, 2009) in which the composition of grain and/or other raw agricultural commodities of the biotechnology-derived crop are compared to the appropriate conventional counterpart that has a history of safe use. Compositional assessments are performed using the princi ples and analytes outlined in the OECD consensus document for canola composition (OECD, 2001).

A recent review of compositional assessments conducted according to OECD guidelines that encompassed a total of seven biotechnology-derived cr op varieties, nine countries and eleven growing seasons concluded th at incorporation of biotechnology-derived agronomic traits has had little impact on na tural variation in crop composition. Most compositional variation is attributable to growing region, agronomic practices, and genetic background (Harrigan et al., 2010). Co mpositional quality, therefore, implies a very broad range of endogenous levels of indi vidual constituents. Numerous scientific publications have further documented the extensive variability in the concentrations of crop nutrients, anti-nutrients, and secondary metabolites that reflec t the influence of environmental and genetic factors as well as extensive conventiona l breeding efforts to improve nutrition, agronomics, and yield (Harri gan et al., 2010; Ma iler and Pratley, 1990; Marwede et al., 2004; Naczk et al., 1998; OECD , 2001; Pritchard et al., 2000; Reynolds et al., 2005; Ridley et al., 2004; Werteker et al., 2010).

Compositional equivalence between biotech nology-derived and conventional crops provides an “equal or increased assurance of the safety of f oods derived from genetically modified plants” (OECD, 1998). OECD consensus documents on compositional considerations for new crop varieties emphasi ze quantitative measurem ents of essential

nutrients and known anti-nutrients . This is based on the premise that such comprehensive and detailed analyses will most effectively discern any compositional changes that imply potential nutritional or safety ( e.g ., anti-nutritional) concerns. Levels of the components in seed and/or other raw agricultural co mmodities of the biotechnology-derived crop are compared to: 1) corresponding levels in a conventional comparator, the genetically similar conventional line, grown concurrently, in the same field trial, and 2) natural ranges generated from an evaluation of commercial reference varieties grown concurrently and from data published in the scientific literature. The comparison to data published in the literature places any potenti al differences between the assessed crop and its comparator in the context of the well-doc umented variation in the concentrations of crop nutrients, toxicants, and anti-nutrients.Monsanto Company 11-CA-233U 77 of 500 This section provides analyses of concentr ations of key nutrients, toxicants, and antinutrients of MON 88302 compared with e quivalent analyses of a conventional counterpart grown and harveste d under the same conditions, as appropriate. In addition, commercial conventional canola reference varieties were included in the composition analyses to establish a range of natural va riability for each analyte, defined by a 99% tolerance interval. The production of material s for the compositional analyses used field designs to allow accurate assessments of compositional characteristics over a range of

environmental conditions under which MON 88302 is expected to be grown. Design parameters included a sufficient number of trial sites to allow adequate exposure to the variety of conditions met in nature. Field site s were replicated with an adequate number of plant samples, and the methods of analysis were sufficiently sens itive and specific to detect variations in the components measured and to allow statistica lly rigorous analyses. The information provided in this section al so addresses the releva nt factors in Codex Plant Guidelines, Section 4, paragraphs 44 and 45 for compositional analyses (Codex Alimentarius, 2009).

VI.A. Compositional Equivalence of MO N 88302 Seed to Conventional Canola

Compositional analysis comparing MON 883 02 to the conventional control variety (Ebony) and commercial reference varie ties demonstrated that MON 88302 is compositionally equivalent to conventional canola. Seed sa mples were collected from

MON 88302 and the conventional control grown in a 2009 North American field production. Canola forage is rarely consumed by animals and is not a source of nutrition for humans. Therefore, the OECD consensu s document on compositional considerations for canola (OECD, 2001) does not recommend analysis of canola forage, and forage samples were not analyzed. The background genetics of the conventional control were similar to that of MON 88302, but did not contain the cp4 epsps expression cassette.

Seven different commercial reference varieties were included across all sites of the field production to provide data on natural vari ability of each compositional component analyzed. The samples utilized for compositi onal analysis were obtained from two U.S. sites [Wilkin County, Minnesota (MNC A) and McHenry County, North Dakota (NDVA)] and three Canadian sites [Portage la Prairie, Manitoba (MBPL); Newton, Manitoba (MBNW); and Saskatoon, Saskatchewan (SKSA)]. The sites were planted in a randomized complete block design with f our replicates per site. MON 88302, the conventional control, and commercial referenc e varieties were treated with maintenance pesticides as necessary throughout the growi ng season. In addition to the conventional weed control programs, MON 88302 plots were treated at the 5-6 leaf stage with a glyphosate application at a target rate of 1.6 lb acid equivalent s per acre (1800 g a.e./ha).

Compositional analyses were conducted as recommended for canola seed (OECD, 2001)

to assess whether levels of ke y nutrients, toxicants and anti -nutrients in MON 88302 were equivalent to levels in the conventional control and to the composition of commercial

reference varieties. Nutrients assessed in this analysis incl uded proximates (ash, carbohydrates by calculation, moisture, protein, a nd total fat), fibers (acid detergent fiber [ADF], neutral detergent fiber [NDF], to tal dietary fiber [TDF ]), amino acids (18 components), fatty acids (FA; C8-C24,), vitamin E ( α -tocopherol), and minerals (calcium, copper, iron, magnesium, manga nese, phosphorus, potassium, sodium, andMonsanto Company 11-CA-233U 78 of 500 zinc) in seed. The toxicants assessed in seed included eruc ic acid and glucosinolates (alkyl glucosinolates [including 3-buten yl, 4-pentenyl, 2-hydr oxy-3-butenyl, and 2- hydroxy-4-pentenyl glucosinolates ], indolyl glucosinolates [including 3-indolylmethyl and 4-hydroxy-3-indoylmethyl], and total glucosi nolates [sum of alkyl and indolyl]). The anti-nutrients assessed in seed included phy tic acid and sinapine (as sinapic acid).

Methods used in the assessments of nutrients, toxicants and anti-nutrients are found in Appendix E. The toxicant and anti-nutrien t results are discussed together under the general heading of anti-nutrients. In all, 70 different components were measured. Of those 70 components, 18 nutrients and one toxi cant (18 fatty acids, including erucic acid, and one mineral) had more than 50% of the observations below the assay limit of quantitation (LOQ) and, as a result, were excluded from the statistical analyses. Therefore, 51 components were statistically assessed using a mixed model analysis of variance method. Values for all components we re expressed on a dry weight basis with the exception of moisture, expressed as percent fresh weight, and fatty acids, expressed as percent of total FA.

For MON 88302, six statistical co mparisons to the conventional control were conducted for each compositional component. One co mparison was based on compositional data combined across all five field sites (com bined-site analysis) and five separate comparisons were conducted on data from each of the individual field sites. Statistically significant differences were id entified at the 5% level ( α = 0.05). Data from the

commercial reference varieties were combined across all sites and used to calculate a 99% tolerance interval for each compositional co mponent to define the natural variability of each component in canola varieties that ha ve a history of safe consumption, and that were grown concurrently with MON 88302 and the conventional control in the same trial.

For the combined-site analysis, significant differences in nutrient, toxicant, and antinutrient components were further evaluated usi ng considerations rele vant to the safety and nutritional quality of MON 88302 when co mpared to the convent ional control, which is the conventional counterpart that has a history of safe consumption. Considerations

used to assess the relevance of each combined-site statis tically significant difference included: 1) the relative magnitude of the difference in the mean values of nutrient, toxicant, and anti-nutrient components of MON 88302 and the conventional control; 2) whether the MON 88302 component mean value is within the range of natural variability of that component as represented by the 99% tolerance interval of the commercial reference varieties grown concurrently in the same trial; 3) evaluation of the reproducibility of the stat istically significant ( α = 0.05) combined-site component

differences at individua l sites; and 4) an assessment of the differences within the context of natural variability of commercial canola composition published in the scientific literature. If statistically signi ficant differences detected in the individual site analyses were not observed in the combined-site analys is, they were not considered further for the compositional assessment of safety. Statistical summaries of nutrients , toxicants and antinutrients for individual sites are found in Appendix E.

This analysis provides a comprehensive comparative assessment of the levels of key nutrients, toxicants, and anti- nutrients in seed of MO N 88302 and the conventional control, discussed in the context of natura l variability in composition of commercialMonsanto Company 11-CA-233U 79 of 500 canola. Results of the comparison indica te that the composition of the seed of MON 88302 is equivalent to that of the c onventional control and within the natural variability of commercial reference varieties.

VI.A.1. Nutrient Levels in Canola Seed

In the combined-site analysis of nutrient levels in seed, the following components showed no significant differences in mean values between MON 88302 and the conventional control: proximates, two type s of fiber (ADF and NDF), 18 amino acids (alanine, arginine, aspartic acid, cystine, gl utamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine , proline, serine, th reonine, tryptophan, tyrosine, and valine), four fatty acids (16:0 palmitic acid, 20:1 eicosenoic acid, 24:0 lignoceric acid, and 24:1 nervonic acid), eight minerals (calcium, copper, iron, magnesium, manganese, phosphorus, potassium, and zinc), and vitamin E (Table VI-2).

The components that showed significant differences in mean values between

MON 88302 and the conventional control in the combined-site analysis were: total dietary fiber (TDF) and seven fatty acids (1 6:1 palmitoleic acid, 18:0 stearic acid, 18:1 oleic acid, 18:2 linoleic acid, 18:3 linolenic acid, 20:0 arac hidic acid, and 22:0 behenic acid) (Tables VI-1 and VI-2).

1) The statistically significant differen ces in nutrients were evaluated using

considerations relevant to the nutriti onal quality of MON 88302 when compared to the conventional control: eight combined-site nutrient significant differences ( α = 0.05) between MON 88302 and the conventi onal control were attributable to TDF (expressed as % dry weight) and seven fatty acids (expressed as % total FA). The relative magnitudes of differences between the combined-site mean values for MON 88302 and the conventional control showed an increase for TDF, 18:2 linoleic acid, and 18:3 linolenic acid , (13.81%, 8.98%, and 20.01%, respectively) and a decrease for 16:1 palmitoleic acid, 18:0 stearic acid, 18:1 oleic acid, 20:0 arachidic acid, and 22:0 beheni c acid (7.56%, 15.06%, 4.52%, 10.68%, and 6.01%, respectively). The relative differences in these components in the combined-site analysis and at indivi dual sites were be tween 3.48% and 28.69% (Table VI-2 and Tables E-3, E-5, E- 7, E-9, and E-11). The magnitudes of

differences observed between MON 88302 and the conventional control were small relative to the natural variability of these components as determined by the

99% tolerance interval established by the concurrently grown commercial reference varieties that have a history of safe consumption, as presented in the

tables referenced above.

2) Mean values for all of the nutrient co mponents found to be significantly different ( α = 0.05) from the combined-site analysis of MON 88302 were within the 99%

tolerance interval established from th e commercial reference varieties grown concurrently and were, therefore, within the range of natural variability of that component in commercial canola varieties th at have a history of safe consumption (Table VI-1).Monsanto Company 11-CA-233U 80 of 500 3) Assessment of the reproduc ibility of the combined-site differences at the five individual sites demonstrated no signi ficant differences for TDF; however, significant differences ( α = 0.05) were observed for 18: 0 stearic acid, 18:1 oleic acid, and 18:2 linoleic acid at all five sites; significant differences for 16:1 palmitoleic acid and 18:3 linolenic acid at four sites, significant differences for

20:0 arachidic acid at three sites, and significant differe nces for 22:0 behenic acid at two sites (Table VI-1). The magnitude s of differences between the mean fatty

acid values for MON 88032 and the conventional control were small relative to the variability of these components as de termined by the 99% tolerance interval established by the concurre ntly grown commercial reference varieties, and relative to the variability of fatty acid components in canola due to environment (Pritchard et al., 2000). Individual site mean values of MON 88302 for all nutrient components with si gnificant differences fell within the 99% tolerance

interval established from the commercial reference varieties grown concurrently and were, therefore, within the range of natural variabili ty of that component in

commercial canola varieties that have a hist ory of safe consumption (Table VI-2).

4) With the exception of TDF, for which no commercial reference values have been published, all of the compositional co mponents identified as significantly

different from the conventional control were within the natural variability of these components in commercial canola composition as published in the scientific literature (Table VI-4).

In summary, the combined-site statistical analysis identified eight significant differences ( α = 0.05) that were small in magnitude relative to their natural variability as determined by the 99% tolerance interval establishe d by the concurrently grown commercial reference varieties that have a history of safe consumption. Of these significant differences, only 18:0 stearic ac id, 18:1 oleic acid, and 18:2 linoleic acid were observed consistently at all of the individual sites. All of the components identified as significantly different in the combined-site analysis an d corresponding individual site analyses, were within the natural variability of commercial canola defined by the 99 % tolerance interval established by the concurrently grown commerci al reference varieties, and were within the published literature ranges (there is no published refere nce data for TDF) for these components in conventional canola. Therefor e, these significant differences are not meaningful to food and feed safety or nutrition. These findings support the conclusion

that nutrients in seed from MON 88302 are compositionally equivalent to those in

conventional canola varieties with a history of safe usage.

VI.A.2. Anti-Nutrient Levels in Canola Seed

According to OECD (2001), canola seed cont ains toxicants includ ing erucic acid and glucosinolates, and anti-nutrients, including ph ytic acid and sinapine. Erucic acid has

been shown to have card iopathic potential resulting in a weakening of the heart muscle in experimental animals (Bozcali et al., 2009; Chien et al., 1983). Glucosinolates in canola

seed can be characterized into two main ch emical groups, alkyl a nd indolyl, with alkyl being the most common (CCC, 2009). Upon enzymatic hydrolysis with myrosinase, certain glucosinolates form compounds that can depress growth and thyroid functionMonsanto Company 11-CA-233U 81 of 500 (Bell, 1984). The standard for glucosi nolates in canola seed is <18 μmoles/g (Szmigielska et al., 2000). Phytic acid is pr esent in canola seed. Phytic acid chelates mineral nutrients, including calcium, magne sium, potassium, iron, and zinc, rendering them biologically unavailable to monogastric animals consuming the seed (Liener, 2000). Sinapine is the choline este r of sinapic acid, the primar y phenolic component in canola seed. Sinapine imparts a bitter taste and redu ces palatability of the seed (OECD, 2001). Sinapine levels were determined based on qua ntitation of the hydrol ysis product, sinapic

acid.

MON 88302 levels of 22:1 erucic acid were be low the level of det ection (0.04% total FA) in canola seed, and therefore, 22:0 erucic acid was excluded from statistical analysis. In the combined-site analysis, no significant difference ( α = 0.05) was observed between

MON 88302 and the conventional control (Tables VI-1 and VI-3) for indolyl glucosinolates, total glucosinolates, phytic acid, and sinapine. One statistically significant difference was identified for alkyl glucosinolates, and the net effect was a slight reduction of this an ti-nutrient in MON 88302. The fo llowing considerations show that this difference is not a meaningful conc ern from a food and feed nutritional or safety perspective:

1) The magnitude of the difference between the combined-site mean value for alkyl glucosinolates in MON 88302 and the conventional control showed a 27.59% decrease. This magnitude of difference was small relative to the natural variability of these components as dete rmined by the 99% tolerance interval established by the concurrently grown comm ercial reference varieties that have a history of safe consumption.

2) The MON 88302 mean alkyl glucosinolates value from the combined-site analysis

was within the 99% tolerance interval established from the commercial reference varieties grown concurrently. The mean va lue was, therefore, within the range of natural variability for alkyl glucosinolat es in commercial canola varieties that have a history of safe consump tion (Tables VI-1 and VI-3).

3) The combined-site difference for alkyl glucosinolates wa s not consistently observed at all individual sites. A signi ficant difference for alkyl glucosinolates

was observed at one of the individual site s. However, the mean value for alkyl glucosinolates in MON 88302 at this indivi dual site was within the 99% tolerance interval established from the concurrently grown commercial reference varieties.

4) An assessment based on of the natural variability of alkyl glucosinolates in

commercial canola varieties could not be made because a range was not available

in the scientific literature.

In summary, the statistical analyses found a combined-site significant difference in alkyl glucosinolates that was lower than the conve ntional mean value, and not consistently

observed at the individual sites. The m ean alkyl glucosinolates value for MON 88302 was within the natural variability of comm ercial canola defined by the 99% tolerance interval established from the concurrently gr own commercial reference varieties that haveMonsanto Company 11-CA-233U 82 of 500 a history of safe consumption, and the value was within the safety threshold for canola. Total glucosinolate levels in seed from MON 88302 ranged from 1.73 to 11.42 μmoles/g (Table VI-3 and Tables E-4, E-6, E-8, E-10, a nd E-12), within the standard for canola. Thus, an evaluation of anti-nutrient compone nts in seed supports the conclusion that MON 88302 is as safe as and compositionally equivalent to conventional canola.Monsanto Company 11-CA-233U 83 of 500Table VI-1. Summary of Differences (p < 0.05) for the Comparison of Canola Seed Component Levels for MON 88302 vs. theConventional ControlMean Difference(MON 88302 minus Control)Analytical Component (Units)1MON 883022Mean3Control4MeanMean Difference(% of Control)Significance(p-Value)MON 88302RangeCommercialTolerance Interval5Statistical Differences Observed in Combined-Site AnalysisSeed Fiber (% dw)Total Dietary Fiber20.9018.3713.810.00416.91 - 27.8113.97, 24.85Seed Fatty Acid (% Total FA)16:1 Palmitoleic0.220.24-7.560.0080.20 - 0.260.17, 0.3018:0 Stearic1.681.98-15.06<0.0011.54 - 1.870.90, 3.0518:1 Oleic62.8265.79-4.52<0.00160.51 - 65.2056.13, 70.6918:2 Linoleic19.2617.678.98<0.00117.78 - 20.6612.60, 24.4918:3 Linolenic9.587.9820.01<0.0018.71 - 11.236.96, 11.7320:0 Arachidic0.540.60-10.68<0.0010.50 - 0.570.45, 0.8022:0 Behenic0.270.28-6.010.0160.24 - 0.290.19, 0.43Monsanto Company 11-CA-233U 84 of 500Table VI-1.Summary of Differences (p<0.05) for the Comparison ofCanola Seed Component Levels for MON 88302 vs. theConventional Control (continued)Mean Difference(MON 88302 minus Control)Analytical Component (Units)1MON 883022Mean3Control4MeanMean Difference(% of Control)Significance(p-Value)MON 88302RangeCommercialTolerance Interval5Statistical Differences Observed in Combined-Site AnalysisSeed Anti-nutrientAlkyl Glucosinolate (μmole/g dw)3.685.08-27.590.0351.19 - 5.870, 29.02Statistical Differences Observed in More than One Individual SiteSeed Fatty Acid (% Total FA)18:0 Stearic Site MBNW1.731.97-12.230.0281.64 - 1.870.90, 3.0518:0 Stearic Site MBPL1.581.87-15.64<0.0011.55 - 1.590.90, 3.0518:0 Stearic Site MNCA1.671.86-10.010.0221.65 - 1.710.90, 3.0518:0 Stearic Site NDVA1.772.11-16.060.0041.71 - 1.840.90, 3.0518:0 Stearic Site SKSA1.662.08-20.140.0011.54 - 1.720.90, 3.0518:1 Oleic Site MBNW63.4065.71-3.510.00462.94 - 64.0356.13, 70.69Monsanto Company 11-CA-233U 85 of 500Table VI-1.Summary of Differences (p<0.05) for the Comparison ofCanola Seed Component Levels for MON 88302 vs. theConventional Control (continued)Mean Difference(MON 88302 minus Control)Analytical Component (Units)1MON 883022Mean3Control4MeanMean Difference(% of Control)Significance(p-Value)MON 88302RangeCommercialTolerance Interval5Statistical Differences Observed in More than One Individual SiteSeed Fatty Acid (% Total FA)18:1 Oleic Site MBPL62.0664.30-3.48<0.00161.82 - 62.3556.13, 70.6918:1 Oleic Site MNCA61.6764.86-4.920.00561.70 - 61.8756.13, 70.6918:1 Oleic Site NDVA65.1468.38-4.740.00364.90 - 65.2056.13, 70.6918:1 Oleic Site SKSA61.9165.69-5.750.00160.51 - 62.2956.13, 70.6918:2 Linoleic Site MBNW19.2717.897.710.01118.82 - 19.6612.60, 24.4918:2 Linoleic Site MBPL20.4319.186.50<0.00120.13 - 20.6612.60, 24.4918:2 Linoleic Site MNCA20.2018.3510.070.00120.00 - 20.3212.60, 24.4918:2 Linoleic Site NDVA17.8615.7113.670.00917.78 - 18.0212.60, 24.49Monsanto Company 11-CA-233U 86 of 500Table VI-1.Summary of Differences (p<0.05) for the Comparison ofCanola Seed Component Levels for MON 88302 vs. theConventional Control (continued)Mean Difference(MON 88302 minus Control)Analytical Component (Units)1MON 883022Mean3Control4MeanMean Difference(% of Control)Significance(p-Value)MON 88302RangeCommercialTolerance Interval5Statistical Differences Observed in More than One Individual SiteSeed Fatty Acid (% Total FA)18:2 Linoleic Site SKSA18.4917.227.360.01918.08 - 19.4812.60, 24.49Seed Vitamin (mg/100g dw)Vitamin E (a-tocopherol) Site MBNW13.069.3639.510.00412.22 - 13.473.88, 17.28Vitamin E (α-tocopherol) Site MBPL11.507.6350.83<0.00110.70 - 12.203.88, 17.28Vitamin E (α-tocopherol) Site MNCA13.3910.8223.730.00612.58 - 14.623.88, 17.28Vitamin E (α-tocopherol) Site NDVA15.899.4368.390.01015.23 - 16.553.88, 17.28Vitamin E (α-tocopherol) Site SKSA1.496.91-78.470.0191.30 - 1.663.88, 17.28Seed Anti-nutrientSinapic Acid (% dw) Site MBNW1.020.9210.340.0010.99 - 1.060.57, 1.13Monsanto Company 11-CA-233U 87 of 500Table VI-1. Summary of Differences (p<0.05) for the Comparison of Canola SeedComponent Levels for MON 88302 vs. theConventional Control (continued)Mean Difference(MON 88302 minus Control)Analytical Component (Units)1MON 883022Mean3Control4MeanMean Difference(% of Control)Significance(p-Value)MON 88302RangeCommercialTolerance Interval5Statistical Differences Observed in More than One Individual SiteSeed Anti-nutrientSinapic Acid (% dw) Site MBPL0.970.8612.04<0.0010.95 - 0.990.57, 1.13Sinapic Acid (% dw) Site MNCA1.060.9610.660.0011.02 - 1.080.57, 1.13Sinapic Acid (% dw) Site NDVA1.020.8323.560.0011.00 - 1.040.57, 1.13Sinapic Acid (% dw) Site SKSA0.220.81-73.120.0010.16 - 0.280.57, 1.13Seed Fatty Acid (% Total FA)16:1 Palmitoleic Site MBNW0.210.23-9.710.0150.20 - 0.210.17, 0.3016:1 Palmitoleic Site MBPL0.230.25-10.100.0080.22 - 0.230.17, 0.3016:1 Palmitoleic Site MNCA0.210.24-10.880.0010.21 - 0.210.17, 0.3016:1 Palmitoleic Site NDVA0.200.22-11.050.0360.20 - 0.200.17, 0.30Monsanto Company 11-CA-233U 88 of 500Table VI-1. Summary of Differences (p<0.05) for the Comparison of Canola SeedComponent Levels for MON 88302 vs. theConventional Control (continued)Mean Difference(MON 88302 minus Control)Analytical Component (Units)1MON 883022Mean3Control4MeanMean Difference(% of Control)Significance(p-Value)MON 88302RangeCommercialTolerance Interval5Statistical Differences Observed in More than One Individual SiteSeed Fatty Acid (% Total FA)18:3 Linolenic Site MBNW9.198.1213.270.0048.88 - 9.426.96, 11.7318:3 Linolenic Site MBPL9.287.7419.89<0.0019.12 - 9.436.96, 11.7318:3 Linolenic Site NDVA8.827.3120.67<0.0018.71 - 8.946.96, 11.7318:3 Linolenic Site SKSA10.788.3828.69<0.00110.39 - 11.236.96, 11.73Seed Fatty Acid (% Total FA)20:0 Arachidic Site MBPL0.530.60-11.73<0.0010.52 - 0.540.45, 0.8020:0 Arachidic Site NDVA0.570.65-12.58<0.0010.56 - 0.570.45, 0.8020:0 Arachidic Site SKSA0.540.62-13.28<0.0010.52 - 0.550.45, 0.80Monsanto Company 11-CA-233U 89 of 500Table VI-1. Summary of Differences (p<0.05) for the Comparison of Canola Seed Component Levels for MON 88302 vs.the Conventional Control (continued)Mean Difference(MON 88302 minus Control)Analytical Component (Units)1MON 883022Mean3Control4MeanMean Difference(% of Control)Significance(p-Value)MON 88302RangeCommercialToleranceInterval5Statistical Differences Observed in More than One Individual SiteSeed MineralCopper (mg/kg dw) Site MBNW3.723.419.280.0133.61 - 3.832.00, 4.43Copper (mg/kg dw) Site MBPL3.473.97-12.500.0163.35 - 3.562.00, 4.43Copper (mg/kg dw) Site MNCA4.404.116.910.0274.16 - 4.572.00, 4.43Seed Fatty Acid (% Total FA)22:0 Behenic Site MBPL0.270.30-13.00<0.0010.26 - 0.270.19, 0.4322:0 Behenic Site NDVA0.270.30-9.830.0070.27 - 0.270.19, 0.43Seed MineralIron (mg/kg dw) Site MBPL44.1351.01-13.480.00142.80 - 45.0923.39, 86.23Iron (mg/kg dw) Site MNCA42.5750.64-15.930.00740.56 - 44.1823.39, 86.23Monsanto Company 11-CA-233U 90 of 500Table VI-1. Summary of Differences (p<0.05) for the Comparison of Canola SeedComponent Levels for MON 88302 vs. theConventional Control (continued)Mean Difference(MON 88302 minus Control)Analytical Component (Units)1MON 883022Mean3Control4MeanMean Difference(% of Control)Significance(p-Value)MON 88302RangeCommercialTolerance Interval5Statistical Differences Observed in More than One Individual SiteSeed MineralPotassium (g/100g dw) Site MBPL0.700.77-8.910.0230.63 - 0.760.39, 0.96Potassium (g/100g dw) Site SKSA0.820.7115.32<0.0010.77 - 0.900.39, 0.96Zinc (mg/kg dw) Site MBPL31.2533.88-7.760.02430.45 - 32.0520.19, 48.23Zinc (mg/kg dw) Site SKSA41.5833.1025.610.01039.33 - 45.4920.19, 48.23Statistical Differences Observed in One Individual SiteSeed Proximate (% dw)Carbohydrates Site MNCA27.3125.995.070.03526.27 - 27.9023.12, 30.77Moisture (% fw) Site MNCA5.526.69-17.46<0.0015.37 - 5.614.33, 6.91Protein Site SKSA23.8222.147.580.03823.62 - 24.5817.20, 30.08Monsanto Company 11-CA-233U 91 of 500Table VI-1. Summary of Differences (p<0.05) for the Comparison of Canola SeedComponent Levels for MON 88302 vs. theConventional Control (continued)Mean Difference(MON 88302 minus Control)Analytical Component (Units)1MON 883022Mean3Control4MeanMean Difference(% of Control)Significance(p-Value)MON 88302RangeCommercialTolerance Interval5Statistical Differences Observed in One Individual SiteSeed Proximate (% dw)Total Fat Site NDVA48.0445.176.350.01447.20 - 48.8739.65, 51.24Seed Fiber (% dw)Acid Detergent Fiber Site MBPL16.7514.1918.000.00515.17 - 18.196.95, 23.92Neutral Detergent Fiber Site MBPL19.4516.8715.310.01718.35 - 20.0210.07, 25.94Seed Amino Acid (% dw)Tyrosine Site MBPL0.720.712.460.0280.72 - 0.730.57, 0.81Valine Site MNCA1.151.24-7.320.0481.13 - 1.150.92, 1.55Seed Fatty Acid (% Total FA)16:0 Palmitic Site SKSA4.514.0710.90<0.0014.46 - 4.572.84, 5.2620:1 Eicosenoic Site SKSA1.241.139.550.0051.22 - 1.260.83, 1.68Monsanto Company 11-CA-233U 92 of 500Table VI-1. Summary of Differences (p<0.05) for the Comparison of Canola SeedComponent Levels for MON 88302 vs. theConventional Control (continued)Mean Difference(MON 88302 minus Control)Analytical Component (Units)1MON 883022Mean3Control4MeanMean Difference(% of Control)Significance(p-Value)MON 88302RangeCommercialTolerance Interval5Statistical Differences Observed in One Individual SiteSeed Fatty Acid (% Total FA)24:0 Lignoceric Site MBPL0.160.19-12.240.0290.16 - 0.170.033, 0.2524:1 Nervonic Site MBPL0.130.16-20.370.0310.12 - 0.130.041, 0.18Seed Anti-nutrientAlkyl Glucosinolate (μmole/g dw) SiteSKSA1.615.82-72.320.0051.19 - 2.170, 29.02Indolyl Glucosinolate (μmole/g dw) SiteSKSA0.863.30-73.880.0010.49 - 1.311.37, 6.62Total Glucosinolate (μmole/g dw) SiteSKSA2.539.22-72.580.0021.73 - 3.510, 32.201dw = dry weight; fw = fresh weight; FA = fatty acid.2MON 88302 treated with glyphosate.3Mean = least-square mean.4Control refers to the genetically similar, conventional control Ebony.5With 95% confidence, interval contains 99% of the values expressed in the populationof commercial conventional reference varieties. Negative limits setto zero.Monsanto Company 11-CA-233U 93 of 500Table VI-2. StatisticalSummary of Combined-site Canola Seed Nutrients for MON 88302 vs. Conventional ControlDifference (MON 88302 minus Control)Analytical Component (Units)1MON 883022Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95% CI3Lower, UpperSignificance(p-Value)CommercialTolerance Interval5(Range)Proximate (% dw)Ash3.96 (0.18)3.90 (0.18)0.055 (0.095)-0.14, 0.250.5653.32, 4.66(3.31 - 4.45)(3.20 - 5.10)(-0.21 - 0.64)(2.98 - 4.52)Carbohydrates25.96 (0.68)26.13 (0.68)-0.17 (0.54)-1.42, 1.090.76523.12, 30.77(21.83 - 28.81)(23.91 - 28.73)(-4.18 - 1.94)(22.53 - 29.96)Moisture (% fw)5.35 (0.34)5.45 (0.34)-0.10 (0.24)-0.65, 0.450.6884.33, 6.91(3.90 - 6.08)(4.41 - 6.98)(-1.53 - 0.87)(4.09 - 8.48)Protein23.04 (0.70)23.14 (0.69)-0.10 (0.52)-1.32, 1.110.84717.20, 30.08(19.68 - 25.98)(20.29 - 27.02)(-2.29 - 2.50)(18.68 - 28.32)Total Fat47.06 (0.83)46.82 (0.83)0.24 (0.52)-1.00, 1.480.65939.65, 51.24(43.96 - 49.26)(43.65 - 50.24)(-2.28 - 4.10)(40.71 - 50.26)Fiber (% dw)Acid Detergent Fiber15.32 (1.36)14.47 (1.36)0.84 (0.41)-0.14, 1.830.0826.95, 23.92(9.19 - 20.24)(8.94 - 18.71)(-2.71 - 3.57)(9.75 - 21.22)Monsanto Company 11-CA-233U 94 of 500Table VI-2. Statistical Summary of Combined-Site Seed Nutrient Content for MON 88302 vs. the Conventional Control(continued)Difference (MON 88302 minus Control)Analytical Component (Units)1MON 883022Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95% CI3Lower, UpperSignificance(p-Value)CommercialTolerance Interval5(Range)Fiber (% dw)Neutral Detergent Fiber17.43 (1.38)16.70 (1.38)0.74 (0.57)-0.58, 2.050.23110.07, 25.94(9.48 - 21.36)(11.56 - 19.58)(-2.74 - 4.43)(10.93 - 22.75)Total Dietary Fiber20.90 (0.79)18.37 (0.78)2.54 (0.84)0.85, 4.230.00413.97, 24.85(16.91 - 27.81)(14.58 - 23.00)(-0.49 - 9.96)(12.64 - 26.47)Amino Acid (% dw)Alanine1.02 (0.025)1.04 (0.025)-0.015 (0.022)-0.066, 0.0350.5020.77, 1.34(0.88 - 1.15)(0.93 - 1.19)(-0.12 - 0.069)(0.87 - 1.27)Arginine1.45 (0.054)1.51 (0.054)-0.063 (0.032)-0.13, 0.000820.0521.10, 1.93(1.23 - 1.72)(1.29 - 1.77)(-0.27 - 0.15)(1.23 - 1.96)Aspartic Acid1.65 (0.067)1.71 (0.067)-0.055 (0.043)-0.16, 0.0450.2381.33, 2.12(1.40 - 1.93)(1.46 - 1.97)(-0.37 - 0.12)(1.42 - 2.23)Cystine0.57 (0.027)0.58 (0.027)-0.0044 (0.015)-0.040, 0.0310.7810.38, 0.83(0.48 - 0.73)(0.49 - 0.79)(-0.054 - 0.053)(0.45 - 0.79)Monsanto Company 11-CA-233U 95 of 500Table VI-2. Statistical Summary of Combined-Site Seed Nutrient Content for MON 88302 vs. the Conventional Control(continued)Difference (MON 88302 minus Control)Analytical Component (Units)1MON 883022Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95% CI3Lower, UpperSignificance(p-Value)CommercialTolerance Interval5(Range)Amino Acid (% dw)Glutamic Acid4.06 (0.18)4.24 (0.17)-0.19 (0.10)-0.43, 0.0490.1032.73, 5.89(3.37 - 5.06)(3.64 - 5.26)(-0.68 - 0.36)(3.26 - 5.43)Glycine1.14 (0.040)1.19 (0.040)-0.041 (0.025)-0.10, 0.0180.1420.96, 1.47(1.02 - 1.32)(1.01 - 1.38)(-0.18 - 0.044)(1.01 - 1.50)Histidine0.63 (0.023)0.65 (0.023)-0.015 (0.011)-0.038, 0.00740.1810.47, 0.86(0.55 - 0.77)(0.57 - 0.78)(-0.065 - 0.044)(0.54 - 0.80)Isoleucine0.93 (0.028)0.96 (0.028)-0.024 (0.021)-0.074, 0.0260.2990.70, 1.22(0.81 - 1.08)(0.82 - 1.12)(-0.13 - 0.041)(0.78 - 1.15)Leucine1.64 (0.049)1.68 (0.049)-0.042 (0.039)-0.13, 0.0480.3081.21, 2.18(1.40 - 1.90)(1.46 - 1.95)(-0.25 - 0.086)(1.36 - 2.07)Lysine1.39 (0.041)1.41 (0.041)-0.019 (0.023)-0.064, 0.0270.4101.02, 1.90(1.22 - 1.63)(1.25 - 1.65)(-0.12 - 0.086)(1.20 - 1.68)Monsanto Company 11-CA-233U 96 of 500Table VI-2.Statistical Summary of Combined-Site Seed Nutrient Content for MON 88302 vs. the Conventional Control(continued)Difference (MON 88302 minus Control)Analytical Component (Units)1MON 883022Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95% CI3Lower, UpperSignificance(p-Value)CommercialTolerance Interval5(Range)Amino Acid (% dw)Methionine0.46 (0.015)0.46 (0.015)-0.0018 (0.0089)-0.022, 0.0190.8470.30, 0.65(0.40 - 0.54)(0.40 - 0.56)(-0.038 - 0.034)(0.36 - 0.57)Phenylalanine0.98 (0.029)1.00 (0.028)-0.024 (0.024)-0.079, 0.0310.3480.77, 1.26(0.84 - 1.11)(0.87 - 1.15)(-0.17 - 0.044)(0.84 - 1.25)Proline1.40 (0.054)1.42 (0.054)-0.028 (0.027)-0.093, 0.0360.3350.90, 2.01(1.20 - 1.71)(1.20 - 1.73)(-0.16 - 0.17)(1.12 - 1.78)Serine1.02 (0.030)1.05 (0.030)-0.035 (0.019)-0.080, 0.00950.1050.81, 1.32(0.87 - 1.14)(0.94 - 1.18)(-0.17 - 0.052)(0.88 - 1.30)Threonine0.98 (0.030)1.00 (0.030)-0.025 (0.018)-0.066, 0.0160.1920.82, 1.20(0.86 - 1.11)(0.88 - 1.12)(-0.12 - 0.065)(0.84 - 1.22)Tryptophan0.23 (0.010)0.24 (0.010)-0.013 (0.0093)-0.032, 0.00590.1720.13, 0.35(0.17 - 0.26)(0.19 - 0.31)(-0.063 - 0.036)(0.17 - 0.32)Monsanto Company 11-CA-233U 97 of 500Table VI-2.Statistical Summary of Combined-Site Seed Nutrient Content for MON 88302 vs. the Conventional Control(continued)Difference (MON 88302 minus Control)Analytical Component (Units)1MON 883022Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95% CI3Lower, Upper lSignificance(p-Value)CommercialTolerance Interval5(Range)Amino Acid (% dw)Tyrosine0.67 (0.019)0.69 (0.019)-0.017 (0.013)-0.048, 0.0150.2490.57, 0.81(0.59 - 0.75)(0.61 - 0.77)(-0.11 - 0.028)(0.60 - 0.84)Valine1.20 (0.035)1.22 (0.035)-0.025 (0.025)-0.084, 0.0340.3520.92, 1.55(1.04 - 1.37)(1.05 - 1.41)(-0.16 - 0.054)(1.01 - 1.46)Fatty Acid (% Total FA)16:0 Palmitic4.23 (0.078)4.10 (0.077)0.13 (0.067)-0.027, 0.280.0942.84, 5.26(3.95 - 4.57)(3.94 - 4.41)(-0.22 - 0.48)(3.55 - 4.69)16:1 Palmitoleic0.22 (0.0081)0.24 (0.0081)-0.018 (0.0053)-0.030, -0.00590.0080.17, 0.30(0.20 - 0.26)(0.22 - 0.26)(-0.039 - 0.0074)(0.19 - 0.27)18:0 Stearic1.68 (0.044)1.98 (0.044)-0.30 (0.031)-0.37, -0.23<0.0010.90, 3.05(1.54 - 1.87)(1.78 - 2.19)(-0.48 - -0.059)(1.50 - 2.64)18:1 Oleic62.82 (0.62)65.79 (0.62)-2.97 (0.31)-3.69, -2.26<0.00156.13, 70.69(60.51 - 65.20)(63.72 - 68.44)(-4.30 - -1.52)(57.86 - 68.53)Monsanto Company 11-CA-233U 98 of 500Table VI-2.Statistical Summary of Combined-Site Seed Nutrient Content for MON 88302 vs. the Conventional Control(continued)Difference (MON 88302 minus Control)Analytical Component (Units)1MON 883022Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95% CI3Lower, UpperSignificance(p-Value)CommercialTolerance Interval5(Range)Fatty Acid (% Total FA)18:2 Linoleic19.26 (0.51)17.67 (0.51)1.59 (0.17)1.20, 1.97<0.00112.60, 24.49(17.78 - 20.66)(15.72 - 19.29)(0.40 - 2.42)(14.12 - 22.57)18:3 Linolenic9.58 (0.27)7.98 (0.27)1.60 (0.21)1.12, 2.07<0.0016.96, 11.73(8.71 - 11.23)(7.19 - 8.99)(0.76 - 2.64)(7.99 - 10.94)20:0 Arachidic0.54 (0.011)0.60 (0.011)-0.064 (0.0074)-0.081, -0.047<0.0010.45, 0.80(0.50 - 0.57)(0.54 - 0.65)(-0.091 - -0.0032)(0.53 - 0.71)20:1 Eicosenoic1.13 (0.024)1.09 (0.024)0.036 (0.017)-0.0034, 0.0760.0680.83, 1.68(1.06 - 1.26)(1.00 - 1.18)(-0.042 - 0.14)(1.04 - 1.56)22:0 Behenic0.27 (0.0072)0.28 (0.0072)-0.017 (0.0056)-0.030, -0.00410.0160.19, 0.43(0.24 - 0.29)(0.24 - 0.31)(-0.047 - 0.016)(0.27 - 0.38)24:0 Lignoceric0.16 (0.016)0.16 (0.015)0.0038 (0.017)-0.030, 0.0380.8230.033, 0.25(0.049 - 0.23)(0.045 - 0.22)(-0.14 - 0.11)(0.044 - 0.21)Monsanto Company 11-CA-233U 99 of 500Table VI-2.Statistical Summary of Combined-Site Seed Nutrient Content for MON 88302 vs. the Conventional Control(continued)Difference (MON 88302 minus Control)Analytical Component (Units)1MON 883022Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95% CI3Lower, UpperSignificance(p-Value)CommercialTolerance Interval5(Range)Fatty Acid (% Total FA)24:1 Nervonic0.12 (0.015)0.11 (0.015)0.013 (0.014)-0.020, 0.0470.3770.041, 0.18(0.046 - 0.20)(0.045 - 0.17)(-0.072 - 0.081)(0.044 - 0.20)MineralCalcium (g/100g dw)0.41 (0.030)0.40 (0.030)0.015 (0.012)-0.0089, 0.0390.2100.16, 0.61(0.30 - 0.51)(0.28 - 0.49)(-0.068 - 0.081)(0.25 - 0.53)Copper (mg/kg dw)3.78 (0.17)3.65 (0.17)0.14 (0.14)-0.19, 0.460.3612.00, 4.43(3.27 - 4.57)(2.96 - 4.18)(-0.83 - 0.57)(2.52 - 4.93)Iron (mg/kg dw)48.73 (4.28)54.01 (4.24)-5.28 (2.89)-11.85, 1.300.10223.39, 86.23(40.55 - 69.61)(41.65 - 77.74)(-20.41 - 14.87)(39.16 - 77.92)Magnesium (g/100g dw)0.37 (0.014)0.36 (0.014)0.0048 (0.0070)-0.011, 0.0210.5080.32, 0.43(0.31 - 0.42)(0.31 - 0.42)(-0.032 - 0.043)(0.30 - 0.45)Manganese (mg/kg dw)41.44 (2.02)40.34 (1.99)1.10 (1.83)-2.62, 4.820.55114.85, 61.05(35.28 - 51.55)(33.12 - 50.97)(-8.36 - 12.63)(25.00 - 54.11)Monsanto Company 11-CA-233U 100 of 500Table VI-2.Statistical Summary of Combined-Site Seed Nutrient Content for MON 88302 vs. the Conventional Control(continued)Difference (MON 88302 minus Control)Analytical Component (Units)1MON 883022Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95% CI3Lower, UpperSignificance(p-Value)CommercialTolerance Interval5(Range)MineralPhosphorus (g/100g dw)0.72 (0.042)0.72 (0.041)-0.0090 (0.022)-0.055, 0.0370.6920.38, 1.06(0.56 - 0.87)(0.56 - 0.93)(-0.095 - 0.16)(0.44 - 0.87)Potassium (g/100g dw)0.64 (0.053)0.64 (0.052)0.0016 (0.025)-0.056, 0.0600.9510.39, 0.96(0.48 - 0.90)(0.53 - 0.81)(-0.097 - 0.14)(0.50 - 0.92)Zinc (mg/kg dw)35.58 (1.78)33.01 (1.76)2.57 (1.83)-1.66, 6.800.19820.19, 48.23(29.81 - 45.56)(28.46 - 40.66)(-4.50 - 11.44)(22.18 - 47.61)Vitamin (mg/100g dw)Vitamin E (α-tocopherol)11.06 (2.08)8.85 (2.08)2.21 (1.66)-1.61, 6.030.2183.88, 17.28(1.30 - 16.55)(3.33 - 11.77)(-6.92 - 8.09)(2.62 - 14.84)1dw = dry weight; fw = fresh weight; FA = fatty acid.2MON 88302 treated with glyphosate.3Mean (S.E.) = least-square mean (standard error); CI –confidence interval.4Control refers to the genetically similar, conventional control Ebony.5With 95% confidence, interval contains 99% of the values expressed in the populationof commercial conventional reference varieties. Negative limits wereset to zero.Monsanto Company 11-CA-233U 101 of 500Table VI-3. Statistical Summary of Combined-site Canola Seed Anti-nutrientsfor MON 88302 vs. Conventional ControlDifference (MON 88302 minus Control)Analytical Component (Units)1MON 883022Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95% CI3Lower, UpperSignificance(p-Value)CommercialTolerance Interval5(Range)Anti-nutrientAlkyl Glucosinolate (μmole/g dw)3.68 (0.43)5.08 (0.42)-1.40 (0.59)-2.69, -0.110.0350, 29.02(1.19 - 5.87)(2.45 - 8.28)(-6.11 - 1.43)(2.32 - 28.33)Indolyl Glucosinolate (μmole/g dw)3.50 (0.51)3.89 (0.50)-0.39 (0.45)-1.42, 0.640.4081.37, 6.62(0.49 - 5.76)(1.83 - 5.89)(-3.05 - 2.83)(1.84 - 7.18)Phytic Acid (% dw)1.95 (0.18)2.11 (0.18)-0.16 (0.083)-0.33, 0.0100.0640.70, 3.52(1.20 - 2.58)(1.46 - 2.77)(-0.67 - 0.68)(1.10 - 2.71)Sinapic Acid (% dw)0.86 (0.12)0.88 (0.12)-0.023 (0.11)-0.27, 0.220.8370.57, 1.13(0.16 - 1.08)(0.65 - 0.97)(-0.76 - 0.21)(0.48 - 0.99)Total Glucosinolate (μmole/g dw)7.35 (0.87)9.08 (0.86)-1.73 (1.01)-4.06, 0.610.1270, 32.20(1.73 - 11.42)(4.38 - 12.72)(-9.21 - 3.58)(5.52 - 31.98)1dw = dry weight.2MON 88302 treated with glyphosate.3Mean (S.E.) = least-square mean (standard error); CI = confidence interval.4Control refers to the genetically similar, conventional control Ebony.5With 95% confidence, interval contains 99% of the values expressed in the populationof commercial conventional reference varieties. Negative limits wereset to zero.Monsanto Company 11-CA-233U 102 of 500 Table VI-4. Literature Ranges for Components in Canola Seed

Component 1 Literature Range 2

Proximates (% dw)

Ash 4.067 – 5.917 a
Carbohydrates N
Moisture (% fw) 3.177 – 8.045 a ; 7.4 – 10.0 b

Protein 21.30 – 28.125 a ; 18.7 – 26.0 b ; 17.4 – 23.0 c ; 21.1 – 26.7 d

Total Fat 35.59 – 44.93 a ; 24.0 – 43.6 b ; 42.0 – 49.5 d

Fiber (% dw)
Acid Detergent Fiber (ADF) 11.934 – 26.799 a ;11.6 f ; 12.4 g ; 22.2 h

Neutral Detergent Fiber (NDF) 18.653 – 34.720 a ; 17.8 f ; 16.49 g ; 31.3 h

Total Dietary Fiber N

Amino Acids (% dw)

Alanine 0.93 – 0.96 b ; 1.15 – 1.38 e
Arginine 1.13 – 1.21 b ; 2.23 – 2.46 e
Aspartic acid 1.54 – 1.59 e
Cystine/Cysteine 0.52 – 0.54 b

Glutamic acid 4.60 – 4.71 e
Glycine 1.04 – 1.06 b ; 2.20 – 2.22 e

Histidine 0.51 – 0.66 b ; 0.80 – 0.82 e

Isoleucine 0.80 – 0.86 b ; 0.96 – 1.03 e

Leucine 1.35 – 1.47 b ; 1.83 – 1.99 e
Lysine 1.03 – 1.19 b ; 1.67 – 1.85 e

Methionine 0.42 – 0.44 b

Phenylalanine 0.75 – 0.82 b ; 0.90 – 1.03 e

Proline 1.19 – 1.33 b ; 3.36 – 3.74 e

Serine 0.90 – 0.94 b ; 1.44 – 1.55 e

Threonine 0.87 – 0.94 b ; 1.28 – 1.30 e
Tryptophan 0.23 – 0.27 b ; Tyrosine 0.51 – 0.59 b ; 0.81 – 0.92 e

Valine 1.02 – 1.13 b ; 1.45 – 1.55 e

Vitamins (mg/kg dw)
Vitamin E ( α -tocopherol) 71.1 – 108.4 iMonsanto Company 11-CA-233U 103 of 500 Table VI-4. Literature Ranges for Components in Canola Seed (continued)

Component 1 Literature Range 2

Minerals
Calcium (% dw) 0.29 – 0.48 b ; 0.348 – 0.729 a
Copper (mg/kg dw) 7 b ; 1.388 – 5.492 a
Iron (mg/kg dw) ND b ; 0.0 – 965.6 a
Magnesium (% dw) 0.29 – 0.31 b ; 0.272 – 0.402 a
Manganese (mg/kg dw) ND b ; 33.813 – 64.757 a
Phosphorus (% dw) 0.48 – 0.85 b ; 0.581 – 0.895 a
Potassium (% dw) 0.83 – 0.91 b ; 0.681 – 1.016 a
Sodium (% dw) 0.05 b ; 0.003 – 0.030 a
Zinc (mg/kg dw) 62 b ; 0 – 126.953 a

Fatty Acids (% total)

16:0 Palmitic 3.3 – 6.0 b

16:1 Palmitoleic 0.1 – 0.6 b

18:0 Stearic 1.1 – 2.5 b

18:1 Oleic 52.0 – 66.9 b

18:2 Linoleic 16.1 – 24.8 b

18:3 Linolenic 6.4 – 14.1 b

20:0 Arachidic 0.2 – 0.8 b

20:1 Eicosenoic 0.1 – 3.4 b

20:2 Eicosadienoic 0.0 – 0.1 b

20:3 Eicosatrienoic N

20:4 Arachidonic N

22:0 Behenic 0.0 – 0.5 b

22:1 Erucic 0.0 – 2.0 b

24:0 Lignoceric 0.0 – 0.2 b

24:1 Nervonic 0.0 – 0.04 b

Anti-nutrients
Total Glucosinolates (μmol/g) 6 – 29 b ; 7.8 – 26.8 c ; 18 – 57 j

Phytic Acid (% dw) 2.0 – 5.0 b

Sinapine (% dw) 0.6 – 1.8 b

1 fw = fresh weight; dw = dry weight; dm = dry matter; ND defined as below the level of detection; N defined as not reported.

2 Literature Range = Values published for low erucic acid oilseed rape (canola).

Citations = a (Dairy One Forage Lab, 2010); b (OECD, 2001); c (Pritchard et al., 2000); d (Barthet and Daun, 2005); e (Wang et al., 1999); f (NRC, 2001); g (Mustafa et al., 2000); h (Leupp et al., 2006); i (Marwede et al., 2004);. j (Mailer and Pratley, 1990). Conversions: mg/100g dw × 10 = mg/kg dw; g/100g dw× 10 = mg/g dw.Monsanto Company 11-CA-233U 104 of 500 VI.B. Compositional Assessment of MON 88302 Conclusion

Analyses of nutrient, toxicant, and an ti-nutrient levels in MON 88302 and the

conventional control were conducted to asse ss compositional equivalence. The tissue analyzed was seed harvested from plants grown at five field sites in the U.S. and Canada during the 2009 field season. The compositiona l analysis, conducted in accordance with OECD guidelines, included measurement of nutrient, toxicant, and anti-nutrient components in a genetically similar conven tional control variety, Ebony and also in

commercial conventional referen ce varieties that have a hist ory of safe consumption to establish the natural range of variability. MON 88302, the conventional control, and

commercial reference varieties were treated w ith conventional weed control programs. In addition, MON 88302 plots were treated with gl yphosate herbicide at a target rate of 1.6 lb a.e./acre (1800 g a.e./ha).

The following components showed no significant differences in mean values between

MON 88302 and the conventional control: proximates, ADF, NDF, amino acids, 16:0 palmitic acid, 20:1 eicosenoic acid, 24:0 lignoceric acid, and 24:1 nervonic acid, minerals, and vitamin E.

The significant differences ( α = 0.05) in nutrient and anti -nutrient (including toxicant) components were evaluated usi ng considerations relevant to the safety and nutritional quality of MON 88302 when compared to the conventional control:

1) The relative magnitudes of differences for nutrients that were statistically significant in the combined-site analys is were small (4.52% to 20.01%), when

considered relative to the natural vari ability determined by the 99% tolerance interval established by the concurrently gr own commercial reference varieties that have a history of safe consumption. Th e relative magnitude of difference for the anti-nutrient alkyl glucosinol ate that was statistically si gnificant in the combined site analysis was small (27.59%) when considered relativ e to the natural variability determined by the 99% tolerance interval established by the concurrently grown commercial reference va rieties that have a history of safe consumption.

2) Mean values for these nutrient and anti-nutrient components from the combinedsite analysis of MON 88302 fell within the 99% tolerance interval established

from the commercial reference varietie s grown concurrently. Therefore, the

differences were within th e range of natural variabil ity of those components in

commercial canola varieties that have a hi story of safe consumption (Tables VI-2 and VI-3).

3) Assessment of the reproduc ibility of the combined-site differences at the five individual sites showed similar significant differences ( α = 0.05) at multiple sites. In all instances the individual site mean values for these components in

MON 88302 were within the 99% tolera nce interval esta blished from the concurrently grown commercial reference varieties. Therefore, these componentsMonsanto Company 11-CA-233U 105 of 500 were within the range of natural variab ility in commercial canola that have a history of safe consumption.

4) With the exception of TDF and alkyl glucosinolates, al l of the components identified as significantly different from the conventional control were within the natural variability of these components in commercial canola as published in the

scientific literature. There are no rele vant published values for TDF and alkyl glucosinolates.

This analysis provides a comprehensive comparative assessment of the levels of key nutrients, toxicants, and anti- nutrients in seed of MO N 88302 and the conventional control, discussed in the context of natural variability of commercial canola. Results of the comparison indicate that the seed of MON 88302 is compositionally equivalent to that of the conventional canola control. The genetic modification in MON 88302 does not meaningfully impact seed composition an d therefore the food and feed safety and nutritional quality of this product is comparab le to conventional canola that has a history of safe consumption.

Canola seed is processed to oil and meal. The processing of MON 88302 is not expected to be any different from that of conventional canola. As described in this section, detailed compositional analyses of key co mponents of MON 88302 have been performed

and have demonstrated that MON 88302 is co mpositionally equivalent to conventional canola. Additionally, the mode of action of CP4 EPSPS protein, as described in Section V.A., is well understood, and there is no reason to expect interactions with important nutrients or endogenous toxicants or anti-nutrients that may be present in canola. Therefore, when MON 88302 and its progeny are used on a commercial scale as a source of food or feed, these products are not expected to be different from the equivalent foods or feeds originat ing from conventional canola.Monsanto Company 11-CA-233U 106 of 500 VII. PHENOTYPIC, AGRO NOMIC, AND ENVIRONMENTAL

INTERACTIONS ASSESSMENT

This section provides an assessment of th e phenotypic, agronomic, and environmental interaction characteristics of MON 88302 comp ared to the conventi onal control Ebony. The data support a conclusion th at MON 88302 is similar to conventional canola with the exception of the glyphosate-tolera nce trait and, therefore, is no more likely to pose a plant pest risk or have a signifi cant environmental impact. Thes e conclusions are based on the results of multiple evaluations reported here.

Phenotypic, agronomic, and environmental interaction characte ristics of MON 88302 were evaluated in a comparative manner to assess plant pest potential. These assessments included evaluation of seed germination char acteristics, plant growth and development characteristics, observations for plant responses to abiotic stress, plant-disease and plantarthropod interactions, and pollen characteri stics. Results from the phenotypic, agronomic, and environmental interaction a ssessment demonstrate that MON 88302 does not possess a) increased weediness characteristic s; b) increased susceptibility or tolerance to specific abiotic stress, dise ases, or arthropods; or c) charact eristics that would confer a plant pest risk or a significant environmen tal impact compared to the conventional

control.

VII.A. Characteristic s Measured for Assessment

In the phenotypic, agronomic, and envi ronmental interactions assessment of MON 88302, data were collected to evaluate specific aspects of altered plant pest potential. A detailed description of the regulated article phenot ype is requested as part of the petition for determination of nonregul ated status in 7 CFR § 340. 6 including differences from the unmodified recipient or ganism that would “s ubstantiate that the regulated article is unlikely to pose a greater plant pest risk than the unmodified organism from which it was derived...” As part of the characterization of MON 88302, data were collected to provide a detail ed phenotypic, agronomic, a nd environmental interaction description of MON 88302. A su bset of these data were in cluded in an evaluation of specific characteristics related to altered plant pest potential.

As part of the phenotype description, the plant characteri zation of MON 88302 encompassed five general categories: 1) germination, dormancy, and emergence; 2) vegetative growth; 3) reproductive growth (i ncluding pollen characteristics); 4) seed retention on the plant an d lodging; and 5) plant response to abiotic stress a nd interactions with diseases and arthropods. An overview of the characteristics assessed is presented in

Table VII-1.

The phenotypic, agronomic, and environmental interactions data were evaluated from a basis of familiarity (OECD, 1993) and were comprised of a combination of field and laboratory studies conducted by scientists who are familiar with the production and

evaluation of canola. In each of these assessments, MON 88302 was compared to a conventional control that had a genetic background similar to MON 88302 but did not contain the glyphosate-tolerance trait. In addition, multiple commercial canola varieties,Monsanto Company 11-CA-233U 107 of 500 (see Appendix F, G, and H and Tables F-1, G- 1, and H-1) were included to provide a range of comparative values that are representative of commercial canola varieties for each measured phenotypic, agronomic, and environmental interaction characteristic.

Reference varieties used in these studies were selected to represen t a range of genetic backgrounds and phenotypic characteristics and have been grown in the canola production regions of Canada and the U.S. Th e commercial referenc e varieties reflect a range of values for the agronomic, phenotypic and environmental interaction characteristics assessed within the crop and th erefore can provide context for interpreting experimental results.Monsanto Company 11-CA-233U 108 of 500 Table VII-1. Phenotypic, Agronomic and Environmental Interaction Characteristics

Evaluated in U.S. and Canadian Fi eld Trials or Laboratory Studies

Data
category Characteristics

measured (associated section where discussed) Evaluation ti ming Evaluation description

Seed germination, dormancy,

and

emergence Normal germinated (VII.C.1) Day 7 (15/25 °C) Percentage of seed producing seedlings exhibiting normal developmental

characteristics 2

Abnormal germinated (VII.C.1) Day 7 (15/25 °C) Percentage of seed producing seedlings

that could not be cl assified as normal germinated 2

Germinated (VII.C.1) Day 7 and 14 (5, 15, 25, 30, and 5/25 °C) Percentage of seed that had germinated normally or abnormally

Dead (VII.C.1) Day 7 and 14 (15/25 °C); Day 7, 14 and 21 (5, 15, 25, 30, and 5/25 °C) Percentage of seed that had visibly

deteriorated and become soft to the touch or did not germinate and was determined to be non-viable by tetrazolium test 2

Viable non-dormant (VII.C.1) Day 21 (5, 15, 25, 30, and 5/25 °C) Percentage of seed that did not germinate at sub-optimal temperatures but whensubsequently moved to the AOSArecommended temperature (15/25°C) they did germinate 2
Dormant (VII.C.1) Day 14 (15/25 °C); Day 21 (5, 15, 25, 30, and 5/25 °C) Percentage of seed that did not germinate when incubated at any of the tested temperatures (viability determined by a

tetrazolium test 2 ) Early stand count (VII.C.2.1) Seedling (G.S. 12 – 14) 1 Number of emerged plants estimated bycounting plants from three nonsystematically chosen linear meter rows per plot

Final stand count (VII.C.2.1) Post-harvest Number of plants at harvest estimated by counting the stems after harvest from three non-systematically chosen linear meter rows per plot

Vegetative growth Seedling vigor (VII.C.2.1) Seedling (G.S. 12 – 14) 1 Rated on a 1-9 scale, where 1 = excellent and 9 = poor vigor

Plant height (VII.C.2.1) Maturity (G.S. 71 – 89) 1 Distance (inches) from the soil surface to the top of the main raceme of 15 representative plants per plot

Reproductive growth Days to 50% flowering (VII.C.2.1) Flowering (G.S. 60 – 69) 1 Number of days after planting when approximately 50% of the plants in each plot had one or more flowers Seed maturity (VII.C.2.1) Maturity (G.S. 80 – 89) 1 Number of days after planting when 30%

or more of the seed in the lower 1/3 of the main raceme had changed from a green

color to black/brown/tan color Pollen viability (VII.C.3) Flowering Percentage of vi able pollen based on pollen grain staining characteristics Pollen morphology (VII.C.3) Flowering Diameter (μm) of viable pollen grains

Seed moisture (VII.C.2.1) Harvest Percent moisture content of harvested seed

Seed quality (VII.C.2.1) Harvest Percentage of distinctly green seeds from a 100 seed subsample of the harvested seed

Yield (VII.C.2.1) Harvest Bushels of harvested seed per acre, adjusted to 8% moistureMonsanto Company 11-CA-233U 109 of 500 Table VII-1. Phenotypic, Agronomic and En vironmental Interaction Characteristics

Evaluated in U.S. and Canadian Field Tr ials or Laboratory Studies (continued)

Data
category Characteristics

measured (associated section where discussed) Evaluation timing Evaluation description

Seed

retention and lodging Lodging (VII.C.2.1) Maturity (G.S. 80 – 89) 1 Rated on 1 - 9 scale, where 1 = completely upright and 9 = completely flat or lodged

Quantitative pod shattering (VII.C.2.1) Maturity (G.S. 83 – 89) 1 A collection tray placed in the plant canopy of each plot and seed losses from shattering counted once per week for three

weeks

Visual pod shattering (VII.C.2.1 Maturity (G.S. 83 – 89) 1 A rating scale of 1 - 9 scale, where 1 = 0 to 10% shatter, and each subsequent value on the scale increasing in 10% increments up to 9 = greater than 80% shatter.

Plantenvironment

interactions Plant response to

abiotic stress (VII.C.2.2) Four times during growing

season Qualitative assessment of each plot, with rating on a 0 - 9 scale, where 0 = no symptoms and 9 = severe symptoms Disease damage (VII.C.2.2) Four times during growing

season Qualitative assessment of each plot, with rating on a 0 - 9 scale, where 0 = no symptoms and 9 = severe symptoms Arthropod-related damage (VII.C.2.2) Four times during growing

season Qualitative assessment of each plot, with rating on a 0 - 9 scale, where 0 = no symptoms and 9 = severe symptoms Flea beetle damage (VII.C.2.2) Two times from seedlings (G.S.

12 - 16) 1 Damage was assessed quantitatively from 10 plants per plot using a 0 - 10 rating scale where 0 = no damage and 10 = 100% of leaf area damaged Seedpod weevil damage (VII.C.2.2) Ripening (G.S. 80 – 89) 1 Damage was assessed quantitatively from 10 plants (5 pods per plant) by counting the number of exit holes in each of 50 pods per plot Arthropod

abundance (VII.C.2.2) Four times during growing

season Identification and enumeration of pest and beneficial arthropods abundance in cone beat sheet samples

1 Canola plant growth stages (G.S.) were determined using descriptions and guidelines outlined in the Canola Council of Canada Growers Manual (Thomas, 2003a): 12 = two true leaves unfolded; 14 = four true leaves unfolded; 16 = six true leaves unfolded; 60 – 69 = flowering; 70 -79 = pods begin to develop to nearly all pods have reached final size; 80 = ripening begins (seed green, filling pod cavity); 81 = 10% of pods ripe, seeds black and hard; 83 = 30% of seeds have changed color; 89 = fully ripe, nearly all seeds have changed color.

2 Methods for testing seed were consistent with AOSA guidelines (AOSA, 2009a; 2009b).Monsanto Company 11-CA-233U 110 of 500 VII.B. Interpretation of Phenotypic and Environmental Interaction Data

Plant pest risk assessments for biotechnology-derived crops ar e comparative assessments. Familiarity provides a basis from which th e potential environmental impact of a biotechnology-derived plant can be evaluated. The concept of familiarity is based on the fact that the biotechnology-derived plant is developed from a well-characterized conventional plant variety. Familiarity considers the biology of the crop, the introduced trait, the receiving environment and the inter action of these factors, and provides a basis for comparative environmental risk assessm ent between a biotechnology-derived plant and its conventional counterpart.

Expert knowledge and experience with c onventionally bred canola was the basis for selecting appropriate endpoints and estimatin g the range of responses that would be considered typical for canola. As such, MON 88302 was compared to the conventional control in the assessment of phenotypic, ag ronomic, and environmental interaction

characteristics. An overview of the characte ristics assessed is presented in Table VII-1. A subset of the data relating to well-unde rstood weediness criteria (Baker, 1974) ( e.g., seed dormancy, pre-harvest seed loss characteristics, lodging) was used to assess whether there was an increase in weediness potenti al of MON 88302, an element of Animal and Plant Health Inspection Service’s (APHIS) pl ant pest determination. Evaluation of environmental interaction characteristics ( e.g., plant abiotic stress, plant-disease, and plant-arthropod interactions) was also considered in the plant pest risk assessment. Based

on all of the data colle cted, an assessment was made to determine if MON 88302 is likely to pose an increased plant pest risk compared to conventional canola. Prior to analysis,

the overall dataset was evaluated for evidence of biologically relevant changes, and for possible evidence of an unexpect ed plant response. No unexp ected observations or issues

were identified.

VII.B.1. Interpretation of Detected Differences Criteria

Comparative plant characteriz ation data between a biotec hnology-derived crop and the conventional control are interpre ted in the context of contribu tions to increased plant pest potential as assessed by APHIS. Under the fr amework of familiarity, characteristics for which no differences are detected support a co nclusion of no increased pest potential of the biotechnology-derived crop compared to the conventional crop. Characteristics for

which differences are detected are considered in the step-wise method described below.

All detected differences for a characteristic are considered in the context of whether or not the difference would increase the plan t pest potential of the biotechnology-derived crop. Ultimately, a weight of evidence appr oach considering all characteristics and

studies was used for the final risk assessment of differen ces and their significance. Figure VII-1 illustrates the step-wise assessment process employed in detail:Monsanto Company 11-CA-233U 111 of 500 Note: A “no” answer at any step indicates that the characteristic does not contribute to a biological or environmental change for the crop in terms of plant pest potential and subsequent steps are not considered. The subsequent step is considered if the answer is “y es” or uncertain. It should be noted that some steps are not necessarily applicable for all data. For example, some data are not statistically analyzed ( e.g., abiotic stress response, disease damage data) or are only statistically analyzed within individual sites ( e.g., arthropod abundance data); however, the method for interpreting these data follows the same general approach.

Figure VII-1. Schematic Diagram of Agronomic and Phenotypic Data Interpretation Methods

Steps 1 and 2 - Evaluate Detected St atistically Significant Differences

Data on each measured characteristic are sta tistically analyzed, where appropriate, within

each individual site and in a combined-sit e analysis, in which data are pooled among

sites. Differences detected in the indivi dual site analysis must be observed in the combined-site analysis to be considered further for plant pest potential. Any difference detected in the combined-site analysis is assessed further.

Step 3 - Evaluate Differences Relative to Range of Commercial Reference Varieties

If a difference for a characteristic is dete cted in the combined-site analysis across multiple environments or when only an individual site analysis is conducted and a Hazard identification and risk

assessment on difference Outside variation for crop? No

Yes

No

Yes Adverse in terms of plant pest potential?

Yes Outside variation of study references? No Not adverse; the direction or magnitude of the detected

difference in the measured

characteristic does not contribute to a biological or environmental change for the

crop in terms of plant pest potential Yes Statistical differences detected in combined-site analysis? No Differences detected in the combined-site and

individual site anal yses are evaluated*

Step 2

Step 3

Step 4 The measured characteristic

does not contribute to a biological or ecological change for the crop in terms of plant pest potential

Step 5

Step 6

*See text for interpretation of differences detected in the individual site analysis Step 1Monsanto Company 11-CA-233U 112 of 500 difference for a characteristic is detected, then the m ean value for the biotechnologyderived crop is assessed re lative to the commercial reference varieties.

Step 4 - Evaluate Differences in the Context of the Crop

If the mean value of the biotechnology-deri ved crop is outside the variation of the commercial reference varieties ( e.g., reference range), the mean value of the biotechnology-derived crop for th e characteristic is assesse d relative to known values common for the crop ( e.g ., published values).

Step 5 - Plant Pest Potential

If the mean value of the biotechnology-deri ved crop is outside the range of values common for the crop, the detected difference is then assessed for whether or not it is adverse in terms of pl ant pest potential.

Step 6 - Conduct Risk Assessment on Identified Hazard

If an adverse effect (hazard) is identifi ed, a risk assessment on the difference is

conducted. The risk assessment considers cont ributions to enhanced plant pest potential of the crop itself, the impact of differences detected in othe r measured characteristics, and potential for, and effects of trait transfer to feral populations of th e crop or a sexually compatible species.Monsanto Company 11-CA-233U 113 of 500 VII.C. Comparative Assessments of the Phenotypic, Agronomic, and

Environmental Interaction Characteristics of MON 88302

This section provides the results of compar ative assessments condu cted in re plicated laboratory and/or multi-site field experiments to provide a detailed phenotypic, agronomic, and environmental interact ion description of MON 88302. The

characteristics of MON 88302 evaluated in th ese assessments included: seed dormancy and germination characteristics (Section VII.C.1), plant phenotypic, agronomic, and

environmental interaction characteristics unde r field conditions (Section VII.C.2), and pollen characteristics (Section VII.C.3). Additional details for each assessment are provided in Appendices F, G, and H.

VII.C.1. Seed Dormancy and Germination Characteristics

USDA-APHIS considers the potential for weedin ess to constitute a plant pest risk (7 CFR § 340.6). Seed germination and dormancy mechanisms vary with species and their genetic basis tends to be complex. Seed dorma ncy is an important characteristic that is often associated with plants that are c onsidered weeds (Anderson, 1996; Lingenfelter and Hartwig, 2007). Canola seed dormancy is know n to occur under field conditions and can contribute to seed persistence in soil (G ulden et al., 2004) and Section IX.C). Standardized germination assays are rou tinely used to measure the germination

characteristics of canola seed. The Associat ion of Official Seed Analysts (AOSA), an internationally recognized seed testing orga nization, recommends a temperature regime of 15/25 °C for testing the ge rmination characteristics of canola seed (AOSA, 2009a).

Comparative assessments of seed dormanc y and germination characteristics were conducted on MON 88302 and the conventional control. In addition, four commercial reference varieties were included to provide a range of comparative values that are representative of commerci al canola varieties. The seed lots for MON 88302, conventional control, and the commercial refe rence varieties were produced under field conditions in Grand Forks County, ND in 2009, a geographic area which represents an environment with conditions suitable for canola production. In addition to the AOSA recommended temperature regime of 15/25 °C, seed was tested at five additional temperature regimes of constant 5, 15, 25 or 30 °C, and alternating 5/25 °C to assess seed germination properties. The details of th e experimental materials and methods are presented in Table VII- 1 and Appendix F.

No statistically signi ficant differences ( α = 0.05) were detected between MON 88302 and the control at any of the te mperature regimes for normal and abnormal germination and dead seed (Table VII-2). No statistically significant differences ( α = 0.05) were detectedbetween MON 88302 and the control at the 5 oC temperature regime for viable nondormant seed (defined in Table VII-1; Tabl e VII-2). Analysis of variance was not conducted on dormant seed in all temperatures and viable non-dormant seed (defined in Table VII-1) seed at the 15 oC, 25 oC, 30 oC and 5/25 oC temperature regimes due to low numbers of seed in these categories.Monsanto Company 11-CA-233U 114 of 500 Although statistical comparisons were not made due to a low number of dormant seed in the AOSA-recommended temperature regime (15/25 oC), MON 88302 numerically had

fewer dormant seeds than the conventional co ntrol (0.0% vs. 0.3%). A reduction in the

number of dormant seed would not increas e plant weediness since non-dormant seed would be more likely to germinate reducing th e potential for persistence in the soil seed bank (Gulden et al., 2003). Although, no statistical comparisons were made for viable non-dormant seed in the 15 oC, 25 oC, 30 oC and 5/25 oC temperature regimes, the magnitude of the difference for seed of MON 88302 and the conve ntional control was small and there were no observable trends in the mean difference in this category across the different temperature regimes.

The biological characterist ics evaluated were used to characterize MON 88302 in the context of plant pest risk assessment. Based on the assessed characteristics, the results of this study demonstrate that there were no changes in the dormancy or germination

characteristics that are indicative of increas ed plant weediness or pest potential of MON 88302 compared to the conventional control.Monsanto Company 11-CA-233U 115 of 500 Table VII-2. Germination Characteristic s of MON 88302 and Conv entional Control

Temperature Germination 1 Mean% (S.E.) 2

Regime (°C) Category MON 88302 Control Reference Range 3

5 Germinated 95.3 (1.0) 96.5 (0.6) 99.0-99.8
Dead 2.3 (0.3) 1.8 (0.9) 0.0-0.5
Viable non-dormant 2.5 (1.0) 1.8 (0.9) 0.3-0.5

Dormant † 0.0 (0.0) 0.0 (0.0) 0.0-0.0

15 Germinated 97.5 (1.4) 99.0 (0.4) 98.5-100.0
Dead 2.5 (1.4) 0.8 (0.3) 0.0-1.5

Viable non-dormant † 0.0 (0.0) 0.3 (0.3) 0.0-0.0

Dormant † 0.0 (0.0) 0.0 (0.0) 0.0-0.0

25 Germinated 98.5 (0.6) 98.8 (0.6) 98.5-99.8
Dead 1.3 (0.5) 1.0 (0.7) 0.3-1.5

Viable non-dormant † 0.3 (0.3) 0.3 (0.3) 0.0-0.0

Dormant † 0.0 (0.0) 0.0 (0.0) 0.0-0.0

30 Germinated 98.8 (0.6) 97.3 (0.9) 97.0-99.5
Dead 1.0 (0.7) 2.8 (0.9) 0.5-2.8

Viable non-dormant † 0.3 (0.3) 0.0 (0.0) 0.0-0.3

Dormant † 0.0 (0.0) 0.0 (0.0) 0.0-0.0

5/25 Germinated 99.3 (0.5) 99.0 (0.4) 96.8-99.5
Dead 0.8 (0.5) 1.0 (0.4) 0.5-3.0

Viable non-dormant † 0.0 (0.0) 0.0 (0.0) 0.0-0.3

Dormant † 0.0 (0.0) 0.0 (0.0) 0.0-0.0

15/25 Normal germinated 98.0 (1.1) 98.0 (0.7) 96.3-98.5 (AOSA) Abnormal germinated 1.3 (1.0) 1.3 (0.3) 1.5-3.8
Dead 0.8 (0.5) 0.5 (0.5) 0.0-1.3

Dormant † 0.0 (0.0) 0.3 (0.3) 0.0-0.0

Note: experimental design was a randomized complete block with four replicates (n = 4). No statistically significant differences were detected ( α = 0.05) between MON 88302 and the conventional

control.

1 Categories evaluated were modified from Association of Official Seed Analysts (AOSA, 2007; 2009a; 2009b). Further details and definitions are provided in Table F-2.

2 In some instances, the total percentage for both MON 88302 and the conventional control did not equal 100% due to numerical rounding of the means.

3 Minimum and maximum means determined from among the commercial reference varieties. †Analysis of variance (ANOVA) was not conducted due to low numbers of seed within these categories.Monsanto Company 11-CA-233U 116 of 500 VII.C.2. Field Phenotypic, Agronomi c, and Environmental Interactions

Characteristics

Phenotypic, agronomic, and environmental inte ractions characterist ics were evaluated under field conditions as part of the plant characterization assessment of MON 88302. These data were developed to provide US DA-APHIS with a detailed description of

MON 88302 relative to the conve ntional control and commercia l reference varieties. According to 7 CFR § 340.6, as part of the pe tition to seek deregula tion, a petitioner must submit “a detailed description of the phenot ype of the regulated article.” This information is being provided to assess whet her there are phenotypi c differences between

MON 88302 and the conventional control that may impact its pest potential. Certain growth, reproduction, and pre-harves t seed loss characteristics ( e.g., lodging, pod shattering) were used to asse ss whether there is an increas e in weediness of MON 88302, an element of APHIS’s plant pest risk dete rmination. Environmental interactions were

also assessed as an indirect indicator of phenotypic changes to MON 88302 compared to the same comparators described above and are also considered in the plant pest risk

assessment.

Data were collected from field trials located at eight field sites in th e U.S. and nine field sites in Canada during 2009 to evaluate phenotypic, agronomic, and environmental interaction characteristics (Table VII-3). Th ese 17 field sites provide d a diverse range of environmental and agronomic conditions repr esentative of comme rcial canola production areas in North America. The sites were pl anted in a randomized complete block design with four replicates per site. All plots of MON 88302, the conventional control, and the

commercial reference varieties at each site were uniformly managed in order to assess

whether the introduction of the glyphosate-tolerance trai t altered the phenotypic and agronomic characteristics or the environmen tal interactions of MON 88302 compared to the conventional control. Glyphosate herbicide was not applied to any of the plots in the trial. A description of the evaluated phenotypic and envir onmental interaction characteristics and the designated developmen tal stages when evaluations occurred are

listed in Table VII-1. The methods and deta iled results of the individual site data comparisons are presented and discussed in Appendix G, while the combined-site analyses are summarized below. The results of this assessment demonstrated that the introduction of the glyphosate-tolerance tra it did not alter MON 88302 compared to the conventional control in terms of weediness pot ential. The lack of differences in plant response to abiotic stress, disease damage , arthropod-related damage, and pest and beneficial arthropod abundance further support the conclusion that the introduction of the glyphosate-tolerance trait is not likely to resu lt in increased plant pest potential or an adverse environmental impact from MON 88302 compared to conventional canola.

VII.C.2.1. Field Phenotypic and Agronomic Characteristics

A total of 12 phenotypic and agronomic charac teristics were evaluated (Table VII-4 and Table G-4 of Appendix G). In the combined-site analysis in which the data were pooled among the sites, no statistically signif icant differences were detected ( α = 0.05) between

MON 88302 and the conventional control for early stand c ount, seedling vigor, seed maturity, lodging, plant height, visual ra ting for pod shattering, quantitative podMonsanto Company 11-CA-233U 117 of 500 shattering, seed quality, yield, and final sta nd count (Table VII-4). Two statistically significant differences were detected be tween MON 88302 and the conventional control in the combined-site analysis. MON 88302 reached first flowering later than the conventional control (61.1 vs. 56.2 days). However, the mean value of MON 88302 for days to first flowering was within the natu ral variability of the commercial reference varieties (45.9 – 67.5 days). Ther efore, the difference in days to first flower is unlikely to be biologically meaningful in terms of increas ed weediness potenti al. MON 88302 also had higher harvested seed moisture than th e conventional control (13.2% vs. 11.7%). However, the mean value of MON 88302 for ha rvested seed moisture was within the natural variability of the commercial referen ce varieties (7.5% – 14.8%). Therefore, the difference in seed moisture is unlikely to be biologically meaningful in terms of increased weediness potential.

The plant phenotypic and agronomic character istics and environmental interactions evaluated in this study were used to characte rize the plant and its in teractions with the environment and to assess the plant pest or weed potential of MON 88302 compared to

the conventional canola control. Based on the assessed characteristics, the results of this study demonstrate that there were no unexpect ed changes indicative of increased plant pest potential or adverse environmental im pact of MON 88302 compared to conventional

canola.Monsanto Company 11-CA-233U 118 of 500 Table VII-3. Field Phenotypic E valuation Sites for MON 88302 during 2009

Location (County or RM, State or Province) 1 Country Location

Code USDA-APHIS

Notification/CFIA

Authorization Number Leduc, AB Canada ABLE 09-MON1-378-CAN Brookings, SD U.S. IARL 09-048-105n Elton, MB Canada MBBR 09-MON1-378-CAN Whitewater, MB Canada MBMI 09-MON1-378-CAN Portage le Prairie, MB Canada MBNW 09-MON1-378-CAN Otter Tail, MN U.S. MNCA 09-048-105n
Stearns, MN U.S. MNPY 09-048-105n Wilkins, MN U.S. MNRO 09-048-105n Grand Forks, ND U.S. NDBI 09-069-101n McHenry, ND U.S. NDBO 09-044-107n Brookings, SD U.S. NDCL 09-048-105n McHenry, ND U.S. NDVA 09-044-107n Flett’s Springs, SK Canada SKME 09-MON1-378-CAN Corman Park, SK Canada SKRA 09-MON1-378-CAN Wallace, SK Canada SKRO 09-MON1-378-CAN Rosthern, SK Canada SKSA 09-MON1-378-CAN Viscount, SK Canada SKWA 09-MON1-378-CAN

1 RM = rural municipality, AB Alberta. SD South Dakota, MB Manitoba, MN Minnesota, ND North Dakota, SK Saskatchewan.Monsanto Company 11-CA-233U 119 of 500Table VII-4. Combined-Site Comparison of MON 83302 to Conventional Control During 2009 for Phenotypic and AgronomicCharacteristicsMON 88302 ControlReference Range1Phenotypic Characteristic (units) Mean(S.E.) Mean (S.E.) Minimum MaximumEarly stand count (# plants per linear meter)218.2 (0.8) 19.2 (0.7) 12.7 30.1Seedling vigor (1-9 scale)35.1(0.2) 4.5 (0.2) 1.9 7.0Days to first flowering (Days after planting)461.1 (1.5)* 56.2 (1.4) 45.9 67.5Seed maturity (Days after planting)5102.6 (1.4) 101.3 (1.4) 84.0 108.0Lodging (1-9 scale)61.7 (0.3) 1.8 (0.2) 1.0 4.6Plant height (inches)745.4 (0.9) 44.3 (0.8) 29.5 47.5Visual rating for pod shattering (0-9 visual scale)81.2 (0.1) 1.4 (0.1) 1.0 5.8Quantitative pod shattering (seeds per ft.2)964.7 (22.7) 132.5 (52.2) 13.5 590.1Seed moisture (%)1013.2 (0.5)* 11.7 (0.4) 7.5 14.8Seed quality (%)113.3 (0.5) 2.8 (0.5) 0.0 7.8Yield (bushels per acre)1243.4 (2.2) 44.5 (2.2) 11.7 72.3Final stand count (# plants per linear meter)1318.3 (0.7) 18.1 (0.6) 12.5 28.7Note: The experimental design was a randomized complete block with four replicates. S.E. = Standard Error. The number of plots (n) used in the statisticalanalysis was 68 except where noted.*Indicates a statistically significant difference between MON 88302 and the conventional canola control (α= 0.05).1Reference range = Minimum and maximum mean values among the 24 commercial canola reference varieties.2Early stand count data plot number (n)=68 for MON 88302 and n=67 for the conventional control.3Rated on a scale of 1-9, where 1 =excellent vigor, and 9 = poor vigor. Plot number (n)=48 for MON 88302 and the conventional control.4Days to first flowering was determined from the number of daysafter planting when 50% of the plants in a plot had one or moreflowers. Plot number (n)=58for MON 88302 and n=59 for the conventional control.5Seed maturity was determined as the number of days after planting when 30% or more of the seed in the lower 1/3 of the main raceme had changed from a greento black/brown/tan color. Plot number (n)=41 for MON 88302 and n=43 for the conventional control.6Rated on a 0 - 9 scale, where 0 = completely upright plants and 9 = completely flat. Plot number (n)=64 for MON 88302 and theconventional control.7Plant height data plot number (n)=60 for MON 88302 and the conventional control.Monsanto Company 11-CA-233U 120 of 5008Visual pod shattering was estimated with a rated on a 1 - 9 scale, where 1 = 0 to 10%shatter, and each subsequent value on thescale increasing in 10%increments up to 9 = greaterthan 80% shatter. Plot number (n)=56 forMON 88302 and the conventional control.9Collection trays placed within the crop canopy and seed losses from shattering counted once per week for three weeks. Plot number (n)=12 for MON 88302 andthe conventional control.10Seed moisture data plot number (n)=57 for MON 88302 and n=60 for the conventional control.11Seed quality was determined at harvest by counting the percentage of green seeds froma 100 seed subsample from each plot. Plot number (n)=64 forMON 88302 and the conventional control.12Yield data plot number (n)=61 for MON 88302 and n=64 for the conventional control.13Final stand counts plot number (n)=64 for MON 88302 and the conventional control.Monsanto Company 11-CA-233U 121 of 500 VII.C.2.2. Environmental Interaction Characteristics

To determine the potential for increased plant pest characteristics, USDA-APHIS

considers the environmental in teraction of the biotechnology-derived crop compared to its conventional counterpart. Evaluations of environmental interactions were conducted as part of the plant characterization for MON 88302. In the 2009 North American field trials conducted for evaluation of phenot ypic and agronomic characteristics of MON 88302, data were also collected on plan t response to abiotic stress (drought, wind, nutrient deficiency, etc.), disease dama ge, arthropod damage, and arthropod abundance (Tables VII-5 and VII-6; Appendix G; Tables G-5, G-6, G-7, G-8, G-9 and G-10, respectively). These data were used as part of the environmental consequences (Section IX) to assess plant pest poten tial and provide an indicati on of potential effects of MON 88302 on non-target organisms (NTOs) as well as threaten ed and endangered species compared to the conventional control. In addition, multiple commercial reference varieties were included in the analysis to establish a range of natural variability for each

assessed characteristic. The results of the field evaluations show ed that the glyphosatetolerance trait did not unexpect edly alter the assessed environmental interactions of MON 88302 compared to the conventional contro l. The lack of si gnificant biologicallymeaningful differences in plant response to abiotic stress, dis ease damage, arthropod damage, and pest and beneficial arthropod abundance support the c onclusion that the introduction of the glyphosat e-tolerance trait in MON 88302 is unlikely to result in increased plant pest potential or cause an adverse environmental impact compared to

conventional canola.

In the 2009 field trials, the qualitative observa tions of plant response to abiotic stress, disease damage, and arthropod-related damage were performed four times during the growing season at all 17 site s. At four of the 17 site s (MBMI, MNPY, NDBI and SKSA) quantitative assessments were conducted four times for arthropod a bundance, twice for flea beetle damage and once for seedpod w eevil damage during the growing season.

In a qualitative assessment of plant respons e to abiotic stress, no differences were

observed between MON 88302 and the conv entional control for 130 out of 131 comparisons involving 9 assessed abiotic stre ssors (Table VII-5; Appendix G; Table G5). One difference was observed in abiotic stress response in Observation 1 for frost damage at the MBBR site (where MON 88302 was severe and the conventional control was moderate). However, the observed fr ost damage to MON 88302 was within the range of the damage observed among the commercia l reference varieties (slight – severe). In addition, the difference was not observe d during any of the other 12 frost damage observations among the sites. Thus, the diffe rence in frost damage rating during the single observation was not indicative of a cons istent response associated with the trait and was considered not biologically meaningful in terms of plant pest potential or an adverse environmental impact from MON 88302 compared to conventional canola.

In a qualitative assessment of plant response to disease damage and arthropod damage, no

differences were observed between MON 88302 and the conventional control for any of the 141 comparisons involving 16 assessed diseas es or for any of the 165 comparisons forMonsanto Company 11-CA-233U 122 of 500 any of the 13 assessed arthropods among all observations at the sites (Table VII-5, Appendix G; Tables G-6 and G-7).

In the combined-site analysis for a quantitati ve assessment of flea beetle (Chrysomelidae) damage and seedpod weevil ( Ceutorhynchus obstrictus ) damage, no statistically significant differences were detected ( α = 0.05) between MON 88302 and the

conventional control from the four sites evaluated (Table VII-6).

In a quantitative assessment of pest and be neficial arthropod abundance, no statistically significant differences ( α = 0.05) were detected between MON 88302 and the conventional control for any of the 51 co mparisons, including 36 arthropod pest comparisons and 15 beneficial arthropod compar isons, among the observations at the four sites (Table VII-5; Appendix G; Tables G-9 andG-10).

The results of the field evaluations showed that the glyphosate-to lerance trait did not unexpectedly alter the assessed environmenta l interactions of MON 88302 compared to the conventional control. The lack of signif icant biological differenc es in plant responses to abiotic stress, di sease damage, arthropod -related damage, and pest and beneficial arthropod abundance support the conclusion th at the introduction of the glyphosatetolerance trait in MON 88302 is unlikely to resu lt in increased plant pest potential or an adverse environmental impact as comp ared to conventional canola.Monsanto Company 11-CA-233U 123 of 500 Table VII-5. Summary of Qualitative and Quantitative Environmental Interaction Assessments During 2009

Assessments 1 Number

of sites Number of

observations across

sites Number of observations

where no differences

were detected between

MON 88302 and the

conventional control
Qualitative Plant response to abiotic

stress 2 17 131 130

Disease damage 17 141 141
Arthropod damage 17 165 165

Quantitative
Pest arthropod abundance 4 36 36 Beneficial arthropod

abundance 4 15 15

1 For each qualitative assessment, MON 88 302 was considered different from the conventional control if the severity of injury to MON 88302 did not overlap with the severity of injury to the conventional control across all four replicates. Quantitative assessments were statistically analyzed ( α = 0.05).

2 A single difference was observed for frost damage between MON 88302 and the conventional control during Observation 1 at the MBBR site: MON 88302 = severe, Conventional Control = moderate, Reference range = slight-severe.Monsanto Company 11-CA-233U 124 of 500Table VII-6. Combined-Site Analysis: Quantitative Assessment of Flea Beetle and Seedpod Weevil Damage to MON 88302Compared to the ConventionalControl in 2009 Field Trials1Mean (S.E)Arthropod Pest Damage assessment MON 88302 ControlReferencerange2Flea beetles3(Chrysomelidae)Mean (S.E.) damage of 10 plantsper plot (0-10 rating scale) –Observation 11.18 (0.24) 1.13 (0.24) 0.33 – 2.20Mean (S.E.) damage of 10 plantsper plot (0-10 rating scale) –Observation 21.22 (0.19) 1.23 (0.20) 0.15 – 1.90Seedpod weevil4(Ceutorhynchusobstrictus)Mean (S.E.) number of holes inpods from 10 plants per plot0.03 (0.02) 0.05 (0.02) 0.00 – 0.02No statistically significant differences were detected between MON 88302 and the conventional control (α= 0.05).Note: The experimental design was a randomized complete block withfour replicates. S.E. = Standard Error. The number of plots (n) used in the statisticalanalysis was 16 except where noted.1Sites are as follows: MBMI = Whitewater,MB; MNPY = Stearns, MN; NDBI = Grand Forks, ND; SKSA = Rosthern, SK.2Reference range = minimum and maximum mean values among the commercial reference varieties.3Damage assessments for flea beetle were conducted 1-2 times during growing season. The first assessment was conducted during two to three weeks followingcrop emergence at all four sites. The second assessment was conducted approximately 1 week later at the MBMI, MNPY, and NDBI sites. Damage wasassessed quantitatively using a 0 - 10 rating scale where 0 = no damage and 10 = 100% of leaf area damaged. The first flea beetle assessment at SKSA could notbe conducted due to a severe rain storm (n = 12 plots).4Damage assessments for seedpod weevilwere conducted at the ripening stage.Monsanto Company 11-CA-233U 125 of 500 VII.C.3. Pollen Characteristics

To determine the potential for MON 88302 to im pact weediness of th e receiving species, the USDA-APHIS considers the potential fo r gene flow and introgression of the biotechnology derived trait into other canola varieties and wild relatives. An assessment of pollen morphology and viabilit y provides information to char acterize the plant and are also pertinent to assessments of gene flow and, therefore, were assessed for MON 88302. In addition, characterization of polle n produced by MON 88302 and the conventional control is relevant to the plant pest risk assessment because it adds to the detailed description of the phenotype of MON 88302 comp ared to the convent ional control.

The purpose of this evaluation was to as sess the morphology and viability of pollen collected from MON 88302 compared to that of the conventional control. Pollen was collected from non-glyphosate-treated MON 88302, the conventional control, and four commercial reference varieties. All plants were grown in pots in a growth chamber established at 21 °C day/18 °C night with a 16 h photoperiod. The plants were arranged in a randomized complete block design with fi ve replicates with 1 plant of each entry (MON 88302, conventional control and commercial reference varieties) per replicate. Once the plants started flower ing a total of three newly ope ned flowers were collected from each plant. Pollen was extracted, comb ined among flowers collected from the same plot, and stained with Alex ander’s stain (Alexander, 19 80). Pollen viability was evaluated for each sample, and pollen grain diameter was measured for ten representative viable pollen grains per replicate. Gene ral morphology of the pollen was observed for each of the five replicates of MON 88302, the conventional control, and the four

commercial reference varie ties (see Appendix H).

No statistically significant differences were detected ( α = 0.05) between MON 88302 and the conventional control for percent viable polle n or pollen grain diamet er (Table VII-7). Furthermore, no visual differences in gene ral pollen morphology were observed between

MON 88302 and the conventional control. These results demonstrate that the introduction of the glyphosate-tolerance tra it did not alter the overall morphology or pollen viability of MON 88302 compared to the conventional control. The pollen

characterization data contribute to the detailed phenotypic desc ription of MON 88302 compared to the conventional control. The result supports an overa ll conclusion that MON 88302 is comparable to conventional canol a and is no more likely to pose a plant pest risk than conventional canola.Monsanto Company 11-CA-233U 126 of 500 Table VII-7. Pollen Characteristics of MON 88302 Compared to the Conventional

Control

Pollen

Characteristic Mean (S.E.) 1 Reference Range 2

MON 88302 Control Minimum Maximum

Viability (%) 99.8 (0.2) 100.0 (0.0) 98.0 99.8

Diameter (μm) 24.8 (0.1) 25.1 (0.1) 24.6 25.8

Note: No statistically significant differences were detected between MON 88302 and the conventional control ( α = 0.05).

1 Means based on n = 5. S.E. = Standard Error

2 Reference ranges were determined from the minimum and maximum mean value from among the four

commercial reference varieties.

VII.D. Conclusions for Phenotypic, Agro nomic, and Environmental Interactions

Evaluation

An extensive and robust set of information and data were used to assess whether the introduction of the glyphosate-tolerance tra it altered the plant pest potential of MON 88302 compared to the conventional control. Phenotypic, agronomic, and

environmental interaction char acteristics of MON 88302 were evaluated and compared to

those of the conventional cont rol and considered within the variation among commercial

reference varieties. These assessments in cluded five general data categories: 1) germination, dormancy, and emergence; 2) vegetative growth; 3) reproductive growth (including pollen characteristics) ; 4) seed retention on the pl ant and lodging; and 5) plant response to abiotic stress and interactions w ith diseases and arthropods. Results from the phenotypic, agronomic, and environmental interaction assessment demonstrate that MON 88302 does not possess: a) increased w eediness characteristics; b) increased susceptibility or tolerance to specific abio tic stress, diseases, or arthropods; or c) characteristics that would confer a plant pest risk or a significant environmental impact compared to the conventional control.Monsanto Company 11-CA-233U 127 of 500 VIII. U.S. AGRO NOMIC PRACTICES

VIII.A. Introduction

As part of the plant pest assessment re quired by 7 CFR § 340.6(c)(4), impacts to agricultural and cultivation practices must be considered. This section provides a summary of current agronomic practices in the U.S. and North America for producing canola, and is included in this petition as a baseline to assess possible impacts to agricultural practices due to the cult ivation of MON 88302. Di scussions include canola production, canola seed production, plant growth and development, general management practices during the se ason, management of weeds, insect s and diseases, canola rotational crops, and volunteer canola management. In formation presented in Section VII.C.2

demonstrated that MON 88302 is no more su sceptible to diseas es or pests than conventional canola. Additionally data presen ted in Section VII.C show that, with the exception of tolerance to the herbicid e glyphosate, MON 88302 is phenotypically equivalent to conventional canola. Thus, th ere are no changes to the inputs needed for MON 88302, and no impacts to most of the agronomic practices employed for production of canola. Discussion will also be provided on the current use of herbicide-tolerant canola and the expected use of MON 88302. In the areas where there is potential for impact on agronomic practices from the deregulation of MON 88302, the scope and magnitude of those impacts will be discussed.

Almost all the canola production in the U.S. is reported from eight states with the majority (88%) being produced in North Dakota. Spring and winter canola varieties have been developed which permit production in the northern Great Plains, the southern Great Plains and Pacific Northwest regions of the U.S. The soil types, moisture and temperature requirements for producing canola ar e generally similar to those for wheat. Spring canola is generally produced in areas with dry weather and shorter growing seasons while winter varieties can be produced further south to take advantage of longer growing seasons. Proper seedbed preparati on, good genetics, proper planting dates and density and good integrated pest management practices are important to optimizing the yield potential and economi c returns of canola.

Annual and perennial weed infestations are a serious obstacle to maximum yield potential in canola. Weeds compete with canola for water, nutrients and light resulting in substantial yield losses when le ft uncontrolled. Weed species in canola vary from region to region. Tillage, crop rotations, cultural practices and herbicides are all employed for weed management in canola. Early season w eed control, particular ly for spring canola varieties, is most important to minimize weed competition and yield losses in canola.

Several selective herbicides are available for pre-emergence and post-emergence control of annual and perennial weeds in canola. Herbicide-tolerant canola varieties are utilized extensively which permit the in-crop post-em ergence use of glyphosate, glufosinate and

imazamox.

When canola volunteer plants are found in a field, they can easily be controlled using mechanical and chemical methods. Insect pe sts and diseases can also be consideredMonsanto Company 11-CA-233U 128 of 500 problematic in canola fields and can be ma naged using chemical means and crop rotation practices.

VIII.B. Overview of U.S. Canola Production

VIII.B.1. Canola Production

Brassica napus or oilseed rape is believed to ha ve originated in the Mediterranean (Brown et al., 2008) and was cultivated by ancient civilizations in Asia and the Mediterranean and its oil was used for lighting (Colton and Sykes, 1992). It was reportedly grown in Europe for lamp oil and lubrication in the 13th century, and in Asia for cooking oil for thousands of year s (Boyles et al., 2009). Demand for B. napus industrial grade oil increased during World War II when it became the oil of choice for the lubrication of steam engines. Diesel engines replaced steam engines at the end of World War II and demand plummeted. Resear chers began investigating other market opportunities for oilseed rape. The high erucic acid content of the i ndustrial oil was not desirable for human consumption. In addition, the meal derived from oilseed rape had relatively high concentrations of glucosinolat es which reduced the animal feed value of the meal. In the 1960s, through intensive breeding programs, Canadian scientists made two important genetic modifications to oils eed rape which lead to the first double-low (low-erucic acid and low glucosinolate) va riety (Brown et al., 2008). In 1978, to distinguish this new edible variety of B. napus oil from industrial B. napus oil, the Canola Council of Canada (formerly known as the Ra peseed Association of Canada) chose the word “canola” (Canadian oil, low acid) to b ecome the registered trademark for edible B. napus oil (Brown et al., 2008) with less than 2% erucic acid in the oil (Codex Alimentarius, 2005). In 1985, the U.S. F ood and Drug Administration (FDA) granted Generally Recognized As Safe (GRAS) status to canola oil (U.S. FDA, 1988) with these same characteristics which greatly increased the edible canola oil market in the U.S. In addition, canola varieties contain less than 30 μmol of aliphatic gluc osinolates per gram of oil-free seed meal, making canola meal more attractive as an animal feed (Brown et al., 2008).

There are three species of Brassica that produce canola-quality oil: Brassica napus L. (oilseed rape), Brassica rapa (field mustard; also known as Brassica campestris L.) and Brassica juncea L. (mustard greens) (Codex Alimenta rius, 2005). Both spring and winter

canola varieties are available in B. napus and B. rapa while B. juncea has spring canola varieties only. Spring canola is a cool season crop that is grown in the northern states while winter canola varieties that are planted in the fall and require vernalization (winter chilling) to flower, are planted more in th e Great Plains, Pacific Northwest, and Midwest regions of the U.S. Brassica napus is currently grown as an oilseed crop in Canada, China, Europe, India, Pakistan, Australia, and in the U.S. (Brown et al., 2008).

Canola oil is high quality oil that is used in a variety of foods including frying and baking oils, salad oils, margarines and shortenings a nd is the most valuable component of canola seed. It is the world’s third largest s ource of vegetable oi l with 15% of world vegetable oil consumption after soybean oi l at 28% and palm o il at 32% (ASA, 2010; USDA-ERS, 2010). Canola oil appeals to health conscious consumers because itMonsanto Company 11-CA-233U 129 of 500 contains a low level (7%) of saturated fatt y acids which have b een shown to increase blood cholesterol levels; a high level (approximately 60%) of the monounsaturated fatty acid, oleic acid, which has been shown to reduce serum cholesterol levels; a moderate level (approximately 20%) of linoleic acid, and an appr eciable amount (approximately 10%) of alpha-linolenic acid relative to ot her oils (CCC, 2010) that are essential to human health and must be supplied in the di et. Canola oil has we ll established heart

health benefits. The FDA has confirmed GR AS status for canola oil (U.S. FDA, 1988), and has issued a qualified health claim based on its ability to reduce the risk of coronary heart disease (U.S. FDA, 2006). Approximately 70% of Canada’s ca nola oil is exported to the U.S. (CRB, 2008). Canola seed is also processed into canola meal which is used as high protein animal feed and is the second largest protein meal source produced in the world (CCC, 2009). However, it is relatively small compared to soybean meal. Global production of canola meal was 30.8 million metr ic tons in 2008/2009 compared to 151.6 million metric tons for soybean meal (USDA-ERS, 2010).

The European Union, Canada, and China are th e largest producers of oilseed rape with 19.0, 12.64, and 12.10 million metric tons, respectiv ely. The U.S. produced only about 0.66 million metric tons (1.1%) of the worl d’s canola production (57.88 million metric tons) in the growing season 2008/2009 (USDA-FAS, 2010).

U.S. canola acreage in the past ten years has varied from approximately 827,000 to 1,524,000 acres with the lowest acreage reco rded in 2009 and th e highest in 2010 (Table VIII-1). Average canola yields for the U.S. have varied from 1,197 to 1,811 pounds per acre. Canola production ra nged from 1.34 to 2.45 billion pounds over the past ten years with 2010 being the largest production year on record (Table VIII-1). According to data from USDA-NASS (USD A-NASS, 2011a), canola was planted on approximately 1.5M acres in the U.S. in 2010 producing 2.45 bil lion pounds of canola (Table VIII-2). The average yield in 2010 of 1,713 pounds per acre was the second highest yield per acre ever reported. The value of canola reached $486.9 million in the U.S. in 2010. In comparison, maize, soyb ean and wheat values in 2010 were $66.65, 38.92, and $12.99 billion, respectively (USDA-NASS, 2011b).

Canola can be grown on a wide range of soil ty pes, but well-drained, clay-loam soils that do not crust are ideal for canola (NDSU, 2005). Canola cannot tolerate standing water or water-logged soils, and has moisture requiremen ts similar to small grains, but is less tolerant to drought conditions. Only 13,535 (1.1%) of canola acres were irrigated in 2007 (USDA-ERS, 2010). Due to these factors canola fits well into rotatio ns with small grain cereal crops (winter and spring wheat and spring barley).

U.S. canola is grown in three geographical regions – the Northern and Southern Great Plains and the Pacific Northwest (Table VI II-2). Approximately 730,000 acres or 88.3% of the U.S. canola production was grown in North Dakota in 2009 where a dryer, shorter growing season makes maize and soybean pro duction less attractive (Figure VIII-1). Nearly all the canola grown in North Dakota is spring-sown B. napus (NDSU, 2005). Oklahoma had the second largest acreage wi th 42,000 acres which is planted primarily

with winter canola varieties.Monsanto Company 11-CA-233U 130 of 500 In the remaining acres reported, Minnesota and Montana tend to plant spring canola varieties, while Oregon, Colorado, Kansas, and Washington usually plant winter canola varieties. In past years, limited acres of canola were grown in many of the Midwest states and Southeastern states. However, the profit potential of maize and soybean has

been more favorable than canola in these ar eas in recent years and canola acreage has declined accordingly. Oregon produced th e highest average yield of 2,550 pounds per acre in 2009 with North Dakota coming in second with 1,840 pounds per acre. The lowest average yield was recorded in Oklahoma with 1,300 pounds per acre.Monsanto Company 11-CA-233U 131 of 500 Figure VIII-1. Planted Canola Acres by County in the U.S. in 2009
Source: http://www.nass.usda.gov/Charts _and_Maps/Crops_County/cn-pl.asp.

Table VIII-1. Canola Production in the United States, 2001 – 2010 1

Year Acres

Planted Acres

Harvested Yield
(lbs/acre) Production
(thousand pounds) Value
(millions of

dollars)

2010 1,524,000 1,431,000 1,713 2,450,947 486.9

2009 827,000 814,000 1,811 1,474,130 238.9

2008 1,011,000 989,000 1,461 1,445,064 271.0

2007 1,176,000 1,156,000 1,238 1,430,734 260.3

2006 1,044,000 1,021,000 1,366 1,394,312 165.5

2005 1,159,000 1,114,000 1,419 1,580,985 152.0

2004 865,000 828,000 1,618 1,336,530 143.9

2003 1,082,000 1,068,000 1,416 1,512,250 159.9

2002 1,460,000 1,281,000 1,197 1,533,420 162.7

2001 1,494,000 1,455,000 1,374 1,998,515 175.4

1 Source: (USDA-NASS, 2011c)Monsanto Company 11-CA-233U 132 of 500 Table VIII-2. U.S. Canola Production by State in 2010

Region/State Acres

Planted 1 Acres

Harvested 1 Average

Yield 1

(pounds/acre) Total Production 1

(thousands pounds) Value 2

(millions of

dollars ) Idaho 19,500 18,400 1,800 33,120 6.2

Minnesota 46,000 45,000 1,530 68,850 (ND)

Montana 17,500 17,400 1,730 30,102 6.0

North Dakota 1,280,000 1,270,000 1,720 2,184,400 436.9

Oklahoma 60,000 56,000 1,600 89,600 15.5

Oregon 6,000 5,700 2,450 13,965 2.5

Other States 3 19,800 18,500 1,671 30,910 19.8

U.S. Totals 1,448,800 1,431,000 1,713 2,450,947 486.9 (ND) Not disclosed

1( (USDA-NASS, 2011a)

2 (USDA-NASS, 2011b)

3 Other states include Colorado, Kansas, and Washington.

Managing input costs is a major component to the economics of pr oducing a canola crop. The key decisions on input costs include choosi ng what seed or canola varieties to plant, amounts of fertilizer to appl y and what herbicide program to use. The average gross return for producing canola in North Dakota was $250.89 per acre for the years 2005- 2009 according to statistics compiled by Farm Ma nagement Specialists (Table VIII-3).

The total direct and overh ead expenses were $179.52 and $27.44 per acre, respectively. The net return for the five-year period averaged $43.94 per acre. With an average government payment of $11.33 per acre th e net return was $55.27 per acre.Monsanto Company 11-CA-233U 133 of 500 Table VIII-3. North Dakota Canola Production Costs and Returns from 2005 –

2009 1

Yield:1546 lbs./Acre Market Value: $15.27 per cwt

Product Income 237.62 Crop Insurance & Other Income 13.27

Total Income 250.89

Direct Expenses:

Seed 31.06

Fertilizer 43.56 Crop Chemicals 22.69 Crop Insurance 13.14

Fuel and Oil 13.19 Repairs 13.60

Custom Hire 3.74

Land Rent 34.50 Operating Interest 3.67

Miscellaneous 0.36 Total Direct Expenses 179.52 Return Over Direct Expenses 71.38

Overhead Expenses:

Hired Labor 3.61 Machinery Leases 2.35

Farm Insurance 2.26

Utilities 1.63

Dues & Professional Fees 0.53

Interest 3.12 Machinery & Building Depreciation 11.86

Miscellaneous 2.09 Total Overhead Expenses 27.44

Total & Direct Overhead Expenses 206.95

Net Return 43.94 Net Return with Government Payment ($11.33) 55.27

1 (FINBIN, 2010). Supporting Information: Cash rent farms only, total number of fields: 540, total number of farms: 315, and average size of fields: 166 acresMonsanto Company 11-CA-233U 134 of 500 VIII.B.2. Canola Seed Production

Standardized seed production practices are responsible for maintaining high-quality seed stocks, an essential basis for U.S. agricultu re. By the early 20th century, agronomists learned how to develop specific plant varieties with desirable traits. In the U.S., state agricultural experiment stations developed many seed varietie s that were distributed to growers for use. Seed was saved by grower s and later sold to ne ighbors; however, the

desirable traits of the varieties often we re lost through random genetic changes and contamination with other crop a nd weed seed (Sundstrom et al., 2002). The value of seed quality (including genetic purity, vigor, weed seed presence, seed-borne diseases, and inert materials, such as dirt) was quickly id entified as a major fact or in crop yields. States in the U.S. developed seed laws a nd certification agencies to ensure that purchasers who received certified seed could be assured that the seed met established seed quality standards (Bradford, 2006). The federal government passed the U.S. Federal Seed Act of 1939 to recognize seed certification and offici al certifying agencies. Regulations first adopted in 1969 under the Fede ral Seed Act recognize land history, field isolation, and varietal purity standards for foundation, registered, an d certified seed. Under international agreements such as the Organization for Economic Co-Operation and Development (OECD) scheme, the U.S. an d other countries mutually recognize minimum seed quality standards (Bradford, 2006). The Association of Official Seed Certifying Agencies (AOSCA) represents state and private seed certification in the U.S.,

and includes international member countries in North and South Amer ica, Australia, and

New Zealand.

Canola seed is broadly separate d into three seed classes: 1) breeder; 2) foundation; and 3) certified (CCC, 2011). Breeder seed is seed directly controlled by the originating or sponsoring plant breeding organi zation or firm responsible for the maintenance of that variety. Foundation seed is first-generation seed increased from breeder seed and is handled to maintain specific varietal purity a nd identity. Certified se ed is the progeny of breeder or foundation seed, and is the class recommended for commercial canola production. Not all canola seed sold may be officially certified; however, commercial canola seed sold and planted for normal ca nola production is produced predominately to

meet or exceed certified seed standards. Th is section of the petiti on will provide a broad overview of the standards (AOSCA, 2009) and practices used in producing certified

canola seed.

Seed breeders and producers have put in place practical meas ures to assure the quality and genetic purity of seed varieties for commer cial planting. The need for such systems arose from the recognition that the quality of improve d seed varieties qui ckly deteriorated in the absence of monitoring for quality and genetic purity (CAST, 2007). Seed certification programs were initiated in the early 1900s in the U.S. to preserve the genetic identity and varietal purity of seed. Th ere are special land requi rements, seed stock eligibility requirements, field inspections and seed labeling standards for seed

certification. Seed certification services are available through va rious state agencies affiliated with the AOSCA. Large seed pr oducers implement their own seed quality assurance programs. However, large seed producers often will utilize the services ofMonsanto Company 11-CA-233U 135 of 500 state certifying agencies as a third party sour ce to perform certain field inspections and

audits.

The U.S. canola production fo r all purposes has varied fr om approximately 0.8 to 1.5 million acres in the past ten years (USDA- NASS, 2011c) Table VIII-1). Based on an average seeding rate of five pounds per ac re, approximately 7.5 million pounds of canola seed would be required to plant 1.5 million acres. Additional seed volume would be required to make allowances for seed losses due to weather, poor yields, and quality issues. Additional allowances would be requi red for distribution excess, seed returns, replants, and potential incr eases in canola acreage. Approximately 5,000 acres of commercial seed production would supply sufficien t seed to plant the entire U.S. canola acreage with the various production and di stribution allowances mentioned above (Bateman, Monsanto - Persona l Communication, 3/17/11).

Canola seed production involves a three stage pr ocess. The first stage or pre-foundation seed stage occurs in isolated tents for comple te pollination control. Individual inbred plants are grown and tested to ensure the pl ants meet the desired genetic characteristics and profile. The next stage or foundation seed stage occurs in highly isolated open fields. During this stage inbred seed lines are further tested to ensure the desired genetic characteristics and purity are maintained under field conditions. Th e final stage involves the production of hybrid seed or open-pollinated seed for commercial seed sales. Most of the hybrid and open-pollinated seed is produ ced in Southern Alberta, Canada and Northwestern U.S. These are preferred lo cations for seed production because of ideal heat units, accessibility to irrigation, and distance from commercial canola fields (Monsanto Company, 2009). To meet the de mand for hybrid seed and to minimize production risks, most seed companies have off-season seed producti on locations in the

Southwestern U.S. and Chile.

Canola is a relatively easy crop to manipulat e genetically and many new varieties with new quality and agronomic characteristics are available from universities and private seed companies (NDSU, 2005). Canola seed va rieties are developed from three different breeding techniques, namely open-pollinate d, synthetic hybrids, and hybrids (NDSU, 2005). Over 95% of the acreage in the U.S. is planted with hybr id canola varieties (Bonnetta, Monsanto Company – Personal Co mmunication, 8/22/2010). Hybrid canola varieties generally have highe r yield potentials, but also have higher seed production costs. Hybrid canola varieties are produ ced from a cross betw een two genetically different inbred parent lines provi ding hybrid vigor (heterosis).

Producing hybrid canola requires using a pollin ation control system to prevent unwanted self-pollination. Three primary pollination c ontrol methods used in North America: 1) the biotechnology derived male sterility syst em; 2) the NPZ MSL (Lembke) system ; and 3) the INRA-ogura cytoplasmic male sterilit y (CMS) system (Stiewe et al., 2010). The biotechnology-derived system uses tapetal cell-specific male sterility and fertility restorer genes to facilitate hybrid seed production (Mariani et al., 1992). The MSL-system (Male Sterility Lembke) is a private system ow ned by NPZ/Lembke based on a spontaneous mutant selected in the NPZ nursery (F rauen and Paulmann, 1999). The INRA-ogura system uses a CMS and restorer gene transferred from Raphanus (Buzza, 1995). TheMonsanto Company 11-CA-233U 136 of 500 CMS system is composed of three lines, usually referred to as the A line (male sterile or female parent line), B line (maintainer line) a nd R line (restorer or male parent line). The A and B lines have similar genotypes, with the exception of the CMS gene, that confers male sterility to the A line. The B line has a cytoplasm which allows normal pollen production facilitating the breeding process (Bu zza, 1995). The first step in the hybrid seed production process involve s foundation seed cr ossing block where female parent Line A is pollinated by the genetically identic al Line B to produce sufficient quantities of Line A seed. The second step involves pollin ation of Line A with restorer Line R to produce fully fertile F1 hybrid seed (Thomas, 2003b).

Special planting methods are utilized in th e production of hybrid canola seed involving planting alternating strips of ma le and female lines. The ratio for male to female varies with seed producer and variety. The male rows are destroyed after flowering and only

the female blocks are harvested for seed.

Canola seed is produced by several companies that produce and sell seed, such as Monsanto Company, Pioneer Hi-Bred Inte rnational, Bayer CropScience, Croplan Genetics, and Dow AgroSciences. Seed compan ies in turn contract acreage with growers to produce the required amount of canola s eed. Seed companie s identify top canola growers to produce the seed and also mon itor and inspect seed fields throughout the growing season. Seed companies have processing facilities and/or a network of tollers to clean, condition, and bag the harvested canola s eed as well as monitor and inspect all the processes at the plant.

The entire seed production process for the major ity of the seed companies and tollers is certified by the International Organization for Standardization (ISO) and includes internal and external audits (ISO, 2009). ISO standard s ensure desirable characteristics of seeds and services, such as quality, safety, reli ability, and efficiency. The ISO standards represent an international consensus on good management practices with the aim of ensuring that the organization can consistently deliver excellent product and services.

The standards must meet the customer’s re quirements and applicable seed regulatory requirements, and must aim to e nhance customer satisfaction.

The field operations and management practic es for producing canola seed are similar to normal canola production. However, special attention is needed in certain areas to produce seed with high quality, high germina tion rates, and high ge netic purity. General guidelines specific for seed produc tion are discussed below.

A seed production field should not have been planted with canola the preceding five years in order to avoid voluntee r canola plants and to ensure genetic purity. All seed

stock should be treated with fungicides and insecticides to protect seedlings from various seedling diseases and flea beetle. Very early planting into cold soil conditions should be avoided because this can result in poor em ergence and uneven stands. Every effort

should be made to eliminate weeds in a seed field through the use of herbicides, cultivation, and hand weeding to prevent w eed seed in the harvested canola seed. Roguing should be performed on all seed produ ction fields to remove volunteer canola and off-type plants prior to or during flowering. Fields sh ould be scouted frequently forMonsanto Company 11-CA-233U 137 of 500 insect pests and insecticides should be app lied when insect pest infestations reach economic threshold levels. Foliar-applied fungi cides should be consid ered when disease infestations are predicted in th e area. Honey bee hives should be placed in the seed fields to encourage pollination of the female parent line with pollen of the male parent line. Swathing (cutting the crop and plac ing it in rows directly on the cut stubble to facilitate drying rate, ensure even ripening and reduce th e possibility of seed losses from wind and hail) and combining at harvesting should occur at optimum times to avoid seed shattering and damage to the seed. With large acreage of canola, swathing s hould start when seed color change is approximately 20% to 25% and the majority of the crop should be cut at or near the optimum seed color. Swathing should begin as early as 15% seed color change (NDSU, 2005). Combining should begi n when the seed moisture drops below 10% and no green pods are visi ble (Boyles et al., 2009). Ha rvesting equipment should be adjusted to minimize or avoid seed damage and should be cleaned before entering the seed fields to minimize genetic contamination. Certain handling equipment, such as auger elevators, should be avoided becau se they can increase seed damage.

Field inspections are vital to ensure canola seed meets seed certification requirements, ISO certification standards, regulatory standard s, and trait licensing agreement standards. Field inspections are conducted on seed production fields throughout the growing season to evaluate variety purity, ensure canola plan ts are developing prope rly, and fields are maintained free of weeds, insects, and diseases.

Production plant personnel make every effort to avoid mechanical damage to the harvested seed during the scr eening, cleaning, and bagging process. Specific methods are used to assure the genetic purity and identi ty of the seed is maintained throughout the handling and storage operation. Bin inspections and sample collections are conducted at storage locations at the plant to examine the physical characteristics of the canola seed and to ensure proper bin cleanout. Seed is inspected for appearance, disease, discoloration, seed coat, mechanical damage, inert matter, and weed seed. Germination, hybridity, and impurity are tested and quantif ied on all seed lots to verify acceptable levels and meet labeling requirements.

Commercially certified canola seed must meet state and federal seed standards and labeling requirements. AOSCA standards for cert ified canola seed require all seed fields be inspected in the early flowering stage to ensure the fields are isolated from other canola crop fields by at l east 2640 feet (AOSCA, 2009). Fields must be free of prohibited noxious weeds. Inspections should also ensure that the number of off-type plants and plants of other varieties do not exceed 1.5 plants per 10,000 canola plants.

Plants of other Brassica crop species must not exceed 1 plant per 10,000 canola plants. The percentage of hybrid seed shall not be less than 75%. St ate seed certification standards vary slightly from state to state and can be more restrictive than the seed

standards of AOSCA.

When deregulated, MON 88302 seed will be produced in the same manner as commercially certified canola seed, such that it will meet all state and federal seed standards and labeling requirements.Monsanto Company 11-CA-233U 138 of 500 VIII.C. Production Management Considerations

Pre-Season

There are generally three types of canola crop s that can be grown in the U.S. and the grower must decide which of these types is best suited for his area and cropping system (Brown et al., 2008). Winter canola is planted in the fall, overwinters, requires vernalization (winter-chilling) to produce flowers, and is harvested the following summer. Winter canola is generally produced in the Pacific Northwest, southern Great Plains and Midwest regions of the U.S. Ther e is a second type of winter canola that is planted in the fall and overwint ers, but does not require verna lization to produce flowers. This winter type is produced in the southeast region of the U. S. The third type is spring canola which is planted in the early spring, requires no vernalizati on to flower, and is harvested in late summer. Spring canola is grown primarily in the northern Great Plains states including North Dakota, South Dakota, Minnesota, Montana, Idaho, and also in Washington. Winter and spring canola may require different agr onomic practices and can be affected by different insect pests and diseases.

Crop rotation, tillage system, seed or vari ety selection(s), and soil fertility are management issues which require production de cisions well in advance of planting the canola crop. Many of the deci sions in this area are made immediately after harvest of the previous crop or sooner, particularly w ith winter canola production. There are many benefits to crop rotation and the majority of the canola acreage is planted in rotations with small grain cereal crops. Crop rotati on is generally a long term decision, but the rotation sequence can be modified to take a dvantage of a particular economic or market opportunity. Canola is commonly grown follo wing small grain cereal crops (winter or spring wheat, barley, maize or sorghum) (Bro wn et al., 2008). Growers are advised not to plant canola following canola to avoid a build-up of soil-bor ne diseases like Sclerotinia stem rot, blackleg, or club root. Ideally, ca nola should be planted only once every four years to minimize diseases. Also, growers are advised to avoid planting canola after legume crops or sunflower which is susceptible to Sclerotinia stem rot, or Rhizoctonia

and Fusarium root rots.

Canola is highly sensitive to certain herbicide residues in the soil, particularly sulfonylurea herbicide residues (Boyles etal., 2009). Therefore only sulfonylureatolerant varieties should be planted where sulfonylurea herbicides were used in the previous year. Additionally, growers must know the herbicide use history and plant back restrictions before pl anting canola in any field to avoid severe crop injury and/or yield loss due to carryover herbic ide residues. For example, bromoxynil has only a 30-day plant back restriction for canola, but imazetha pyr has a restriction of 40 months for most varieties. In addition to the plant back rest riction, some herbicide labels require a field bioassay to determine whether canola can be planted without risk of crop injury. Imazethapr herbicide is used often in legum e crops in the Pacifi c Northwest and crop injury to canola has been repo rted six years after an imazet hapyr application (Brown et al., 2008).Monsanto Company 11-CA-233U 139 of 500 Canola can be grown with conven tional tillage, conservation til lage, or direct-seeded into small grain straw stubble in a no-tillage cropping system (Brown et al., 2008). The benefits of conservation tillage or no-till sy stems include reduced soil erosion, reduced fuel and labor costs, soil moisture conserva tion, and improved soil structure and organic matter content (Brown et al., 2008).

Primary and secondary pre-plant tillage he lps remove crop residue , control weeds and volunteer plants from the prev ious crop and is an importa nt consideration prior to adopting no-till or direct-seed ing systems for canola. Regardless of the tillage system utilized by the grower, the tillage system mu st facilitate good stand establishment and emergence. Good seed-to-soil contact is e ssential for good seed ge rmination and uniform emergence that are critical to obtaining ma ximum yields (Brown et al., 2008). Good,

uniform stands of canola will also be an important component of weed management during the growing season. Conservation and no -tillage systems leave crop residue such as small grain straw from the previous crop which is desirable fo r erosion control and moisture conservation. However, no-till plan ting or direct seedi ng into heavy crop residue can make it difficult to achieve good seed-to-soil contact and result in poor emergence of canola. These factors must be weighed against the fact that additional tillage will generally result in additional soil moisture loss, and an increased probability of soil crusting before seedling emergence.

Spring canola has provided good performance under conservation tillage while winter canola has generally performed poorly with conservation tillag e systems (Brown et al., 2008). Winter canola is not recommended when planting into excessi ve amounts of fresh straw, or when soil temperatures are lower th an average in the fall months, because it can result in poor emergence. Seed yields w ith direct-seeded winter canola have been significantly lower than with conventional tillage systems (B rown et al., 2008). Spring canola is better suited to conservation tillage due to better soil mo isture conservation and availability. Spring canola has fewer probl ems dealing with heavy straw residue and, residue from the previous crop has usually d ecomposed to some de gree during the winter months prior to planting spri ng canola. In addition, cool er soil temperature can be advantageous to spring canol a. The Conservation Tillage Information Center (CTIC, 2008) reports that conservation tillage (no-till or mulch-till) is used on approximately

32% of the canola acres in North Dakota. Crop Specialists at North Dakota State University indicate that the amount of direct-seeded or no -till canola in North Dakota

varies across the state based on rainfall. In the eastern or Red River Valley area where rainfall is relatively high, only about 5% is direct-seeded into wheat straw (Kandel – NDSU, Personal Communication, 7/23/2010). Th e drier, central area and even drier,

western area of the state are estimated to have approximately 80% and 100% of the acreage direct-seeded, respectively.

Effective nutrient management and maintainance of good soil fertility is essential for high yielding and high quality canol a. Nitrogen, phosphorus, pot assium, sulfur, and boron, depending on the region, are the most limiti ng nutrients for successf ul canola production (Brown et al., 2008). Soil sampli ng and testing is the first step in assessing soil nutrient levels and the requirements for supplemental fert ilizers. The availabi lity of soil nutrients is dependent on soil acidity or the pH level a nd must be included in this assessment. TheMonsanto Company 11-CA-233U 140 of 500 ideal soil pH for growing canola is between 6.0 and 7.0. Canola yields will be adversely affected when the soil pH is below 5.5 (Brown et al., 2008).

Nitrogen is the most limiting of all plant nut rients in canola and sufficient nitrogen must be available to the plant at every growth stage. Supplemental nitrogen requirements for North Dakota are based on the yield potential of canola, nitrate nitrogen available in the soil at 0-24 inch depth and th e previous crop credit for ni trogen (Franzen and Lukach, 2007).

Research studies have shown that nitrogen recommendations can be capped at 150 lbs. per acre in the cooler, moister areas of the state without impacting yield. In the drier, warmer areas of the state nitrogen rates can be capped at 120 lbs per acre. Canola is very

sensitive to fertilizer salts and no more than 5 lbs. of nitrogen per acre is recommended for placement with the seed at planting on me dium-textured soils (Franzen and Lukach, 2007).

Canola has a moderate requirement for phosphor us and phosphorus fertilizer rates are based on soil tests. Phosphorus is not mobile in the soil and should be banded with the seed at planting or incorporated into the soil before planting (B rown et al., 2008). A

starter fertilizer rate of 20-30 lbs. of P 2 O 5 per acre is generally sufficient for most soil test levels unless the grower intends to build up phos phorus levels in th e soil (Franzen and Lukach, 2007). Potassium requirements are also based on soil tests. Many soils contain sufficient levels of potassium requiring no potas sium fertilizers. Potassium applications

are not needed when the soil test indicates 160 ppm or more of potassium (Franzen and Lukach, 2007). Potassium can also be applied as a starter fertilizer with or alongside the seed at planting.

Canola has special requirements for sulfur and it is often the second most limiting nutrient in canola production (Bro wn et al., 2008). Sulfur defi ciencies result in yellowing between leaf veins, cupped leaves and stunti ng (Franzen and Lukach, 2007). In addition, in the presence of sulfur deficiency floweri ng is delayed, seed often does not set, and pods will be barren or poorly developed. North Dakota studies have demonstrated significant yield increases from sulfur appl ications (Franzen and Lukach, 2007). Since

soil tests tend to overestimate available su lfate and are highly variable, North Dakota specialists recommend 20 to 30 pounds of sulfur per acre when medium to low levels of

sulfur are detected and 10 to 15 lbs of sulfur per acre when high levels of sulfur are detected (Franzen and Lukach, 2007). The sulfur fertilizer should be in the form of ammonium sulfate, ammonium thiosulfate or potassium thiosulfate since canola takes up

sulfate sulfur.

Canola requires more boron than most other crops. Boron at 1 to 2 pounds per acre

should be broadcast when the soil tests s how less than 0.5 ppm boron (Brown et al., 2008). Canola has not shown yi eld responses to applications of micronutrients such as chlorine, copper, iron, manganese, molybde num or zinc (Brown et al., 2008).

Many varieties are available for each canola type , and variety selection is one of the most important decisions a grower makes in gr owing a successful crop (NDSU, 2005). TheMonsanto Company 11-CA-233U 141 of 500 performance of a given variety may differ from year to year and location to location due to changing environmental conditions. Ther efore, growers rely on variety performance trials conducted by universities and private companies across a number of locations and years to make a selection. There are severa l factors to consider in choosing canola varieties, namely: yield, maturity, plant he ight and lodging, disease tolerance, and seedling vigor (NDSU, 2005).

Planting and Early Development

An understanding of the growth stages of ca nola is important for the proper timing of certain management practices such as herbicid e and insecticide appli cations. In addition, the impact of certain weather conditions a nd diseases on canola yi eld is dependent on growth stage. Temperature, moisture, light, nutrition and variety influence the length of each growth stage (NDSU, 2005). The growth stage key utilized by BBCH (2001) outlines nine main growth stages: Stage 0 - Germination, Stage 1 – Leaf Development, Stage 2 – Formation of Side Shoots, Stag e 3 –Stem Elongation, Stage 5 – Inflorescence Emergence, Stage 6 – Flowering, Stage 7 – Development of the Fruit, Stage 8 – Ripening and Stage 9 - Senescence.

The pre-emergence stage or germination proce ss typically takes from four to ten days depending on soil temperature and moisture, seed-soil contact and depth of planting (NDSU, 2005). Canola is susceptible to many soil-borne pathogens during this stage. Planting high quality seed is essential for controlling diseases. University specialists recommend growers always plant certified canola seed that is free of diseases to prevent the spread of seed-borne blackleg ( Leptosphaeria maculans ), Sclerotinia stem rot ( Sclerotinia sclerotinium ), and Alternaria black spot ( Alternaria spp .) (Brown et al., 2008). Fungicide seed treatments are economic ally beneficial for protecting seedlings from seed rot, damping off, seedling bli ght, and early season root rot caused by Pythium,

Rhizoctonia , and Fusarium . The fungicide seed treatments are also effective protection against seed-borne blackleg a nd Alternaria black spot.

During the seedling stage, canola plants are also very susceptible to flea beetle species ( Phyllotreta cruciferae and P. strilolata ) infestations. Insecticide seed treatments provide protection from flea beetle species feeding for most of the susceptible seedling stage (NDSU, 2005). Some of the seed treatme nts registered for canola contain multiple fungicides for broad spectrum disease protection plus an insecticide for control of flea beetles. For example, Prosper 400 cont ains the fungicides thiram, carboxin, and metalaxyl plus the insecticide clothianidi n. Helix XTra and Helix Lite contain the fungicides difenoconazole, mefenoxam, and fludioxinil plus the insecticide thiamethoxam (Brown et al., 2008).

The exposed growing point of spring canola makes the seedlings more susceptible to spring frosts, soil drifting, and hail damage. Freezing temperatures in the spring can cause serious injury to canola plants. Th e amount of frost injury will depend on soil moisture conditions, the rate at which thawing occurs, the growth stage of the plants and the amount of cold temperature-hardening that the plant is exposed to prior to freezing temperatures (NDSU, 2005). Spring canola that is exposed to several days of nearMonsanto Company 11-CA-233U 142 of 500 freezing temperatures will undergo a gradual hardening process th at will allow the plants to withstand freezing temperatures without serious damage.

The minimum soil temperature for canola to germinate is 38 °F (NDSU, 2005). The length of time needed for emergence is de pendent on soil temperature. Canola will emerge in 17 to 21 days from planting when the average soil temperature is in the low 40s. When the soil temperature averages in the low 50s, canola will emerge in approximately 10 days. In the early years of practicing conservation tillage, if was found that heavy straw residues from the previous cr op can result in lower soil temperature and higher soil moisture which could delay plan ting (Brown et al., 2008). However, water infiltration improves after utilizing no-tillage or direct-seeded systems for several years. Frequently, that allows grower s to enter fields sooner, plan t earlier and establish the crop earlier. For optimum seed germination, seed ing should be delayed until soil temperatures exceed 49 °F (Brown et al., 2008). Seeding into cooler soil temperatures will cause slow and uneven germination, thin or uneven stands and fosters weed competition.

Seeding date of spring canola will have an effect on time of germination, crop emergence and establishment, days from planting to flowering, plant hei ght, maturity, and final yield (Brown et al., 2008). Spring ca nola should be planted in Ap ril to early May in North Dakota to maximize yields (NDSU, 2005). Sp ring canola should be planted prior to planting spring cereal grains. Research studies have shown significant reductions in yield when planting is delayed beyond May 1 in southwestern North Dakota and May 15 in other regions of the state (NDSU, 2005). Delaying planting much beyond these dates will increase the chances of encountering high temperatures and/or drought conditions during flowering when canola is suscep tible to heat and drought stress.

Since winter canola is planted in the fall, planting date s should be selected to provide time for plants to develop sufficient growth be fore the onset of winter to minimize winter damage (Brown et al., 2008). Generally, wi nter canola should be planted six weeks before the first killing frost in most states (Boyles et al., 2009; Br own et al., 2008). The goal is to have at least 45 days of growth or to have plants with four to six fully opened leaves (in a rosette stage of growth) before wi nter. Planting too early in the fall can result in crop failure as overly large plants deplete soil moisture. This moisture stress may cause the plants to flower in the fall which ma kes them more susceptible to winter kill. On the other hand, planti ng too late will result in seedli ngs that are too small that have

insufficient reserves to survive winter condi tions and/or compete well with weeds in the spring. Planting dates for optimum yields with winter canola in the Pacific Northwest are August 14 to 24 although plantin g can be in early August to the second week of September (Brown et al., 2008). Recommende d planting dates for winter canola in the Great Plains vary according to latitude. For example, optim um planting dates for Kansas and Oklahoma are August 26 through Sept ember 25 and August 20 to September 21 respectively (Brown et al., 2008).

Canola is adapted to a wide range of plan t population densities. The optimum seeding rate for spring canola is 600,000 pure live seed s (PLS) per acre which equates to 16 PLS per square foot (NDSU, 2005). This seeding rate should result in an optimum established stand of 8 to 14 plants per s quare foot. Similar seeding ra tes are recommending in winterMonsanto Company 11-CA-233U 143 of 500 canola. The seeding rate for winter canola should result in 10 to 16 establ ished seedlings per square foot (Brown et al., 2008). Signi ficant yield differences do not occur unless populations at harvest are less than one or grea ter than 15 plants per square foot (Boyles et al., 2009). Low plant populations of canol a will not compete as effectively with weeds, and can produce lower yields although canola plants in low planting densities have the ability to compensa te (Brown et al., 2008). Indi vidual plants in low plant densities will have broader stems and greater raceme branching because of less intra-crop competition. Plant populations that are too high will result in thin-stemmed plants, high intra-crop competition, crop lodging at maturity and decreased yields. Planting into poor

soil and seedbed conditions will require increased seeding ra tes to compensate for poor seedling emergence. Direct-seeding or no-ti lling spring canola into standing wheat straw requires a 10 to 15% increase in seeding rate to achieve the same plant stand compared to

well-cultivated and firm seedbed co nditions (Brown et al., 2008).

To determine pounds of canola seed to plant in order to achieve the optimum seeding rates presented above, it is important to know the number of seeds per pound for the specific variety of canola be ing planted. Canola seed s are small and round with approximately 75,000 to over 200,000 seeds per pound dependent on the type of canola and variety (NDSU, 2005). Gene rally, the hybrid varieties of B. napus canola will contain 75,000 to 100,000 seeds per pound. Open-pollinated B. napus canola varieties will vary from 135,000 to 160,000 seeds per pound. Brassica rapa canola varieties usually contain more than 200,000 seeds per pound. Certified canola seed bags will indicate the number of seeds per pound and s eed germination percentage which is vital information in determining the actual seeding ra te. Seeding rates will generally be 4 to 8 pounds per acre (NDSU, 2005).

Most commercial grain drills us ed to plant small grain cereals are acceptable for planting canola (Brown et al., 2008). The narrower row spacing provides quicker canopy closure, reduces weed competition, and lessens wind sh attering before harv est (Boyles et al., 2009). Canola should be seeded to a depth that allows the s eed to be covered and must be planted into moist soil. The best germ ination and emergence usually occurs with seeding depths of 0.5 to 1 inch (Boyles et al., 2009; NDSU, 2005). Canola has difficulty emerging through thick soil covers or cr usted soil (Boyles et al., 2009).

Early season weed control is important to minimize yield losses in canola (Boyles et al., 2009). Canola in the seedling stage is a poor competitor with weed s and becomes more competitive as it approaches the late rose tte and bolting stage (NDSU, 2005). Planting

canola into a weed-free seedbed is essential a nd can be achieved with tillage, herbicides,

or a combination of both methods. Seedbe d conditions that provide rapid germination and early emergence are important for uniform stand establishment and minimize competition with weeds. Uniform stands of canola will be an effective weed control management tool as the growing season prog resses. Effective weed management in canola relies heavily on selective post-emerge nt herbicides. The list of registered

herbicides in the U.S. is re latively small compared to ot her major crops. Sethoxydim and clethodim are post-emergent in-crop herbic ides for control of annual grasses while quizalofop will control annual grass and quackgra ss (perennial). Clopyralid will control several broadleaf species in canola. Herbicid e-tolerant canola varieties are also availableMonsanto Company 11-CA-233U 144 of 500 which allow the use of broads pectrum post-emergent herbic ides such as glyphosate, glufosinate, and imazamox. Early canopy clos ure can reduce the number of in-crop herbicide applications.

Mid- to Late Season

The first true leaves of canola develop 4 to 8 days after emergence and plants quickly establish a rosette. The older leaves develop at the base increasing in size while smaller, younger leaves develop in the center. During the rosette stage rapid and abundant leaf growth occurs producing more dry matter pe r day and increasing yield potential. Bud formation is triggered as the days lengthen a nd temperatures rise. The plant reaches its maximum leaf area index in the late bud stag e. The vegetative stag es (seedling to first flower) for B. napus generally range from 40 to 60 days, depending on environmental conditions. Flowering begins with the opening of the lowest bud on the main stem and continues for the next 14 to 21 days. High te mperatures together with moisture stress during this stage can severely reduce yield pot ential. Ripening begins when the petal on

the last formed flower on the main stem falls . Seed fill is complete approximately 35 to 45 days after flower initiation.

Most insect pests of canola damage the flow ers, leaves, developing buds, seed pods, or developing seed and cause yield reductions and often an associated reduction in oil content (Brown et al., 2008). Spring and wint er canola is impacted by the same insect pests, but the degree of damage may be diffe rent. New generations of adult flea beetles can emerge in mid-July to early August ( NDSU, 2005). Populations usually are not high enough to cause serious damage to canola. However, extremely high populations can feed on green pods causing pods to shatter and seeds to remain green and require

treatment with inse cticides. Cabbage seedpod weevil ( Ceutorhynchus assimilis ) is the major insect pest of winter canola (Brown et al., 2008). Other insect pests which occasionally will infest canola are diamondback moth ( Plutella xylostella ), bertha armyworm ( Mamestra configurata ), lygus bugs ( Lygus spp .), cutworms, grasshoppers, blister beetles, and aphids (NDSU, 2005). Infestations may exceed economic threshold levels requiring insecticide treatments.

As previously mentioned, blackleg, Sclerotini a stem rot and Alternaria black spot are

seed-borne diseases which can be effectivel y managed by planting certified seed with seed fungicide treatments. The first step in minimizing these diseases is to avoid growing canola within three years of another host crop species (Brown et al., 2008). Where blackleg infestations exist, planting blackleg-resistant varieties is an effective management practice. Foliar fungicide applic ations for control th ese diseases are not economical under most situations, although foliar applications of boscalid and azoxystrobin fungicides are effective on Sclero tinia stem rot and Alternaria black spot respectively as needed.

Harvest

Spring canola is potentially at risk for frost da mage in the fall. It is important to have stands of canola that ripen early and unifo rmly since the amount of fall frost damageMonsanto Company 11-CA-233U 145 of 500 depends on the stage of maturity (NDSU, 2005) . A temperature of 27 °F is enough to kill immature seeds containing 50% to 60% mois ture while seeds at 35% moisture will escape damage (NDSU, 2005). Frost fixes th e chlorophyll or green color in immature seed and can increase the cost of refining the oil (Brown et al., 2008; NDSU, 2007). Canola seeds in all pods on a pl ant reach physiological maturity and complete filling at about 40% moisture. They th en slowly turn from green to light yellow, or reddishbrown, brown or black (NDSU, 2005). With dry weather and temperatures of 90°F,

canola seed can have a 10% to 50% seed color ch ange in three to five days or less. Seeds

lose moisture at the rate of about 2% to 3% or more each day depe nding on the weather. Spring varieties of canola usually mature 85 to 110 days after planting, depending on the variety and environmental conditions (NDSU, 2005). Pod shattering in standing canola due to excessive wind, rain, an d hail can result in yield lo sses of greater than 50% when the crop is ripe and ready to harvest (Boyles et al., 2009). Th erefore, if possible, canola should be harvested immediately upon ripe ning to prevent pre-harvest losses.

Most canola is harvested using one of two methods: 1) direct combining of standing canola, and 2) swathing and combining. In the latter method, the canola swath is allowed to cure and ripen for a minimum of 10 to 14 days before harvesting which hastens maturity and avoids frost damage in areas wi th a short growing season (Boyles et al., 2009). The direct combining method does not require additional equipment (swather) for growers. There has been considerable rese arch and debate on which method is best for harvesting canola. Direct combining standing canola has resulted in less green seed and generally higher oil content and test weight than the swathing and combining method in multi-year research studies in North Dakot a (NDSU, 2007). However, canola is more

vulnerable to seed and shatter loss when di rect combining is delayed past the optimum time. Swathing can be advantageous since it facilitates harvest 8 to 10 days earlier (Boyles et al., 2009). Earlier harvest can be be neficial for spring canola if a hard frost is expected. It is important to harvest at th e optimum stage of ripe ning using the swathing and combining method to reduce green seed problems and seed shatter losses (NDSU, 2007). The quantity of green seed in the harv ested crop affects qual ity and subsequently the market price of canola. Chlorophyll in seed is extracted with the oil during the crushing process and can increase the cost of refining the oil (Bro wn et al., 2008). Swathing can hasten the loss of green seed by about 2 days comp ared to seeds in standing crops (Brown et al., 2008). Direct combin ing is generally recommended for winter canola in the southern Great Plains region because dry-down is accelerated by higher air temperatures during the ripening stage a nd harvest period (Boyles et al., 2009). However, both methods have been used successf ully for harvesting cano la in this region. Swathing is generally recomme nded if harvest cannot be co mpleted in a timely manner with direct combining.

Pushing, a new procedure being considered for harvesting ca nola, is reportedly a faster and less expensive alternative to swathing (B oyles et al., 2009). The pushing procedure pushes the stalks over, but does not cut or break the stalks off. When the canola matures, it is combined in the opposite direction of th e pushing procedure. Experience with the pushing procedure is very limited at this time.Monsanto Company 11-CA-233U 146 of 500 When direct combining, the ideal time to harvest is when the average seed moisture of canola is 8 to 10% and no green pods are visible on the plants (Boyles et al., 2009). Canola is an indeterminate crop and will retain a few immature pods and seed at harvest. Growers are advised not to delay harvest to allow these smaller im mature pods and seeds to mature because waiting will result in the larger, higher yielding seed pods to shatter and reduce potential yield. To reduce the poten tial for pod shatter, gr owers can harvest at a slightly higher moisture (10% to 15%) and then artificially dry the seed.

The decision of when to swath is critical and is based on the correct seed maturity stage and favorable weather conditions (Brown et al., 2008). Canola seed color is a good indication of the maturity of the crop, and is the factor used to determine the time to swath. Growers must examine only the pods on main stem for seed color change. When

the overall moisture content of seed from the total plant av erages 30% to 35%, about 30% to 40% of the seeds in pods on the main st em will have changed color or started to change color (NDSU, 2005). In the case of canola, growers should try to avoid swathing when the air temperatures are above 82 °F par ticularly when humidity is low (Brown et al., 2008). These weather conditions can re sult in higher shat ter losses and some immature green seed. Swathing in the evening or at night when air temperatures are cooler allows the seed to dry at a slower rate helping prevent green seed and low oil content. Swathing over-ripe ca nola (80 % seed color change) re sults in fluffy cut rows of canola that are more suscepti ble to blowing and increased shattering. Seed moisture content and green seed counts s hould be checked to determine the proper time to begin combining. Under normal weather conditions, this is generally 10 to 14 days after swathing for spring canola in North Dakota ( NDSU, 2007) and 5 to 14 days for winter

canola in the southern Great Plains region (Boyles et al., 2009).

Canola should be stored under c ool, dry conditions. The optimum storage conditions for

canola are 55°F and 7% seed moisture and th e storage life of canol a doubles with every 10° reduction below 77° and every 1% reduction in seed moisture below 9%. (Boyles et al., 2009). Canola seed is very sensitive to heating in storage (Brown et al., 2008) and storage bins must be properly aerated or vented for heat and moisture to escape.

VIII.D. Management of Insects

Insect pest infestations are a limiting factor in successful canola production (Brown et al., 2008). Insect pests can reach infestation leve ls that cause yield reductions and often a corresponding reduction in the oil content of canola. Insect pests ca n cause damage throughout the growing season (Boyles et al., 2009) on flowers, leaves, developing buds, seed pods, and developing seeds of canola. Seedling canola is espe cially vulnerable to chewing insects. A severe in festation of flea beetles can co mpletely destroy a stand of canola seedlings. Late season insect pests can typically cause yiel d losses of 20 to 50% in spring canola when left uncontrolled (Brown et al., 2004). Some insects, such as aphids act as vectors for plant viruses like turnip mosaic, cauliflower mosaic virus, aster yellows and beet western yellows. Damage from insect pests is more severe during periods of stress, especially drought stress. Spring and winter canola varieties are impacted by the same insect pests. Howeve r, some pests negativel y impact one varietyMonsanto Company 11-CA-233U 147 of 500 more than the other. Crop rotation, volunt eer and wild mustard control, and managing crop residues are all important cultural practices for insect control.

The insect pests of spring and winter canola va rieties in the U.S. ar e listed in Table VIII4. Flea beetle is a serious insect pest in sp ring canola wherever it is grown (Brown et al., 2008). This insect pest is less important in winter canola, except where winter and spring canola are grown in the same area. The most significant plant in jury and subsequent yield loss is caused by adult beetles in the early spring when the plants are in the cotyledon to two-leaf stage (Weiss et al., 2009). An insecticide seed treatment (imidacloprid or clothianidin) is advisabl e in spring canola to protect the seedlings (NDSU, 2005; 2007). Seed treatments usually pr ovide at least 3 to 4 weeks of protection.

If a seed treatment is not used or does not provide adequate protection, a foliar insecticide application is beneficial wh en 25% defoliation occurs on th e cotyledons and true leaves (NDSU, 2005). Minimum or no-tillage systems generally have lower infestations of flea beetles (Weiss et al., 2009). In addition, pl anting early can allow establishment of the canola prior to beetle emergence.

Insect pests causing serious damage to winter canola include flea be etles, grasshoppers, army cutworms, diamondback moth larvae, aphids and maggots (Brown et al., 2008). Aphids have become the most important insect pest of canola in this region. The green peach and turnip aphid survive mild winters and increase to damaging levels during the early spring. Fields should be scouted from the seedli ng stage through pod development for damaging infestation levels of aphids . Aphid populations are sometimes high enough to cause significant stand reduction and yield losses (Brown et al., 2008). Predatory and parasitic insects are beneficial in control ling aphid populations. Ho wever, these insects alone will not prevent aphid populations from reaching damaging levels. An insecticide seed treatment (clothianidi n, imidacloprid, or thiamethoxamare) is recommended in winter canola as a preventive management practice due to the frequency of damaging aphid populations in the fall in the southern Great Plains (Brown et al., 2008). Fields should be scouted in the spring for any s ubsequent build up of aphid populations.

Cabbage seedpod weevil is a major insect pest in winter canola in the Pacific Northwest (Brown et al., 2008). However, it has not been reported in the major canola production

area of North Dakota and Minne sota (Weiss et al., 2009). Pl ant injury is caused by the adults feeding on buds. Yield losses of 20 to 40% are common from second generation feeding of cabbage seed pod weevil on the developing buds and s eeds (Weiss et al., 2009). A foliar insecticide (bifenthrin or lambda-cyhalothrin) is recommended when

three to four weevils are co llected in a sampling sweep dur ing scouting at bud stage.Monsanto Company 11-CA-233U 148 of 500 Table VIII-4. Insect Pests by Canola Development Stage in Spring and Winter

Canola in the U.S. 1

Common Name Latin Name Seedling to Rosette Stage Crucifer and striped flea beetle Phyllotreta cruciferae, P. striolata

Cutworm Euxoa species

Diamondback moth Plutella xylostella Grasshopper Various species Green peach and turnip aphid Myzus persicae, Lipaphis erysimi

Rosette to Flowering Stage

Cutworm Diamondback moth Plutella xylostella Grasshopper Various species Green peach and turnip aphid M. persicae , L. erysimi Lygus bugs Lygus species

Flowering to Pod Development Bertha armyworm Mamestra configurata

Blister beetles Lytta nuttalli, Epicauta species

Diamondback moth Plutella xylostella False chinch bug Nysius raphanus Grasshopper Various species Green peach, turnip and cabbage aphids M. persicae, L. erysimi, Brevicoryne brassicae Lygus bugs Lygus species

Pod Development to Harvest Bertha armyworm Mamestra configurat a Cabbage aphid Brevicoryne brassicae Cabbage seedpod weevil Ceutorhynchus assimilis Crucifer and striped flea beetle Phyllotreta cruciferae, P. striolata False chinch bug Nysius raphanus Grasshopper Various species Harlequin bug Murgantia histrionica Lygus bugs Lygus species

1 Source: (Boyles et al., 2009; NDSU, 2005)

.

Other insect pests can reach populations that cause serious damage in spring and winter canola. Fields should be scouted throughout the growing season for economic threshold levels of insect pests. Economic threshold le vels have been established for most insect pests by university entomologist to determine whether an insecticide treatment is justified. Foliar insecticides currently registered in the U.S. for use in canola include bifenthrin, deltamethrin, gammacyhalothrin, lamba cyhalothrin and methyl parathion (Brown et al., 2008). Insecticides were us ed only on approximately 3% of the canola acres in North Dakota in 2008 (Zollinger et al ., 2009). Growers must be careful in the selection and applicatio n of insecticides. Most insect icides are non-selective and may damage non-pest insects. Bee keepers frequen tly set their hives close to flowering winterMonsanto Company 11-CA-233U 149 of 500 canola fields because canola is an early and ve ry rich source of pollen for bees (Brown et al., 2008). Therefore, growers need to exerci se special care to pr otect honey bees from insecticide applications.

VIII.E. Management of Diseases and Other Pests

Plant diseases can be a serious problem in canola production. Diseases attack canola at all stages of development and can be soilbor ne, seedborne, or airbor ne, and also spread from infected crop residue (Boyles et al., 2009). Proper management of diseases is critical to maximize yields in canola.

Diseases that affect canola include blackleg ( Leptosphaeria maculans ), Sclerotinia stem rot ( Sclerotinia sclerotiorum ), Alternaria black spot ( Alternaria spp .), downy mildew ( Peronospora parasitica ), powdery mildew ( Erysiphe cruciferarum ), black rot ( Xanthomonas campestris ) and aster yellow (Boyles et al., 2009; NDSU, 2005). In addition, there is a fungal seedling disease complex that includes Pythium spp., Fusarium

spp ., and Rhizoctonia spp . Blackleg and Sclerotinia stem rot are the most serious diseases in spring and winter canola.

Blackleg fungus is a seedborne disease that is commonly intr oduced by planting infected seeds (Brown et al., 2008). Once introduced in to an area, it will remain as long as canola is grown there. The virulent or aggressive strains can infe ct early growth causing leaf spots any time from the seedling stage to maturity (NDSU, 2005). Seed pods and seeds also may be infected resulting in split pods a nd seed loss. Temperatures in the 70s and extended periods of wet plant canopy favor infection. Temperatures above 86°F and below 50°F inhibit development of the dis ease (NDSU, 2005). Several management practices are available to control or mini mize this disease (NDS U, 2005). The most important practice is planting disease-free, cer tified seed where blackleg has not been introduced. Blackleg has yet to be introduc ed in the Pacific Northwest region and growers are advised to plant locally produced seed to pr event the introduction of the disease (Brown et al., 2008). A fungicide s eed treatment should be used such as Helix Liteor Helix XTra which contains fungicides a nd an insecticide for flea beetle control. Growers should plant blackleg resi stant varieties. Many of the B. napus canola varieties have moderate to good blackleg resistance. Deep tillage is recommended to bury the canola residue and speed decomposition of the re sidue and prevent the release of blackleg ascospores to nearby fields. Foliar applica tions of azoxystrobin fungicide are effective on blackleg.

Sclerotinia stem rot is present in most of the canola growing regions and has been the

most serious disease of ca nola in North Dakota and Mi nnesota (NDSU, 2005). This disease has caused estimated losses up to 13 % in North Dakota and Minnesota and yield reductions approaching 50% have been report ed on seriously infected fields (NDSU, 2005). Canola primarily is susceptible duri ng all bloom stages and shortly thereafter (NDSU, 2007). Wet weather immediatel y preceding and at flowering favors development of the Sclerotinia fungus. The fi rst step in minimizing this disease is to plant canola in a four-year rotation with non-su sceptible crops (Brown et al., 2008). The

Sclerotinia fungus may survive four to six years in the soil (NDSU, 2005). Field peas andMonsanto Company 11-CA-233U 150 of 500 flax are much less susceptible to the Scle rotinia fungus and are good rotational crops as are small grains and maize which are immune to the disease (NDSU, 2005). Sunflowers, dry beans, crambe, chickpeas, lentils and soybe ans are some of the most susceptible host crops and should be avoided in the rota tion. Many broadleaf weeds, including lambsquarters, Canada thistle, ragweed and ma rshelder are also susceptible host plants. All canola varieties are suscep tible to Sclerotinia fungus, butsome varieties are less susceptible than others and will perform bette r in moderate to severe disease pressure (NDSU, 2005). Foliar applications of a f ungicide (bosclid or azoxystrobin) provide effective suppression of Sclerotinia stem rot. Since fungicides are expensive, applications should be limited to fields wher e: 1) the yield potential is above normal (2,000 lbs per acre or more); 2) weather preceding early bloom has been wet, 3) additional rain and high humidity is expecte d, and 4) Sclerotinia has been a problem in recent years (NDSU, 2005). Foli ar fungicides were applied to approximately 19% of the

canola acres in North Dakota in 2008 (Zollinger et al., 2009).

VIII.F. Weed Management

Weeds compete with canola for light, nutrients , and soil moisture and can be a major limiting factor in canola production. Canola yi elds can be reduced by as much as 50% due to weed competition with canola (CCC, 2006). The primary factors affecting the amount of yield loss are the weed species , weed density and the duration of the competition (CCC, 2006). Research studies have shown that w eeds that emerge before or with the crop such as wild oats result in gr eater yield loss than weeds that emerge after the crop (CCC, 2006). Canola in the seedli ng stage is a poor competitor with weeds (NDSU, 2005). However, once established a nd with proper management, winter canola suppresses and out-competes most annual weeds with proper management (Boyles et al., 2009). Both Canada thistle and wild oats ar e very competitive with canola early in the growing season. Canada thistle is three or four times more compe titive with canola than wild oats (CCC, 2006). Ten Cana da thistle plants per square meter resulted in 10% yield loss while forty plants per square meter resu lted in over 50% yield loss (CCC, 2006). A wild oat density of thirty plants per squa re meter caused between 15 and 20% yield loss (CCC, 2006). However, an average infestati on of wild oats will range from 60 to 100 plants per square meter.

Knowing the critical period for weed control (CPWC) is useful in determining the need for and timing of herbicide applications (Knezevic et al., 2002). The CPWC is defined as the period in the crop growth cycle during wh ich weeds must be controlled to prevent yield losses. Since the morphology, physiology, and development of each crop is different, the CPWC is likely to be unique for every crop.

Field studies conducted in 1998 and 1999 indicate that canola must be maintained weedfree up to the four-leaf stage ( 17 to 38 days after emergence) to consistently prevent a yield loss greater than 10% (Martin et al., 2001) . However, the critical weed-free period is influenced by crop seeding date. Early- seeded canola may result in extending the CPWC to the six-leaf stage (41 days after em ergence). Originally pre-emergent herbicide applications in canola provided weed control for the duration of this CPWC. With the

advent of herbicide-tolerant canola varieties, post-emergent applications have been theMonsanto Company 11-CA-233U 151 of 500 dominate method of herbicide application. Th e goal in timing post-em ergent applications is to delay application for as long as practic al to capture the most weed flushes without incurring yield loss. Frequently the crop ca nopy will prevent the establishment of lateemerging weeds and decrease the need for post-emergent herbicide applications (Martin et al., 2001). The same studies on CPWC dete rmined that weeds need to be removed by the four-leaf stage of canola to prevent a yield loss of greater than 10%. Studies

conducted in 1998 to 2000 at diffe rent locations with three di fferent herbicide-tolerant canola systems generally confirmed these fi ndings (Harker et al., 2006). Based on these studies, the first post-emergent applications would need to be applied prior to the fourleaf stage. Clayton et al., (2002) reported a yield benefit wh en removing weeds as early

as the one- to two-l eaf stage of canola in certain conditions. Although late-emerging weeds are not always competitive with th e crop, controlling these weeds may be

beneficial to reduce contaminants such as cha ff, stems, weed seeds etc. that could lower the grade of the canola seed and prevent weeds from producing seed and infesting subsequent crops (Har ker et al., 2006).

Crop rotations and environment have a significant impact on the adaptation and

occurrence of weeds in canola. The most common weeds in canola for each growing region are presented in Table VIII-5, 6 a nd 7. Common weeds are those species which can be found abundantly infesting a signi ficant portion of the acreage throughout the region. These weeds are most commonly present when no weed management

intervention has occurred.Monsanto Company 11-CA-233U 152 of 500 Table VIII-5. Common Weeds in Spring Canola Production in North Dakota 1

Annual Grass Weeds Annual Broadleaf Weeds Biennial and Perennial Weeds Foxtail, green and yellow Buckwheat, wild Quackgrass
Volunteer Cereals Kochia Thistle, Canada Wild Oat Field Pennycress Lambsquarters, common Mustard, wild Pigweed species Thistle, Russian

1 Source: (Zollinger, 2003; Jenks-NDSU Personal Communication, 8/2/2010).

Table VIII-6. Most Common Weeds in Winter Canola in the Great Plains 1

Annual Grass Weeds Annual Broadleaf Weeds Winter Annuals Japanese brome Blue mustard Henbit Cheat Bushy wallflower Common chickweed Downy brome Wild mustard Cheatgrass

Rescuegrass Tumble mustard Down bromegrass

Feral rye Tansy mustard Mustards Jointed goatgrass Flixweed Volunteer cereals

Italian ryegrass Field pennycrest Wild oat Shepherd’s purse

Volunteer cereals

1 Source: (Boyles et al., 2009).

Table VIII-7. Most Common Weeds in Spr ing and Winter Canola in the Pacific

Northwest 1

Annual Grasses Annual Broadleaf Weeds Perennial Weeds

Downy brome Field pennycress Canada thistle

Wild oat Catchweed bedstraw Italian ryegrass Prickly lettuce Volunteer cereals Chamomile mayweed

Rattail fescue Common lambsquarters Redroot Pigweed

Russian thistle

Wild mustard

Tumble mustard Birdsrape mustard

1 Source: (Davis – University of Idaho, Personal Communication, 7/30/2010; Wysocki – Oregon State University, Personal Communication, 7/30/2010).

Mechanical methods of weed control including tillage have been used for centuries to control weeds in crop production. Spring or fall pre-plant till age can effectively reduce the competitive ability of weeds by burying th e plants, disturbing or weakening the rootMonsanto Company 11-CA-233U 153 of 500 systems or causing sufficient physical injury to kill the plants. Additionally selective herbicides are an effective management tool to supplement cultural methods for controlling annual and perennial weeds in canola. The number of registered herbicides for canola in-crop application is relatively li mited compared to the number available for use in other crops such as maize, soyb ean, and small grain ce reals. Sethoxydim, clethodim, quizalofop and imazamo x are post-emergence in-crop herbicides for control of annual grasses and broadleaf weeds in c onventional canola variet ies (NDSU, 2005). In addition, along with pre-pl ant incorporated herbicides ethalfluralin and trifluralin, glyphosate and glufosinate herbicides can be applied in conventi onal canola as a preplant burndown treatment for control of emerged weeds in no-tillage systems. Table VIII-8 provides a summary of the herbicides used in conventional and biotechnology-derived canola in North Dakota in 2008.

Table VIII-8. Herbicide Usage in Canola in North Dakota in 2008 1

Number of Applications

Herbicide Acres

Treated % Acres

Treated 1X 2X

Clethodim 12,600 13.6 100.0 Glufosinate 354,300 38.9 99.4 0.6 Glyphosate 660,300 72.6 58.0 42.1
Imazamox 21,200 2.3 48.4 51.6 Quizalofop 31,700 3.5 100.0

Other herbicides 29,200 3.2 100.0

1 Source: (Zollinger et al., 2009).

Herbicide-tolerant canola was introduced to provide farmers with additional options to improve crop safety and/or improve weed contro l. The herbicide-tolerance traits enable

the use of certain herbicides in canola that previously would not pr ovide satisfactory crop safety when applied post-emergence to conventional canola. Roundup Ready, Liberty Link, and Clearfield-tolerant varieties allow the in-crop use of broad spectrum postemergence herbicides, glyphosate, glufosin ate, and imazamox, re spectively. In 2008

herbicide-tolerant canola varieties were planted on approximately 97% of the canola acreage in North Dakota (Zollinger et al., 2009). Glyphosate-to lerant canola was commercially introduced in 1999. Roundup Ready canola is tolerant to post-emergence applications of glyphosate. Glyphosate was a pplied to approximately 73% of the canola acreage in 2008 which includes pre-plant burn down and in-crop applications (Table VIII-8). Liberty Link-tolerant canol a was introduced in 1995 and is tolerant to post-emergence applications of glufosinate. Glufosinate is the second most commonly applied herbicide used in canol a (38% of acres) (Table VIII-8 ). Clearfield varieties are

tolerant to imazamox herb icide. Roundup Ready and Li berty Link are biotechnology

derived traits while Clearfie ld was developed through traditi onal breeding techniques.

Tables VIII-9 and 10 provide a summary of th e efficacy of herbicides on weed species commonly present in canola as well as their persistence in soil. Glyphosate is rated excellent on all but one annual grass (wild oat – good to excellent). Glufosinate andMonsanto Company 11-CA-233U 154 of 500 imazamox are rated good and/or excellent on annual grasses, except for quackgrass where the rating is poor and fair, respectively. Glyphosate is the only herbicide in canola that is rated excellent on quackgrass, a perennial grass species . Glufosinate and

imazamox are rated fair to excellent on all th e annual and biennial broadleaf weeds with the exception of Canada th istle, buckwheat, mallow, ragweed and wormwood. The ratings for glyphosate are more variable on broadleaf weeds ranging from excellent to poor-excellent for all annual a nd biennial weeds and good to ex cellent on Canada thistle. It is important to note that glyphosate, glufosinate and imazamox, the three main herbicides used in herbicide-tolerant canola are rated as not persisting in soil (NDSU, 2005).

The benefits of growing herbic ide-tolerant canola varieties in clude superior weed control, higher yield, less dependence on tillage and summer-fallow, and higher net return on profits (CCC, 2001; Harker et al., 2000) . Roundup Ready and Liberty Link hybrid

varieties are the dominate varieties in canola production with 56% and 39% of the acreage in North Dakota, respec tively (Zollinger et al., 2009). The acreage planted with Clearfield varieties is only 1.9%. The superior weed co ntrol ratings presented for glyphosate and glufosinate in Tables VII I-9 and VIII-10 explain the reason for the success of the Roundup Ready and Liberty Link weed control systems (Harker et al., 2004). The consistency of monocot weed c ontrol was usually greater for the Roundup Ready system than for the Liberty Link or Clearfield systems. The researchers also concluded that the management risks were usually less apparent for the Roundup Ready system. The yield and performance of the canola variety is also an im portant factor in the selection of the herbicide-tolerant canola system. Determining the best combination of weed management and variety performance is important to the profitability and net

returns in canola.

Current label directions indica te glyphosate agricultural herbicides can be applied postemergence on glyphosate-tolerant canola fro m emergence to the 6-leaf stage of development in spring canola varieties. To prevent early weed competition and maximize yield potential, the fi rst application of glyphosate to glyphosate-tolerant canola should be made at the two- to three-leaf stag e. An additional application can be made up to the six-leaf stage to control any late-emerging weeds. Two applications of glyphosate are made on approximately 42% of the canola ac reage (Table VIII-8). Most of the acres receiving two applications are no-till seeded acres where the first application applied is for burn down of emerged weeds and the seco nd application is in-c rop to control weeds emerging later.

.Monsanto Company 11-CA-233U 155 of 500Table VIII-9. Grass Weed Species Responses and Persistence in Soil to Herbicides Applied in Canola Production1Product Barnyardgrass Field Sandbur Foxtail, Green Foxtail, Yellow Volunteer Cereals Wild Oat Wild Proso Millet Quackgrass Herbicide PersistencePreplant IncorporatedEthalfluralinE G E E G F P-F N STrifluralinE G E E N P-F P-F N SPostemergenceClopyralidN N N N N N N N SImaxamoxE F-G E G-E G-E E G-E F NGlyphosateE E E E E G-E E E NGlufosinateE G E G F-G G-E E P NQuizalofopE E E G-E E G-E E G-E NSethoxydimE E E E E G-E E F NClethodimE E E E E E E G N1Source: (NDSU, 2005). Weed control ratings based on the following scale: E = Excellent = 90 to 99% control, G = Good = 80 to 90% control, F = Fair = 65 to80% control, P = Poor = 40 to 65 % control, and N = None = no control. Herbicide persistence ratings are for residues present12 months after application: O =Often, S = Seldom, and N = None.Monsanto Company 11-CA-233U 156 of 500Table VIII-10. Broadleaf Weed Species Responsesto Herbicides Applied in Canola Production1Product Buckwheat Cocklebur Flixweed Kochia Lambsquarters Lancelleaf Sage Mallow, Common Mallow, Venice Marshelder Mustard, Wild Nightshade, E. Black Nightshade, Hairy Redroot Pigweed Prickly Lettuce Ragweed, Common Smartweed, Annual Sunflower Thistle, Russian Wormwood, Biennial Thistle, CanadaPreplant Incorporated OnlyEthalfluralinP-F P P F-G E N - F-G N N P P E P P P N G-E N NTrifluralinP-F N P F G-E N - F-G N N N N E N P P N G N NPostemergenceClopyralidF-G E N N P-F F P P E N E E P E G-E G-E G-E P-F E EImaxamoxP G-E E E F E P P G-E E E E E - P G-E E G-E P N-PGlyphosateF-G E G-E F-E P-E E F-G E G-E G-EF-G F-G E E E F-E G-E G G-E G-EGlufosinateE E G-E E F-G E E E E E E E E G-E E E E G-E E PQuizalofopN NNNNNNNNNNNNNNNNNNNSethoxydimN NNNNNNNNNNNNNNNNNNNClethodimN NNNNNNNNNNNNNNNNNNN1Source: (NDSU, 2005). Weed control ratings based on the following scale: E = Excellent = 90 to 99% control, G = Good = 80 to 90% control, F = Fair = 65 to80% control, P = Poor = 40 to 65 % control, and N = None = no control.‘-‘ = No data availableMonsanto Company 11-CA-233U 157 of 500 VIII.G. Second Generation Glyphosate-tolerant Canola – MON 88302

Monsanto Company has developed a sec ond-generation glyphosate-tolerant canola product, MON 88302, designed to provide growers with impr oved weed control through greater flexibility of gly phosate herbicide application. MON 88302 produces the same 5- enolpyruvylshikimate-3

- phosphate synthase (CP4 EPSPS) protein that is produced in commercial Roundup Ready crop products , via the incorporation of a cp4 epsps coding sequence. The CP4 EPSPS prot ein confers tolerance to th e herbicide glyphosate, the active ingredient in the family of Roundup agricultural herbicides.

MON 88302 utilizes an improved promoter sequence to enhance CP4 EPSPS expression in male reproductive tissues, compared to the FMV 35S promoter used to drive CP4 EPSPS production in the first-generati on product, Roundup Ready canola (RT73). Glyphosate is a systemic herbicide and is tran slocated in the plant, generally in the direction of strong source ( e.g., leaf) to rapidly developing, or sink tissue. Sink tissues, such as pollen, that accumulate glyphosate and that are deficient in CP4 EPSPS expression are considered to be at risk fo r glyphosate injury. By virtue of enhanced CP4 EPSPS expression in male reproductive tissues, MON 88302 provides tolerance to glyphosate during the sensitive reproductive stages of growth, and enables the application of glyphosate at later stages of development and at higher rate s than is possible with the current product.

It is anticipated that canol a production practices with M ON 88302 will remain the same as they are for the first-generation Roundup R eady canola system. This includes the use of rotational crops, tillage systems, planting a nd harvesting operations and equipment. In addition to having similar agronomic prac tices, MON 88302 will continue to provide growers flexibility and simplicity in weed control, and allow them to realize the environmental benefits associated with the use of conservation-tillage and integrated weed management practices that are fac ilitated by the first-generation Roundup Ready canola system.

The current label directions for Roundup Ready spring canola allow for a single application of 0.39 to 0.56 lbs. of glyphosate a.e. per acre up to the 6-leaf growth stage. However, applications greater than 0.39 lbs a.e. per acre afte r the 4-leaf stage can result in crop injury. As an alte rnative, two sequent ial applications of up to 0.39 lbs. of glyphosate a.e. per acre may be applied up to the 6-leaf stage. Due to crop tolerance concerns, the total in-crop app lication of glyphosate cannot exceed 0.78 lbs. a.e. per acre. MON 88302 provides greater crop tolerance wh ich will permit higher rates and a wider period for application of glyphosate in spring canola compared to the first-generation Roundup Ready canola system. Once the gly phosate label is amended by the EPA, MON 88302 will permit two in-crop sequentia l glyphosate applications up to 0.77 lbs. a.e. per acre each in spring canola or one applic ation prior to the 6-leaf growth stage up to 1.55 lbs of glyphosate a.e. per ac re. Total in-crop applications will be increased from 0.78 to 1.55 lbs of glyphosate a.e. per acre for spring canola.

In winter varieties of Roundup Ready canola, a single application up to 0.77 lbs of glyphosate a.e. per acre can be made in the fall up to the 6-leaf growth stage. However,Monsanto Company 11-CA-233U 158 of 500 crop injury can result at appli cation rates greater than 0.56 lbs a.e. per acre prior to the 6- leaf stage in the fall. A sequential application can be made the following spring up to 0.77 lbs a.e. per acre prior to the bolting stage. Two in-crop applications not to exceed a total of 1.55 lbs. a.e. per acre of glyphosate can be made in winter varieties of the first generation Roundup Ready canola. Recomme nded application rates for glyphosate in winter canola will likely remain uncha nged with the introduction of MON 88302.

However application timing will be extended up to the first flower stage for both spring

and winter varieties with MON 88302. The higher glyphosate rates and extended timing for applications will provide improved control of weed species such as Canada thistle, dandelion, sow thistle, common lambsquarters, kochia, smartweed and wild buckwheat. The increased maximum rate limit in spring canola will provide gr eater flexibility to utilize the appropriate glyphosat e rate for difficult to control weed species when making sequential application of glyphosate

Weed competition can be a major limiting factor in canola pr oduction leading to significant yield reductions ( CCC, 2006). Certain perennial weeds, such as Canada thistle, are known to be particularly impor tant to control in canola production. For example, studies have demonstrated that onl y 10 Canada thistle plan ts per square meter have resulted in 10% yield lo ss while 40 plants per square meter have resulted in over 50% yield loss (CCC, 2006). Gl yphosate is highly effective against the majority of annual and perennial grasses and broad-leaf weeds including Canada thistle (NDSU, 2005; 2010; Padgette et al., 1996). Glyphosate has been shown to have a favorable safety profile by the U.S. EPA (1993b) which has concluded that use of glyphosate will not pose unreasonable risks to human or the environment.

VIII.H. Crop Rotation Practices in Canola

The well-established farming pr actice of crop rotation is a key management tool for growers. One of the main pur poses of growing canola in rota tion with other crops is to mitigate or break canola disease and insect cycles (Brown et al., 2008; NDSU, 2005). Including canola in wheat rotations has resulte d in 10% to 20% yiel d increases in wheat compared to continuous wheat (Brown et al., 2008). Incorporating ca nola in small grain

rotations has also decreased disease incide nce, increased quality of cereal crops and provides better herbicide opti ons for controlling certain weeds such as downy brome, jointed goat grass, wild oat, Italian ryegrass a nd feral rye, which are difficult to control in small grains (Brown et al., 2008). Although the benefits of crop rotations can be substantial, the grower stil l must make cropping decisions based on both the agronomic and economic returns of various cropping system s. Crop rotations also afford the grower the opportunity to diversify farm production in order to minimize market risks.

Growers should not plant canola following canola to avoid a bui ldup of soilborne diseases like Sclerotinia stem rot, blackle g or club root (Brown et al., 2008). Where fungal diseases are present, canola should be planted only once every four years. In addition, growers should avoid planting canola af ter other crops like legumes (pea, lentil chickpea, soybean, field and dry been) or sunf lower that are susceptible to Sclerotinia,Monsanto Company 11-CA-233U 159 of 500 Rhizoctonia and Fusarium root rots. Ca nola is most commonly grown following small grain cereals such as winter or spring wheat, barley, mai ze or sorghum (Brown et al., 2008).

Crop rotations with canola may change due to economic conditions and market opportunities. Agronomic practices such as rotations for canola vary from region to region. This section provides a detailed desc ription and quantification of the rotational cropping practices immediately following canol a, by state, that account for 99+% of the total canola acreage. These data ar e presented in Table VIII-11.

In the northern Great Plains (North Dakota and Minnesota), canola is generally grown in a 3- or 4-year rotation with small grains (wheat, barley, oats) (Hoefing – North Dakota State University, Minot - Pe rsonal Communication, 8/2/2010; Miller – Montana State University – Personal Communication, 8/27/2010) . However, flax, soybean, dry beans, dry peas and sunflower could also be adde d into the rotation on occasion. A typical 4- year rotation sequence could be; canola-wheat- flax-barley with oat, soybean or dry beans substituted for flax. Barley could also be replaced w ith wheat depending on economic conditions. A typical 3-year rotation sequen ce could be canola-wheat-wheat with barley replacing a wheat crop on occasion.

In Oklahoma, approximately 95% of the winter canola is grown in a 3-year rotation with winter wheat (wheat-canola-wheat) (Peep er, Oklahoma State University – Personal Communication, 8/3/2010). Soybean or sorghu m may follow canola in place of winter wheat in a 3-year rotation.

In the Pacific Northwest states (Idaho and Oregon), spring canola is generally grown in a 3-year rotation with wheat or barley – spring canola- winter wheat-spring cereal (wheat or barley) (Davis - University of Idaho; Wysocki – Oregon State University - Personal Communication, 7/29/2010). The rotation seque nces in winter canola could be as

follows: winter canola-winter wheat-spring cereal-fallow or winter canola-fallow-winter wheat-fallow. Spring and winter canola is almost always followed with wheat.

The majority of the U.S. canola acreage (81%) is rotated to wheat (Table VIII-11). The second largest rotational crop following canola is barley (14%). The remaining canola acres, approximately 5%, are rotated to dr y bean, field bean, fl ax, oats, sorghum, soybean, and sunflower.

Column J of Table VIII-11provides the acreage of canola in each state as a percentage of the total rotational crop acreage to indicate the level that canola is the primary crop preceding rotational crops. For the U.S., (Tab le VIII-11), this percentage is 0.5%. The percentage of canola preceding each state’s rotational crop acreage ranges from 0.1% (MN and MT) to 4.6% (ND) (Table VIII-11).

Table VIII-11 also provides an assessment of the Roundup Ready canola acreage that will be rotated to another Roundup Ready cr op in the U.S. Roundup Ready soybean is the only Roundup Ready crop rotated with canol a and it has been deregulated by the USDA. For the purposes of this assessmen t, the adoption rates used for Roundup ReadyMonsanto Company 11-CA-233U 160 of 500 soybean in 2010 were obtained from the US DA-NASS Acreage report - June 2010 (USDA-NASS, 2010a). The percentage of the total rotational crop acreage rotated from Roundup Ready canola to another Roundup Ready crop (Table VIII-11 - Column K) is 0.0005% for the U.S. and ranges from none (ID , MT, OR) to 0.04% (ND) across the canola growing states.Monsanto Company 11-CA-233U 161 of 500Table VIII-11. Rotational Practices in the U.S. Following Canola ProductionA B C D E F G H I J KStateTotalCanolaAcres1Major CropsThat FollowCanola InRotationTotal Acreageof RotationCrop in States1Percent ofRotationalCrop RotatedFrom Canola2RotationalCrop AcresFollowingCanola3PercentRotationalCrop ofTotalCanola4PercentRoundupReadyRotationalCropOption5Acreage ofRoundupReadyRotationalCropOption6Percent ofCanolaAcresPrecedingMajorRotations7EstimatedPercentageof RoundupReadyCrops asMajorRotations8UnitedStates811BarleyDry BeanField PeaFlaxOatsSorghumSoybeanSunflowerWheat35676548633173404663377451203059133Total: 154,3693.21.10.022.20.20.020.010.31.111370.277187659Total: 80913.90.90.020.90.90.11.00.981.3NANANANANANA93%NANA7.4Total: 7.40.5%0.0005%ID 15Wheat1,310Total: 1,3101.115Total: 15100NA0Total: 01.1%0%MN 13BarleyDry BeanFlaxOatsSoybeanSunflowerWheat956832507200451655Total: 9,3162.10.23.30.040.0010.20.620.10.10.10.10.110Total: 13151111180NANANANA93%NANA0.1Total: 0.10.1%0.001%MT 6.5BarleyField PeaWheat8702405520Total: 6,6300.10.040.110.15Total: 6.515283NANANATotal: 00.1%0%Monsanto Company 11-CA-233U 162 of 500Table VIII-11. Rotational Practices in the U.S. Following Canola Production (continued)A B C D E F G H I J KStateTotalCanolaAcres1Major CropsThat FollowCanola InRotationTotalAcreage ofRotationCrop inStates1Percent ofRotationalCrop RotatedFromCanola2RotationalCrop AcresFollowingCanola3PercentRotationalCrop ofTotalCanola4PercentRoundupReadyRotationalCropOption5Acreage ofRoundupReadyRotationalCropOption6Percent ofCanolaAcresPrecedingMajorRotations7EstimatedPercentageof RoundupReadyCrops asMajorRotations8ND 730BarleyDry BeanFlaxOatsSoybeanSunflowerWheat121053329535039007708680Total: 15,7388.31.32.420.20.96.711077777584Total: 730151111180NANANANA94%NANA6.6Total: 6.64.6%0.04%OK 42SorghumSoybeanWheat2504055700Total:63550.40.30.71140Total: 422.52.595NA90%NA0.9Total: 0.90.70.01%OR 4.9Wheat890Total: 8900.6 4.9Total: 4.9100NA 0Total: 00.6%0%The United States summary was developed by compiling the data from all states. All acreages areexpressed as 1000s of acres.Unlike the individual state data, the data inColumn G for the US were obtained by dividing Column F by Column B. NA denotes not applicable.1Acreage planted of the specific crops is based on 2009 planting data from the USDA-NASS, Crop Production - 2009 Summary, January 2010 (2010b).2Column E obtained by dividing Column F by Column D and multiplying by 100.3Column F obtained by multiplying Column B by Column G.4Rotational crop percentages based on estimates from personal communications with individual state canola Extension Crop Production Specialists (Hoefing, North Dakota StateUniversity, 8/2/2010; Peeper, Oklahoma StateUniversity, 8/3/2010; Davis, University ofIdaho, 7/29/2010; Wysocki, Oregon StateUniversity, 7/29/2010).5Roundup Ready rotational crop adoption rates for soybean are based on 2010 planting data taken from USDA-NASS, Acreage Report,June 2010 (2010a).6Column I obtained by multiplying Column F by Column H except as noted above for U.S.7Column J obtained by dividing Column B by Column D Total and multiplying by 100.8Column K obtained by dividing Column I Total by Column D Total and multiplying by 100.Monsanto Company 11-CA-233U 163 of 500 VIII.I. Canola Volunteer Management

Volunteer canola is defined as a plant that has germinated and emerged unintentionally during the production of a subse quent rotational crop. Canola seeds can remain in a field after canola harvest as a result of pods shatte ring before or at harvest time. Canola seeds can also remain in a field when pod placement on the plants is too close to the ground for the combine head to collect all the pods, or the combine is improperly adjusted for efficient harvesting. Canola seeds can also be transported to other fields with the harvested equipment. Volunteer canola will compete with the succeeding rotational crop and may affect yield depending on the volunteer density (NDS U, 2005). Although

canola can volunteer for seve ral years following a canol a crop, management problems with volunteer canola are not common (Boyles et al., 2009). In spring canola, seeds that remain on or near the soil surface may germinate in the fall and be killed by frost (NDSU, 2005). In winter canola, the seed typically germinates af ter summer rains and can be controlled by tillage. A Canadian study indicate s that the incidence of fields with canola volunteers in the subsequent rotation al crop is about 10% (CCC, 2005).

Precautions need to be taken to minimize canola seed losses during swathing and combining operations. With spring canola, time should be allowed in the spring for the seeds to germinate in order to control the vo lunteers with tillage prior to planting. With no-till small grains, growers should add a labe led herbicide (2,4-D) to the glyphosate preemergence application for cont rol of glyphosate-resistant canol a volunteer plants. Canola

volunteers will be controlled best when the herbicide is applied by the 5-leaf stage (NDSU, 2005). Canola volunteers become much more difficult to control with herbicides when they reach the 6-leaf to bolting stage. Table VIII-12 provides a listing of labeled

herbicides that control vol unteer glyphosate-tolerant canol a in the various rotational crops, and their effectiveness.

No changes are anticipated from the in troduction of MON 88302 on current volunteer management practices in the cul tivation of commercial canola.Monsanto Company 11-CA-233U 164 of 500 Table VIII-12. Ratings for Post-emergence Control of Volunteer Canola in Labeled Rotational Crops 1

Product Rate

Product/Acre Preemergence Canola 3-leaf Stage Canola 6-leaf Stage

Maize

Accent 0.5 oz
- E E
Balance Pro 3 fl oz E

- - Callisto 3 fl oz

- E G Option 1.5 oz - E E

Steadfast 0.75 oz
- E E Soybean Extreme 1.5 pt E - - Flexstar 0.75 pt - E E Raptor 4 fl oz - E G-E

Valor 2.5 oz E - - Dry Pea Basagran 0.5 pt - G-E F MCPA amine 0.5 pt - G-E P

Pursuit 0.72 oz G-E G-E G Raptor 4 fl oz - E G-E

Sencor 4 oz E - -

Sunflower Assert 0.8 pt - E G Spartan 4 oz P-F - -

Flax

Bronate

Advanced 0.8 pt - E F-G

MCPA ester 0.5 pt - E F-G Spartan 4 oz P-F - -

Wheat

Bronate

Advanced 0.8 pt - E F-G

2,4-D ester 0.5 pt - G-E P-G Express 0.167 oz - E G-E Harmony GT 0.3 oz - F-E P-F MCPA ester 0.5 pt - G-E F-G

Finesse 2 0.3 oz G-E

Amber 2 0.56 oz G-E Agility 2 3.2 oz G-E

1 Sources: Modified from NDSU (2005). Weed control ratings: E = Excellent (90 to 99% control), G = Good (80 to 90% control), F = Fair (65 to 80 control), and P = Poor (40 to 65% control).

2 Source: (Boyles et al., 2009). Control rating in winter wheat applied in fall or spring.Monsanto Company 11-CA-233U 165 of 500 VIII.J. Weed Resistance to Glyphosate Herbicide

The risk of weeds developing resistance and the potential impact of resistance on the usefulness of a herbicide vary greatly across different modes of action, and is dependent

on a combination of different factors. Mons anto considers product stewardship to be a fundamental component of customer serv ice and business practices, and invests considerably in research to understand the proper uses and stewardship of our herbicidetolerant soybean systems. This research includes an evaluation of the factors that can contribute to the development of weed re sistance. Detailed information regarding herbicide resistance is presented in Appendix J.

VIII.K. Stewardship of MON 88302

Monsanto Company develops effective produc ts and technologies and is committed to assuring that its products and technologies are safe and e nvironmentally responsible. Monsanto demonstrates this commitment by implementing product stewardship processes throughout the lifecycle of a product and by pa rticipation in the Excellence Through Stewardship SM (ETS) Program. These policies a nd practices include rigorous field compliance and quality management systems and verification through auditing. Monsanto’s Stewardship Princi ples are also articulated in Technology Use Guides that are distributed annually to growers w ho utilize Monsanto branded traits.

As an integral action of fulfilling this st ewardship commitment, Monsanto will seek biotechnology regulatory approvals for MON 88302 in all key canola import countries with a functioning regulatory sy stem to assure global compliance and support the flow of

international trade. These actions will be consistent with the Biotechnology Industry Organization (BIO) Policy on Product Launch. Monsanto continues to monitor other countries that are key importers of canola fr om the U.S. for the development of formal biotechnology approval processe s. If new functioning regulatory processes are developed, Monsanto will make appropria te and timely regulatory submissions.

Monsanto is committed to utilizing the best industry practices on seed quality assurance and control to ensure the purity and integr ity of MON 88302 seed. As with all of Monsanto’s products, before commer cializing MON 88302 in any country, a

MON 88302 detection method will be made ava ilable to canola producers, processors and buyers.

Glyphosate is a non-selective he rbicide registered with the U.S. EPA for the postemergence control of annual and perennial gr asses and broadleaf weeds in agriculture. Glyphosate has a long history of effective us e in U.S. crop production, including maize, soybean, small grains, sorghum, cotton and canola production. Monsanto is seeking regulatory approvals with the U.S. EPA for an amended label for use of glyphosate on MON 88302 which incorporates hi gher application rates and la ter timing for applications. Although the amended labeling increases the rate of application and widens the application period relative to canola developm ent, this use of glyphosate does not present any new environmental exposure scenarios. Glyphosate residue levels for this productMonsanto Company 11-CA-233U 166 of 500 fall within the existing U.S. and Codex Maximum Residue Leve l of 20 ppm (Codex, 2011).

As with all U.S. EPA registered herbicides fo r agricultural use, it is possible that offsite movement during and/or following application can occur such that non-target plants may be exposed to direct spray or to spray drif t. Potential impacts from drift have been mitigated through specific label use restric tions for glyphosate when applied with aerial equipment. Good management practices for application of all pesticides are also promoted by state university extension se rvices (Jordan et al ., 2009; University of Illinois, 2010).

VIII.L. Impact of the Introduction of MON 88302 on Agricultural Practices

Canola fields are typically highly managed ag ricultural areas that are dedicated to crop production. MON 88302 is likely to be used in common rota tions on land previously used for agricultural purposes. Certified seed production will continue to use wellestablished industry practices to deliver high quality seed c ontaining MON 88302 to growers. Cultivation of MON 88302 is not expected to differ from current canola cultivation using first-generation glyphosate-to lerant canola, with the exception of an opportunity to use glyphosate during an expanded window of application and at rates higher than those currently recommended and au thorized for the first- generation product.

MON 88302 is similar to conventional canola in its agronomic, phenotypic, environmental, and compositional characteristic s and has levels of tolerance to insects and diseases comparable to conventional canola (Sections V, VI and VII). Therefore, no significant impacts on current cultivation an d management practices for canola are expected following the introduction of MON 88302. Based on this assessment, the introduction of MON 88302 will not impact curre nt U.S. canola cultivation practices or weed management practices , other than intended w eed control benefits.Monsanto Company 11-CA-233U 167 of 500 IX. ENVIRONMENTAL CONSEQUENCES

IX.A. Introduction

This section provides a brief review and assessment of the plant pest potential of MON 88302 and its impact on ag ronomic practices as well as the environmental impact of the introduced CP4 EPSPS protein. USDA -APHIS has responsib ility, under the Plant Protection Act (PPA) (7 U.S.C. § 7701- 7772), to prevent the introduction and dissemination of plant pests into the U.S. Regulation 7 CFR § 340.6 provides that an applicant may petition APHIS to evaluate submitted data to determine that a particular regulated article does not present a plant pest risk and should no longer be regulated. If APHIS determines that the regulated article doe s not present a plant pe st risk, the petition is granted, thereby allowing unrestricte d introduction of the article.

According to the PPA, the definition of “plant pest” includes living organisms that can directly or indirectly injure, damage, or cau se disease in any plant or plant product (7 U.S.C. § 7702[14]).

The regulatory endpoint under the PPA for bi otechnology-derived crop products is not zero risk, but rather a determination that de regulation of the article in question is not likely to pose a plant pest risk. Information in this petition related to plant pest risk characteristics includes: 1) mode of ac tion and changes to plant metabolism; 2) composition; 3) expression and characteristic s of the gene product; 4) potential for weediness of the regulated article; 5) impacts to NTOs; 6) disease and pest susceptibilities; 7) impacts on agronomic pr actices; and 8) impacts on the weediness of any other plant with which it can interbreed, as well as the potential for gene flow.

The following lines of evidence form the basis for the plant pest risk assessment in this petition: 1) insertion of a single functional copy of the cp4 epsps expression cassette; 2) characterization of the CP4 EPSPS protein e xpressed in MON 88302; 3) safety and mode of action of the CP4 EPSPS protein; 4) co mpositional equivalence of MON 88302 seed to conventional canola; 5) phenotypic and ag ronomic characteristics demonstrating no increased plant pest potential including disease and pest suscep tibilities; 6) negligible risk to non target organisms (NTOs) and threat ened or endangered species (TES); 7) familiarity with canola as a cu ltivated crop; and 8) no greater likelihood to significantly impact agronomic practices, including land us e, cultivation practices, or the management of weeds, diseases, and insects compar ed to commercially grown canola.

Using the assessment above, the data and analys is presented in this petition leads to a conclusion that MON 88302 is unlikely to be a plant pest, and therefore should no longer be subject to regulation under 7 CFR § 340.

In 2008, APHIS proposed amendments to 7 CFR § 340 that included provisions to utilize its noxious weed authority in regulating genetically engineer ed plants (73 FR 600008). Because the data presented in this petiti on demonstrate that MON 88302 has no potential to cause injury or damage to protected interests under the noxious weed authority,Monsanto Company 11-CA-233U 168 of 500 MON 88302 would not be considered a “n oxious weed” as defined by the Plant

Protection Act.

IX.B. Plant Pest Assessment of MON 88302 Insert and Expressed Protein

This section summarizes the details of the genetic insert, characteristics of the genetic modification, and safety and expression of the CP4 EPSPS protein expressed in MON 88302 used to evaluate the food, feed, and environmental safety of MON 88302.

IX.B.1. Characteristics of the Gene tic Insert and Expressed Protein

IX.B.1.1. Genetic Insert

MON 88302 was developed through Agrobacterium -mediated transformation of canola hypocotyls utilizing the plasmid vector PV-BNHT2672. PV-BNHT2672 contains one transfer DNA (T-DNA) that is delineated by Le ft Border and Right Border regions. The

T-DNA contains the cp4 epsps coding sequence under the control of the FMV/Tsf1 chimeric promoter, the Tsf1 leader and intron sequences, and the E9 3 ′ untranslated region. The chloroplast transit peptide CTP2 directs transport of the CP4 EPSPS protein to the chloroplast a nd is derived from CTP2 target sequence of the Arabidopsis thaliana

shkG gene (Figure III-1; Table III-1). The ch aracterization of the genetic modification demonstrated that a single copy of the T- DNA was inserted at a single locus of the genome, and no additional genetic elemen ts, including backbone sequences, from PV-BNHT2672 were detected in MON 88302 (F igures IV-2 to IV-6). DNA sequence analyses confirmed integrity of the inserted cp4 epsps expression cassette and identified

the 5’ and 3’ insert-to- genomic DNA junctions.

Additional sequence analysis of conventi onal canola genomic DNA confirmed a 9 base pair insertion immediately adjacent to the 3' end of the MON 88302 insert and a 29 base pair deletion immediately adja cent to the 5' end of the MON 88302 from the conventional genomic DNA. Those changes most likely occurred in MON 88302 upon insertion of the T-DNA. Such changes are quite common dur ing plant transforma tion; these changes presumably resulted from double-stranded brea k repair mechanisms in the plant during

the Agrobacterium -mediated transformation process (Salomon and Puchta, 1998). Finally, Southern blot analysis demonstrat ed that the DNA fingerprint of the T-DNA

insert in MON 88302 has been maintained through multiple genera tions of breeding, thereby confirming the stability of th e insert in multiple generations.

These data demonstrate that there are no unintended changes in the MON 88302 genome

as a result of the insertion of the cp4 epsps expression cassette.

IX.B.1.2. Mode of Action

MON 88302 produces the same 5-enolpyruvylshikimate-3

- phosphate synthase (CP4 EPSPS) protein that is produced in commercial Roundup Ready crop products, including the first generation product Roundup R eady canola, which confers tolerance to glyphosate. In most plants, glyphosate binds to the endogenous EPSPS enzyme and blocks the biosynthesis of EPSP thereby de priving the plant of essential amino acidsMonsanto Company 11-CA-233U 169 of 500 (Steinrücken and Amrhein, 1980). In Roundup Ready plants, requirements for aromatic amino acids and other metabolites are met by the continued action of the CP4 EPSPS enzyme in the presence of glyphos ate (Padgette et al., 1996).

IX.B.1.3. Expressed Protein Safety

Several Roundup Ready crops that produc e the CP4 EPSPS protein have been deregulated by the USDA and been review ed during the biotec hnology consultation process by the FDA. The CP4 EPSPS protei n expressed in MON 88302 is identical to the CP4 EPSPS proteins in other R oundup Ready crops including Roundup Ready soybean, Roundup Ready 2 Yield soybea n, Roundup Ready corn 2, Roundup Ready canola, Roundup Ready sugar beet, Roundup Ready cotton, Roundup Ready Flex cotton and Roundup Ready alfalfa. The safety of CP4 EPSPS proteins present in such biotechnology-derived crops has been extens ively assessed (Harri son et al., 1996). Results from the protein char acterization studies included in this petition confirmed the identity of the MON 88302-produced CP4 EPSPS protein and established the equivalence of MON 88302- produced protein to the E. coli -produced CP4 EPSPS protein (Section V.B.) used previous ly to demonstrate the safe ty of the CP4 EPSPS protein produced in other Roundup Ready crops. The Environmental Protection Agency (EPA) also reviewed the safety of the CP4 E PSPS protein and has esta blished a tolerance exemption for the protein and the genetic materi al necessary for its production either in or on all raw agricultural commodities (U.S. EPA, 1996a). This exemption was based on a safety assessment that incl uded rapid digestion in simula ted gastric fluids, lack of homology to known toxins and allergens, and l ack of toxicity in an acute oral mouse gavage study. A history of safe use is s upported by the lack of any documented reports

of adverse effects since the introduction of Roundup Ready crops in 1996. Therefore, it is concluded that the CP4 EPSPS protein poses no risk to human or animal health.

IX.B.1.4. Protein Expression Levels

CP4 EPSPS protein levels in MON 88302 ranged from 22 to 500 μg/g dw in root, forage, harvested seed and over-season leaf (Section V.C.). The levels of CP4 EPSPS from all

tissues tested and across all sites are co mparable to other commercialized Roundup Ready crops containing the CP4 EPSPS protein that have been cleared for environmental release by regulatory agencies around the world.

IX.B.2. Compositional Characteristics

Compositional analyses were conducted as recommended for canola seed (OECD, 2001) to assess whether levels of key nutrients, toxicants, and anti-nutrients in MON 88302 were equivalent to levels in the conve ntional control and to the composition of commercial reference varieties (Section V I). The background genetics of the conventional control (Ebony) we re similar to that of MON 88302, but did not contain the cp4 epsps expression cassette. Seed was harveste d from five individual sites in which MON 88302, the conventional control, and a range of commercial reference varieties were grown concurrently in the same field tria l. The commercial re ference varieties were used to calculate a 99% tolerance interval for each compositional component to defineMonsanto Company 11-CA-233U 170 of 500 the natural variability of each component in canola varieties that have a history of safe consumption.

Nutrients assessed in this analysis in cluded proximates (ash, carbohydrates by calculation, moisture, protein, and total fat), fi bers (acid detergent fiber [ADF], neutral detergent fiber [NDF], and total dietary fibe r [TDF]), amino acids (18 components), fatty acids (FA; C8-C24,), vitamin E (  -tocopherol), and minerals (calcium, copper, iron, magnesium, manganese, phosphorus, potassium , sodium, and zinc) in seed. The

toxicants assessed in seed included erucic aci d and glucosinolates (a lkyl glucosinolates [including 3-butenyl, 4-pe ntenyl, 2-hydroxy-3-butenyl , and 2-hydroxy-4-pentenyl glucosinolates], indolyl glucosinolates [including 3-indolylmethyl and 4-hydroxy-3- indoylmethyl], and total glucosi nolates). The anti-nutrients assessed in seed included phytic acid and sinapine (as sinapic acid). All components analyzed in MON 88302 seed were either not statistically significantly different ( α = 0.05) compared to the conventional control, or, if significantly differe nt, were within the natural variability of canola composition as expressed in the 99 % tolerance interval. Collectively, the compositional analyses data support the conc lusion that MON 88302 is as safe as and compositionally equivalent to conventional canola.

IX.B 3. Phenotypic and Agronomic and En vironmental Interaction Characteristics

An extensive and robust set of information and data were used to assess whether the introduction of the glyphosate-tolerance tra it altered the plant pest potential of MON 88302 compared to the conventional control. Phenotypic, agronomic, and

environmental interaction char acteristics of MON 88302 were evaluated and compared to

those of the conventional cont rol and considered within the variation among commercial

reference varieties. These assessments in cluded five general data categories: 1) germination, dormancy, and emergence; 2) vegetative growth; 3) reproductive growth (including pollen characteristics) ; 4) seed retention on the pl ant and lodging; and 5) plant response to abiotic stress and interactions w ith diseases and arthropods. Results from the phenotypic, agronomic, and environmental interaction assessment demonstrate that MON 88302 does not possess: a) increased w eediness characteristics; b) increased susceptibility or tolerance to specific abiotic stresses, dis eases or arthropods; or c) characteristics that would confer a plant pest risk or a significant environmental impact compared to the conventional control. Taken together, the results of the analysis support

a determination that MON 88302 is no more likel y to pose a plant pest risk or have a biologically meaningful change in environmental impact than conventional canola.

IX.B.3.1. Seed Dormancy and Germination

Seed germination and dormancy mechanisms va ry with species and their genetic basis tends to be complex. Seed dormancy is an important characteristic that is often associated with plants th at are considered weeds (A nderson, 1996; Lingenfelter and Hartwig, 2007). There were no changes in th e dormancy or germina tion characteristics that are indicative of increased plant w eediness or pest pot ential of MON 88302 compared to the conventional control (Section VII.C.1.).Monsanto Company 11-CA-233U 171 of 500 IX.B.3.2. Plant Growth and Development

Evaluations of plant growth and development characteristics in the field are useful for assessing potential weediness characteristics such as lodgi ng and pod shattering (Section VII.C.2.). Phenotypic characteristics such as early stand count, seedling vigor, days to first flowering, lodging, plant height, visual rating for pod shattering, quantitative pod shattering, seed maturity, seed quality, yield, seed moisture, and final stand count were assessed. Of the growth and development characteristics assessed between MON 88302 and the conventional control, no statistically si gnificant differences we re detected at a 5% level of significance ( α = 0.05) with the exception of days to first flowering and harvested

seed moisture in a combined-s ite analysis of the data. The mean values of MON 88302 were within the natural variability of the commercial reference varieties for these characteristics. Therefore, the differences in these parameters are not considered to be biologically meaningful in te rms of increased weediness or plant pest potential of MON 88302 compared to conventional canola.

IX.B.3.3. Response to Abiotic Stressors

No biologically meaningful differences were observed during comparative field

observations between MON 88302 and the conven tional control and th eir response to abiotic stressors, such as drought, wind, nutrien t deficiency, etc. (Sec tion VII.C.2.). The lack of significant biological differences in plant responses to abiotic stress support the conclusion that the introduction of the glyphosat e-tolerance trait is unl ikely to result in increased plant pest potential or an adve rse environmental impact from MON 88302 compared to conventional canola.

IX.B.3.4. Pollen Morphology and Viability

Evaluations of pollen morphology and viability of non-glyphosate treated plants provide information useful in a plant pest assessment as it relates to the poten tial for gene flow to, and introgression of, the biotechnology-derived trait into other canol a varieties and wild relatives (Section VII.C.3.). Pollen morphol ogy and viability evaluations demonstrated no statistically significant differences ( α = 0.05) between MON 88302 and the conventional control. Taken together, thes e comparative assessments indicate that MON 88302 is not fundamentally different fr om conventional canola or more or less likely to have increased weedin ess or plant pest potential.

IX.B.3.5. Interactions with Non-target Organisms Including Threatened and Endangered Species

Evaluation of MON 88302 for potential advers e impacts on non target organisms (NTOs) is a component of the plant pest risk asse ssment. The safety of CP4 EPSPS proteins present in biotechnology-derived crops has be en extensively assessed (Harrison et al., 1996; ILSI-CERA, 2010). A mouse gavage st udy demonstrated no acute oral toxicity (Harrison et al., 1996), and consequently th e low potential for impact to terrestrial vertebrate NTOs including threatened and en dangered vertebrate species. Impacts of deregulation of MON 88302 to NTOs and th reatened and endangered species areMonsanto Company 11-CA-233U 172 of 500 discussed in greater detail in Appendix I. Data from the 2009 North American phenotypic and agronomic study including observational data on environmental interactions such as plant-disease in teraction, arthropod da mage and arthropod abundance, were collected at select site s for MON 88302 and the conventional control (Section VII.C.2.; Appendix G). These resu lts support the conclusion of no adverse environmental impact from cultivation of MON 88302 to non-target organisms and no increased incidence of disease in MON 88302. Additionally, after numerous environmental risk assessments on a range of plant species expressing the CP4 EPSPS protein, data indicate no correlation betw een CP4 EPSPS protein expression and any increased tendency for persistence or spread in the environment, alterations in reproductive biology affecting ge ne flow, or negative impact s on other organisms in the environment (ILSI-CERA, 2010).

MON 88302 produces the identical CP4 EPSP S protein as is found in commercial Roundup Ready crop products including Roundup R eady canola (RT73). This protein confers tolerance to glyphosate. The CP4 EPSPS protein is a member of the larger family of EPSPS proteins that are ubiquitous in plants and microbes in the environment,

and have been isolated from both sources (Harrison et al., 1996; Klee et al., 1987; Schönbrunn et al., 2001; Steinrücken and Amrhei n, 1984). The CP4 EPSPS protein is an enzyme in the shikimate metabolic pathwa y in plants and microorganisms and is important in the production of aromatic ami no acids that are essential to growth and development. The mode of action of this fa mily of proteins is well known (Alibhai and Stallings, 2001).

The shikimate pathway and EPSPS enzyme are found in plants and microorganisms but are absent in animals including mammals, fis h, birds and reptiles (Alibhai and Stallings, 2001; Haslam, 1993). This explains the sele ctive activity of glyphosate in plants and contributes to the low animal toxicity of glyphosate. Additionally the U.S. EPA has concluded that, when used according to label directions, glyphosate does not pose

unreasonable risks or adverse effects to humans or the environment and has a favorable NTO safety profile (U.S. EPA, 1993a). APHIS also concluded in the final environmental impact statement for glyphosate tolerant alfalfa (USDA-APHIS, 2010) that glyphosate does not pose a risk to birds, mammals, fi sh, terrestrial invert ebrates or aquatic

invertebrates. APHIS further concluded that potential impacts to non-target plants may occur from drift resulting fr om the aerial application of glyphosate, but that these potential impacts have been mitigated th rough specific label us e restrictions for glyphosate when applied with aerial equipm ent. Good management practices for application of all pesticides are also promoted by state un iversity extension services (Jordan et al., 2009; University of Illinois, 2010). In addition, the majority (> 60% in 2008 (Zollinger et al., 2009) of U.S. canola ac res are planted with glyphosate-tolerant canola and are routinely tr eated with glyphosate.

Taken together, these data support the conclusion that MON 88302 has no reasonable mechanism for harm to NTOs, or impact to threatened and endangered species compared

to the cultivation of commercial canola. In addition, the U.S. EPA has taken measures (specified label language regarding glyphosat e application) to minimize any potential effect to nontarget plants.Monsanto Company 11-CA-233U 173 of 500 IX.C. Weediness Potential of MON 88302

Brassica napus is generally regarded as an oppor tunistic species, that is, a species adapted to take advantage of temporary condi tions such as distur bed areas (CFIA, 1994), not as an environmentally hazardous coloni zing (EC, 2000) or invasive species in undisturbed natural ecosystems (C rawley et al., 2001). Although B. napus has some characteristics typical of weedy species such as a high reproduc tive capacity, rapid growth and multiple pollination mechanisms (self, wind, insect), it also has many characteristics typical of domesticated speci es including low genetic diversity, lack of long-distance seed dispersal mechanisms, lim ited population persistence, lack of primary seed dormancy and an inability to compete well with perennial species (Hall et al., 2005). Brassica napus is not listed as an invasive weed in the Catalog of Invasive Plant Species of the U.S. (NYBG, 2011), nor is it pres ent on the lists of noxious weed species maintained by the federal government (7 CFR § 360).

Brassica napus has been documented to be present in disturbed areas such as roadsides and railways used for transporta tion of seed and the margins of fields where it has been previously grown (Aono et al., 2006; Knispel et al., 2008; Nishizawa et al., 2009; Pivard et al., 2008; Saji et al., 2005). However, popula tions of canola outside agricultural fields do not effectively compete with perennial ve getation, and usually persist only for a few years in the absence of ongoi ng seed introductions into areas from spillage during handling and transport or processing (B aker and Preston, 2008; Crawley and Brown, 1995; 2004; Crawley et al., 1993; Kn ispel et al., 2008). The pe rsistence of canola outside the agroecosystem has been studied extensively and is summarized in Table IX-1. Canola plants outside agricultural fields can produce seed (Crawley and Brown, 1995; Knispel et al., 2008) but this is often prevented because most plants do not survive to maturity. This is due to competition from other vegetation (Crawley and Brown, 1995), management operations such as roadside mowi ng, the use of broadleaf herbicides, animal predation, diseases and environmental conditi ons (Crawley and Brow n, 1995; Knispel et al., 2008; Norris and Sweet, 2002; Yoshimura et al., 2006). Additionally, biotechnologyderived canola populations have not been found to be more invasive or more persistent than conventional canola populations (Crawley et al., 2001).

The viability of the large majority of canola se ed in soil declines over time (Gulden et al., 2003; Gulden et al., 2004; Hails et al., 1997). Biotechnology-d erived canola seed has not been demonstrated to persist longer than conventional canol a seed (Gruber et al., 2004; Hails et al., 1997). However, canola seed can persist in the so il for several years becoming secondarily dormant, and then germ inate as volunteers (G ruber et al., 2004). Canola that has germinated and emerged uni ntentionally in a subs equent crop, also known as volunteer canola, may compete with the succeeding rotational crop. However, problems controlling volunteer canola are not common (Boyles et al., 2009). Volunteers, including volunteers with herbicide-tolerant traits, can be managed with pre-plant or selective post-emergent herbicide applicati ons or by mechanical means (Boyles et al., 2009; EC, 2000).

In comparative studies between MON 88302 a nd the conventional control, phenotypic, agronomic and environmental interaction da ta were evaluated (Section VII.C.2.) forMonsanto Company 11-CA-233U 174 of 500 changes that would impact the plant pest pot ential, and, in particular, plant weediness potential. Results of these evaluations s how that there is no biologically significant

difference between MON 88302 and the conven tional control for traits potentially associated with weediness such as germ ination, dormancy, reproductive growth and pollen characteristics, response to abiotic stress and disease or pest susceptibilities. Furthermore, comparative field observati ons between MON 88302 and the conventional control and their response to abiotic stressors, such as cold, drought, heat, nutrient deficiency, soil compaction, wet soil, and wind, indicated no differences and, therefore, no increased weediness potential. Collectivel y, these findings support the conclusion that MON 88302 has no increased weed potential co mpared to conventional canola and it is no more likely to become a weed than conventional canola.Monsanto Company 11-CA-233U 175 of 500Table IX-1. Summary of Literature on the Persistence of Canola Outside of Cultivated FieldsReference Methods Results(Nishizawa et al.,2009)Three years of monitoring forcanola on roadsides along routes fortransportation of canola seed fromthe shipping port of Kashima (1 of11 shipping ports for canola seed inJapan). Plants ofB.junceawerealso observed, and tested for thepresence of glyphosate- andglufosinate-tolerance proteins andgenes.The number of plants foundeach year; along a 20 km section ofroadway varied from 2162 plants in2005, 4066 plants in 2006 and 278 plants in 2007.Brassicanapuspopulations were found alongroadsides or sidewalks, with somefound in vacant lots and flowerbeds. Most plants found wereB.napusbut a fewB.junceaplants were also observed. In all three years herbicide-tolerant canolaplants were found, with the percentage of canola plants with either the glyphosate- or glufosinatetolerance trait varying from 0 to 1.8%. The authors concluded that no massive expansion or longterm persistence of canola populations had occurred and that environmental impact from canola seedspillage along transportation routes would be negligible.(Baker andPreston, 2008)Monitoring of canola seedpersistence in managed fields inAustralia for up to 3.5 years afterthe crop was grown over 3geographical regions from a total of66 fields. Canola seeds wereisolated and counted from soilsamples taken from commercialfields.The overall mean number of seeds recovered per meter squared that germinated was 3% for seedcollected 6 months after harvest and for seed collected 1.5 and 2.5 years after the last canola crop, itwas 1.4% and 1.5%, respectively. No seed collected3.5 years after the lastcanola crop germinated.Seed viability was not tested in the study.(Knispel et al.,2008)Observations of canola plantdensities along field edges androadways in Manitoba Canada from2004 to 2006. Sampling of seedfrom 16 canola populations andtesting for herbicide tolerance. Soilsampling and testing for canolaseed viability and dormancy.Many canola plants along roadways died from mowing and most did not survive to set seed. Only25% of the populations had plants that set seed over 2 years, and only one population set seed in all3 years of the study. Herbicide-tolerant canola plants were detected at a high frequency in thepopulations sampled, and evidence of pollen-mediated gene flow of herbicide-tolerance traits wasdetected. Soil sampling from along roadways indicated that the seedbanks were small (less than 5germinable seeds per square meter) and had little dormancy. The authors concluded that canolapopulations outside of cultivated areas are highly transient.Monsanto Company 11-CA-233U 176 of 500Table IX-1. Summary of Literature on the Persistence of Canola Outside of Cultivated Fields (continued)Reference Methods Results(Pivard et al.,2008)Observations of canola alongroadsides and field margins inFrance in a large-scale study over 4years. Regression modeling wasused to estimate the contribution ofecological processes involved incanola presence and persistence.The presence of 35–40 % of the ruderal canola populations was associated with seed immigrationfrom neighboring fields. Approximately 15 % of the populations were attributed to seed lossesduring transportation. The presence of no more than10 % of the populations was attributed to seedproduced by ruderal canola populations in the previous year. The remaining canola populations, 35–40 %, were attributed to recruitment from seed that had been in the seed bank for 1 or more years.(Aono et al., 2006)Screening of seed collected fromcanola (B. napus),B.junceaandB. rapaplants from 95 sites from 7port areas in Japan for herbicidetolerance.No biotechnology-derived seeds were found inB. junceaandB. rapasamples. Seeds were collectedfrom canola plants and tolerance to glyphosate and glufosinate was found. A portion of the progenyof two individual plants had both the glyphosate-tolerance and glufosinate-tolerance traits.(Yoshimura et al.,2006)Monitoring of sites inSaskatchewan, where canola isgrown, and in Vancouver, BritishColumbia, where most canoladestined for export is transported byrail. Observations at both areas ofrailway and along side roads forBrassicaspecies were conductedPlants were tested for glyphosateand glufosinate herbicide-toleranceproteins.The glyphosate and glufosinate tolerance traits were found in approximately 2/3 of the canola plantssampled in Saskatchewan and British Columbia.In Saskatchewan and Vancouver plants ofcultivated (163 samples) and weedy relatives of canola (223 samples) were screened for glyphosateand glufosinate tolerance traits. These relatives included wild mustard (Sinapisarvensis), orientalmustard (B.juncea), dog mustard (Erucastrumgallicum) and bird’s rape (B. rapa). Among thesesamples only a singleB. rapaB.napushybrid with the glyphosate-tolerance trait was detected inBritish Columbia.Monsanto Company 11-CA-233U 177 of 500Table IX-1. Summary of Literature on the Persistence of Canola Outside of Cultivated Fields (continued)Reference Methods Results(Lutman et al.,2005)Comparison of seed losses byshattering at harvest and persistencein soil of glyphosate-, glufosinateand imidazolinone-tolerant andconventional canola varieties at 5sites over 4 years.No difference between glyphosate-tolerant, glufosinate-tolerant, imidazolinone-tolerant, andconventional canola varieties for seed shattering at harvest and seed persistence in soil. The numberof canola seeds of all varieties declined rapidly inthe soil in the first few months after harvest with amean loss of 60 %. The numberof canola seeds in the soil declined by approximately 20 % per yearfor the remainder of the experiment.(Saji et al., 2005)Collection of seeds from canola,B. rapaandB.junceafoundgrowing near ports, along roadsidesand along riverbanks. Seeds weregrown and tested with either ofglufosinate or glyphosate herbicide,and survivors were tested forpresence of both herbicidetolerance proteins and genes.Seeds were collected from 7500 feralB. napus, 300B. rapaand 5800B. junceaplants from 143locations. No herbicide-tolerance genes or proteins were detected inB. rapaorB.junceanor inB. napusplants found along riverbanks.(Crawley andBrown, 2004)Survey ofB. napuspopulations in100-m quadrants along roadside inthe U.K. for 10 years (1993–2002)Seed spillage from trucks was a significant contributor to the observedB.napuspopulations alongthe roadside. Plants were mostly observed within 1 m of the roadside and seldom in dense grass orin deep shade. In the years following road worksB. napuspopulation densities were significantlyhigher in 9 of 12 cases. Overall the populationswere not self-replacing. Canola populations wereextinct after 1 to 4 years. The most frequent pattern was for quadrants to have canola present for 1to 2 years out of 10 with only 1 % of quadrants surveyed having one or more canola plants in all 10years.(Gruber et al.,2004)Study of seed viability after burialin two separate years in the UK,with evaluation of seed viability ofconventional and transgenic seedsafter burial.No differences between biotechnology-derived and the near isogenic counterparts were found forseed survival in soil. Mean canola seed viability of buried seed was 66 % and 33 % for the first andsecond years of the study, respectively.Monsanto Company 11-CA-233U 178 of 500Table IX-1. Summary of Literature on the Persistence of Canola Outside of Cultivated Fields (continued)Reference Methods Results(Gulden et al.,2004)Laboratory and field-based studiesof canola secondary dormancy andseed persistence in soil.In the field experiments, canola viability after 4 weeks in soil ranged from 0.1% to 6.3%. Canolaseedling recruitment from seed in the shallow soil seedbank was low, ranging from 0.1% to 1.5%.The authors conclude that germination may have contributed to loss of seed from the deep buriedseedbank but because seedlings were not recruited from the deep seedbank depth (10 cm)germination at these deeper depths must have been lethal.(Gulden et al.,2004)Seeds were planted at two sites inCanada and persistence wasmeasured each spring for 3 years.Different canola varieties persisted for different lengths of time. After 1, 2 and 3 winters themaximum persistence was 44, 1.4 and 0.2 % of the original buried seeds, respectively, over allvarieties. In 2001, canola genotypes with high potential for dormancy tended to exhibit 6- to 12-foldgreater persistence than medium dormancy potential canola genotypes, indicating lower seedbankmortality in high potential dormancy varieties canola.(Norris and Sweet,2002)Monitoring along field margins of aglufosinate-tolerant canola trial inthe UK which had not previouslybeen cropped to biotechnologyderived canola.Only one persistent population of feralB.napus was found at any site in any year. The populationpersisted for 3 years. In the third year of monitoring all of the plants in the population were mowedlate in the season, and no plants were found in the fourth year of monitoring. Feral populations ofoilseed rape were rare in this study, and where populations were found in one year, these did notgenerally persist to following years. Individual plants growing in roadsides and verges rarelysurvived to maturity and set seed. Many factors were involved in the mortality of these plants, themain ones being competition with perennial weeds (especially grasses), predation by animals (slugs,birds and insects) and susceptibility to diseases of canola.(Crawley et al.,2001)Conventional and biotechnologyderived canola planted at 12habitats and monitored for 10 years.No difference in recruitment between conventional and biotechnology-derived canola. None of theconventional or biotechnology-derived canola increased in abundance. Biotechnology-derivedcanola did not persist longer than conventional canola. Establishment of seedlings was lower forbiotechnology-derived canola than conventional at 6 of the 12 sites,and was not significantly greaterat any site. Survival of biotechnology-derived canola relative to conventional canola was lower in 3of 12 cases and higher in 2 of 12 cases. All populations were completely extinct after 4 years.Monsanto Company 11-CA-233U 179 of 500Table IX-1. Summary of Literature on the Persistence of Canola Outside of Cultivated Fields (continued)Reference Methods Results(Hails et al., 1997)Seed burial studyfor 2 years at 12four habitats (sites) in each of 3different geographic sites in the UK(12 total habitats). The source ofcanola seed was the variety Westarfor the conventional andbiotechnology-derived lines (oneantibiotic-tolerant, one herbicidetolerant and a third with both traits).A wild relative,Sinapis arvensis(charlock or wild mustard), wasalso included.After burial in soil only 1.5 % of the canola seedspersisted after the firstyear and 0.2% after thesecond year at all sites. The survival rateof biotechnology-derived seeds was lower than theconventional seeds at both burial depths at all 12 habitats. However, when individual habitats wereexamined this trend was only detectable at 5 habitats and no detectable difference at the other 7habitats. The weedS. arvensispersisted significantly longer than any of the canola seeds in allhabitats and sites.(Crawley andBrown, 1995)Monitoring for canola plants inroad verges adjacent to the M25highway (UK).Canola plants were not seen in dense grass. Populations established by seed losses duringtransportation in the first year of the study were extinct within 4 years. In the absence of disturbancelocal canola populations were typically extinct within 2–4 years. In the year following soildisturbance canola densities were often high.(Crawley et al.,1993)Experiment to examine whetherbiotechnology-derived herbicidetolerance affects the likelihood ofB. napusbecoming invasive ofnatural habitats.No evidence of invasiveness of canola in undisturbed natural habitats. No difference in invasivenessor persistence between biotechnology-derived and conventional varieties. Local canola populationsmeasured were declining and localextinction in all habitats was likely. The authors concluded thatthere is no evidence thatB. napusis invasive of undisturbed natural habitats.Monsanto Company 11-CA-233U 180 of 500 IX.D. Potential for Pollen Mediated Gene Flow and Introgression

Pollen mediated gene flow (often referred to as cross pollination) occurs when pollen of one plant fertilizes ovules of a second plant. Pollen mediated gene flow is affected by

both biotic and abiotic factors such as plant biology, pollen bi ology/volume, plant phenology, overlap of flowering times, proximity of the pollen source and sink, ambient conditions such as temperature and humid ity and field architecture. Because pollen mediated gene flow is a natural biologic al process, it does not constitute an

environmental risk in and of itself.

Introgression is a process wher eby one or more genes successfully incorporate into the genome of a recipient plant. Pollen mediated gene flow and gene introgression must be considered in the context of the transgenes inserted into the biot echnology-derived plant, and the likelihood that the presen ce of the transgenes and th eir subsequent transfer to recipient plants and plant populations will resu lt in increased plant pest potential. The potential for gene flow and introgression from deregulatio n of MON 88302 is discussed in greater detail below.

IX.D.1. Potential for Pollen Mediated Gene Flow and Introgression of B. napus

Brassica napus is predominantly self-pollinating alt hough interplant (plants are touching one another) cross pollination rates range from 12% to 55% with a mean of 30% (Beckie et al., 2003). Pollen of B. napus is heavy and sticky (OECD, 1997) and pollen movement is primarily by insects, such as honey bees (Thompson et al., 1999) although wind is also responsible for some pollen movement. Most (98.8%) of pollen travel s less than twelve meters from its source (Scheffler et al., 1993) although dispersal due to pollinators may occur over greater distances at low frequency (Thompson et al., 1999). In general, the percentage of pollen flow and potential for cross pollination diminishes with increasing distance from the source (Table IX-2). This information is useful for managing pollen flow during canola breeding, seed produc tion and for identity preservation.

Brassica napus produces a large amount of pollen (OGTR, 2008) which can remain viable up to four to five days under field conditions (R antio-Lehtimäki, 1995). This, coupled with the potential for B. napus pollen movement, suggests the possibility for hybridization between B. napus and related species.Monsanto Company 11-CA-233U 181 of 500 Table IX-2. Summary of Representative Studies of Brassica napus Cross Pollination

Rates at Various Distances

Reference Distance from Pollen Source (meters) % Cross Pollination Observed (Stringam and Downey, 1982) 47

137

366 2.1

1.1

0.6 (Manasse and Kareiva, 1991) 50

100 0.022

0.011 (Scheffler et al., 1993) 0

1

3

6

12

24

36

47

70 4.8

1.4 – 1.6

0.35 – 0.4

0.033 – 0.11

0.016 – 0.025

0.0 – 0.0041

0.0011 – 0.0031

0.0 – 0.00034

0.0 (Morris et al., 1994) 0

0.3

0.6

3

4.6 ~2.0 – 3.5 §

~1.0 – 1.5 §

~0.75 – 1.2 §

~0.5 – 0.75 §

~0.5 – 0.7 §

(Scheffler et al., 1995) 200

400 0.0156

0.0038 (Downey, 1999) 33

66

100 0.1 – 1.5

0.0 – 0.4

0.1 – 0.4 (Staniland et al., 2000) 0

2.5

5

10

15

20

25

30 0.69

0.29

0.14 φ

0.07 φ

0.08 φ

0.07 φ

0.04 φ

0.02 φ (Rieger et al., 2002) 100

1500

>3000 0.012 – 0.014

0 – 0.197

None detected

§ Frequencies estimated from Figure within the publication.

φ Values calculated fro m publication.Monsanto Company 11-CA-233U 182 of 500 IX.D.2. Potential for Pollen Mediated Gene Flow and Introgression of B. napus to Closely Related Brassica Species

A survey of the literature provides informa tion on the potential for hybridization (cross pollination) and introgression from B. napus to related species. Reports of unassisted (without human intervention or as sistance) field hybridization of B. napus to related species provide biologically relevant inform ation that can be useful in predicting the potential for hybridization under field conditions. However, the majority of the published information used to assess hybridization of B. napus with related species is from the breeding literature where human assistance has occurred. Often these studies involve emasculation or male-sterility of the female parent, assisted pollination by hand or the use of cages and high numbers of pollinators ( e.g., bees). In addition, cross-species hybrids have also been obtained using in vitro methods like embryo or ovary culture. Although, methods using assistance or in vitro techniques may not be indicative of the possibility of hybridization potential under fi eld conditions, they do provide information about hybrid viability, fertility, chromosome behavior and the potential for introgression into a B. napus relative, if hybrids were to form.

The frequency of hybridization between B. napus and several of its relatives is often

more successful when B. napus is the male parent in some crosses or the female parent in

others. This directional bias has been obser ved in a number of studies (Scheffler and Dale, 1994). In the case of MON 88302, transfer of characteristics from other species to B. napus is not a primary concern in evaluating environmental impacts since it is highly unlikely that transfer of characteristics wi ll result in an increase in the weediness of B. napus . If such a transfer were to occur, the resulting hybrids will likely be present in cultivated fields and subject to the same agricultural management practices as existing

herbicide-tolerant canola varieties that would c ontrol their ability to grow and reproduce. Thus, for the purposes of MON 88302 risk assessment, hybridizations in which B. napus is the male parent were carefully reviewe d. There are reports of hybridization under field

conditions with B. napus as the pollen donor w ith six species including B. rapa

, B. juncea

, B. oleracea

, H. incana

, R. raphanistrum and S. arvensis (Table IX-3). The species B. rapa

, B. juncea and B. oleracea are cultivated for crop production. The other species listed, H. incana

, R. raphanistrum and S. arvensis are not cultivated for crop production, but are found in the environment. In all cases the resulting hybrids had decreased environmental fitness evidenced by a variety of characteristics including decreased pollen viability, seed production, seedling survival, etc . when compared to parental varieties.

Brassica rapa

Brassica rapa is widespread throughout temperate North America, and may be found in areas where canola is grown (BONAP, 2010a). Brassica napus (AACC) was derived from the hybridization of diploid species, B. rapa (AA) and B. oleracea (CC) (Section II). Therefore B. napus and B. rapa have a common set of chromosomes, are known to be sexually compatible, and can form hybrid s under field conditions (Bing et al., 1991; Warwick et al., 2003). For pollination, B. rapa is self-incompatible, and is not likely to form hybrids when in close proximity to con- specifics (members of the same species)Monsanto Company 11-CA-233U 183 of 500 (Warwick et al., 2003). Hybr idization frequencies between B. napus and B. rapa in neighboring fields or inter-pla nted in large plots vary fr om study to study, ranging from 0% up to 69% (as reviewed in (Devos et al ., 2009), but can be as high as 93% (Jørgensen et al., 1996). For example, Warwick (2003) measured hybridization frequencies between these two species with B. rapa as the maternal parent in field experiments, including

natural environments and commercial B. napus fields. The higher ra te of hybridization in commercial fields (13.6%) vs. natu ral environments (7%) is due to the higher pressure of B. napus pollen on isolated plants of self-incompatible species, like B. rapa , favoring inter-specific hybridization. Further eviden ce for this were th e lower hybridization frequencies (0.4% to 1.5%) when B. napus was the male parent with B. rapa female parent plants located outside of cultivated fields of B. napus (Scott and Wilkinson, 1998).

F 1 hybrids between B. napus and B. rapa had reduced pollen viability with an average viability of approximately 50% (Warwick et al., 2003). F 1 hybrids between B. napus and B. rapa produced approximately half as many germinable seeds as their respective parents, and had very low (< 2%) hybrid s eedling survival rates (Scott and Wilkinson, 1998). Additionally, unlike the B. rap a parent, under field conditions, seeds of F 1 hybrids between these two species lacked seed dor mancy (Jørgensen et al., 1998) which may

limit F 1 hybrid persistence. In a field study backcrosses with B. rapa as the female parent

with F 1 and BC 1 hybrids occurred at very low fre quencies with an overall mean of 0.088% (Halfhill et al., 2004).

The presence of a herbicide-tolerance trait (glufosinate) introgressed experimentally

under controlled conditions from B. napus into B. rapa did not increase its survival or number of seeds per plant compared to parental B. rapa (Snow et al., 1999). In fact, F 1 hybrids, F 2 plants

, and backcross generations with B. rapa as the female parent, had fewer pods and viable seeds than those of the B. rapa parent due in part to reduced vegetative vigor and photosynthetic capability (Ammitzboll et al., 2005; Hauser et al., 2003; Hauser et al., 1998). F 1 hybrids between B. napus and wild B. rapa demonstrated a lower vegetative performance (seedling height and seedling biomass) than the parents when compromised by selection pressures simulate d herbivory and inters pecific competition (Sutherland et al., 2006).

Introgression, i.e. , the stable incorporation of genes from one differentiated gene pool into another, of a biotec hnology-derived trait from B. napus to B. rapa has been demonstrated on a single occasion under commer cial field conditions (Warwick et al., 2008). Populations of B. rapa located close to commercial fields of glyphosate-tolerant B. napus were monitored for multiple years. The number of hybrids with the glyphosatetolerance trait declined drastically from the firs t to fifth year of m onitoring, but persisted

at low levels at one of the two sites. A single glyphosate-tolerant diploid individual with 29% pollen viability was discovered five years after the last planting of glyphosatetolerant B. napus confirming the presence of the herbicide tolerance trait over time (Warwick et al., 2008).

These data support the conclusi on that hybridization between B. napus and B. rapa is possible under field conditions, but resulting pr ogeny have decreased fitn ess. In the rare occurrence that the glyphosat e-tolerance trait introgresse d and persistence of hybrids

between MON 88302 and B. rapa were to occur, these plants could be controlled byMonsanto Company 11-CA-233U 184 of 500 mechanical or chemical means (Section VIII.H. ). Additionally, in Canada, with twelve years of experience growing millions of acres of herbicide-tolerant canola each year, no significant agronomic problems with herbicide tolerant B. rapa have been encountered (Beckie, 2006).

Brassica juncea

Brassica juncea is sparsely but widely distributed in temperate North America in

cultivated and disturbed ar eas, and may be found in ar eas where canola is grown (BONAP, 2010a). Brassica juncea is an amphidiploid ( AABB) derived from the hybridization of B. nigra (BB) and B. rapa (AA) (Section II). Brassica napus and B. juncea have a common set of chromosome s (AA), are known to be sexually compatible, and can form hybrids under open pollination conditions (Bing et al., 1996; Frello et al., 1995; Jørgensen et al., 1998). Brassica juncea is a predominantly selfpollinating species with approximately one thir d of pollination due to insect pollination (Duke, 1997). As reviewed in Devos (2009) , in field plots w ith interplanted B. napus and B. juncea interspecific hybridization frequencie s were low ranging from 0.3% to 3%. F 1 hybrids between B. juncea and B. napus had reduced male and fe male fertility, very low pollen viability ranging from 0% to 28% and low seed set (Bi ng et al., 1996; Frello et al., 1995). The majority of studi es of hybridization between B. napus and B. juncea have

shown some directional effects such that F 1 hybrid seed production is much lower in

some studies when B. napus is the female parent, but lower in other studies when B. juncea is the female parent. Bing et al., (1996) conducted fiel d experiments with neighboring B. juncea and B. napus plants to assess the poten tial for hybridization under

field conditions. When B. napus was the female, the authors found five hybrid seeds from 469 plants (1.1%); when B. juncea was the female, they found three hybrid seeds from 990 plants (0.3%). H ybrid seeds formed when B. napus was the female parent were often deformed ( e.g., without an embryo) or they star ted to germinate in the seed pod (Frello et al., 1995). Thus, B. napus and B. juncea can hybridize at a low rate under field conditions, but the resulting hybrids have reduc ed male and female fertility significantly decreasing the likelihoo d of introgression.

Brassica oleracea

Brassica oleracea is distributed primarily in northeas tern and midwestern states, and may be found in areas where canola is grow n (BONAP, 2010a). The CC genome of B. napus

was derived from B. oleracea (Section II). Therefore it is not surprising that B. napus

and B. oleracea are known to have some limite d sexual compatibility under open pollination conditions (Ford et al., 2006). Brassica oleracea is predominately a selfpollinating species (Devos et al., 2009) lim iting its potential for hybridization with B. napus . In field plots with inter-planted B. napus and B. oleracea , formation of F 1 hybrid seed was reported when B. napus was the male parent, but th e viability of the seed was not assessed (Chevre et al., 1998). In a field survey of co-localized B. napus and

B. oleracea populations no hybrids with B. napus as the female parent were reported (Wilkinson et al., 2000). At a site in the UK where B. napus grew near wild B. oleracea, one triploid hybrid (0.1%) was detected out of 842 seed samp les from two sites (Ford etMonsanto Company 11-CA-233U 185 of 500 al., 2006). Thus, hybridization between these two species under field conditions appears to be relatively rare and introgression under field conditi ons is unlikely to occur.

Brassica nigra

Brassica nigra is widespread in temperate North America, and may be found in areas where canola is grown (BONAP, 2010a). No hybrids were produced in field plots with inter-planted B. napus and B. nigra (Bing et al., 1996). Even under controlled conditions, hybrids between these two species were not obtained using hand pollination, only when embryo rescue was performed and only when B. napus was the female parent. Reduced pollen fertility (0-1.9%) in the resulting hybrids (Kerlan et al., 1992) ensures that even if such a cross were to occur, reduced repr oductive success makes introgression highly unlikely. Further, no hybrid plants were obtained when B. napus was the male parent (Kerlan et al., 1992). Therefore, th e likelihood of gene flow from B. napus to B. nigra under field conditions is extremely low.

Brassica carinata

No field studies of the hybr idization potential between B. napus and B. carinata have been reported (FitzJohn et al., 2007). Ma nual pollination results in only 0.08 seeds produced per pollination (Getin et et al., 1997) making gene flow under field conditions highly unlikely.

IX.D.3. Potential for Pollen Mediated Gene Flow and Introgression of B. napus to

Brassica Vegetables

Gene flow from B. napus canola to B. napus vegetables ( e.g., Swedes or rutabaga, Siberian kale) is possible as they are memb ers of the same species. As previously described, since B. napus has chromosomes in common with B. rapa and B. oleracea

, B.

napus , gene flow to B. rapa vegetables ( e.g., turnip and Chinese cabbage) and

B. oleracea vegetables ( e.g., cabbage, cauliflower, broccoli, collards, kale, Brussels sprouts) is less likely but may occur. However, B. napus

, B. rapa and B. oleracea vegetables are not considered weedy, and ar e generally harvested prior to flowering, preventing cross-pollination, hybridization and seed formation. Thus the potential for B. napus gene flow and introgression into closely related vegetable species is low. Coexistence of cultivated B. napus with closely related vege table species is discussed in greater detail in Appendix I..

IX.D.4. Potential for Pollen Mediated Gene Flow and Introgression of B. napus to Related Species in the Family Brassicaceae

Other species in the family Brassicaceae with which B. napus is sexually compatible

under field conditions includes Hirschfeldia incana, Raphanus raphanistrum and Sinapis

arvensis . A survey of the literature provides information on the potential for hybridization (cross po llination) and in trogression from B. napus to these species.Monsanto Company 11-CA-233U 186 of 500 Hirschfeldia incana

Hirschfeldia incana is found primarily along roadsides, in ditches and waste areas in California, Arizona, Oregon and Nevada (BONAP, 2010b). With the exception of Oregon, canola is not usually grown in thes e states. In Oregon in 2010 approximately 6000 acres, or less than 1% of total U.S. canola acres, were planted (USDA-NASS, 2011a).

In a field study with interplanted B. napus and H. incana plants the mean hybridization frequency with the H. incana as the female parent was 1.9 % (16 hybrids from a total of 853 seedlings) (Lefol et al., 1996b). The fitness of the F 1 hybrids produced was approximately 10 -6 relative to the H

. incana parent (Lefol et al., 1996b). In a two year field study evaluating the potenti al for introgression of a herb icide-tolerance gene from B. napus to H. incana , from 0.17% to 0.79% hybrid seed was produced (Darmency and Fleury, 2000). F 1 hybrid plants obtained from both th e field experiment and from a hand pollination experiment in a greenhouse were backcrossed to H. incana . The herbicidetolerant progeny in each generation were selected for further backcrossing. In each subsequent backcross generation, fewer seed s were produced and by the fifth backcross generation no viable seed were produced (Darmency and Fleury, 2000). The chromosome numbers of the plants in each backcross generation were ir regular (Darmency and Fleury, 2000), and it has been suggested that genetic factors in H. incana limit homoeologous pairing, resulting in an elimination of B. napus chromosomes (Eber et al., 1994). In a subsequent evaluation of the F 1 hybrid seed produced from fi eld studies, the survival and emergence after burial in soil under field conditions was asse ssed. Prior to burial in soil

the F 1 hybrid seed had 97% germination, similar to the B. napus parent. However, hybrid plants behaved similarly to B. napus , and seedling emergence decreased to 1% after three years (Chadoeuf et al ., 1998). Thus, although F 1 hybrids may occur with B. napus in

areas where H. incana is found, introgression of B. napus genes into H. incana is not likely to be a significant phenomen on (Darmency and Fleury, 2000).

Raphanus raphanistrum

Raphanus raphanistrum is widely distributed in North America with the exception of the central Great Plains and may be found where canola is grown (BONAP, 2010c). In field experiments where both B. napus and R. raphanistrum were planted, interspecific hybrids were detected at very low frequencies in France at 0.001 -0.00001% (Chevre et al., 2004), Australia at 0.000004% (Rieger et al., 2001), and Canada at 0.003% (Warwick et al., 2003). However, in surveys of large experi mental fields where herbicide-tolerant B. napus and R. raphanistrum were both present, no hybrids between B. napus and R. raphanistrum were detected over five years in the UK, and over two years in Canada (Warwick et al., 2003). The fr equency of cro ssing between B. napus canola and R. raphanistrum is extremely low especially with R. raphanistrum as the female parent (Gueritaine et al., 2002). As reviewed in De vos et al. (2009), seed dormancy of hybrids

of B. napus and R. raphanistrum was within the range of th eir original parents and the hybrid plants had delayed seedling emergence, lower survival compared to both parents and produced less than two seeds per plant. Hybrids between these two species have a reduced pollen viability of less th an 1% (Warwick et al., 2003).Monsanto Company 11-CA-233U 187 of 500 Studies of inheritance of B. napus genes in hybrids between herbicide-tolerant B. napus

and R. raphanistrum over four backcross generations to R. raphanistrum demonstrated that each successive generation had a reductio n in chromosome number. By the fourth backcross generation, the hybrids had a chromosome number close to R. raphanistrum

and the percentage of herbicide to lerant plants also decreased (Chevre et al., 1997). In another study (Gueritaine et al., 2002) DNA content and seed production were measured in sixth generation hybrids of B. napus and R. raphanistrum . Herbicide-tolerant hybrids were found to have higher DNA content and produce half the numbe r of seeds than nonherbicide-tolerant plants and the R. raphanistrum control.

The potential for hybridization between B. napus and R. raphanistrum under field conditions is extremely low, and, if it were to occur, the hybrid s would have reduced reproductive success. Therefore, introgression of a herbicide tolerance gene is very unlikely.

Sinapis arvensis

Sinapis arvensis is found abundantly in temperate agricultural areas of North America, and may be found in areas where canola is grown (BONAP, 2010d). Hybrids between B. napus and S. arvensis were not found in the field ex cept under controlled conditions (Bing et al., 1996; Chèvre et al., 1996; Moyes et al., 2002). Hybrids in which S. arvensis is the female parent have not been reported without the use of in vitro techniques (Chèvre et al., 1996; Moyes et al., 2002). Hybrids de veloped using hand polli nation demonstrated poor pollen viability and fertility an d were unable to backcross with S. arvensis (Moyes et al., 2002).

In one report a single hybrid plant was found along the margins of a field trial containing biotechnology-derived herbicide-tolerant B. napus canola (Daniels et al., 2005). It demonstrated herbicide tolerance and the pres ence of the gene was confirmed using PCR techniques. In the following growing season seeds were harvested from the S. arvensis population at this same location, but none were found to be tolerant to the herbicide (Daniels et al., 2005). T hus, hybridization between B. napus and S. arvensis occurs at an extremely low frequency making introgression very unlikely under field conditions.

Eruca vesicaria

Eruca vesicaria is distributed throughout North America with the exception of southeastern U.S. and may be found in areas where canola is grown (USDA-NRCS, 2010a). No field studies on the hybridization pote ntial between B. napus and E. vesicaria have been reported. Using protoplast fusi on, hybrids can be produced (Fahleson et al., 1988). F 1 hybrids have limited pollen fertility and seed set relative to their parents.

Therefore the likelihood of introgression of genes from B. napus into E. vesicaria is virtually non-existent and has not b een reported in the literature.

Other Species in the Family Brassicaceae

Under field conditions hybrids ha ve not been reported between B. napus and Diplotaxus

muralis

, Diplotaxus tenuifolia (Salisbury, 2002), Erucastrum gallicum (Warwick et al.,Monsanto Company 11-CA-233U 188 of 500 2003), R. sativus or S. alba (Daniels et al., 2005; No rris and Sweet, 2002). Cross pollination of B. napus with any of these specie s is highly unlikely.Monsanto Company 11-CA-233U 189 of 500Table IX-3. Summary of Published Literature on Unassisted Hybridization under Field Conditions withB. napusas the MaleParentSpecies F1Hybrids F2or Backcross (BC) Hybrids Comments ReferenceB. rapa160 hybrids N/ABrassica napusandB. rapacross readily when either isthe male or female parent. Self-sterileB. rapaproduced many hybrid progeny when compatiblepollen from otherB. rapaplants was not available andfew hybrids whenB. rapapollen was available.(Palmer,1962)B. rapa18 putative F1hybrids.All F1s were self-sterile butbackcrosses with both parents yieldedsome seed.Seed formed on recipientsfrom the crosses was plantedand the progeny visually assessed for morphologicalvariants indicative of putative hybrids.(Leckie etal., 1993)B. rapaSome populations did not haveany hybrids. In one population,flowering near a winterB. napuscrop, identified 33 hybrids amongthe 952 plantsexamined.N/AB. rapaplants with well developed seed pods werecollected from six Danish localities and planted ingreenhouse. Seeds were harvested from eachindividual plant at maturity. Potential hybrids withB. napuswere selected based on morphology and asubset characterized with isozymes.(Landbo etal., 1996)B. rapa9% to 93% depending onexperimental design.N/A but pollen fertility of the F1hybrids under field conditions rangedform 12 to 54% (pollen fertility of theB. rapaandB. napusparents were89%-99%, and 99%-100%,respectively)WhenB. rapawas the female parent F1seedgermination was 55% witha seed dormancy breakingtreatment. The frequency of hybrids whenB. rapawasplanted at low density (93%) amongB. napusplantswas much higher than when the two were planted in 1:1ratio (13%).(Jørgensen etal., 1996)B. rapaMore than 4000 seeds from 32hybrids.F1hybrids crossed withB. rapahadpronounced seed dormancy. BC1crossed withB. raparesulted in anaverage 6.4 viable BC2offspringplants per pollination.From 44 hybrids selected withB. rapa-like morphologysome had 20 chromosomes (likeB. rapa) and greaterthan 90% pollen fertility.(Mikkelsenet al., 1996)Monsanto Company 11-CA-233U 190 of 500Table IX-3. Summary of Published Literature on Unassisted Hybridization under Field Conditions withB. napusas the MaleParent (continued)SpeciesF1HybridsF2or Backcross (BC) HybridsCommentsReferenceB. rapa0.09% and 1.5% F1hybridseed of the total harvestedseed.BC1failed under field conditionswhenB. rapawas the female.Field trials produced F1seed.B. rapawas sown in thefield with interspecific F1 hybrids, but backcrossesbetween the hybrid andB. rapaas the female were notobserved. Under field conditions, backcrosses withB. rapaas the female parent were attempted but did notproduce seed. F1hybrids produced approximately 2.5seeds per pod, whereas, the original parents had 16 to23. The seeds of the F1hybrids showed almost no signof dormancy, which the authors suggest could limitinterspecific gene flow because germination underunfavorable conditions (e.g.,with effective weedmanagement) may occur.(Jørgensenet al., 1998)B. rapa1 F1hybrid from 505(0.2%) plants screenedfrom sympatric locations.N/ARemote sensing was used to find potential areas ofsympatry betweenB.napusand populations ofB.rapain England. TwoB.rapapopulations sympatric withB. napuswere found. NoB.rapaplants were observedin any cultivated fields in the survey, indicating there isnegligible scope for transgene movement into naturalB. rapafromB. napusxB. rapahybrids formed inagricultural fields.(Wilkinsonet al., 2000)B. rapaF1hybrid frequency withdifferentB. napuslinesranged from 0.7% to16.9%.Backcross seed could only beproduced with half of theB. napuslines in the greenhouse.Field experiments with 1200:1 ratio ofB. napustoB. rapa.(Halfhill etal., 2002)B. rapaF1hybrid frequency was7% and 13.6% in fieldexperiments andcommercial fields,respectively.F1hybrids had ~54% (range of twopopulations was 20-77% and 10-86%) pollen viability.From field experiments a total of 32,154 seedlings werescreened and from the commercial fields 9567seedlings were screened.(Warwick etal., 2003)Monsanto Company 11-CA-233U 191 of 500Table IX-3. Summary of Published Literature on Unassisted Hybridization under Field Conditions withB. napusas the MaleParent (continued)SpeciesF1HybridsF2or Backcross (BC) HybridsCommentsReferenceB. rapaF1hybrid frequency withdifferentB. napuslinesranged from 4% to 22%with an overall mean of10%.WhenB. rapawas the female parentbackcross frequencies from twolocations were 0.088% and 0.06%.A 600:1 ratio ofB. napustoB. rapawith severaldifferent transformed lines of the former. Screened12,388B. rapaseedlings.(Halfhill etal., 2004)B. junceaMean F1hybrid frequencyof 3% under fieldconditions.Two F1hybrids with ~24% and 28%pollen fertility were used inbackcrosses toB. juncea. Thebackcrosses inthe greenhouseproduced 148 seeds with 75%germination compared to 90-100%for the initial cross.F1hybrid seed germination was 93% and pollen fertilityranged from 0% to 31%. BC1pollen fertility rangesfrom 24% to 90%, but note the study only used thehighest pollen fertility F1hybrids for these crosses.(Frello et al.,1995)B. juncea3 F1hybrids from 990B. junceaplants as thefemale parent.N/ATwo experiments consisting of (1) five-row plots with61 cm inter-row spacing with a 50:50 mixture of the cocultivating species sown in the center row, or (2) fourrow plots with 25 cm inter-row spacing, 6 m long, withfemale plants transplanted at low density inter-row.(Bing et al.,1996)B. juncea2.3%, 1.1% and 0.3%hybrids in co-cultivationplots with 1:3, 1:10 and1:15 ratio ofB. napustoB.junceaplants,respectively.BC1withB. junceaas the femaleparent.Results from backcrosses reported in Frello (1995).(Jørgensenet al., 1998)B. oleracea0 F1hybrids from oneB.oleraceapopulationsympatric with aB. napusfield.N/ARemote sensing was used to identify potential sites ofsympatry betweenB. napusproduction fields andpopulations ofB. oleraceain England. Every newlyrecruited plant in the population was screened forhybrid status using flow cytometry and molecularanalyses.(Wilkinsonet al., 2000)Monsanto Company 11-CA-233U 192 of 500Table IX-3. Summary of Published Literature on Unassisted Hybridization under Field Conditions withB. napusas the MaleParent (continued)SpeciesF1HybridsF2or Backcross (BC) HybridsCommentsReferenceB. oleracea1 triploid F1hybrid,.Twelve tentative introgessants wereidentified withB. napusspecificmarkers from 842 (1.4%). Of thesenine were diploid and two neartriploid hybridsA single F1 hybrid from 842 samples did not providesufficient statistical power to provide an accurateestimate of hybridization frequency. The 12 tentativeintrogressed genotypes with crop-specific markers (3triploids and nine diploids) out of 842 provided afrequency of 1.4%.(Ford et al.,2006)B. nigra0 F1hybrids from 198B. nigraplants.N/AMale parents were sown in four-row plots, 6 m long,with female plants transplanted at low density inter-row.(Bing et al.,1996)B. nigraNo hybrids. N/ANo hybrids were observed formB. nigraplantssurrounded byB. napusplants in cages with blowfliesas pollinators(Leckie etal., 1993)H. incana0 F1in experiment withcages from 36, 900 seedsscreened and 16 F1 hybridsfrom 1061 in open field(~1/plant)N/AAn experiment with cages and bees withB. napusandH. incanaplants produced no hybrids.F1hybrids produced almost no pollen and the pollengrains were aborted. On average F1hybrids produced40% fewer flowers, 20x fewer pods/flower, 50x fewerseeds/pod and 2x105fewer seeds/plant thanH. incana.Of the 168 F1hybrids only 32 seeds were collected ofwhich only 5 germinated. The seeds that did germinate3 were completely sterile, 1 produced 5 viable seedsand 1 produced 57 viable seeds.(Lefol et al.,1996b)H. incanaMean of 0.6 F1hybridseeds per plant.Backcrossing interspecific hybrids toH. incanaover five generationsshowed that introgression was notsuccessful.Seeds output in each backcross generation decreasedand no viable seeds were produced by the 5thgeneration.(Darmencyand Fleury,2000)Monsanto Company 11-CA-233U 193 of 500Table IX-3. Summary of Published Literature on Unassisted Hybridization under Field Conditions withB. napusas the MaleParent (continued)SpeciesF1HybridsF2or Backcross (BC) HybridsCommentsReferenceR. raphanistrum2 F1seeds from 147,671total seeds.Seed production of F1hybrids andtheir F2descendants was up to 0.4%and 2%, respectively, of that ofR. raphanistrum.Male-fertileB. napusin cages with differentproportions ofR. raphanistrumand bees did notproduce hybrids. Two hybrids produced under fieldconditions without cages.(Darmencyet al., 1998)R. raphanistrum1 F1hybrid from 189,084seedlings obtained fromseeds harvested fromR.raphanistrumplants.N/AThe 1 F1hybrid obtained in the study had pollenfertility of 6.5%.(Chevre etal., 2000)R. raphanistrum0 F1hybrids from 25,000seeds harvested fromR.raphanistrumplants.N/ATwo hybrids were obtained whenB. napuswas thefemale parent from 52-million seeds screened(frequency of 4 x 10-8).(Rieger etal., 2001)R. raphanistrum1 F1hybrid from 32,821seedlings screened.N/A F1hybrid had <1% pollen viability.(Warwick etal., 2003)R. raphanistrum0 F1hybrids from 19,274seeds harvested.N/A Ratio of 6001 ofB. napustoR. raphanistrum.(Halfhill etal., 2004)S. arvensis1 putative F1hybrid. N/ABlowflies were added to cages to encourage pollenmovement. The chromosome number and fertility ofthe single hybrid was not confirmed in this publication.(Leckie etal., 1993)S. arvensis0 F1hybrids from 61S.arvensisplants.N/AMale parents were sown in four-row plots, 6 m long,with female plants transplanted at low density interrow.(Bing et al.,1996)S. arvensis0 F1hybrid seeds from 2.9million seeds harvested.Artificial hybrids grown in the presence ofS. arvensis,or hand-crossed, produced a few aborted seeds..(Lefol et al.,1996a)S. arvensis1 F1hybrid.The F1hybrid did not have viablepollen. The F1hybrid produced noseed.The authors concluded that the risk of gene transferfromB. napustoS. arvensisis minimal.(Moyes etal., 2002)S. arvensis0 F1hybrids. N/AScreened approximately 43,000 seedlings ofS. arvensisand no hybrids were detected.(Warwick etal., 2003)Monsanto Company 11-CA-233U 194 of 500 IX.D.5. Transfer of Genetic Information to Species with Which Brassica napus Cannot Interbreed (Horizontal Gene Flow)

Monsanto is not aware of any reports confir ming the transfer of genetic material from B. napus to other species with which B. napus cannot sexually interbreed. The probability for horizontal gene flow to occur is judged to be exceedingly small. Even if it were to occur, the consequences would be no greater than fromany other herbicidetolerant canola currently av ailable. Furthermore, M ON 88302 produces the identical CP4 EPSPS protein as is found in commer cial Roundup Ready crop products including Roundup Ready canola (RT73) and has been show n to have no meaningful toxicity to

humans and to other NTOs unde r the conditions of use.

IX.E. Potential Impact on Canola Agronomic Practices

An assessment of current canola agronomic practices was conducted to determine

whether the cultivation of MON 88302 has the potential to impact current canola and weed management practices (Section VIII). Canola fields are typically managed agricultural areas that are dedicated to crop production. MON 88302 is likely to be used in common rotations on land pr eviously used for agricultura l purposes. Certified seed production will continue to use well-established industry practices to deliver high quality seed containing MON 88302 to growers. Cu ltivation of MON 88302 is not expected to

differ from current canola cultivation us ing glyphosate-tolerant canola, with the exception of an opportunity to use glyphosate during an expanded pe riod of application and at rates higher than those curren tly recommended and authorized.

MON 88302 is similar to conventional canola in its agronomic, phenotypic, ecological and compositional characteristics and has levels of resistance to insects and diseases comparable to conventional canola. Ther efore, no significant impacts on current cultivation and management practices for ca nola are expected following the introduction of MON 88302. Based on this assessment, the introduction of MON 88302 will not impact current U.S. canola cultivation prac tices or weed management practices, other

than intended weed c ontrol benefits.

IX.F. Summary of Plant Pest Assessments

Plant pests, as defined in th e Plant Protection Act, are livi ng organisms that can directly or indirectly injure, cause damage to or cau se disease to any plan t or plant product (7 U.S.C. § 7702[14]). Data presented in Sectio ns V through VII of this petition confirm that MON 88302, with the exception of glyphosate tolerance, is not significantly different from conventional canola, in terms of pest pot ential. Monsanto is not aware of any study

results or observations associated with M ON 88302 that would suggest that an increased plant pest risk would result from its introduction.

The plant pest assessment was based on multiple lines of evidence developed from a detailed characterization of MON 88302 comp ared to conventional canola, followed by a risk assessment on detected differences. The pl ant pest risk assessment in this petition was based on the following lines of evidence: 1) insertion of a single functional copy ofMonsanto Company 11-CA-233U 195 of 500 the cp4 epsps expression cassette; 2) characte rization of the CP4 EPSPS protein expressed in MON 88302; 3) safety of th e CP4 EPSPS protein; 4) compositional equivalence of MON 88302 seed as compared to a conventional c ontrol; 5) phenotypic and agronomic characteristics demonstratin g no increased plant pest potential; 6) negligible risk to NTO and threatened or e ndangered species; 7) familiarity with canola as a cultivated crop and 8) no greater likelihood to impact agronomic practices, including land use, cultivation practices, or the management of weeds, diseases and insects, than

conventional canola.

Based on the data and information presented in this petition, it is concluded that, similar to the currently deregulated canola products, MON 88302 is high ly unlikely to be a plant pest. Thus, the results s upport a conclusion of no increa sed weediness potential of MON 88302 compared to conventional canola. In addition, APHI S has proposed to amend 7 CFR § 340 to include its noxious weed authority. M ON 88302 would not be considered a “noxious weed” as defined by th e Plant Protection Act because the data in this petition show that it has no potential to cause direct inju ry or damage (physical harm) to any protected interest. Therefore, Mons anto Company requests a determination from

APHIS that MON 88302 and a ny progeny derived from cr osses between MON 88302

and other commercial canola be granted non-regulated st atus under 7 CFR § 340.Monsanto Company 11-CA-233U 196 of 500 X. ADVERSE CONSEQUENCES OF INTRODUCTION

Monsanto is not aware of a ny study results or observati ons associated with MON 88302 or the CP4 EPSPS protein indicating that th ere would be an adverse environmental consequence from the introduction of MON 88302. MON 88302 utilizes an improved promoter sequence to enhance CP4 EPSPS ex pression in male reproductive tissues ( i.e.

, pollen), compared to the promoter used to drive CP4 EPSPS production in the firstgeneration product, Roundup Ready canola (RT73) . Enhanced CP4 EPSPS expression in the male reproductive tissues of MON 88302 a llows the greater flexibility of glyphosate herbicide applications possi ble with MON 88302. Glyphosate is a systemic herbicide

and is translocated in the plant, generally from a strong source tissue ( e.g., leaf) to rapidly developing, or sink tissue. Si nk tissues, such as pollen, that accumulate glyphosate and lack sufficient CP4 EPSPS expression are consid ered to be at risk for glyphosate injury. MON 88302 plants can be sprayed with higher ra tes of glyphosate and at later stages of development with no detectable impact to male fertility. By virtue of enhanced CP4 EPSPS expression in male reproductive tissues, MON 88302 provides tolerance to glyphosate during the sensitive reproductive stages of growth, and enables the application of glyphosate at higher rates and at later stages of developmen t than is possible with the current product. The CP4 EPSPS protein produced in MON 88302 is identical to the CP4 EPSPS protein present in Roundup Ready crop products that were previously granted a determination of nonre gulated status by APHIS, a nd have been widely planted in the U.S. and globally. As demonstrated by field results and labor atory tests, the only phenotypic difference between MON 88302 a nd conventional canola is glyphosate

tolerance.

The data and information presented in th is petition demonstrate that MON 88302 is unlikely to pose an increased plant pest risk or to have an adverse environmental consequence compared to conventional canola. This conclusion is reached based on multiple lines of evidence developed from a detailed characterization of the product compared to conventional canola, followed by risk assessment on detected differences.

The characterization evaluation included mo lecular analyses, which confirmed the insertion of a single functiona l copy of the CP4 EPSPS expre ssion cassette at a single locus within the canola genome. Additionally, protein expression analyses demonstrate the CP4 EPSPS protein is expressed in vegeta tive and male reproductive tissues. The CP4 EPSPS protein produced by MON 88302 is similar and functi onally identical to endogenous plant EPSPS enzymes, but has a much reduced affinity for glyphosate, relative to endogenous plant EPSPS. The ami no acid sequence of th e CP4 EPSPS protein expressed in MON 88302 is identical to th e amino acid sequence of the recombinant E.

coli -produced CP4 EPSPS protein standard previously utilized. Analyses of key nutrients, anti-nutrie nts and toxicants of MON 88302 seed demonstrate that MON 88302 is compositionally equivalent to conventional canola. Th e phenotypic evaluations of MON 88302, including an assessment of seed germination and dormancy characteristics, plant growth and development characteristics, pollen characteristics and environmental

interactions also indicated MON 88302 is unc hanged compared to conventional canola.

There is no indication that MON 88302 would have an advers e impact on beneficial or non-target organisms, including threatened or endangered species. Therefore, based on the lack of increased pest pot ential or adverse environmental consequences compared toMonsanto Company 11-CA-233U 197 of 500 conventional canola, the risks for humans, animals, and other NTOs from MON 88302 are negligible under the conditions of use.

The introduction of MON 88302 will not adversel y impact cultivation practices or the management of weeds, diseases and insect s in canola production systems. Growers familiar with the Roundup Ready canola system would continue to employ the same crop rotational practices, weed contro l practices and/or volunteer c ontrol measures currently in place for the first-generation Roundup Ready canola product.Monsanto Company 11-CA-233U 198 of 500 REFERENCES

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North Dakota.Monsanto Company 11-CA-233U 217 of 500 APPENDICESMonsanto Company 11-CA-233U 218 of 500 Appendix A: USDA Notifications

Field trials of MON 88302 have been conducted in the U.S. since 2005. The protocols

for these trials include fiel d performance, breeding and observation, agronomics, and generation of field materials and data necessary for this petition. In addition to the MON 88302 phenotypic assessment data, observati onal data on pest and disease stressors were collected from these product development trials. The majority of the final reports have been submitted to the USDA. However, some final reports, mainly from the 2010 season, are still in preparati on. A list of trials conducte d under USDA notifications and the status of the final reports for th ese trials are provi ded in Table A-1.

Table A-1. USDA Notifica tions Approved for MON 88302 and Status of Trials

Conducted under These Notifications

USDA Number Effective Date Release Site (State) Trial Status

2005

05-021-10n 2/28/2005 WA Submitted to USDA

05-041-07n 4/1/2005 MN, ND (2) Submitted to USDA

05-213-05n 8/31/2005 CA Submitted to USDA

2006

06-053-06n 4/7/2006 MN (3), ND (3) Submitted to USDA

06-054-09n 4/7/2006 ND(2) Submitted to USDA

06-055-07n 5/15/2006 MN (2), MT, ND (3) Submitted to USDA

2008

08-016-111n 2/15/2008 MT, ND (5) Submitted to USDA

08-016-101n 2/20/2008 MN (5), ND (5) Submitted to USDA

08-051-102n 3/21/2008 WA (3) Submitted to USDA

2009

09-034-111n 3/5/2009 ND (2) Submitted to USDA

09-044-107n 3/15/2009 ND (2) Submitted to USDA

09-048-105n 3/19/2009 MN (3), ND, SD (2) Submitted to USDA

09-058-121n 3/29/2009 MN (4), ND (3) Submitted to USDA

09-064-109n 4/4/2009 MN, ND (2) Submitted to USDA

09-069-101n 4/9/2009 ND Submitted to USDA

09-076-101n 4/16/2009 ID, MN (2), ND Submitted to USDA

09-079-101n 4/19/2009 ID (3), MN, ND (3), NJ Submitted to USDA

09-215-104n 9/2/2009 MN (2), ND Submitted to USDA

09-257-102n 10/14/2009 CA Submitted to USDA

2010

10-042-102n 3/12/2010 MN In Progress

10-050-112n 3/21/2010 MN (2), ND, SD In Progress

10-055-104n 3/26/2010 MN, ND In Progress

10-061-101n 3/31/2010 MN (4), ND (6) In ProgressMonsanto Company 11-CA-233U 219 of 500 Appendix B: Materials, Methods, and Results for Molecular Analyses of

MON 88302

B.1. Materials

The genomic DNA used in molecular analyses was isolated from leaf tissue of the R 3 generation of MON 88302 and the conventional control (Ebony). The leaf tissue was harvested from a greenhouse production in 20 09. For generational stability analysis, genomic DNA was extracted from leaf tissue of the R 2 , R 3 , R 4 , R 5a and R 5b generations of

MON 88302. The leaf tissue was harv ested from production plan PPN-09-523. The reference substance, PV-BNHT2672 (Figure IV -1), was used as a positive hybridization control in Southern blot analyses. Probe templates generated from PV-BNHT2672 were used as additional positive hybrid ization controls. As additio nal reference standards, the

1 Kb DNA Extension Ladder and λ DNA/ Hind III Fragments from Invitrogen (Carlsbad, CA) were used for size estimations on agarose gels and subsequent So uthern blots. The 1 Kb DNA Ladder from Invitrogen was used for size estimations on agarose gels for PCR analyses.

B.2. Characterization of the Materials

The identity of the source materials was verified by methods used in molecular characterization to confirm the presence or absence of MON 88302. The stability of the genomic DNA was confirmed by observation of interpretable signals from digested DNA samples on ethidium bromide stained agarose gels and/or specific PCR products, and the samples did not appear visibly degraded on the ethidium bromide stained gels.

B.3. DNA Isolation for South ern Blot and PCR Analyses

Genomic DNA was isolated from MON 88302 l eaf tissue using a modified sarkosyl method. The leaf tissue was ground to a fine powder in liquid nitrogen using a mortar and a pestle. Approximately 4 ml of ground leaf tissue was transf erred to each 50 ml conical tube. Twenty milliliter of the lysis buffer (50 mM Tris HCl, 2% w/v PVP, 20 mM EDTA, 500 mM NaCl, 1.2% w/v SDS, 0.5% w/v sarkosyl, an d 0.4% w/v sodium bisulfate) and 300 μg of RNase A were adde d to each tube. After suspending the powder in the buffer, the samples were incubated at 60-70 °C for 60-90 min with intermittent mixing. Following the completion of the inc ubation, the samples were allowed to come to room temperature and 20 ml of ph enol/chloroform/isoamyl alcohol (PCI) [25:24:1(v/v)] were added to each sample. The samples were then mixed by inversion with hand for 2-3 minutes followed by centrifugation at 2,000 x g for 20-25 min at 2-8 °C to separate the phases. The upper aqueous phase was transferred to a clean tube and the PCI extraction and centrifugation process wa s repeated at least once followed by a chloroform extraction. The aqueous phase wa s transferred to a clean tube and the amount of aqueous phase was recorded. The DNA was precipitated with an equal amount of 100% ethanol and spooled into a tube with 10-12 ml of 70% ethanol to wash the DNA. The DNA was pelleted by cen trifugation at 4,000 x g for 5 min at 2-8 °C and the 70% ethanol was discarded. After being ai r-dried, the DNA pellet was suspended inMonsanto Company 11-CA-233U 220 of 500 appropriate amount of TE buffer (10 mM Tris HCl, 1 mM EDTA, pH8.0) and stored in a 4 oC refrigerator or a -20 °C freezer.

B.4. Quantification of DNA

Genomic DNA was quantified using a DyNA Quant 200 Fluorometer (Hoefer, Inc., Holliston, MA). Molecular Size Marker IX (R oche, Indianapolis, IN) was used as the

calibration standard.

B.5. Restriction Enzyme Digestion of DNA

Approximately ten micrograms (μg) of genomic DNA extracted from MON 88302 and conventional control were digest ed with restriction enzyme Ase I (New England Biolabs, Inc. Ipswich, MA) and a combination of restriction enzymes Sal I and Sca I (New England Biolabs, Inc.). All digests were conducted in 1X NEBuffer 3 (New England Biolabs, Inc.) at 37°C in a total volume of ~5 00 microliter (μl) with ~50 units of each restriction enzyme. Digests conducted with the combina tion of restriction enzymes Sal I

and Sca I also included 1X BSA (New England Bi olabs, Inc.) in the reaction. For the purpose of running positive hybrid ization controls, ~10 μg of genomic DNA extracted from the conventional control was dige sted with the restriction enzyme Ase I and the appropriate positive hybridization control(s) were added to these digests prior to loading the agarose gel.

B.6. Agarose Gel Electrophoresis

Digested DNA was resolved on ~0.8% (w/v) agarose gels. For T-DNA insert/copy number and backbone analyses, individua l digests containing ~10 μg each of MON 88302 and conventional control genomic DNA were loaded on the same gel in a long run/short run format. Th e long run allows for greater resolution of large molecular weight DNA, whereas the short run allows fo r retaining the small molecular weight DNA on the gel. The positive hybridization controls were only run in the short run format. For the insert stability analysis, individual digests of ~10 μg each of genomic DNA extracted from five leaf samples from four ge nerations of MON 88302 and the conventional control along with the positive hybridization co ntrols were loaded on the agarose gel in a single run format.

B.7. DNA Probe Preparation fo r Southern Blot Analyses

Probe templates were prepared by PCR amp lification using the PV-BNHT2672 DNA as a template. The PCR products were separate d on an agarose gel by electrophoresis and purified from the gel using QIAquick Ge l Extraction Kit (Qiagen, Valencia, CA) according to manufacturer’s instruction. The probe templates were designed based on the nucleotide composition (%GC) of the sequence in order to optimize the detection of DNA sequences during hybridization. When po ssible, probes possessing similar melting temperature (Tm) were combined in th e same Southern blot hybridization. Approximately 25 ng of each probe templa te were radiolabeled with either [ α

32 P] deoxycytidine triphosphate (d CTP) (6000 Ci/mmol) or [ α

32 P] deoxyadenosineMonsanto Company 11-CA-233U 221 of 500 triphosphate (dATP) (6000 Ci/mmol) us ing RadPrime DNA Labeling System (Invitrogen, Carlsbad, CA) according to manufacturer’s instruction

B.8. Southern Blot Analyses of DNA

Genomic DNA isolated from MON 88302 and th e conventional control was digested and evaluated using Southern blot analys es (Southern, 1975). The PV-BNHT2672 DNA, previously digested with the co mbination of rest riction enzymes Bam HI and Sca I was

added to conventional control genomic DNA digested with Ase I to serve as positive hybridization control on each Southern blot . When multiple probes were hybridized simultaneously to one Southern blot, the prob e templates were spiked in the digested conventional control genomic DNA to serve as additional positive hybridization controls

on the Southern blot. The DNA was then se parated by agarose gel electrophoresis and transferred onto a nylon membrane. Southe rn blots were hybridized and washed at 55 °C, 60 °C, or 65 °C, depending on the cal culated melting temperature (Tm) of the probes that were used. Table A-1 lists th e radiolabeling conditi ons and hybridization temperatures of the probes used in this study. Multiple exposures of each blot were then generated using Kodak Biomax MS film (Eas tman Kodak, Rochester, NY) in conjunction

with one Kodak Biomax MS intensif ying screen in a -80 °C freezer.Monsanto Company 11-CA-233U 222 of 500 Table B-1. Hybridization Conditions of Utilized Probes

Probe DNA Probe Element Sequence Spanned by DNA

Probe Probe labeled

with dNTP ( 32 P) Hybridization/Wash Temperature (  C)

1 T-DNA Probe 1 B-Right Border,

P- FMV/Tsf1 , L- Tsf1

, I- Tsf1

, TS- CTP2 (portion) dATP 55

2 T-DNA Probe 2 TS- CTP2 (portion),

CS- cp4 epsps (portion) dATP 60

3 T-DNA Probe 3 CS- cp4 epsps (portion), T- E9,

B-Left Border dATP 55

4 Backbone Probe 4 Backbone sequence dCTP 65

5 Backbone Probe 5 Backbone sequence dCTP 60

6 Backbone Probe 6 Backbone sequence dCTP 60Monsanto Company 11-CA-233U 223 of 500 B.9. DNA Sequence Analyses of the Insert

Overlapping PCR products, denoted as Product A and Product B, were generated that span the insert and adjacent 5' and 3' flanking genomic DNA sequences in MON 88302. These products were sequenced to determine the nucleotide sequence of the MON 88302 insert as well as the nucleotide sequence of the genomic DNA flanking the 5' and 3' ends

of the insert.

The PCR analyses were performed accordi ng to SOP BR-ME-0486-01. To generate both Product A and Product B, the PCR reactions were conducted using 30 ng of genomic DNA template in a 50 μl reaction volume co ntaining a final concentration of 1.5 mM MgSO 4 (Novagen, Madison, WI), 0.3 μM of each primer, 0.2 mM of each dNTP (Novagen, Madison, WI), and 2.0 units of KOD Hot Start DNA polymerase (Novagen, Madison, WI). For the generation of Product B, a final concentration of 1 M betaine (USB Corp. Cleveland, OH) was also include d in the reaction. The amplification of Product A was performed under th e following cycling conditions : 1 cycle at 94 oC for 2 minutes; 35 cycles at 94 oC for 30 seconds, 65 oC for 15 seconds, 72 oC for 2 minutes; 1 cycle at 72 oC for 5 minutes. The amplif ication of Product B was performed under the following cycling conditions: 1 cycle at 94 oC for 2 minutes; 35 cycl es at 94 oC for 30 seconds, 60 oC for 15 seconds, 72 oC for 2 mi nutes; 1 cycle at 72 oC for 5 minutes.

Aliquots of each PCR product were separated on 1.0% (w/v) ag arose gels and visualized by ethidium staining to verify that the produc ts were of the expected size. Prior to sequencing, each verified PCR product was purified with th e QIAquick PCR Purification Kit (Qiagen, Inc., Valencia, CA) according to manufacturer’s instruction and gel quantified according to SOP BR-ME-1222-0 1. The purified PCR products were sequenced using multiple primers, including primers used for PCR amplification. All sequencing was performed by the Monsanto Ge nomics Sequencing Ce nter using BigDye terminator chemistry (Applied Biosystems, Foster City, CA).

Numerous sequencing reactions performe d on the overlapping PCR products were compiled and a consensus sequence was ge nerated. This consensus sequence was aligned with the sequence of plasmid vect or PV-BNHT2672 to determine the integrity and genomic organization of the integrated DNA and the junctions of th e 5' and 3' flanks

in MON 88302.

B.10. PCR and DNA Sequence Analysis to Examine the MON 88302 Insertion Site

To determine the integrity and genomic orga nization of the insertion site in MON 88302 and to demonstrate that the DNA sequences flanking the insert in MON 88302 are native to the canola genome, PCR analyses were performed on the genomic DNA from both MON 88302 and the conventional control Ebony. The primers used in this analysis were designed from the genomic DNA sequences flanking the insert in MON 88302. A forward primer specific to the genomic DNA sequence flanking the 5' end of the insert was paired with a reverse primer specific to the genomic DNA sequence flanking the 3'

end of the insert.Monsanto Company 11-CA-233U 224 of 500 The PCR analyses were performed according to SOP BR-ME-0486-01. The PCR reactions were conducted using 30 ng of ge nomic DNA template in a 50 μl reaction volume containing a final concentration of 2 mM MgSO 4 , 0.4 μM of each primer, 0.2 mM of each dNTP, and 2.0 units of KOD Hot Start DNA polymerase. The amplification was performed under the following cycling conditions: 1 cycle at 94oC for 2 minutes; 35 cycles at 94oC for 15 seconds, 64oC for 30 seconds, 72oC for 2 minutes; 1 cycle at 72oC for 5 minutes.

Aliquots of each PCR product were separated on 1.0% (w/v) ag arose gels and visualized by ethidium staining to verify that a unique PCR product was produced from MON 88302 or the conventional control Ebony genomic DNA. Prior to sequencing, only the verified PCR product from the conven tional control Ebony was purified with the QIAquick PCR Purification Kit according to manufacturer’s instruction and gel quantified according to SOP BR-ME-1222-01. The purified PCR product was sequenced using multiple primers, including primers used for PCR amplification. All sequencing was performed by the Monsanto Genomics Se quencing Center using BigDye terminator chemistry.

Numerous sequencing reactions performed on the PCR product were compiled and a consensus sequence was generated. This cons ensus sequence was aligned with the 5' and 3' flanking sequences of the MON 88302 insert to determine the integrity and genomic organization of the insertion site in MON 88302.Monsanto Company 11-CA-233U 225 of 500 References for Appendix B

Southern, E.M. 1975. Detection of specifi c sequences among DNA fragments separated by gel electrophoresis. Journal of Molecular Biology 98: 503-517.Monsanto Company 11-CA-233U 226 of 500 Appendix C: Materials, Methods, an d Results for Characterization of

CP4 EPSPS Protein Pr oduced in MON 88302

C.1. Materials

The MON 88302-produced CP4 EPSPS protein (lot 11266369) was purified from seed of MON 88302 (lot 11225246). The MON 88302-pr oduced CP4 EPSPS protein was stored

in a -80 oC freezer in a buffer solution cont aining 50 mM Tris-HCl, pH 7.5, 50 mM KCl, 2 mM dithiothreitol (DTT), 1 mM ben zamidine-HCl, and 25% glycerol.

The E. coli -produced CP4 EPSPS protein (lot 10000739) was used as the reference substance. The CP4 EPSPS pr otein reference substance wa s generated from cell paste produced by large-scale fermentation of E. coli containing the pMON21104 expression plasmid. The coding sequence for cp4 epsps contained on the expression plasmid (pMON21104) was confirmed prior to and after fermentation. The E. coli -produced CP4 EPSPS protein was prev iously characterized.

C.2. Description of Assay Controls

Protein MW standards (Precision Plus Protei n Standards Dual color; Bio-Rad, Hercules, CA) were used to calibrate some SDS-PAGE gels and verify protein transfer to polyvinylidene difluoride (PVDF) and nitrocel lulose membranes. Broad Range SDSPAGE molecular weight standards (Bio-Rad, Hercules, CA) were used to generate a standard curve for the appa rent MW estimation. The E. coli -produced CP4 EPSPS

reference standard was used to construct a standard curve for the estimation of total protein concentration using a Bio-Rad pr otein assay. A phenylthiohydantoin (PTH) amino acid standard mixture (Applied Biosystems , Foster City, CA) was used to calibrate the instrument for each analysis. A peptide mixture (Sequazyme Peptide Mass Standards kit, Applied Biosystems, Foster City, CA) was used to calibrat e the MALDI-TOF mass spectrometer for tryptic mass and a bovine serum albumin (BSA) standard (NIST, Gaithersburg, MD) was used to calibrate th e MALDI-TOF mass spectrometer for intact mass analysis. Transferrin (Sigma-Aldrich, St. Louis, MO) was used as positive control for glycosylation analysis.

C.3. CP4 EPSPS Protein Purification

The plant-produced CP4 EPSPS protein was purified from seed of MON 88302. The purification procedure was not performed under a GLP plan; however, all procedures were documented on worksheets and, where a pplicable, SOPs were followed. The CP4 EPSPS protein was purified at ~4 °C from an extract of ground seed using a

combination of ammonium sulfate fractionation, hydrophobic interaction chromatography, anion exchange chromatogr aphy, and cellulose phosphate affinity chromatography. The purification proce dure is briefly described below.

Approximately 500 g of seed of MON 88302 was frozen with liquid nitrogen in a mortar and ground with a pestle. The partially crus hed seed was further ground using a Magic Bullet grinder. The ground seed was then defatted by extraction with heated hexaneMonsanto Company 11-CA-233U 227 of 500 (~50 °C) followed by vacuum filtration. This was repeated three times at a ground seed (g) to hexane volume (ml) ratio of appr oximately 1:5. The defatted ground seed was allowed to dry overnight at room temperat ure in a fume hood. The following day the defatted ground seed was mixed with extr action buffer (100 mM Tris-HCl, pH 7.5, 2 mM EDTA, 2 mM benzamidine-HCl, 4 mM DTT, 1 mM phenylmethylsulfonyl fluoride, 1% polyvinylpolypyrrolidone, and 10% glycerol) for ~1.5 h at a sample weight (g) to buffer volume (ml) ratio of approximately 1:10. The slurry was centrifuged at 15,182 x g for 1 h at ~ 4 oC. The supernatant (~3.5 liters) was collected and brought to 45% ammonium sulfate saturation by slow ad dition of 903 g of ammonium sulfate in a cold room (~4 oC). The solution was stirred for ~1 h at ~4 oC and then centrifuged at 15,182 x g for 1 h. The supernatant (~3.8 lite rs) was again collected and 592 g of

ammonium sulfate was added to bring the solution to 70% a mmonium sulfate saturation. The solution was stirred for ~1 h in a cold room and the pellet was collected by centrifugation at 15,182 x

 g for 1 h. The pellet was re-suspended in 1 liter of PS(A) buffer [50 mM Tris-HCl, pH 7.5, 1 mM DT T, 10% (v/v) glycerol,1.5 M ammonium sulfate]. The sample was loaded onto a 46 0 ml column (5 cm x 23 cm) of Phenyl Sepharose Fast Flow (GE Healthcare, Piscataw ay, NJ) equilibrated with PS(A) buffer. Proteins were eluted with a linear salt gr adient that decreased from 1.5 M to 0 M

ammonium sulfate over a volume of 2.3 liters . Fractions containing the CP4 EPSPS protein, identified based on immunoblot analys is and SDS-PAGE analysis, were pooled to a final volume of ~440 ml. The pooled sample was desalted by dialysis against 20 liters of QS(A) buffer ( 50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 1 mM benzamidineHCl, 4 mM DTT) at ~4 °C using a dialysis tubing [Spectrum Laboratories, Inc., Rancho Dominguez, CA; Molecular Weight Cutoff (MWCO): 3.5 kDa] for a total of 16 h.

The desalted sample (600 ml) was loaded onto a 180 ml column (5 cm x 9.2 cm) of Q Sepharose Fast Flow anion exchange resin (GE Healthcare, Piscataway, NJ) equilibrated with QS(A) buffer. The bound CP4 EPSPS was el uted with a linear salt gradient that increased from 0 M to 0.4 M KCl in QS(A) bu ffer over 2.1 liters. Fractions containing CP4 EPSPS, identified by imm unoblot analysis, were pool ed to a final volume of ~ 280 ml. The pooled sample was dialyzed against 20 liters CP(A) buffer (10 mM sodium citrate, pH 5.0, 1 mM benzamidine-HCl, 2 mM DTT) for a tota l of 18 h at ~4 °C using a dialysis tubing (Spectrum Laborator ies, Inc. Rancho Dominguez, CA; MWCO: 3.5 kDa).

Of the 350 ml recovered after dialysis, approxi mately 50 ml of the dialyzed sample was loaded onto a 5 ml column (1.6 x 2.5 cm) of cellulose phosphate P11 cation exchange (Whatman, Kent, UK) pre-equili brated with CP(A) buffer. After an initial wash with 40 ml of CP(A) buffer, the column was washed with 50 ml of CP(B) buffer [CP(A) buffer with pH adjusted to 5.2 and supplemented with 0.5 mM phosphoenolpyruvate (PEP)]. The column was further washed with CP(C) buffer [CP(A) buffer with pH adjusted to 5.4 and supplemented with 0.5 mM PEP]. The bound CP4 EPSPS protein was eluted over 90 ml of CP(D) buffer [CP( A) buffer with pH ad justed to 5.7 and supplemented with 0.5 mM PEP and 0.5 mM shikimate-3-phosphate (S3P)]. Fractions containing CP4 EPSPS protein, based on SD S-PAGE analysis and confirmed by immunoblot analysis, were pooled (~22 ml), supplemented with 10% glycerol, labeled
Pool 1, and stored at -20  C. Approximately 200 ml of the remaining dialyzed sampleMonsanto Company 11-CA-233U 228 of 500 was then loaded onto a fres hly prepared 20 ml column (2.6 x 3.7 cm) of cellulose phosphate P11 cation exchange (Whatman, Ke nt, UK) pre-equilibr ated with freshly prepared CP(A) buffer. After an initial wa sh with 200 ml of CP(A ) buffer, the column was washed with 160 ml of freshly prepared CP(B) buffer. The column was further washed with freshly prepared CP(C) buffe r. The bound CP4 EPSPS protein was eluted with freshly prepared CP(D) buffer. Frac tions containing CP4 E PSPS protein, based on SDS-PAGE analysis and confirmed by immunobl ot, were pooled (Pool 2). Pool 1 and Pool 2 were combined (~82 ml) and divided between four iCon  concentrators (MWCO: 20 kDa; size: 20 ml; Pierce, Rockford, IL) and concentrated by cen trifugation at 4,000 x

 g

for 30 min at ~4 °C. Buffer exchange was carried out in the same concentrators by the

addition of ~19 ml an initial buffer cont aining 50 mM Tris-HCl, pH 7.5, 50 mM KCl, 2 mM DTT, 1 mM benzamidine-HCl followed by centrifugation at 4,000 x

 g for 30 min at ~4 °C repeated four times. After the fourth buffer exchange the remaining sample (~10 ml) was transferred to a new iCon con centrator (MWCO: 20 kDa; size: 20 ml; Pierce, Rockford, IL), supplemented with equal volume of the buffer containing 50 mM Tris-HCl pH 7.5, 50 mm KCl, 2 mm DTT, 50% glycerol and 1 mm benzamidine-HCl, and the sample was concentrated to ~2.4 ml. The final buffer composition of the sample was: 50 mM Tris-HCl, pH 7.5, 50 mM KCl, 2 mM DTT, 1 mM benzamidine-HCl and 25% glycerol. This CP4 EPSPS protein purified from the seed of MON 88302 was aliquoted and stored in a -80 °C freezer.

C.4. N-Terminal Sequencing

C.4.1. Methods

N-terminal sequencing, carried out by automa ted Edman degradation chemistry, was used to confirm the identity of th e MON 88302-produced CP4 EPSPS.

MON 88302-produced CP4 EPSPS was separate d by SDS-PAGE and transferred to a

PVDF membrane. The blot wa s stained using Coomassie Bl ue R-250. The major band at ~44 kDa containing the test protein was ex cised from the blot and was used for Nterminal sequence analysis. The analysis was performed for 15 cycles using automated Edman degradation chemistry (Hunkapiller et al., 1983). An Applied Biosystems 494 Procise Sequencing System with a 140C Mi crogradient pump and a 785 Programmable

Absorbance Detector was controlled with Procise  Control (version 1.1a) software. Chromatographic data were collected using Atlas 2003 software (version 3.59a, LabSystems, Altrincham, Cheshire, England). A control protein,  -lactoglobulin, (Applied Biosystems, Foster City, CA) was analyzed before and after the sequence analysis of the CP4 EPSPS protein to verify that the sequencer met performance criteria for repetitive yield and sequence identity. Identity was established if ≥ 8 amino acids, consistent with the predicted sequence of the N-terminus of the MON 88302-produced CP4 EPSPS, were observe d during analysis.Monsanto Company 11-CA-233U 229 of 500 C.4.2. Results of the N-t erminal Sequence Analysis

N-terminal sequencing of the first 15 amino acids was performed on MON 88302- produced CP4 EPSPS protein. The expected sequence for the CP4 EPSPS protein

deduced from the cp4 epsps gene present in MON 88302 was observed. The data obtained correspond to the deduced CP4 EPSPS protein beginning at amino acid positions 2 and 4 (Table C-1, Experimental Sequence 1 and 2, respectively). Hence, the sequence information confirms the identity of the CP4 EPSPS protein isolated from the

seed of MON 88302.

Amino acid

residue #

from the Nterminus → 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

Expected Sequence → M L H G A S S R P A T A R K S S G L

│ │ │ │ │ │ │ │ │ │ │ │ │ │ │ │ │

Experimental

Sequence 1 → - L H G A X X X P A T X X X X X ^ ^

│ │ │ │ │ │ │ │ │ │ │ │ │ │ │

Experimental

Sequence 2 → - - - G A S X R P A T A X K S X G X

Table C-1. N-Terminal Sequence of the MON 88302-produced CP4 EPSPS The expected amino acid sequence of the N- terminus of CP4 EPSPS protein was deduced

from the cp4 epsps coding region present in MON 88302. The experimental sequences obtained from the MON 88302-produced CP4 E PSPS protein were compared to the expected sequence. The single letter IUPAC-IU B amino acid code is M, methionine; L, leucine; H, histidine; G, glycine; A, alanin e, S, serine; R, arginine; P, proline; T, threonine; K. lysine; (X) indicates that the residue was not identifiable; (-) indicates the residue was not observed; (^) indicates not done, i.e. , sequencing cycle was not

conducted.

C.5. MALDI-TOF Tryptic Mass Map Analysis

C.5.1. Methods

MALDI-TOF tryptic mass fingerprint analysis was used to confirm the identity of the MON 88302-produced CP4 EPSPS protein. MON 88302-produced CP4 EPSPS protein was subjected to SDS-PAGE and the gel was stained using Brilliant Blue G Colloidal stain. Each ~44 kDa band was excised and transferred to a microcentrifuge tube. The gel

bands were washed in 100 mM ammonium bi carbonate and then, to reduce the protein in each, gel bands were incubated in 100 μl of 10 mM DTT at ~37°C for 2 h. The protein was then alkylated in the dark for 25 min with 100 μl of 20 mM iodoacetic acid and washed with 200 μl of 25 Mm ammonium bi carbonate for 3 x 20 min washes. Gel bands were dried with a Speed-Vac  concentrator (Thermo Fisher Scientific, Waltham, MA) and then rehydrated with 20 μl of trypsin solution (20 μg/m l). After 1 h, excess liquid was removed and the gel was incubated at ~37 °C for 16 h in 40 μl of 10% acetonitrile inMonsanto Company 11-CA-233U 230 of 500 25 mM ammonium bicarbonate. Gel bands were sonicated for 5 min to further elute proteolytic fragments. The resulting extrac ts were transferred to new microcentrifuge tubes labeled Extract 1 and dried using Speed -Vac concentrator. The gel bands were reextracted twice with 30 μl of a 60% acet onitrile, 0.1% trifluoroacetic acid, 0.1% β -octylglucopyranoside solution and sonicated fo r 5 min. Both 60% acetonitrile, 0.1% trifluoroacetic acid, 0.1% β -octyl-glucopyranoside extracts were pooled into a new

tube labeled Extract 2 and dried with a Speed-Vac concentrator. A solution of 0.1% trifluoroacetic acid (TFA) was added to all Extract 1 and 2 tubes and they were dried as before. To acidify the extracts, a solution of 50% acetonitrile, 0.1% TFA was

added to each tube and all were sonicated for 5 min. Each extract (0.3 μl) was spotted to three wells on an analysis plate. For each extract 0.75 μl of 2, 5-dihydroxybenzoic acid (DHB), α -cyano-4-hydroxycinnamic acid ( α Cyano), or 3,5-dimethoxy-4-hydroxycinnamic acid (sinapin ic acid) (Waters Corp., Milford, MA) was added to one of the spots. The samples in DHB matrix were analyzed in the 300 to 7000 Da range. Samples in α -Cyano and Sinapinic acid were analyzed in the 500 to 5000 and 500 to 7000 Da range, respectively. Protonated peptide masses were monoisotopically resolved in re flector mode (Aebersold, 1993; Billeci and Stults, 1993).

CalMix 2 was used as the external calibrant (Sequazyme Peptide Mass Standards kit, Applied Biosystems, Foster City, CA) for the analysis. GPMAW32 software (Lighthouse Data, Odense M, Denmark) was used to gene rate a theoretical tr ypsin digest of the CP4 EPSPS protein sequence. Those experime ntal masses within 1 Da of a theoretical mass were matched. All matching masses were tallied and a coverage map was generated for the mass fingerprint. The tryptic mass fingerprint cove rage was considered acceptable if  40% of the protein sequence was identified by matching experimental masses observed for the tryptic peptide fr agments to the expected masses for the fragments.

C.5.2. Results of MALDI-TOF Tryptic Mass Map Analysis

The identity of the MON 88302-produced CP4 EPSPS protein was also confirmed by MALDI-TOF MS analysis of peptide fragment s produced from tryptic digestion of the MON 88302-produced CP4 EPSPS protein. The ab ility to identify a protein using this method is dependent upon matching a suffi cient number of observed tryptic peptide fragment masses with predicted tryptic peptid e fragment masses. In general, protein identification made by peptide mapping is cons idered to be reliable if the measured coverage of the sequence is 15% or higher with a minimum of five matched peptides (Jensen et al., 1997).

There were 34 unique peptides identified that corresponded to the masses (Table C-2) expected to be produc ed by tryptic digestion of the CP 4 EPSPS protein. The identified

masses were used to assemble a covera ge map of the entire CP4 EPSPS protein (Figure C-1). The experimentally determin ed mass coverage of the CP4 EPSPS protein was 85.5% (389 out of 455 amino acids). This analysis serves as additional identity confirmation for the MON 88302-produced CP4 EPSPS protein.Monsanto Company 11-CA-233U 231 of 500Table C-2. Summary of the TrypticMasses Identified for the MON 88302-produced CP4 EPSPS Using MALDI-TOF MassSpectrometry1Only experimental masses that matched expected masses are listed in the table.2The difference between the expected mass and the first column mass.Other masses shown within a row are also within 1 Da of the expected mass.3Position refers to amino acid residues within the predicted CP4 EPSPS sequence as depicted in Figure C-1.DHB = 5-dihydroxybenzoic acid matrix,α-cyano =α-cyano-4-hydroxycinnamic acid matrix; Sinapinic acid = 3, 5-dimethoxy-4-hydroxycinnamic acid matrix;Ave = experimental mass average (for large peptides the monoisotopic mass is poorly resolved, therefore the mass average valueis used forcomparison).-Cyano-Cyano DHB DHBSinapinicacidSinapinicacidExpectedMass1Diff2Position3SequenceExtract 1 Extract 2 Extract 1 Extract 2 Extract 1 Extract 2389.28 389.25 0.03 225-227 TIR474.32 474.27 0.05 228-231 LEGR506.29506.22 0.07 354-357 ESDR529.36 529.30 0.06 24-28 IPGDK599.43 599.51 599.41 599.33 0.10 29-33 SISHR616.44 616.48 616.41 616.24 616.34 0.10 128-132 RPMGR629.44 629.45 629.29 0.15 201-205 DHTEK629.44 629.45 629.34 0.10 383-388 GRPDGK711.57 711.62 711.56 711.61 711.45 0.12 133-138 VLNPLR790.55 790.48 0.07 306-312 VRSSTLK790.55 790.41 0.14 139-145 EMGVQVK805.54 805.43 0.11 447-453 IELSDTK835.54 835.58 835.53 835.39 0.15 62-69 AMQAMGAR863.61 863.68 863.60 863.46 0.15 15-23 SSGLSGTVR872.61 872.66 872.61 872.67 872.53 872.45 0.16 313-320 GVTVPEDR872.61 872.66 872.61 872.67 872.53 872.52 0.09 358-366 LSAVANGLK930.66 930.51 0.15169-177 VPMASAQVK948.68 948.74 948.68 948.75 948.52 0.16 161-168 TPTPITYR991.72 991.71 991.55 0.17 14-23 KSSGLSGTVR1115.75 1115.831115.77 1115.86 1115.69 1115.57 0.18 295-305 LAGGEDVADLR1357.94 1358.011357.97 1358.05 1357.89 1357.71 0.23 146-157 SEDGDRLPVTLR1359.88 1359.96 1359.91 1360.00 1359.81 1359.87 1359.72 0.16 354-366 ESDRLSAVANGLK1359.88 1359.96 1359.91 1360.00 1359.81 1359.87 1359.64 0.24 34-46 SFMFGGLASGETR1559.11 1559.18 1559.13 1559.01 1558.83 0.28 47-61 ITGLLEGEDVINTGK1647.10 1647.241647.16 1647.24 1646.84 0.26 389-405 GLGNASGAAVATHLDHR1764.10 1764.26 1764.16 1764.06 1763.81 0.29 367-382 LNGVDCDEGETSLVVR1994.31 1994.43 1994.35 1994.55 1994.21 1994.35 1993.97 0.34 206-224 MLQGFGANLTVETDADGVR2183.54 2183.67 2183.57 2183.80 2183.45 2183.53 2183.17 0.37 275-294 TGLILTLQEMGADIEVINPR2367.73 2367.87 2367.77 2367.85 2367.65 2367.8 2367.33 0.40 178-200 SAVLLAGLNTPGITTVIEPIMTR2450.65 2450.83 2450.80 2450.51 2450.6 2450.23 0.42 24-46 IPGDKSISHRSFMFGGLASGETR2450.65 2450.83 2450.80 2450.51 2450.6 2450.22 0.43 105-127 LTMGLVGVYDFDSTFIGDASLTK3247.10 (Ave) 3247.05 (Ave) 3246.89 (Ave) 3246.97 (Ave) 3246.54 (Ave) 0.56 73-104 EGDTWIIDGVGNGGLLAPEAPLDFGNAATGCR3251.94 (Ave) 3252.18 (Ave) 3252.06 (Ave) 3253.42 (Ave) 3252.58 (Ave) 3252.04 (Ave) 3251.75 (Ave) 0.19 321-351 APSMIDEYPILAVAAAFAEGATVMNGLEELR4191.34 (Ave) 4191.48(Ave) 4191.89 (Ave) 4191.63 (Ave) 4190.89 (Ave) 0.37 234-274 LTGQVIDVPGDPSSTAFPLVAALLVPGSDVTILNVLMNPTRMonsanto Company 11-CA-233U 232 of 500 Figure C-1. MALDI-TOF MS Coverage Map of the MON 88302-produced

CP4 EPSPS Protein The amino acid sequence of the mature CP4 EPSPS protein was deduced from the cp4 epsps gene present in MON 88302. Boxed re gions correspond to regions covered by tryptic peptides that were identified from the MON 88302-produced CP4 EPSPS protein sample using MALDI-TOF MS. In total, 85.5% (389 of 455 total amino acids) of the expected protein sequence was cove red by the identified peptides.

C.6. Western Blot An alysis-Immunoreactivity

C.6.1. Methods

Western blot analysis was performed to confirm the identity of the CP4 EPSPS protein purified from seed of MON 88302 and to compare the immunoreactivity of the

MON 88302- and E. coli -produced proteins.

The MON 88302- and E. coli -produced CP4 EPSPS proteins were analyzed concurrently on the same gel using three loadings of 1, 2 and 3 ng. Loadings of the three concentrations were made in duplicate on the ge l. Aliquots of each protein were diluted in water and 5X Laemmli buffer (LB) containing 312 mM Tris-HCl, 20% (v/v) 2-mercaptoethanol, 10% (w/v) SDS, 0.025% (w/v) bromophenol blue, 50% (v/v) glycerol, pH 6.8), heated at ~99 °C for 3 min, and applied to a 15-well pre-cast Trisglycine 4-20% polyacrylamide gradient gel (Invitrogen, Carlsbad, CA). Pre-stained molecular weight markers (Precision Plus Protein Standards Dual color; Bio-Rad, Hercules, CA) were loaded in parallel to veri fy electrotransfer of the proteins to the

membrane and to estimate the size of the immunoreactive bands observed. Electrophoresis was performed at a constant voltage of 130 V for 90 min. Electrotransfer to a 0.45 μm nitrocellulose membrane (Invi trogen, Carlsbad, CA) was performed for 90 min at a constant voltage of 30 V. Afte r electrotransfer, the membrane was blocked

001 MLHGASSRPA TARKSSGLSG TVRIPGDKSI SHRSFMFGGL ASGETRITGL

051 LEGEDVINTG KAMQAMGARI RKEGDTWIID GVGNGGLLAP EAPLDFGNAA

101 TGCRLTMGLV GVYDFDSTFI GDASLTKRPM GRVLNPLREM GVQVKSEDGD

151 RLPVTLRGPK TPTPITYRVP MASAQVKSAV LLAGLNTPGI TTVIEPIMTR

201 DHTEKMLQGF GANLTVETDA DGVRTIRLEG RGKLTGQVID VPGDPSSTAF

251 PLVAALLVPG SDVTILNVLM NPTRTGLILT LQEMGADIEV INPRLAGGED

301 VADLRVRSST LKGVTVPEDR APSMIDEYPI LAVAAAFAEG ATVMNGLEEL

351 RVKESDRLSA VANGLKLNGV DCDEGETSLV VRGRPDGKGL GNASGAAVAT

401 HLDHRIAMSF LVMGLVSENP VTVDDATMIA TSFPEFMDLM AGLGAKIELS

451 DTKAAMonsanto Company 11-CA-233U 233 of 500 for 1 h with 5% (w/v) non-fat dried milk (NFDM) in 1X phosphate buffered saline containing 0.05% (v/v) Tween-20 (PBST). The membrane was then probed with a 1:1000 dilution of goat anti-CP4 EPSPS antibod y (lot 10000787) in 5% NFDM in PBST overnight at 4  C. Excess antibody was removed using three 10 min washes with PBST. Finally, the membrane was probed with horse radish peroxidase (HRP)-conjugated rabbit anti-goat IgG (Thermo, Rockford, IL) at a d ilution of 1:10,000 in 5% NFDM in PBST for 1 h at room temperature. Excess HRP-conj ugate was removed using three 10 min washes with PBST. All washes were performed at room temperature. Immunoreactive bands were visualized using the ECL detection syst em (GE, Healthcare, Piscataway, NJ) with exposure (1 and 3 min) to Amersham Hyperfilm ECL (GE, Healthcare , Piscataway, NJ). The film was developed using a Konica SR X-101A automated film processor (Tokyo, Japan).

Quantification of the bands on the blot was performed using a Bio-Rad GS-800 densitometer with the supplied Quantity On e software (version 4.4.0, Hercules, CA) using the lane finding and contour tools. The signal intensities of the immunoreactive

bands observed for the MON 88302- and E. coli -produced proteins migrating at the expected position on the blot film were quant ified as “contour quantity” values. The raw data was exported to a Microsoft Excel (2007) file for the pair wise comparison of the average contour quality of the load replicates. An average difference was calculated for each comparison to assess the immunoreactivity equivalence. The immunoreactivity of

the MON 88302- and E. coli -produced CP4 EPSPS proteins were considered equivalent if the signal intensity of th e CP4 EPSPS bands were within 35% of one another.Monsanto Company 11-CA-233U 234 of 500 C.6.2. Results of CP4 EPSPS Prot ein Immunoreactivity Equivalence

Western blot analysis was conducted using goat anti-CP4 EPSPS polyclonal antibody to 1) confirm the identity of the CP4 EPSPS pr otein isolated from the seed of MON 88302 and 2) to determine the relative i mmunoreactivity of the MON 88302- and the E. coli

produced CP4 EPSPS proteins. The results demonstrated that the anti-CP4 EPSPS antibody recognized the MON 88302-produced CP4 EPSPS protein that migrated to an identical position as the E. coli -produced CP4 EPSPS protein (Figure C-2). Furthermore, the immunoreactive signal increased with increasing amounts of CP4 EPSPS protein

loaded.

Densitometric analysis was conducted to compare the immunoreactivity of MON 88302-

and E. coli -produced CP4 EPSPS proteins

. The average signal intensity (OD x mm2) from the MON 88302-produced CP4 EPSPS bands and the E. coli -produced CP4 EPSPS bands at each amount of protein loaded are shown in Table C-3. The percent differences in the average signal intensity from th e MON 88302-produced CP4 EPSPS bands and

from the E. coli -produced CP4 EPSPS bands for each amount analyzed was calculated.

These values as well as the overall average percent differen ce (24.1%) are al so shown in Table C-3. The acceptance criterion for equi valence of immunoreac tivity (±35%) of the MON 88302-produced CP4 EPSPS bands and E. coli -produced CP4 EPSPS bands was met. Thus, the western blot analysis es tablished identity of the MON 88302-produced

CP4 EPSPS and demonstrated that the MON 88302- and E. coli -produced CP4 EPSPS proteins have equivalent immunoreactivity with a CP 4 EPSPS-specific antibody.Monsanto Company 11-CA-233U 235 of 500 Figure C-2. Western Blot Analysis of MON 88302- and E. coli -produced

CP4 EPSPS Proteins Aliquots of the MON 88302-produced CP4 EPSPS protein and the E. coli -produced CP4 EPSPS protein were separated by SDS- PAGE and electrotransferred to a PVDF

membrane. The membrane was incubated with anti-CP4 EPSPS antibodies and immunoreactive bands were visualized using an ECL system (GE Healthcare, Piscataway, NJ). Approximate molecular weights (kDa) are shown on the left and correspond to the markers loaded in lane 1. The 1 min exposure is shown.

Lane Sample Amount (ng)

1 Precision Plus Protein Standards Dual color

- 2 E. coli -produced CP4 EPSPS protein 1

3 E. coli -produced CP4 EPSPS protein 1

4 E. coli -produced CP4 EPSPS protein 2

5 E. coli -produced CP4 EPSPS protein 2

6 E. coli -produced CP4 EPSPS protein 3

7 E. coli -produced CP4 EPSPS protein 3

8 Empty

- 9 MON 88302-produced CP4 EPSPS protein 1

10 MON 88302-produced CP4 EPSPS protein 1

11 MON 88302-produced CP4 EPSPS protein 2

12 MON 88302-produced CP4 EPSPS protein 2

13 MON 88302-produced CP4 EPSPS protein 3

14 MON 88302-produced CP4 EPSPS protein 3

15 Empty

-Monsanto Company 11-CA-233U 236 of 500 Table C-3. Comparison of Immunorea ctive Signal Between MON 88302- and E.

coli -produced EPSPS Proteins

Sample Gel

lane Amount (ng) Contour Qty (OD ×

mm 2 ) Average

Contour

Qty 1 Percent

Difference 2

(%) Average

Difference 3

(%)

E. coli CP4 EPSPS 2 1 1.257 1.408

30.8

24.1 E. coli CP4 EPSPS 3 1 1.558

MON 88302 CP4 EPSPS 9 1 2.064 2.033 MON 88302 CP4 EPSPS 10 1 2.002

E. coli CP4 EPSPS 4 2 3.296 3.748

26.5 E. coli CP4 EPSPS 5 2 4.199

MON 88302 CP4 EPSPS 11 2 4.979 5.101 MON 88302 CP4 EPSPS 12 2 5.222

E. coli CP4 EPSPS 6 3 6.264 6.407

14.9 E. coli CP4 EPSPS 7 3 6.549

MON 88302 CP4 EPSPS 13 3 7.737 7.527 MON 88302 CP4 EPSPS 14 3 7.317

1 Average Contour Quantity = ∑ (Contour Quantity)/2; contour quantity is average pixel density × band area.

2 Percent Difference (%) = ((|Average Contour Quantity MON 88302–Average Contour Quantity

E. coli |)/(Average Contour Quantity MON 88302))  100% .

3 Average difference (%) = ∑ 〖 % difference 〗 /3.

C.7. Molecular Weight and Purity Estimation using SDS-PAGE

C.7.1. Methods

An aliquot of the MON 88302-produ ced CP4 EPSPS protein and an E. coli -produced CP4 EPSPS protein was mixed with 5X LB and diluted with H 2 O to a final total protein concentration of 0.2 μg/μl. Bio-Rad broad ra nge Molecular Weight Standards (Hercules, CA) were diluted to a final to tal protein concentration of 0.9  g/  l. The MON 88302-produced CP4 EPSPS protein was an alyzed in duplicate at 1, 2, and 3 μg protein per lane. The E. coli -produced CP4 EPSPS reference standard was analyzed at 1 μg total protein in a single lane. The samp les were loaded onto a 10-well pre-cast Tris glycine 4-20% polyacrylamide gradient mini-gel (Invitrogen, Carlsbad, CA) and electrophoresis was performed at a constant voltage of 130 V for 95 min. Proteins were fixed by placing the gel in a so lution of 40% (v/v) methanol and 7% (v/v) acetic acid for ~30 min, stained for ~16 h with Brilliant Bl ue G-Colloidal stain (Sigma-Aldrich, St. Louis, MO). Gels were destained for 30 to 45 sec with a soluti on containing 10% (v/v) acetic acid and 25% (v/v) methanol, and for ~7 h with 25% (v/v) methanol. Analysis of the gel was performed using a Bio-Rad GS-800 densitometer with the supplied Quantity One software (version 4.4.0, Hercules, CA). The apparent MW of each observed band was estimated from a standard curve generated by the Quantity One software which was based on the MWs of the markers and their migration distance on the gel. To determine purity, all visible bands within each lane we re quantified using Quantity One software. The purity of the MON 88302-produced CP4 E PSPS protein was reported as the percentMonsanto Company 11-CA-233U 237 of 500 of the total of all quantified bands in a lane . Apparent MW and pur ity were reported as an average of all six lanes cont aining the MON 88302-produced CP4 EPSPS

C.7.2. Results of CP4 EPSPS Protei n Molecular Weight Equivalence

For molecular weight and purity analysis , the MON 88302-produced CP4 EPSPS protein was separated using SDS-PAGE. The gel was stained with Brilliant Blue G Colloidal stain and analyzed by densitometry (Figure C-3). The MON 88302-produced CP4 EPSPS protein (Figure C-3, lanes 3-8) mi grated to the same position on the gel as

the E. coli -produced CP4 EPSPS protein (Figure C-3, lane 2) and had an apparent molecular weight of 43.1 kDa (Table C-4). The apparent molecular weight of the E. coli

produced CP4 EPSPS protein as reported on its Certificate of Analysis was 43.8 kDa (Table C-4). The apparent mol ecular weights of the MON 88302- and E. coli -produced CP4 EPSPS proteins were consider ed equivalent if they were within 10% of one another. Because the experimentally determined a pparent molecular weight of the MON 88302- produced CP4 EPSPS protein was within 10% of the E. coli -produced CP4 EPSPS protein (Table C-4), the MON 88302- and E. coli -produced CP4 EPSPS proteins were determined to have equivalent apparent molecular weights.

The purity of the MON 88302-produced CP4 E PSPS protein was calculated based on the six loads on the gel (Figure C- 3, lanes 3 to 8). The average purity was determined to be

99%.

Table C-4. Molecular Weight Comp arison Between the MON 88302- and E. coli

produced CP4 EPSPS Proteins
Molecular Weight

of MON 88302-Produced

CP4 EPSPS Protein Molecular Weight of

E. coli -Produced

CP4 EPSPS Protein 1 % Difference from

E. coli -Produced

CP4 EPSPS Protein

43.1 kDa 43.8 kDa 1.6%

1 The molecular weight of the E

. coli -produced CP4 EPSPS protein as re ported on its Certificate of Analysis.Monsanto Company 11-CA-233U 238 of 500 Figure C-3. Molecular Weight and Puri ty Analysis of the MON 88302-produced

EPSPS Protein Aliquots of the MON 88302- and the E. coli -produced CP4 EPSPS proteins were separated on a 4-20% Tris glycine polyacryl amide gradient gel and then stained with Brilliant Blue G-Colloidal stain. Approxima te molecular weights are shown on the left and correspond to the markers loaded in Lanes 1 and 9.

Lane Sample Amount (μg) 1 Broad Range Molecular Weight Markers 4.5

2 E. coli -produced CP4 EPSPS protein 1 3 MON 88302-produced CP4 EPSPS protein 1 4 MON 88302-produced CP4 EPSPS protein 1 5 MON 88302-produced CP4 EPSPS protein 2 6 MON 88302-produced CP4 EPSPS protein 2 7 MON 88302-produced CP4 EPSPS protein 3 8 MON 88302-produced CP4 EPSPS protein 3 9 Broad Range Molecular Weight markers 4.5 10 Empty -Monsanto Company 11-CA-233U 239 of 500 C.8. Glycosylation Analysis

C.8.1. Methods

Glycosylation analysis was used to determine whether the MON 88302-produced CP4 EPSPS was post-translationally modi fied with covalent ly bound carbohydrate moieties. Aliquots of the MON 88302-produced CP4 EPSPS protein, the E. coli

produced CP4 EPSPS and the positive control, transferrin (Sigma-Aldrich, St Louis, MO), were each diluted with water and mixed with 1X LB. These samples were heated

at ~95 °C for 3 min. The MON 88302- and the E. coli - produced CP4 EPSPS proteins were loaded at approximately 100 and 200 ng per lane and transferrin was loaded at approximately 50, 100, 150 and 200 ng on a Tris-g lycine 10-well 4- 20% polyacrylamide gradient mini-gel (Invitrogen, Carlsbad, CA ). Precision Plus Protein Dual color Standards (Bio-Rad, Hercules, CA) were also loaded to verify electrotransfer of the proteins to the membrane and as markers for molecular weight. Electrophoresis was performed at a constant voltage of 155 V fo r 75 min. Electrotransfer to a 0.45 μm PVDF membrane (Invitrogen, Carlsbad, CA) was perfor med for 35 min at a constant voltage of

100 V.

Carbohydrate detection was performed dir ectly on the PVDF me mbrane at room temperature using the Amersham ECL glycopr otein Detection Module (GE, Healthcare, Piscataway, NJ). With this module, carbohydr ate moieties of protei ns are oxidized with sodium metaperiodate and are then biot inylated with bio tin-X-hydrazide. The biotinylated proteins can be de tected on the blot by addition of streptavidin conjugated to HRP for luminol-based detection using ECL re agents (GE, Healthcare, Piscataway, NJ) and with subsequent exposure (1, 2 and 3 min) to Amersham Hyperfilm (GE, Healthcare). The film was developed using a Konica SRX-101A automated film processor (Tokyo, Japan).

A second identical blot run in parallel to that used for th e glycosylation analysis was stained to visualize the proteins present on the membrane. Proteins were stained for 30 sec to 2 min using Coomassie Brilliant Blue R-250 staining solution (Bio-Rad, Hercules, CA) and then destained with 1X Coomassie Brilliant Blue R-250 Destaining Solution (Bio-Rad) for 5 min. After washing with water, the blot was dried and scanned using Bio-Rad GS-800 densitometer with th e supplied Quantity One software (version 4.4.0).

C.8.2. Results of Glyco sylation Analysis

Some eukaryotic proteins are post-translationally modified by the addition of carbohydrate moieties (Rademacher et al., 19 88). To test whether the CP4 EPSPS protein was glycosylated when expresse d in the seed of MON 88302, the MON 88302- produced CP4 EPSPS protein was analyzed using an ECL Glycoprotein Detection Module (GE, Healthcare, Piscataway, NJ). Tr ansferrin, a glycosylat ed protein, was used as a positive control in the assay. To assess equivalence of the MON 88302- and E. coli

produced CP4 EPSPS proteins, the E. coli -produced CP4 EPSPS protein, previously been shown to be free of glycosylation (Harrison et al., 1996), was also analyzed. The positiveMonsanto Company 11-CA-233U 240 of 500 control was clearly detected at expected molecular weight ( ~ 76 kDa) and the band intensity increased with increasing concentrat ion (Figure C-4, Panel A, lanes 2-5). In contrast, signals were not observed in the lanes containing the MON 88302- or E. coli

produced protein at the expected molecular weight for the CP4 EPSPS protein (Figure C4 panel A, lanes 6-9). To conf irm that sufficient MON 88302- and E. coli -produced CP4 EPSPS proteins were present for glycos ylation analysis, a second membrane (with identical loadings and transfer times) was st ained with Coomassie Blue R250 for protein detection (Figure C-4 Panel B). Both the MON 88302- and E. coli -produced CP4 EPSPS proteins were clearly detected (Figure C-4, Panel B, Lanes 6-9) . These data indicate that the glycosylation status of MON 88302-produced CP4 EPSPS protein is equivalent to

that of the E. coli -produced CP4 EPSPS protein and that neither is glycosylatedMonsanto Company 11-CA-233U 241 of 500 Figure C-4. Glycosylation Analysis of the MON 88302-produced CP4 EPSPS

Protein Aliquots of the transfe rrin (positive control), E. coli -produced CP4 EP SPS protein and MON 88302-produced CP4 EPSPS protein were separated by SDS-PAGE (4-20%) and electrotransferred to PVDF membranes. (A ) Where present, the labeled carbohydrate moieties were detected using the ECL-based system with exposure to Hyperfilm. A 2 min exposure is shown. (B) An equivalent bl ot was stained with Coomassie Blue R250 to confirm the presence of proteins. Th e signal was captured us ing a Bio-Rad GS-800 with Quantity One software (version 4.4.0) . Approximate molecular weights (kDa) correspond to the Precision Plus, dual color mark ers (used to verify transfer and MW) in

Lane 1. Arrows indicate the band co rresponding to CP4 EPSPS protein.

Lane Sample Amount (ng) 1 Precision Plus, dual color MW markers

- 2 Transferrin (positive control) 50 3 Transferrin (positive control) 100 4 Transferrin (positive control) 150 5 Transferrin (positive control) 200

6 E. coli -produced CP4 EPSPS (negative control) 100

7 E. coli -produced CP4 EPSPS (negative control) 200 8 MON 88302-produced CP4 EPSPS 100 9 MON 88302-produced CP4 EPSPS 200 10 Empty -Monsanto Company 11-CA-233U 242 of 500 C.9. Functional Activity Analysis

C.9.1. Methods

Prior to functional activity analysis, both MON 88302- and E. coli -produced proteins were diluted to a purity corrected concen tration of ~50 μg/ml with 50 mM HEPES, pH 7.0 buffer. Assays for both proteins were conducted in triplicate. The reactions were performed in 50 mM HEPES, pH 7.0, 0.1 mM ammonium molybdate, 1 mM PEP and 5 mM potassium fluoride with or wi thout 2 mM S3P for 2 min at ~25  C. The reactions were initiated by the addition of PEP. After 2 min, the reactions were quenched with phosphate assay reagent (0.033% malachite gr een, 1.1% ammonium molybdate) and then fixed with 33% (w/v) sodium citrate. A standard curve was prepared using 0 to 10 nmoles of inorganic phosphate in water tr eated with the phosphate assay reagent and 33% (w/v) sodium citrate. The absorban ce of each reaction and each standard was measured in duplicate at 660 nm using a PowerWave Xi  (Bio-Tek, Richmond, VA) microplate reader. The amount of inorganic phosphate rel eased from PEP in each reaction was determined using the standard curve. For CP4 EPSPS, the specific activity was defined in unit per mg of protein (U/mg) , where a unit (U) is defined as 1 μmole of inorganic phosphate released from PEP per min at 25  C. Calculations of the specific activities were performed usi ng Microsoft Excel (2007).

C.9.2. Results of Functional Activity

The functional activities of the MON 88302- and E. coli -produced CP4 EPSPS proteins were determined using a colorimetric assa y that measures formation of inorganic phosphate (Pi) from the EPSPS-catalyzed r eaction between shikimate-3-phosphate (S3P) and phosphoenolpyruvate (PEP). In this assay, protein-specific activity is expressed as units per milligram of protein (U/mg), where a unit is defined as one μmole of inorganic phosphate released from PEP per mi nute at 25 °C. The MON 88302- and E. coli

produced CP4 EPSPS proteins were considered to have equivalent functional activity if the specific activities were within 2-fold of one another.

The experimentally determined speci fic activities for the MON 88302- and E. coli

produced CP4 EPSPS proteins are presented in Table C-5. The sp ecific activities of

MON 88302- and E. coli -produced CP4 EPSPS proteins were 4.93 U/mg and 2.79 U/mg of CP4 EPSPS protein, respectively. B ecause the specific activity of the MON 88302- produced CP4 EPSPS protein falls within the pr eset acceptance criterion (Table C-5), the MON 88302-produced CP4 EPSPS protein was cons idered to have equivalent functional activity to that of the E. coli -produced CP4 EPSPS protein.Monsanto Company 11-CA-233U 243 of 500 Table C-5. CP4 EPSPS Functional Activity Assay

MON 88302-produced

CP4 EPSPS Protein 1

(U/mg) E. coli -produced

CP4 EPSPS Protein 1

(U/mg) Previously set acceptance

limits 2

(U/mg)

4.93 ± 0.36 2.79 ± 0.26 1.40 – 5.58

1 Value refers to mean and standard deviation calcula ted based on n = 6 which includes three replicate assays spectrophotometrically.

2 Within 2-fold of the E.coli -produced CP4 EPSPS specific activity (2.79 ÷ 2 U/mg to 2.79 x 2 U/mg)Monsanto Company 11-CA-233U 244 of 500 References for Appendix C

Aebersold, R. 1993. Mass spectr ometry of proteins and peptides in biotechnology. Current Opinion in Biotechnology 4: 412-419.

Billeci, T.M. and J.T. Stults. 1993. Tryptic mapping of recombinant proteins by matrixassisted laser desorption/ionization mass spectrometry. Analytical Chemistry 65: 1709-

1716.

Harrison, L.A., M.R. Bailey, M.W. Naylor, J.E. Ream, B.G. Hamm ond, D.L. Nida, B.L. Burnette, T.E. Nickson, T.A. Mitsky, M.L. Taylor, R.L. Fuchs and S.R. Padgette. 1996. The expressed protein in glyphosate-to lerant soybean, 5-enolpyruvylshikimate-3- phosphate synthase from Agrobacterium sp. strain CP4, is rapidly digest ed in vitro and is not toxic to acutely gavaged mice. Journal of Nutrition 126: 728-740.

Hunkapiller, M.W., R.M. Hewick, L.E. Hood and W.J. Dreyer. 1983. High-sensitivity sequencing with gas-phase sequenato r. Methods in Enzymology 91: 399-413.

Jensen, O.N., A.V. Podtelejnikov and M. Mann. 1997. Identification of the components of simple protein mixtures by high-accuracy peptide mass mapping and database searching. Analytical Chemistry 69: 4741-4750.

Rademacher, T.W., R.B. Parekh and R.A. Dwek. 1988. Glycobiology. Annual Review of Biochemistry 57: 785-838.Monsanto Company 11-CA-233U 245 of 500 Appendix D: Materials and Methods Used for the Analysis of the Levels of

CP4 EPSPS Protei n in MON 88302

D.1. Materials

Seed, forage, over-season leaf (OSL-1-4), an d root (Root-1-2) tissue samples from

MON 88302 were harvested from three field si tes in the U.S. and three field sites in Canada during the 2009 growing seas on from starting seed lot 11225246. An

E. coli -produced CP4 EPSPS protein (lot 10000739) wa s used as the analytical reference

standard.

D.2. Characterization of the Materials

The identity of MON 88302 was confirme d by verifying the chain of custody documentation prior to analysis. To furt her confirm the ident ities of MON 88302 event-specific polymerase chain reaction (PCR) analyses were conducted on the harvested seed from each site. Any seed sample and its associated tissues, for which three or more pools out of four tested une xpectedly during PCR ve rification, were not analyzed in this study.

D.3. Field Design and Tissue Collection

Field trials were initia ted during the 2009 planting s eason to generate MON 88302 samples at various canola growing locations in the U.S. and Canada. The forage, seed, OSL-1-4, and Root-1-2 tissue samples from the following field sites were analyzed: Power County, Idaho, U.S. (IDAF), Wilkin County, Minnesota, U.S (MNCA), McHenry County, North Dakota, U.S. ( NDVA), Portage la Prairie, Manitoba, Canada (MBPL), Newton, Manitoba, Canada (MBNW) and Sa skatoon, Saskatchewan, Canada (SKSA). These field sites were repres entative of canola producing re gions suitable for commercial production. At each site, four replicated plots of plants containing MON 88302 were planted using a randomized complete block field design. OSL-1- 4, forage, seed and Root-1-2 samples were collected from each re plicated plot at all field sites. See Table V-1 for a detailed description of when the samples were collected.

From the IDAF site, seed and Root-2 samples were excluded from the study due to inclement weather during collection which im pacted sample quality and quantity.

D.4. Tissue Processing and Protein Extraction

Tissue samples were shipped to Monsanto, St . Louis. The following tissues were not received by Sample Management: all OSL-2 tissue samples from sites MBPL, MBNW, and MNCA, and one Root-2 sample from site NDVA. The following tissues were received but not processed by sample manageme nt due to compromised sample integrity: all OSL-1 samples from site MBNW, one Root -1 sample from site SKSA, all Root-2 samples from site SKSA, and one Root-2 sa mple from site NDVA. The processed tissue samples were stored in a -80 °C freezer.Monsanto Company 11-CA-233U 246 of 500 CP4 EPSPS protein was extracted from the ti ssue samples as described in Table D-1. CP4 EPSPS protein was extracte d from all tissues samples using a Harbil Mixer with the appropriate amount of Tris-borate buffer w ith L-ascorbic acid (1 × TBA) [0.1 M Tris,

0.1 M Na 2 B 4 O 7 • 10H 2 O, 0.005 M • 6H 2 O MgCl 2 , 0.05% (v/v) Tween-20 at pH 7.8, 0.2% (w/v) L-ascorbic acid]. Insoluble material was removed from all tissue extracts using a serum filter (Fisher Scie ntific, Pittsburgh, PA). The extr acts were aliquotted and stored frozen in a -80 °C freezer until ELISA analysis.Monsanto Company 11-CA-233U 247 of 500 Table D-1. Protein Extraction Methods for Tissue Samples

Sample Type Tissue-to-Buffer Ratio Extraction Buffer

Leaf 2 1:100 1× TBA

Root 3 1:100 1× TBA

Forage 1:100 1× TBA

Seed 1:100 1× TBA

1 Over- season leaf (OSL-1, OS L-2, OSL-3, and OSL-4).

2 Root (Root-1 and Root-2).Monsanto Company 11-CA-233U 248 of 500 D.5. CP4 EPSPS Antibodies

Mouse monoclonal antibody clone 39B6.1 (IgG2a isotype, kappa light chain; lot 10002190) specific for the CP4 EPSPS protein wa s purified from mouse ascites fluid using Protein-A Sepharose affi nity chromatography and was used as the capture antibody

in the CP4 EPSPS ELISA. The concentration of the purified IgG was determined to be 2.3 mg/ml by spectrophotometric methods . Production of the 39B6.1 monoclonal antibody was performed by Strategic Biosolutio ns (Newark, DE). The purified antibody was stored in a buffer (pH 7.2) containing 20 mM sodium phosphate, 150 mM NaCl, and 15 ppm Proclin 300 (Sigma-Aldrich, St. Louis, MO).

The detection reagent was goat anti-CP4 EPSPS antibody, otherwise known as anti-protein 4 (Sigma-Aldrich, catalog number P-5867) conjugated to horseradish peroxidase (HRP).

D.6. CP4 EPSPS ELISA Method

Mouse anti-CP4 EPSPS antibodies were diluted in coating buffer (15 mM Na 2 CO 3 , 35 mM NaHCO 3 , and 150 mM NaCl, pH 9.6) to a final concentration of 2.0 μg/ml, and immobilized onto 96-well microtiter plates foll owed by incubation in a 4 °C refrigerator

for ≥ 8 hours. Prior to each step in the assa y, plates were washed with 1× PBST. CP4 EPSPS protein standard or sample extract was added at 100 μ l per well and incubated for 1 hour at 37 °C. The capture d CP4 EPSPS protein was detected by the

addition of 100 μ l per well of anti-CP4 EPSPS HRP conj ugate. Plates were developed by adding 100 μl per well of 3,3',5,5' tetramet hyl-benzidine (TMB; Kirkegaard & Perry, Gaithersburg, MD). The enzymatic reaction wa s terminated by the addition of 100 μl per

well of 6 M H 3 PO 4 . Quantification of the CP4 E PSPS protein was accomplished by interpolation from a CP4 EPSPS protei n standard curve that ranged from 0.456-14.6 ng/ml.

D.7. Moisture Analysis

Tissue moisture content was determined us ing an IR-200 Moisture Analyzer (Denver Instrument Company, Arvada, CO). A homogeneous tissue -specific site pool (TSSP) was prepared consisting of samples of a give n tissue type grown at a given site. The average percent moisture for each TSSP was cal culated from triplicate analyses. A TSSP Dry Weight Conversion Factor (D WCF) was calculated as follows:

DWCF ൌ 1 െ ൬ Mean% TSSP Moisture

100 ൰

The DWCF was used to convert protein levels assessed on a μg/g fres h weight (fw) basis into levels reported on a μg/g dry weight (dw) basis using the following calculation:Monsanto Company 11-CA-233U 249 of 500 Protein Level in Dry Weight ൌ ൬ Protein Level Fresh Weight

DWCF ൰

The protein levels (ng/ml) that were reported to be less than or equal to the limit of detection (LOD) or less than the limit of quantitation (LOQ) on a fresh weight basis were not reported on a dry weight basis.

D.8. Data Analyses

All CP4 EPSPS ELISA plates were analyzed on a SPECTRAmax Plus 384 (Molecular Devices, Sunnyvale, CA) microplate spec trophotometer, using a dual wavelength detection method. All protein concentrations were determined by optical absorbance at a wavelength of 450 nm with a simultaneous reference reading of 620-650 nm. Data reduction analyses were performed usi ng Molecular Devices SOFTmax PRO GxP version 5.0.1. Absorbance readings and protein standard concentrations were fitted with a four-parameter logistic curve. Following th e interpolation from the standard curve, the amount of protein (ng/ml) in the tissue was c onverted to a μg/g fw basis for data that were greater than or equal to the LOQ. This conversion utilized a sample dilution factor and a tissue-to-buffer ratio. The protein values expressed as μg/g fw were also converted to μg/g dw by applying the DWCF. Microsoft Excel 2007 (Version (12.0.6535.5002) SP2 MSO (12.0.6535.5002) Microsoft, Redmond, WA) was used to calculate the CP4 EPSPS protein level in canola tissues. Th e sample means, standard deviations, and ranges were also calculated by Microsoft Excel 2007.

Any MON 88302 sample extracts that resulte d in unexpectedly negative results by ELISA analysis were re-extracted twice for the protein of interest and re-analyzed by ELISA to confirm the results. Samples with confirmed unexpected results were omitted

from all calculations.Monsanto Company 11-CA-233U 250 of 500 Appendix E: Materials, Methods, and I ndividual-Site Results for Compositional Analysis of MON 88302 Canola Seed

E.1. Materials

Seed from MON 88302 (Seed Lot Number 11225246) and the conventional control (Seed Lot Number 11225244) was evaluated. The conventional control has background genetics similar to that of MON 88302 but does not contain the cp4 epsps expression

cassette. The commercial reference variet ies were seven conventional canola varieties (Table E-1).

Table E-1. Commercial Reference Canola Varieties

Material Name Seed Lot Number Field Sites 1

Q2 10001931 MBPL, MBNW, SKSA, NDVA, MNCA Hyola 401 10001850 NDVA, MBPL, SKSA SP Armada 10001932 MBPL, SKSA, NDVA Croplan 601 10001849 MBPL, SKSA, NDVA SValof Sponsor 10002116 MNCA, MBNW SValof Senator 10002115 MNCA, MBNW DSV Ability 10002117 MNCA, MBNW

1 Field sites described in Section E.3.

E.2. Characterization of the Materials

The identities of MON 88302, the conventional control, and commercial reference varieties were confirmed by verifying th e chain of custody documentation prior to analysis. To further confirm the identitie s of MON 88302, the conventional control, and commercial reference varieties, event-specific polymerase chain reaction (PCR) analyses were conducted on the harvested seed from each site to confirm the presence or absence

of the cp4 epsps expression cassette.

E.3. Field Production of the Samples

Seeds from the MON 88302, the conventional co ntrol and commercial reference varieties were collected from replicated plots at each of two U.S. sites [Wilkin County, Minnesota (MNCA); and McHenry County, North Dako ta (NDVA)] and thre e Canadian sites [Portage la Prairie, Manitoba (MBPL); Newton, Manitoba (MBNW); and Saskatoon, Saskatchewan (SKSA) ]. Seeds were planted in a random ized complete block design with four replicates per site. The MON 88302 plots were treated with glyphosate applications between the 5-6 leaf stage, at a target rate of 1800 g a.e./ha. All samples at the field sites were grown under normal agronomic field c onditions for their respective geographic regions. Seed samples were harvested from all plots and shipped at ambient temperature

from the field sites to Monsan to Company (St. Louis, MO). Sub-samples were ground to a powder, stored in a freezer set to maintain -20 °C located at Monsanto Company (St.Monsanto Company 11-CA-233U 251 of 500 Louis, MO), and then shipped on dry ice to Covance Laboratories Inc. (Madison, WI) for analysis.

E.4. Summary of Analytical Methods and Reference Standards

Ground grain samples were analyzed by Cova nce Laboratories Inc. Upon receipt, the samples were stored in a freezer set to maintain -20 °C until their use. Nutrients assessed in this analysis included proximates (ash, carbohydrates by calcula tion, moisture, protein, and fat), acid detergent fiber (ADF), neutral detergent fiber (NDF), total dietary fiber (TDF), amino acids, fatty acids (C8-C24), vitamin E ( α -tocopherol) and minerals (calcium, copper, iron, magnesium, manga nese, phosphorus, potassium, sodium, and zinc) in the grain. The toxicants assessed in grain included erucic acid and glucosinolates (alkyl glucosinolates, indolyl glucosinolates, and total glucos inolates). The anti-nutrients assessed in grain included phytic acid and sinapic acid.

E.4.1. Acid Detergent Fiber

The ANKOM2000 Fiber analyzer automated the process of removal of proteins, carbohydrates, and ash. Fats and pigments were removed with an ace tone wash prior to analysis. The fibrous residue that is prim arily cellulose, lignin, and insoluble protein complexes remained in the Ankom filter ba g, and were determined gravimetrically. (Komarek et al., 1994; USDA, 1970). The resu lts are reported on fresh weight basis. The limit of quantitation was 0.100%.Monsanto Company 11-CA-233U 252 of 500 E.4.2. Amino Acid Composition

The following 18 amino acids were analyzed:

Total threonine Total aspartic acid (including asparagine) Total serine Total tyrosine Total phenylalanine Total glut amic acid (including glutamine) Total proline Total histidine Total glycine Total lysine Total alanine Total arginine Total valine Total tryptophan

Total isoleucine Total methionine Total leucine Total cystine (including cysteine)

The sample was assayed by three methods to obtain the full profile. Tryptophan required a base hydrolysis with sodium hydroxide. Th e sulfur-containing ami no acids required an oxidation with performic acid prior to hydrolysis with hydrochloric acid. Analysis of the samples for the remaining amino acids was accomplished through direct acid hydrolysis with hydrochloric acid. Once hydrolyzed, the individual amino acids were then quantified using an automated amino aci d analyzer (AOAC, 2005a). The limit of quantitation was 0.100 %.

Reference Standards:

 Thermo Scientific, K18 amino acid standard, H,, 2.5 ± 0.1 μmol/mL per constituent (except cystine 1.25 ± 0.1 μmol/mL), Lot Number KG137091

 Sigma, L-Tryptophan, 100%, Lot Number 097K0119

 Sigma/BioChemika, L-Cysteic Acid M onohydrate, 99.5% (used as 100%),

Lot Number 1305674

 Sigma, L-Methionine Sulfone, 100%, Lot Number 047K1321

 Sigma, L-Norvaline, 100%, 087K1954

E.4.3. Ash

The sample was placed in an electric fu rnace at 550 °C and i gnited. The nonvolatile matter remaining was quantified gravimetrically and calculated to determine percent ash (AOAC, 2005b). The limit of quantitation was 0.100%.Monsanto Company 11-CA-233U 253 of 500 E.4.4. Carbohydrates

The total carbohydrate level was calculated by difference using the fresh weight-derived data and the following equation:

% carbohydrates = 100% - (% protein + % fat + % moisture + % ash)

The results are reported on fr esh weight basis (USDA, 1973). The limit of quantitation

was 0.100%.

E.4.5. Fat by Soxhlet Extraction

The sample was weighed into a cellulose thim ble containing sodium sulfate and dried to remove excess moisture. Pentane was dripped through the sample to re move the fat. The extract was then evaporated, dried, and weighed (AOAC, 2005c). The results are reported on fresh weight basis. Th e limit of quantitation was 0.100%.

E.4.6. Fatty Acids as Triglycerides

The lipid was extracted, saponified with 0.5 N methanolic sodium hydroxide, and methylated with 14% boron trifluoride in meth anol. The resulting methyl esters of the fatty acids were extracted with heptane cont aining an internal standard. The methyl esters of the fatty acids we re analyzed by gas chromatogr aphy using external standards for quantitation (AOAC, 2005d; AOCS, 1997; 2007) . The results are reported on fresh weight basis. The limit of quantitation was 0.0400%.

Reference Standards:

 Nu Chek Prep GLC Reference Standard Hazleton No. 1, *, Lot Number MA30-U

 Nu Chek Prep GLC Reference Standard Hazleton No. 2, *, Lot Number AU24-T

 Nu Chek Prep GLC Reference Standard Hazleton No. 3, *, Lot Number JY17-T

 Nu Chek Prep GLC Reference Standard Hazleton No. 4, *, Lot Number MA30-U

 Nu Chek Prep Methyl Gamma Linolena te, used as 100%, Lot Number U-63M08-T

 Nu Chek Prep Methyl Tridecanoate, us ed as 100%, Lot Number N-13M-MA25-T

 Nu Chek Prep Methyl Erucate, used as 100%, Lot Numbers U-79M-JA28-T

 Nu Chek Prep Methyl Lignocerate, us ed as 100%, Lot Number N-24M-S8-T

 Nu Chek Prep Methyl Docosapentaenoa te, used as 100%, Lot Number U-101MD4-T

 Nu Chek Prep Methyl Docosahexaenoa te, used as 100%, Lot Number U-84MJA15-UMonsanto Company 11-CA-233U 254 of 500  Nu Chek Prep Methyl Eicosapentaenoa te, used as 100%, Lot Number U-99MS22-T

 Nu Chek Prep Methyl Nervonate, us ed as 100%, Lot Number U-88M-MA31-U

 Cayman Chemicals Steariodonic Acid Methyl Ester, 100%, Lot Number 0407775

*Overall purity of the su m of the mixture of components was used as 100%

E.4.7. Glucosinolates

Glucosinolates were extracted using 70% meth anol at 75 °C. They were then purified and enzymatically desulfatated on ion-excha nge resin. Determination was by reversedphase high performance liquid chromatography with gradient elution and ultraviolet detection using an internal standard. Quantification was performed based on the relative responses to the internal st andards. Peak id entification was made based on retention times determined by comparing the chromatogr ams of internal standard(s) and three BCR certified oilseed rape controls (ISO, 1992). The results are repo rted on fresh weight basis. The limit of quant itation was 0.00300 μmole/g.

Reference Standard:

 Chromadex, Glucotropaeolin Potassium Salt, 98.7%, Lot Number 07300-304

E.4.8. ICP Emission Spectrometry

The sample was dried, precharred, and ashed overnight in a muffle furnace set to

maintain 500  C. The ashed sample was re-ashed with nitric acid, treated with hydrochloric acid, taken to dryne ss, and put into a solution of 5% hydrochloric acid. The amount of each element was determined at appropriate wavelengths by comparing the emission of the unknown sample, measur ed on the inductively coupled plasma spectrometer, with the emissi on of the standard solutions (AOAC, 2005e). The results are reported on fresh weight basis.Monsanto Company 11-CA-233U 255 of 500 Reference Standards:

Inorganic Ventures Reference Standa rds and Limits of Quantitation:

E.4.9. Moisture

The sample was dried in a vacuum oven at approximately 100  C to a constant weight. The moisture weight loss was determined and converted to percent moisture (AOAC, 2005f). The results are reported on fresh wei ght basis. The limit of quantitation was

0.100%.

E.4.10. Neutral Detergent Fiber, Enzyme Method

The ANKOM2000 Fiber Analyzer automated the process of the removal of proteins, carbohydrates, and ash. The fats and pigments were removed with an acetone wash prior to analysis. Hemicellulose, cellulose, lignin a nd insoluble protein frac tion was left in the filter bag and determined gravimetrically (AACC, 1998; Komare k et al., 1994; USDA, 1970). The results are reported on fresh weight basis. Th e limit of quantitation was

0.100%.

E.4.11. Phytic Acid

The sample was extracted using 0.5 M HCl with ultrasonication. Purification and concentration were accomplished on a silica- based anion-exchange column. The sample was analyzed on a polymer high-performan ce liquid chromatography column PRP-1, 5 μm (150 x 4.1mm) with a refractive index detector (Lehrfeld, 1989 ; Lehrfeld, 1994). The results are reported on fresh weight ba sis. The limit of quantitation was 0.100%.

Mineral Lot Numbers Concentration (μg/mL) Limit of Quantitation (ppm) Calcium D2-MEB322092MCA, D2-MEB322094 200, 1000 20.0

Copper D2-MEB322092MCA, D2-

MEB322093MCA 2.00, 10.0 0.500

Iron D2-MEB322092MCA, D2-MEB322095 10.0, 50.0 2.00

Magnesium D2-MEB322092MCA, D2-

MEB322093MCA 50.0, 250 20.0

Manganese D2-MEB322092MCA, D2-

MEB322093MCA 2.00, 10.0 0.300

Phosphorus D2-MEB322092MCA, D2MEB322094 200, 1000 20.0 Potassium D2-MEB322092MCA, D2-MEB322094 200, 1000 100 Sodium D2-MEB322092MCA, D2-MEB322094 200, 1000 100

Zinc D2-MEB322092MCA, D2-

MEB322093MCA 10.0, 50.0 0.400Monsanto Company 11-CA-233U 256 of 500 Reference Standard:

Sigma-Aldrich, Phytic Acid Sodium Salt Hydrate, 96%, Lot Number 089K0159

E.4.12. Protein

The protein and other organic nitrogen in the sample were converted to ammonia by digesting the sample with sulfur ic acid containing a catalyst mixture. The acid digest was

made alkaline. The ammonia was distille d and then titrated with a previously standardized acid. The per cent nitrogen was calculated a nd converted to equivalent protein using the factor 6.25 (AOAC, 2005g; AOCS, 1998). The results are reported on fresh weight basis. The lim it of quantitation was 0.100%.

E.4.13. Sinapic Acid

The ground sample was extracted with meth anol followed by alkaline hydrolysis and buffering prior to injection on an analyti cal high-performance liquid chromatography (HPLC) system for quantification of sina pic acid by ultra violet (UV) detection (Hagerman and Nicholson, 1982). The results are reported on fresh weight basis. The limit of quantitation was 200 ppm.

Reference Standard:

Sigma, Sinapic Acid, 99.3%, Lot No. 079K1171.

E.4.14. Total Dietary Fiber

Duplicate samples were gelatinized with  -amylase and digested with enzymes to break down starch and protein. Ethanol was added to each sample to precipitate the soluble fiber. The sample was filtered, and the residue was rinsed with ethanol and acetone to remove starch and protein degradation pr oducts and moisture. Protein content was determined for one of the duplicates; ash conten t was determined for the other. The total dietary fiber in the sample was calculated using protein an d ash values (AOAC, 2005h). The results were reported on fresh weight ba sis. The limit of quantitation was 1.00%.

E.4.15. Vitamin E

The sample was saponified to break down any fat and release vitamin E. The saponified mixture was extracted with ethyl ether a nd then quantified by high-performance liquid chromatography using a silica column (Cort et al., 1983; McMurray et al., 1980; Speek et al., 1985). The results are reported on fresh we ight basis. The limit of quantitation was 0.500 mg/100g.

Reference Standard:

USP, α -Tocopherol, 98.9%, Lot Number N0F068Monsanto Company 11-CA-233U 257 of 500 E.5. Data Processing and Statistical Analysis

After compositional analyses were performed, data spreadsheets containing individual values for each analysis were sent to Monsanto Company for review. Data were then transferred to Certus International (Chesterfi eld, MO) where they were converted into the appropriate units and statistically analyzed . The formulas that were used for reexpression of composition data for statistical analysis are listed in Table E-2.Monsanto Company 11-CA-233U 258 of 500 Table E-2. Re-expression Formulas for St atistical Analysis of Composition Data

Component From (X) To Formula 1
Proximates (excluding Moisture), Fiber, Phytic Acid % fw % dw X/d

Alkyl Glucosinolate, Indolyl Glucosinolate, Total Glucosinolate μmole/g fw μmole/g dw X/d

Sinapic Acid ppm fw % dw X/(10 4 d) Calcium, Magnesium, Phosphorus, Potassium, Sodium ppm fw g/100g dw X/(10 4 d)

Copper, Iron, Manganese, Zinc ppm fw mg/kg dw X/d

Vitamin E mg/100g fw mg/100g dw X/d Amino Acids (AA) mg/g fw % dw X/(10d)

Fatty Acids (FA) % fw % Total FA (100)X j /  X, for each FA j where  X is over all the FA

1 ‘X’ is the individual sample value; ‘d’ is the fraction of the sample that is dry matter

.

In order to complete a statistical analysis for a compositional component in this study, at

least 50% of the values for a component had to be grea ter than the assay limit of quantitation (LOQ). Components with more than 50% of ob servations below the assay LOQ were excluded from summar ies and analysis. The following 19 components with

more than 50% of the observations below the assay LOQ were excluded: 8:0 caprylic acid, 10:0 capric acid, 12:0 la uric acid, 14:0 myristic aci d, 14:1 myristoleic acid, 15:0 pentadecanoic acid, 15:1 pentadecenoic acid, 17:0 heptadecanoic acid, 17:1 heptadecenoic acid, 18:3 gamma-linolenic acid, 18:4 octadecatetraenoic acid; 20:2 eicosadienoic acid, 20:3 eicosatrienoic acid, 20:4 arachidonic acid, 20:5 eicosapentaenoic acid, 22:1 erucic acid, 22:5 docosapentae noic acid, 22:6 docosahexaenoic acid, and

sodium.

If less than 50% of the observations for a component we re below the LOQ, individual analyses that were below the LOQ were assign ed a value equal to one-half the LOQ. In this study 24 values for 24:0 lignoceric acid and 34 values for 24:1 nervonic acid were assigned a value of 0.02% total fw.

The data were assessed for potential outliers using a studentized PRESS (Predicted

Re sidual Sum of Squares) calc ulation. A PRESS residual is the difference between any value and its value predicted from a statistic al model that excludes the data point. The

studentized version scales these residuals so that the values tend to have a standard normal distribution when outliers are absent. Thus, most values are expected to be

between  3. Extreme data points that are also outside of the  6 studentized PRESS residual range are considered for exclusion, as outliers, from the final analyses. One 18:3

linolenic value from MON 88302 at the MNCA site, one alkyl glucosinolate value and one total glucosinolate value from one comm ercial reference at the MBPL site were identified as outliers, but the values were either similar to other nearby data points or were not the extreme highest or lowest value, and were not removed from statistical analysis. One carbohydrate value and one tota l fat value from one commercial reference

at the MBNW site were extreme da ta points that were outside the  6 studentized PRESS residual range and were removed fr om the statistical analysis.Monsanto Company 11-CA-233U 259 of 500 All canola components were st atistically analyzed using a mixed model analysis of variance. The five replicated field sites we re analyzed individually and as a combined data set. Individual replicated s ite analyses used model (1).

(1) Y ij = U + T i + B j + e ij ,

where Y ij = unique individual observation, U = overall mean, T i = substance effect,

B j = random block effect, and e ij = residual error.

Combined-site analyses used model (2).

(2) Y ijk = U + T i + L j + B(L) jk

+ LT ij + e ijk ,

where Y ijk = unique individual observation, U = overall mean, T i = substance effect,

L

j = random site effect, B(L) jk

= random block within site effect, LT ij = random site by substance interaction effect, and e ijk = residual error.

For each compositional component, a range of observed values and a 99% tolerance interval were calculated. A tolerance interval is an interval that one can claim, with a specified degree of confidence, contains at least a specifi ed proportion, p, of an entire sampled population for the parameter measure d. The calculated tole rance intervals are expected to contain, with 95% confidence, 99% of the quantitie s expressed in the population of conventional canola. Each to lerance interval estim ate was based upon the average observation for each unique reference material. Because negative quantities are not possible, negative calculated lower tolerance bounds were set to zero.

SAS (Version 9) software was used to gene rate all summary statistics and perform all analyses.

Report tables present p-values from SAS as either <0.001 or the actual value truncated to three decimal places.Monsanto Company 11-CA-233U 260 of 500Table E-3. Statistical Summary of SiteMBNW Canola Seed Nutrient Contentfor MON 88302 vs. the Conventional ControlDifference (MON 88302 minus Control)Analytical Component (Units)1MON 883022Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%ConfidenceInterval3Significance(p-Value)CommercialTolerance Interval5(Range)Proximate (% dw)Ash3.98 (0.087)3.84 (0.10)0.14 (0.13)-0.28, 0.560.3673.32, 4.66(3.72 - 4.10)(3.66 - 4.03)(0.076 - 0.41)(2.98 - 4.52)Carbohydrates27.18 (0.29)26.02 (0.33)1.16 (0.40)-0.13, 2.450.06323.12, 30.77(26.75 - 28.02)(25.81 - 26.35)(1.08 - 1.67)(22.53 - 29.96)Moisture (% fw)5.26 (0.16)4.90 (0.18)0.36 (0.24)-0.39, 1.120.2254.33, 6.91(4.99 - 5.56)(4.69 - 5.13)(0.12 - 0.87)(4.09 - 8.48)Protein21.00 (0.62)20.78 (0.71)0.22 (0.94)-2.78, 3.220.83017.20, 30.08(19.68 - 22.64)(20.29 - 21.61)(-0.86 - 0.49)(18.68 - 28.32)Total Fat47.84 (0.47)49.35 (0.54)-1.51 (0.72)-3.79, 0.770.12539.65, 51.24(46.87 - 49.26)(48.89 - 49.93)(-2.28 - -0.61)(40.71 - 50.26)Fiber (% dw)Acid Detergent Fiber16.26 (0.63)14.93 (0.71)1.32 (0.81)-1.25, 3.900.1996.95, 23.92(15.05 - 17.66)(13.64 - 16.34)(0.019 - 3.09)(9.75 - 21.22)Monsanto Company 11-CA-233U 261 of 500TableE-3.Statistical Summary of Site MBNWCanola Seed Nutrient Content for MON 88302 vs. the Conventional Control(continued)Difference (MON 88302 minus Control)Analytical Component (Units)1MON 883022Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%ConfidenceInterval3Significance(p-Value)CommercialTolerance Interval5(Range)Fiber (% dw)Neutral Detergent Fiber19.08 (0.75)17.16 (0.87)1.92 (1.07)-1.50, 5.330.17110.07, 25.94(17.16 - 21.36)(16.68 - 17.45)(-0.29 - 3.97)(10.93 - 22.75)Total Dietary Fiber22.93 (1.65)19.30 (1.87)3.63 (2.10)-3.05, 10.320.18113.97, 24.85(19.17 - 27.81)(15.21 - 22.29)(-0.29 - 7.36)(12.64 - 26.47)Amino Acid (% dw)Alanine0.95 (0.026)0.95 (0.030)0.0018 (0.040)-0.13, 0.130.9670.77, 1.34(0.88 - 1.02)(0.93 - 0.98)(-0.047 - 0.011)(0.87 - 1.27)Arginine1.35 (0.042)1.37 (0.049)-0.019 (0.065)-0.23, 0.190.7841.10, 1.93(1.23 - 1.44)(1.36 - 1.38)(-0.12 - 0.025)(1.23 - 1.96)Aspartic Acid1.60 (0.057)1.58 (0.066)0.018 (0.087)-0.26, 0.300.8461.33, 2.12(1.44 - 1.72)(1.55 - 1.64)(-0.10 - 0.090)(1.42 - 2.23)Cystine0.52 (0.020)0.51 (0.023)0.0048 (0.031)-0.093, 0.100.8860.38, 0.83(0.48 - 0.59)(0.50 - 0.54)(-0.043 - 0.0090)(0.45 - 0.79)Monsanto Company 11-CA-233U 262 of 500Table E-3.StatisticalSummary of Site MBNW Canola Seed Nutrient Content for MON 88302 vs.the Conventional Control(continued)Difference (MON 88302 minus Control)Analytical Component (Units)1MON 883022Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%ConfidenceInterval3Significance(p-Value)CommercialTolerance Interval5(Range)Amino Acid (% dw)Glutamic Acid3.68 (0.12)3.71 (0.14)-0.030 (0.19)-0.64, 0.580.8862.73, 5.89(3.37 - 4.02)(3.64 - 3.84)(-0.27 - 0.043)(3.26 - 5.43)Glycine1.09 (0.028)1.09 (0.032)0.0020 (0.043)-0.13, 0.140.9650.96, 1.47(1.02 - 1.16)(1.06 - 1.12)(-0.046 - 0.014)(1.01 - 1.50)Histidine0.59 (0.017)0.58 (0.019)0.0052 (0.026)-0.076, 0.0870.8510.47, 0.86(0.55 - 0.64)(0.57 - 0.60)(-0.023 - 0.0092)(0.54 - 0.80)Isoleucine0.87 (0.028)0.86 (0.032)0.010 (0.042)-0.12, 0.140.8200.70, 1.22(0.81 - 0.94)(0.82 - 0.90)(-0.029 - 0.0069)(0.78 - 1.15)Leucine1.51 (0.044)1.51 (0.051)0.00056 (0.067)-0.21, 0.210.9931.21, 2.18(1.40 - 1.62)(1.48 - 1.56)(-0.082 - 0.026)(1.36 - 2.07)Lysine1.31 (0.034)1.28 (0.040)0.033 (0.052)-0.13, 0.200.5731.02, 1.90(1.22 - 1.41)(1.25 - 1.32)(-0.030 - 0.057)(1.20 - 1.68)Monsanto Company 11-CA-233U 263 of 500Table E-3.StatisticalSummary of Site MBNW Canola Seed Nutrient Content for MON 88302 vs.the ConventionalControl(continued)Difference (MON 88302 minus Control)Analytical Component (Units)1MON 88302Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%ConfidenceInterval3Significance(p-Value)CommercialTolerance Interval5(Range)Amino Acid (% dw)Methionine0.41 (0.012)0.42 (0.014)-0.0029 (0.019)-0.063, 0.0570.8870.30, 0.65(0.40 - 0.45)(0.40 - 0.44)(-0.038 - 0.0034)(0.36 - 0.57)Phenylalanine0.91 (0.025)0.91 (0.029)0.0043 (0.038)-0.12, 0.120.9160.77, 1.26(0.84 - 0.97)(0.90 - 0.93)(-0.053 - 0.019)(0.84 - 1.25)Proline1.27 (0.038)1.24 (0.044)0.029 (0.058)-0.16, 0.210.6590.90, 2.01(1.20 - 1.35)(1.20 - 1.29)(-0.030 - 0.036)(1.12 - 1.78)Serine0.96 (0.029)0.95 (0.033)0.0032 (0.044)-0.14, 0.140.9450.81, 1.32(0.87 - 1.03)(0.94 - 0.97)(-0.077 - 0.051)(0.88 - 1.30)Threonine0.94 (0.022)0.94 (0.025)0.0053 (0.034)-0.10, 0.110.8840.82, 1.20(0.88 - 0.98)(0.92 - 0.96)(-0.044 - 0.058)(0.84 - 1.22)Tryptophan0.21 (0.017)0.21 (0.020)-0.0040 (0.026)-0.088, 0.0800.8890.13, 0.35(0.17 - 0.26)(0.19 - 0.25)(-0.037 - -0.0025)(0.17 - 0.32)Monsanto Company 11-CA-233U 264 of 500Table E-3.StatisticalSummary of Site MBNW Canola Seed Nutrient Content for MON 88302 vs.the ConventionalControl(continued)Difference (MON 88302 minus Control)Analytical Component (Units)1MON 883022Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%ConfidenceInterval3Significance(p-Value)CommercialTolerance Interval5(Range)Amino Acid (% dw)Tyrosine0.64 (0.015)0.63 (0.017)0.0046 (0.022)-0.066, 0.0750.8500.57, 0.81(0.59 - 0.66)(0.63 - 0.64)(-0.037 - 0.016)(0.60 - 0.84)Valine1.12 (0.036)1.10 (0.041)0.021 (0.054)-0.15, 0.190.7190.92, 1.55(1.04 - 1.21)(1.05 - 1.15)(-0.032 - 0.014)(1.01 - 1.46)Fatty Acid (% Total FA)16:0 Palmitic4.10 (0.029)4.00 (0.033)0.10 (0.044)-0.039, 0.240.1052.84, 5.26(4.02 - 4.16)(3.97 - 4.06)(0.0047 - 0.18)(3.55 - 4.69)16:1 Palmitoleic0.21 (0.0031)0.23 (0.0036)-0.022 (0.0044)-0.036, -0.00810.0150.17, 0.30(0.20 - 0.21)(0.22 - 0.23)(-0.028 - -0.015)(0.19 - 0.27)18:0 Stearic1.73 (0.039)1.97 (0.045)-0.24 (0.060)-0.43, -0.0490.0280.90, 3.05(1.64 - 1.87)(1.93 - 2.01)(-0.35 - -0.059)(1.50 - 2.64)18:1 Oleic63.40 (0.19)65.71 (0.22)-2.30 (0.29)-3.24, -1.370.00456.13, 70.69(62.94 - 64.03)(65.55 - 65.93)(-3.00 - -1.52)(57.86 - 68.53)Monsanto Company 11-CA-233U 265 of 500Table E-3.StatisticalSummary of Site MBNW Canola Seed Nutrient Content for MON 88302 vs.the ConventionalControl(continued)Difference (MON 88302 minus Control)Analytical Component (Units)1MON 883022Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%ConfidenceInterval3Significance(p-Value)CommercialTolerance Interval5(Range)Fatty Acid (% Total FA)18:2 Linoleic19.27 (0.16)17.89 (0.19)1.38 (0.25)0.59, 2.170.01112.60, 24.49(18.82 - 19.66)(17.70 - 18.17)(0.65 - 1.96)(14.12 - 22.57)18:3 Linolenic9.19 (0.091)8.12 (0.10)1.08 (0.14)0.64, 1.520.0046.96, 11.73(8.88 - 9.42)(7.98 - 8.25)(0.76 - 1.43)(7.99 - 10.94)20:0 Arachidic0.52 (0.010)0.56 (0.012)-0.042 (0.015)-0.091, 0.00640.0690.45, 0.80(0.50 - 0.54)(0.54 - 0.58)(-0.081 - -0.0032)(0.53 - 0.71)20:1 Eicosenoic1.08 (0.021)1.03 (0.024)0.055 (0.032)-0.046, 0.160.1800.83, 1.68(1.06 - 1.15)(1.00 - 1.08)(-0.016 - 0.064)(1.04 - 1.56)22:0 Behenic0.25 (0.0058)0.26 (0.0067)-0.010 (0.0089)-0.038, 0.0180.3370.19, 0.43(0.24 - 0.26)(0.24 - 0.27)(-0.030 - 0.00017)(0.27 - 0.38)24:0 Lignoceric0.15 (0.0053)0.15 (0.0061)0.0020 (0.0081)-0.024, 0.0280.8240.033, 0.25(0.14 - 0.16)(0.15 - 0.15)(-0.0041 - 0.0063)(0.044 - 0.21)Monsanto Company 11-CA-233U 266 of 500Table E-3.StatisticalSummary of Site MBNW Canola Seed Nutrient Content for MON 88302 vs.the ConventionalControl(continued)Difference (MON 88302 minus Control)Analytical Component (Units)1MON 883022Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%ConfidenceInterval3Significance(p-Value)CommercialTolerance Interval5(Range)Fatty Acid (% Total FA)24:1 Nervonic0.096 (0.027)0.090 (0.031)0.0065 (0.041)-0.13, 0.140.8840.041, 0.18(0.046 - 0.12)(0.046 - 0.12)(-0.069 - 0.072)(0.044 - 0.20)MineralCalcium (g/100g dw)0.48 (0.010)0.44 (0.012)0.038 (0.016)-0.012, 0.0890.0950.16, 0.61(0.45 - 0.51)(0.43 - 0.46)(0.017 - 0.081)(0.25 - 0.53)Copper (mg/kg dw)3.72 (0.040)3.41 (0.046)0.32 (0.061)0.12, 0.510.0132.00, 4.43(3.61 - 3.83)(3.36 - 3.44)(0.22 - 0.40)(2.52 - 4.93)Iron (mg/kg dw)42.22 (1.46)46.51 (1.66)-4.30 (1.90)-10.33, 1.740.10823.39, 86.23(40.55 - 43.60)(41.65 - 51.30)(-8.99 - -1.10)(39.16 - 77.92)Magnesium (g/100g dw)0.34 (0.010)0.33 (0.012)0.0078 (0.016)-0.042, 0.0570.6510.32, 0.43(0.31 - 0.35)(0.31 - 0.35)(0.0036 - 0.039)(0.30 - 0.45)Manganese (mg/kg dw)39.62 (1.70)39.54 (1.96)0.078 (2.60)-8.18, 8.340.97714.85, 61.05(35.28 - 43.84)(37.35 - 41.11)(-3.95 - 6.49)(25.00 - 54.11)Monsanto Company 11-CA-233U 267 of 500Table E-3.StatisticalSummary of Site MBNW Canola Seed Nutrient Content for MON 88302 vs the Conventional Control(continued)Difference (MON 88302 minus Control)Analytical Component (Units)1MON 883022Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%ConfidenceInterval3Significance(p-Value)CommercialTolerance Interval5(Range)MineralPhosphorus (g/100g dw)0.72 (0.041)0.72 (0.047)0.0064 (0.062)-0.19, 0.200.9250.38, 1.06(0.60 - 0.78)(0.61 - 0.79)(-0.034 - 0.16)(0.44 - 0.87)Potassium (g/100g dw)0.56 (0.0096)0.56 (0.011)-0.0054 (0.015)-0.052, 0.0410.7340.39, 0.96(0.54 - 0.57)(0.54 - 0.58)(-0.0098 - 0.021)(0.50 - 0.92)Zinc (mg/kg dw)34.91 (1.09)30.24 (1.26)4.66 (1.66)-0.63, 9.950.06720.19, 48.23(32.40 - 37.15)(28.46 - 32.84)(-0.44 - 7.72)(22.18 - 47.61)Vitamin (mg/100g dw)Vitamin E (-tocopherol)13.06 (0.31)9.36 (0.36)3.70 (0.48)2.17, 5.230.0043.88, 17.28(12.22 - 13.47)(8.89 - 10.15)(3.07 - 4.46)(2.62 - 14.84)1dw = dry weight; fw = fresh weight; FA = fatty acid.2 MON 88302 treated with glyphosate.3Mean (S.E.) = least-square mean (standard error); CI = confidence interval.4Control refers to the geneticallysimilar, conventional control.5With 95% confidence, interval contains 99% of the values expressed in the populationof commercial conventional references. Negative limits were set tozero.Monsanto Company 11-CA-233U 268 of 500Table E-4. Statistical Summary of Site MBNW Canola Seed Anti-nutrient Content for MON 88302 vs. the ConventionalControlDifference (MON 88302 minus Control)Analytical Component (Units)1MON 883022Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%ConfidenceInterval3Significance(p-Value)CommercialTolerance Interval5(Range)Anti-nutrientAlkyl Glucosinolate (μmole/g dw)5.19 (0.28)5.43 (0.31)-0.23 (0.28)-1.13, 0.670.4720, 29.02(4.47 - 5.87)(4.85 - 6.16)(-0.38 - 0.091)(2.32 - 28.33)Indolyl Glucosinolate (μmole/g dw)4.23 (0.57)4.01 (0.65)0.22 (0.86)-2.53, 2.970.8171.37, 6.62(2.92 - 5.75)(2.92 - 5.31)(-1.32 - 2.83)(1.84 - 7.18)Phytic Acid (% dw)2.06 (0.17)2.27 (0.20)-0.21 (0.26)-1.05, 0.630.4890.70, 3.52(1.73 - 2.46)(1.77 - 2.56)(-0.67 - 0.68)(1.10 - 2.71)Sinapic Acid (% dw)1.02 (0.014)0.92 (0.014)0.095 (0.0093)0.066, 0.120.0010.57, 1.13(0.99 - 1.06)(0.92 - 0.94)(0.076 - 0.11)(0.48 - 0.99)Total Glucosinolate (μmole/g dw)9.60 (0.77)9.61 (0.88)-0.014 (1.05)-3.35, 3.320.9900, 32.20(7.60 - 11.42)(8.44 - 11.56)(-1.54 - 2.98)(5.52 - 31.98)1dw = dry weight.2 MON 88302 treated with glyphosate3Mean (S.E.) = least-square mean (standard error); CI = confidence interval.4Control refers to the geneticallysimilar, conventional control.5With 95% confidence, interval contains 99% of the values expressed in the populationof commercial conventional references. Negative limits were set tozero.Monsanto Company 11-CA-233U 269 of 500Table E-5. Statistical Summary of SiteMBPL Canola Seed Nutrient Contentfor MON 88302 vs. the Conventional ControlDifference (MON 88302 minus Control)Analytical Component (Units)1MON 883022Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%ConfidenceInterval3Significance(p-Value)CommercialTolerance Interval5(Range)Proximate (% dw)Ash4.15 (0.070)4.28 (0.080)-0.14 (0.089)-0.38, 0.110.2013.32, 4.66(3.99 - 4.38)(4.17 - 4.38)(-0.21 - 0.020)(2.98 - 4.52)Carbohydrates27.51 (0.48)28.11 (0.56)-0.59 (0.74)-2.64, 1.450.46623.12, 30.77(26.55 - 28.81)(26.87 - 28.73)(-2.18 - 1.94)(22.53 - 29.96)Moisture (% fw)5.68 (0.12)5.24 (0.14)0.44 (0.19)-0.078, 0.950.0774.33, 6.91(5.45 - 5.93)(4.93 - 5.47)(0.12 - 0.67)(4.09 - 8.48)Protein23.70 (0.26)23.46 (0.30)0.23 (0.40)-0.87, 1.330.59017.20, 30.08(23.17 - 24.33)(23.03 - 24.12)(-0.95 - 1.30)(18.68 - 28.32)Total Fat44.66 (0.36)44.20 (0.41)0.46 (0.49)-0.91, 1.830.40539.65, 51.24(43.96 - 45.72)(43.65 - 44.85)(-0.84 - 1.11)(40.71 - 50.26)Fiber (% dw)Acid Detergent Fiber16.75 (0.70)14.19 (0.73)2.55 (0.46)1.28, 3.830.0056.95, 23.92(15.17 - 18.19)(12.59 - 16.16)(2.03 - 3.57)(9.75 - 21.22)Monsanto Company 11-CA-233U 270 of 500Table E-5.StatisticalSummary of Site MBPL Canola Seed Nutrient Content for MON 88302vs. the ConventionalControl (continued)Difference (MON 88302 minus Control)Analytical Component (Units)1MON 883022Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%ConfidenceInterval3Significance(p-Value)CommercialTolerance Interval5(Range)Fiber (% dw)Neutral Detergent Fiber19.45 (0.50)16.87 (0.57)2.58 (0.66)0.74, 4.430.01710.07, 25.94(18.35 - 20.02)(15.44 - 18.06)(1.50 - 4.43)(10.93 - 22.75)Total Dietary Fiber22.61 (1.30)17.91 (1.50)4.70 (1.99)-0.82, 10.230.07713.97, 24.85(18.67 - 24.98)(14.58 - 20.42)(3.52 - 9.96)(12.64 - 26.47)Amino Acid (% dw)Alanine1.06 (0.013)1.04 (0.015)0.017 (0.020)-0.040, 0.0730.4600.77, 1.34(1.04 - 1.08)(1.02 - 1.09)(-0.049 - 0.059)(0.87 - 1.27)Arginine1.57 (0.033)1.54 (0.039)0.031 (0.051)-0.11, 0.170.5781.10, 1.93(1.51 - 1.64)(1.48 - 1.65)(-0.14 - 0.15)(1.23 - 1.96)Aspartic Acid1.84 (0.024)1.79 (0.027)0.047 (0.029)-0.034, 0.130.1791.33, 2.12(1.81 - 1.89)(1.73 - 1.85)(-0.023 - 0.086)(1.42 - 2.23)Cystine0.55 (0.016)0.57 (0.019)-0.013 (0.025)-0.082, 0.0560.6250.38, 0.83(0.50 - 0.59)(0.53 - 0.60)(-0.054 - 0.052)(0.45 - 0.79)Monsanto Company 11-CA-233U 271 of 500Table E-5. Statistical Summary of Site MBPLCanola Seed Nutrient Content for MON 88302vs. the ConventionalControl (continued)Difference (MON 88302 minus Control)Analytical Component (Units)1MON 883022Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%ConfidenceInterval3Significance(p-Value)CommercialTolerance Interval5(Range)Amino Acid (% dw)Glutamic Acid4.26 (0.084)4.24 (0.097)0.017 (0.13)-0.34, 0.370.9032.73, 5.89(4.15 - 4.41)(4.05 - 4.53)(-0.37 - 0.36)(3.26 - 5.43)Glycine1.23 (0.013)1.21 (0.015)0.013 (0.020)-0.043, 0.0700.5420.96, 1.47(1.21 - 1.24)(1.19 - 1.26)(-0.047 - 0.044)(1.01 - 1.50)Histidine0.65 (0.0098)0.65 (0.011)0.0039 (0.015)-0.037, 0.0450.8060.47, 0.86(0.63 - 0.67)(0.62 - 0.68)(-0.041 - 0.044)(0.54 - 0.80)Isoleucine0.99 (0.011)0.98 (0.012)0.0090 (0.016)-0.036, 0.0540.6090.70, 1.22(0.95 - 1.01)(0.96 - 1.00)(-0.050 - 0.034)(0.78 - 1.15)Leucine1.73 (0.021)1.71 (0.024)0.027 (0.032)-0.063, 0.120.4481.21, 2.18(1.70 - 1.76)(1.66 - 1.78)(-0.076 - 0.086)(1.36 - 2.07)Lysine1.41 (0.019)1.40 (0.022)0.0064 (0.029)-0.073, 0.0860.8331.02, 1.90(1.37 - 1.45)(1.36 - 1.45)(-0.067 - 0.086)(1.20 - 1.68)Monsanto Company 11-CA-233U 272 of 500Table E-5. Statistical Summary of Site MBPLCanola Seed Nutrient Content for MON 88302vs. the ConventionalControl(continued)Difference (MON 88302 minus Control)Analytical Component (Units)1MON 883022Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%ConfidenceInterval3Significance(p-Value)CommercialTolerance Interval5(Range)Amino Acid (% dw)Methionine0.46 (0.0083)0.47 (0.0095)-0.0073 (0.013)-0.042, 0.0280.5930.30, 0.65(0.43 - 0.48)(0.45 - 0.49)(-0.024 - 0.030)(0.36 - 0.57)Phenylalanine1.05 (0.011)1.03 (0.013)0.014 (0.017)-0.032, 0.0600.4430.77, 1.26(1.04 - 1.05)(1.01 - 1.07)(-0.032 - 0.039)(0.84 - 1.25)Proline1.39 (0.033)1.36 (0.038)0.021 (0.050)-0.12, 0.160.6960.90, 2.01(1.32 - 1.47)(1.30 - 1.45)(-0.12 - 0.17)(1.12 - 1.78)Serine1.08 (0.016)1.07 (0.018)0.012 (0.018)-0.039, 0.0630.5590.81, 1.32(1.05 - 1.09)(1.04 - 1.12)(-0.037 - 0.052)(0.88 - 1.30)Threonine1.06 (0.013)1.02 (0.015)0.031 (0.016)-0.013, 0.0750.1190.82, 1.20(1.04 - 1.06)(0.99 - 1.07)(-0.0054 - 0.065)(0.84 - 1.22)Tryptophan0.25 (0.0075)0.26 (0.0087)-0.011 (0.011)-0.043, 0.0210.3880.13, 0.35(0.24 - 0.25)(0.25 - 0.27)(-0.014 - -0.0041)(0.17 - 0.32)Monsanto Company 11-CA-233U 273 of 500Table E-5. Statistical Summary of Site MBPLCanola Seed Nutrient Content for MON 88302vs. the ConventionalControl(continued)Difference (MON 88302 minus Control)Analytical Component (Units)1MON 883022Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%ConfidenceInterval3Significance(p-Value)CommercialTolerance Interval5(Range)Amino Acid (% dw)Tyrosine0.72 (0.0065)0.71 (0.0069)0.017 (0.0052)0.0030, 0.0320.0280.57, 0.81(0.72 - 0.73)(0.69 - 0.73)(0.0078 - 0.028)(0.60 - 0.84)Valine1.26 (0.014)1.25 (0.016)0.016 (0.021)-0.041, 0.0740.4740.92, 1.55(1.21 - 1.29)(1.23 - 1.27)(-0.058 - 0.054)(1.01 - 1.46)Fatty Acid (% Total FA)16:0 Palmitic4.25 (0.031)4.34 (0.035)-0.090 (0.047)-0.22, 0.0400.1262.84, 5.26(4.20 - 4.29)(4.23 - 4.41)(-0.22 - 0.030)(3.55 - 4.69)16:1 Palmitoleic0.23 (0.0035)0.25 (0.0040)-0.026 (0.0053)-0.040, -0.0110.0080.17, 0.30(0.22 - 0.23)(0.24 - 0.26)(-0.031 - -0.015)(0.19 - 0.27)18:0 Stearic1.58 (0.023)1.87 (0.026)-0.29 (0.028)-0.37, -0.22<0.0010.90, 3.05(1.55 - 1.59)(1.79 - 1.93)(-0.34 - -0.22)(1.50 - 2.64)18:1 Oleic62.06 (0.11)64.30 (0.13)-2.24 (0.13)-2.59, -1.88<0.00156.13, 70.69(61.82 - 62.35)(64.19 - 64.56)(-2.40 - -1.84)(57.86 - 68.53)Monsanto Company 11-CA-233U 274 of 500Table E-5. Statistical Summary of Site MBPL Canola SeedNutrient Content for MON 88302 vs. the Conventional Control(continued)Difference (MON 88302 minus Control)Analytical Component (Units)1MON 883022Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%ConfidenceInterval3Significance(p-Value)CommercialTolerance Interval5(Range)Fatty Acid (% Total FA)18:2 Linoleic20.43 (0.088)19.18 (0.099)1.25 (0.11)0.95, 1.55<0.00112.60, 24.49(20.13 - 20.66)(19.01 - 19.26)(0.92 - 1.41)(14.12 - 22.57)18:3 Linolenic9.28 (0.085)7.74 (0.091)1.54 (0.073)1.34, 1.74<0.0016.96, 11.73(9.12 - 9.43)(7.52 - 7.92)(1.35 - 1.67)(7.99 - 10.94)20:0 Arachidic0.53 (0.0046)0.60 (0.0049)-0.070 (0.0042)-0.082, -0.059<0.0010.45, 0.80(0.52 - 0.54)(0.59 - 0.61)(-0.079 - -0.063)(0.53 - 0.71)20:1 Eicosenoic1.09 (0.0059)1.08 (0.0068)0.011 (0.0089)-0.014, 0.0350.2980.83, 1.68(1.08 - 1.10)(1.06 - 1.09)(-0.013 - 0.042)(1.04 - 1.56)22:0 Behenic0.27 (0.0027)0.30 (0.0032)-0.040 (0.0039)-0.050, -0.029<0.0010.19, 0.43(0.26 - 0.27)(0.29 - 0.31)(-0.047 - -0.030)(0.27 - 0.38)24:0 Lignoceric0.16 (0.0046)0.19 (0.0054)-0.023 (0.0068)-0.042, -0.00380.0290.033, 0.25(0.16 - 0.17)(0.18 - 0.19)(-0.024 - -0.022)(0.044 - 0.21)Monsanto Company 11-CA-233U 275 of 500Table E-5. Statistical Summary of Site MBPL Canola SeedNutrient content for MON 88302 vs. the Conventional Control(continued)Difference (MON 88302 minus Control)Analytical Component (Units)1MON 883022Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%ConfidenceInterval3Significance(p-Value)CommercialTolerance Interval5(Range)Fatty Acid (% Total FA)24:1 Nervonic0.13 (0.0066)0.16 (0.0076)-0.033 (0.010)-0.061, -0.00470.0310.041, 0.18(0.12 - 0.13)(0.15 - 0.17)(-0.052 - -0.014)(0.044 - 0.20)MineralCalcium (g/100g dw)0.36 (0.0051)0.34 (0.0059)0.021 (0.0078)-0.00057, 0.0430.0530.16, 0.61(0.35 - 0.37)(0.32 - 0.34)(0.0015 - 0.035)(0.25 - 0.53)Copper (mg/kg dw)3.47 (0.082)3.97 (0.094)-0.50 (0.12)-0.84, -0.150.0162.00, 4.43(3.35 - 3.56)(3.68 - 4.18)(-0.83 - -0.23)(2.52 - 4.93)Iron (mg/kg dw)44.13 (0.64)51.01 (0.73)-6.87 (0.90)-9.38, -4.370.00123.39, 86.23(42.80 - 45.09)(49.75 - 52.89)(-9.20 - -4.80)(39.16 - 77.92)Magnesium (g/100g dw)0.41 (0.0070)0.41 (0.0081)-0.0029 (0.011)-0.033, 0.0270.7970.32, 0.43(0.39 - 0.42)(0.40 - 0.42)(-0.022 - 0.021)(0.30 - 0.45)Manganese (mg/kg dw)39.33 (0.90)37.78 (1.03)1.55 (1.37)-2.25, 5.340.32114.85, 61.05(37.24 - 41.46)(36.29 - 39.99)(-1.82 - 4.38)(25.00 - 54.11)Monsanto Company 11-CA-233U 276 of 500Table E-5. Statistical Summary of Site MBPL Canola SeedNutrient Content for MON 88302 vs. the Conventional Control(continued)Difference (MON 88302 minus Control)Analytical Component (Units)1MON 883022Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%ConfidenceInterval3Significance(p-Value)CommercialTolerance Interval5(Range)MineralPhosphorus (g/100g dw)0.78 (0.0092)0.81 (0.011)-0.031 (0.014)-0.069, 0.00770.0900.38, 1.06(0.75 - 0.80)(0.80 - 0.82)(-0.033 - -0.0099)(0.44 - 0.87)Potassium (g/100g dw)0.70 (0.025)0.77 (0.027)-0.068 (0.019)-0.12, -0.0150.0230.39, 0.96(0.63 - 0.76)(0.77 - 0.81)(-0.097 - -0.017)(0.50 - 0.92)Zinc (mg/kg dw)31.25 (0.49)33.88 (0.56)-2.63 (0.75)-4.70, -0.560.02420.19, 48.23(30.45 - 32.05)(32.82 - 35.76)(-4.50 - -1.02)(22.18 - 47.61)Vitamin (mg/100g dw)Vitamin E (-tocopherol)11.50 (0.24)7.63 (0.27)3.88 (0.36)2.87, 4.89<0.0013.88, 17.28(10.70 - 12.20)(7.50 - 7.72)(3.20 - 4.23)(2.62 - 14.84)1dw = dry weight; fw = fresh weight; FA = fatty acid.2 MON 88302 treated with glyphosate.3Mean (S.E.) = least-square mean (standard error); CI = confidence interval.4Control refers to the geneticallysimilar, conventional control.5With 95% confidence, interval contains 99% of the values expressed in the populationof commercial conventional references. Negative limits were set to zero.Monsanto Company 11-CA-233U 277 of 500Table E-6. Statistical Summary of SiteMBPL Canola Seed Anti-nutrient Content for MON 88302 vs. the ConventionalControlDifference (MON 88302 minus Control)Analytical Component (Units)1MON 883022Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%ConfidenceInterval3Significance(p-Value)CommercialTolerance Interval5(Range)Anti-nutrientAlkyl Glucosinolate (μmole/g dw)2.98 (0.64)5.03 (0.74)-2.05 (0.98)-4.76, 0.660.1030, 29.02(1.91 - 4.03)(3.06 - 6.50)(-2.68 - -1.15)(2.32 - 28.33)Indolyl Glucosinolate (μmole/g dw)3.90 (0.69)4.29 (0.79)-0.39 (0.93)-2.98, 2.200.6971.37, 6.62(1.67 - 5.76)(3.26 - 5.89)(-1.59 - 0.96)(1.84 - 7.18)Phytic Acid (% dw)2.36 (0.066)2.39 (0.076)-0.027 (0.10)-0.31, 0.250.8030.70, 3.52(2.19 - 2.58)(2.35 - 2.41)(-0.15 - 0.17)(1.10 - 2.71)Sinapic Acid (% dw)0.97 (0.0080)0.86 (0.0090)0.10 (0.010)0.076, 0.13<0.0010.57, 1.13(0.95 - 0.99)(0.86 - 0.86)(0.085 - 0.12)(0.48 - 0.99)Total Glucosinolate (μmole/g dw)7.01 (1.30)9.40 (1.49)-2.39 (1.83)-7.45, 2.680.2610, 32.20(3.66 - 9.77)(6.42 - 12.59)(-2.82 - -0.44)(5.52 - 31.98)1dw = dry weight; fw = fresh weight; FA = fatty acid.2 MON 88302 treated with glyphosate.3Mean (S.E.) = least-square mean (standard error); CI = confidence interval.4Control refers to the geneticallysimilar, conventional control.5With 95% confidence, interval contains 99% of the values expressed in the populationof commercial conventional references. Negative limits were set to zeroMonsanto Company 11-CA-233U 278 of 500Table E-7. Statistical Summary of SiteMNCA Canola Seed Nutrient Content for MON 88302 vs. the Conventional ControlDifference (MON 88302 minus Control)Analytical Component (Units)1MON 883022Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%ConfidenceInterval3Significance(p-Value)CommercialTolerance Interval5(Range)Proximate (% dw)Ash4.35 (0.23)4.18 (0.20)0.17 (0.30)-0.60, 0.940.5913.32, 4.66(4.20 - 4.45)(3.76 - 5.10)(0.33 - 0.64)(2.98 - 4.52)Carbohydrates27.31 (0.35)25.99 (0.30)1.32 (0.46)0.13, 2.510.03523.12, 30.77(26.27 - 27.90)(25.57 - 26.55)(-0.29 - 1.92)(22.53 - 29.96)Moisture (% fw)5.52 (0.13)6.69 (0.11)-1.17 (0.17)-1.60, -0.74<0.0014.33, 6.91(5.37 - 5.61)(6.33 - 6.98)(-1.53 - -0.72)(4.09 - 8.48)Protein22.00 (0.70)23.23 (0.61)-1.23 (0.81)-3.30, 0.850.18917.20, 30.08(21.51 - 22.03)(21.50 - 24.27)(-2.29 - 0.53)(18.68 - 28.32)Total Fat46.04 (0.72)46.59 (0.69)-0.55 (0.46)-1.72, 0.620.28039.65, 51.24(45.76 - 47.55)(45.26 - 48.05)(-0.78 - -0.21)(40.71 - 50.26)Fiber (% dw)Acid Detergent Fiber17.89 (0.87)17.66 (0.75)0.23 (1.15)-2.73, 3.200.8476.95, 23.92(15.99 - 20.24)(16.11 - 18.71)(-2.71 - 2.09)(9.75 - 21.22)Monsanto Company 11-CA-233U 279 of 500Table E-7. Statistical Summary of Site MNCA Canola SeedNutrient Content for MON 88302 vs. the Conventional Control(continued)Difference (MON 88302 minus Control)Analytical Component (Units)1MON 883022Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%ConfidenceInterval3Significance(p-Value)CommercialTolerance Interval5(Range)Fiber (% dw)Neutral Detergent Fiber19.55 (0.82)18.78 (0.71)0.76 (1.08)-2.01, 3.540.51110.07, 25.94(17.90 - 21.19)(17.83 - 19.58)(-1.56 - 3.36)(10.93 - 22.75)Total Dietary Fiber20.18 (1.55)19.75 (1.35)0.44 (2.06)-4.85, 5.720.83913.97, 24.85(16.91 - 22.24)(17.40 - 23.00)(-0.49 - 3.79)(12.64 - 26.47)Amino Acid (% dw)Alanine0.98 (0.027)1.05 (0.023)-0.070 (0.032)-0.15, 0.0140.0840.77, 1.34(0.97 - 0.98)(0.98 - 1.10)(-0.12 - -0.014)(0.87 - 1.27)Arginine1.40 (0.054)1.53 (0.048)-0.13 (0.063)-0.29, 0.0290.0891.10, 1.93(1.38 - 1.39)(1.40 - 1.65)(-0.27 - -0.010)(1.23 - 1.96)Aspartic Acid1.59 (0.067)1.79 (0.058)-0.20 (0.082)-0.41, 0.00980.0571.33, 2.12(1.57 - 1.60)(1.61 - 1.97)(-0.37 - -0.045)(1.42 - 2.23)Cystine0.57 (0.025)0.55 (0.022)0.015 (0.031)-0.064, 0.0940.6500.38, 0.83(0.53 - 0.58)(0.52 - 0.61)(0.012 - 0.035)(0.45 - 0.79)Monsanto Company 11-CA-233U 280 of 500Table E-7. Statistical Summary of Site MNCA Canola SeedNutrient Content for MON 88302 vs. the Conventional Control(continued)Difference (MON 88302 minus Control)Analytical Component (Units)1MON 883022Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%ConfidenceInterval3Significance(p-Value)CommercialTolerance Interval5(Range)Amino Acid (% dw)Glutamic Acid3.89 (0.15)4.25 (0.13)-0.36 (0.17)-0.80, 0.0830.0912.73, 5.89(3.84 - 3.87)(3.98 - 4.45)(-0.59 - -0.11)(3.26 - 5.43)Glycine1.12 (0.034)1.22 (0.030)-0.099 (0.044)-0.21, 0.0140.0730.96, 1.47(1.11 - 1.12)(1.13 - 1.30)(-0.18 - -0.0061)(1.01 - 1.50)Histidine0.62 (0.018)0.65 (0.016)-0.031 (0.022)-0.087, 0.0250.2140.47, 0.86(0.61 - 0.62)(0.61 - 0.67)(-0.058 - -0.00063)(0.54 - 0.80)Isoleucine0.90 (0.027)0.97 (0.024)-0.076 (0.031)-0.16, 0.00440.0590.70, 1.22(0.88 - 0.90)(0.89 - 1.03)(-0.13 - -0.0068)(0.78 - 1.15)Leucine1.56 (0.052)1.70 (0.045)-0.15 (0.064)-0.31, 0.0150.0661.21, 2.18(1.55 - 1.55)(1.58 - 1.80)(-0.25 - -0.034)(1.36 - 2.07)Lysine1.37 (0.032)1.40 (0.029)-0.033 (0.036)-0.13, 0.0590.3971.02, 1.90(1.35 - 1.36)(1.35 - 1.44)(-0.055 - -0.0095)(1.20 - 1.68)Monsanto Company 11-CA-233U 281 of 500Table E-7. Statistical Summary of Site MNCA Canola SeedNutrient Content for MON 88302 vs. the Conventional Control(continued)Difference (MON 88302 minus Control)Analytical Component (Units)1MON 883022Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%ConfidenceInterval3Significance(p-Value)CommercialTolerance Interval5(Range)Amino Acid (% dw)Methionine0.44 (0.020)0.44 (0.017)0.0058 (0.024)-0.056, 0.0670.8190.30, 0.65(0.43 - 0.45)(0.42 - 0.48)(0.011 - 0.019)(0.36 - 0.57)Phenylalanine0.93 (0.030)1.02 (0.026)-0.089 (0.036)-0.18, 0.00430.0570.77, 1.26(0.92 - 0.93)(0.94 - 1.08)(-0.17 - -0.011)(0.84 - 1.25)Proline1.36 (0.045)1.40 (0.040)-0.040 (0.053)-0.18, 0.0960.4790.90, 2.01(1.29 - 1.39)(1.30 - 1.46)(-0.16 - 0.076)(1.12 - 1.78)Serine0.98 (0.038)1.08 (0.033)-0.095 (0.050)-0.22, 0.0330.1140.81, 1.32(0.98 - 0.99)(1.02 - 1.16)(-0.17 - -0.037)(0.88 - 1.30)Threonine0.95 (0.027)1.02 (0.024)-0.073 (0.031)-0.15, 0.00740.0670.82, 1.20(0.94 - 0.94)(0.97 - 1.06)(-0.12 - -0.027)(0.84 - 1.22)Tryptophan0.22 (0.020)0.24 (0.017)-0.022 (0.026)-0.089, 0.0440.4260.13, 0.35(0.18 - 0.24)(0.22 - 0.27)(-0.063 - 0.024)(0.17 - 0.32)Monsanto Company 11-CA-233U 282 of 500Table E-7. statistical Summary of Site MNCA Canola SeedNutrient Content for MON 88302 vs. the Conventional Control(continued)Difference (MON 88302 minus Control)Analytical Component (Units)1MON 883022Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%ConfidenceInterval3Significance(p-Value)CommercialTolerance Interval5(Range)Amino Acid (% dw)Tyrosine0.65 (0.019)0.71 (0.017)-0.061 (0.024)-0.12, 0.00170.0540.57, 0.81(0.64 - 0.65)(0.66 - 0.75)(-0.11 - -0.0055)(0.60 - 0.84)Valine1.15 (0.031)1.24 (0.027)-0.091 (0.035)-0.18, -0.000820.0480.92, 1.55(1.13 - 1.15)(1.15 - 1.31)(-0.16 - -0.017)(1.01 - 1.46)Fatty Acid (% Total FA)16:0 Palmitic4.27 (0.051)4.14 (0.045)0.13 (0.061)-0.029, 0.280.0902.84, 5.26(4.27 - 4.28)(4.07 - 4.19)(0.087 - 0.21)(3.55 - 4.69)16:1 Palmitoleic0.21 (0.0030)0.24 (0.0026)-0.026 (0.0039)-0.036, -0.0160.0010.17, 0.30(0.21 - 0.21)(0.23 - 0.25)(-0.039 - -0.020)(0.19 - 0.27)18:0 Stearic1.67 (0.044)1.86 (0.038)-0.19 (0.057)-0.33, -0.0390.0220.90, 3.05(1.65 - 1.71)(1.78 - 1.92)(-0.26 - -0.074)(1.50 - 2.64)18:1 Oleic61.67 (0.59)64.86 (0.52)-3.19 (0.69)-4.98, -1.410.00556.13, 70.69(61.70 - 61.87)(63.72 - 65.52)(-3.81 - -3.11)(57.86 - 68.53)Monsanto Company 11-CA-233U 283 of 500Table E-7. Statistical Summary of Site MNCA Canola SeedNutrient Content for MON 88302 vs. the Conventional Control(continued)Difference (MON 88302 minus Control)Analytical Component (Units)1MON 883022Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%ConfidenceInterval3Significance(p-Value)CommercialTolerance Interval5(Range)Fatty Acid (% Total FA)18:2 Linoleic20.20 (0.24)18.35 (0.21)1.85 (0.30)1.07, 2.630.00112.60, 24.49(20.00 - 20.32)(17.90 - 19.29)(1.86 - 2.42)(14.12 - 22.57)18:3 Linolenic9.79 (0.58)8.40 (0.50)1.39 (0.75)-0.54, 3.320.1226.96, 11.73(9.76 - 9.79)(8.16 - 8.64)(1.31 - 1.60)(7.99 - 10.94)20:0 Arachidic0.53 (0.013)0.57 (0.012)-0.042 (0.018)-0.088, 0.00270.0600.45, 0.80(0.52 - 0.54)(0.55 - 0.60)(-0.081 - -0.019)(0.53 - 0.71)20:1 Eicosenoic1.08 (0.012)1.07 (0.011)0.016 (0.016)-0.025, 0.0580.3540.83, 1.68(1.06 - 1.09)(1.05 - 1.09)(-0.024 - 0.041)(1.04 - 1.56)22:0 Behenic0.27 (0.0056)0.27 (0.0049)0.0022 (0.0074)-0.017, 0.0210.7760.19, 0.43(0.27 - 0.28)(0.26 - 0.29)(-0.021 - 0.016)(0.27 - 0.38)24:0 Lignoceric0.11 (0.033)0.14 (0.029)-0.032 (0.044)-0.15, 0.0810.4990.033, 0.25(0.049 - 0.16)(0.049 - 0.19)(-0.14 - 0.069)(0.044 - 0.21)Monsanto Company 11-CA-233U 284 of 500Table E-7. Statistical Summary of Site MNCA Canola SeedNutrient Content for MON 88302 vs. the Conventional Control(continued)Difference (MON 88302 minus Control)Analytical Component (Units)1MON 883022Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%ConfidenceInterval3Significance(p-Value)CommercialTolerance Interval5(Range)Fatty Acid (% Total FA)24:1 Nervonic0.10 (0.025)0.099 (0.021)0.0042 (0.032)-0.079, 0.0880.9020.041, 0.18(0.049 - 0.15)(0.049 - 0.12)(-0.072 - 0.062)(0.044 - 0.20)MineralCalcium (g/100g dw)0.47 (0.016)0.45 (0.014)0.018 (0.022)-0.038, 0.0740.4380.16, 0.61(0.46 - 0.47)(0.42 - 0.49)(-0.0037 - 0.053)(0.25 - 0.53)Copper (mg/kg dw)4.40 (0.074)4.11 (0.064)0.28 (0.093)0.046, 0.520.0272.00, 4.43(4.16 - 4.57)(4.06 - 4.18)(0.056 - 0.39)(2.52 - 4.93)Iron (mg/kg dw)42.57 (1.72)50.64 (1.53)-8.07 (1.87)-12.89, -3.250.00723.39, 86.23(40.56 - 44.18)(46.23 - 54.03)(-12.92 - -4.82)(39.16 - 77.92)Magnesium (g/100g dw)0.38 (0.0074)0.37 (0.0067)0.014 (0.0076)-0.0049, 0.0340.1130.32, 0.43(0.36 - 0.40)(0.36 - 0.38)(0.0074 - 0.018)(0.30 - 0.45)Manganese (mg/kg dw)38.70 (2.68)40.94 (2.32)-2.24 (3.54)-11.34, 6.860.55414.85, 61.05(37.83 - 39.93)(33.70 - 46.19)(-8.36 - -2.11)(25.00 - 54.11)Monsanto Company 11-CA-233U 285 of 500Table E-7. Statistical Summary of Site MNCA Canola SeedNutrient Content for MON 88302 vs. the Conventional Control(continued)Difference (MON 88302 minus Control)Analytical Component (Units)1MON 883022Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%ConfidenceInterval3Significance(p-Value)CommercialTolerance Interval5(Range)MineralPhosphorus (g/100g dw)0.81 (0.039)0.79 (0.034)0.022 (0.044)-0.090, 0.130.6330.38, 1.06(0.74 - 0.87)(0.72 - 0.93)(-0.011 - 0.098)(0.44 - 0.87)Potassium (g/100g dw)0.65 (0.027)0.64 (0.023)0.012 (0.035)-0.079, 0.100.7460.39, 0.96(0.58 - 0.70)(0.60 - 0.72)(-0.020 - 0.098)(0.50 - 0.92)Zinc (mg/kg dw)39.18 (2.73)35.29 (2.41)3.90 (3.15)-4.21, 12.000.27120.19, 48.23(35.19 - 45.56)(32.63 - 36.66)(-1.44 - 10.33)(22.18 - 47.61)Vitamin (mg/100g dw)Vitamin E (-tocopherol)13.39 (0.48)10.82 (0.42)2.57 (0.57)1.11, 4.030.0063.88, 17.28(12.58 - 14.62)(10.15 - 11.77)(2.18 - 3.99)(2.62 - 14.84)1dw = dry weight; fw = fresh weight; FA = fatty acid.2 MON 88302 treated with glyphosate.3Mean (S.E.) = least-square mean (standard error); CI = confidence interval.4Control refers to the geneticallysimilar, conventional control.5With 95% confidence, interval contains 99% of the values expressed in the populationof commercial conventional references. Negative limits set to zero.Monsanto Company 11-CA-233U 286 of 500Table E-8. Statistical Summary of Site MNCA Canola Seed Anti-nutrient Content for MON 88302 vs. the ConventionalControlDifference (MON 88302 minus Control)Analytical Component (Units)1MON 883022Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%ConfidenceInterval3Significance(p-Value)CommercialTolerance Interval5(Range)Anti-nutrientAlkyl Glucosinolate (μmole/g dw)4.64 (0.62)4.88 (0.54)-0.24 (0.79)-2.28, 1.800.7750, 29.02(4.35 - 4.88)(2.92 - 6.16)(-1.48 - 1.43)(2.32 - 28.33)Indolyl Glucosinolate (μmole/g dw)4.17 (0.52)4.35 (0.45)-0.19 (0.69)-1.96, 1.590.7981.37, 6.62(3.79 - 4.47)(3.28 - 5.66)(-1.42 - 0.13)(1.84 - 7.18)Phytic Acid (% dw)2.28 (0.12)2.36 (0.10)-0.078 (0.15)-0.47, 0.310.6300.70, 3.52(2.14 - 2.47)(2.15 - 2.77)(-0.011 - 0.12)(1.10 - 2.71)Sinapic Acid (% dw)1.06 (0.015)0.96 (0.014)0.10 (0.015)0.063, 0.140.0010.57, 1.13(1.02 - 1.08)(0.94 - 0.97)(0.082 - 0.12)(0.48 - 0.99)Total Glucosinolate (μmole/g dw)9.08 (0.69)9.42 (0.61)-0.33 (0.81)-2.41, 1.740.6960, 32.20(8.35 - 9.36)(7.15 - 10.65)(-1.29 - 1.19)(5.52 - 31.98)1dw = dry weight.2 MON 88302 treated with glyphosate.3Mean (S.E.) = least-square mean (standard error); CI = confidence interval.4Control refers to the geneticallysimilar, conventional control.5With 95% confidence, interval contains 99% of the values expressed in the populationof commercial conventional references. Negative limits set to zero.Monsanto Company 11-CA-233U 287 of 500Table E-9. Statistical Summary of SiteNDVA Canola Seed Nutrient Content for MON 88302 vs. the Conventional ControlDifference (MON 88302 minus Control)Analytical Component (Units)1MON 883022Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%ConfidenceInterval3Significance(p-Value)CommercialTolerance Interval5(Range)Proximate (% dw)Ash3.31 (0.31)3.25 (0.26)0.063 (0.40)-1.22, 1.350.8863.32, 4.66(3.31 - 3.31)(3.20 - 3.35)(0.11 - 0.11)(2.98 - 4.52)Carbohydrates24.01 (0.95)25.49 (0.78)-1.48 (1.22)-5.37, 2.420.31423.12, 30.77(21.83 - 26.20)(24.69 - 26.01)(-4.18 - 0.45)(22.53 - 29.96)Moisture (% fw)5.72 (0.23)5.61 (0.20)0.12 (0.22)-0.58, 0.820.6334.33, 6.91(5.72 - 6.08)(5.24 - 6.18)(-0.10 - 0.090)(4.09 - 8.48)Protein24.66 (0.74)26.12 (0.61)-1.46 (0.96)-4.52, 1.600.22617.20, 30.08(23.33 - 25.98)(25.33 - 27.02)(-1.99 - -0.028)(18.68 - 28.32)Total Fat48.04 (0.44)45.17 (0.36)2.87 (0.57)1.07, 4.670.01439.65, 51.24(47.20 - 48.87)(44.77 - 45.78)(1.42 - 4.10)(40.71 - 50.26)Fiber (% dw)Acid Detergent Fiber15.20 (0.37)15.22 (0.31)-0.016 (0.39)-1.26, 1.230.9696.95, 23.92(14.53 - 15.86)(14.88 - 15.35)(-0.62 - 0.52)(9.75 - 21.22)Monsanto Company 11-CA-233U 288 of 500Table E-9. Statistical Summary of Site NDVA Canola SeedNutrient Content for MON 88302 vs. the Conventional Control(continued)Difference (MON 88302 minus Control)Analytical Component (Units)1MON 883022Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%ConfidenceInterval3Significance(p-Value)CommercialTolerance Interval5(Range)Fiber (% dw)Neutral Detergent Fiber17.32 (0.81)17.74 (0.66)-0.42 (1.05)-3.76, 2.930.71810.07, 25.94(15.91 - 18.74)(17.16 - 18.65)(-2.74 - 1.58)(10.93 - 22.75)Total Dietary Fiber18.71 (1.39)17.17 (1.15)1.54 (1.65)-3.70, 6.790.41813.97, 24.85(17.08 - 21.08)(14.88 - 19.61)(-0.30 - 1.47)(12.64 - 26.47)Amino Acid (% dw)Alanine1.08 (0.042)1.14 (0.034)-0.061 (0.054)-0.23, 0.110.3440.77, 1.34(1.01 - 1.15)(1.11 - 1.19)(-0.10 - 0.031)(0.87 - 1.27)Arginine1.60 (0.076)1.72 (0.062)-0.12 (0.098)-0.43, 0.190.3161.10, 1.93(1.47 - 1.72)(1.68 - 1.77)(-0.21 - 0.030)(1.23 - 1.96)Aspartic Acid1.79 (0.067)1.83 (0.054)-0.040 (0.086)-0.31, 0.230.6751.33, 2.12(1.65 - 1.93)(1.79 - 1.89)(-0.14 - 0.12)(1.42 - 2.23)Cystine0.70 (0.040)0.71 (0.033)-0.018 (0.046)-0.16, 0.130.7150.38, 0.83(0.64 - 0.73)(0.66 - 0.79)(-0.028 - 0.037)(0.45 - 0.79)Monsanto Company 11-CA-233U 289 of 500Table E-9. Statistical Summary of Site NDVA Canola SeedNutrient Content for MON 88302 vs. the Conventional Control(continued)Difference (MON 88302 minus Control)Analytical Component (Units)1MON 883022Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%ConfidenceInterval3Significance(p-Value)CommercialTolerance Interval5(Range)Amino Acid (% dw)Glutamic Acid4.66 (0.25)5.04 (0.20)-0.38 (0.32)-1.41, 0.640.3192.73, 5.89(4.25 - 5.06)(4.92 - 5.26)(-0.68 - 0.13)(3.26 - 5.43)Glycine1.24 (0.048)1.33 (0.039)-0.094 (0.062)-0.29, 0.100.2290.96, 1.47(1.16 - 1.32)(1.30 - 1.38)(-0.15 - 0.0093)(1.01 - 1.50)Histidine0.72 (0.034)0.75 (0.027)-0.032 (0.043)-0.17, 0.110.5090.47, 0.86(0.67 - 0.77)(0.73 - 0.78)(-0.065 - 0.032)(0.54 - 0.80)Isoleucine1.01 (0.041)1.07 (0.034)-0.067 (0.053)-0.24, 0.100.2990.70, 1.22(0.94 - 1.08)(1.04 - 1.12)(-0.099 - 0.0095)(0.78 - 1.15)Leucine1.77 (0.073)1.88 (0.059)-0.11 (0.094)-0.40, 0.190.3361.21, 2.18(1.64 - 1.90)(1.83 - 1.95)(-0.19 - 0.051)(1.36 - 2.07)Lysine1.55 (0.066)1.60 (0.054)-0.054 (0.085)-0.32, 0.220.5661.02, 1.90(1.46 - 1.63)(1.58 - 1.65)(-0.12 - 0.052)(1.20 - 1.68)Monsanto Company 11-CA-233U 290 of 500Table E-9. Statistical Summary of Site NDVA Canola SeedNutrient Content for MON 88302 vs. the Conventional Control(continued)Difference (MON 88302 minus Control)Analytical Component (Units)1MON 883022Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%ConfidenceInterval3Significance(p-Value)CommercialTolerance Interval5(Range)Amino Acid (% dw)Methionine0.52 (0.019)0.53 (0.016)-0.0059 (0.020)-0.068, 0.0560.7820.30, 0.65(0.49 - 0.54)(0.50 - 0.56)(-0.013 - 0.0079)(0.36 - 0.57)Phenylalanine1.04 (0.038)1.11 (0.031)-0.066 (0.049)-0.22, 0.0910.2720.77, 1.26(0.97 - 1.11)(1.08 - 1.15)(-0.12 - 0.031)(0.84 - 1.25)Proline1.59 (0.065)1.68 (0.053)-0.086 (0.083)-0.35, 0.180.3770.90, 2.01(1.46 - 1.71)(1.62 - 1.73)(-0.16 - 0.041)(1.12 - 1.78)Serine1.09 (0.036)1.16 (0.029)-0.064 (0.046)-0.21, 0.0830.2580.81, 1.32(1.05 - 1.14)(1.13 - 1.18)(-0.12 - 0.0094)(0.88 - 1.30)Threonine1.05 (0.035)1.09 (0.029)-0.037 (0.045)-0.18, 0.110.4700.82, 1.20(0.99 - 1.11)(1.06 - 1.12)(-0.065 - 0.020)(0.84 - 1.22)Tryptophan0.26 (0.023)0.26 (0.019)0.0021 (0.025)-0.079, 0.0830.9380.13, 0.35(0.25 - 0.26)(0.21 - 0.31)(0.0061 - 0.036)(0.17 - 0.32)Monsanto Company 11-CA-233U 291 of 500Table E-9. Statistical Summary of Site NDVA Canola SeedNutrient Content for MON 88302 vs. the Conventional Control(continued)Difference (MON 88302 minus Control)Analytical Component (Units)1MON 883022Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%ConfidenceInterval3Significance(p-Value)CommercialTolerance Interval5(Range)Amino Acid (% dw)Tyrosine0.71 (0.023)0.74 (0.019)-0.031 (0.029)-0.13, 0.0620.3640.57, 0.81(0.67 - 0.75)(0.72 - 0.77)(-0.060 - 0.025)(0.60 - 0.84)Valine1.29 (0.050)1.36 (0.041)-0.073 (0.064)-0.28, 0.130.3400.92, 1.55(1.21 - 1.37)(1.32 - 1.41)(-0.12 - 0.020)(1.01 - 1.46)Fatty Acid (% Total FA)16:0 Palmitic3.98 (0.065)3.95 (0.053)0.036 (0.084)-0.23, 0.300.6992.84, 5.26(3.95 - 4.02)(3.94 - 3.96)(-0.016 - 0.083)(3.55 - 4.69)16:1 Palmitoleic0.20 (0.0057)0.22 (0.0046)-0.025 (0.0068)-0.047, -0.00310.0360.17, 0.30(0.20 - 0.20)(0.22 - 0.23)(-0.029 - -0.023)(0.19 - 0.27)18:0 Stearic1.77 (0.033)2.11 (0.027)-0.34 (0.042)-0.47, -0.200.0040.90, 3.05(1.71 - 1.84)(2.10 - 2.12)(-0.41 - -0.28)(1.50 - 2.64)18:1 Oleic65.14 (0.35)68.38 (0.29)-3.24 (0.37)-4.43, -2.050.00356.13, 70.69(64.90 - 65.20)(68.11 - 68.44)(-3.24 - -3.21)(57.86 - 68.53)Monsanto Company 11-CA-233U 292 of 500Table E-9. Statistical Summary of Site NDVA Canola SeedNutrient Content for MON 88302 vs. the Conventional Control(continued)Difference (MON 88302 minus Control)Analytical Component (Units)1MON 883022Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%ConfidenceInterval3Significance(p-Value)CommercialTolerance Interval5(Range)Fatty Acid (% Total FA)18:2 Linoleic17.86 (0.31)15.71 (0.25)2.15 (0.36)1.00, 3.290.00912.60, 24.49(17.78 - 18.02)(15.72 - 15.77)(2.06 - 2.26)(14.12 - 22.57)18:3 Linolenic8.82 (0.070)7.31 (0.057)1.51 (0.091)1.22, 1.80<0.0016.96, 11.73(8.71 - 8.94)(7.19 - 7.40)(1.35 - 1.54)(7.99 - 10.94)20:0 Arachidic0.57 (0.0047)0.65 (0.0046)-0.082 (0.0023)-0.089, -0.074<0.0010.45, 0.80(0.56 - 0.57)(0.64 - 0.65)(-0.085 - -0.077)(0.53 - 0.71)20:1 Eicosenoic1.15 (0.011)1.16 (0.0088)-0.0098 (0.014)-0.054, 0.0350.5310.83, 1.68(1.13 - 1.17)(1.15 - 1.18)(-0.042 - 0.020)(1.04 - 1.56)22:0 Behenic0.27 (0.0036)0.30 (0.0029)-0.029 (0.0044)-0.043, -0.0150.0070.19, 0.43(0.27 - 0.27)(0.30 - 0.30)(-0.031 - -0.027)(0.27 - 0.38)24:0 Lignoceric0.17 (0.053)0.16 (0.043)0.012 (0.068)-0.20, 0.230.8730.033, 0.25(0.16 - 0.18)(0.049 - 0.22)(-0.045 - 0.11)(0.044 - 0.21)Monsanto Company 11-CA-233U 293 of 500Table E-9. Statistical Summary of Site NDVA Canola SeedNutrient Content for MON 88302 vs. the Conventional Control(continued)Difference (MON 88302 minus Control)Analytical Component (Units)1MON 883022Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%ConfidenceInterval3Significance(p-Value)CommercialTolerance Interval5(Range)Fatty Acid (% Total FA)24:1 Nervonic0.12 (0.028)0.090 (0.023)0.031 (0.037)-0.086, 0.150.4600.041, 0.18(0.11 - 0.13)(0.049 - 0.12)(-0.0061 - 0.081)(0.044 - 0.20)MineralCalcium (g/100g dw)0.34 (0.049)0.31 (0.040)0.030 (0.063)-0.17, 0.230.6630.16, 0.61(0.30 - 0.37)(0.28 - 0.34)(-0.038 - 0.068)(0.25 - 0.53)Copper (mg/kg dw)3.80 (0.092)3.53 (0.076)0.28 (0.11)-0.084, 0.640.0922.00, 4.43(3.72 - 3.89)(3.33 - 3.67)(0.16 - 0.22)(2.52 - 4.93)Iron (mg/kg dw)51.55 (4.68)63.44 (3.82)-11.89 (6.04)-31.12, 7.340.14323.39, 86.23(46.78 - 56.32)(60.79 - 67.18)(-20.41 - -6.03)(39.16 - 77.92)Magnesium (g/100g dw)0.36 (0.012)0.35 (0.0099)0.0074 (0.016)-0.043, 0.0580.6680.32, 0.43(0.34 - 0.37)(0.33 - 0.37)(-0.012 - 0.043)(0.30 - 0.45)Manganese (mg/kg dw)50.88 (6.56)50.07 (5.45)0.81 (7.57)-23.29, 24.900.92114.85, 61.05(46.85 - 51.55)(47.11 - 50.97)(-0.26 - 0.58)(25.00 - 54.11)Monsanto Company 11-CA-233U 294 of 500Table E-9. Statistical Summary of Site NDVA Canola SeedNutrient Content for MON 88302 vs. the Conventional Control(continued)Difference (MON 88302 minus Control)Analytical Component (Units)1MON 883022Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%ConfidenceInterval3Significance(p-Value)CommercialTolerance Interval5(Range)MineralPhosphorus (g/100g dw)0.57 (0.077)0.57 (0.063)0.0026 (0.094)-0.30, 0.300.9790.38, 1.06(0.56 - 0.59)(0.56 - 0.58)(0.0027 - 0.014)(0.44 - 0.87)Potassium (g/100g dw)0.48 (0.037)0.54 (0.030)-0.053 (0.048)-0.20, 0.0990.3460.39, 0.96(0.48 - 0.49)(0.53 - 0.54)(-0.053 - -0.048)(0.50 - 0.92)Zinc (mg/kg dw)30.46 (1.73)32.41 (1.43)-1.94 (2.07)-8.52, 4.630.41620.19, 48.23(29.81 - 31.08)(28.89 - 34.23)(-3.15 - 0.93)(22.18 - 47.61)Vitamin (mg/100g dw)Vitamin E (-tocopherol)15.89 (0.88)9.43 (0.72)6.45 (1.14)2.82, 10.080.0103.88, 17.28(15.23 - 16.55)(8.46 - 10.20)(5.03 - 8.09)(2.62 - 14.84)1dw = dry weight; fw = fresh weight; FA = fatty acid.2 MON 88302 treated with glyphosate.3Mean (S.E.) = least-square mean (standard error); CI = confidence interval.4Control refers to the geneticallysimilar, conventional control.5With 95% confidence, interval contains 99% of the values expressed in the populationof commercial conventional references. Negative limits set to zero.Monsanto Company 11-CA-233U 295 of 500Table E-10. Statistical Summary of Site NDVA Canola SeedAnti-nutrient Content for MON 88302 vs. the ConventionalControlDifference (MON 88302 minus Control)Analytical Component (Units)1MON 883022Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%ConfidenceInterval3Significance(p-Value)CommercialTolerance Interval5(Range)Anti-nutrientAlkyl Glucosinolate (μmole/g dw)3.90 (0.65)4.22 (0.54)-0.32 (0.76)-2.73, 2.100.7050, 29.02(3.74 - 3.88)(2.45 - 5.22)(-1.34 - 1.29)(2.32 - 28.33)Indolyl Glucosinolate (μmole/g dw)4.51 (0.57)3.40 (0.49)1.11 (0.58)-0.74, 2.960.1521.37, 6.62(3.99 - 4.48)(1.83 - 4.23)(0.25 - 2.16)(1.84 - 7.18)Phytic Acid (% dw)1.58 (0.097)1.59 (0.088)-0.0050 (0.082)-0.27, 0.260.9540.70, 3.52(1.41 - 1.57)(1.46 - 1.68)(-0.052 - -0.012)(1.10 - 2.71)Sinapic Acid (% dw)1.02 (0.027)0.83 (0.025)0.19 (0.018)0.14, 0.250.0010.57, 1.13(1.00 - 1.04)(0.83 - 0.88)(0.18 - 0.21)(0.48 - 0.99)Total Glucosinolate (μmole/g dw)8.59 (1.20)7.72 (1.00)0.87 (1.33)-3.37, 5.100.5600, 32.20(7.96 - 8.57)(4.38 - 9.61)(-1.04 - 3.58)(5.52 - 31.98)1dw = dry weight.2 MON 88302 treated with glyphosate.3Mean (S.E.) = least-square mean (standard error); CI = confidence interval.4Control refers to the geneticallysimilar, conventional control.5With 95% confidence, interval contains 99% of the values expressed in the populationof commercial conventional references. Negative limits set to zero.Monsanto Company 11-CA-233U 296 of 500Table E-11. Statistical Summary of SiteSKSA Canola Seed Nutrient Content for MON 88302 vs. the Conventional ControlDifference (MON 88302 minus Control)Analytical Component (Units)1MON 883022Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%ConfidenceInterval3Significance(p-Value)CommercialTolerance Interval5(Range)Proximate (% dw)Ash3.95 (0.18)4.01 (0.17)-0.058 (0.10)-0.34, 0.220.5943.32, 4.66(3.68 - 4.44)(3.70 - 4.33)(-0.20 - 0.12)(2.98 - 4.52)Carbohydrates23.46 (0.51)25.05 (0.44)-1.59 (0.59)-3.23, 0.0560.05523.12, 30.77(23.11 - 23.84)(23.91 - 26.81)(-2.97 - -0.39)(22.53 - 29.96)Moisture (% fw)4.36 (0.21)4.69 (0.18)-0.33 (0.28)-1.12, 0.450.3064.33, 6.91(3.90 - 4.82)(4.41 - 4.88)(-0.75 - 0.010)(4.09 - 8.48)Protein23.82 (0.55)22.14 (0.50)1.68 (0.55)0.15, 3.210.03817.20, 30.08(23.62 - 24.58)(21.03 - 24.16)(0.42 - 2.50)(18.68 - 28.32)Total Fat48.83 (0.65)48.81 (0.59)0.022 (0.64)-1.76, 1.800.97439.65, 51.24(47.91 - 49.22)(46.96 - 50.24)(-1.02 - 0.95)(40.71 - 50.26)Fiber (% dw)Acid Detergent Fiber10.40 (0.63)9.85 (0.61)0.54 (0.35)-0.42, 1.510.1936.95, 23.92(9.19 - 11.50)(8.94 - 10.78)(-0.011 - 1.18)(9.75 - 21.22)Monsanto Company 11-CA-233U 297 of 500Table E-11. Statistical Summary of Site SKSA Canola SeedNutrient Content for MON 88302 vs. the Conventional Control(continued)Difference (MON 88302 minus Control)Analytical Component (Units)1MON 883022Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%ConfidenceInterval3Significance(p-Value)CommercialTolerance Interval5(Range)Fiber (% dw)Neutral Detergent Fiber11.61 (0.73)12.59 (0.67)-0.98 (0.63)-2.72, 0.770.19410.07, 25.94(9.48 - 12.75)(11.56 - 13.91)(-2.08 - 0.013)(10.93 - 22.75)Total Dietary Fiber18.68 (0.54)17.21 (0.48)1.48 (0.56)-0.075, 3.030.05713.97, 24.85(17.17 - 19.24)(16.57 - 17.89)(0.60 - 2.52)(12.64 - 26.47)Amino Acid (% dw)Alanine1.05 (0.033)1.01 (0.028)0.040 (0.043)-0.080, 0.160.4050.77, 1.34(1.02 - 1.07)(0.93 - 1.10)(-0.031 - 0.069)(0.87 - 1.27)Arginine1.33 (0.052)1.39 (0.045)-0.062 (0.069)-0.26, 0.130.4201.10, 1.93(1.27 - 1.38)(1.29 - 1.55)(-0.18 - -0.018)(1.23 - 1.96)Aspartic Acid1.45 (0.037)1.53 (0.032)-0.085 (0.049)-0.22, 0.0520.1591.33, 2.12(1.40 - 1.50)(1.46 - 1.65)(-0.15 - -0.084)(1.42 - 2.23)Cystine0.55 (0.022)0.55 (0.019)-0.0025 (0.025)-0.072, 0.0670.9230.38, 0.83(0.53 - 0.57)(0.49 - 0.62)(-0.050 - 0.053)(0.45 - 0.79)Monsanto Company 11-CA-233U 298 of 500Table E-11. Statistical Summary of Site SKSA Canola SeedNutrient Content for MON 88302 vs. the Conventional Control(continued)Difference (MON 88302 minus Control)Analytical Component (Units)1MON 883022Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%ConfidenceInterval3Significance(p-Value)CommercialTolerance Interval5(Range)Amino Acid (% dw)Glutamic Acid3.84 (0.14)3.98 (0.12)-0.14 (0.18)-0.64, 0.360.4932.73, 5.89(3.66 - 3.97)(3.65 - 4.39)(-0.50 - 0.094)(3.26 - 5.43)Glycine1.05 (0.023)1.08 (0.020)-0.026 (0.030)-0.11, 0.0580.4360.96, 1.47(1.02 - 1.07)(1.01 - 1.15)(-0.084 - -0.016)(1.01 - 1.50)Histidine0.59 (0.015)0.61 (0.013)-0.016 (0.019)-0.070, 0.0370.4430.47, 0.86(0.57 - 0.60)(0.57 - 0.65)(-0.058 - 0.0030)(0.54 - 0.80)Isoleucine0.91 (0.021)0.90 (0.018)0.011 (0.028)-0.067, 0.0880.7160.70, 1.22(0.89 - 0.93)(0.84 - 0.96)(-0.049 - 0.041)(0.78 - 1.15)Leucine1.61 (0.045)1.58 (0.039)0.029 (0.059)-0.13, 0.190.6441.21, 2.18(1.55 - 1.65)(1.46 - 1.71)(-0.073 - 0.085)(1.36 - 2.07)Lysine1.33 (0.037)1.37 (0.032)-0.034 (0.050)-0.17, 0.100.5271.02, 1.90(1.27 - 1.37)(1.30 - 1.48)(-0.12 - 0.024)(1.20 - 1.68)Monsanto Company 11-CA-233U 299 of 500Table E-11. Statistical Summary of Site SKSA Canola SeedNutrient Content for MON 88302 vs. the Conventional Control(continued)Difference (MON 88302 minus Control)Analytical Component (Units)1MON 883022Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%ConfidenceInterval3Significance(p-Value)CommercialTolerance Interval5(Range)Amino Acid (% dw)Methionine0.45 (0.015)0.44 (0.013)0.0082 (0.016)-0.037, 0.0540.6430.30, 0.65(0.44 - 0.47)(0.40 - 0.49)(-0.022 - 0.034)(0.36 - 0.57)Phenylalanine0.96 (0.023)0.94 (0.020)0.026 (0.031)-0.059, 0.110.4430.77, 1.26(0.93 - 0.98)(0.87 - 1.01)(-0.028 - 0.044)(0.84 - 1.25)Proline1.39 (0.035)1.44 (0.030)-0.050 (0.046)-0.18, 0.0770.3350.90, 2.01(1.34 - 1.42)(1.35 - 1.52)(-0.13 - 0.0026)(1.12 - 1.78)Serine0.97 (0.025)0.99 (0.022)-0.022 (0.033)-0.11, 0.0700.5460.81, 1.32(0.93 - 0.99)(0.94 - 1.06)(-0.067 - -0.0013)(0.88 - 1.30)Threonine0.89 (0.022)0.93 (0.019)-0.038 (0.029)-0.12, 0.0430.2630.82, 1.20(0.86 - 0.92)(0.88 - 0.99)(-0.072 - -0.031)(0.84 - 1.22)Tryptophan0.20 (0.0091)0.22 (0.0082)-0.018 (0.0093)-0.043, 0.00840.1340.13, 0.35(0.19 - 0.22)(0.21 - 0.24)(-0.035 - 0.0041)(0.17 - 0.32)Monsanto Company 11-CA-233U 300 of 500Table E-11. Statistical Summary of Site SKSA Canola SeedNutrient Content for MON 88302 vs. the Conventional Control(continued)Difference (MON 88302 minus Control)Analytical Component (Units)1MON 883022Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%ConfidenceInterval3Significance(p-Value)CommercialTolerance Interval5(Range)Amino Acid (% dw)Tyrosine0.64 (0.014)0.64 (0.012)-0.0084 (0.018)-0.060, 0.0430.6720.57, 0.81(0.61 - 0.65)(0.61 - 0.68)(-0.037 - 0.0079)(0.60 - 0.84)Valine1.16 (0.025)1.15 (0.022)0.0090 (0.034)-0.085, 0.100.8010.92, 1.55(1.13 - 1.19)(1.08 - 1.23)(-0.073 - 0.046)(1.01 - 1.46)Fatty Acid (% Total FA)16:0 Palmitic4.51 (0.047)4.07 (0.042)0.44 (0.049)0.31, 0.58<0.0012.84, 5.26(4.46 - 4.57)(4.05 - 4.10)(0.41 - 0.48)(3.55 - 4.69)16:1 Palmitoleic0.26 (0.0047)0.25 (0.0043)0.0062 (0.0044)-0.0061, 0.0190.2350.17, 0.30(0.25 - 0.26)(0.24 - 0.25)(0.0043 - 0.0074)(0.19 - 0.27)18:0 Stearic1.66 (0.055)2.08 (0.049)-0.42 (0.057)-0.58, -0.260.0010.90, 3.05(1.54 - 1.72)(1.91 - 2.19)(-0.48 - -0.35)(1.50 - 2.64)18:1 Oleic61.91 (0.55)65.69 (0.51)-3.78 (0.47)-5.08, -2.480.00156.13, 70.69(60.51 - 62.29)(64.73 - 66.86)(-4.30 - -2.59)(57.86 - 68.53)Monsanto Company 11-CA-233U 301 of 500Table E-11. Statistical Summary of Site SKSA Canola SeedNutrient Content for MON 88302 vs. the Conventional Control(continued)Difference (MON 88302 minus Control)Analytical Component (Units)1MON 883022Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%ConfidenceInterval3Significance(p-Value)CommercialTolerance Interval5(Range)Fatty Acid (% Total FA)18:2 Linoleic18.49 (0.37)17.22 (0.34)1.27 (0.34)0.33, 2.200.01912.60, 24.49(18.08 - 19.48)(16.64 - 18.01)(0.40 - 1.90)(14.12 - 22.57)18:3 Linolenic10.78 (0.23)8.38 (0.20)2.40 (0.25)1.72, 3.09<0.0016.96, 11.73(10.39 - 11.23)(7.94 - 8.99)(2.08 - 2.64)(7.99 - 10.94)20:0 Arachidic0.54 (0.0079)0.62 (0.0069)-0.082 (0.0090)-0.11, -0.057<0.0010.45, 0.80(0.52 - 0.55)(0.59 - 0.63)(-0.091 - -0.070)(0.53 - 0.71)20:1 Eicosenoic1.24 (0.015)1.13 (0.013)0.11 (0.020)0.052, 0.160.0050.83, 1.68(1.22 - 1.26)(1.10 - 1.17)(0.050 - 0.14)(1.04 - 1.56)22:0 Behenic0.28 (0.0031)0.29 (0.0027)-0.0088 (0.0037)-0.019, 0.00140.0730.19, 0.43(0.28 - 0.29)(0.28 - 0.30)(-0.014 - 0.0025)(0.27 - 0.38)24:0 Lignoceric0.20 (0.029)0.15 (0.026)0.050 (0.030)-0.032, 0.130.1630.033, 0.25(0.20 - 0.23)(0.045 - 0.22)(-0.016 - 0.062)(0.044 - 0.21)Monsanto Company 11-CA-233U 302 of 500Table E-11. Statistical Summary of Site SKSA Canola SeedNutrient Content for MON 88302 vs. the Conventional Control(continued)Difference (MON 88302 minus Control)Analytical Component (Units)1MON 883022Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%ConfidenceInterval3Significance(p-Value)CommercialTolerance Interval5(Range)Fatty Acid (% Total FA)24:1 Nervonic0.17 (0.020)0.12 (0.018)0.055 (0.021)-0.0021, 0.110.0550.041, 0.18(0.16 - 0.20)(0.045 - 0.16)(0.034 - 0.055)(0.044 - 0.20)MineralCalcium (g/100g dw)0.41 (0.015)0.44 (0.013)-0.031 (0.020)-0.086, 0.0250.1990.16, 0.61(0.40 - 0.42)(0.40 - 0.47)(-0.068 - 0.0021)(0.25 - 0.53)Copper (mg/kg dw)3.52 (0.16)3.23 (0.15)0.29 (0.18)-0.21, 0.780.1812.00, 4.43(3.27 - 3.85)(2.96 - 3.48)(-0.21 - 0.57)(2.52 - 4.93)Iron (mg/kg dw)63.21 (8.79)59.66 (7.61)3.56 (11.63)-28.72, 35.840.77423.39, 86.23(55.62 - 69.61)(50.11 - 77.74)(-13.33 - 14.87)(39.16 - 77.92)Magnesium (g/100g dw)0.36 (0.017)0.36 (0.016)-0.0044 (0.014)-0.042, 0.0330.7640.32, 0.43(0.31 - 0.39)(0.34 - 0.39)(-0.032 - 0.026)(0.30 - 0.45)Manganese (mg/kg dw)41.77 (2.44)34.73 (2.12)7.04 (3.03)-1.37, 15.450.08014.85, 61.05(38.56 - 47.24)(33.12 - 37.61)(0.95 - 12.63)(25.00 - 54.11)Monsanto Company 11-CA-233U 303 of 500Table E-11. Statistical Summary of Site SKSA Canola SeedNutrient Content for MON 88302 vs. the Conventional Control(continued)Difference (MON 88302 minus Control)Analytical Component (Units)1MON 883022Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%ConfidenceInterval3Significance(p-Value)CommercialTolerance Interval5(Range)MineralPhosphorus (g/100g dw)0.68 (0.051)0.74 (0.048)-0.057 (0.043)-0.18, 0.0620.2540.38, 1.06(0.57 - 0.80)(0.67 - 0.78)(-0.095 - 0.040)(0.44 - 0.87)Potassium (g/100g dw)0.82 (0.030)0.71 (0.030)0.11 (0.012)0.077, 0.14<0.0010.39, 0.96(0.77 - 0.90)(0.67 - 0.80)(0.084 - 0.14)(0.50 - 0.92)Zinc (mg/kg dw)41.58 (2.06)33.10 (1.90)8.48 (1.84)3.36, 13.600.01020.19, 48.23(39.33 - 45.49)(29.75 - 40.66)(4.84 - 11.44)(22.18 - 47.61)Vitamin (mg/100g dw)Vitamin E (-tocopherol)1.49 (1.08)6.91 (0.94)-5.43 (1.43)-9.40, -1.450.0193.88, 17.28(1.30 - 1.66)(3.33 - 9.22)(-6.92 - -1.67)(2.62 - 14.84)1dw = dry weight; fw = fresh weight; FA = fatty acid.2 MON 88302 treated with glyphosate.3Mean (S.E.) = least-square mean (standard error); CI = confidence interval.4Control refers to the geneticallysimilar, conventional control.5With 95% confidence, interval contains 99% of the values expressed in the populationof commercial conventional references. Negative limits set to zero.Monsanto Company 11-CA-233U 304 of 500Table E-12. Statistical Summary of SiteSKSA Canola Seed Anti-nutrient Content for MON 88302 vs. the ConventionalControlDifference (MON 88302 minus Control)Analytical Component (Units)1MON 883022Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%ConfidenceInterval3Significance(p-Value)CommercialTolerance Interval5(Range)Anti-nutrientAlkyl Glucosinolate (μmole/g dw)1.61 (0.71)5.82 (0.63)-4.21 (0.78)-6.36, -2.060.0050, 29.02(1.19 - 2.17)(4.87 - 8.28)(-6.11 - -3.28)(2.32 - 28.33)Indolyl Glucosinolate (μmole/g dw)0.86 (0.34)3.30 (0.32)-2.44 (0.29)-3.24, -1.640.0011.37, 6.62(0.49 - 1.31)(2.68 - 4.35)(-3.05 - -2.19)(1.84 - 7.18)Phytic Acid (% dw)1.58 (0.20)1.95 (0.18)-0.37 (0.24)-1.03, 0.290.1910.70, 3.52(1.20 - 1.91)(1.69 - 2.20)(-0.62 - 0.22)(1.10 - 2.71)Sinapic Acid (% dw)0.22 (0.059)0.81 (0.051)-0.60 (0.075)-0.80, -0.390.0010.57, 1.13(0.16 - 0.28)(0.65 - 0.95)(-0.76 - -0.49)(0.48 - 0.99)Total Glucosinolate (μmole/g dw)2.53 (1.02)9.22 (0.93)-6.69 (1.02)-9.51, -3.860.0020, 32.20(1.73 - 3.51)(7.85 - 12.72)(-9.21 - -5.78)(5.52 - 31.98)1dw = dry weight.2 MON 88302 treated with glyphosate.3Mean (S.E.) = least-square mean (standard error); CI = confidence interval.4Control refers to the geneticallysimilar, conventional control.5With 95% confidence, interval contains 99% of the values expressed in the populationof commercial conventional references. Negative limits set tozero.Monsanto Company 11-CA-233U 305 of 500 References for Appendix E

AACC. 1998. Official method 32-20. American Association of Ce real Chemists, St. Paul,

Minnesota.

AOAC. 2005a. AOAC official method 982.30. Association of Analytical Communities International, Gaithersburg, Maryland.

AOAC. 2005b. AOAC offi cial method 923.03. Association of Analytical Communities International, Gaithersburg, Maryland.

AOAC. 2005c. AOAC official method 960.39 a nd 948.22. Association of Analytical Communities International, Gaithersburg, Maryland.

AOAC. 2005d. AOAC offi cial method 983.23. Association of Analytical Communities International, Gaithersburg, Maryland.

AOAC. 2005e. AOAC official methods 984.27 and 985.01. Association of Analytical Communities International, Gaithersburg, Maryland.

AOAC. 2005f. AOAC offici al method 926.08 and 925.09. Association of Analytical Communities International, Gaithersburg, Maryland.

AOAC. 2005g. AOAC official method 955.04 and 979.09. Association of Analytical Communities International, Gaithersburg, Maryland.

AOAC. 2005h. AOAC offi cial method 985.29. Association of Analytical Communities International, Gaithersburg, Maryland.

AOCS. 1997. Official Methods and Recommended Practices of the American Oil Chemists Society, 5th Edition, Methods Ce 2-66 and Ce 1b-89. Methods Ce 2-66 and Ce 1b-89, American Oil Chemists Society, Champaign, Illinois.

AOCS. 1998. Official Methods and Recommended Practices of the American Oil Chemists Society, 5th Edition, Method Ac 4-91. Method Ac 4-91, American Oil Chemists Society, Champaign, Illinois.

AOCS. 2007. Official Methods and Recommended Practices of the American Oil Chemists Society, 5th Edition, Methods Ce 1d-91 and Ce 1i-07. Methods Ce 1d-91 and Ce 1i-07, American Oil Chemists Society, Champaign, Illinois.

Cort, W., T.S. Vicente, E.H. Waysek a nd B.D. Williams. 1983. Vitamin E content of feedstuffs determined by high-performance liquid chromatographic fluorescence. Journal of Agriculture and Food Chemistry 31: 1330-1333.Monsanto Company 11-CA-233U 306 of 500 Hagerman, A.E. and R.L. Nicholson. 1982. High-performance liquid chromatographic determination of hydroxycinnamic acids in the maize mesocotyl. Journal of Agricultural and Food Chemistry 30: 1098-1102.

ISO. 1992. Rapeseed – Determination of gluconsi nolates content. Part 1: Method using high-performance liquid chromatography. Inte rnational Standards Organization, Geneva,

Switzerland.

Komarek, A.R., J.B. Robertson and P.J. Va n Soest. 1994. Improved efficiency of ADF analysis using a filter bag procedure. Journal of Animal Science 72 114.

Lehrfeld, J. 1989. High-performance liquid chro matography analysis of phytic acid on a pH-stable macroporous polymer column. Cereal Chemistry 66: 510-515.

Lehrfeld, J. 1994. HPLC separation and quant itation of phytic acid and some inositol phosphates in foods: Problems and solutions. Jo urnal of Agricultural and Food Chemistry

42: 2726-2731.

McMurray, C.H., W.J. Blanchflower and D.A. Rice. 1980. Infl uence of extraction techniques on determination of  Tocopherol in animal feedstuffs. Journal of the

Association of Official Analytical Chemists 63: 1258-1261.

Speek, A.J., J. Schrijver and W.H.P. Sc hreurs. 1985. Vitamin E composition of some seed oils as determined by high-performa nce liquid chromatography with fluorometric

detection. Journal of Food Science 50: 121-124.

USDA. 1970. Forage and fiber analyses (app aratus, reagents, procedures, and some applications). Agricultur al Handbook No. 379. U.S. Department of Agriculture, Washington, DC.

USDA. 1973. Energy value of foods. Pages 2- 11 in Agriculture Handbook No. 74. U.S. Department of Agriculture, Washington, D.C.Monsanto Company 11-CA-233U 307 of 500 Appendix F: Materials, Methods a nd Results for Seed Dormancy and

Germination Assessment of MON 88302

F.1. Materials

Starting seed of MON 88302, the conventiona l control and commercial reference varieties, were produced in the field in Grand Forks County, ND in 2009 (Table F-1).

F.2. Characterization of the Materials

For the MON 88302, conventional control and commercial reference varieties starting seed lots, the presence or absence of MO N 88302 was confirmed by event-specific polymerase chain reaction analyses.

F.3. Germination Testing Facili ty and Experimental Methods

Seed dormancy and germination evaluations we re conducted at BioDiagnostics, Inc. in River Falls, WI. The principal investigator is qualified to conduc t seed dormancy and germination testing consistent with the st andards established by the Association of Official Seed Analysts (AOSA), a seed tr ade association (AOSA, 2009a; AOSA, 2009b).

Seed lots of MON 88302, the conventional control and four co mmercial reference varieties were tested under six different temp erature regimes. Six germination chambers were used in this study, and each chamber was maintained with one of the six temperature regimes: constant temperature of approximately 5, 15, 25, 30 or alternating temperatures of approximately 5/25, or 15/25 °C . The constant temperature regimes were

maintained with 16 h of dark and 8 h of li ght and the alternating temperatures regimes were maintained for 16 h at the lower temp erature in the dark and 8 h at the higher temperature in the light. The temperature inside each germination chamber was monitored and recorded every 15 min. throughout the duration of the study. Starting seed for each entry was treated with Helix XT ra (insecticide 20.7% thiamethoxam and fungicides 1.25% difenoconazole, 0.40% me fenoxam and 0.13% fludioxonil) at the recommended rate of 23 fl. oz. per 100 lbs of seed. Four moist blotters in a 9” x 6” germination box for each entry were prepared per facility SOPs for each temperature regime. A target of 100 seed s per entry were placed in a germination tray on moist blotters (one entry per germination tray) usin g a vacuum planting syst em. Four replicates of each entry of MON 88302, conventional cont rol and commercial reference varieties were arranged in a randomized complete block design within each germination chamber for each temperature regime.

A description of each germination characterist ic evaluated, and the timing of evaluations are presented in Table F-2. The types of data collected depended on the temperature regime. Each germination tray in the AOSA-recommended temperature regime (15 °C/25 °C) was assessed periodically during the study for normal germinated, abnormal germinated, dead (visual or ungerminated nonviable) and dormant (ungerminated viable). AOS A only provides guidelines ( AOSA, 2009a) for testing seed under a recommended temperature (15 °C/25 °C). Additional temperature regimes wereMonsanto Company 11-CA-233U 308 of 500 included in this study to test diverse environmental conditions. Seeds in the five additional temperature regimes (5, 15, 25, 30, an d 5/25 °C) were assessed 7 and 14 days after planting (DAP) and categorized as eith er germinated, dead, viable non-dormant or dormant. For the additional temperature re gimes, no distinction was made between normal and abnormal germinated seed. Any s eedling with a radicle of 1 mm or more was classified as germinated. The distinction between normal and abnormal germinated seedlings was not made because emergence and/ or development of essential structures of seedlings that otherwise would be categor ized as “normal germinated” under the AOSArecommended temperature conditions may not be so at other temp eratures. Fourteen DAP seeds that had not germinated were moved to the AOSA recommended temperature regime (15 °C/25 °C; 16 h dark and 8 h light ) for 7 days. This additional seven day

treatment was included to allow seeds that did not germinate under sub-recommended temperatures to germinate under the AOSA -recommended temperature. Twenty-one DAP (7 days at the AOSA temperature) germ inated seeds were counted as “viable nondormant” seeds. Ungerminated seeds were subject to a tetrazolium (Tz) test to assess for

dormant and dead seed. Dead and dormant s eed were defined the same as in the AOSA temperature regime.

F.4. Statistical Analysis

Analysis of variance (ANOVA) was conducte d according to a randomized complete block design with four replicates using SAS (version 9). MON 88302 was compared to the conventional control for germination char acteristics. The le vel of statistical significance was predetermined to be 5% ( α = 0.05). MON 88302 was not statistically compared to the commercial reference vari eties and no comparisons were made across temperature regimes. The minimum and ma ximum mean values (r eference range) were

determined from the commercial reference varieties.Monsanto Company 11-CA-233U 309 of 500 Table F-1. Starting Seed of MO N 88302, Control and Commercial Canola Reference Varieties Used in Dormancy Assessment

Material Material

Substance Type Phenotype Monsanto Lot #

MON 88302 Test Glyphosate-tolerant 11263712
Ebony Control Conventional 11263709 Q2 Reference Conventional 11263207 InVigor 5550 Reference Glufosinate-tolerant 11263706 71-45 RR Reference Glyphosate-tolerant 11263710

46A65 Reference Conventional 11263707Monsanto Company 11-CA-233U 310 of 500Table F-2. Dormancy and Germination Characteristics of MON 88302 and the Conventional Control SeedTemperature Regimes and Seed Characteristics EvaluatedEvaluation AOSA2, 3Additional Temperatures4Timing115/25 °C 5, 15, 25, 30, 5/25 °C followed by15/25 °C7 DAP5Normal germinatedAbnormal germinatedDeadGerminatedDead14 DAP5Dormant4DeadGerminatedDead21 DAP5No Data CollectedViable non-dormantDormantDead1Seed in the AOSA recommended (15/25 °C) temperature regime were evaluated 7 and 14 days after planting, while seed in the additional temperatures regimeswere evaluated on Days 7, 14 and 21 days after planting. Ungerminated seeds in the AOSA temperature regime on day 14 were subject to tetrazolium test (Tz)to determine viability. Ungerminated seeds in the additional temperature regimes on day 14 were moved to the AOSA recommended(15/25 °C) temperatureregime for 7 days (21 days after planting) and then subject to tetrazolium test (Tz) to determine viability.2The Association of Official Seed Analysts.3AOSA Category definitions:Normal germinated seed: Seedlings that exhibited normal developmental characteristics and possessed both a root and a shoot.Abnormal germinated seed: Seedlings that could not be classified as normal germinated (i.e., insufficient root and shoot development, lack of a shoot, shoot withdeep cracks or lesions or exhibited mechanical damage).Dead seed: Seeds that had visibly deteriorated and had become soft to the touch, or ungerminatedseeds that when tested with Tz had a negative result.Dormant seed: Ungerminated seeds that when tested with Tz had a positive result.4Non-AOSA category definitions:Germinated: Any seedling with a radical of 1 mm ormore was classified as germinated.Viable and not dormant:Ungerminated seeds in the additional temperature regimes at day 14 that when moved to the AOSA temperature regime germinated.Dead seed: Seeds that hadvisibly deteriorated and had become soft to the touch or ungerminated seeds that when tested with Tz had a negative result.Dormant seed: Ungerminated seedsthat when tested with Tz had a positive result.5DAP; Days after planting.Monsanto Company 11-CA-233U 311 of 500 References for Appendix F

AOSA. 2009a. AOSA Rules for testing seeds. Volume 1. Principl es and Procedures.

Association of Official Seed Analysts, Lincoln, Nebraska.

AOSA. 2009b. AOSA Rules for testing s eeds. Volume 4. Seedling Evaluation.

Association of Official Seed Analysts, Lincoln, Nebraska.Monsanto Company 11-CA-233U 312 of 500 Appendix G: Materials, Methods and Indi vidual Site Results from Phenotypic, Agronomic and Environmental Intera ction Assessment of MON 88302 under

Field Conditions

G.1. Materials

The materials for phenotypic assessment in cluded: MON 88302, a conventional control, and 24 unique, commercial reference varieties. The list of varieties planted at each of 17 sites is presented in Table G-1.

G.2. Characterization of the Materials

For the MON 88302 and conventional control star ting seed lots, the presence or absence of the glyphosate-tolerance trait was confir med by event-specific polymerase chain reaction analyses.

G.3. Field Sites and Plot Design

Data were collected at eight field sites in the U.S. and ni ne sites in Canada during 2009 (Section VII, Table VII-3). These 17 locations provided a diverse range of environmental and agronomic conditions repr esentative of comme rcial canola production

areas in North America. The researchers at each field site were familiar with the growth, production and evaluation of ca nola characteristics.

The experiment was established at each of the 17 sites in a randomized complete block design with four replicates. Variation in equipment and planting methods among the sites meant some variation in seeding rate occurre d. However, the seeding rates were within commercially acceptable ranges for canola ( NDSU, 2005). At sites with arthropod collections (MBMI, MNPY, NDBI, and SKSA), the seeding rates were approximately 12 to 17 seeds per ft 2 over an area for each plot of 551 to 754 ft 2 . At the remaining sites, the seeding rate was approximately 12 to 18 seeds per ft 2 over an area for each plot of 85 to

131 ft 2 (Table G-2).

At sites without arthropod collection, each pl ot was planted with a single pass of the seeder drill. One additional seeder pass wa s planted at the beginning and end of each replicate with a commercial canola variety. The purpose of the planted borders was to minimize edge effects. Phenotypic and qualita tive environmental interactions data were

collected from the middle of the seeder pass in each plot.

At sites with arthropod collecti on, each plot was planted with four passes of the seeder

drill. Two additional seeder passes were planted at th e beginning and end of each replicate with a commercial canola variety to minimize edge effects. Alleys between replicates and in the front and back of rep licate 1 and 4 were planted with a commercial canola variety. The purpose of the planted border areas was to create a continuous canola stand across the study area to ensure collecti on of more robust arthropod abundance data. The four seeder passes in each plot were dedi cated to specific types of data collection. Seeder pass 1 was used as a buffer. Seeder pass 2 was designated for the collection of theMonsanto Company 11-CA-233U 313 of 500 phenotypic and qualitative environmental interac tions data. Seeder pass 3 was used for the collection of the quantitative environmental interactions data. Seeder pass 4 was used to collect quantitative shattering data.

G.4. Planting and Field Operations

Field and planting information are listed in Table G-2. Agronomic practices used to prepare and maintain each study site were characteristic of those used in each respective geographic region. All maintenance operations were performed uniformly over the entire

trial area.Monsanto Company 11-CA-233U 314 of 500 Table G-1. Starting Seed for Phenotypic, Agronomic and Environmental

Interaction Assessment

Material 1 Material Type Phenotype Monsanto

Seed Lot # Sites 2

MON 88302 Test Glyphosate- tolerant 11225246 All sites

Ebony Control Conventional 11225243 All sites

Hyola 401 Reference Conventional 10001850 MNCA, NDBI 3 , SKME

46A65 Reference Conventional 11220680 ABLE, MBBR, MNCA, MNPY 3

, MNRO, NDBI 3 , NDCL, SKRA,

SKSA 3 , SKWA

71-45 RR Reference Glyphosate-tolerant 4 11225379 MBBR, MBMI 3 , MNRO, NDBI 3

, SKRA, SKSA 3

In Vigor 5630 Reference Glufosinate-tolerant 4 11225748 MBMI 3 , NDBI 3 , SKWA

71-30 CL Reference Conventional 10001852 ABLE, IARL, MNPY 3 , NDBO, SKSA 3

71-40 CL Reference Conventional 11261966 SKRA

Hyola 357 Magnum Reference Glyphosate-tolerant 4 10001727 IARL, MBBR, MNPY 3 , NDBO

In Vigor 5550 Reference Glufosinate-tolerant 4 10001855 MNPY 3 , NDCL, SKME

In Vigor 5020 Reference Glufosinate-tolerant 4 11225944 ABLE, MBNW, SKSA 3

In Vigor 5030 Reference Glufosinate-tolerant 4 11225943 SKRA, SKRO

Python 1651H Reference Conven tional 11225749 MBBR, NDBO, SKWA

Q2 Reference Conventional 11220679 MBMI 3 , MBNW, NDVA, SKRO,

SKME

45H73 Reference Conventional 10008476 MBMI 3 , MBNW, NDCL, SKRO

Cropland 601 Reference Conventional 10001849 IARL

In Vigor 8440 Reference Glufosinate-tolerant 4 11225747 IARL

50 Calibur Reference Glyphosate-tolerant 4 11225744 NDCL

72-55 RR Reference Glyphosate-tolerant 4 11225751 NDBO, SKME

HiLite 618 Reference Conventional 10001851 MNRO, NDVA

52-41 RR Reference Glyphosate-tolerant 4 11225752 NDVA, SKRO

45H28 Reference Glyphosate-tolerant 4 11225746 MNCA

30 Calibur Reference Glyphosate-tolerant 4 11225745 MNCA

34-65 RR Reference Glyphosate-tolerant 4 10001423 MNRO, SKWA

30-42 RR Reference Glufosinate-tolerant 4 11225750 ABLE

45H26 Reference Glyphosate-tolerant 4 10008474 MBNW, NDVA

1 MON 88302 and conventional control were planted at all field sites; the commercial reference varieties were site-specific

2 Site codes are as follows: ABCA = Camrose, AB; ABFS = Sturgeon, AB; ABLE = Leduc, AB; IARL = Brookings, SD; MBBR = Elton, MB; MNCA = O tter Tail, MN; MBMI = Whitewater, MB; MBNW = Portage le Prairie, MB; MNPY = Stearns, MN; MNRO = Wilkins, MN ; NDBI = Grand Forks, ND; NDBO = McHenry, ND; NDCL = Brookings, SD; NDGF = Grand Forks, ND; NDVA = McHenry, ND; SKME = Flett’s Springs, SK; SKRA = Corman Park, SK; SK RO = Wallace, SK; SKSA = Rosthern, SK; SKWA = Viscount, SK.

3 Sites with arthropod collections.

4 Commercially availabl e herbicide-tolerant canola varieties.Monsanto Company 11-CA-233U 315 of 500Table G-2. Field andPlanting InformationSite1PlantingDate2Plantingrate(seeds/ft.2)Plantingdepth(inches)Plotarea(ft.2)‡Seeder passes/plot Soil series, organic matter, pHCropping History2007 2008ABLE 05/15/09 14.0 0.6 108 1 Morinville clay loam, 10%, 6.3 Wheat BarleyIARL 05/15/09 14.0 1.0 114 1 Barnesclay loam,.3%, 7.1 Maize SoybeanMBBR 05/21/09 18.0 0.4 85 1 Newdale clay loam, 5.5%, 7-8.4 Wheat WheatMBMI 05/28/09 12.0 0.5 754 4 Ryerson clay loam, 5.4%, 7.3 Canola WheatMBNW 05/26/09 18.0 0.4 85 1 Read Rome clay loam, 5.2%, 7.2-7.9 Dry Bean WheatMNCA 05/30/09 12.0 0.5 130 1 Sisseton Heimdal loam, 3%, 7 Soybean WheatMNPY 05/21/09 12.0 0.8 751 4 Estherville loam, 3.4%, 6.4 Maize FallowMNRO 05/24/09 12.0 0.5 130 1 Aazdahl clay loam, 4-6%, 6.6-7.3 Soybean WheatNDBI 05/22/09 14.0 0.8 665 4 Gardena silt loam, 4.5%, 6.8 Fallow WheatNDBO 05/20/09 12.0 0.5 131 1 Svea-Barnes loam, 3.3%, 6.9 Flax WheatNDCL 05/15/09 14.0 1.0 114 1 Barnes clay loam , 4.5%, 7.1 Soybean MaizeNDVA 05/19/09 12.0 0.5 131 1 Williams loam, 2.4%, 5.4 Durum DurumSKME 05/28/09 15.0 0.8 106 1 Melfort clay loam, 9.1%, 6.5 Canola WheatSKRA 05/17/09 15.0 0.8 106 1 Weyburn loam, 3.3%, 7.5 Fallow WheatSKRO 05/19/09 15.0 0.5 103 1 Canora silty clay loam, 6.5%, 7.7 Wheat OatsSKSA 05/27/09 17.0 0.5 551 4 Hamlin loam , 2.19%, 6.35 Canola BarleySKWA 05/22/09 15.0 0.8 106 1 Weyburn loam, 4.6%, 6.7 Flax Barley1Site codes are as follows: ABCA = Camrose, AB; ABFS = Sturgeon,AB; ABLE = Leduc, AB; IARL = Brookings, SD; MBBR = Elton, MB; MNCA = OtterTail, MN; MBMI = Whitewater, MB; MBNW = Portage le Prairie, MB; MNPY = Stearns, MN; MNRO = Wilkins, MN; NDBI = Grand Forks, ND;NDBO =McHenry, ND; NDCL = Brookings, SD; NDGF = Grand Forks, ND; NDVA = McHenry, ND; SKME = Flett’s Springs, SK; SKRA = Corman Park,SK; SKRO= Wallace, SK; SKSA = Rosthern, SK; SKWA = Viscount, SK.2Month-day-year (MM/DD/YY).‡Sites with arthropod collection (MBMI, MNPY, NDBI and SKSA) had larger plot areas.Monsanto Company 11-CA-233U 316 of 500 G.5. Phenotypic Observations

The description of the charac teristics measured and the de signated developmental stages

where observations occurred are listed in Table VII-1.

G.6. Environmental Interaction Observations

Environmental interactions ( i.e. , interactions between the cr op plants and their receiving environment) were used to characteri ze MON 88302 by evaluating plant response to abiotic stress, disease damage, arthropod-r elated damage and pest and beneficial arthropod abundance in the plots using the methods described in sections G.7 and G.8.

G.7. Abiotic Stress Response, Disease Damage, and Arthropod-Related Damage

Qualitative Assessments: Abiotic Stress Response, Disease Damage and Arthropod Damage

MON 88302 and the conventional control were ev aluated at all 17 sites for differences in plant response to abiotic stressors, disease damage and arthropod-related damage. Three abiotic stressors, three diseases and thre e arthropod pests were evaluated four times during the growing season at the following intervals:

Observation 1: Seedling to rosette stage Observation 2: Bud to first flowering stage Observation 3: Full flowering to flower completion stage Observation 4: Pod development stage

The principal investigator at each site chose abiotic st ressors, diseases and arthropod pests that were either actively causing plant injury in the study area or were likely to occur in canola during a given observation period. Therefore, abiotic stressors, diseases and arthropod pests assessed of ten varied between observati ons at a site and between

sites.

Abiotic stressor and disease damage observatio ns were collected from a single seeder pass of each plot (sites without arthropod collec tion) and from seeder pass 2 of each plot (sites with arthropod collection) using a con tinuous 0 – 9 scale of increasing severity. Data were collected numerically and then placed into one of the following categories for reporting purposes:

Rating Severity of plant damage 0 none (no symptoms observed) 1 – 3 slight (symptoms not damaging to plant development) 4 – 6 moderate (intermediate between slight and severe) 7 – 9 severe (symptoms damaging to plant development)Monsanto Company 11-CA-233U 317 of 500 Quantitative Assessments: Flea Beetle and Seedpod Weevil Damage Assessment

Flea beetle and seedpod weevil damage were evaluated quantitatively at the MBMI, MNPY, NDBI and SKSA sites. Flea beetle damage was assessed 1-2 times during growing season. The first assessment was conducted during two to three weeks following emergence. The second assessment was conducted approximately 1 weeklater. At each assessment time, flea beetle damage was assessed by examining 10 nonsystematically selected plants from the middl e of seeder pass 3 of each plot using a 0 - 10 rating scale adapted from (Palaniswamy a nd Lamb, 1992) where 0 = no damage and 10 = 100% of leaf area damaged.

For seedpod weevil damage assessment, pods were sampled from 10 non-systematically selected plants (five pods per plant) from th e middle of seeder pass 3 of each plot at the ripening stage. Seedpod weevil damage wa s assessed by counting the number of exit holes in each pod.

G.8. Arthropod Abundance

Pest and beneficial arthropods were colle cted at the MBMI, MNPY, NDBI and SKSA sites four times during the growing s eason at the following time intervals:

Collection 1: Bud to first flowering stage Collection 2: Approximately 2 weeks after collection 1 Collection 3: Approximately 2 weeks after collection 2 Collection 4: Approximately 2 weeks after collection 3

Arthropods were collected using a cone beat sheet sampling method. The cone beat sheet consists of a white sheet approximately 91 cm long, 46 cm wide at the top and 10 cm wide at the base, to which a container lid has been attached. The attached container lid has a hole in the middle to allow arthropods to pass through. Plants were shaken vigorously into a cone beat sheet (larger plan ts were folded in half), to collect the dislodged insects into the container. Arthropods were colle cted from five nonsystematically selected plants from the mi ddle of seeder pass 3 of each plot. The arthropods from all plants were combined into one pre-labeled container and placed on frozen ice packs. The samples collected from U.S. field sites and Canadian field sites were then sent overnight to Monsanto Co mpany, St. Louis, MO and Department of Agricultural, Food and Nutritional Science at University of Albe rta, respectively for arthropod identification and enumeration.

A maximum of the five most abundant pest a nd five most abundant beneficial arthropods were determined from four non-systematica lly selected samples for each collection from each individual site ( e.g., Collection 1, MBMI site). These specific arthropods were then enumerated across all samples ( i.e. , one sample per plot) from a given collection at each individual site. The arthropods assessed ofte n varied between collections from a site and between sites due to differences in temporal activity and geographical distribution of the

taxa.Monsanto Company 11-CA-233U 318 of 500 G.9. Environmental Interact ions Evaluation Criteria

For the qualitative assessments of abiotic stress response, disease damage, and arthropod damage MON 88302 and the conventional cont rol were considered different in susceptibility or tolerance to an abiotic stre ssor, disease or arthropod pest on a particular observation date if the range of injury severity to MON 88302 did not overlap with the range of injury severity to the conventional co ntrol across all four re plicates. These data are categorical and were not subjected to st atistical analysis. For each observation at a site, the range of injury severity across the commercial reference varieties provided a range of comparative values that are re presentative of commercially-released canola varieties. Arthropod abundance, flea beetle damage and seedpod weevil damage data were quantitatively evaluated and subjected to statistical analys is (Section G.11.).

G.10. Data Assessment

Experienced scientists familiar with the expe rimental design and evaluation criteria were involved in all components of data collection, summariza tion, and analysis. Study personnel assessed that measurements were ta ken properly, data were consistent with expectations based on experi ence with the crop, and the experiment was carefully monitored. Prior to analysis, the overall dataset was evaluated for evidence of biologically relevant changes and for possible evidence of an unexpected plant response. Any unexpected observations or issues dur ing the study that would impact the study objectives were noted. Data were then subj ected to statistical analysis as indicated

below.

G.11. Statistical Analysis

Analysis of variance (ANOVA) was conducte d according to a randomized complete block design using SAS (Version 9) to compare MON 88302 and the conventional control for the phenotypic characteristics listed in Table VII-1. Comparisons of

MON 88302 and the conventional control were conducted within si te (individual site analysis) and across sites (combined-site analys is). The level of statistical significance was predetermined to be 5% ( α = 0.05). MON 88302 and the conventional control were not statistically compared to the commercial reference varieties. Minimum and

maximum mean values were calculated fo r each characteristic from the 24 unique

commercial reference varieties th at were included in this study. Data excluded from the study and the reasons for their exclusion are lis ted in Table G-3. Exclusion of these data did not adversely affect th e quality of the study.

An analysis of variance (ANOVA) was c onducted according to a randomized complete block design using SAS (Version 9) for th e flea beetle damage, seedpod weevil damage and the arthropod abundance. Th e level of statistical signi ficance was predetermined to be 5% ( α = 0.05). MON 88302 was compared to the conventional control at each site (individual site analysis) for flea beetle damage, seedpod weevil damage and arthropod abundance. Additionally, flea beetle dama ge and seedpod weevil damage data were pooled across sites (c ombined-site analysis) for a statistical comparison between

MON 88302 and the conventional control. Minimum and maximum mean values wereMonsanto Company 11-CA-233U 319 of 500 calculated for flea beetle damage and s eedpod weevil damage from 10 commercial

reference varieties that were included at the MBMI, MNPY, NDBI and SKSA sites. The reference range for the arthr opod abundance evaluated from a given collection and site was determined from the minimum and maximum damage values collected from the

commercial reference varieties at the site. Data excluded from the study and the reasons

for their exclusion are listed in G-3. Exclusi on of these data did not adversely affect the quality of the study.

G.12. Individual Field Site Plant Gr owth, Development and Environmental

Interactions Results and Discussion

In the individual site analysis, a total of 45 statistically significant differences were detected out of 148 comparisons between MON 88302 and the conventional control (Table G-4). These differences were distributed among all 12 of the phenotypic characteristics. Early stand count was lower for MON 88302 than the conventional control at the SKME site (23.6 vs. 29.0 plants/linear meter). MON 88302 was less vigorous ( i.e. , higher rating indicates less vigor) than the conventional control at MBMI (4.0 vs. 3.0 rating), MNPY (6.0 vs. 5.0 ra ting) and NDBO (5.3 vs. 3.5 rating). MON 88302 reached days to first flowering later than the conv entional control at all sites with the exception of the SKRA site where it reached first flowering in the same number of days. MON 88302 took longer to reach seed maturity than the conventional control at the SKSA site (106.5 vs. 99.5 days). M ON 88302 had less lodging than the conventional control at the MNRO site (1.0 vs. 1.5 rating) , but had more lodging than the conventional control at the NDBI site (3.3 vs. 2.0). MON 88302 was taller than the conventional

control at the MNCA site (51.3 vs. 43.5 inches), MNRO (55.8 vs. 52.3 inches), NDBI (55.8 vs. 51.8 inches) and the SKME (43.3 vs. 40.8 inches) sites. However, MON 88302

was shorter than the conven tional control at the IARL site (41.8 vs. 44.0 inches). MON 88302 had less visual pod shattering than the conventional control at the MNCA site (4.0 vs. 4.8 rating). MON 88302 had lo wer quantitative pod shattering than the

conventional control at the MBMI site (155.9 vs. 365.0 seeds/ft. 2 ). MON 88302 had higher seed moisture than the conventional control at all sites except ABLE, NDBI, SKRA and SKWA. MON 88302 had lower seed quality ( i.e. , more green seed) than the conventional control at IARL (6.5% vs. 1.8%), MNPY (0.5% vs. 0.0%), and MNRO (11.3% vs. 6.8%). MON 88302 had a higher yield than the c onventional control at the MNCA (19.4 vs. 15.5 bu/ac) and MNRO ( 50.1 vs. 43.6 bu/ac) sites. MON 88302 had a

lower final stand count than the conventiona l control at the MBNW site (13.6 vs. 19.8 plants/linear meter).

The statistical differences dete cted in the individual site an alyses for early stand count, seedling vigor, seed maturity, lodging, plan t height, visual pod sh attering, quantitative pod shattering, seed quality, yi eld and final stand counts we re not detected in the combined-site analysis. Thus, the diffe rences detected for these phenotypic

characteristics are not indicativ e of a consistent response asso ciated with th e trait and are unlikely to be biologically meaningful in terms of increased weediness potential of MON 88302 compared to the conventional contro l (Figure VII-1, step 2, “no” answer).Monsanto Company 11-CA-233U 320 of 500 While a statistical difference was detected for da ys to first flowering and seed moisture in both the individual site analys is and the combined-site analysis, the mean values of MON 88302 for days to first flowering and seed moisture in the combined-site analysis were within the range of values for commerci al reference varieties (Figure VII-1, step 3, “no” answer). Therefore, the difference in da ys to first flowering and seed moisture are unlikely to be biologically meaningful in te rms of increased weediness potential.

In the individual site analys is of arthropod damage among a ll observations at the four sites, one statistically significant difference was detected out of 9 comparisons between MON 88302 and the conventional control (Table G-8). Lack of variability in the data precluded statistical comparisons between MON 88302 and the conventional control for two additional comparisons involving seedpod weevil; however, the means for

MON 88302 and the conventional control were the same value for these comparisons, indicating no biological differe nces. MON 88302 had more flea beetle damage than the conventional control during Observation 1 at the MBMI site (1.18 vs . 0.53 rating). The

statistical difference detected for flea beetle damage was not detected when the data were pooled across all four sites. Thus, this diffe rence was not indicative of a consistent plant response associated with the trait and is unlikel y to be biologically meaningful in terms of an adverse environmental impact of MO N 88302 compared to conventional canola (Figure VII-1, step 2, “no” answer).Monsanto Company 11-CA-233U 321 of 500Table G-3 Data Missing orExcluded from AnalysisSite1Substance Rep CharacteristicReason for exclusionABLE 46A65 3 Seed maturity Protocol revision to increase the precision of this assessment.ABLE MON 88302 1 & 2 Seed maturity Protocol revision to increase the precision of this assessment.IARL All All Days to first flowering Data was not recorded correctly.IARL All All Visual pod shattering Data assessment added to the protocol by amendment afterharvest was completed.IARL All All Abiotic stressors, disease and arthropod damageratingsData not recorded.MBBR All All Seed maturity, lodging, plant height, visual podshattering, seed moisture, seed quality, yield andfinal stand countA hail storm destroyed the plants after flowering started.MBBR All All Seedling vigor Data were not recorded or was recorded incorrectly.MBBR Ebony 2 All The results of the seeding quality testing indicated that onecontrol plot may have contained plants of MON 88302.MBNW All All Seedling vigor Data were notrecorded or were recorded incorrectly.MNCA All All Seedling vigor Data were notrecorded or were recorded incorrectly.MNCA All All Abiotic stressorsand disease damage ratings Data was not recorded correctly.MNPY All All Plant height, abiotic stressors ratings Data was not recorded correctly.MNRO All All Seedling vigor Data were notrecorded or were recorded incorrectly.MNRO All All Abiotic stressorsand disease damage ratings Data was not recorded correctly.NDBO All All Seed maturity Data was not recorded correctly.NDBO MON 88302 1 & 4 Seed moisture Instrument failure.NDCL ALL ALL Visual pod shattering Data assessment added to the protocol by amendment afterharvest was completed.NDCL ALL ALL Abiotic stressors, disease and arthropod damageratingsData not recorded.Monsanto Company 11-CA-233U 322 of 500Table G-3.Data Missing or Excludedfrom Analysis (continued)Site1Substance Rep CharacteristicReason for exclusionNDCL All All Days to first flowering Data was not recorded correctly.NDCL MON 88302& Ebony1 Seed maturity Protocol revision to increase the precision of this assessment.NDVA All All Seed maturity Data was not recorded correctly.NDVA Q2 All Seed moisture Instrument failure.NDVA HiLite 618 1 & 3 Seed moisture Instrument failure.NDVA 45H26 4 Seed moisture Instrument failure.NDVA MON 88302 4 Seed moisture Instrument failure.SKME All All Seed maturity Data was not recorded correctly.SKRA MON 88302 3 & 4 Days to first flowering Data not recorded.SKRA All All Seed maturity Data was not recorded correctly.SKRA All All Abiotic stressors, disease and arthropod damageratingsFirst two of the four total ratings were not recorded.SKSA All All Seedling vigor and seed moisture Data was not recorded correctly.SKSA All All Quantitative pod shattering Snowstorm prevented the collection of this data.SKSA All All First arthropod collection and second flea beetleassessmentWeather prevented the collection of arthropods and assessmentof flea beetle damage.SKWA All All Seed maturity Data was not recorded correctly.SKWA All All Abiotic stressors, disease and arthropod damage First of four ratings was not recorded.1Site codes are as follows: ABCA=Camrose, AB; ABFS=Sturgeon, AB; ABLE=Leduc, AB; IARL=Brookings, SD; MBBR=Elton, MB; MNCA=OtterTail, MN;MBMI=Whitewater, MB; MBNW=Portage le Prairie, MB; MNPY=Stearns, MN; MNRO=Wilkins, MN; NDBI=Grand Forks, ND; NDBO=McHenry, ND;NDCL=Brookings, SD; NDGF=Grand Forks, ND; NDVA=McHenry, ND; SKME=Flett’s Springs, SK; SKRA=Corman Park, SK; SKRO=Wallace, SK;SKSA=Rosthern, SK; SKWA=Viscount, SK.Monsanto Company 11-CA-233U 323 of 500Table G-4. Individual Site Phenotypic Comparison of MON 88302 to Conventional ControlPhenotypic Characteristic (units)Early stand count2(number of plants per linear meter)Seedling vigor3(1-9 scale)Days to first flowering4(days after planting)MON 88302ControlMON 88302ControlMON 88302ControlSite1Mean (S.E.)Mean (S.E.)Mean (S.E.)Mean (S.E.)Mean (S.E.)Mean (S.E.)ABLE12.8 (1.9)18.3 (1.6)6.3 (0.5)5.5 (0.3)61.0 (0.0) *59.0 (0.0)IARL21.7 (1.9)21.5 (2.4)6.0 (0.0)6.0 (0.0)––MBBR7.1 (1.1)6.8 (0.6)––68.0 (0.0) *63.0 (0.0)MBMI17.8 (1.4)19.1 (0.6)4.0 (0.0)*3.0 (0.0)57.0 (0.0)*51.0 (0.0)MBNW14.8 (1.6)20.1 (1.7)––62.0 (0.0) *54.3 (0.8)MNCA23.7 (1.2)20.8 (2.2)––63.3 (0.8)*57.0 (0.0)MNPY26.8 (1.1)24.5 (0.5)6.0 (0.0)*5.0 (0.0)100.0 (0.0)*94.0 (0.0)MNRO22.6 (0.8)20.2 (0.9)––52.0 (0.0) *47.5 (0.5)NDBI19.9 (1.5)17.9 (1.7)7.0 (0.0)6.3 (0.5)58.3 (0.5)*50.5 (0.9)NDBO17.8 (2.1)16.6 (0.8)5.3 (0.8)*3.5 (0.3)53.0 (0.0)*48.5 (0.6)NDCL23.0 (0.5)24.9 (0.5)7.0 (0.0)†6.0 (0.0)––NDVA14.3 (1.3)13.2 (1.5)6.5 (0.5)5.8 (0.6)54.0 (0.0)*49.0 (0.0)SKME23.6 (1.2)*29.0 (1.7)3.3 (0.5)3.3 (0.3)56.0 (0.0)*53.0 (0.0)SKRA22.0 (0.9)21.8 (1.9)3.3 (0.3)3.0 (0.0)54.0 (0.0)54.0 (0.0)SKRO8.8 (1.9)10.6 (1.9)3.0 (0.0)3.5 (0.3)59.0 (0.0)*57.8 (0.5)SKSA10.5 (1.1)14.3 (0.8)––53.8 (0.3)*50.5 (0.3)SKWA23.0 (3.2)23.3 (2.3)3.5 (0.3)3.0 (0.0)61.0 (0.0)*56.0 (0.0)Monsanto Company 11-CA-233U 324 of 500Table G-4. Individual Site Phenotypic Comparison of MON 88302 to Conventional Control (continued)Phenotypic Characteristic (units)Seed maturity5(days after planting)Lodging6(1 – 9 scale)Plant height7(inches)MON 88302ControlMON 88302ControlMON 88302ControlSite1Mean (S.E.)Mean (S.E.)Mean (S.E.)Mean (S.E.)Mean (S.E.)Mean (S.E.)ABLE108.0 (0.0)†108.0 (0.0)1.0 (0.0)†1.0 (0.0)36.3 (0.8)36.0 (0.4)IARL90.3 (1.4)88.8 (0.8)1.0 (0.0)†1.0 (0.0)41.8 (1.0)*44.0 (1.2)MBBR––––––MBMI101.0 (0.0)†101.0 (0.0)1.0 (0.0)2.0 (0.0)52.5 (1.3)53.0 (0.8)MBNW105.0 (0.0)104.0 (1.0)1.0 (0.0)1.8 (0.5)54.5 (0.9)51.5 (2.7)MNCA101.0 (0.0)†101.0 (0.0)1.0 (0.0)†1.0 (0.0)51.3 (0.5)*43.5 (0.5)MNPY118.0 (0.0)118.0 (0.0)9.0 (0.0)†9.0 (0.0)––MNRO91.0 (0.0)†86.0 (0.0)1.0 (0.0)*1.5 (0.3)55.8 (0.5)*52.3 (1.9)NDBI104.0 (0.0)101.8 (0.8)3.3 (0.5)*2.0 (0.4)55.8 (1.8)*51.8 (0.9)NDBO––1.0 (0.0)1.0 (0.0)42.8 (0.9)42.5 (1.2)NDCL92.3 (1.7)92.3 (1.7)1.0 (0.0)1.5 (0.3)36.3 (2.2)36.3 (2.3)NDVA––1.3 (0.3)1.0 (0.0)45.0 (0.6)44.8 (0.9)SKME––1.0 (0.0)1.5 (0.3)43.3 (0.5)*40.8 (0.8)SKRA––1.0 (0.0)†1.0 (0.0)38.0 (0.8)38.5 (0.9)SKRO112.0 (0.0)†112.0 (0.0)1.0 (0.0)1.0 (0.0)39.0 (0.6)41.0 (1.6)SKSA106.5 (2.3)*99.5 (1.9)2.3 (0.3)1.8 (0.3)42.5 (0.5)41.0 (0.7)SKWA––1.0 (0.0)†1.0 (0.0)47.0 (1.5)47.8 (0.3)Monsanto Company 11-CA-233U 325 of 500Table G-4. Individual Site Phenotypic Comparison of MON 88302 to Conventional Control (continued)Phenotypic Characteristic (units)Visual pod shattering8(0-9 scale)Quantitative pod shattering9(seeds per ft.2)Seed moisture10(%)MON 88302ControlMON 88302ControlMON 88302ControlSite1Mean (S.E.)Mean (S.E.)Mean (S.E.)Mean (S.E.)Mean (S.E.)Mean (S.E.)ABLE1.0 (0.0)†1.0 (0.0)––8.6 (0.1)8.4 (0.1)IARL––––14.9 (0.6)*12.1 (0.4)MBBR––––––MBMI1.0 (0.0)1.0 (0.0)155.9 (38.6)*365.0 (53.7)14.5 (0.4)*12.8 (0.2)MBNW1.0 (0.0)1.0 (0.0)––11.8 (0.4)*9.9 (0.1)MNCA4.0 (0.4)*4.8 (0.5)––13.2 (0.2)*11.9 (0.1)MNPY1.0 (0.0)†1.0 (0.0)18.4 (0.8)12.6 (0.9)16.0 (0.4)*15.2 (0.2)MNRO1.3 (0.3)1.8 (0.3)––18.1 (0.1)*15.4 (0.1)NDBI1.0 (0.0)1.0 (0.0)19.9 (4.8)20.0 (6.5)10.3 (0.2)10.2 (0.1)NDBO1.0 (0.0)1.0 (0.0)––24.2 (0.7)*17.6 (1.6)NDCL––––9.1 (0.4)*7.7 (0.0)NDVA1.0 (0.0)1.5 (0.3)––14.4 (0.4)*13.4 (0.4)SKME1.0 (0.0)†1.0 (0.0)––13.0 (0.4)*10.2 (0.2)SKRA1.0 (0.0)†1.0 (0.0)––7.5 (0.2)7.1 (0.2)SKRO1.0 (0.0)†1.0 (0.0)––15.1 (0.5)*12.5 (0.6)SKSA1.0 (0.0)1.0 (0.0)––––SKWA1.0 (0.0)†1.0 (0.0)––12.7 (0.2)11.9 (0.3)Monsanto Company 11-CA-233U 326 of 500Table G-4. Individual Site Phenotypic Comparison of MON 88302 to Conventional Control (continued)Phenotypic Characteristic (units)Seed quality11(%)Yield12(bu per ac)Final stand count13(number of stubs per linear meter)MON 88302ControlMON 88302ControlMON 88302ControlSite1Mean (S.E.)Mean (S.E.)Mean (S.E.)Mean (S.E.)Mean (S.E.)Mean (S.E.)ABLE0.8 (0.5)0.5 (0.3)29.5 (1.2)26.6 (1.3)13.8 (2.2)15.3 (0.5)IARL6.5 (1.6)*1.8 (0.6)21.0 (1.8)26.9 (2.4)21.7 (1.9)21.5 (2.4)MBBR––––––MBMI1.0 (1.0)3.3 (1.7)62.1 (3.5)66.9 (2.3)17.8 (1.4)19.0 (0.7)MBNW0.5 (0.3)1.5 (0.6)72.3 (4.9)66.2 (4.0)13.6 (1.1)*19.8 (1.6)MNCA3.3 (0.9)3.0 (0.4)19.4 (0.4)*15.5 (0.7)19.3 (0.6)17.7 (2.3)MNPY0.5 (0.3)*0.0 (0.0)24.7 (0.2)23.8 (0.3)26.7 (1.0)24.5 (0.5)MNRO11.3 (0.9)*6.8 (1.4)50.1 (1.2)*43.6 (1.0)20.6 (0.8)18.6 (0.6)NDBI0.3 (0.3)0.3 (0.3)64.5 (3.2)67.9 (4.7)19.1 (2.6)16.2 (1.9)NDBO8.5 (3.1)4.5 (1.0)29.6 (1.5)35.1 (1.0)17.4 (1.5)16.2 (1.5)Monsanto Company 11-CA-233U 327 of 500Table G-4. Individual Site Phenotypic Comparison of MON 88302 to Conventional Control (continued)Phenotypic Characteristic (units)Seed quality11(%)Yield12(bu per ac)Final stand count13(number of stubs per linear meter)MON 88302ControlMON 88302ControlMON 88302ControlSite1Mean (S.E.)Mean (S.E.)Mean (S.E.)Mean (S.E.)Mean (S.E.)Mean (S.E.)NDCL3.5 (0.6)7.5 (3.1)23.4 (2.1)30.3 (3.5)23.0 (0.5)24.9 (0.5)NDVA9.3 (0.9)10.3 (2.1)40.4 (2.0)38.4 (2.8)16.7 (1.0)13.8 (1.7)SKME1.3 (0.5)0.0 (0.0)45.3 (2.0)52.7 (1.6)20.9 (1.5)21.3 (0.9)SKRA0.5 (0.3)0.8 (0.5)47.4 (1.5)47.7 (2.1)22.4 (1.3)21.3 (2.0)SKRO3.8 (0.8)2.5 (0.5)58.1 (4.3)66.6 (4.8)9.1 (2.0)9.9 (2.0)SKSA0.5 (0.3)0.0 (0.0)39.4 (1.0)41.2 (3.6)10.5 (0.9)12.0 (0.5)SKWA1.0 (0.4)2.5 (1.6)60.3 (2.1)62.6 (0.9)19.7 (0.7)17.7 (1.0)Note: The experimental design was a randomized complete block with four replicates. S.E. = Standard Error. The number of plots (n) used in the statisticalanalysis was 4 except where noted..(–) = Data not collected or excluded.* Indicates a statistically significant difference between MON 88302 and the conventional canola control (α= 0.05).†No statistical comparisons were made due to lack of variability in the data.‡Data not planned for collection at these sites.1Site codes are as follows: ABCA=Camrose, AB; ABFS=Sturgeon, AB; ABLE=Leduc, AB; IARL=Brookings,SD; MBBR=Elton, MB; MNCA=OtterTail,MN; MBMI=Whitewater, MB; MBNW=Portage le Prairie, MB; MNPY=Stearns, MN; MNRO=Wilkins, MN; NDBI=Grand Forks, ND; NDBO=McHenry, ND;NDCL=Brookings, SD; NDGF=Grand Forks, ND; NDVA=McHenry, ND; SKME=Flett’s Springs, SK;SKRA=Corman Park, SK; SKRO=Wallace, SK;SKSA=Rosthern, SK; SKWA=Viscount, SK.2Early stand counts were conducted by counting the number of plants from three separate linear meter rows data. At MBBR plot number (n)=4 for MON 88302and N=3 for the conventional control.3Rated on a scale of 1-9, where 1 =excellent vigor and 9 = poor vigor.4Days to first flowering was determined from the number of days after planting when 50% of the plants in a plot had one or moreflowers. Days to first floweringdata were at IARL and NDCL plot number (n)=2 for MON 88302 and n=4 for the conventional control and at MBBR the plot number (n)=4 for MON 88302 andn=3 for the conventional control.Monsanto Company 11-CA-233U 328 of 5005Seed maturity was determined as the numberof days after planting when 30% or more of the seed in the lower third of the main raceme had changed from agreen to black/brown/tan color. Seed maturity data at ABLE plot number (n)=2 for MON 88302 and n=4 for the conventional control, at NDCL plot number(n)=3 for MON 88302 and the conventional control.6Rated on a 0 - 9 scale, where 0 = completelyupright plants and 9 = completely flat.7Plant heights were measured from the soil surface to the top of the main raceme after flowering was completed.8Visual pod shattering was estimated with a rated on a 1 - 9 scale,where 1 = 0 to 10% shatter, and each subsequent value on thescale increasing in 10%increments up to 9 = greater than 80% shatter.9Collection trays placed within the crop canopyand seed losses from shattering counted once per week for three weeks. This data was intended for collection atMBMI, MNPY, NDBI and SKSA.10Seed moisture data at NDBO plot number (n)=2 for MON 88302 and n=4 for the conventional control and at NDVA plot number (n)=3 for MON 88302 andn=4 for the conventional control.11Seed quality was determined at harvest bycounting the percentage of green seeds froma 100 seed subsample from each plot.12Yield data at NDBO plot number (n)=2 for MON 88302 and n=4 for the conventional control at NDVA plot number (n)=3 for MON 88302and n=4 for theconventional control.13Final stand counts were conducted after harvest by measuring plant stems from three separate linear meter rows.Monsanto Company 11-CA-233U 329 of 500 Table G-5. Abiotic Stress Response E valuations of MON 88302 and Conventional Control Using an Observational Severity Scale

Abiotic stressor Number of observations

across all sites Number of observations where no

differences were observed between

MON 88302 and the control

Total 131 130

Cold 9 9 Compaction 4 4 Drought 34 34

Flood 3 3

Frost 13 12 *

Hail 19 19

Heat 22 22 Nitrogen deficiency 4 4

Wind 23 23

* A single difference was observed between MON 88302 and the conventional control during Observation 1 at the MBBR site: Test = severe, Control = moderate, Reference range = slight-severe. Data were not subjected to statistical analysis. Note: The experimental design was a randomized complete block with four replicates. Observational data were collected at four crop develo pment stages: Observation 1: seedling to rosette stage; Observation 2: bud to first flowering stage; Observation 3: full flowering to flower completion stage; Observation 4: pod development stageMonsanto Company 11-CA-233U 330 of 500 Table G-6. Disease Damage Evaluation s of MON 88302 and Conventional Control Using an Observational Severity Scale

Disease Number of observations

across all sites Number of observations

where no differences were

observed between

MON 88302 and the control

Total 141 141

Alternaria 12 12 Aster yellow 10 10 Bacterial leaf spot 3 3 Black leg 39 39 Cercospera leaf spot 4 4

Clubroot 8 8 Downy mildew 11 11

Fusarium 10 10 Phytophthora 2 2 Powdery mildew 1 1

Root rot 1 1

Sclerotina 31 31 Seedling blight 2 2 Seedling disease complex 1 1

White mold 4 4

White rust 2 2

No differences were observed between MON 88302 and the conventional control during any observation for damage caused by any of the assessed diseases. Da ta were not subjected to statistical analysis. Note: The experimental design was a randomized complete block with four replicates. Observational data were collected at four crop develo pment stages: Observation 1: seedling to rosette stage; Observation 2: bud to first flowering stage; Observation 3: full flowering to flower completion stage; Observation 4: pod development stage.Monsanto Company 11-CA-233U 331 of 500 Table G-7. Arthropod-Related Da mage Evaluations of MON 88302 and Conventional Control Using an Observational Severity Scale

Arthropod Number of

observations

across all sites Number of observations where

no differences were observed

between MON 88302 and the

conventional control

Total 165 165

Alfalfa loopers ( Autographa californica ) 2 2

Aphids (Aphididae) 18 18

Bertha armyworms ( Mamestra configurata ) 15 15

Blister beetles ( Spicauta spp.) 15 15

Cabbage seedpod weevils ( Ceutorhynchus obstrictus ) 4 4

Cabbage worms 1 (Pieridae) 8 8

Cutworms (Noctuidae) 10 10

Diamondback moth larvae ( Plutella xylostella) 24 24

Flea beetles (Chrysomelidae) 25 25

Grasshoppers (Acrididae) 24 24

Lygus bugs (Miridae) 15 15

Red turnip beetles ( Entomoscelis Americana) 4 4

Wireworms (Elateridae) 1 1

No differences were observed between MON 88302 and the conventional control during any observation for damage caused by any of the assessed arthropods. Data were not subjected to statistical analysis. Note: The experimental design was a randomized complete block with four replicates. Observational data were collected at four crop develo pment stages: Observation 1: seedling to rosette stage; Observation 2: bud to first flowering stage; Observation 3: full flowering to flower completion stage; Observation 4: pod development stage.

1 Includes cabbage white caterpillarsMonsanto Company 11-CA-233U 332 of 500 Table G-8. Individual Site An alysis: Quantitative Asse ssment of Flea Beetle and Seedpod Weevil Damage to MON 88302 Compa red to the Conventional Control in

2009 Field Trials

Pest Damage assessment Site 1 MON 88302 Control

Flea beetles 2
(Chrysomelidae) Mean (S.E.) damage of 10 plants per plot (0-10 rating scale) – Observation 1 MBMI 1.18 (0.25)* 0.53 (0.06)

MNPY 0.63 (0.06) 0.83 (0.12)

NDBI 2.63 (0.18) 2.70 (0.07)

SKSA 0.30 (0.07) 0.48 (0.09)

Mean (S.E.) damage of 10 plants per plot (0-10 rating scale) – Observation 2 MBMI 0.43 (0.13) 0.40 (0.09)

MNPY 1.43 (0.05) 1.48 (0.08)

NDBI 1.80 (0.21) 1.80 (0.22)

SKSA

— —

Seedpod weevil 3
( Ceutorhynchus

obstrictus ) Mean (S.E.) number of holes in pods from 10 plants per plot) MBMI 0.00 (0.00) † 0.00 (0.00)

MNPY 0.13 (0.03) 0.02 (0.00)

NDBI 0.00 (0.00) ‡ 0.00 (0.00)

SKSA 0.00 (0.00) † 0.00 (0.00)

*Indicates a significant difference between MON 88302 and the conventional control ( α = 0.05).

† No statistical comparisons were made due to lack of variability in the data.

‡ Mean values reported are zeros based on ro unding of values that were small. Note: The experimental design was a randomized complete block with four replicates. S.E. = Standard Error

1 Site codes are as follows: MBMI = Whitewater, MB; MNPY = Stearns, MN; NDBI = Grand Forks, ND; SKSA = Rosthern, SK.

2 Damage assessments for flea beetle were conducted 1-2 times during growing season. The first assessment was conducted during two to three weeks following crop emer gence at all four sites. The second assessment was conducted approximately 1 week later at the MBMI, MNPY and NDBI sites. The

first flea beetle assessment at SKSA could not be conducted due to a severe rain storm.

3 Damage assessments for seedpod weevil were conducted at the ripening stage. A dash (—) indicates information not available.Monsanto Company 11-CA-233U 333 of 500Table G-9. Abundance of Pest Arthropods in Cone BeatSheet Samples Collected fromMON 88302, Conventional Controland the Commercial Reference VarietiesPest Arthropod2Aphids(Aphididae)Bertha armyworms(Mamestra configurata)Diamondback moth larvae(Plutella xylostella)Col. Site1MON 88302Mean (S.E.)ControlMean (S.E.)ReferenceRange3MON 88302Mean (S.E.)ControlMean (S.E.)ReferenceRange3MON 88302Mean (S.E.)ControlMean (S.E.)ReferenceRange31 MBMI−−−−−−1.00 (0.00) 0.50 (0.29) 0.00 – 1.25MNPY 1.00 (1.00) 0.25 (0.25) 0.25 – 1.25−−−−−−NDBI−−−−−−−−−SKSA−−−−−−−−−2 MBMI−−−−−−0.25 (0.25) 0.50 (0.29) 0.00 – 0.25MNPY 0.50 (0.29) 1.50 (0.87) 2.75 – 4.50−−−−−−NDBI 0.00 (0.00) 0.25 (0.25) 0.00 – 0.25−−−−−−SKSA−−−−−−−−−3 MBMI 0.50 (0.50) 0.00 (0.00) 0.00 – 0.5−−−1.00 (0.58) 0.25 (0.25) 0.00 – 0.25MNPY 0.50 (0.29) 0.75 (0.75) 0.00 – 17.00 0.00 (0.00) 0.50 (0.29) 0.00 – 0.50−−−NDBI 0.00 (0.00) 0.25 (0.25) 0.00 – 3.00−−−−−−SKSA−−−−−−−−−4 MBMI 2.75 (1.60) 2.50 (1.26) 0.75 – 9.25−−−2.50 (1.44) 3.00 (0.71) 0.75 – 1.25MNPY 0.25 (0.25) 0.25 (0.25) 0.00 – 0.25−−−0.25 (0.25) 0.25 (0.25) 0.00 – 0.25NDBI−−−−−−−−−SKSA−−−−−−0.00 (0.00) 0.75 (0.48) 0.00 – 0.50Monsanto Company 11-CA-233U 334 of 500Table G-9. Abundance of Pest Arthropods in Cone BeatSheet Samples Collected fromMON 88302, Conventional Controland the Commercial Reference Varieties (continued)Pest Arthropod2Flea beetles(Chrysomelidae)Lygus bugs(Miridae)Thrips(Thripidae)Col. Site1MON 88302Mean (S.E.)ControlMean (S.E.)ReferenceRange3MON 88302Mean (S.E.)ControlMean (S.E.)ReferenceRange3MON 88302Mean (S.E.)ControlMean (S.E.)ReferenceRange31 MBMI−−−−−−0.50 (0.50) 0.25 (0.25) 0.00 – 0.50MNPY−−−0.00 (0.00) 0.00 (0.00) 0.00 – 0.25−−−NDBI 0.00 (0.00) 0.00 (0.00) 0.00 – 0.75−−−1.75 (0.75) 0.25 (0.25) 4.00 – 13.50SKSA−−−−−−−−−2 MBMI−−−−−−0.75 (0.75) 0.00 (0.00) 0.00 – 0.25MNPY−−−−−−−−−NDBI−−−−−−0.25 (0.25) 0.75 (0.75) 2.00 – 5.00SKSA−−−0.00 (0.00) 0.00 (0.00) 0.00 – 0.00 8.25 (5.12) 5.00 (0.58) 2.75 – 4.003 MBMI 0.25 (0.25) 0.00 (0.00) 0.00 – 0.25−−−4.25 (2.46) 0.25 (0.25) 0.00 – 0.75MNPY−−−0.25 (0.25) 1.25 (0.63) 0.50 – 2.25 1.00 (0.71) 0.00 (0.00) 0.00 – 0.25NDBI 0.50 (0.29) 2.00 (0.82) 0.00 – 0.75−−−4.00 (1.68) 2.75 (1.89) 0.50 – 2.75SKSA−−−−−−0.50 (0.50) 3.25 (2.36) 0.00 – 3.504 MBMI 1.25 (0.95) 1.25 (0.48) 0.25 – 0.50 0.00 (0.00) 0.00 (0.00) 0.00 – 0.50 0.00 (0.00) 0.00 (0.00) 0.00 – 0.00MNPY−−−2.5 (0.50) 3.75 (0.85) 0.25 – 5.75−−−NDBI 4.25 (0.25) 3.25 (2.36) 2.00 – 3.50−−−−−−SKSA−−−−−−2.25 (1.32) 1.50 (1.19) 0.00 – 2.75No statistically significant differences were detected between MON 88302 and the conventional control (α= 0.05).Note: The experimental design was a randomized complete block with four replicates. S.E. = Standard Error.A dash (−) indicates arthropod not evaluated.1Site codes are as follows: MBMI = Whitewater, MB; MNPY = Stearns, MN; NDBI = Grand Forks, ND; SKSA = Rosthern, SK.2Arthropod collection 1 was made at bud tofirst flowering stage and the three subsequent collections at approximately two weekintervals thereafter. The firstarthropod collection could not be conducted at SKSA due to a severe rain storm.3Reference range = minimum and maximum mean values among the commercial reference varieties.Monsanto Company 11-CA-233U 335 of 500Table G-10. Abundance of BeneficialArthropods in Cone Beat SheetSamples Collected from MON 88302, ConventionalControl and the Commercial Reference VarietiesBeneficial Arthropod2Chironomid midgeLacewings(chrysopidae)Ladybird beetles(Coccinellidae)Col. Site1MON 88302Mean (S.E.)ControlMean (S.E.)ReferenceRange3MON 88302Mean (S.E.)ControlMean (S.E.)ReferenceRange3MON 88302Mean (S.E.)ControlMean (S.E.)ReferenceRange31 MBMI−−−−−−−−−MNPY−−−−−−−−−NDBI−−−−−−−−−SKSA−−−−−−−−−2 MBMI−−−−−−−−−MNPY−−−−−−−−−NDBI−−−−−−−−−SKSA−−−−−−0.00 (0.00) 0.00 (0.00) 0.00 – 0.003 MBMI−−−−−−−−−MNPY−−−−−−0.00 (0.00) 0.00 (0.00) 0.00 – 0.50NDBI−−−−−−−−−SKSA 0.00 (0.00) 0.00 (0.00) 0.00 – 0.25−−−−−−4 MBMI−−−0.00 (0.00) 0.00 (0.00) 0.00 – 0.25−−−MNPY−−−−−−−−−NDBI−−−−−−−−−SKSA−−−−−−−−−Monsanto Company 11-CA-233U 336 of 500Table G-10. Abundance of Beneficial Arthropods in Cone Beat Sheet Samples Collected from MON 88302, ConventionalControl and the Commercial Reference Varieties (continued)Beneficial Arthropod2Micro-parasitic hymenoptera Macro-parasitic hymenopteraOriusspp.Col. Site1MON 88302Mean (S.E.)ControlMean (S.E.)ReferenceRange3MON 88302Mean (S.E.)ControlMean (S.E.)ReferenceRange3MON 88302Mean (S.E.)ControlMean (S.E.)ReferenceRange31 MBMI−−−−−−−−−MNPY−−−0.00 (0.00) 0.00 (0.00) 0.00 – 0.25−−−NDBI−−−−−−−−−SKSA−−−−−−−−−2 MBMI 0.00 (0.00) 0.00 (0.00) 0.00 – 0.25−−−−−−MNPY−−−−−−−−−NDBI−−−−−−0.00 (0.00) 0.25 (0.25) 0.00 – 0.00SKSA−−−−−−−−−3 MBMI 0.75 (0.75) 0.25 (0.25) 0.00 – 0.50−−−−−−MNPY−−−−−−2.00 (1.08) 1.75 (0.85) 0.50 – 2.25NDBI 0.25 (0.25) 0.00 (0.00) 0.00 – 0.00−−−−−−SKSA 0.00 (0.00) 0.00 (0.00) 0.00 – 0.00−−−−−−4 MBMI−−−−−−−−−MNPY−−−−−−1.50 (1.19) 0.25 (0.25) 0.00 – 0.00NDBI−−−−−−−−−SKSA 0.00 (0.00) 0.25 (0.25) 0.00 – 0.25−−−−−−Monsanto Company 11-CA-233U 337 of 500Table G-10. Abundance of Beneficial Arthropods in Cone Beat Sheet Samples Collected from MON 88302, ConventionalControl and the Commercial Reference Varieties (continued)Beneficial Arthropod2Spiders(Araneae)Sphecid wasps(Sphecidae)Col. Site1MON 88302Mean (S.E.)ControlMean (S.E.)ReferenceRange3MON 88302Mean (S.E.)ControlMean (S.E.)ReferenceRange31 MBMI−−−−−−MNPY−−−0.00 (0.00) 0.00 (0.00) 0.00 – 0.25NDBI−−−−−−SKSA−−−−−−2 MBMI−−−−−−MNPY−−−−−−NDBI−−−−−−SKSA−−−−−−3 MBMI−−−−−−MNPY−−−−−−NDBI−−−−−−SKSA−−−−−−4 MBMI−−−−−−MNPY−−−−−−NDBI−−−−−−SKSA 0.00 (0.00) 0.00 (0.00) 0.00 – 0.25−−−No statistically significant differences were detected between MON 88302 and the conventional control (α= 0.05).Note: The experimental design was a randomized complete block with four replicates. S.E. = Standard Error.A dash (−) indicates arthropod not evaluated.1Site codes are as follows: MBMI = Whitewater, MB; MNPY = Stearns, MN; NDBI = Grand Forks, ND; SKSA = Rosthern, SK.2Arthropod collection 1 was made at bud tofirst flowering stage and the three subsequent collections at approximately two weekintervals thereafter. The firstarthropod collection could not be conducted at SKSA due to a severe rain storm.3Reference range = minimum and maximum mean values among the commercial reference varieties.Monsanto Company 11-CA-233U 338 of 500 References for Appendix G

NDSU. 2005. Canola production field guide. A-1280, North Da kota State University Extension Service, Fargo, North Dakota.

Palaniswamy, P. and R.J. Lamb. 1992. Screen ing for antixenosis resistance to flea beetles, Phyllotreta crucifer ae (Goeze) (Coleoptera: Chrysomelidae), in rapeseed and related crucifers. Canadian Entomologist 124: 895-906..Monsanto Company 11-CA-233U 339 of 500 Appendix H: Materials and Method s for Pollen Morphology and Viability

Assessment

H.1. Plant Production

MON 88302, a conventional control, and four commercial reference va rieties were grown in pots in a growth chamber established at 21 °C day/18 °C night with a 16 h photoperiod (Table H-1). The plants were arranged in a randomized complete block design with five replicates with 1 plant of each entry per replicate. Prior to planting, the starting seed was treated with Helix XTra (insecticide 20.7% thiamethoxam and fungicides 1.25% difenoconazole, 0.40% mefenoxam, and 0.13% fl udioxonil) at the r ecommended rate. Canola was seeded into 4” pots with Metro-Mix 200 containing 14 oz/yds 3 Micromax soil medium. Three seeds of MON 88302, th e conventional control or commercial reference varieties were planted in each pot at an approximate depth of ~ 0.25” (~ 0.6 cm). The pots were thinned to one pl ant per pot when all of the plants had approximately one true leaf. Pots were plac ed in watering trays with capillary mats and irrigated as needed. Fertilizer was applied approximately weekly at a rate of 75 to 100 parts per million (Peter’s 20-20-20). Plants were not treated with glypho sate herbicide.

H.2. Flower Collection

A total of three newly opened flowers were co llected from each plant. Anthers from each

flower were removed and transferred to a container labeled with the pot number and containing 400 μl of Alexander’s stain so lution (Alexander, 1980). The stain solution was prepared on the day anthers were collected w ith a final concentration of lactic acid of 0.15%. Each tube was gently agitated with a vortex for approximately 30 seconds to release the pollen to the stain solution and stor ed for at least 24 h at approximately 4 °C prior to preparati on and analysis.

H.3. Pollen Sample Preparation

Pollen samples were prepared in a laboratory using microscope slides labeled with the pot number. The pot number identifier was cr oss-referenced with the study notebook to verify the original entry. A 20 μl aliquot of the pollen/stain solution was then added to the slide and covered with a cover slip.

H.4. Data Collection

Pollen samples were assessed for viabilit y, pollen grain diameter and general morphology. Samples were viewed under an Olympus Provis BX51TRF light/fluorescence microscope equipped with an Olympus DP70 digital color camera. The microscope and camera were connected to a computer runni ng Microsoft Windows 2000 Professional (©1981-1999, Microsoft Corp.) and installed with associated camera software [DP Controller v1.2.1.108 and DP Manager v1.2.1.107, respectively (© 2001- 2003, Olympus Optical Co., Ltd.)] and imagi ng software [Image-Pro Plus v6.2.1.491 (© 1993-2007, Media Cybernetics, Inc.)].Monsanto Company 11-CA-233U 340 of 500 H.4.1. Pollen Viability

When exposed to the stain so lution, viable polle n grains had a round to oval shape and stained red to purple due to the presence of living cytoplasmic content. Non-viable pollen grains stained blue to green and appeared round to collapsed in shape, depending on the degree of hydration (Alexander, 1980). For each pollen sample, the number of viable and non-viable pollen grains was c ounted from a minimum of 75 pollen grains

from a random field of view under the micros cope. Dense clusters of pollen or pollen grains adhering to flower part s were not counted because they may not have absorbed the stain solution uniformly.

H.4.2. Pollen Diameter

Micrographs (400X resolution) of ten representativ e pollen grains from one sample per plant were taken and imported into the imaging software. The software was used to measure pollen grain diameter along two perpendicular axes for each selected pollen grain. The mean of the twenty diameter va lues was calculated for each pollen sample.

H.4.3. General Pollen Morphology

General pollen morphology of MON 88302, conventional control and commercial

reference varieties was observed from the micr ographs that were used for pollen diameter

measurements.

H.5. Statistical Analysis

Analysis of variance (ANOVA) was conducte d according to a randomized complete block design with five replicates using SAS (Version 9). The level of statistical significance was predetermined to be 5% ( α = 0.05). MON 88302 was compared to the conventional control for percent viable pol len and pollen grain diameter. MON 88302 was not statistically different when compared to the commercial reference varieties. A reference range for each measured characteri stic was determined from the minimum and maximum mean values from among the four commercial reference varieties. General pollen morphology was qualitatively assessed and not subjected to statistical analysis.Monsanto Company 11-CA-233U 341 of 500 Table H-1. Starting Seed for Pollen Morphology and Viability Assessment

Substance Substance Type Phenotype Monsanto Lot #

MON 88302 Test Glyphosate-tolerant 11263712
Ebony Control Conventional 11263709

Dekalb 71-30 CL Reference Conventional 10001852
InVigor 5020 Reference Glufosinate-tolerant 11225944 DK71-45 Reference Glyphosate-tolerant 11225379

46A65 Reference Conventional 11220680Monsanto Company 11-CA-233U 342 of 500 References for Appendix H

Alexander, M.P. 1980. A versat ile stain for pollen fungi, yeast and bacteria. Stain Technology 55: 13-18.Monsanto Company 11-CA-233U 343 of 500 Appendix I: Petitioner’s Environmental Report

I.1. Summary

Since its launch in 1997, Roundup Ready canola has been an excellent weed management tool for growers offering both a simple and cost-effectiv e solution for broad spectrum weed control. The current Roundup Ready canola product RT73 has re strictions in application rates and timing which make the ability to control some importa nt annual and perennia l weeds challenging. Because application of glyphosate is limited to the 6-leaf stage and earlier in current Roundup Ready canola, weed control can become a signi ficant issue when weather conditions prevent

herbicide treatment until after the 6-leaf stage. MON 88302 is a second generation glyphosatetolerant canola product that allows for a wider period of applic ation up to first flower instead of the 6-leaf growth stage and pr ovides tolerance that allows fo r a glyphosate application rate similar to the rates for glyphosate-tolerant maize and soybeans. The proposed maximum glyphosate application rate on MON 88302 is twice the currently labeled maximum application rate for RT73 and offers improved control of difficult weeds such as dandelion and Canada thistle. Monsanto has submitted a request for a review by the U.S. EPA of an amended label for use of glyphosate on glyphosate-tole rant canola, which incorporates the higher application rates and later timing of application. Under th e proposed expanded glyphosate use pattern with MON 88302, glyphosate residue levels fall within the U.S. EP A’s existing tolera nce of 20 parts per million (ppm) for canola. In accordance with U.S. FDA’s consultation policy (57 FR 22984- 23005), Monsanto has also submitted to the U.S. FDA a food and feed safety assessment and nutritional assessment summary for MON 88302.

This environmental report (ER), which has be en prepared to support an anticipated APHIS environmental assessment, evaluates two alterna tives: the “deregulation in whole” alternative

and the “no action” alternative. Under the deregul ation in whole alte rnative, MON 88302 and any progeny derived from crosses between MON 88302 and conventional Brassica species, and crosses of MON 88302 with other biotechnology-der ived canola that has been deregulated would no longer be a regulated article under 7 CFR Part 340 and would be widely available for planting. Under the no action alternative, MON 88302 would rema in a regulated article and plantings could be conducted under APHIS notificat ion or permit. Interstate movements and

field trials of MON 88302 have been conducte d under permits issued or notifications acknowledged by APHIS since 2005.

U.S. Production of Canola: Canola is a low erucic acid type of oilseed rape that has been produced in the U.S. since the 1980s, and is used primarily for edible oil and meal for feed. Before the development of canola, oilseed rape wa s unsuited for food or feed use because of the high erucic acid and high glucosinolate conten t. In 2010, approximately 1.45 million acres of canola were planted in the U.S. The major U.S. canola production area is in North Dakota and northwestern Minnesota. This production area in the U.S. is in the southern part of the major North American canola production area, which is pr imarily in Canada. This area is planted exclusively with spring canola, a type that is planted in spring and harvested in fall. In 2010, North Dakota and Minnesota produced 92% of th e 1.1 million-metric ton U.S. canola crop, with

almost all of this in North Dakota. After the North Dakota/Minnesota area, the highest 2010 canola production was in Oklahoma (4% of U.S. production in 2010). The southern plains production area uses winter canola varieties, which are planted in the fall and harvested in lateMonsanto Company 11-CA-233U 344 of 500 spring or early summer. The remaining 4% of U.S. production is both wi nter and spring canola grown primarily in Idaho, Montana, Oregon, Co lorado, Kansas and Washington. Winter canola has higher yield potential than spring canola, but can be plante d only where the plants can survive the winter. Although Brassica rapa and Brassica juncea varieties are also available and can be used to produce canola quality oil, almost all U.S. canola production is from Brassica

napus varieties. U.S. production of canola is far s hort of U.S. demand, and the shortfall is made up primarily by imports of oil, seed, and meal from Canada. Biotechnology-derived canola varieties have been commercially available in the U.S. sin ce 1999. In 2008 approximately 95% of the North Dakota canola crop was produced us ing biotechnology-derived varieties containing a herbicide tolerance trait, with approximately 56% glyphosate tolerant and 39% glufosinate

tolerant.

Canola Seed Production: Approximately 5,000 acres is needed for production of the U.S. canola seed crop. Most canola seed gr own for sale for crop production fo r the North American market is produced in the summer in a relatively small geographic area in southern Alberta, Canada and the northwestern U.S. states of Washingt on, Oregon, Idaho, North Dakota and Colorado.

Specialty and Organic Canola Production: Specialty canola includes hi gh erucic acid varieties used in industrial processes, organic, and vari eties with improved fatty acid profiles including high oleic and high oleic, low linoleic varieties. Based on the most recent USDA Census of Agriculture, which was conducte d in 2008, there were approximat ely 232 acres of organic canola grown in the U.S., all grown in four states, none of which are ma jor canola production states. As noted above, the vast majority of canola curren tly produced in the U.S. contains a herbicidetolerant trait.

Other Brassica Production: Because of potential for cros s pollination between canola ( B. napus )

and related Brassica species, this ER describes other Brassica crop and seed production. Some of these crops are organic; however, the av ailable information on acreage and areas of production does not different iate areas of organic a nd non-organic production.

The primary Brassica vegetable crops in the U.S. are B. napus (rutabaga and Siberian kale),

Brassica oleracea (cabbage, broccoli, cauliflower, Brusse ls sprouts, collard, kale, kohlrabi), B. juncea (red giant mustard, sawtooth mustard, and others) and B. rapa crops (Chinese cabbage, pak choi, choi sum, turnip, mizuna, mibuna, tat soi and others). While Brassica vegetable crops are grown throughout the U.S., they are harveste d before they produce se ed, and are therefore not able to cross pollinate with other Brassica species.

The majority of the Brassica vegetable seed production occurs in three distinct areas: the Willamette Valley in Oregon; Skagit County in we stern Washington; and the Columbia Basin in eastern Washington. To maintain the desired ve getable seed quality, these areas have strict regulations or prohibitions on growing canola and other oilseed rape crops. Brassica vegetable seed crops are also produced in California and Arizona, and brocco li seed is grown primarily in California and Arizona. According to the 2007 Ce nsus of Agriculture, canola was grown on 16 acres in California and was not grown in Arizona.

Brassica and related field crops grown for seed in a ddition to canola include high erucic acid or industrial oilseed rape, mustard seed and biofum igant mustard seed. High erucic acid oilseed rape is grown on very limited acres in Washi ngton, Oregon, and Idaho. Th ese states all regulateMonsanto Company 11-CA-233U 345 of 500 where canola and oilseed rape can be grown, to pr event cross pollination. The great majority of mustard seed grown in the U.S. is yellow mustard ( Sinapsis alba ); only very small acreages of

Brassica mustard is grown.

Agronomic Practices in Canola Including Weed Management: Canola productio n typically involves the extensive use of agronomic input s and cultivation pract ices to maximize productivity and grower profitabili ty. Weeds are one of the primary pests that limit productivity.

The major weeds in canola are wild mustard, Canada thistle and wild oat, with volunteer cereal, flixweed, wild buckwheat, perennial sowthistle , dandelion and quackgrass also contributing to yield loss. The majority of canola acres are pl anted with glyphosate- a nd glufosinate-tolerant varieties and application of in-crop herbicides plus tillage practi ces are used to control weeds. In combination with appropriate agronomic practices , herbicide-tolerant ca nola volunteers can be controlled in the subsequent rotational crop with a vari ety of herbicides.

While some of these weeds have developed resist ance to other herbicid es in canola production states, no glyphosate-resistant popula tion of these weed species has been reported in the major U.S. canola production area in North Dakota/Mi nnesota. The three- to four-year rotations commonly used for canola (recommended primarily for disease control), the fact that other

herbicide modes of action are used in the rotati onal crops and that most rotational crops do not have glyphosate-tolerant varieties, plus the option of glyphosate- or glufosinate-tolerant canola

varieties all combine to minimize the potential for development of glyphosate resistant weeds.

Interactions between Canola and the Environment

Canola is grown in highly manage d agricultural settings and can interact with the environment directly, via pollen movement to sexually comp atible plants and potential hybridization/gene introgression, or via persisten ce of canola plants themselves in non-agricultural settings.

Cross Pollination Potential – Other Canola Crops: Canola is predominantely self-pollinating, although interplant (plants are touching one anot her) cross pollination can occur at a rate of approximately 30%. Most canola pollen (98%) trav els less than 12 meters from its source. However, canola pollen dispersal due to wind and insects can occur over greater distances at low frequency.

Cross Pollination Potential – Brassica Vegetable Species: In areas where plants are allowed to flower, canola could cross pollinate with B. napus vegetables such as rutabaga and Siberian kale. Cross pollination with B. rapa and B. oleracea vegetables is also possi ble, but less likely to
occur. However, B. napus

, B. rapa and B. oleracea vegetables are not considered weedy, and are generally harvested prior to flowering mini mizing cross pollination, hybridization and seed formation. Additionally, cross pollination between canola and vegetable seed crops, whether organic or non-organic, is expect ed to be non-existent or negligib le given that there are strict controls on canola prod uction in the major Brassica vegetable seed production areas (Oregon and Washington). In other Brassica vegetable seed production ar eas (California and Arizona), there is negligible, if any, canola production or canola seed production.

Cross Pollination Potential – Native Brassica: No native Brassica species have been identified in North America; therefore there is no poten tial for cross pollinati on to native species.Monsanto Company 11-CA-233U 346 of 500 Cross Pollination Potential – Naturalized or Weedy Brassica 2 : There are several Brassica weed species in the U.S. and Canada, including seve ral that are common in canola production areas; however, none of these are B. napus , and the potential for introgression between B. napus canola and all other species except B. rapa is expected to range from ve ry low to extremely low. Cross pollination between canola and B. rapa is rare, and gene introgression has been documented in only one case to our knowledge; in that case th e hybrid population declined rapidly and was gone in a few years.

Ruderal Canola: Brassica napus is regarded as an opportunistic species; that is, a species adapted to take advantage of temp orary conditions such as disturbe d areas. It is not considered to be a colonizing or invasive sp ecies capable of establishing in undisturbed natural ecosystems. Therefore, if canola seeds, which are very small, are lost from transport vehicles, canola may be found growing at the edge s of roadways and other transporta tion routes. Multiple studies of

roadside or ruderal canola have found that canola populations generally persist only for a year or two, and are usually found within a few feet of a roadway in disturbed so il not yet colonized by grasses and other more competitive plants.

Public Health, Worker Sa fety and Animal Health: The primary food use for canola is canola oil, which has well established heart health benefits. Canola meal is used as an animal feed source and the canola plant has lim ited use as a forage crop. Pesticide use on canola including glyphosate is regulated by the U.S. EPA, whic h registers specific uses after finding no

unreasonable adverse effects to human health and the environment resulting from that use. Glyphosate has been registered for use in canol a production since the intr oduction of the first glyphosate-tolerant product RT73 in 1999.

Animal, Plant and Microbial Communties: The affected environment for growing canola plants can generally be considered the agroecosystem (managed agricultural fields) plus areas extending beyond the intended plantings that mi ght be affected by agricultural operations.

Canola can interact with the environment direct ly, via pollen movement to sexually compatible plants or via persistence of canola plants themselves in non-agricultural settings.

Animals that consume the canola seed or plant, inhabit the fields, prey on the small animals inhabiting the field, or live in streams draini ng the canola field are part of the affected environment. Plants on adjacent land may be aff ected by fertilizer runoff, water runoff, and/or herbicide drift. Threatened or endangered an imal or plant species that may inhabit the surrounding area are also a part of the affected environment. Signifi cant variation in soil microbial populations within and among agricultural fields is also expected due to fertilization

and cultivation.

Land Use: Acreage planted in canola in the U.S. has in creased in recent year s, and may continue to increase based on U.S. demand exceeding U.S. production. Recent increases in canola production have resulted primarily from canol a replacing wheat in wheat monocultures or cropping systems in which wheat is the primary cr op, and this trend may continue. While canola

2 As defined by the USDA Plant Database. A naturalized plant is any non-native plant that can survive on its own, and includes

both weeds and non-weeds.Monsanto Company 11-CA-233U 347 of 500 has potential as a biodiesel crop, soybean oil is cu rrently substantially less expensive than canola oil in the U.S., limiting that potential use for canola.

Surface Water and Groundwater Quality: Surface water may be impacted from canola production by pesticide and herbicide runoff from canola fields. Based on the states’ water quality reports to the U.S. EPA, pesticides and herbicides are relatively minor contributors to impairment of surface water in the U.S. compared to sedimentation and turbidity. Additionally, researchers have found significan t increases in conservation til lage with the introduction of

herbicide-tolerant canola.

Economics: Canola was planted on approximately 1.45M acres in the U.S. producing 1.1 million

metric tons or 1.9% of the world’s 60.6 million me tric tons of canola in the 2010 growing season with a value of approximately $487 million. In the same year the U.S. imported 0.7 million metric tons from Canada. Total global U.S. imports of canola oil continue to increase steadily

from 0.5 million metric tons in 2000 to 1.2 million metric tons in 2010.

Environmental Consequences and Comparison of Alternatives: Table I-1 presents a summary of the potential impacts associated with selection of either of the alternatives evaluated in this

ER.Monsanto Company 11-CA-233U 348 of 500 Table I-1. Summary of Pote ntial Environmental Impacts

Attribute/Measure No Action Alternative Deregulation in Whole

Commercial Canola

Production Glyphosate-tolerant canola already planted

and traded for over a

decade Same as no action; glyphosatetolerant canola will continue to

be traded; glyphosate residue levels will fall within existing

tolerance Canola Seed Production Seed production practices well established; cross pollination from B. napus possible Same as no action; no change anticipated in seed production practices; B. napus cross pollination possible

Specialty Canola Production ( i.e ., organic, modified oil, etc.) Seed production practices in place; cross pollination from B. napus possible Same as no action; no changes anticipated in specialty canola production; cross pollination

from B. napus possible

Other Brassica Crop and Seed Production Including

Brassica Vegetables Seed and crop production practices in place; cross pollination from B. napus unlikely Same as no action; no changes anticipated in Brassica crop and seed production including vegetables ; cross pollination

from B. napus unlikely Agricultural Practices Gr owers will continue to use glyphosate-tolerant

and other herbicidetolerant canola Same as no action for cropping practices, disease and insect management and volunteer

management; growers may use a higher rate of glyphosate for weed management (comparable to rates used on maize, cotton, soybean) and a wider window of application Cross pollination and ruderal canola populations Cross pollination occurs in canola fields planted in close proximity; ruderal populations are

short-lived unless the seed bank is replenished Same as no action: no change in cross pollination from B. napus or establishment of populations

of ruderal canola

Human Health Canola oil will continue to be widely consumed for its health benefits; exposure to glyphosate from glyphosate-tolerant

canola will continue MON 88302 oil composition comparable to conventional control; exposure to glyphosate

increases, (but comparable to current maize, cotton, soybean levels)Monsanto Company 11-CA-233U 349 of 500 Table I-1. Summary of Potential Environmental Impacts (continued) Attribute/Measure No Action Alternative Deregulation in Whole

Plant and Animal Communities Including

Threatened and Endangered Species (TES) Exposure of

communities and TES to glyphosate will continue

at a level U.S. EPA has determined is acceptable

in terms of human health

and environmental impact Exposure to glyphosate in canola may increases, (but comparable to current maize, cotton, soybean levels) which U.S. EPA has determined are acceptable in

terms of human health and environmental impact

Soil Microbes Soil microbes will continue to be exposed to glyphosate Exposure to glyphosate in canola may increases, (but comparable to current maize, cotton, soybean levels); no change in soil microbial populations anticipated Land Use Glyphosate-tolerant canola (RT73) will continue to be grown on

land devoted to crop production; acreage is driven by demand Same as no action; growers may replace RT73 with MON 88302 or other deregulated events; overall canola acreage not expected to change

Climate (air, water) Growers will continue to use glyphosate-tolerant

and other herbicidetolerant canola; glyphosate will continue

to be used with glyphosate-tolerant

canola May increase glyphosate use (but comparable to rates used on

maize, cotton, soybean )

Non-crop and Nonagricultural Areas Growers will continue to use glyphosate-tolerant

and other herbicidetolerant canola; areas

will continue to be exposed to glyphosate

drift; non-persistent populations of feral

canola will continue to

be established May increase exposure to glyphosate (but comparable to

rates used on maize, cotton, soybean), which U.S. EPA has

determined are acceptable in

terms of human health andenvironmental impact; nonpersistent feral populations of

canola will be established

Economic Growers continue to use glyphosate-tolerant

canola and derive

economic benefit due to improved weed control Same as no action; growers will

use MON 88302 and derive

similar economic benefits from improved weed controlMonsanto Company 11-CA-233U 350 of 500 Table I-1. Summary of Potential Environmental Impacts (continued) Attribute/Measure No Action Alternative Deregulation in Whole

Cumulative Impacts Glyphosate-tolerant canola and glyphosate will continue to be used; stacked glyphosate and

glufosinate-tolerant canola varieties may be developed Same as no action with t he exception that more glyphosate may be used (but comparable to

what is used on maize, cotton and soybean), on the 1-1.5M

acres in the U.S. currently devoted to canola productionMonsanto Company 11-CA-233U 351 of 500 I.2. Background and Rationale

Monsanto Company has developed a sec ond-generation glyphosate-tolerant canola product, MON 88302, designed to provide growers with impr oved weed control through greater flexibility and tolerance to higher ra tes of glyphosate herb icide application. MON 88302 produces the same 5-enolpyr uvylshikimate-3-phosphate synthase (CP4 EPSPS) protein that is produced in commercial R oundup Ready crop products, via the incorporation of a cp4 epsps coding sequence. The CP 4 EPSPS protein confers tolerance to the herbicide glyphosate, the ac tive ingredient in th e family of Roundup agricultural herbicides.

I.3. Purpose and Need for Action

I.3.1. Regulatory Authority

“Protecting American agriculture" is the ba sic charge of the U.S. Department of Agriculture's (USDA) Animal and Plant H ealth Inspection Service (APHIS). APHIS provides leadership in ensuring the health and care of plants and animals. The agency improves agricultural productivity and competitiveness, and contributes to the national economy and the public health. USDA asserts th at all methods of agricultural production (conventional, organic, or the use of gene tically engineered va rieties) can provide benefits to the environment, consumers, and farm income.

In 1986, the Federal Government’s Office of Science and Technology Policy (OSTP) published a policy document know n as the Coordinated Framework for the Regulation of Biotechnology. This document specifies three Fe deral agencies that are responsible for regulating agricultural biotechnology in the U.S.: USDA APHIS, the U.S. FDA, and U.S. EPA. Products are regulated according to their intended use and some products are regulated by more than one agency. The USDA, U.S. EPA, and U.S. FDA enforce agency-specific regulations to products of biotechnology that are based on the specific nature of each biotechnology-derived organism. Together, these agencies ensure that the products of modern biotechnology are safe to grow, safe to eat, and safe for the environment. APHIS regulates biotec hnology-derived organisms under the Plant Protection Act of 2000 (PPA). 3 U.S. FDA regulates biotec hnology-derived food products under the authority of the Federal Food, Drug, and Cosmetic Act (FFDCA). 4 The U.S. FDA policy statement concerning regulatio n of products derived from new plant varieties, including t hose genetically engineered, was pub lished in the Fe deral Register on May 29, 1992 5 . Under this policy, U.S. FDA uses a consultation process to ensure that human food and animal feed safety, nutri tion and other regulatory issues are resolved prior to commercial distribution of foods and feeds derived from new plant varieties. The U.S. EPA regulates plant-incorporated pr otectants under the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) and certain biological control organisms under

3 7 USC § 7701 et seq.

4 21 USC § 301 et seq.

5 57 FR 22984- 23005Monsanto Company 11-CA-233U 352 of 500 the Toxic Substances Control Act (TSCA). 6 Together, these agencies ensure that the products of modern biotechnology are safe to grow, safe to eat, and safe for the

environment.

In addition to its authority with respect to plant-incorporated protectants, the U.S. EPA also regulates the use of herb icides under FIFRA, and establ ishes tolerances for herbicide

residues on food under the FFDCA. In order to be registered as a pesticide under FIFRA, it must be demonstrated that when used with common practices, a pesticide will not cause

unreasonable adverse effects in the environmen t. Under the FFDCA, pesticides added to (or contained in) raw agricultural commodities generally are considered to be unsafe unless a tolerance or exemption from the requirement of a to lerance has been established. Residue tolerances for pesticides are esta blished by U.S. EPA under the FFDCA; the U.S.

FDA enforces the toleran ces set by the U.S. EPA.

I.3.2. Regulated Organisms

The APHIS Biotechnology Regulatory Service’s (BRS) mission is to protect America’s agriculture and environment using a science-ba sed regulatory framew ork that allows for the safe development and use of biotechnol ogy-derived organisms. APHIS regulations 7

, which were promulgated pursuant to auth ority granted by the PPA, regulate the introduction (importation, interstate movement, or release into the environment) of certain biotechnology-derived organism s and products. A bi otechnology-derived organism is considered a regulated articl e if the donor organism, recipient organism, vector, or vector agent used in engineering th e organism belongs to one of the taxa listed in the regulation (7 CFR § 340.2) and is also considered a plant pest. A biotechnologyderived organism may also be regulated under Part 340 when APHIS has reason to believe that the biotechnology- derived organism may be a pl ant pest or APHIS does not

have sufficient information to determine if the biotechnology-derived organism is unlikely to pose a plant pest risk (7 CFR § 340.1).

A person may petition the agency under 7 CFR 340.6, “Petition for Determination of Nonregulated Status” to evaluate submitted data and determine that a particular regulated article is unlikely to pose a plant pest risk, and, therefore, should no longer be regulated. The petitioner is required to provide inform ation under § 340.6(c)(4) related to plant pest risk that the agency may use to determine whether the regulated article is unlikely to present a greater plant pest risk than th e unmodified organism . A biotechnology-derived organism is no longer subject to the regul atory requirements of 7 CFR part 340 when

APHIS determines that it is unlik ely to pose a plant pest risk.

I.3.3. Petition for Determination of Nonregulated Status: Monsanto 88302

In July 2011, Monsanto Company (Monsanto) has submitted a petition to APHIS seeking a determination of nonregulated status for MON 88302 (Petition #11-CA-233U). As detailed in the petition, MON 88302 produc es the same 5-enolpyruvylshikimate-3-

6 7 USC § 136a et seq.; 15 USC § 2601 et seq.

7 7 CFR part 340Monsanto Company 11-CA-233U 353 of 500 phosphate synthase (CP4 EPSPS) protein that is produced in other Monsanto commercial glyphosate-tolerant crop products, including Monsanto’s current glyphosate-tolerant canola, RT73. The CP4 EPSPS protein confers tolerance to the herbicide glyphosate.

MON 88302 allows for a wider window of glyphosate application than RT73 and higher

rates of use. The extended window and hi gher rates of glyphosat e application with

MON 88302 result from an increased leve l of glyphosate tolerance in the male reproductive tissues of MON 88302, which is achieved through use of improved promoter sequences that regulate expressi on of the CP4 EPSPS coding sequence.

I.3.4. APHIS Action

Under the authority of 7 CFR part 340, APHIS has the responsibility for the safe development and use of biotechnology-derive d organisms under the provisions of the PPA. APHIS must respond to pe titioners that request a dete rmination of the regulated status of biotechnology-derived organisms, including genetically engineered crop plants such as MON 88302. If a petition for nonregulat ed status is submitted, APHIS must make a determination if the genetically engineered organism is not likely to pose a plant pest

risk.

MON 88302 has been field tested in the U.S. since 2005 under APHIS authority and oversight. Regulatory notific ations of these field tria ls acknowledged by APHIS are listed in the petition in Appendix A. These fi eld tests allow for evaluation in agricultural settings under confinement measures desi gned to minimize the lik elihood of the test crop’s persistence in the environment after co mpletion of the field trial. Under confined field trial conditions, data are gathered on multiple parameters and used by the developer to evaluate agronomic characteristics and product performance. In summary, the phenotypic, agronomic, and environmental interaction data we re evaluated to characterize MON 88302, and to assess wh ether the introduction of the trait in MON 88302 alters the plant pest potential compared to conventional canola. The evaluation, using a weight of evidence ap proach, considered the reproducibility, magnitude, and direction of detected di fferences (trends) between MON 88302 and the conventional control, and comparison to the ra nge of the commercial re ference varieties. Results from the phenotypic, agronomic, an d environmental interactions assessment indicated that MON 88302 does not possess weedy characteristics, increased susceptibility or tolerance to specific ab iotic stress, diseases, or arthropods, or characteristics that would confer a plant pest risk or a significant environmental impact compared to conventional canola. These data are also valuable to APHIS for assessing the potential for a biotechnology-derived pl ant to pose a plant pest risk.

As a Federal agency subject to compliance with the National Environmental Policy Act (NEPA), 8 APHIS must consider the potent ial environmental effects of its

actions/decisions and reasonabl e alternatives to those acti ons, consistent with NEPA regulations 9 and the USDA and APHIS NEPA implementing regulations and

8 42 USC §4321 et seq.

9 40 CFR Parts 1500-1508Monsanto Company 11-CA-233U 354 of 500 procedures. 10 This environmental report (ER) has been prepared to support an anticipated environmental assessment to be prepared by APHIS.

I.3.5. Other Regulatory Submissions

In accordance with the U.S. FDA’s consulta tion policy (discussed in Petition Section I.C.1.), Monsanto submitted a food and feed safety assessment and nutritional assessment summary to U.S. FDA for MON 88302 in Ma rch 2011. In the first quarter of 2011, Monsanto also submitted a request for a joint review by the U.S. EPA and Health Canada’s Pest Management Regulatory Agency (PMRA) of an amended label for use of glyphosate on MON 88302 glyphosate-tolerant canola, which incorporates higher application rates and later tim ing of the application. The glyphosate residue levels for MON 88302 fall within existing tolerances. 11

As indicated herein, although certain issues related to the usage of glyphosate in conjunction with biotechnology- derived canola such as weed resistance and potential impacts of glyphosate on animals or plants are addressed by the U.S. EPA based on its authority under FIFRA, this ER also discusses those issues.

I.4. Affected Environment

I.4.1. Commercial Canola Production and Use

While canola oil can be derived fr om any one of three species: B. napus, B. rapa, and B. juncea (U.S. FDA, 1988; U.S. FDA, 2000; OGTR, 2008)

, most canola oil is derived

from Brassica napus . Oilseed rape is a member of the mustard (Brassicaceae) family, and has been cultivated by ancient civilizations in Asia and the Mediterranean primarily for its use as oil in lamps (Colton and Sykes, 1992). Later B. napus oil was used as an industrial lubricant, and today there is stil l demand for high erucic oil in a variety of industrial applications.

Until recently, the presence of the naturally occurring toxicants, erucic acid in the oil fraction and glucosinolates in the meal has ma de oilseed rape oil and meal derived from B. napus unattractive for human consumption a nd as an animal feed, respectively, particularly in western countri es. High erucic acid oilseed rape oil (as much as 50% of total fatty acids) has been shown to have ca rdiopathic potential resulting in a weakening

of the heart muscle in experimental animals (Bozcali, et al., 2009; Chien, et al., 1983) and high levels of glucosinolates made oilseed rape meal unsuitable for use in animal nutrition because of anti-nutritional, goi trogenic (suppresses thyroid function), reproductive, and palatability problems (Fenwic k, et al., 1989). Ho wever, in the 1960s intensive breeding programs resulted in the development and introduction of low erucic acid or canola (Can adian o il, l ow a cid) varieties of oilseed rape (OECD, 2001; OGTR, 2008). At approximately the same tim e low erucic acid varieties of B. rapa were introduced (OECD, 2001). Slightly later, in the 1980s, low erucic acid varieties of

10 7 CFR § 1b and 7 CFR part 372

11 40 CFR § 180.364Monsanto Company 11-CA-233U 355 of 500 B. juncea were developed (CCC, 1999). However, B. napus varieties are the most commonly grown canola oil-producing varietie s in the U.S. (Boyles, et al., 2009).

In North America canola is grown primarily in prairie areas that ha ve high quality soil, but where shorter, drier gr owing seasons make maize a nd soybean production less attractive (Figure I-1). As shown in Figur e I-1, the major canola production region is primarily in Canada and exte nds into North Dakota.

I.4.1.1. Canola Types

There are generally three types of canola crop s that can be grown in the U.S. and the grower must decide which of these types is best suited for his ar ea and cropping system (Brown, et al., 2008). Winter canola is pl anted in the fall, ove rwinters, requires vernalization (winter-chilling) to produce flowers, and is harvested the following summer. Winter canola is generally produced in the Pacific Northwest, southern Great Plains and Midwest regions of the U.S. Ther e is a second type of winter canola that is planted in the fall and overwint ers, but does not require verna lization to produce flowers. This winter type is produced in the southeast region of the U. S. The third type is spring canola which is planted in the sp ring and is harvested in late summer of that same year. Spring canola is grown primarily in the north ern Great Plains states including North Dakota, South Dakota, Minnesota , Montana, Idaho, and also in Washington. Winter and spring canola varieties may require different ag ronomic practices and can be affected by different insect pest s and diseases.Monsanto Company 11-CA-233U 356 of 500 Figure I-1. Canola Growing Regions of Canada and the U.S.

Source: Canola Council of Canada http://www.canola-c ouncil.org/gallery/726/canol a_growing_region_map.aspxMonsanto Company 11-CA-233U 357 of 500 In a few areas, either crop may be grown. For example, Washington State University Extension staff reported in 2007 that 5,000 acres of irrigated winter and spring canola was grown in Washington, primarily in the Co lumbia Basin (Hang, et al., 2009). Winter and spring canola varieties requi re slightly different agronomic practices and can be affected by different insect pe sts and diseases (Brown et al ., 2008). Winter canola has a higher yield potential than spring canola (Boyl es et al., 2009) but can only be grown in areas with relatively mild winters.

I.4.1.2. Herbicide Tolerance

The great majority of the canola grown in Nort h America is herbicide- tolerant. In 2008 an estimated 95% of the canola grown in North Dakota, where approximately 88% of U.S. canola is grown, and 86% of canola gr own in Canada was herbicide-tolerant (USDA-NASS, 2011c; Zollinger, et al., 2009; CCC, 2010b). An additional 2% of the North Dakota crop in 2008 wa s tolerant to the herbicide imazamox. The imazamox tolerance was developed through conv entional breeding techniques.

In North Dakota in 2008, 56% of the canol a planted was glyphosate-tolerant and 39% was glufosinate-tolerant (Zollinger et al., 2009). Glufosinate-tolerant canola was commercially introduced in the U.S. in 1998 and glyphosate-tolerant canola was commercially introduced in the U.S. in 1999.

I.4.1.3. Uses

Canola oil is currently the world’s third larges t source of vegetable oi l after palm oil and soybean oil (ASA, 2010; USDA-ERS, 2010b). Canola oil appeals to health conscious consumers because it contains a low level (7%) , of saturated fatty acids which have been

shown to increase blood chol esterol levels; a high level (approximately 60%) of the monounsaturated fatty acid, oleic acid, which has been shown to reduce serum cholesterol levels; a moderate level (app roximately 20%) of linoleic acid, and an appreciable amount (approximately 10%) of alpha-linolenic aci d, relative to other oils, (CCC, 2010a) that are essential to human health and must be supplied in the diet. Canola seed is also processed into canola meal which is used as high protein animal feed. Canola meal is the second largest protein meal source produced in the world. However, it is relatively small compared to soybean meal. Global production of canola meal was 30.8 million metric tons in 2008/2009 compared to 151.6 million metric tons for soybean meal (USDA-ERS, 2010b).

I.4.1.4. U.S. Production and Demand

In 2009-2010 the European Union, China and Canada were the largest producers of canola with 36%, 23%, and 20% of world share, respectively (USDA-FAS, 2011c). The U.S. produced 1.9% (USDA-NASS, 2011a) of the world’s 60.6 million metric tons of canola in the 2010 growing season (USDA-FA S, 2011c), and imported 0.7 million metric tons from Canada (Statistics Canada, 2011). 12 Total global U.S. imports of canola oil

12 The USDA FAS reports do not distingui sh canola but report all rapeseed.Monsanto Company 11-CA-233U 358 of 500 continue to increase steadily from 0.5 milli on metric tons in 2000 (USDA-FAS, 2011a) to 1.2 million metric tons in 2010 (USDA-FAS, 2011b).

In 2010 canola was planted on approximately 1.4 5M acres in the United States producing 1.1 million metric tons of canola, with a value of approximately $487 million (USDANASS, 2011a; USDA-NASS, 201 1b). In a 2010 dollar-value comparison with other major crops, canola represents 0.7% of the value of the maize crop, 1.3% of the value of the soybean crop and 3.7% of the value of the wheat crop (USDA- NASS, 2011b). Figure I-2 shows U.S. canola production and demand from 1987 to 2009. Although canola production in the U.S. has increased dramatic ally since the 1980s, it has always been

short of demand. The U.S. shortfall is made up with imports primarily from Canada.

Figure I-2. U.S. Canola Oil Production and Demand

Source: USDA-ERS, 2010b.

Figure I-3 shows acres of U.S. canola planted steadily rose through the 1990s until they peaked between 2000 and 2002, and varied th roughout the 2000s. The planted acres in 2010 (1.45M) were also near the 2000 peak, however, production (metric tons) was substantially higher, reflecting a trend of ge nerally increasing yields since that time (USDA-NASS, 2011c).Monsanto Company 11-CA-233U 359 of 500 Figure I-3. U.S. Canola Harvested Seed Production 1991-2010

Source: USDA NASS 2011c.

Figure I-4 shows U.S. Canola production by state. As shown, the bulk of U.S. canola is produced in North Dakota and Minnesota. U.S. canola production is summarized in

Petition Tables VIII-1 and VIII-2.

The 2007 Census of Agriculture reports canola harvested acreage in 22 states in addition to those specified in Figure I-4. In twelve of these stat es, canola production occurred on 3 or fewer farms and acreage was not reported (Alaska, Arkansas, Illinois, Indiana, Iowa, New York, North Carolina, South Dakota, Ut ah, Vermont, Virginia and Wyoming). Harvested acres were reported for the following states (harvested acres in parentheses): Washington (10,449), Kansas (3,362), Wisc onsin (1,996), Colorado (1,757), Maine (1,364), Texas (486), Michigan (152), Pennsylva nia (108), Nebraska (95), and California (16) (USDA-NASS, 2009). 0 200,000 400,000 600,000 800,000 1,000,000 1,200,000 1,400,000 1,600,000 1,800,000 

### Canola Production in the United States, 1991

‐ 2010

Acres Planted

Production (Metric Tons)Monsanto Company 11-CA-233U 360 of 500 Figure I-4. U.S. 2010 Canola Harvested Seed Production by State (metric tons)

Source: USDA-NASS, 2011c. * See text for discussion of other states.

I.4.2. Seed Production for Crop Production

In addition to the need for fertile agricultural land, favorable conditions for canola seed production include dry conditions, access to irrigation, appropriate temperatures (measured as heat units that are determined by the maximum and minimum daily temperature through the growing season), and adequate distance from commercial canola production areas (Monsanto Company, 2009). Ba sed on these constraints, most of the canola seed produced for sale for crop produc tion for the North American market is produced in the summer in a small geographic area in southern Alberta, Canada and the northwestern U.S., with the bulk of the produc tion in the area around Lethbridge, Alberta (Monsanto Company, 2009; Bewley 2008). Among the areas of U.S. summer seed production are the Columbia Basin in east ern Washington, the Grand Ronde Valley in Union County in northeastern Oregon, the Magic Valley along the Snake River Plain in south-central Idaho, the Idaho Falls area in far eastern Idaho near the Wyoming border,

the Park River area of northeastern Nort h Dakota (and possibly nor thwestern Minnesota) in the Red River Valley, and the San Luis Va lley in south central Colorado (Bateman, Monsanto Company, Personal Communication, 2011; Bewley 2008).

To meet the demand for seed and to mini mize production risks, most seed companies have off-season seed production locations in the southwestern U.S. (Imperial Valley, California and Yuma Valley, Ariz ona) and/or Chile, with the Central Valley of Chile the main off-season location (Monsanto Company, 2009, Bateman, Monsanto Company, Personal Communication, 2011). Canola seed pr oduction is discussed in more detail in

Petition Section VIII.B.2.Monsanto Company 11-CA-233U 361 of 500 I.4.2.1. Canola Seed Needed for Crop Production

Based on an average canola seeding rate of five pounds per acre, approximately 7.5 million pounds (3,400 metric tons) of canola seed is required to plant the approximately 1.5 million acre U.S. crop (Monsanto Company, 2009). With allowances for seed losses due to weather, poor yields, quality issues, di stribution excess, seed returns and replants, approximately 5,000 acres of commercial seed production are needed to supply sufficient seed to plant the entire U.S. canola acreage.

I.4.2.2. Certified Seed Production

Many growers choose to use certified seed when planting their fields. Certified seed is seed of a known variety produced under stri ct state and federal seed certification standards to maintain varietal purity. Seed lots must also meet specified standards for other crop seed, inert matter, weed seeds, an d germination. All certified seed must pass field inspection and be sampled and pass labora tory testing before it can be labeled and

sold as certified seed. In the U.S. the stat e seed certifying agencies are often associated with land grant universities. These various state agencies along with certifying agencies

from other countries are members of the Association of Offici al Seed Certifying Agencies (AOSCA). The AOSCA coordinates the efforts of seed certification agencies, which, through the certification process, establish standards for genetic purity and seed quality (AOSCA, 2011).

AOSCA identifies three seed classes: 1) breed er; 2) foundation; and 3) certified. Breeder seed is seed directly controlled by the originating or sponsoring plant breeding organization or firm responsible for the main tenance of that variety. Foundation seed is first-generation seed increased from breeder seed and is handled to maintain specific varietal purity and identity. Certified seed is the progeny of breeder or foundation seed,

and is the class recommended for commercia l canola production. AOSCA standards for certified canola seed require all seed fields be inspected in the early flowering stage to

ensure the fields are isolated from other canola crop fields by at least 660 feet for the production of open-pollinated varieties and 2,640 feet for production of hybrid varietal seed (AOSCA, 2009). Inspections ensure that th e number of off-type plants and plants of other varieties do not exceed 1.5 plants pe r 10,000 canola plants. Plants of other Brassica crop species must not exceed 1 plant per 10,000 canola plants. The percentage of hybrid

seed shall not be less than 75%.

State seed certification standards and isolati on distances vary slightly from state to state

and can be more restrictive than the seed st andards of AOSCA (Table I-2). In states where canola seed is produced, foundation seed fields must be isolated from canola and other cross pollinated varieties by 1,320 feet to 2 miles, while certified fields must be isolated by 660 feet to 2 miles. In order to cultivate foundation canola seed, no canola or oilseed rape must have been grown on the land in the previous three to five years depending on the variety and state. This fiel d history restriction is utilized to limit the spontaneous germination and sprouting of s eeds left in the seedbank from previous canola or oilseed rape cultiv ation. All volunteer plants and noxious weeds must be

eradicated and definite boundaries to the field se t before field use.Monsanto Company 11-CA-233U 362 of 500 Note that Table I-2 does not in clude all standards for all states listed; different states have requirements not shown in the table.

The first stage or pre-foundation seed stage occurs in isolation tents for complete pollination control. Individual inbred plants are grown and tested to ensure the plants meet the desired genetic characteristics. Th e next stage or foundati on seed stage occurs in highly isolated open fields. During this stage seed lines are further tested to ensure the desired genetic characterist ics and purity are maintain ed under field conditions.

I.4.2.3. Production of Open-Pollinated and Hybrid Seed

Producing hybrid canola requires using a pollin ation control system to prevent unwanted self-pollination. Three primary pollination c ontrol methods used in North America: 1) the biotechnology derived male sterility syst em; 2) the NPZ MSL (Lembke) system; and 3) the INRA- ogura cytoplasmic male sterility (CMS) system (Stiewe, et al., 2010). The biotechnology-derived system uses tapetal cell-specific male sterility and fertility restorer genes to facilitate hybr id seed production (Mariani, et al., 1992). The MSL-system (Male Sterility Lembke) is a private system ow ned by NPZ/Lembke based on a spontaneous mutant selected in the NPZ nursery (Frauen and Paulmann, 1999). The INRA- ogura system uses a CMS and restorer gene transferred from Raphanus ( Buzza, 1995)

. The CMS system is composed of three lines, usually referred to as the A line (male sterile or female parent line), B line (maintainer line) a nd R line (restorer or male parent line). The A and B lines have similar genotypes, with the exception of the CMS gene, that confers male sterility to the A line. The B line has a cytoplasm which allows normal pollen production facilitating the breeding process (Bu zza, 1995). The first step in the hybrid seed production process involve s foundation seed cr ossing block where female parent Line A is pollinated by the genetically identic al Line B to produce sufficient quantities of Line A seed. The second step involves pollin ation of Line A with restorer Line R to produce fully fertile F1 hybrid seed (Thomas, 2003).Monsanto Company 11-CA-233U 363 of 500Table I-2. Certified Canola Seed StandardsCategory CharacteristicColoradoa(Hybrid/Nonhybrid Varieties) IdahobMinnesotac(Hybrid/Non-hybridVarieties)NorthDakotadOregoneField IsolationDistancesFields of any Variety of the Same Kind(feet) 2,640 / 660 660 2,640 / 660 660 2 milesFields of any Variety from AnotherKind(feet) NA / 660 330 NA / NA 660 NAFieldStandardsYears Field Has Not Produced Oilseedrape, Mustard and/or Canola 5 / 3 3 5 / 3 3 5Ratio of Plants - other varieties 1.5:10,000 / 1:500 NA 1.5:10,000 / 1:500 1:500 1.5:10,000Plants of otherBrassica& InseparableOther Crops 1:10,000 / 1:10,000 NA 1:10,000 / 1:10,000 1:500 1:10,000Seed StandardsPure Seed (Min) 99% / 99% 99% 99% / 99% 99% 99%Inert Matter (Max) 1% / 1% 1% 1% / 1% 1% 1%Other Crop Seeds (Max) 0.25% / 0.25% 40/50 kg 0.25% / 0.25% 0.25% 0.01%Weed Seeds (Max)33/kg / 33/kg 400/50 kg 0.10% / 0.10% 33/kg 0.25%Objectionable Weed Seeds(Max)211/kg / 11/kg 40/50 kg 31/kg / 31/kg 11/kg 2/sampleSclerotia bodies (Max)15/kg / 15/kg None 15/kg / 15/kg 15/kg NAGermination Rate (Min) 85% / 85% 85% 85% / 85% 85% 85%aColorado Seed Growers Association (CSGA, 2010).bIdaho Crop Improvement Association (ICIA, 2006).cMinnesota Crop ImprovementAssociation (MCIA, 2010).dNorth Dakota State Seed Department (NDLB, 2008).eOregon Seed Certification Service (OSU, 2008).Monsanto Company 11-CA-233U 364 of 500 I.4.2.4. Canola Seed Manufacturing

Canola seed is produced by several companies that produce and sell seed, such as Monsanto Company, Pioneer Hi-Bred Inte rnational, Bayer CropScience, Croplan Genetics, and Dow AgroSciences. Seed comp anies contract with growers to produce the specified amount of canola seed. Seed comp anies have processing facilities and/or a network of contractors to clean, condition, and bag the harvested canola seed as well as monitor and inspect all f acility processes.

For most of the major seed companies, the en tire seed production process is International Organization for Standardizati on (ISO) certified and; therefore, includes internal and external audits (ISO, 2009). The ISO standa rds represent an inte rnational consensus on good management practices with the aim of ensuring that the organization can consistently deliver excelle nt product or services.

While field operations and management practic es for producing canola seed are similar to those used for canola crop production, additional measures specific to seed production are sometimes needed and are described briefly below.

A seed production field should not have been planted with canola the preceding five years in order to avoid voluntee r canola plants and to ensure genetic purity. All seed stock is treated with fungicides and insect icides to protect seedlings from various seedling diseases and flea beetle. Very early planting into cold soil conditions should be avoided because this can result in poor emerge nce and uneven stands. Every effort must

be made to eliminate weeds in a seed field through the use of herbic ides, cultivation and hand weeding to prevent weed seed in the harvested canola seed. Roguing is performed on all seed production fields to remove volunteer canola and off-type plants prior to or during flowering. Fields are scouted frequent ly for insect pests and insecticides are applied when insect pest infestations reach economic threshold levels. Foliar-applied fungicides should be considered when disease infestations are predicted in the area. Honey bee hives are placed in the seed fiel ds to encourage pollination of the female parent line with pollen of the male parent line. Swathing (cutting the crop and placing it in rows directly on the cut stubble to facilitate drying rate, ensure even ripening and reduce the possibility of seed losses from wind and hail) and combining at harvesting should occur at optimum times to avoid seed shattering and damage to the seed. With large acreage of canola, swat hing should start when seed color change is approximately 20% to 25% and the majority of the crop can be cut at or near the optimum seed color. Swathing can begin as early as 15% seed color change (NDSU, 2005). Combining should begin when the seed moisture drop s below 10% and no gr een pods are visible (Boyles et al., 2009). Harvesting equipment must be adjusted to minimize or avoid seed damage and must be cleaned before enteri ng the seed fields to minimize genetic contamination. Certain handling equipment, su ch as auger elevators, should be avoided because they can increase seed damage.

Field inspections are vital to ensure canola seed meets seed certification requirements, ISO certification standards, regu latory standards and trait licensing agreement standards. Field inspections are conducted on seed production fields throughout the growing seasonMonsanto Company 11-CA-233U 365 of 500 to evaluate variety purity, ensure canola pl ants are developing pr operly and fields are maintained free of weeds, insects and diseases.

Production plant personnel make every effort to avoid mechanical damage to the harvested seed during the scre ening, cleaning and bagging proce ss. Specific methods are used to assure the genetic purity and identi ty of the seed is maintained throughout the handling and storage operation. Bin inspections and sample collections are conducted at storage locations at the plant to examine the physical characteristics of the canola seed and to ensure proper bin cleanout. Seed is inspected for appearance, disease, discoloration, seed coat, mechanical damage, inert matter and weed seed. Germination, hybridity and impurity are tested and quantif ied on all seed lots to verify acceptable levels and meet labeling requirements.

I.4.2.5. Variety Trials

Many varieties are available for each canola type and growers rely on variety performance trials conducted by universities and private companies across a number of locations and years to help assist them in making variety selections. The North Dakota State University (NDSU) Exte nsion Office, for example, ha s conducted variety trials for many years, and publishes the results each year . The 2010 trials incl uded 58 varieties: 49 glyphosate-tolerant varieties (representi ng ten different seed companies), five glufosinate-tolerant varieties (one seed company) and four imidazolinone (imazamox)- tolerant varieties (three seed companies). Researchers obtain and report the following data by variety and location, for growers to use in variety sel ection: first flower, flower duration, maturity, seed weight, seed yield, 3- year average for seed yield (if applicable), plant height, and lodging (Kandel, 2010).

I.4.3. Specialty Canola Production

Specialty canola products, whic h include high erucic acid, high oleic and high oleic, low linoleic as well as organic canola require identity preservation throughout the process from seed to end product, in cluding planting, harvesting, tr ansporting, processing, and marketing in order to preserve their value. While standard s eed production practices preserve variety identity through planting, se gregation in the remainder of the production process (including the need for special cleaning of harvesting and transportation equipment and custom crushes) adds increm ental costs that, for the production to be worthwhile, must be recovered in a pri ce premium for the end product (Phillips and Smyth, 2003).

In some cases, and particularly for organic production, specialty pr oduction is required which utilizes oil processing f acilities other than the very la rge, regional facilities where commodity canola is processed. Reproducing th e complex multi-step process used in the larger facilities would be cost-prohibitive on a small scale. Small-scale specialty canola, including organic, is generally processed usi ng a screw-type mechanical press (expeller). Expellers are available in a range of sizes su itable for commercial production to backyard

use.Monsanto Company 11-CA-233U 366 of 500 I.4.3.1. Organic Canola Farming

Organic farming operations as described by the National Organic Program (NOP), which is administered by USDA’s Agricultural Ma rketing Service (AMS), requires organic production operations to have distinct, defi ned boundaries and buffer zones to prevent unintended contact with prohi bited substances or products of excluded methods from adjoining land that is not under an organic production management plan. 13 Organic production operations must also develop and maintain an or ganic production system plan approved by an accredited certifying agent. This plan enables the production operation to achieve and document compliance with the Na tional Organic Standa rds, including the prohibition on the use of excluded methods. 14 Excluded methods in clude a variety of methods used to genetically engineer or ganisms or influence their growth and development by means that are not possible und er natural conditions or processes. The use of biotechnology such as that used to produce MON 88302 is an excluded method under the National Organic Program. 15

Organic certification involves oversight by an accredited certifying ag ent of the materials and practices used to produce or handle an or ganic agricultural produc t. This oversight

includes an annual review of the certified operation’s organic syst em plan and on-site inspections of the certified operation and its records. Although the National Organic Standards prohibit the use of excluded methods , they do not require testing of inputs or products for the presence of excluded methods. The presence of a detectable residue of a product of excluded methods alone does not n ecessarily constitute a violation of the National Organic Standards. The unintentiona l presence of the pr oducts of excluded

methods will not affect the status of an or ganic product or operation when the operation

has not used excluded methods and has taken reasonable steps to a void contact with the products of excluded methods as detailed in an approved organic system plan. Organic

certification indicates that organic production and hand ling processes have been followed, not that the product itself is “fr ee” from any particular substance. As USDA AMS has recently re-iterated in a policy me morandum, organic certification is process based. The NOP regulations do not allo w the use of excluded methods such as biotechnology; however, the in advertent presence of produc ts of biotechnology “does not

constitute a use because there was no intent on the part of the certified ope rator to use excluded methods” (USDA-AMS Organic, 2011).

Many crop management practices are sim ilar to those used by non-organic canola growers, except that organic growers may no t use synthetic fertilizers or pesticides. Because synthetic herbicides are not allowe d, organic growers use cover crops and/or tillage to control weeds (Myers, 2002).

Organic producers use producti on practices designed to pr event commingling of their crop with neighboring crops treate d with herbicides and other pe sticides (spray drift), or that may be using plant varieties produced by excluded methods (pollen movement).

13 7 CFR 205.202(c)

14 7 CFR Part 205.

15 7 CFR § 205.2.Monsanto Company 11-CA-233U 367 of 500 These well established practices include isol ation zones, use of buffer rows surrounding the organic crop, adjusted planting dates, and varietal selection (Kuepper 2006).

I.4.3.2. U.S. Organic Canola Production

Comprehensive nationwide data on organic prod uction in the U.S. was collected for the first time as part of USDA’s 2007 Census of Agriculture. 16 The USDA reported 8 farms in the U.S. in 2008 that produced organic canola, with 2 farms in Iowa, one in Michigan, 2 in New York and 3 in Washington. A total of 184 metric tons of canola with a value of $92,752 was harvested on 232 acres in 2008 (U SDA-NASS, 2008). To avoid reporting data for individual farms, the only state-sp ecific data reported was 8.5 metric tons harvested in Washington, with a value of $3,560. Based on the 2007 Census of Agriculture, there were only two farms in Iowa and two in New York that harvested canola (USDA-NASS 2009); these are likely the f our organic canola farms in those states reported in 2008. The census reported 5 farm s growing canola in Michigan and 56 in Washington, the other two states with or ganic canola producti on in 2008 (USDA-NASS 2009).

There is little specific information availabl e about organic canola pr oduction in the U.S. before the USDA 2008 organic census. USDA- ERS has data on certified organic oilseed acreage by state for 1997 and 2000 to 2008. Howeve r, only the larger organic crops (flax and sunflowers) are differentiated; canola is grouped in the “Oth er” category along with safflower and unclassified acreage (USDA-ERS, 2010a). 17 In 1997, the year glufosinatetolerant canola was introduced in the U.S. and the year before the introduction of glyphosate-tolerant canola, USDA-ERS repor ted 12,487 acres of “Unc lassified/other” organic oilseed production, of which 35% (4,411 acres) was in California and 39% (4,857 acres) was in Utah. Based on other sources, for both states, this unclassified/other acreage was probably mostly, if not entirely, safflower. California (994 acres) and Utah (3,454 acres) were the only two states with reported acreage for safflower in the 2008 organic census (USDA-NASS 2008). The State of California has data for organic oilseed production for jojoba, safflower, sesame, sunflower and “other” for 2000 to 2009 (Klonsky and Richter, 2007; Klonsky and Rich ter, 2011). California safflower acreage for that period ranged from zero to 4,875 acr es. For the period 2000 to 2009, safflower was by far the largest California organic oilseed crop, with jojoba second. The reported acreage for the “other” category was zero for 6 of the 10 years, and except for a high of 285 acres in 2002, ranged from 4 to 97 acres in the other years. If any canola had been grown in California during the reported time period, it woul d have been included in the “other” category.

While organic canola may have been grown in North Dakota in the past, we found only two North Dakota growers who previously grew it or considered growing it, both of them long-term organic farmers. One grower ha s a 3,500-acre farm in Windsor, North Dakota, that he has operated as or ganic since 1980 (Leopold Center for Sustained Agriculture

16 The organic survey was part of the 2007 census, but was done in 2008. Data were collec ted by mail, with a overall response rate of 87%. The mailing lis t was developed primarily through data from the 2007 Census of Agriculture.

17 USDA ERS data is obtained through USDA-accredited State and private certification groups.Monsanto Company 11-CA-233U 368 of 500 2000). The grower reported th at canola had been a “very go od crop” in his rotation, but that he had previously made a firm decision that he would not grow any crops on his farm that “have a counterpart that has a GMO crop.” Therefore, when GE canola was introduced in the U.S., he stopped grow ing canola (Kirschenmann, 2009). The other grower has a 640-acre farm in the Red Rive r Valley in eastern No rth Dakota and grows organic wheat, clover, sunflowers, rye, barle y, flax, alfalfa and crambe (an oilseed crop), which he has chosen to grown instead of canola. According to the grower, crambe is better suited for organic production than canola because it is resistant to diseases and tolerant of flea beetles (Bowman, 2002).

I.4.4. Other Specialty Canola Production

A fairly recent development in canola producti on is the developmen t of high-oleic canola varieties (CCC, 2006a), which produce an oil higher in monounsaturated fat and lower in polyunsaturated fat. High-oleic canola oil is more stable than commodity canola oil, allowing for a longer shelf life and greater heat tolerance than trad itional or commodity canola oil (CanolaInfo, 2007). High-oleic canola oil is so ld most commonly to food companies and food service operations (C anolaInfo, 2007). In the 2010 NDSU variety trials, five of the 58 varieties test ed were high-oleic (Kandel, 2010).

Canola products labeled “GMO-Free” have no detectable presence of genetically modified material, and may be specialty pr oducts, depending on how they are produced. Some products, such as oils, are labeled GMO-Free even though they may or may not have been made from genetically engineered crops. Since oil does not contain proteins no detectable levels of genetic material are present (North Dakota Organics, 2011).

I.4.5. Other Brassica Production

This section describes Brassica crops other than canola th at are grown in the U.S. Because of potential concerns with canola re lated to cross pollination and spread of disease among related species, this section fo cuses on seed production for vegetables and

Brassica crops grown for seed such as mustard.

I.4.5.1. Brassica Vegetable Production

The primary Brassica vegetable crops in the U.S. are B. napus (rutabaga and Siberian kale), B. oleracea (cabbage, broccoli, cauliflower, Br ussels sprouts, collards, kale, kohlrabi), B. juncea (red giant mustard, sawtoot h mustard, and others) and B. rapa crops (Chinese cabbage, pak choi, choi sum, turnip, mizuna, mibuna, tat soi and others). Also grown are radish, daikon, a nd arugula, which are not Brassica species, but are in the Brassicaceae family. All Brassica vegetables grown in the U.S. are harvested in the vegetative stage and are therefore not of c oncern for cross pollination with canola.

Brassica species represent a pproximately 6.8% of the total ve getable acreage in the U.S. (USDA-NASS, 2009). The 2007 Census of Agriculture reports 322,591 acres of

harvested Brassica vegetable crops in the U.S., with broccoli accounting for 40% of theMonsanto Company 11-CA-233U 369 of 500 harvested acreage and head cabbage (cabba ge) and cauliflower combined accounting for and additional 37% of the harvested acreage (USDA-NASS, 2009). 18 Broccoli is commercially grown in most states, with the majority (106,271 acres or 81%) in California. The next-highest production st ate in 2007 was Arizona (11,869 acres or 9% harvested), then Oregon with 1,410 acres or 1% harvested. A ll other states had less than 1,000 acres harvested (USDA-NAS S, 2009). The distributi on of cauliflower production is similar to that of broccoli, with 82% of harvested acres in Califor nia, and with Arizona and Oregon with the next highest production. California is also th e largest producer of cabbage (14,099 acres harvested or 17% of the total), just ahead of New York (13,618 acres). Florida, Georgia, North Carolina, Wisconsin and Texas are also major cabbage producers. California is also the leading producer of Brusse ls sprouts, Chinese cabbage and kale. Georgia is the leadi ng producer of collards and Mi chigan leads in turnips.

I.4.5.2. Brassica Vegetable Seed Production

Because of the wealth of literature a nd detail involved, the description of Brassica and related vegetable seed production is included as Attachment 1. The majority of the

Brassica vegetable seed production occurs in th ree distinct areas: the Willamette Valley in Oregon; Skagit County in western Washi ngton; and the Columbia Basin in eastern Washington (Bateman, Monsanto Comp any, Personal Communication, 2011). These areas have strict regulations or prohibitions on growing canola. Brassica vegetable seed crops are also produced in California and Ariz ona, and broccoli seed is grown primarily in California and Arizona. According to th e 2007 Census of Agriculture, canola was grown on 16 acres in California a nd was not grown in Arizona.

I.4.5.3. Other Brassica Crops Grown for Seed

Brassica and related field crops in addition to canola include high erucic acid oilseed rape, mustard seed and biofumigant mustard seed.

To meet industry requirements, industrial grad e oil from oilseed rape must contain at least 45% erucic acid. It is used primarily as a lubricant, an additive in plastic, and for birdseed (USDA-ERS, 1996). It was grown on 2,200 acres in the U.S. in 2010, with a total crop value of $975,000 (USDA-NASS, 2011b; USDA-NASS, 2011c). In 2007, USDA reported 1,060 acres harvested by 11 operato rs, 9 of whom were in Idaho. Oregon and Washington each had one operator. No breakdown of acreage by state was reported (USDA-NASS, 2009). Partly to prevent cross pollination between industrial oilseed rape and canola, these three states have impl emented regulations on where canola can and cannot be grown (Appendix Section I.4.6).

Mustard seed was harvested on approxi mately 48,000 acres in 2010 (USDA-NASS, 2011c). In 2007, mustard seed was grown in 10 states with North Dakota, Montana,

18 Other Brassica crops reported include Brussels sprouts, Chines e cabbage, mustard cabbage, collards, kale, mustard greens, turnips, and turnip greens, with harvested acr eages ranging from 66 acres (mus tard cabbage) to 11,480 acres (Chinese cabbage). Other ve getable crops in the Brassicaceae family in clude daikon (624 acres), horseradish (3,692 acres), radishes (14,599 acres) and watercress (679 acres).Monsanto Company 11-CA-233U 370 of 500 Washington and Idaho together accounting for 98% of the acreag e (61%, 25%, 9% and 3%, respectively). In North Da kota mustard is grown in rotation with small grains, but is

not recommended for close rotation with ca nola because of similarity of disease susceptibility (NDSU, 2007b). The most common type grown is yellow mustard ( S. alba ), which is used as condiment mustar d. Only small acreages of brown and oriental mustard ( B. juncea ) are grown; these are used for oils and spices (NDSU, 2007b).

Biofumigant mustard is used as a green manur e crop (crop is grown th en plowed into the soil at different times in the growing season) . As it decomposes it releases naturallyoccurring compounds that are toxic to some weeds, nematodes and fungi (McGuire, 2003). Biofumigant mustard species are the sa me as those used for mustard seed crops ( S. alba and B. juncea ).

I.4.6. State Restrictions on Brassica Crops

Oregon, Washington and Idaho all have some rest rictions (spatial, crop rotation, etc.) on growing canola and some other Brassica seed crops. No other states were found that

have state-based restrictions on growing canola and other Brassica crops. These

restrictions are described in this section. Pa rt of the reason for th ese restrictions is to prevent cross pollination betw een canola and industrial oils eed rape. There is little industrial oilseed rape grown in these states today; however, in the 1980s and early 1990s industrial oilseed rape acreag es were substantially higher (USDA-ERS, 1996). As

discussed in Attachment 1 these states all have Brassica and related vegetable seed production. Blackleg, a fungal disease common to Brassica and related species, is widespread in most canola producing areas, but has not been introduced in Oregon, Washington or Idaho. Thus the regulations are also intended to help keep blackleg out of these states. Finally, given the potential for cross pollination between sexually compatible Brassica species, these states have also adopted regulations to limit cross pollination between canola and Brassica vegetable crops in ve getable seed production

areas.

I.4.6.1. Oregon Restrictions

Since 1989, the Oregon Department of Agri culture has had the authority under Oregon

law to establish control areas for the production of oilseed ra pe (including canola) if it determines control is necessary for protection of “horticultural, agricultural or forestry

industries of the state from diseases , insects, animals or noxious weeds....” 19 The Department of Agriculture first implemented regulations with oilseed rape growing restrictions in 1992 for the purpose of preven ting cross pollination between canola (edible oil) and industrial oilseed rape. 20 The regulations were rewritten in 2005 “to account for

19 Oregon Revised Statues (ORS) §570.450 and §570.405.

20 Oregon regulations 603-052-0860(8) define rapeseed as follows: "Rapeseed" means plants of the species Brassica

napus , Brassica rapa and Brassica juncea , where seeds of high oil content are the economically valuable product. Included are the industrial seed types, with high erucic ac id levels and canola with low erucic acid content used for

edible oils.Monsanto Company 11-CA-233U 371 of 500 conflicts between specialty s eed production and canola [oilse ed rape] grown for biodiesel or edible oil.” The Depart ment of Agriculture made small modifications to the regulations in 2009 and will be reviewing th em again in 2012 (ODA, 2011). The fungal disease blackleg ( Leptosphaeria maculans ), which is a serious disease in the major North American canola production areas, is not present in the Pacific Northwest (Brown et al., 2008), and the regulations includes requirements for seed production (des cribed below) to help prevent blackleg’s introdu ction into Oregon. Long dist ance spread of blackleg is usually by infected seed (NDSU, 2004).

The regulations establish a “general produc tion area” and four “protected districts.” 21

General Production Area: All areas of the state not in a protected district are in the general production area. The following restrictio ns apply to all oilseed rape grown within the general production area:

• All oilseed rape seed that “trades with in commerce” must be certified by AOSCA and state standards, the seed must be certified to be free from blackleg and must be treated with a fungicide approved for blackleg control.

• Oilseed rape cannot be grown in the sa me plot more often than two years (nonconsecutively) in five.

• All unbagged loads of oilseed rape transpor ted through protected districts “must be in

enclosed bins or in contai ners lined and covered in a manner to prevent seed loss.” 22

Protected Districts: The Willamette Valley Protected District includes the Willamette Valley. The Central Oregon Protected Distri ct includes parts of Crook, Jefferson and Deschutes counties. The Northeast Oregon Prot ected District includes part or all of Baker, Union and Wallowa counties. The Ma lheur Protected Distri ct includes a 3 mile strip of northern Malheur County adjacent to the Idaho border (ODA, 2011). Within all the protected districts except the Northeast Di strict, oilseed rape pr oduction for seed or oil is prohibited except under sp ecial permit. Oilseed rape production for seed or oil is

allowed in the Northeast Distri ct without a permit. The same restrictions applicable to the general production area apply to production in the protect ed districts, except that oilseed rape can be grown on the same plot of land only once ever y four years. The following additional restrictions a pply to all protected districts: 23

• Oilseed rape “must be isolated from othe r crops with which it will cross pollinate, by a distance of not less than three mile s,” except that in the Northeast District,

the isolation distance is two miles.

• The locations of oilseed rape fields mu st be recorded with the Oregon State University County Extension Office, or in the Willamette Valley, with the Willamette Valley Specialty Seed Association (WVSSA), at least 10 days prior to planting.

21 Oregon regulations: 603-052-0850. Map is available on website. See Oregon Department of Agriculture 2011.

22 603-052-0880(1)

23 603-052-0880(2)Monsanto Company 11-CA-233U 372 of 500 • Forage and cover crop oilseed rape sh all not be allowed to flower.

• Any volunteer or uncontrolled oilseed ra pe “in and around production fields must be prevented from flow ering by the producer.”

• Violators may be fined or s ubjected to crop destruction. 24

I.4.6.2. Washington Restrictions

Since 2007, Washington state law allows the Stat e Department of Agriculture to establish

Brassica seed production districts, with restrictions on production of Brassica seed. 25

The law is broad and allows the Department of Agriculture to make specific restrictions. The State Department of Agri culture has established two Brassica seed production

districts. Seed Production Di strict 1 includes all or parts of Whatcom, Skagit, Snohomish, Island and Clallam counties (in western Washington) and Seed Production District 2 includes parts of Grant and Adams counties. 26

Within both Brassica seed production districts, all Brassica seed crops grown for seed or oil may be grown only through participatio n in the Washington State University Extension Center pinning process. Within bot h districts, seed must be transported in

“covered containers from which the seed ca nnot leak” and volunteers must be controlled “as soon as feasible but prior to pollen production or blooming.” 27

In Brassica Seed Production District 1, oilseed rape grown for s eed or oil is prohibited unless grown under the conditions of a Brassica production agreement. 28 A production agreement must be developed through the direct or of the State Department of Agriculture and an advisory committee and the agreemen t must contain “terms and conditions that are necessary and sufficient to mitigate reas onably possible risks to the economic wellbeing of growers within the Brassica seed production district from the proposed activity”. The agreement is subject to appeal by any grower or processor within the Brassica production district. 29

In Brassica Seed Production District 2, a 2-mile isolation di stance is required between

Brassica seed crops, except for certain situations where growers make specific written

agreements. 30 A part of Brassica Seed Production District 2 (2A) has an additional

restriction: Brassica seed crops “intended for oil or fu el production” may be planted only

under the conditions of a Brassica production agreement. 31

I.4.6.3. Idaho Restrictions

24 603-052-0880(6)

25 RCW 15.51.030

26 Washington regulations 16-326-010

27 Washington regulations 16-326-020

28 Washington regulations 16-326-030

29 RCW 15.51.40

30 Washington regulations 16-326-040

31 Washington regulations 16-326-050Monsanto Company 11-CA-233U 373 of 500 Idaho has had restrictions on oilseed rape production since 2005. The Idaho regulations

divide the state into seven districts. Within four of these districts, only edible oilseed rape (canola) production is allowed, within two only industrial oilseed rape production is allowed, and within one (D istrict IV) no oilseed rape production is allowed. 32 District IV covers Treasure Valley and includes Ada, Gem, Canyon, and Payette counties and part of Owyhee County, in western Idaho. Except for District IV, exemptions are allowed for planting the restricted oilseed rape with minimum one-mile isolation and written agreement from all adjacent farmers.

Volunteer oilseed rape growing outside of cu ltivated fields “shall be destroyed before flowering” by “the person responsib le for planting the oilseed rape.” 33 Idaho regulations require that any Brassica seed transported in Idaho be sealed or covered to avoid spillage. 34 Idaho also has requirements similar to Oregon’s to prevent the introduction of blackleg fungus into Idaho. 35

I.4.7. Agronomic Practices for Canola

A grower’s goal in canola pr oduction is to produce a crop with high yield, high oil content, low green seed content, low weed seed c ontent, and little seed left in the field, all while managing input costs. This section di scusses those practices that are within a grower’s control that help achie ve this goal. Agronomic pract ices are discussed in detail

in Petition Section VIII.

I.4.7.1. Production Costs

Managing input costs is a major component to the economics of producing a profitable canola crop. The key decisions on input costs include ch oosing what seed or canola varieties to plant, amounts of fertilizer to apply and what herbicide program to use. The average total income for producing canola in North Dakota was $250.89 per acre for the years 2005-2009 according to statistics comp iled by Farm Management Specialists (FINBIN, 2010). The total direct and overhead expenses were $179.52 and $27.44 per acre, respectively. Major direct per acre e xpenses were fertilizer ($43.56), land rent ($34.50), seed ($31.06) and crop chemicals ($22.69) . The net return for the five-year period averaged $43.94 per acre. With an average government payment of $11.33 per

acre the net return wa s $55.27 per acre. For more deta iled information see Table VIII-3

in the Petition.

I.4.7.2. Tillage

Canola can be grown with conven tional tillage, conservation til lage, or direct-seeded into small grain straw stubble in a no-tillage cropping system (Brown et al., 2008). The benefits of conservation tillage or no-ti ll systems in canola production include reduced

32 Idaho regulations 02.06.13 §50 and §100

33 Idaho regulations 02.06.13 §200

34 Idaho regulations 02.06.13 §250

35 Idaho regulations 02.06.13 §150Monsanto Company 11-CA-233U 374 of 500 soil erosion, reduced fuel and labor costs, co nservation of soil moisture, improvement of soil structure, reduction of soil compacti on and improvement of soil organic matter content (Brown et al., 2008).

Spring canola has provided good performance under conservation tillage while winter canola has generally performed poorly with conservation tillag e systems (Brown et al., 2008). Winter canola is not recommended when planting into excessi ve amounts of fresh straw, or when soil temperatures are lower than average in the fall months. This can result in poor emergence. Seed yields w ith direct-seeded winter canola have been significantly lower than with conventional tillage systems (B rown et al., 2008). Spring canola is better suited to conservation tillage due to better soil mo isture conservation and availability. Spring canola has fewer probl ems dealing with heavy straw residue and, residue from the previous crop has usually d ecomposed to some de gree during the winter months prior to planting spri ng canola. In add ition, cooler soil te mperatures can be advantageous to spring canola. The Conser vation Tillage Information Center (CTIC, 2008) reports that conservation tillage (no-till or mulch-till) is used on approximately

32% of the canola acres in North Dakota. Crop specialists at North Dakota State University indicate that the amount of direct-seeded or no -till canola in North Dakota

varies across the state based on rainfall. In the eastern or Red River Valley area where rainfall is relatively high, only about 5% is di rect-seeded into wheat straw (Kandel, NDSU, Personal Communication, 2010). The drie r, central area and ev en drier, western

area of the state are estimated to have approximately 80% and 100% of the acreage direct-seeded, respectively.

I.4.7.3. Planting

Canola is typically seeded in 6 or 7-inch ro ws with a grain drill or air seeder, with optimal seeding depths between one half to one inch. Canola is very susceptible to soil crusting, and the seedbed can easily be damage d by wind erosion. Seed and soil moisture contact is critical for rapid emergence (NDSU, 2007a).

I.4.7.4. Fertilizer

Effective nutrient management and maintenance of good soil fertility is essential for high yielding and high quality canol a. Nitrogen, phosphorus, pot assium, sulfur, and boron, depending on the region, are the most limiti ng nutrients for successf ul canola production (Brown et al., 2008). Soil sampli ng and testing is the first step in assessing soil nutrient levels and the requirements for supplemental fert ilizers. The availabi lity of soil nutrients is dependent on soil acidity or the pH level a nd must be included in this assessment. The ideal soil pH for growing canola is between 6.0 and 7.0. Canola yields will be adversely affected when the soil pH is below 5.5 (Brown et al., 2008).

Nitrogen is the most limiting of all plant nut rients in canola and sufficient nitrogen must be available to the plant at every growth stage. Supplemental nitrogen requirements for North Dakota are based on the yield potential of canola, nitrate nitrogen available in the soil at 0-24 inch depth, and the prev ious crop credit for nitrogen.Monsanto Company 11-CA-233U 375 of 500 Research studies have shown that nitr ogen recommendations can be capped at 150 pounds per acre in the cooler, moister areas of the state without impacting yield. In the drier, warmer areas of the state nitrogen rates can be capped at 120 pounds per acre. Canola is very sensitive to fertilizer salts and no more than 5 pounds of nitrogen per acre is recommended for placement with the seed at planting on medium-textured soils (Franzen and Lukach, 2007).

Canola has a moderate requirement for phosphor us and phosphorus fertilizer rates are based on soil tests. Phosphorus is not mobile in the soil and should be banded with the seed at planting or incorporated into the soil before planting (B rown et al., 2008). A starter fertilizer rate of 20-30 pounds of P 2 O 5 per acre is generally sufficient for most soil test levels unless the grower intends to bu ild up phosphorus levels in the soil (Franzen and Lukach, 2007). Potassium requirements ar e also based on soil tests. Many soils contain sufficient levels of potassium requi ring no potassium fertilizers. Potassium applications are not needed when the soil te st indicates 160 ppm or more of potassium (Franzen and Lukach, 2007). Potassium can also be applied as a starte r fertilizer with or alongside the seed at planting.

Canola has special requirements for sulfur and it is often the second most limiting nutrient in canola production (Bro wn et al., 2008). Sulfur defi ciencies result in yellowing between leaf veins, cupped leaves and stunti ng (Franzen and Lukach, 2007). In addition, in the presence of sulfur deficiency floweri ng is delayed, seed often does not set, and pods will be barren or poorly developed. North Dakota studies have demonstrated significant yield increases from sulfur appl ications (Franzen and Lukach, 2007). Since

soil tests tend to overestimate available su lfate and are highly variable, North Dakota specialists recommend 20 to 30 pounds of sulfur per acre when medium to low levels of

sulfur are detected and 10 to 15 pounds of sulfur per acre wh en high levels of sulfur are detected (Franzen and Lukach, 2007). The sulfur fertilizer should be in the form of ammonium sulfate, ammonium thiosulfate or potassium thiosulfate since canola takes up

sulfate sulfur.

Canola requires more boron than most other crops. Boron at 1 to 2 pounds per acre

should be broadcast when the soil tests s how less than 0.5 ppm boron (Brown et al., 2008). Canola has not shown yi eld responses to applications of micronutrients such as chlorine, copper, iron, manganese, molybde num or zinc (Brown et al., 2008).

I.4.7.5. Harvesting

The grower’s challenge is to harvest canol a during the very brief optimum stage when most seeds are mature but the pods are not so dry that they will shat ter. The brevity of this optimum stage is one of the factors limiting canola production (USDA-ERS, 2010b). Harvesting too early results in higher green s eed content. Green seeds increase refining costs and thus reduce seed value (Brown et al ., 2008). Shattered pods result in seed loss

and volunteer canola in subsequent crops.

Most canola is harvested using one of two methods: 1) direct combining of standing canola, and 2) swathing followed by combining. In the latter method, the canola swath isMonsanto Company 11-CA-233U 376 of 500 allowed to cure and ripen for a minimum of 10 to 14 days before harvesting which hastens maturity and avoids frost damage in areas with a short grow ing season (Boyles et al., 2009). Direct combining is generally r ecommended for winter canola in the southern Great Plains region because dry-down is accel erated by high air temp eratures during seed ripening (Boyles et al., 2009).

Canola seed is sensitive to heating in st orage and must be stored under cool, dry conditions (Boyles et al ., 2009; NDSU, 2007a).

I.4.7.6. Irrigation and Water Use

Canola has similar moisture requirements as sm all grains, but is less tolerant of drought conditions. However, according to the US DA Census of Agriculture, only 13,535 or 1.2% of canola acres were irrigated in the U.S. in 2007 (USDA-NASS, 2009).

I.4.7.7. Insect Pest Management

Insect pests can reach infestation levels that cause yield re ductions and often a corresponding reduction in the oil content of canola. Some aphids act as vectors for plant viruses (Brown et al., 2008). Canola seedli ngs are especially vulnerable to chewing

insects. A severe infestati on of flea beetles can complete ly destroy a stand of canola seedlings. A firm seedbed and adequate fer tilizer will help plants outgrow the beetle damage during the seedling stage (NDSU, 2007a). Late season insect pests can typically cause yield losses of 20 to 50% in canola when left uncontr olled (Brown, et al., 2004). Damage from insect pests is more severe du ring periods of stress, especially drought stress. Spring and winter canola types are imp acted by the same insect pests. However, some pests negatively impact one type more than the other.

Flea beetle ( Phyllotreta cruciferae ) is a serious insect pest in spring canola wherever it is grown in the U.S. (Brown et al., 2008). This in sect pest is less important in winter canola unless the winter crop is planted very early or in regions where winter and spring canola is grown in the same area. An insecticide seed treatment (imidacloprid or clothianidin) may be needed in spring canola to protec t canola during the seedling stage (NDSU, 2007a). Minimum or no-tillage systems generally have lower infestations of flea beetles (Weiss, et al., 2009). In addition, planting ear ly can allow establis hment of the canola prior to beetle emergence.

Insect pests causing serious dama ge to canola in the southern Great Plains in addition to flea beetle include grasshoppers ( Stethophyma sp .), army cutworms ( Euxoa sp. ), diamondback moth larvae ( Plutella xylostella ) , aphids ( Myzus persicae, Lipaphis
erysimi ), and root maggots (Boyles et al., 20 09). Aphids have become the most important insect pest of canola in this region.

Crop rotation, controlling volunteer canola a nd wild mustard plants and managing crop residue are all important cultural pr actices for insect control.

Insect Pests and Herbicide-Tolerant Canola: In a three-year study (2000-2002) in

southwestern Canada research ers compared the results of insect damage in glyphosate-Monsanto Company 11-CA-233U 377 of 500 tolerant, glufosinate-tolerant and conventional canola. Inse ct damage from three pests, the flea beetle ( Phyllotreta cruciferae ), the cabbage seedpod weevil ( Ceutorhynchus

assimilis ) and the lygus bug ( Lygus sp. ), was documented. Researchers found no differences in insect damage among the three cultivars (Cárcamo and Blackshaw, 2007).

I.4.7.8. Disease Management

Disease management is discussed in detail in Petition Section VIII.E. Plant diseases can be a serious problem in canol a production. Diseases attack canola at all stages of development, can be soil borne, seed borne, or airborne and also can spread from infected crop residue (Boyles et al., 2009).

Blackleg ( Leptosphaeria maculans ) and Sclerotinia stem rot ( Sclerotinia sclerotiorum ) are serious diseases in spring and winter canola. Other diseases that affect canola include Alternaria black spot ( Alternaria spp .), downy mildew ( Peronospora parasitica ), powdery mildew ( Erysiphe cruciferarum ), black rot ( Xanthomonas campestris), and aster yellow (Boyles et al., 2009; NDSU, 2005). Di seases are managed primarily through use of genetics, seed treatment, chemi cal application and crop rotation.

Blackleg: Blackleg a fungal disease caused by Leptosphearia maculans, first detected in Saskatchewan in 1975, is a serious dis ease in the major North American canola production areas (SMA, 2009; NDSU, 2004). Th e blackleg fungus is present in most

canola areas in North Dakota and is most common in the northern part of the state (NDSU, 2004), as it is in the western Cana dian provinces. It is less common in Minnesota (Lamey, et al., 2003). Highly aggre ssive strains are present in Saskatchewan, Manitoba, and in some parts of North Dakot a (NDSU, 2004). Black leg was first reported in Oklahoma in 2009 (Dominiak-Olson, et al., 2009). It is not present in the Pacific Northwest (Brown et al., 2008).

Canola varieties currently available for plan ting in North Dakota have been bred for resistance to blackleg (Kandel, 2010). Canol a seeds should be treated with a fungicide that is effective in controlling blackleg (Dominiak-Olson et al., 2009; NDSU, 2004). While the fungus can spread via spore migratio n from adjacent fields and from infected seeds, a study showed that most infection come s from infected residue in the field itself (NDSU, 2004). To prevent spread to a new crop, NDSU Extension specialists recommend using a four year rotation with blackleg suscep tible canola (NDSU, 2005). Oklahoma State University Extension specialists recommend three to five year rotations (Dominiak-Olson et al., 2009). Long-term studies have found that 3- to 4- year rotations and genetic resistance are the most effective means to control blackleg (TopCropManager, 2011b). For crop rotations to be effective, plants that can harbor blackleg, such as volunteer canola and wild mu stard, must also be controlled (NDSU, 2004).

Sclerotinia Stem Rot (SSR): Sclerotinia stem rot ( Sclerotinia sclerotiorum ) is one of the

most serious and common diseases of canola in the northern Great Plains. Wet weather conditions around flowering faci litate fungal infection (Mar kell, et al., 2009). SSR occurs in canola, dry beans, soybeans, peas, lentils and chickpeas, but does not occur inMonsanto Company 11-CA-233U 378 of 500 members of the grass family, including small grains and maize. It is also present in the Great Plains, but its impact has been mini mal (Boyles et al., 2009). Crop rotation is important in managing the disease, and a pr eferred rotation would have canola planted after two or more nonsusceptible crops (Boyl es et al., 2009; Markel l et al., 2009). Many broadleaf weeds, including lambsquarters, Canada thistle, ragweed and marsh elder are also susceptible host plants (Lamey and Meronuck, 1999).

Fungal Disease and Glyphosate Use: S cientific evidence indicat es that at recommended use rates, glyphosate-based herb icides applied in conventio nal or glyphosate-tolerant cropping systems do not result in increased susceptibili ty to diseases (Baley, et al., 2009; Lee, et al., 2000; Njiti, et al., 2003); Sanogo, et al., 2001). This observation has been reinforced through several year s of commercial experience and results of grower surveys which indicate that the overall performance of glyphosate-tolerant crops, as measured by yield and constant increase in acreage, is e qual to or greater than that of conventional

varieties.

Scientists from Purdue University Extens ion recently published an article assessing whether glyphosate use has impacted crop production and disease development (Camberato, et al., 2011). Prior research fr om Purdue University (and research published by others) had previously found that plants sprayed with non-glyphos ate herbicides were more susceptible to biological and physiological disorders, suggesting that plants already weakened by the herbicide application are mo re susceptible to infection. However, research found that glyphosate-tolerant soyb eans and wheat are no more susceptible to soil-borne fungal diseases than conventional glyphosate-sensitive varieties, regardless of whether or not glyphosate is ap plied (Baley et al ., 2009; Njiti et al., 2003). The use of herbicides, which have been protecting crops from weeds for more than 50 years, has not been linked to yield-limiting disease during that time (C amberato, et al., 2011).

I.4.7.9. Weeds and Weed Management

Weeds compete with canola for light, nutrients , and soil moisture and can be a major limiting factor in canola production. Canola yi elds can be reduced by as much as 50% due to weed competition with canola (CCC, 2006b) . Weed control is discussed in detail

in Petition Section VIII.F.

I.4.7.9.1. Problem Weeds in Canola – Major Production Area

Table I-3 lists weeds reported as the most common weeds in spring canola production in North Dakota, the primary U.S. canola production area.Monsanto Company 11-CA-233U 379 of 500 Table I-3. Common Weeds in Spring Canola Production in North Dakota

Annual Grass Weeds Annual Broadleaf Weeds Biennial and Perennial Weeds Foxtail, green and yellow Buckwheat, wild Quackgrass
Volunteer Cereals Kochia Thistle, Canada Wild Oat Field Pennycress Lambsquarters, common Mustard, wild Pigweed species Thistle, Russian
Source: Zollinger, 2003; Jenks, NDSU, Personal Communication, 2010.

In addition to its high potential for causing yi eld losses, wild mustard is a serious seed contaminant in canola and can cause price discounts or rejection in the market. Extension advisors recommend speci al attention to c ontrolling this weed prior to planting in canola fields (NDSU, 2007a).

Research studies have shown that Canada this tle is three or four times more competitive than wild oats in canola fields (CCC, 2006b). Only 10 Canada thistle plants per square meter have resulted in 10% canola yield lo ss, while 40 plants per square meter have resulted in over 50% canola yield loss (CCC, 2006b). An average infestation of wild oats will range from 60 to 100 plants per square me ter. A wild oat density of 30 plants per square meter has caused between 15 and 20% canola yield loss (CCC, 2006b). As can be seen in Table I-7 glyphosate provides good to excellent control of both Canada thistle and

wild oats.

Another weed found on canola acres, but not listed in the table above, is dandelion (TopCropManager, 2011a). Dandelion field coverage is greatest in minimum-tillage fields, and it is correlated with yield loss. For example a dandelion infestation providing 50% ground cover caused between 39% and 64% canola yield loss (University of Manitoba, 2002).Monsanto Company 11-CA-233U 380 of 500 I.4.7.9.2. Problem Weeds in Canola – Minor Production Areas

Much less information specific to the impact s of weeds on canola is available for minor production areas. Boyles et al. (2009) identified the we eds in Table I-4 in canola production in Oklahoma, Kansas and Nebraska:

Table I-4. Most Common Weeds in Winter Canola in the Great Plains

Annual Grass Weeds Annual Broadleaf Weeds Winter Annuals Japanese brome Blue mustard Henbit Cheat Bushy wallflower Common chickweed Downy brome Wild mustard Cheatgrass

Rescuegrass Tumble mustard Down bromegrass

Feral rye Tansy mustard Mustards Jointed goatgrass Flixweed Volunteer cereals

Italian ryegrass Field pennycrest Wild oat Shepherd’s purse

Volunteer cereals

Source: Boyles et al., 2009.

Davis (2010) and Wysocki (2010) identified th e weeds in Table I-5 in canola production

in the Pacific northwest:

Table I-5. Most Common Weeds in Spring and Winter Canola in the Pacific

Northwest

Annual Grass Weeds Annual Broadleaf Weeds Perennial Weeds

Downy brome Field pennycress Canada thistle

Wild oat Catchweed bedstraw Italian ryegrass Prickly lettuce Volunteer cereals Chamomile mayweed

Rattail fescue Common lambsquarters Redroot Pigweed

Russian thistle

Wild mustard

Tumble mustard Birdsrape mustard

Source: Davis, University of Idaho, Personal Communication, 2010; Wysocki, Oregon State University, Personal Communication, 2010.

I.4.7.9.3. Weed Management

Weeds are best managed through a combination of agronomic practices also known as integrated weed management (IWM). Co mponents of IWM in canola production can include the following (Harker, 2011; NDSU, 2007a):Monsanto Company 11-CA-233U 381 of 500 Appropriate Tillage: Spring or fall pre-plant tillag e can effectively reduce the competitive ability of weeds by burying the plants, disturbing or weakening the root systems or causing sufficient physical injury to kill the plants . Harrowing canola seedlings is not recommended. (NDSU, 2007a).

Careful Seeding and Fertilization: Careful planting of canola at the correct depth can help ensure a vigorous and early-emerging crop that can compete well with weeds. Placement of nitrogen fertilizer in or too cl ose to the seed row can damage the crop (Harker, 2011). High seeding rates can al so make canola more competitive. Other researchers have found that applying nitrogen in the spring instead of the fall resulted in

lower weed biomass and a 20% decrease in the weed seedbank (Blacksh aw, et al., 2005).

Use of Competitive Crop Cultivars: Hybrids can be strong competitors with weeds (Harker, 2011). Over 95% of the acreage in the U.S. is planted with hybrid canola varieties (Bonnetta, Monsanto Compa ny, Personal Communication, 2010).

Early Weed Removal: Early weed removal is importa nt to attain high canola yield (Harker, et al., 2008; Harker, 2011).

Crop Diversity and Rotation: Because different crop speci es and their associated management practices present different cha llenges for weeds, crop diversity and rotation can help prevent creating advantages fo r any given species (Harker, 2011).

Combining Optimal Agronomics: Researchers have demons trated optimized weed control associated with applying multiple op timal agronomic practices such as early seeding, higher seeding rates, spring-applied fertilizer and ti mely herbicide applications (Blackshaw et al., 2005 ; Harker, 2011).

Careful Harvesting: Careful harvesting can reduce seed losses and the need to control volunteer canola in subsequent crops. Spring or fall pre-plant tillage can effectively reduce the competitive ability of weeds by burying the plants, disturbing or weakening the root systems or causing sufficient physical in jury to kill the plants. (NDSU, 2007a).

As discussed in Petition Section VIII, the gr eat majority of the canola grown in North

America is herbicide-tolerant. In No rth Dakota in 2008, 56% of the canola was glyphosate tolerant and 39% was glufosinate tolerant (Zollinger et al., 2009).

Herbicides were applied to over 97% of the canola acr es in North Dakota in 2008 (Zollinger et al., 2009). The number of registered herbicid es for canola is relatively limited compared the number available for use in other crops such as maize, soybean, and small grain cereals. Ethalfluralin and trifluralin are pre-plant incorporated herbicides and clopyralid, sethoxydim, clethodim, and quizalof op are post-emergence in-crop herbicides for control of annual grasses and broadlea f weeds in conventional canola varieties (NDSU, 2005). In addition, gl yphosate herbicide can be app lied as a pre-plant burndown treatment for control of emerged weeds in no- tillage systems. Table I-6 summarizes the

herbicides used in canola in North Dakota in 2008. Two applica tions of glyphosate are made on approximately 42% of the canola acreage (Table I-6) . In most cases, glyphosate is commonly used as a burndown treatment to remove weeds prior to planting, and theMonsanto Company 11-CA-233U 382 of 500 second application is in-crop to control weeds that emerge after planting in glyphosatetolerant canola.

Table I-6. Herbicide Usage in Canola in North Dakota in 2008

Number of Applications/year

Herbicide Acres Treated % Acres

Treated % of Acres Receiving One

Application % of Acres Receiving Two

Application

Clethodim 123,600 13.6 100.0

Glufosinate 354,300 38.9 99.4 0.6

Glyphosate 660,300 72.6 58.0 42.1

Imazamox 21,200 2.3 48.4 51.6

Quizalofop 31,700 3.5 100.0

Other

herbicides* 29,200 3.2 100.0

Source: Zollinger et al., 2009. *The broadleaf herbicide clopyralid is included in the “other” category.

Table I-7 summarizes the efficacy of herbicides on weed species commonly present in canola. Glyphosate is rated excellent on a ll but one annual grass (wild oat – good to excellent). Glufosinate a nd imazamox are rated excellent or good-excellent on annual grasses except volunteer cereals and field sandbur, respectivel y, where they are rated fair to good. Glyphosate is the only herbicide in can ola that is rated excellent on quackgrass, a perennial grass species. Gl ufosinate is rated excellent or good-excellent on all the

annual and biennial broadleaf weeds listed while rated poor on Canada thistle. The ratings for glyphosate are more variable on broadleaf weeds ranging from excellent to poor-excellent for all annual a nd biennial weeds and good to ex cellent on Canada thistle. While imazamox is rated excellent on some broadleaf weeds it is also rated poor on

buckwheat and Canada thistle. In terms of control of the main problem weed species, Canada thistle, wild mustard and wild oat, glyphosate is rated good to excellent on all; glufosinate is good to excellent for wild oat, ex cellent for wild mustard, but poor for Canada thistle. Of the herbicides, other th an glyphosate, only clopyr alid is effective on Canada thistle, but it provides no control for wild oat or wild mustard. None of the herbicides, other than glyphosate and gl ufosinate, control wild mustard.Monsanto Company 11-CA-233U 383 of 500Table I-7. Weed Species Responses to Herbicides in Canola1Product Foxtail, Green Foxtail, Yellow Volunteer Cereals Wild Oat Buckwheat Kochia Lambsquarters Mustard, Wild Redroot Pigweed Thistle, Russian Quackgrass Thistle, Canada FlixweedPreplant Incorporated OnlyEthalfluralin E E G F P-F F-G E N E G-E N N PTrifluralin E E N P-F P-F F G-E N E G N N PPostemergenceClopyralid N N N N F-G N P-F N P P-F N E NImazamox E G-E G-E E P E F E E G-E F N-P EGlyphosate E E E G-E F-G F-E P-E G-E E G E G-E G-EGlufosinate E G F-G G-EE E F-G E E G-E P P G-EQuizalofop E G-E E G-E N N N N N N G-E N NSethoxydim E E E G-E N N N N N N F N NClethodim E E E E N N N N N N G N N1Source: NDSU, 2005. Weed control ratingsbased on the following scale: E = Excellent= 90 to 99% control, G = Good = 80 to 90%control, F = Fair = 65 to 80% control, P =Poor = 40 to 65% control,and N = None = no control.Monsanto Company 11-CA-233U 384 of 500 I.4.7.10. Canola Volunteers

Volunteer canola is defined as a plant that ha s germinated and emerged unintentionally in the production of a subsequent rotational crop. Canol a seeds will remain in a field after canola harvest, primarily as a result of pods shattering before or at harvest time. Researchers have estimated that an average of 3,000 seeds per square meter may be lost in harvesting (Harker, 2011). Volunteer canola will compete with the succeeding rotational crop and may affect yield depending on the density (NDSU, 2005). Alt hough canola can volunteer for several years following a canola crop (Beckie and Warwic k, 2010), problems with volunteer canola in rotations are not common (Boyles et al., 2009). Beckie and Warwick (2010), documented “the longest persistence of oilseed ra pe volunteers in Canada” (7 y ears) and concluded that their results supported the findings from previous studies that volunteer oilseed rape populations generally do not persist in the absence of replenishment of the seed bank. Researchers investigating the persistence of canola seeds in the seedbank for several different canola genotypes found, after one, two, and three winters, maximum persistence of 44, 1.4 and 0.2% of the original seedbank, respectively (Gulden, et al ., 2003). In another thr ee-year study in which wheat was planted for three years (2001, 2002 a nd 2003) following canola in 2000, researchers found average canola densities of 6.2, 0.7, and 0.0 plan ts per square meter in the first, second, and third year of the wheat crops. Researchers c oncluded that preventing seed production in new

canola volunteers in 2001 reduced canola densities in subseque nt years (2002 and 2003) below those required to mitigate weed-crop competition infl uences in most crops (H arker, et al., 2006).

Data from Canada suggest that presence of a he rbicide tolerance trait is not a major factor influencing volunteer canola a bundance (Beckie and Warwick, 2010). Beckie and Warwick (2010) also report the ranking of oilseed rape as a weed species in Canadian province field surveys went from 10th in relative abundance in the mid-1990s (when canola was mostly nonherbicide tolerant) to 12th in 2001-2003, when herb icide tolerant canola wa s widely used. Based on estimated crop yield loss, the economic impact of volunteer wheat and oilseed rape is ranked third and eighth, respectively, among wee dy species (Beckie and Warwick, 2010).

In spring canola, seeds that remain on or near the soil surface may germinate in the fall and be killed by frost (NDSU, 2005). Researchers have concluded that producers can expect from 1 to 9% of canola harvest losses to emerge in th e spring immediately following a sown canola crop and that an effective management strategy to limit long-term volunteers ma y be to avoid tilling in the fall and then till in the spring after the seeds have germinated (Lawson and Van Acker, 2005). In winter canola, the seed typically germ inates after summer rains and can be controlled by tillage before planting the next rotational crop.

Many of the same management practices for w eed management discussed in Petition Section VIII.F are applicable to contro l of volunteer canola including: high seeding rates, crop rotation, early weed removal (CCC, 2005) and careful harvesting.

Multiple herbicide options are available for pre-plant burndown that will control herbicidetolerant canola prior to seeding cereal or flax (CCC, 2005) , which are recommended rotational crops following canola in North Dakota. Burndow n options for control of herbicide-tolerant volunteer canola are more limited prior to planting broadleaf crops such as field pea, lentil, sunflower, dry bean or chick pea (CCC, 2005).Monsanto Company 11-CA-233U 385 of 500 However, volunteer canola can be controlled thr ough herbicide applications and with appropriate rotational crop management. Table VIII-11 in the Petition provides a detailed description of the rotational cropping practices immedi ately following canola by state. Table VIII-12 in the Petition provides a listing of herbicides that control vol unteer glyphosate-tolerant canola in the various rotational crops and thei r effectiveness.

I.4.7.11. Herbicide Resistance - General

Herbicide resistance is the inherited ability of a plant to survive and reproduce following exposure to a dose of herbicide normally lethal to the wild type (WSSA , 1998). For a more indepth discussion of herbicide resistance see Appendix J.

In the mid-1950s, Harper (1957) theo rized that annual, repeated us e of any herbicide could lead to shifts in weed species com position within a crop-weed community. Similarly, Bandeen, et al. (1982) suggested that a normal va riability in response to herbic ides exists among plant species

and tolerance can increase with re peated use of a herbicide. To simplify, herbicide resistance in

weeds is a result of natural selection. Plants of a given species are not all identical, but are made up of biotypes with various geneti c traits. Biotypes possess certain traits or characteristics not common to the entire population. Herbicides that suppress or kill weeds ex ert selection pressure on weed populations. When a he rbicide is applied, surviving pl ants, those that had reduced sensitivity to the herbicide, have a competitiv e reproductive advantage with progeny more likely to possess the same or superior herbicide resist ance. With repeated a pplication of the same

herbicide and no other herbicide or weed control practice, the re sistant biotypes can become the dominant biotype in that w eed community. As of May 2011, 358 herbicide re sistant weed biotypes have been reported to be resistant to 21 different herbicide modes of action worldwide (Heap, 2011c). Glyphosate-resistant weeds account for approximately 6% of the herbicide resistant biotypes while weeds resistant to herbic ides that inhibit acetolactate synthase (ALS)

account for 31% of the herbicide resistan t biotypes (Heap, 2011b; Heap, 2011a).

For as long as herbicide resistance has been a known phenomenon, public se ctor weed scientists, private sector weed scientists and growers have been identifying methods to address the problem. For instance, when a farmer uses multiple weed control tools, resistant biotypes generally will not become the dominant biotype within a populati on. By contrast, weed resistance is known to occur most rapidly in areas where there is a sole reliance on a single herbicide used repeatedly over multiple crop generations for the management of a specific weed spectrum.

When a grower encounters a biotyp e that is resistant to a herbicide being us ed, the grower needs

to utilize an alternate method of weed control. Management practi ces that can be used to retard the development of resistance, such as those ro utinely used by canola grow ers, include herbicide mixtures, herbicide rotation, and crop rotation. The WSSA reported that weed scientists know that the best defense against weed resistance is to proactively use a combination of agronomic practices, including the judicious use of herbicides with alternative modes of action either concurrently or sequentially (WSSA, 2010).

I.4.7.12. Herbicide Resistant Weeds of Economic Importance in CanolaMonsanto Company 11-CA-233U 386 of 500 Herbicide resistance in weeds causing yield losses in the major U.S. canola production area are summarized in I-8 along with the he rbicide mode of action (MOA).

Table I-8. Herbicide Resistan t Common Weeds in Top Five U.S. Canola-Producing States

in 2010

State 2010 Total Area Planted (acres) Glyphosate Resistant

Common Weeds in

Canola Resistant Common Weeds in

Canola with Alternative Modes of

Action North Dakota 1,280,000 None Wild mustard, Kochia, Wild oat,

Green foxtail Oklahoma 60,000 None Cheat (rye brome) Minnesota 46,000 None Lambsquarters, Wild oat, Kochia,

Yellow foxtail Idaho 19,500 None Prickly lettuce, Kochia, Russian thistle, Italian ryegrass, Wild oat, Mayweed chamomile Montana 17,500 None Kochia, Russian thistle, Wild oat Source: USDA-NASS, 2011c; Heap, 2011d; Heap, 2011e; Heap, 2011f; Heap, 2011g; Heap, 2011h.

To date only one glyphosate resistant weed has been identified in North Dakota, common ragweed ( Ambrosia artemisiifolia ) (Heap, 2011g). Based on information from NDSU, known or suspected glyphosate-resistant weeds have been iden tified in soybean fields in two counties in eastern central North Dakota (Stachler and Chri stoffers, 2010). These counties are not major production counties for canola (USDA-NASS, 2007). In addi tion, glyphosate resistant populations of giant ragweed ( Ambrosia trifida ), common ragweed ( Ambrosia artemisiifolia ) and common waterhemp ( Amaranthus tuberculatus ) have been identified in Minnesota (Heap, 2011e). None of these weeds are listed as co mmon in spring canola production fields in North Dakota (Table I-3).

Glyphosate-resistant It alian ryegrass ( Lolium multiflorum ) has been identified in Oregon (Heap, 2011b) and glyphosate-resistant horseweed ( Conyza canadensis ) has been identified in Oklahoma (Heap, 2011h).

Development of herbicide-resistan t weeds is of particular concern in the scientific agricultural community. For example, Harker (2011) emphasizes the importance of integrated weed management to help preserve herbicide-tolera nt technology, which has been shown to provide superior weed management, yi eld and environmental perfor mance (see Appendix Section I.4.7.9.3.). In addition, the Herbicide Resistan ce Action Committee’s provides guidelines for prevention and management of herbicide re sistance (HRAC, 2009). These guidelines recommend an integrated approach to weed resistance management including crop management (i.e. row spacing, etc), cultural techniques and herbicides.

Canola is grown in rotation with crops that use herbicides other than glyphosate, and both glyphosate- and glufosinate-tolerant canola is produced. Powles (2008) concludes that where diversity in weed management systems is maintained, weed control by glyphosate can be sustainable. While glufosinate- tolerant canola is not yet availa ble for winter canola, winter canola is grown in rotation primar ily with wheat, which utilizes herbicides with alternate modes

of action.Monsanto Company 11-CA-233U 387 of 500 Glyphosate-resistant weeds may occur in canola fiel ds. Some means of weed management other than glyphosate would need to be used for these weeds. Methods would be similar to those used for glyphosate-tolerant canola volunt eers (Appendix Section I.4.7.10).

I.4.7.13. Crop Rotation

One of the main purposes of growing canola in ro tation with other crops is to mitigate or break disease cycles (Brown et al., 2008; NDSU, 2005) . As discussed in Petition Section VIII.H., canola rotation is very important for managing black leg and Sclerotinia stem rot. Some of the other purposes of rotations are to improve or maintain soil fe rtility, reduce er osion, reduce the build-up of pests, reduce risk of weather-relate d crop damage, improve weed control and increase net profits through yiel d increases (Peel, 1998). Comp ared to continuous cereal production, including canola in small grain rotations has resulted in 17 to 20% yield increases in small grain cereals (Brown et al ., 2008). Incorporati ng canola in small grai n rotations has also decreased disease incidence and increased quality of cereal crops. Incl uding canola in a small grain cereal rotation provides broader, more effective herbicide options for controlling certain weeds such as downy brome, jointe d goat grass, wild oat, Italian ryegrass and feral rye that are difficult to control in winter wheat (Brown et al., 2008).

Table VIII-11 in the Petition provides a descript ion and quantification of the rotational cropping practices immediately following canola, by state, that account for 99+% of the total canola acreage. For each state, the table shows quantita tive data for the various crops that are rotated with canola, and also shows the percent of gly phosate-tolerant crops th at are included in the

rotations.

In a 2008 agronomic survey of canola growers in 11 counties representing the major canola growing areas of North Dakota and Minnesota, re searchers reported that 17 of the 45 fields planted canola once in every five year rotation, with other rotations being canola every other year, every three years and every four years (Mazurek, et al., 2008).

I.4.8. Raw and Processed Ag ricultural Commodities

I.4.8.1. Canola as a Food Source

Canola is grown principally for its oil which is extracted from the seed, and has both food and industrial applications (Petition Section II). Canola is approximately 40% oil and 60% meal (Colton and Sykes, 1992). Canola seeds are first fl aked by a rolling process in preparation for oil

extraction after which the flakes are placed in a cooker. Heating reduces the viscosity of the oil and inactivates certain enzymes that can break down glucosinolates to produce isothiocyanates

and nitriles which are harmful when fed to animals (Booth, 2004) . After heating, canola seeds undergo mechanical extraction to produce a cake w ith an oil content of less than 20%, followed by solvent extraction using hexane to remove the bulk of the remaining oil. Several additional processing steps further refine the oil by rem oval of various undesirable constituents (Booth, 2004; Carr, 1995).

Canola oil is high quality oil that is used in a variety of foods including frying and baking oils, salad oils, margarines and shortenings, and is the most valuable component of canola seed.Monsanto Company 11-CA-233U 388 of 500 Canola oil has well established heart health bene fits and the U.S. FDA has issued a qualified health claim based on its ability to reduce the risk of coronary heart disease (U.S. FDA, 2006).

Recently, methods have been developed for producing a water-soluble canola/oilseed rape protein isolate having at least 90% protein that can be used in products includi ng dairy and grain products, fruit and vegetable juices, salad dressi ngs, meal replacements and nutritional bars, have been developed. The U.S. FDA has no objection to the GRAS notification for canola protein isolate for use in food applications (U.S. F DA, 2010) and the first co mmercial production took place in 2010 (BioExx, 2010).

I.4.8.2. Canola as a Feed Source

The solid residue or meal left after oil extraction of canola is used as high protein animal feed. Canola meal is used in poultry, pig, beef and da iry cattle feeds, and can also be used in aquaculture diets for salmon, catfish and trout (CCC, 2009). Canola meal contains approximately 40% protein, 13% crude fiber and essential amino acids including lysine and sulphur-containing amino acids that are most often deficient in cereal meals (Bell, 1995).

Compared to other oilseed-based feed sources, ca nola meal has slightly less digestible energy value. Therefore, animals with requirements for intermediate energy levels such as dairy cattle and laying chickens perform we ll on canola meal, while high energy animals such as broiler chickens perform better on diets with soybean meal (CCC, 2009).

I.4.9. Cross pollination and Gene Flow to Sexually Compatible Species

This section discusses the potential for gene flow within B. napus (intraspecific) and to closely

related Brassica species and other members of the fam ily Brassicaceae that may be present where canola is grown. The first part of the discussion provides information on the

characteristics of B. napus related to gene flow and the potentia l for gene flow to related species

based on field studies. This is followed by a discussion of the potential for gene flow from B.

napus to specific groups of plants in the envi ronment including other canola plants, other

Brassica crops, canola seed pr oduction areas, other Brassica seed production areas, native

Brassica species, and Brassica and related weeds.

I.4.9.1. Background

I.4.9.1.1. Pollen-Mediated Gene Flow and Introgression

Pollen mediated gene flow (often referred to as cross pollination) occurs when pollen of one plant fertilizes ovules of a second plant. Intr ogression is a multi-genera tion process whereby one or more genes successfully incorporate into the genome of a recipient plant. Gene flow is

discussed in detail in Petition Section IX, with Tabl e IX-3 summarizing th e published literature on unassisted hybridization under field conditions with B. napus as the male parent.

This discussion focuses on B. napus , as almost all the canola grown in the U.S. is B. napus

. Brassica napus is predominantly self-pollinating alt hough interplant (pla nts are touching one another) cross pollination rates range from 12% to 55% with a mean of 30% (Beckie, et al., 2003). Pollen of B. napus is heavy and sticky (OECD, 1997) and pollen movement is primarilyMonsanto Company 11-CA-233U 389 of 500 by insects, such as honey bees (Thompson, et al., 1999) although wind is also responsible for some pollen movement. Most (98.8%) of pollen tr avels less than twelve meters from its source (Scheffler, et al., 1993) although dispersal due to pollinators may occur ove r greater distances at low frequency (Thompson et al., 1999).

Brassica napus produces a large amount of pollen (OGTR, 2008) which can remain viable for up to four to five days under field conditions (Rantio-Lehtimäki, 1995). This, coupled with the potential for B. napus pollen movement, provides the possi bility for hybridization between B. napus and related sexually compatible species.

I.4.9.1.2. Potential for Pollen-Mediated Gene Flow and Introgression of B. napus to other

Brassica Species (Including Brassica Vegetable Species) and Closely Related Non- Brassica Species

A survey of the literature provides inform ation on the potential for hybridization (cross pollination) and introgression from B. napus to related species. Reports of unassisted (e.g.

without human intervention or a ssistance) field hybridization of B. napus to related species provide biologically relevant information that can be useful in assessing the potential for hybridization and gene introgre ssion under field conditions.

There are reports of hybridizat ion under field conditions with B. napus as the pollen donor with six species including B. rapa

, B. juncea

, B. oleracea

, H. incana

, R. raphanistrum and S. arvensis (see Petition, Table IX-3). The species B. rapa

, B. juncea and B. oleracea are cultivated for crop production. The other species listed, H. incana

, R. raphanistrum and S. arvensis are not cultivated for crop production, but are found in the environment. In all cases the resulting hybrids had decreased environmenta l fitness evidenced by a variety of characteristics including decreased pollen viability, seed production, seedli ng survival, etc. when compared to parental

varieties.

Brassica rapa

Brassica rapa is widespread throughout temperate No rth America, and may be found in areas where canola is grown (BONAP, 2010). Brassica napus was derived from the hybridization of diploid species, B. rapa and B. oleracea (Petition Section II.A.). Brassica napus and B. rapa have a common set of chromosomes, are known to be sexually compatible, and can form hybrids under field conditions (Bing, et al., 1996; Warwick, et al., 2003) . Hybridization frequencies

between B. napus and B. rapa in neighboring fields or inter-pla nted in large plots vary from study to study, ranging from 0% up to 69% as re viewed in Devos, et al. (2009), but can be as high as 93% (Jørgensen, et al., 1996). For ex ample, Warwick et al. (2003) measured hybridization frequencies betw een these two species with B. rapa as the maternal parent in field experiments, including natura l environments and commercial B. napus fields. The mean hybridization frequency was 7% in natural e nvironmental field trials and 13.6% in the

commercial fields. The firs t-generation hybrids between B. napus and B. rapa had reduced pollen viability, produced fewer germinable seed s, and had very low (<2%) hybrid seedling survival rates (Scott and Wilkinson, 1998; Wa rwick et al., 2003). A dditionally, unlike the B. rap a parent, under field conditions, seeds of F 1 hybrids between these two species lacked seed dormancy (Jørgensen, et al., 1998).Monsanto Company 11-CA-233U 390 of 500 The presence of a herbicide-tolerance trait (glufosinate tolerance) introgressed experimentally

under controlled c onditions from B. napus into B. rapa did not increase its su rvival or number of seeds per plant compared to parental B. rapa (Snow, et al., 1999).

Collectively, these data support the conclusion that hybridization between B. napus and B. rapa is possible under field conditions, but resulti ng progeny demonstrate decreased fitness and likelihood of gene intr ogression is low.

Brassica juncea

Brassica juncea is sparsely but widely distributed in temperate North America in cultivated and disturbed areas, and may be found in areas where canola is grown (BONAP, 2010). Brassica juncea is an amphidiploid derive d from the hybridization of B. nigra and B. rapa (Petition Section II). Brassica napus and B. juncea have a common set of chromosomes, are known to be sexually compatible, and can form hybrids under open pollination conditi ons (Bing et al., 1996; Frello, et al., 1995; Jørgensen et al., 1998). As reviewed in De vos (2009), in field plots with interplanted B. napus and B. juncea interspecific hybridization frequencies were low ranging from 0.3% to 3%. First-generation hybrids between B. juncea and B. napus had reduced male and female fertility, low pollen viability ranging fr om 0% to 28% and low seed set (Bing et al., 1996; Frello et al., 1995). Brassica napus and B. juncea can hybridize at a low rate under field conditions, but the resulting hybrids have reduced male and female fertility significantly decreasing the likelihoo d of introgression.

Brassica oleracea

Brassica oleracea is distributed primarily in northeaste rn and midwestern states, and may be found in areas where canola is grown (BONAP, 2010) . As noted above and discussed in Petition Section II.A., B. napus was derived from the hybridization of B. rapa and B. oleracea. Therefore it is not surprising that B. napus and B. oleracea are known to have some limited sexual compatibility under open pollination condi tions (Ford, et al., 2006). Hybridization between these two species under field conditions a ppears to be relatively rare and introgression under field conditions is unlikely to occur (F ord et al., 2006; Wilkinson, et al., 2000).

Brassica Vegetable Species

Many common vegetables are found in the genus Brassica

. Brassica napus vegetables include Swedes or rutabaga and Siberian kale. Brassica rapa vegetables include turnip and Chinese cabbage while B. oleracea vegetables include cabbage, cauliflo wer, broccoli, collards, kale and Brussels sprouts. Gene flow from B. napus canola to these vegetable species is highly unlikely but may occur. However, B. napus

, B. rapa and B. oleracea vegetables are not considered weedy, and are generally harvested prior to fl owering, preventing cross pollination, hybridization and seed formation. Thus the potential for B. napus gene flow and introgr ession into closely related vegetable sp ecies is low.

Closely Related Non-Brassica Species

In addition to those reported for B. rapa

, B. juncea

, B. oleracea , there are reports of hybridization under non-cultiva tion field conditions with B. napus as the pollen donor andMonsanto Company 11-CA-233U 391 of 500 H. incana

, R. raphanistrum and S. arvensis as the female parent (Petition Table IX-3). As with B. rapa

, B. juncea

, B. oleracea , in all cases these hybrids had decreased environmental fitness evidenced by a variety of char acteristics including decreased pollen viability, seed production, seedling survival, etc. when compared to parental varieties. Based on fi eld studies, hybridization between these species appears to be rare and introgression under field conditions is unlikely to

occur.

I.4.9.2. Potential for Gene Flow in the Crop and Seed Production Affected Environment

I.4.9.2.1. Potential for Intraspecific Ge ne Flow between Canola Crops

Gene flow may occur between canola crops plan ted close together. Ma ny studies have been performed and are discussed in Petition Secti on IX. Table I-9 is from the petition and summarizes some representative studies. As shown in the table, in general, the percentage of pollen flow and potential for cr oss pollination diminishes with increasing distance from the source. Canola cross pollination rates are highly variable and are influenced by experimental design, size of the pollen donor and recipient popul ations, variety (genotype), presence and activity of insect vectors, environmental cond itions (temperature, wind speed and direction, humidity, etc.) and topography. Information on pollen movement is useful for managing pollen flow during canola breeding, seed producti on and for identity preservation.Monsanto Company 11-CA-233U 392 of 500 Table I-9. Summary of Representative Studies of B. napus Cross Pollination Rates at

Various Distances

Reference Distance from Pollen Source (meters) % Cross pollination Observed Manasse and Kareiva, 1991; Stringam and Downey, 1982 47

137

366 2.1

1.1

0.6 Manasse and Kareiva, 1991 50

100 0.022

0.011 Scheffler et al., 1993 0

1

3

6

12

24

36

47

70 4.8

1.4 – 1.6

0.35 – 0.4

0.033 – 0.11

0.016 – 0.025

0.0 – 0.0041

0.0011 – 0.0031

0.0 – 0.00034

0.0 Morris, et al., 1994 0

0.3

0.6

3

4.6 ~2.0 – 3.5 §

~1.0 – 1.5 §

~0.75 – 1.2 §

~0.5 – 0.75 §

~0.5 – 0.7 §

Scheffler et al., 1995 200

400 0.0156

0.0038 Downey, 1999 33

66

100 0.1 – 1.5

0.0 – 0.4

0.1 – 0.4 Staniland, et al., 2000 0

2.5

5

10

15

20

25

30 0.69

0.29

0.14 φ

0.07 φ

0.08 φ

0.07 φ

0.04 φ

0.02 φ Rieger, et al., 2002 100

1500

>3000 0.012 – 0.014

0 – 0.197

None detected

§ Frequencies estimated from Figure within the publication.

φ Values calculated fro m publication.

Since the development of biotechnology-derived canola researchers ofte n use the biotechnologyderived trait as a marker in th eir cross pollinatio n studies (Beckie et al., 2003; Cai, et al., 2008; Dietz-Pfeilstetter and Zwerger, 2004; Downey, 1999; Scheffler et al., 1993; Scheffler, et al., 1995; Hommel and Pallutt, 2003). Since some cro ss pollination does occur at short distances,Monsanto Company 11-CA-233U 393 of 500 adjacent fields with different herbicide tolerant traits would be expected to produce some seeds with both traits, which studies have confirmed (Beckie et al., 2003).

I.4.9.2.2. Potential for Gene Flow in Canola Seed Production Areas

AOSCA and state seed certification standards for canola provide isolation distances and standards for seed purity. The standards provide a guide for acceptable levels of other varieties of canola seed that might be expected to be found in certified seed based on the required seed planting isolation distances. In no case is that level 0%. For example, Colorado and Minnesota both require a seed planting isolation distan ce for canola hybrids of 2,640 feet from other varieties of canola and impose a standard of 1.5:10,000 (0.015%) for the ratio of plants of

allowable seed of other canola va rieties that can be found in certif ied seed (Table I-2). North Dakota has required isolation dist ance of 660 feet from other varieties of canola and a standard of 1:500 (0.2%) for the ratio of seed of other ca nola varieties (Table I-2). These standards are intended to ensure that the seed produced meet s standards for seed purity and quality that are appropriate for growers’ needs and meet the stated purity standards set forth in state and federal laws. However, if a seed produc er wishes to produce seed with more stringent purity standards than those specified by AOSCA and the indivi dual state certification standards, the seed producer may need to implement greater isolation distances.

I.4.9.2.3. Potential for Gene Flow between Canola and Other Brassica Crops

Vegetable Crops

As discussed in Petition Section IXD.3., there are many Brassica vegetable crops in the U.S. Brassica napus

, B. rapa and B. oleracea vegetables are not consider ed weedy, and are generally harvested prior to flowering, preventing cross pollination, hybridization and seed formation.

Biofumigant Mustard

As discussed in Appendix Sec tion I.4.4.3, biofumigant mustard ( S. alba and B. juncea ) is used as a green manure crop. As discussed in Appendi x Section I.4.9.1.2, the poten tial for introgression

with B. juncea is very low, and the potential for introgression with S. alba is extremely low. In addition, since biofumigant mustard is tilled under before the seeds mature (McGrath and Menasha, 2009), the potential for cross pollination is extremely low.

Industrial Oilseed Rape

The cross pollination rates shown in Table I-9 woul d be generally applicable to cross pollination between industrial oilseed rape and canola. As discussed in Appendix Section I.4.5.3, industrial oilseed rape is grown on very limited acreage in Washington, Oregon and Idaho, with almost all the operations in Idaho, where there are strict geographic restrictions on the proximity of industrial oilseed rape to canola. Washington and Oregon also have regulations to minimize cross pollination.

Mustard Seed (for Food)Monsanto Company 11-CA-233U 394 of 500 As discussed in Appendix Section I.4.5.3, mustar d seed is grown on approximately 48,000 acres, in the same general areas where canola is grow n. As discussed in th at section, because of similarity of disease susceptibility, it is not recommended for rotati on immediately following canola. Nearly all the mustard grown in the U.S. is S. alba , with a very small acreage of B. juncea . As discussed in Petitio n Section IX.D.4., the potential for gene flow between B.

napus and B. juncea is very low, and the poten tial for gene flow with S. alba is extremely low. Based on the extremely low potential for gene flow with S. alba , the relatively small number of

S. alba acres, and the low probab ility of a rotation immediat ely following canola, cross pollination between S. alba mustard and canola is highly un likely. Based on the very small acreage, the low potential for introgression and the low probability of close rotation with canola, cross pollination be tween canola and B. juncea mustard and canola is highly unlikely.

I.4.9.2.4. Potential for Gene Flow in Other Brassica Seed Production Areas

Vegetable Seed Production

The available information on Brassica vegetable seed production is detailed in Attachment 1.

As discussed in Attachment 1, the only B. napus vegetables grown or consumed in the U.S. are rutabaga (root) and Siberian kale (greens). Some seed companies offer B. napus varieties called red monarch kale and red Russian kale, which a ppear to be variations of Siberian kale. Therefore, the greatest possibi lity for cross pollination with B. napus canola in ve getable seed production would be with rutaba ga and Siberian kale. Ruta baga seed is produced on approximately 80 acres in western Washingt on State in Skagit and Snohomish Counties (Attachment Table 1). Based on information from Washington State University and other information presented in Attachment 1, the 80 acres of rutabaga seed production probably accounts for at least 90% of U.S. rutabaga seed production. This rutaba ga seed production area is within that part of western Washington wher e there are strict limitations on growing canola that are intended to prevent a ny cross pollination with related Brassica species (Appendix Section I.4.6). We were unable to find any in formation specifically on Siberian kale seed production in the U.S., although a seed producer in the Yuma Valley of Arizona produces seeds for red monarch and red Russian kale greens (A ttachment 1). Based on USDA census data, no canola is grown in Arizona; therefore, there is no potential for cross poll ination from canola with these seed crops.

For the other Brassica and related vegetables, Table I- 10 summarizes the potential for hybridization and introgre ssion with canola, based on the in formation presented in Petition Section IX and summarized in Appendix Section I.4.9.1.Monsanto Company 11-CA-233U 395 of 500 Table I-10. Cross Pollination Potential for Ca nola with Related Vegetable Seed Species

Species Common Names Potential for Hybridization Potential for Introgression

B. rapa Chinese cabbage, pak choi, choi sum, turnip, Chinese mustard, broccoli raab,

mizuna, mibuna, komatsuna, tai soi (most of these are greens) High (0-70%) Low

B. juncea Greens: red giant mustard, sawtooth mustard, Osaka purple mustard, southern giant curled mustard, Florida broadleaf, leaf mustard Low (0.3 to 3%) Very low

B. oleracea Cabbage, broccoli, kale, cauliflower, Brussels sprouts, kohlrabi, collards, Chinese kale Low (0.1%) Very low

R. raphanistrum Radish, daikon Very low (< 0.003%) Extremely low

Seed production of other Brassica and related species occurs al most entirely in areas with prohibitions or strict limitations on canola production and transpor t (eastern Washington, western Washington, the Willamette Valley in Oregon and Treasure Valley in Idaho), or in areas where canola production is either neglig ible or non-existent (Californi a and Arizona; Appendix Section I.4.5; Attachment 1).

Aside from these regulatory and geographic feat ures that currently minimize cross pollination, the vast majority of Brassica and related seed production is B. oleracea and R. raphanistrum

, which have, respectively, very low and extr emely low potential for introgression with B. napus

. In greenhouse and field experiment s, (Quinn, 2010) found that canola ( B. napus ) cross pollinated

with a B. rapa vegetable species (Chinese cabbage) but did not cross pollinate with a B. oleracea vegetable species (broccoli). The study eval uated only cross pollination potential and not introgression. The field studies were done under a “worst case scenario” of placing the vegetable seed receptor plant in the middle of a canola fi eld during peak flowering of both species (Quinn 2010).

The Willamette Valley Specialty Seed Asso ciation (WVSSA) seed production isolation guidelines for vegetable seed pr oduction are established to mi nimize the potential for cross pollination of B. napus canola with related vegeta ble species (WVSSA, 2008).

I.4.9.3. Potential for Gene Flow from Canola to Species in th e Natural Affected

Environment

I.4.9.3.1. Potential for Gene Flow from Canola to Native Brassica Species

None of the 19 Brassica species in the USDA plant database are native to North America (USDA-NRCS, 2010). Therefore, there is no potential for gene flow to native Brassica species.Monsanto Company 11-CA-233U 396 of 500 I.4.9.3.2. Potential for Gene Flow from Canola to Brassica and Related Weed Species

Based on the USDA Plant Database (USDA-NRCS, 2010), the Brassica species that have been specifically identified as weeds in the U.S. are B. rapa (birdsrape or field mustard). Brassica tourneforti (Asian mustard), B. juncea (Indian mustard) and B. nigra (black mustard). Related species identified as weeds include R. raphanistrum (wild radish) and S. arvensis (wild mustard). The USDA plant database (USDA-NRCS 2010) s hows distributions in the U.S. for these

Brassica and related weed species as follows:

• Indian mustard ( B. juncea ) – throughout most of the U.S. and Canada

• Field mustard or birdsrape ( B. rapa ) – throughout U.S. and Canada

• Black mustard ( B. nigra ) - throughout most of the U.S. and Canada

• Asian mustard ( B. tourneforti ) – CA, NV, AZ, NM, TX

• Wild radish ( R. raphanistrum ) - throughout most of the U.S. and Canada

• Wild mustard ( S. arvensis ) - throughout the U.S. and Canada

Based on the information from Petition Section IX , the likelihood of introgression is low with B. rapa and very low to extremely low with the ot her related weed species on this list.

Introgression, i.e., the st able incorporation of genes from one differentiated gene pool into another, of a biotechnology-derived trait from B. napus to B. rapa has been demonstrated on a single occasion under commercial field conditions (Warwick, et al., 2008). Populations of B. rapa located close to commercial fields of glyphosate-tolerant B. napus were monitored for multiple years. The number of hybrids with th e glyphosate-tolerance trait declined drastically from the first to fifth year of monitoring, but pers isted at low levels at one of the two sites. A single glyphosate-tolerant diploi d individual with 29% pollen viab ility was discovered five years after the last planting of glyphosate-tolerant B. napus confirming the presence of the herbicide

tolerance trait ove r time (Warwick et al., 2008).

Warwick et al. (2008) noted that they had no data to suggest thatthe presence of a herbicidetolerant trait in a weedy species poses a specif ic risk and that the trait would have positive selection value only in the presence of the herbicide. However, the selection potential of herbicide application could extend beyond the bounda ry of the field because of the potential advantage that drift may confer to weeds that have cross pollinated with herbicide-tolerant canola (Londo, et al., 2010).

While Brassica and related weeds can be found in canola, no instances or reports of introgression

of traits from herbicide-tolerant canola with w eeds have been found in the literature or in information from university extension offices a nd industry sources other than that reported by Warwick et al. (2008). To date, herbicide-tolerant canola has prove d a useful tool in controlling

weeds of Brassica and related species. For example, as discussed in Appendix Section I.4.7.9, wild mustard ( S. arvensis ) is a problem weed in canola in Nort h Dakota, as it is in the Canadian provinces. The NDSU recommends that conventiona l canola not be planted on fields with heavy infestations of wild mustard. However, glyphos ate, glufosinate and imazamox tolerant canolaMonsanto Company 11-CA-233U 397 of 500 can all be planted on heavily infested wild mu stard fields (NDSU, 2007a) to achieve good weed

control.

I.4.10. Ruderal Canola

Canola is sometimes found growing in locations where it was not intentionally planted. When that occurs in an agricultural field, it’s called volunteer canola (App endix Section I.4.7.10). When it occurs outside an agricultural field, th e term ruderal canola applies. Ruderal canola could potentially be a weed control issue if it was invasive and/or hard to control. This section

addresses these issues and also assesses wh ether herbicide-tolera nce has affected the survivability of ruderal canola. A literature revi ew is included in Table IX-1 of the Petition and

results are summarized below.

Locations of Ruderal Canola

Canola seeds are very small and round, and can spill from trucks and rail cars during transport. Therefore, most ruderal canola is found in dist urbed areas along roadways and railroads where canola has been transported (A ono, et al., 2006; Crawley and Brow n, 2004; Knispel, et al., 2008; Nishizawa, et al., 2009; Pivard, et al., 2008; Saji, et al., 2005). Crawley and Brown (2004) reported that the plants typically grow within one meter of the road. Nishizawa et al. (2009) reported canola in sidewalk cracks and flowerbeds ; however, this was all within a one-to three meter wide monitoring zone immediately adja cent to the roadway pavement. Some ruderal canola is found along the edges of canola fields (Knispel et al., 2008; Pivard et al., 2008).

Persistence of Ruderal Canola

Most researchers have conclude d that ruderal canola populations are not self-sustaining. In a ten-year (1993 to 2003) study of 3,658 quadrants (each 100 meters long) along the edges of roadways near London, Crawley and Brown (2004) found that one percent of the quadrants were occupied (had one or more canola plants) for the entire 10-year period, but none of these had constant population density. Th e most frequent pattern observ ed was for quadrants to have canola plants present for one or two years out of ten. Canola plants outside agricultural fields can produce seed (Crawley and Brown, 1995; Knispe l et al., 2008) but this is often prevented because most plants do not survive to maturity. This is due to competition from other vegetation (Crawley and Brown, 1995), management operations such as roadside mowing, the use of broadleaf herbicides, animal predation, diseas es and environmental conditions (Crawley and Brown, 1995; Knispel et al., 2008; Norris a nd Sweet, 2002; Yoshimura, et al., 2006).

Herbicide-Tolerant Traits and Ruderal Canola

Frequency of occurrence of biot echnology-derived plants in ruderal areas was similar to the proportion of the canola area planted with herbicid e-tolerant canola varie ties in recent preceding years (Yoshimura et al., 2006). Biotechnology-derived canola popul ations have not been found to be more invasive or more persistent than conventional canola populations (Crawley, et al., 2001). The viability of the large majority of canol a seed in soil declines over time (Gulden et al., 2003; Gulden, et al., 2004; Ha ils, et al., 1997) and biotechnol ogy-derived canola seed has not been demonstrated to persist long er than conventional canola seed (Gruber, et al., 2004; Hails et al., 1997). Populations of glypho sate-tolerant ruderal canola can be found along roadsides.Monsanto Company 11-CA-233U 398 of 500 Although glyphosate is frequently the herbicide of choice for roadside weed control (Powles, 2008), there are multiple other herbicide and mechan ical options that are available to control ruderal canola (OGTR, 2010; Gover, 2011).

I.4.11. Public Health

I.4.11.1. Canola Oil

As discussed in Petition Section II.A., the primary food use of ca nola is canola oil, which has

well established heart health be nefits. According to the OECD (2001) canola oil contains one key toxicant, erucic acid. A key toxicant is a potentially significant t oxic compound known to be inherently present in the species, in this case, B. napus (OECD, 2001). Canola oil, which by definition has less than 2% of its fatty acid as er ucic acid has been affirmed as GRAS by U.S. FDA. The Codex Standard for Named Vegetable Oils (Codex Alimentari us, 2005) also specifies

that canola oil cannot contain more than 2% er ucic acid. In addition the U.S. FDA has no objections to the recent GRAS notifica tion for canola prot ein isolate.

Canola oil derived from the current glyphosate-to lerant canola RT73 ha s been shown to be compositionally equivalent to canola oil de rived from conventional canola (Nickson and Hammond, 2002). As noted by the National Research Council (NRC), unexpected and unintended compositional changes arise with a ll forms of genetic mo dification, including both conventional breeding and geneti c engineering (NRC, 2004). However, the NRC also noted that no adverse health effects attributed to genetic engineering had been documented in the human population. Reviews on the nutri tional quality of biotechnology-de rived foods have generally

concluded that there are no si gnificant nutritional differenc es in conventional versus biotechnology-derived plants for food or animal feed (Faust, 2002; Flachowsky, et al., 2005).

I.4.11.2. Pesticide Use and Worker Safety

As discussed in Appendix Section I.3.1, the U. S. EPA regulates herbicides under FIFRA and

established tolerances for herbicide levels on f ood, for protection of human and animal health. The U.S. EPA also regulates other pesticides, incl uding insecticides and fungicides, that are used on canola and establishes applicatio n rates that are deemed not to have an unreasonable adverse effect. The herbicides, insec ticides and fungicides typically used on canola are discussed in

Petition Sections VIII.E and VIII.F. This di scussion focuses on glyphosate, as MON 88302 is tolerant to glyphosate. Glyphosate has been wide ly used in canola fields since 1999 when the first generation Roundup Ready canola RT73 was launc hed. The total rate of glyphosate in-crop application to Roundup Ready canola RT73 is 0.78 lb a.e. per acre. With MON 88302 growers will have the option to make applications at 1.55 pounds a.e. per acre of glyphosate in-crop. The rates for MON 88302 are comparable to the rates used for maize and soybean (approximately 2 pounds a.e. per acre of glyphosate in-crop).

Glyphosate is a herbicide approved for use (register ed) by the U.S. EPA for the control of weeds that would interfere with the growth of many food and non-food crops , including biotechnologyderived crops, as well as for c ontrol of weeds growing in non-crop areas. Currently over 70% of canola acres in the major U.S. canola produci ng region are treated with glyphosate as a component of a weed control regimen either in-cr op or pre-plant. A more detailed discussion onMonsanto Company 11-CA-233U 399 of 500 the potential impact of glyphosate on human h ealth and the environment may be found in Appendix K.

According to the Reregistrati on Eligibility Decision (RED) doc ument for glyphosate (U.S. EPA, 1993), glyphosate is of relatively lo w oral and dermal acute toxic ity. For this reason, glyphosate has been assigned to Toxicity Ca tegories III and IV for these e ffects (i.e., Toxicity Category I indicates the highest degree of acu te toxicity, and Category IV the lowest). An acute inhalation study was waived by the U.S. EPA because glyphosate is a non-volatile solid, and the studies conducted on the end-use product formulation ar e considered sufficient (U.S. EPA, 1993). Expert toxicological reviews the World H ealth Organization (WHO-FAO, 2004) are in agreement that glyphosate does not pose any hu man acute exposure concerns for dietary exposures and thus negated the need to establish an acute reference dose.

Based on the toxicity of glyphosate and its regi stered uses, including use on glyphosate-tolerant crops, the U.S. EPA has conclu ded that occupational exposur es (short-term dermal and inhalation) to glyphosate are not of concern because no short-term dermal or inhalation toxicity endpoints have been identified for glyphosat e (U.S. EPA, 2006a; U.S. EPA, 2006b).

Additional evidence to support the U.S. EPA conclusion can be found in the Farm Family Exposure Study (Acquavella, et al., 2004), a bi omonitoring study of pesticide applicators conducted by independent investigators. This biomonitoring study determined that the highest estimated systemic dose of glyphosate for applicator s as the result of routin e labeled applications of registered glyphosate-based ag ricultural herbicides to crops , including glyphosate-tolerant crops, was approximately 400 times lower th an the RfD established for glyphosate. Furthermore, investigators determined that 40% of applicators did not ha ve detectable exposure on the day of application, and 90% of the applicators had an estimated systemic dose of glyphosate less than 0.06% the RfD (Acquavella et al., 2004).

The biomonitoring study also found little evidence of detectable exposure to individuals on the farm who were not actively involved in or located in the immediate vicinity of labeled applications of glyphosate-based ag ricultural herbicides to crops. Considering the similarity of the use pattern and appli cation rates of the glyphosate products in this study compared to those registered for use on glyphosate-tolerant crops, bystander exposure attri buted to the use of glyphosate on glyphosate-tolerant crops is expected to be negligible. Therefore, the use of currently registered products containing glyphosa te in accordance with the labeling will not pose

unreasonable risks and adverse effect s to humans or the environment. In general, the herbicidal activity of glyphosate is due primar ily to a metabolic pathway that does not occur in humans or other animals, and thus, this mechanism of action is not directly relevant to the human health risk assessment. The U.S. EPA considers glyphosate to be of low acute and chronic toxicity.

Glyphosate is not considered a car cinogen; it has been classified by the U.S. EPA as a Group E carcinogen (evidence of non-carcinogenici ty for humans) (U.S. EPA, 1993).

I.4.12. Animal Health

As discussed in Petition Section II.A., canola meal is a high protein animal feed. Canola is by definition low in erucic acid and low in glucosinol ates, and canola meal has a history of safe useMonsanto Company 11-CA-233U 400 of 500 as an animal feed source. A lthough canola can be used as a forage crop, the use is limited because of its potential high levels of sulfate, and potential to cause bloat and other health problems in livestock (NDSU, 2008).

The OECD (2001) identifies one ke y toxicant in canola meal, glucos inolates. Glucosinolates are organic compounds that contain bot h sulfur and nitrogen, are found abundantly in plants of the

Brassica genus and are responsible for the pungent or biting flavors found in closely related

Brassica species such as mustard and horseradish. Th e low levels of glucosinolates in canola as compared to other oilseed rape make the meal sa fe for use as animal feed. The standard for glucosinolates in canola is 30 μmoles/g (OECD, 2001).

Canola meal from biotechnology-deri ved canola has been shown to be as safe and nutritious for

animals as canola meal from conventional canol a (Nickson and Hammond, 2002; Taylor, et al., 2004). In a study of the effects on pigs of canola meal derived from glyphosate-tolerant canola compared to meal derived from the non-biot echnology-derived parent, researchers concluded that the CP4 EPSPS protein has no effect on growth performance, carcass characteristics, or pork quality (Caine, et al., 2007).

I.4.13. Animal, Plant and Microbial Communiti es Including Threatened and Endangered Species

This section discusses animal and plant communiti es that may be affect ed by canola production. Because MON 88302 is expected to serve as an alternative to Roundup Ready canola, RT73, it

also discusses the affected environment of anim al and plant communities as they relate to current, widespread glyphosate ap plication on canola fields.

As a part of the reregistration evaluation unde r FIFRA, the U.S. EPA conducted an ecological assessment for glyphosate. This assessment compared the results from toxicity tests with glyphosate conducted with various plant and animal species to a conservative estimate of glyphosate exposure in the environment. In the Reregistration Eligibility Decision (RED) for glyphosate (U.S. EPA, 1993), the exposure estimates were determ ined assuming an application rate of 5.0625 pounds a.e. glyphosate per acre, 36 which exceeds the maximum labeled use rate for a single application for agricultural purposes . In its analysis, the U.S. EPA used the 5.0625 pounds a.e.per acre rate to calcula te the estimated environmenta l concentrations (EECs) for aquatic plants and animals. Ba sed on this assessment, the U.S. EPA concluded that effects to birds, mammals, fish and invertebrates are minimal (U.S. EPA, 1993).

I.4.13.1. Animal Communities

The affected environment for growing canola plants can generally be considered the agroecosystem (managed agricultu ral fields) plus adjacent ar eas extending beyond the intended plantings that might be affected by agricult ural operations. Mammals and birds, including migratory mammals and birds, may seasonally consume seeds from the planted fields, and invertebrates can feed on the pl ant and surrounding vegetation duri ng the entire growing season.

36 Although the EPA (1993) refers to the maximum application rates as a.i. (active ingredient), it can be determined

that the maximum rate used in the calculations of the EECs was in a.e. (acid equivalents).Monsanto Company 11-CA-233U 401 of 500 Rodents and other small animals may inhabit canol a fields, and the raptor s, snakes and other animals that may prey on them are part of the a ffected environment. Deer may also browse in canola fields on the forage. Fish and other aqua tic organisms in streams draining agricultural fields are also part of the affected environment.

Glyphosate is practically nontoxic to slightly toxic to birds, freshwa ter fish, marine and estuarine species, aquatic invertebrates and mammals and practically nontoxic to honey bees (which are used to assess effects on nontarget insects in general) (U.S. EPA, 1993). Glyphosate has a low octanol-water coefficient, indicating that it has a tendency to remain in the water phase rather than move from the water phase into fatty substan ces; therefore, it is not expected to accumulate

in fish or other animal tissues.

I.4.13.2. Plant Communities

The affected environment for growing canola plants can generally be considered the agroecosystem (managed agricultural fields) ar eas as well as adjacent areas extending beyond the intended plantings that might be affected by agricultural operati ons. Plants, extraneous to the crop, which grow in planted fields can be consid ered weeds and are dealt with in a separate

section in this document. Plan ts not growing in a field amongs t the canola would include those in ditches, hedge rows, fence ro ws, wind breaks, yards, etc.

Glyphosate is a non-selective herb icide with post-emergence activ ity on essentially all annual and perennial plants and has the potential to impact nontarget plants as a result of runoff or spray drift (U.S. EPA, 1993). Regarding runoff, glyphosat e binds strongly to agri cultural soils and has low potential to move offsite dissolved in wate r (U.S. EPA, 1993). More over, glyphosate is not taken up from agricultural soils by plants. Theref ore, any potential effect s to non-target plants from glyphosate use are only attributed to spray dr ift. During the re-registration process in 1993,

additional data on terrestrial non target plants were requested by the U.S. EPA. These additional

data have been utilized in co njunction with an exposure assessm ent to further understand the potential risk to nontarget and threatened a nd endangered plants from the use of glyphosate herbicides in agriculture. Using the met hodology described in a recent U.S. EPA effects determination for glyphosate (U.S. EPA, 2008b), it can be determined that there is minimal risk to terrestrial plants that are not listed as threatened or endangered at the maximum glyphosate single application rate for ground applications (3.75 pounds a.e. glyphos ate per acre), and at rates just below the maximum single aerial applicat ion rate (1.48 pounds a. e. glyphosate per acre compared to 1.55 pounds a.e. glyphosate per acre).

I.4.13.3. Soil Microorganisms

Microbial populations and associated biochemical processes are critical to maintaining soil health and quality. The occurrence and abundance of soil microorganisms are affected by 1) soil characteristics like tilth, organic matter, nutri ent content, and moistu re capacity, 2) typical physico-chemical factors such as temperature, pH, and redox poten tial, and 3) soil management practices. Agricultural practices such as fer tilization and cultivation may also have profound effects on soil microbial populations, species composition, colonization, and associated biochemical processes (Buckley and Schm idt, 2001; Buckley and Schmidt, 2003). Consequently, significant variation in microbi al populations is expected within and amongMonsanto Company 11-CA-233U 402 of 500 agricultural fields. A recent study performed on the effects of glyphosate-tolerant canola on soil microbial biomass, functional diversity and en zyme activity (Lupwayi, et al., 2007) found that effects on soil microorganisms were minor and inconsistent over a wi de range of growing conditions and crop management.

The effects of glyphosate and glyph osate-based formulations on so il microorganisms have been extensively investigated (Cerdeira and Duke, 2006; Sullivan and Sullivan, 2000). Results of standardized tests with glyphosate formulations pe rformed for submission to regulatory agencies indicate no long-term effects on microorganisms in soil even at rates that exceed maximum use rates (up to five times the labeled rate). In addition, independent res earchers have reviewed numerous laboratory and fi eld studies, investigating the eff ects of glyphosate on soil bacteria and fungi (Felsot, 2000; Giesy, et al ., 2000) and have concluded th at glyphosate has a “remarkable level of safety for virtually all organisms test ed”. In a recent gree nhouse investigation, Arango (2009) evaluated the effects of multiple glyphos ate applications on the rhizosphere bacterial community associated with glyp hosate-tolerant soybean. While subtle, transient shifts in community structure were noted after glyphosate applications , effective resilience and no reduction in bacterial diversity were observed for the bacterial community associated with roots of glyphosate-treated versus unsprayed glyphosat e-tolerant soybean. Although some laboratory tests have shown effects on nitrogen-fixing bact eria (Moorman, et al., 1992; Santos and Flores, 1995) and soil fungi (Busse, et al., 2001; Estok, et al., 1989), effects are typically observed only under artificial laboratory conditions and at glyphosate concentra tions well above normal field application rates. Several researchers have concluded that it is difficult to extrapolate results from the laboratory to the natural soil environment (Busse etal., 2001; Wan, et al ., 1998; Wardle and Parkinson, 1990a; (Wardle and Parkinson, 1990b).

Investigations by Haney, et al., (2000; 2002) related to the increased use of glyphosate-tolerant crops indicate that glyphosate was degraded ove r time by soil microbes, even at high application rates, without adversely impacting soil microbial activity. In addition, results from field studies that have evaluated the fungal component of the soil microbial community indicate that glyphosate treatment had no deleterious effects on beneficial soil fungi (Araujo, et al., 2003; Biederbeck, et al., 1997; Busse et al., 2001 ; Wardle and Parkinson, 1990a; Wardle and Parkinson, 1990b). In a 4-year field study, Powe ll, et al. (2009) assesse d effects of glyphosate applications on soil food web properties and crop litter decomposition in a glyphosate-tolerant soybean and maize rotation. The researchers conc luded that: “Permanent responses in soil biota were not observed, suggesting a high level of resili ence in the soil biota and a lack of a persistent effect resulting from the GM cropping system.” Furthermore, Liphadzi, et al. (2005) observed

that the soil microbial and nematode commun ity was similar when glyphosate or conventional herbicides were applied to crop rotations of glyphosate-tolera nt soybean and maize cultivars. Moreover, the history of safe use and yield da ta obtained for nearly 15 years of glyphosatetolerant crop production, combined with in-crop appli cations of glyphosat e-based agricultural herbicides, reinforce the findings that soil micr obes and microbially mediated processes are not adversely impacted by field-rate applications of glyphosate.

I.4.13.4. Threatened and Endangered Species

Plants or animals that may inhabit areas adjacent to agricultural fields and that U.S. Fish and Wildlife Service (FWS) has identified as threat ened or endangered under the Endangered SpeciesMonsanto Company 11-CA-233U 403 of 500 Act may be part of the a ffected environment. No Brassica species in the U.S. are considered to be threatened or endangered. No Brassica species are utilized uniquely by threatened or endangered species for survival.

Like other animals and plants, threatened and endangered species that are present near canola fields could potentially be affect ed by runoff from the fields and by herbicide drift. As discussed in Appendix Section I.4.13.2, for glyphosate runoff is not a concern but herbicide drift may be.

Detailed information on the potential impacts to plant and animal communities (including threatened and endangered species) by glyphosate us e on glyphosate-tolerant crops is included in the USDA APHIS Final Environmental Impact Statement (FEIS) on the Deregulation of Glyphosate-Tolerant alfalfa. The information is presented in Chapter IV.C and Appendix N of the Alfalfa FEIS and is incorporated in this EA by reference, and summarized below.

As documented in the Glyphosate Tolerant Alfa lfa FEIS (USDA-APHIS, 2010), spray drift is one of the pathways of concern for non-target plants; therefore, Monsanto prepared an analysis of the risk to TE species to evaluate the im pacts to plants and animals from the use of glyphosate-based herbicides in conjunction with glyphosate-tolerant plants. The complete analysis was submitted to APHIS and has been reviewed by APHIS scientists to support the petition for deregulation of glyphos ate tolerant alfalfa. The c onclusion from the APHIS review is that threatened or endangere d terrestrial or semi-aquatic plan t species are not at risk from ground applications of glyphosate at rates less than 3.5 lbs a.e. glyphosate per acre, or from aerial applications at rates less than 0.70 lb a.e. glyphosate per acre. To address this issue, Monsanto has developed a web-based mitigation program calle d Pre-Serve that describes location-specific mitigation measures that must be implemen ted when glyphosate is applied at a ground application rate of 3.5 lbs a.e. gl yphosate per acre or more, or an aerial application rate of 0.7 lb a.e. glyphosate per acre or more Monsanto ’s Technology Use Guide (TUG), which is a contractual part of its licensing agreement with growers, requires growers to access the Pre-Serve

website and follow the instructions when their glyphosate ground application rate is 3.5 lbs a.e. glyphosate per acre or more or th eir aerial application rate is 0.7 lb a.e. glyphosate per acre or more (Monsanto Company, 2010). Only a very sma ll percentage of glyphos ate applications will require implementation of management practices. This is because the vast majority of U.S. cropland is not in close proximity to th reatened and endangered plant species.

I.4.14. Physical Environment

I.4.14.1. Land Use

The total U.S. acreage of canola increased mo re than 200% from 367,000 acres in 1996 to 1.1 million acres in 1998, coinciding with passage of the 1996 Federal Agriculture Improvement

Act. 37 Canola acreage peaked at 1.55 million in 2000 and has remained between 0.8 and 1.6 million acres since 2005 (Figure I-3).

37 The 1996 Federal Agriculture Improvement Act, 7 U.S.C. § 7201 et seq. , gave growers almost complete flexibility in selecting the crops they could plant.Monsanto Company 11-CA-233U 404 of 500 The acreage of glyphosate-tolerant canola increa sed rapidly following co mmercial introduction

in 1999 and has remained between 50 and 70% of planted canola acres since 2001. Fluctuations in total canola acreage before and after glyphosat e-tolerant canola was co mmercialized indicates

that factors unrelated to the availa bility of the glyphosate-tolerant tr ait play a larger role in acres planted than the availability of the glyphosate-tolerant trait.

In 2008 herbicide-tolerant canola varieties were planted on approximately 95% of the canola acreage in North Dakota (Zollinger et al., 2009). Glyphosate was applied to approximately 73% of the canola acreage in 2008 which includes pr e-plant burn down and in-crop applications.

Glufosinate-tolerant canola was introduced in 1995 and is tolerant to post-emergence applications of glufosin ate. Glufosinate is the second most commonly applied herbicide used in canola (38% of acres) in 2008.

As canola cropland has increase d, production of wheat, the main rotational crop for canola, has decreased. Future increases in canola production are likely to result from canola replacing some wheat rotations in wheat monocultures or croppi ng systems with wheat as the main crop (Petition Section VIII.H.). Greater increases in production could result if canola is used more widely for biodiesel. Canola may be a better candidate for biodiesel in Ca nada and some European Union countries, where canola production is higher and s oybeans are not grown on significant acreage. In the U.S., soybean oil is currently substant ially less expensive than canola oil and the USDA

ERS has concluded that this re duces the potential of canola as a biodiesel crop (USDA-ERS, 2010b)

I.4.14.2. Air Quality and Climate Change

Many agricultural activities affect air quality including tillage, traffic and harvest emissions, and nitrous oxide emissions from the use of nitroge n fertilizer. These agricultural activities individually have potentially adverse environm ental impacts on air quality and climate and may be impacted positively or negatively by changes in agricultural practices. Issues of concern include, but are not necessarily limited to, atmospheric emi ssion of carbon dioxide, nitrogen oxide, sulfur oxide, and particulate matter. Agricu ltural practices have th e potential to directly and indirectly impact air quality and to contribute emissions which could lead to climate change.

Tillage contributes to the release of greenhous e gases (GHG) because of the loss of carbon dioxide to the atmosphere and the exposure and oxidation of soil organic matter (Baker, et al., 2005). Emissions released from agricultural e quipment (e.g., irrigation pumps and tractors) include carbon monoxide, nitrogen oxi des, particulate matter, and su lfur oxides. Nitrous oxide may also be released following th e use of nitrogen fertilizer. Agriculture, including land-use changes for farming, is responsible for an estimated 17 to 32% of all human-induced GHG emissions. Herro (2008) proposes that if agri culture practices were modified, significant

reductions in the release of GHGs could be achieved.

I.4.14.3. Surface Water and Groundwater Quality

Tillage and Water Conservation

Tillage practices in canola production are disc ussed in Appendix Section I.4.7.2. The USDA

Economic Research Service defines conservation til lage as cultural operati ons that maintain atMonsanto Company 11-CA-233U 405 of 500 least 30% cover of the soil su rface by plant residue at the time of planting (Anderson and Magleby, 1997). Conservation tillage can encomp ass a range of management practices, from notill to ridge- and strip-till cultiva tion to minimum tillage systems that restrict equipment traffic to dedicated zones. Special tillage field equipmen t can often perform the equivalent functions of several standard implements, reducing the necess ity for multiple passes through the field. No-till is defined by USDA ERS (Anderson and Magleby, 1997) to be those practices that leave the soil undisturbed, and thus leave substantial crop re sidues on the surface of the planted field. Implementing conservation tillage practices can lead to both economic and production quality benefits, as well as having pos itive environmental impacts ( www.nrcs.usda.gov ). Use of conservation tillage compared to use of conventi onal tillage in many soils may allow 10 to 40% greater water infiltration into soils (Hoeft, et al., 2000a; Hoeft, et al., 2000b). Crop residues established by conservation tillage on soil surf aces slow water runoff, increase porosity by increasing numbers of wormholes and by means of remnants of crop residue, and reduce evaporation through the insulating ability of surface mulches. No tillage alone, without additional erosion control measures, reduced runoff and sediment loss by approximately 85% when compared with non-conservation chisel plow tillage (Zhou, et al., 2009), and no till can reduce runoff volume 35 fold compared to conventional tillage (Gregory, et al., 2005). Researchers have found significant increases in conservation tillage with the introduction of

herbicide-tolerant canola (Smyth, et al., 2010).

Water Quality

Surface water may be impacted from canola produc tion by runoff from canola fields that carries soil particles and herbicides or other pesticides to streams, rivers, lakes, wetlands and other water bodies. As discussed below, based on existing da ta, the soil component of runoff is a much more important contributor to surface water imp acts than is the pesticide component.

Tillage causes widespread soil disturbance. Thus, erosion, topsoil loss and the resulting sedimentation and turbidity in st reams are likely to increase with increased tillage. In 2009, based on the states’ water quality reports, the U. S. EPA identified sedimentation and turbidity as two of the top 10 causes of impairment to surface water in the U.S. in general; in 2007, the U.S.

EPA identified sedimentation/siltation as a leadi ng cause of impairment to rivers and streams (U.S. EPA, 2007; U.S. EPA, 2009). Although a comprehensive data set has not yet been developed to prove the point, the U.S. EPA has projected conservati on tillage to be the major soil protection method for improving surface water quality (U.S. EPA, 2002). The U.S. EPA identifies conservation tillage as the first of its CORE four agricultural management practices for water quality protection (U.S. EPA, 2008a).

Based on the states’ water quality reports to the U.S. EPA, which the EPA makes available through its National Assessment Database, pesticides in general and herbicides in particular are a relatively minor contributor to impairment of surface water in the U.S., compared to sedimentation/siltation and turbidity (U.S. EPA, 2007; U.S. EPA, 2009). Pesticides accounted for less than one percent of reported causes of surface water impairment in all but three of the nine leading U.S. canola-producing states. In thos e three states, pesticides accounted for three to ten percent of reported causes of impairment. Of the pesticides that were reported as contributing to impairment among the nine l eading canola-producing states, almost all areMonsanto Company 11-CA-233U 406 of 500 previously used, highly persistent chemicals that are no longer re gistered for use in the U.S. (EPA 2008a).

I.4.15. Economics

The affected trade economic environment is de fined as those countries with which the U.S. engages in canola feed, seed and food trade. Th e affected domestic environment is defined as any land in the U.S. that is cu rrently producing crops that could incorporate a canola rotation, as

well as land that could be converted from inac tive cropland to active cropland, and land currently in the Conservation Reserve Program (CRP) that could be removed from the program and farmed. CRP is a voluntary program sponsored by the USDA offering annual rental payments over a 10-year contract period, as well as cost-sha re assistance, to producers establishing specific types of plant cover on marginal farmland. The affected environment for economics is primarily the farming communities where canola is grown. Production costs for canola are discussed in Appendix Section I.4.7.1.

The U.S. produced 1.9% (USDA-NASS, 2011c) of the world’s 60.6 million metric tons of canola in the 2010 growing season (USDA-FAS, 2011c), and imported 0.7 million metric tons from Canada (Statistics Canada, 2011). 38 Total global U.S. imports of canola oil continue to increase steadily from 0.5 million metric tons in 2000 (USDA-FAS, 2011a) to 1.2 million metric tons in 2010 (USDA-FAS, 20111b) . Although canola production in the U.S. has increased dramatically since the 1980s, it has always been far short of demand (F igure I-2). The U.S. shortfall is made up with imports primarilyfrom Canada. In 2010 canola was planted on approximately 1.45M acres in the United States pr oducing 1.1 million metric tons of canola, with a value of approximately $487 million (USDA- NASS, 2011a; USDA-NASS, 2011b). In a 2010 dollar-value comparison with other major crops, ca nola represents 0.7% of the value of the maize crop, 1.3% of the value of the soybean crop an d 3.7% of the value of the wheat crop (USDANASS, 2011b).

Canola has been able to benefit from the sa me price subsidies as other commodity crops, strengthening the profit potential of this crop. In most side-by-si de tests, canola has provided a greater net profit than wheat, t hough ease of marketing and variab ility in yield are off-setting

factors.

Researchers have found that gr owers have realized substantial economic benefits with the adoption of herbicide-tolerant canola. A survey of canola growers in western Canada revealed

that herbicide-tolerant tec hnology generated from $1.063 – 1.192 billion in annual direct and indirect benefits over the 2005-2007 period partly attributed to lower i nput costs and partly

attributed to better weed cont rol (Smyth et al., 2010).

I.5. Alternatives

The decision-making process of deregulati on is governed by 7 CFR § 340.6 (d)(3)(i) which states that APHIS may approve the petition in whole or in pa rt, resulting in three possible outcomes from Monsanto’s petition, described below.

38 The USDA FAS reports do not distingui sh canola but report all rapeseed.Monsanto Company 11-CA-233U 407 of 500 I.5.1. Alternatives Studied in Detail

I.5.1.1. Deregulation in Whole Alternative

Under the “deregulation in w hole” alternative, MON 88302 w ould no longer be a regulated

article under 7 CFR Part 340 and would be wi dely available for planting without prior authorization in a permit or notification. With MON 88302, growers would have increased flexibility for glyphosate application on their canola crops, the option of applying glyphosate later in the growing season and the option to apply at higher rates.

I.5.1.2. No Action Alternative

Under the “no action” alternat ive, MON 88302 would remain a regulated article under 7 CFR Part 340. MON 88302 could be grown under USDA notification or permit and confined release conditions. However, currentl y deregulated herbicide-tolera nt canola events including glyphosate-tolerant RT73 would con tinue to be availabl e and would be expected to be widely

grown.

I.5.2. Alternatives Considered and Dismissed

I.5.2.1. Approval in Part

The “approval in part” alternat ive is dependent upon a finding of potential plant pest risks for MON 88302 in certain geographies or under certain conditions. APHIS should impose conditions upon the cultivation or use of MON 88302 only in specific geographies or with specific conditions to mitigate any such identified plant pest risks. MON 88302 has been thoroughly characterized and the extensive inform ation presented in Petition Sections I through

IX demonstrates that MON 88302 does not present a plant pest risk in any of the geographies or under any conditions where M ON 88302 may be grown. Therefore, there is no basis for imposing geographic or other conditions on MON 88302. Monsanto has requested an

unconditional determination that MON 88302 poses no plant pest risk, and is therefore not subject to regulation under th e Plant Protection Act.

I.6. Environmental Consequences

I.6.1. Commercial Canola Production and Use

No Action Alternative: Under the No Action Alternative, MON 88302 and its progeny would continue to be regulated articles under the regulations at 7 CFR pa rt 340. Under this alternative planted canola acres in the U.S. may continue to increase in response to increases in consumer demand for canola oil. Based on recent trends, if increases in canola acreage occur, they will likely result from continued additions to wheat rotations (i.e., canola would be introduced as a rotation crop with wheat). General locations of production and uses of canola would be expected to continue as they are now. Herbicide-tolerant canola varieties would likely continue to be used on the vast majority of canola acres.

Deregulation in Whole Alternative: Biotechnology improved crops ar e subject to regulation in many countries. In order that canola seed harves ted in the U.S. may be freely traded, MonsantoMonsanto Company 11-CA-233U 408 of 500 will seek regulatory approval for MON 88302 and its combinations with other biotechnologyderived traits, where required, in all key canola import countries with a functioning regulatory system to support the flow of inte rnational trade (P etition Section VIII.K.). Monsanto adheres to the BIO Product Launch Policy 39 including: 1) conducting a mark et and trade assessment, 2) securing regulatory approvals in key export countries prior to full commercial launch, 3) following generally accepted best seed manageme nt practices to prevent unintended low level presence of the event in seed, 4) providing relia ble detection methods to growers, processors and buyers prior to commercialization, and 5) communi cating to stakeholders the company’s product launch stewardship policies. Thes e actions protect against adverse impacts to trade of canola due

to the introduction of new biotechnology-derived canola.

Under deregulation in whole the same potential increases in canola acres planted may be expected for the same reason as for the no action a lternative. The majority of commercial canola grown in the U.S. is already glyphosate-tolera nt, and company stewardship policies described

above ensure there will be no disruption of trade from the in troduction of new biotechnologyderived products. The difference between the no action and the deregulation in whole alternative is expected to be the gradua l replacement of the current Ro undup Ready canola RT73 event with

MON 88302. As described in Petition Secti ons VI. and VII., MON 88302 is compositionally and phenotypically equivalent to conventional canola. MON 88302 al lows for a wider period of glyphosate application than RT73 at rates compar able to those already used for maize and soybean. The change in the biotechnology-derived trait, and the resulting allowa ble increase in glyphosate application, is not expected to have any imp act on canola production and use. Therefore, the no action alternative and the appr oval in whole alternative would not differ in their impact to commercial canola production.

I.6.2. Seed Production

No Action Alternative: Under the No Action Alternative, MON 88302 and its progeny would continue to be regulated articles under the regulations at 7 CFR pa rt 340. Under th is alternative, seed production practices and geographies wher e canola seed is produced are expected to continue as described in Appendix Section I.4 .2. The widespread use of herbicide-tolerant canola would likely continue, a nd canola seed production practices associated with herbicidetolerant varieties is no t expected to change.

Deregulation in W hole Alternative: Certified seed production is a carefully managed process (Petition Section VIII.B.) for maintaining high quali ty seed stocks, an essential basis for U.S. agriculture. Seed producers have learned to acc ount for and manage pollen flow both within a seed production field and between nearby fields . For decades the canola seed industry has created and adopted systems to maintain and pres erve the purity of canola seed developed for commodity and specialty uses. The Associati on of Seed Certifying Agencies (AOSCA) is dedicated to assisting companie s in the production, identification, distribution and promotion of

certified classes of seed. AOSCA establishes mi nimum standards for quality and identity. Its

39 BIO’s Product Launch guidelines can be found at: http://www.excellencethroughstewardship.org/facts/doc uments/Guide%20for%20Product%20Launch%20Stewardsh ip.pdf

.Monsanto Company 11-CA-233U 409 of 500 goal is to standardize certification regulations and procedures interna tionally so companies compete with one set of standards. The as sociation cooperates with the Organization for Economic Cooperation and Development (OECD) and other internationa l organizations to develop standards, regulations, procedures, and policies to expe dite movement of seed and encourage international commerce in improved seed products.

The difference between the no action and the deregulat ion in whole alternative is expected to be the gradual replacement of the current R oundup Ready canola RT73 event with MON 88302. It is anticipated that seed containing MON 88302 will be produced and marketed in accordance

with OECD and AOSCA standards and the U.S. Federal Seed Act, and will have no adverse impact on current canola seed production practices or the ability of bree ders and seed producers to meet these standards. The only change to seed production practices that may result from the deregulation of MON 88302 would be a potential change in weed ma nagement in seed production operations relative to practices used to produce existing glyphosate-tolerant varieties. Impacts on weed management from the introdu ction of MON 88302 are discussed in Appendix Section I.6.5. The change in the biotechnology-der ived trait, and the potential changes in weed management, is not expected to have any imp act on canola seed producti on. Therefore, the no action alternative and the approval in whole altern ative would not differ in their impact to canola seed production.

I.6.3. Specialty Canola Production

No Action Alternative: Under the No Action Alternativ e, MON 88302 and its progeny would continue to be regulated articles under the regulations at 7 CFR pa rt 340. Under th is alternative, specialty canola production and other Brassica production, including Brassica vegetable production, is expected to conti nue as described in Appendix Sections I.4.3., I.4.4. and I.4.5. Specialty canola growers would c ontinue to manage their produc tion fields to avoid excluded methods including drift from pesticides

Additional new types of specialty canola may be introduced in the future. The very small acreages of organic canola produc tion are likely to continue. Herbicide-tolerant canola will continue to be widely grown, and practices utilized to assure identity preservation of specialty canola would continue to be implemented.

Deregulation in Whole: Production systems designed prior to the introduction of MON 88302 or even prior to the introduction of biotechnology -derived canola have allowed for production of

canola to meet varied customer demands. For example, organic canola producers use production practices designed to specifically avoid the presence of canola pr oducts that use herbicides or other pesticide treatments, as well as biot echnology-derived canola. These well established practices to avoid “excluded methods” will con tinue with the introduction of MON 88302. Currently, organic canola production occurs on a very small scale in the U.S., in areas removed from the major canola production areas. The change in the biotec hnology-derived trait to MON 88302 will have no impact on organic canola production practic es relative to the no action

alternative

The primary Brassica vegetable crops in the U.S. are B. napus (rutabaga and Siberian kale),

B. oleracea (cabbage, broccoli, caulifl ower, Brussels sprouts, collard, kale, kohlrabi), B. junceaMonsanto Company 11-CA-233U 410 of 500 (red giant mustard, sawtooth mustard, and others) and B. rapa crops (Chinese cabbage, pak choi, choi sum, turnip, mizuna, mibuna, tat soi and others). Brassica vegetables grown in the U.S. are harvested in the vegetative stage thus minimizing the potential for cross pollination with canola. Additionally, Brassica vegetable species are gr own primarily in states such as California, Arizona, New York, Florida, Georgia, North Ca rolina, Wisconsin, Texas and Michigan where canola production is minimal. In stat es such as Washington and Oregon where Brassica vegetable crops and canola may both be grown, all Brassica seed crops are grown only through participation in a USDA extension pinning pro cess that maps production sites and ensures adherence to appropriate isolation distances.

The difference between the no action and the deregulat ion in whole alternative is expected to be the gradual replacement of the current R oundup Ready canola RT73 event with MON 88302. The adoption of biotechnology-derived trait M ON 88302 will have no impact on the current processes employed for planting and identity pres ervation in specialty canola, including organic

canola or canola with modi fied oil composition or Brassica vegetables. Therefore, the no action alternative and the approval in whole alternative would not differ in thei r impact to specialty

canola or Brassica vegetable production.

I.6.4. Agronomic Practices – Tillage, Irrigatio n, Crop Rotations, Insect and Disease Management

No Action Alternative: Under the No Action Alternative, MON 88302 and its progeny would continue to be regulated arti cles under the regulations at 7 CFR part 340 and would not be widely grown. Under this alternative, the ag ronomic practices for tillage, irrigation, crop rotation, insect pest management and disease mana gement are expected to remain the same as those described in Appendix Section I.4.7.

Deregulation in Whole: MON 88302 has been shown to be no different from conventional canola in its agronomic and ecologi cal characteristics (Petition Sect ions VII, VIII and IX), and

has the same levels of resistance to insects and diseases as conven tional canola. Therefore, other than applications of glyphosate for weed control, at rates comparable to those currently used for maize and soybean and a wider application window , there are no anticipated changes to current agricultural practices due to the introduction of MON 88302

The difference between the no action and the deregulat ion in whole alternative is expected to be the gradual replacement of the current R oundup Ready canola RT73 event with MON 88302. The adoption of biotechnology-derived trait M ON 88302 will have no impact on the current agronomic practices of tillage, ir rigation, crop rotations and in sect and disease management. Therefore, the no action alternative and the a pproval in whole alternative would not differ significantly in their impact to canola agronomic practices.

I.6.5. Agronomic Practices - Weed Management

No Action Alternative: Under the No Action Alternative, MON 88302 and its progeny would continue to be regulated articles and would not be widely grown. Under this alternative, weed management in canola is expected to continue as described in Appendix Section I.4.7.9. Under the no action alternative, growers will likely continue to use the current Roundup Ready canolaMonsanto Company 11-CA-233U 411 of 500 RT73 and associated glyphosate use on approximately the same percentage of acres as currently. Other weed control pr actices would continue to be utilized at current rate s. Based on U.S. EPA approved labels for Roundup agricultural herb icides, users of Roundup Ready canola RT73 can apply a maximum of 0.78 pounds acid equivalent (a .e.) per acre of glyphosate, from emergence to the 6-leaf stage. Growers can apply glyphosat e once at a rate of up to 0.56 pound a.e. per acre or twice, at a rate of up to 0.39 pounds a.e. per acre. Weeds such as dandelion and Canada thistle would continue to be problematic in canola fields.

Deregulation in Whole Alternative: The difference between the no action and the deregulation in whole alternative is expected to be the gra dual replacement of the current Roundup Ready canola

RT73 event with MON 88302. As described in Petition Section VII. MON 88302 is phenotypically equivalent to conventional canola. Therefore, other than changes in herbicide use, weed management practices would not change. MON 88302 gives the grower additional options for glyphosate use. Monsanto has subm itted amended labeling to the U.S. EPA that proposes to modify the current use pattern of glyphosate in canola based on MON 88302. If approved, MON 88302, growers will have the option to apply up to 1.55 pounds a.e. per acre of glyphosate from emergence to first flower. If the full 1.55 pounds a.e. per acre is applied at once, it must be done before the 6-leaf stage. Alternatively the gr ower may apply glyphosate twice, at rates up to 0.77 pounds a.e. per acre, up to first flower. These rates are similar to those currently allowed for soybeans (2.0 pounds a.e. per acre of glyphosate) and maize (2.0 pounds a.e. per acre of glyphosate), and based on weed control results in soybeans and maize at these glyphosate rates, it is expected th at control of problematic weed s such as dandelion and Canada thistle would be improved. In addition, the in creased window of glyphosate application will provide growers with greater flexibility in when they can apply glyphosate.

With the deregulation of MON 88302 glyphosate could be used for w eed control in seed production acres which may result in a decrease in the application of some currently used herbicides. However Monsanto recommends, for weed control in hybrid seed production acres, and for all canola production acres that glyphosate be used in conjunction with pre-emergent herbicides to achieve the best possible control and mitigate concerns over the development of weed resistance (discussed in Appendix J). Add itionally 3 to 4 year crop rotation practices are not expected to change as they are in place to minimize fungal disease cycles. These rotation practices also minimize the potential for the development of glyphosate resistant weeds.

Given the above, the adoption of biotechnology-d erived trait MON 88302 will have no impact on the current weed management practices other than higher rates of glyphosate may be applied over a wider window. Therefore, the no action alternativ e and the approval in whole alternative would not differ significantly in their imp act to canola weed management practices.

I.6.6. Volunteers

No Action Alternative: Under the No Action Alternative, MON 88302 and its progeny would continue to be regulated articles and would not be widely grown. Under this alternative, growers are expected to continue to use the management practices for control of canola volunteers as described in Appendi x Section I.4.7.10.Monsanto Company 11-CA-233U 412 of 500 Deregulation in Whole: The difference between the no action and the deregulation in whole alternative is expected to be the gradual re placement of the current Roundup Ready canola RT73

event with MON 88302. As desc ribed in Petition Section VII. , MON 88302 is equivalent to conventional canola in terms of seed germina tion, dormancy and emergence. In addition no

differences were detected between MON 88302 a nd the conventional control for early stand count, seedling vigor, pod shatte ring, seed quality, yield and fi nal stand count. Based on the assessed characteristics, the re sults of this study demonstrate that there were no unexpected changes indicative of increased plant pest pot ential or adverse environmental impact of MON 88302 compared to conventional canola. Th erefore, control of volunteer canola is not expected to change from that described in Appendix Section I.4.7.10. In addition to using integrated weed management and cultural pr actices, growers may c ontrol volunteer MON 88302

canola with herbicides other th an glyphosate and cultural practices just as they now control Roundup Ready canola RT73 volunteer canola with he rbicides other than glyphosate and cultural practices.

I.6.7. Weed Resistance

No Action Alternative: Under the No Action Alternative, MON 88302 and its progeny would continue to be regulated article s and would not be widely grown. For the reasons described in Appendix Section I.4.7.11. and Appendix J, weeds th at are glyphosate resist ant are not currently an issue in canola production. With appropriate integrated weed mana gement practices, this condition would be expected to co ntinue. Under the no action alte rnative, growers are expected to continue to use current weed management practices.

Deregulation in Whole: The difference between the no action and the deregulation in whole alternative is expected to be the gradual re placement of the current Roundup Ready canola RT73 event with MON 88302. Canola, including glyphosat e-tolerant canola, is produced in a crop rotation system that has relatively low susceptib ility for development of weeds with herbicide

resistance. As discussed in A ppendix Section I.4.7.13, canola is ty pically grown in a three- to four-year rotation with sm all grains (wheat, barley and oats) th at use herbicides with a different mode of action than glyphosate. The change from RT73 to MON 88302 will not affect these agronomic practices. Thus, the higher rate and wider period of glyphosat e application that can be used with MON 88302 is not expected to have an impact on development of herbicide

resistance in weeds.

I.6.8. Raw and Processed Ag ricultural Commodities

No Action Alternative: Under the No Action Alternativ e, MON 88302 and its progeny would continue to be regulated articles and would not be widely grown. Under this alternative the raw and processed agricultural commodities derived from canola are expected to be the same as described in Appendix Section I.4.8.

Deregulation in Whole: Biotechnology-derived canola pr oducts like MON 88302 undergo a voluntary food and feed consultatio n process with the U.S. FDA prior to release on the market. Monsanto has already initiated this process a nd will complete the consultation prior to a

commercial introduction of MON 88302.Monsanto Company 11-CA-233U 413 of 500 The difference between the no action and the deregulat ion in whole alternative is expected to be the gradual replacement of the current R oundup Ready canola RT73 event with MON 88302. Compositional assessments (Petition Sec tion VI) conducted on MON 88302 seed support a conclusion that the composition of the seed is equiva lent to that of the c onventional control. The genetic modification in MON 88302, has no imp act on the composition, and therefore on the food and feed safety or nutriti onal quality of this product co mpared to conventional canola. Residue tolerances for glyphosat e in canola will not change.

Based on this information it is unlikely that the deregulation of MON 88302 would cause a significant impact on either raw or processed ca nola commodities. Therefore, the no action alternative and the approval in w hole alternative would not differ si gnificantly in their impact to canola weed manage ment practices.

I.6.9. Potential for Cross Pollination and Gene Flow to Sexually Compatible Species

No Action Alternative: Under the No Action Alternative, MON 88302 and its progeny would continue to be regulated articl es and would not be widely grow n. As such, any plantings of MON 88302 would take place under APHIS notif ication or permit under conditions that minimize the potential for persistence of the regulated article in the environment. The potential for canola to cross pollinate in canola crops, canola seed production, other Brassica crop production, other Brassica seed production and the potential for cross pollination to native species and weeds, is discussed in Appendix Secti on I.4.9. As discussed in that section, there are reports of unassisted (e.g. without human interv ention) hybridization under field conditions with B. napus as the pollen donor with six other species including B. rapa

, B. juncea

, B. oleracea

, H.

incana

, R. raphanistrum and S. arvensis (see Petition, Table IX-3). In all cases the resulting hybrids had decreased environmenta l fitness evidenced by a variety of characteristics including decreased pollen viability, seed production and s eedling survival, when compared to parental

varieties.

As a result of state and federal regulatory requ irements, seed certification requirements, seed grower agreements, geography, and/or biological c onstraints provide barriers to cross pollination

between B. napus and other closely related species during seed production. Thus cross pollination during seed production is minimal to non-existent. Under the no action alternative growers are expected to conti nue to use biotechnol ogy-derived, herbicide-tolerant canola having the same potential for cross pollina tion with sexually compatible sp ecies as conventional canola.

Deregulation in Whole: The difference between the no action and the deregulation in whole alternative is expected to be the gradual re placement of the current Roundup Ready canola RT73

event with MON 88302. As described in Peti tion Section VII. , MON 88302 is phenotypically equivalent to conventional canola and is not e xpected to have a greater potential for cross pollination with related species than conventiona l canola. Therefore, the no action alternative and the approval in whole alternative would not differ significantly in their impact to cross pollination in canola.Monsanto Company 11-CA-233U 414 of 500 I.6.10. Ruderal Canola

No Action Alternative: Under the No Action Alternativ e, MON 88302 and its progeny would continue to be regulated article s and would not be widely grow n. Most ruderal canola results from seed loss during transportation and is f ound growing at the edges of roadways and along alternative transportation routes . Multiple studies of roadside or ruderal canola have found that canola populations generally persist only for a y ear or two, and are usually found within a few feet of a roadway in disturbe d soil not yet supplanted by gras ses and other more aggressive plants. The current status of ruderal canola, is not expected to change under the no action alternative, and is discussed in de tail in Appendix Section I.4.10.

Deregulation in Whole: The difference between the no action and the deregulation in whole alternative is expected to be the gradual re placement of the current Roundup Ready canola RT73 event with MON 88302. In studies biotechnology- derived canola populati ons have not been found to be more invasive or more persis tent than conventional canola populations, and biotechnology-derived canola seed has not been demonstrated to persist longer th an conventional

canola seed. The introduction of MON 88302 is not expected to impact canola survival or control as compared to conventional canola. T hus, the no action alterna tive and the approval in

whole alternative would not di ffer significantly in their impact on ruderal canola.

I.6.11. Public Health and Worker Safety

No Action Alternative: Under the No Action Alternative, MON 88302 and its progeny would continue to be regula ted articles and would not be widely grown. Under th is alternative, potential affects to public health relating to the production of canola are expected to remain unchanged and similar to those described in A ppendix Section I.4.11. Ca nola is expected to continue to be grown at appr oximately the same scale and in the same geographic areas. Herbicides, including glyphosate, will continue to be used at current rates as authorized by the

U.S. EPA with reasonable certainty of no harm.

Deregulation in Whole Alternative: Prior to the introduction of a biotechnology-derived crop product to the marketplace, Monsanto conducts tests to assure that the product is as safe as its conventional counterpart under the intended use conditions. Biotechnol ogy-derived crops for food and feed use undergo a voluntary consultation process with the U.S. FDA prior to release into the market. Monsanto will complete th e U.S. FDA consultation process prior to the

introduction of MON 88302.

Under the Federal Food, Drug, and Cosmetic Act (FFDCA), pesticide residues in or on raw agricultural commodities or processed foods are allowed only after a tolerance or exemption

from tolerance has been established. Residue tolerances and exemptions for pesticides are established by the U.S. EPA under the FFDCA. The U.S. FDA enfor ces the tolerances set by the U.S. EPA. The U.S. EPA also reviews the propos ed use pattern for all herbicides and prior to approval and placement on herbicide labels determines that no unreasonable risk exists for the

environment.

The difference between the no action and the deregulat ion in whole alternative is expected to be the gradual replacement of the current Roundup Ready canola RT73 event with MON 88302. AsMonsanto Company 11-CA-233U 415 of 500 summarized in Petition Section V.F, the CP 4 EPSPS protein expressed in MON 88302, as in other Roundup Ready crops, does not pose a significant health risk. MON 83302 expresses the CP4 EPSPS protein throughout the plant conferring tolerance to glyphosate, which is the active ingredient in the Roundup family of agricultural herbicides. It is stru cturally homologous to EPSPS proteins that are part of the amino acid synthesis pathway of all plants (Devine, et al., 1993). The safety of any protein(s) newly introduced into a biotechnology-derived crop needs to be assessed (Delaney, et al., 2008; ILSI, 2004). The safety of CP4 EP SPS protein present in biotechnology-derived crops has been extensivel y evaluated (Harrison et al., 1996; ILSI-CERA, 2010). The U.S. EPA has also reviewed the safe ty of the CP4 EPSPS prot ein and has established a tolerance exemption for the protein and the genetic material necessary for its production in or on all raw agricultural commodities (40 CFR § 1 74.523). This exemption was based on a safety assessment that included rapid digestion in simula ted gastric fluids, lack of homology to known toxins and allergens, and lack of toxicity in an acute oral mouse gavage study. A history of safe use is supported by the lack of any documented reports of adverse eff ects since the introduction of other Roundup Ready crops in 1996.

Roundup Ready canola RT73 is planted on greaterthan 50% of U.S. canola acreage (USDANASS, 2008). Consequently glyp hosate is the most widely used herbicide in canola. The toxicology of glyphosate has b een extensively reviewed. A summary of food and feed tolerances, regulatory approvals with associated dietary exposure assessments and recent chronic and short-term aggregate risk assessments for glyphosate has been conducted by the U.S. EPA.

In regards to canola nutritional and compositional as pects, as described in Petition Section VI, extensive compositional analyses of canola seed were conducted on samples from replicated, multi-site field trials to compare the co mposition of MON 88302 to a conventional canola control and to commercially available canola varieties. The compositional analyses confirmed that for the components analyzed in MON 88302 seed, that MON 88302 is compositionally equivalent to conventional canola th at has a history of safe use.

Workers may be exposed to higher levels of glyphosate with MON 88302 because of the proposed higher glyphosate applica tion rates relative to current ly registered use patterns on canola. However, recall that the proposed rate s of glyphosate applications are comparable to rates already used in maize and soybean (2 po unds a.e. per acre of glyphosate in-crop). The affected environment for pesticide use and hu man health is discussed in Appendix Section

I.4.11.2. That section describes th e U.S. EPA’s conclusions and th e conclusions of other studies regarding worker exposure, and incidental exposure to non-worker s who may be in the vicinity where glyphosate is being applied. Currently th e U.S. EPA is undergoing a separate analysis related to the glyphosate use pattern requested for use with MON 88302.

I.6.12. Animal Health

No Action Alternative: Under the No Action Alternativ e, MON 88302 and its progeny would continue to be regulated articles and would not be widely grown. Under this alternative, the use of canola and its processed fractio ns as a feed source is expected to continue as described in Appendix Section I.4.12. Under th e no action alternative, farmers will likely con tinue to plant Roundup Ready canola RT73 as a feed source at a pproximately the same rate as currently,Monsanto Company 11-CA-233U 416 of 500 continue to apply glyphosate at label rates to co ntrol weeds and the harvested seed will continue

to be used as a feed source.

Deregulation in Whole Alternative: The difference between the no action and the deregulation in whole alternative is expected to be the gra dual replacement of the current Roundup Ready canola

RT73 event with MON 88302. As described in Petition Section V, th e CP4 EPSPS protein expressed in MON 88302, as in other Roundup Rea dy crops, does not pose a significant animal health risk. As discussed previously, there is no meaningful risk to animal health from dietary exposure to CP4 EPSPS produced in MON 88302. There are no toxic properties associated with the CP4 EPSPS protein (Harrison, et al., 1996). The estimated animal dietary feed mean intake of CP4 EPSPS protein from MON 88302 ranges from 0.23 to 0.80 mg/kg body weight per day depending on the animal species. No adverse effects were observed at very high CP4 EPSPS consumption levels (572 mg/kg body weight) (H arrison et al., 1996). Furthermore, the composition of the seed produced by MON 88302 is unchanged compared to conventional

canola. This information indicat es that there would be no negativ e effects to animals consuming

fractions derived from MON 88302.

The food and feed safety assessment Monsanto submitted to U.S. FDA (discussed in Appendix Section I.3.1.) concluded that f eed derived from MON 88302 is as safe and nutritious as feed

derived from conventional canola.

I.6.13. Animal, Plant and Microbial Communi ties Including Threatened and Endangered Species

I.6.13.1. Animal Communities

No Action Alternative: Under the No Action Alternative, MON 88302 and its progeny would continue to be regulated article s and would not be widely grow n. Under this alternative, the effect of canola on animal communities is expected to continue as described in Appendix Section I.4.13. Canola is expected to continue to be gr own in approximately the same amounts and areas and glyphosate would continue to be used on canola acres.

Deregulation in Whole Alternative: The difference between the no action and the deregulation in whole alternative is expected to be the gra dual replacement of the current Roundup Ready canola

RT73 event with MON 88302. As summarized in Petition Section V.F, the CP4 EPSPS protein expressed in MON 88302, as in other Roundup Ready crops, does not pose a significan t health risk to animals. The safety of CP4 EPSPS pr otein present in biotec hnology-derived crops has been extensively evaluated (Harrison et al ., 1996) and reviewed by the U.S. EPA which established a tolerance exemption for the protei n and the genetic materi al necessary for its production in or on all raw agricultural commoditi es (40 CFR § 174.523). A history of safe use is supported by the lack of any documented report s of adverse effects si nce the introduction of the first Roundup Ready crop in 1996.

The composition of the harvested seed produced by canola containing MON 88302 is comparable to conventional canola (Petition Section VI). Furthermore, it is unlikely that canola fields available for foraging would contain high amounts canola seed after harvest. This

information indicates that there would be no negative effects to mammals that forage onMonsanto Company 11-CA-233U 417 of 500 MON 88302. Similarly, it is expected that there w ould be no impact to birds or other animals, including migratory birds and animals, that may consume MON 88302 seed.

During field trials no changes in insect feedin g damage were observed (Petition Section VII.) indicating similar insect susceptibility for MON 88302 compared to conventional canola. Additionally, in a quan titative assessment of pest and be neficial arthropod abundance, no statistically significant differences were de tected between MON 88302 and the conventional control. As MON 88302-containing canola exhibits no toxic effect s on animals or pollinators of other plants in or around fields cultivated wi th MON 88302, it is unlikely insects and animals will be significantly affected.

MON 88302 gives the grow er additional options for glyphosat e use. Growers will have the option to apply up to 1.55 pounds a.e. per acre of glyphosate from emergence to first flower. This maximum rate is similar to rates currently allowed for soybean and ma ize and is less than one third the rate (5.0625 pounds a. e. per acre) used in the U.S. EPA’s ecological assessment for glyphosate (Appendix Section I.4.13.). In its analysis, the U.S. EPA used the 5.0625 pounds a.e.per acre rate to calculate the estimated e nvironmental concentrations (EECs) for aquatic plants and animals. Based on this assessment, it was concluded that effects to birds, mammals, fish and invertebrates are minimal (U.S. EPA, 1993).

Based on the analyses presented Appendix Secti on I.4.13. and the maximum application rate of glyphosate at 1.55 pounds a.e. per acre, glyphosate use a ssociated with MON 88302 is not expected to have any impacts on animal communiti es in or adjacent to canola fields. Thus, the no action alternative and the approva l in whole alternative would not differ significantly in their impact on animal communities.

I.6.13.2. Plant Communities

No Action Alternative: Under the No Action Alternative, MON 88302 and its progeny would continue to be regulated ar ticles and would not be widely grown. Unde r the no action alternative, the effect of canola on plant commun ities is expected to continue as described in Appendix Section I.4.13. Canola an d glyphosate-tolerant canola are ex pected to continue to be grown in approximately the same amounts and areas and glyphosate would continue to be used

on canola acres.

Deregulation in Whole Alternative: The potential for MON 88302 to impact nearby vegetation is related to its weediness potential, which could result in the uncontrolled spread into surrounding environments, as well as its ability to interbreed with nearby sexually compatible plants. In addition, off-target movement from spray or vapor drift occurs with all herbicide applications, and effects on non-target plants do occasionally o ccur as a result of their use. The degree of injury to non-target plants that may occur from off-target movement is dependent on the sensitivity of the plant to the herbicide; how ever these impacts can be minimized through good management practices such as decreasing spra y pressure, lowering boom height, increasing nozzle size, avoiding making applications du ring high winds, etc. (Jordan, et al., 2009; University of Illinois, 2010).Monsanto Company 11-CA-233U 418 of 500 The potential for canola to outcross in ca nola crops, canola seed production, other Brassica crop production, other Brassica seed production and the potential for cross pollination to native species and weeds, is discussed in Appendix Secti on I.4.9. As discussed in that section, there are reports of unassisted (e.g. without human interv ention) hybridization under field conditions with B. napus as the pollen donor with six other species including B. rapa

, B. juncea

, B. oleracea

, H.

incana

, R. raphanistrum and S. arvensis (see Petition Table IX-3). In all cases the resulting hybrids had decreased environmenta l fitness evidenced by a variety of characteristics including decreased pollen viability, seed production and s eedling survival, when compared to parental

varieties.

There is also the potential for ruderal canola popul ations to form at the edges of roadways and fields where canola is grown. Multiple studies of ruderal canola have found that canola populations generally persist only for a year or two, and are usua lly found in disturbed soil not yet supplanted by grasses and other more aggre ssive plants. In studi es biotechnology-derived canola populations have not been fo und to be more invasive or mo re persistent than conventional canola populations, and biotechnology- derived canola seed has not been demonstrated to persist longer than conventional canola seed.

The difference between the no action and the deregulat ion in whole alternative is expected to be the gradual replacement of the current R oundup Ready canola RT73 event with MON 88302.

MON 88302 does not exhibit characteristics associ ated with weedy growth and will not compete with plants found outside of agricultural production. Thus the introduction of MON 88302 does not provide increased potential fo r cross pollination with closely related species or increased potential for the development of ruderal popula tions over conventional canola or the current Roundup Ready canola RT73.

As discussed in Appendix Section I.4.11.2, gly phosate is a non-selective herbicide with postemergence activity on essentia lly all annual and perennial pl ants. As such, exposure to glyphosate could affect aquatic an d terrestrial nontarget plants (U.S. EPA, 1993). In general, plants may potentially be affected from applications of herbicides as a result of spray drift or runoff. Glyphosate, however, bind s strongly to agricultural soils and has low potential to move offsite dissolved in water (U.S. EPA, 1993). Moreover, glyphosate is not taken up from agricultural soils by plants. Th erefore, any potential glyphosate effects to nontarget plants are only due to exposure via spray drift. As described in Appendi x Section I.4.13., using methodology from a recent U.S. EPA effects determin ation, it can be determined that terrestrial plants that are not threatened or endangered are unlikely to be significantly impacted by glyphosate applications to MON 88302. In additi on, glyphosate labels include specific risk management measures to manage spray drift, including mandatory requirements for aerial applications.

Therefore, the no action alternative and the a pproval in whole alternative would not differ significantly in their impact on plant communities.Monsanto Company 11-CA-233U 419 of 500 I.6.13.3. Soil Microorganisms

No Action Alternative: Under the No Action Alternative, MON 88302 and its progeny would continue to be regulated articl es and would not be widely grow n. Under this alternative the effect of canola on soil microorganisms is expected to continue as descri bed in Appendix Section I.4.13. Canola and glyphosate-tolerant canola is expected to continue to be grown in approximately the same amounts and areas and gl yphosate would continue to be used on canola

acres.

Deregulation in Whole Alternative: The difference between the no action and the deregulation in whole alternative is expected to be the gra dual replacement of the current Roundup Ready canola

RT73 event with MON 88302. No adverse effects on soil microorganisms are associated with

MON 88302 nor do the characteristics of the CP 4 EPSPS protein pose any concern to soil microorganisms (ILSI-CERA, 2010). Therefor e, MON 88302 is not expected to have any impacts on soil microorganisms.

The effects of glyphosate on soil mi croorganisms have been extensiv ely investigated (Giesy et al., 2000; Sullivan and Sullivan, 2000). Long-term studies following repeated applications of glyphosate in the field for six (Olson and Lindwa ll, 1991) or over ten years (Biederbeck, et al., 1997; Hart and Brookes, 1996) have shown no dete ctable adverse effect s on soil microbes. Investigations by Haney (2002; 2000) related to the increased use of gl yphosate-tolerant crops demonstrated that glyphosate was degraded over time by soil microorganisms without adversely impacting soil microbial communities. Based on this body of evidence, no significant impacts on soil microorganisms are anticipated from the deregulation of MON 88302 and subsequent applications of glyphosate.

I.6.13.4. Threatened and Endangered Species

No Action Alternative: Under the No Action Alternative, MON 88302 and its progeny would continue to be regulate d articles and would not be widely grown. Under this alternative, the potential impact of canola on threatened or enda ngered (TE) species as defined by the U.S. FWS is expected to remain the same. Canola itself does not impact TE species. Canola is not sexually compatible with any listed TE plant or plant pr oposed for listing, or a host of any TE species because there are no listed species or sp ecies proposed for listing in the genus Brassica or that

would use Brassica species as a host. As summarized in Petition Section V.F, the CP4 EPSPS protein expressed in RT73, as in other Roundup Ready crops has no effect on any TE species.

No reports of any impacts to TE species from th e use of glyphosate applied to canola have been found, and none would be expected, based on the a pplication rates, as discussed in Section 1.4.13. Under the no action alternative, growers are expected to continue to use RT73 in approximately the same amounts and areas, with potentially some increase in acreage.

Deregulation in Whole Alternative: The difference between the no action and the deregulation in whole alternative is expected to be the gra dual replacement of the current Roundup Ready canola

RT73 event with MON 88302.

As with the no action alternativ e, canola itself is not sexually compatible with any TE plant species; therefore there is no potential for a direct effect of MON 88302 on TE plants.Monsanto Company 11-CA-233U 420 of 500 As with the no action alternative, MON 88302 expresses the CP4 EPSPS protein. As summarized in Petition Section V.F, the CP4 EPSPS protein does not pose a significant allergenic risk to humans or animals and the donor organism for the CP4 EPSPS coding

sequence, Agrobacterium sp. strain CP4, is ubiquitous in th e environment and is not commonly known for human or animal pathogenicity or allergenicity. The CP4 EPSPS protein lacks structural similarity to allerg ens, toxins or other proteins known to have adverse effects on mammals. The CP4 EPSPS protein is rapidly di gested in simulated digestive fluids and demonstrates no oral toxicity in mice at the le vel tested. Based on the above information, the consumption of the CP4 EPSPS protein from MON 88302 or its progeny are considered safe for

humans and animals.

As the action agency for pesticide registrations , the U.S. EPA has the responsibility to conduct an assessment of effects of a registration actio n on threatened and enda ngered species (TES). The U.S. EPA Endangered Species Protection Program web site, http://www.epa.gov/espp/

, describes the U.S. EPA assessment process for enda ngered species. Some of the elements of that process, generally taken from th e web site, are summarized below.

When registering a pesticide or reassessing the potential ecological risks from use of a currently registered pesticide, the U.S. EPA evaluates ex tensive exposure and ecological effects data to determine how a pesticide will move through a nd break down in the environment. Risks to birds, fish, invertebrates, mamma ls and plants are routinely assessed and used in the U.S. EPA’s determinations of whether a pesticide may be licensed for use in the U.S.

The U.S. EPA’s core pesticide risk assessment a nd regulatory processes en sure that protections are in place for all populations of nontarget species, including TE species. These assessments provide the U.S. EPA with information needed to de velop label use restrictions for the pesticide. These label restrictions carry the weight of la w and are enforced by U.S. EPA and the states (Federal Insecticide, Fungicide , and Rodenticide Act 7 USC 136j (a)(2)(G) Unlawful acts). Because threatened and endangered species may need specific protection, the U.S. EPA has developed risk assessment procedures described in the Overview of the Ecological Risk

Assessment Process (U.S. EPA, 2004) to determine whethe r individuals of a listed species have the potential to be harmed by a pe sticide, and if so, what specific protections may be appropriate. The U.S. EPA’s conclusion regarding the potential risks a pesticide may pose to a listed species and any designated critical habitat for the spec ies, after conducting a thorough ecological risk assessment, results in an "effects determinatio n" in accordance with Section 7 (a)(2) of the Endangered Species Act.

As a part of the U.S. EPA’s threatened an d endangered species effects assessment for the California red-legged frog (U.S. EPA 2008b), the U.S. EPA evaluated the effect of glyphosate use at rates up to 7.95 lbs a.e. glyphosate per ac re on fish, amphibians, aquatic invertebrates, aquatic plants, birds, mammals, and terrestrial invertebrates. This assessment determined that at the maximum application rate for in-crop ap plications of glyphosat e to MON 88302 (1.55 lbs a.e. glyphosate per acre) there would be no effe cts of glyphosate use on the following taxa of threatened and endangered species: fish, amphib ians, birds, and mammals. The U.S. EPA

assessment was uncertain of the effects on terrestri al invertebrates, citing the potential to affect small insects at all appl ication rates and large insects at the higher application rates. The U.S. EPA considered these potential effects as part of their review process a nd label use restrictionsMonsanto Company 11-CA-233U 421 of 500 imposed under authority of FIFRA. To mitigat e potential adverse effects to threatened and endangered species, the U.S. EPA has imposed sp ecific label use restrictions for glyphosate use when applied with aerial equipment including “The product should only be applied when the potential for drift to adjacent sensitive areas (e.g., residential areas, bodies of water, known habitat for threatened or endangered species, no n-target crops) is minimal (e.g., when wind is blowing away from the sensitive areas).”

In conclusion, there are legal precautions in place (U.S. EPA label use restrictions) and “best practice” guidance to reduce the pos sibility of exposure and adverse impacts to TE species from glyphosate application to MON 88 302; the U.S. EPA has consider ed potential imp acts to TE species as part of thei r registration and labeling process for glyphosate; and adhere nce to the U.S. EPA label use restrictions by the pe sticide applicator will ensure that the use of glyphosate will not adversely affect threatened a nd endangered species or critical habitat. Based on these factors and the legal requirements for pesticide applicator s to follow the U.S. EPA label use restrictions, the use of the U.S. EPA registered glyphosat e for MON 88302 producti on will not adversely impact listed species or species proposed for listing and will not adversely impact designated

critical habitat or habitat proposed for designation.

I.6.14. Physical Environment

I.6.14.1. Land Use

No Action Alternative: Under the No Action Alternative, MON 88302 and its progeny would continue to be regulated articles and would not be widely grown. Under this alternative, impacts of canola production on land use ar e expected to continue as described in Appendix Section I.4.14. Under the no action alternative, grower s will likely continue to use Roundup Ready

canola RT73 in the same areas and with the same weed control options, at approximately the same rate of glyphosate app lications as currently.

Deregulation in Whole Alternative: The difference between the no action and the deregulation in whole alternative is expected to be the gra dual replacement of the current Roundup Ready canola

RT73 event with MON 88302.

Glyphosate-tolerant canola has been deregulat ed and grown in the U.S. since 1999. Roundup Ready canola RT73 currently occupies greater th an 50% of total canola commercial acres. The acreage of glyphosate-tolerant can ola increased rapidly following commercial introduction and has remained between 50 and 70% of planted ca nola acres since 2001. Fluctuations in total canola acreage before and after glyphosate-tolera nt canola was commerci alized (Figure I-3) indicates that factors unrelated to the availability of the glyphosate-tolerant trait play a larger role in acres planted than the availability of the glyphosate-to lerant trait.

Despite the rapid adoption of herb icide-tolerant canola products in the past decade, there has been no significant impact on to tal cropland acreage in the U.S. For example, from 2001 to 2010, the total annual commercial canola acres pl anted averaged between 0.8M to 1.5M acres (Petition Table VIII-1), while in the same time frame, the adoption rate for biotechnologyderived herbicide-tolerant canola in North Dakota increased from 0% to 95% (Zollinger et al., 2009). Agricultural land use, and consequently crop production is dictatedby many factors, theMonsanto Company 11-CA-233U 422 of 500 most significant of which are commodity prices . Accordingly, growers may increase acres dedicated to canola production to meet increased need, but they do so in response to commodity prices and market demand, not in response to av ailability or adoption of biotechnology-derived traits. Thus, the introduction of MON 88302 is not anticipated to facilitat e production of canola

in areas where it is no t currently grown or have signifi cant impact on total canola production

acres.

I.6.14.2. Air Quality and Climate Change

No Action Alternative: Under the No Action Alternative, MON 88302 and its progeny would continue to be regulated article s and would not be widely grown. Potential impacts on air quality and climate change associated with canola prod uction are not expected to change under the no action alternative (Appendix S ection I.4.14.). Under the no acti on alternative, growers will continue to use Roundup Ready canola RT73 and may change tilling practices and no-till adoption rates or use different herbicide regimes, which could have very short-term impacts on air quality.

Deregulation in Whole Alternative: The difference between the no action and the deregulation in whole alternative is expected to be the gra dual replacement of the current Roundup Ready canola RT73 event with MON 88302. Growers may use glyphosate application rates with MON 88302 that are higher than the current Roundup Ready canola RT73, but still comparable to the rates used on maize and soybean at 1.7% and 1.9% the amount of acres respectively. These increased application rates may result in temporary, very short- term increases in herbicide levels in the air. Therefore, the introduction of MO N 88302 is not anticipated to result in si gnificant changes in air quality or climate change.

I.6.14.3. Surface and Groundwater Quality

No Action Alternative: Under the No Action Alternative, MON 88302 and its progeny would continue to be regulated article s and would not be widely grown. Potential impacts on surface water and groundwater, which are not expected to change under the no action alternative, are discussed in detail in Appendi x Section I.4.14. Under the no ac tion alternative, growers will continue to use RT73 and may change tilling rates or use of different herbicides over time, which could have very short-term im pacts on surface and groundwater.

Deregulation in W hole Alternative: Water quality could be impa cted either directly by MON 88302 via plant material imp acts on water resources, or indi rectly via impacts from the use of glyphosate or tillage practices associated with the planting of MON 88302. Conservation tillage, a system that leaves 30% or more of th e previous crop residue c overing the soil when planting another crop has been increasingly em ployed in commercial canola acres, and helps minimize any impacts of canola production on water quality by reducing soil erosion.

In terms of potential direct impacts on wate r quality, the CP4 EPSPS protein contained in MON 88302 is a member of the larger family of EPSPS proteins that are ubiquitous in plants and

microbes in the environment. The mode of acti on of this family of proteins is well known and the introduced CP4 EPSPS protein itself was derived from a common soil bacterium ( Agrobacterium sp . strain CP4). The safety of CP4 E PSPS protein present in other glyphosate-Monsanto Company 11-CA-233U 423 of 500 tolerant crops has been extensively evaluate d, and the U.S. EPA has granted a tolerance exemption for CP4 EPSPS. A history of safe use of CP4 EPSPS is supported by the lack of any documented reports of adverse effects since the introduction of Roundup Ready crops. Under full deregulation of MON 88302, current grower pract ices related to canola production, including weed control and tillage practices would not be significantly altered, as greater than 50% of current canola acres already contain the same glyphosate tole rance trait as in MON 88302. Therefore, it is unlikely that the presen ce of CP4 EPSPS protein in MON 88302 will have a significant impact on water quality.

The difference between the no action and the deregulat ion in whole alternative is expected to be the gradual replacement of the current R oundup Ready canola RT73 event with MON 88302. MON 88302 gives the grower additional options for glyphosate use. Growers will have the option to apply up to 1.55 pounds a.e. per acre of glyphosate from emergence to first flower. These rates are similar to those currently allo wed for soybeans and maize. Glyphosate has been thoroughly reviewed by the U.S. EPA and has been determined to “not pose unreasonable risks

or adverse effects to humans or the envir onment” (U.S. EPA, 1993). Glyphosate is rapidly adsorbed and tightly complexed by soil partic les and, even though it is highly water soluble, it does not leach into ground water in most soils. In intensely farmed areas, herbicides have often been found in surface waters due principally to rainfall runoff. With greater soil sorptivity glyphosate is found at lower concentrations than ot her herbicides such as atrazine and alachlor, and it has been shown to dissipate more rapidly than other herbicides in surface water (Carpenter, et al., 2002; Cerdei ra and Duke, 2006). Therefore, it is unlikely that the use of glyphosate on MON 88302 will have a significant impact on water quality.

I.6.15. Socio-Economic Impacts

No Action Alternative: Under the No Action Alternative, MON 88302 and its progeny would continue to be regulated ar ticles and would not be widely grown. Economic conditions associated with growing canola, which are not expected to change under the no action alternative, are discus sed in Appendix Section I.4.15. Unde r the no action alternative, growers will likely continue to use Roundup Ready ca nola RT73 at approximately the same, or potentially a slightly increased rate as currently , and will continue to use current weed control practices.

We found no cases of organic canola growers who have been affected by the need for increased isolation distances because of the presence of RT73, but did find that one organic canola grower

made the decision to switch from canola to ot her organic crops when biotechnology-derived

canola was introduced in the U.S. Another organic grower c onsidered canola, but chose a different oilseed crop because of concerns about disease and insect management.

Deregulation in Whole Alternative: Total global U.S. imports of ca nola oil continue to increase steadily from 0.5 million metric tons in 2000 (U SDA-FAS, 2011a) to 1.2 million metric tons in 2010 (USDA-FAS, 2011b). Although canola production in the U.S. has increased dramatically since the 1980s, it has always been far short of de mand (Figure I-2). The U.S. shortfall is made up with imports primarily from Canada.Monsanto Company 11-CA-233U 424 of 500 Although canola has been able to benefit from the same price subsidies as other commodity crops, over a ten year period canol a acres have not grown substant ially (Petition Table VIII-1)

even after the introduction of the current Roundup Ready canola RT73. The difference between the no action and the deregulation in whole altern ative is expected to be the gradual replacement of the current Roundup Ready canola RT73 event w ith MON 88302. It is not expected that the

introduction of MON 88302 will re sult in significantly increased acres, and any potential impacts to organic or specialty organic growers would be the same as for the no action alternative. Weed management practices would change slightly as gr owers would be able to apply higher rates of glyphosate for a greater period of time during canol a’s life cycle. Thus the costs of weed management may rise incrementally. However, researchers have f ound that growers have

realized substantial economic benefits with the a doption of herbicide-tolera nt canola. A survey of canola growers in western Cana da revealed that herbicide-to lerant technology generated from
$1.063 – 1.192 billion in annual direct and indire ct benefits over the 2005-2007 period partly attributed to lower input costs and partly attri buted to better weed cont rol (Smyth et al., 2010). Therefore it is unlikely the de regulation of MON 88302 will have a significant negative impact on grower economics.

I.6.16. Cumulative Impacts

CEQ regulations define cumulative impacts as “the impact on the envi ronment which results from the incremental impact of the action when added to other past, present, and reasonably foreseeable future actions regardless of what agency (federal or non- federal) or person

undertakes such other action.” 40 Thus, cumulative impacts are assessed only for those resources that are impacted by the proposed action. Cu mulative impacts may result from individually minor, but collectively significant, actions taking place over time.

Cumulative impacts occur when the effects of an action are added to the effects of other actions occurring in a specific geographic area and time frame. The cumulative impact analysis follows CEQ’s guidance: Considering Cumulative Effect s under the National Environmental Policy Act (CEQ, 1997). The steps associated with the analysis include:

 Specify the class of actions for wh ich effects are to be analyzed.

 Designate the appropriate time and space domain in which the relevant actions

occur.

 Identify and characterize the set of receptors to be assessed.

 Determine the magnitude of effects on the receptors and whether those effects are

accumulating.

This section discusses the cumulative impacts that are associated with the deregulation in whole alternative, when combined with other recent pa st, present, and reasonably foreseeable future

actions within the affected environment.

40 40 CFR 1508.7Monsanto Company 11-CA-233U 425 of 500 Cumulative Impacts of Potential Increased Glyphosate Usage

No Action Alternative: Under the No Action Alternative, MON 88302 and its progeny would continue to be regulated ar ticles and would not be wide ly grown. For MON 88302 the only impacts are those associated with the increased application rate of gl yphosate. Under the no action alternative, weed manageme nt in canola is expected to continue as described in Appendix

Section I.4.7.9. Growers will likely continue to use the current Roundup Ready canola RT73 and associated glyphosate use on approximately the same percentage of acre s as currently. Other weed control practices would con tinue to be utilized at curre nt rates. Based on U.S. EPA approved labels for Roundup agricultural herb icides, users of Roundup Ready canola RT73 can apply a maximum of 0.78 pounds acid equivalent (a .e.) per acre of glyphosate, from emergence to the 6-leaf stage. As shown in Table I-11 canola currently accounts for 0.7% of total glyphosate use and this would not be exp ected to change significantly.

Deregulation in Whole Alternative: The impact of the deregulation in whole alternative results from the option to use increased application ra tes for glyphosate. Users of RT73 can apply a maximum of 0.78 pounds a.e. per acre of glyphosate, from emergence to the 6-leaf stage. With MON 88302, growers will have the option to appl y up to 1.55 pounds a.e. per acre of glyphosate from emergence to first flower. If the full 1.55 pounds a.e. per acre is applied at once, it must be done before the 6-leaf stage. A lternatively the grower may apply glyphosate twice, at rates up to 0.77 pounds a.e. per acre, up to first flower.

Temporal Effects : Cumulative impacts can be important fo r herbicides or pesticides that persist

in the environment and thus can continue to accumulat e with repeated app lications. Glyphosate is not persistent and does not accumulate. In its re-registration decision for glyphosate (U.S. EPA 1993), the U.S. EPA reported a median half- life of 13.9 days for glyphosate, when applied

at the maximum annual rate of 7.95 lb a.e./acre. Colder climates tendedto have higher halflives: the half-life for Minnesota , the closest state to North Dakota that was reported, was 28.7 days. Thus, if all glyphosate used stopped toda y, its effects, both positive and negative, would soon be negligible. Therefore, the temporal cu mulative effects of incr eased glyphosate use are very limited.

Geographic Effects : Because glyphosate binds tightly to soils and drift affects a small area adjacent to a canola field, cumulative geographic eff ects are also very limited. In other words,

the area of influence of a MON 88302 canola field in terms of glyphosate impacts is the field plus a small adjacent area that c ould potentially be impacted by drift. Cumulative effects would occur only when the area of infl uence for some other glyphosate a pplication intersected with the

area of influence for a MON 8830 2 canola field. Thus, the cumulative geographic effects are also very limited.

MON 88302 Use in Context : Table I-11 shows estimated 2010 glyphosate use in the U.S. As shown, canola currently accounts for 0.7% of the total use. Assuming the adoption of the maximum rate (1.55 pounds a.e. per acre, which is an increase from 0.78 pounds a.e. per acre) is used for MON 88302, canola would account for appr oximately 1.6% of the total glyphosate use

in the U.S. In the context of overall U.S. glyphosate use, this is a negligible change.Monsanto Company 11-CA-233U 426 of 500 Table I-11. Glyphosate Use on Roundup Ready Crops

RR Crops lbs

Gl y phosate

per acre 1 RR Adoption 2 Total

Acres (million) 3 RR Acres (million) lbs Glyphosate (thousand) % of

Total

Maize 0.95 0.7 87.9 61.5 58454 25.1

Cotton 1.89 0.78 10.9 8.5 16069 6.9

Soybean 1.36 0.93 79 73.5 99919 42.9

Canola 1.125 0.9 1.5 1.4 1519 0.7

1 Maize, cotton, soybean glyphosate rates from (Benbrook, 2009) canola rate from NDSU (2007b).

2 USDA-NASS, 2010.

3 Zollinger et al., 2009.Monsanto Company 11-CA-233U 427 of 500 Attachment 1: Appendix I. Brassica and Related Vegetable Seed Production

Vegetable seed crops are a very small part of U.S. agricultural output and published data is not widely available. According to the 2007 Census of Agriculture, the total U.S. vegetable seed field acreage in 2007 was 45,309, less than one percent of the tota l harvested vegetable acreage of 4,682,588 (USDA NASS 2009). Based on the analysis below, it appears that nearly all the

non-broccoli Brassica seed production occurs in Washi ngton and Oregon and nearly all the broccoli seed production occurs in California and Arizona.

Washington and Oregon Production

The majority of the Brassica seed grown in the U.S. is prod uced in three areas of Washington and Oregon: in western Washington in the lower-lying areas around th e Puget Sound between the Olympic Mountains and the Cascades; in the Columbia Basin of eastern Washington; and in Oregon’s Willamette Valley, lo cated between the Coastal Range and the Cascades.

The most comprehensive information is availabl e for Washington State. Based on the discussion below, the total estimated Washington acreage of Brassica and related vegeta ble seed production is approximately 4,500 acres, of which appr oximately 43% (1,935 acres) is planted in Brassica species, with the rest being planted with crops such as arugula, daikon and radish, members of the family Brassicaceae but not members of the Brassica genus. Attachment Table 1 summarizes available recent seed crop da ta from 2007 to 2010 for Washington for Brassica and related vegetables. While the reports used to create Attachment Table 1 were not available for

all Brassica and related crops reported to be grown in Washingt on, the acreage of crops for which reports are not available is likely relatively small

. A 2010 summary reported a total of 2,829 acres of vegetable seed crops in western Washington in 2009 (McMoran, 2010). Of this, 2,369 acres (84%) were accounted for as cabbage, spinach and beet seed in Skagit County, leaving only 460 acres of all other seeds in Skagit County and other western Washington counties. A 1997 report of Brassica and related seed producti on in western and eastern Washington listed more crops than are shown in Attachment Table 2, but the totals were similar to those shown in the table (maximum 2,313 acr es in eastern Washi ngton and 2,160 acres in western Washington for an overall total maximum of 4,473 acres) (Thomas, 1997).

McMoran (2010) and du Toit (200 7) both report that Washingt on produces 75% of the U.S. cabbage seed crop. Thomas et al., (1997,) reporte d that Washington produced 100% of the U.S. Brussels sprouts and kale seed crop, 75% of the U.S. cauliflower s eed crop, and 70% of the U.S. radish seed crop. In addition Thomas et al . (1997) reported that the Washington Chinese cabbage, Chinese kale, and Chinese mustard seed cr ops represented 90-100% of the U.S. total.

In Oregon the 100-mile long Willamette Valley has approximately 900,000 acres of crops, of which more than 40% is grass and legume seed pr oduction; most of the remaining acreage is in grains, hay and forage (OSU 2011). While ther e are other seed production areas in Oregon, the Willamette Valley is the major vegetable seed production area. Approximately 13,000 acres or 1.4% of total acres are reported for vegetabl e and flower seed production (OSU, 2011).

According to the Willamette Valley Specia lty Seed Association (WVSSA), common Brassica and related seed crops in the Willamette Valley are: cabbage, Brussels sprouts, turnip, rutabaga, radish, Chinese cabbage, Oriental brassicas, mustard, and kale (WVSSA, 2011). According to Mallory-Smith, et al. (2007) we stern Washington and western Ore gon combined produce nearlyMonsanto Company 11-CA-233U 428 of 500Attachment Table 1. Washington State Brassica and Related Vegetable Seed ProductionWest of Cascades (Western) Columbia Basin(Eastern)Acres Skagit Island Jefferson Snohomish Clallam Lewis San Juan Whatcom Grant Franklin AdamsCabbage (B. oleracea) 600√√√√√Cauliflower (B. oleracea) 100√√√√√Collard (B. oleracea) 100-150√√√√Kale (B. oleracea) 100-150√√√Kohlrabi (B. oleracea) 25-50√√√Chinese kale (B. alboglabra;orB.oleracea ssp. alboglabra)< 100√√√√Rutabaga (B. napus) 80√√Chinese mustard (B. rapa) 350√√√√Turnip (B. rapa) 400√√√√Arugula (Eruca sativa) 100-120√√√√√Daikon (Raphanus sativus) 400-500√√Radish (Raphanus sativus) 1500-2000√√√√√TOTAL FROM RECENT REPORTS3,750-4,500Source: WSU, 2010.Monsanto Company 11-CA-233U 429 of 500 all (~90%) of European cabbage, Brussels sp routs, rutabaga and turnip seed, and a substantial portion (20-30%) of radis h, Chinese cabbage and other Asian Brassica vegetable crops seed.

California and Arizona Production

.

The California Pest Management Center’s (CPMC, 2011) crop management database reports the following Brassica and related vegetable seed crops by region, but with no acreage information:

 Region 2 - Central Valley: broccoli, Chinese cabbage, cabbage, cauliflower, kale, kohlrabi, radish, rutabaga and turnip.

 Region 3 – central coast: broccoli, cabba ge, cauliflower, kale, kohlrabi, mustard

and radish.

 Region 4 – southeast, bordering Nevada a nd Arizona; includes Imperial Valley – broccoli, cabbage, cauliflower, collard gr eens, kale, kohlrabi, mustard, radish and rutabaga.

 Region 5 – south coast (Santa Barbara to Mexican border): broccoli, cabbage, cauliflower, collard greens, kale, kohlrabi, mustard, and radish.

 Region 6 – northern Central Valley – broccoli, Chinese cabbage, cabbage, cauliflower, collard greens, kale , kohlrabi, mustard and radish.

In regions 2 and 4 canola is grown, bu t there is no acreage information.

California and Arizona are the only states fo r which specific menti on of broccoli seed production was found. In 2003 the University of Arizona Extension reported 4,500 acres of seed crops in Yuma County, but did not spec ify the seed types (Uni versity of Arizona, 2003).

A seed company in the Yuma Valley reports it specializes in hybrid broccoli seed production (SeedQuest, 2011). It also offers cauliflower, Chinese cabbage (pak choi), mustard, and Asian types of radish.

Idaho Production

Radish seeds are reportedly produced in Id aho’s Treasure Valley, which is located in western Idaho and is the lower valley of the Snake River (OSA, 2007).Monsanto Company 11-CA-233U 430 of 500 References for Appendix I

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J.1. Introduction

Herbicides are pesticides intended to preven t or kill weeds that can compete with a crop for nutrients, water and in some cases, sun light. By killing weed s, herbicides allow planted crops to grow and thrive, thereby increasing crop yield and allowing these crops to be grown on fewer acres, protecting hab itat and wildlife from unnecessary expansion of cropland production wh erever possible.

Plant populations can develop resistance to a he rbicide due to the selection of individuals that carry altered genetic code(s) producing alleles that can render those individuals

tolerant to the lethal effects of a herbicide. The application of a herbicide to the plant does not, itself, cause a mutation in subsequent generations. Rather, over time, those few plant biotypes that are not susceptible to a herbicide increase in number in the population with repeated use of th e herbicide in the absence of othe r control methods, such as use of

other chemical or mechanical control methods . The development of resistant populations is common to all herbicides. The probability for resistance to develop in a population is a function of: frequency of resistant allele(s ), mechanism of resistance, dominance or recessive nature of the resistant allele(s), relative fitness of the resistant biotype and frequency of herbicide use in the absence of other control methods (Jasieniuk et al., 1996). The probability of resistance is not the same for all herbicides with some herbicides (e.g., ALS and ACCase classes) ex hibiting resistance more quickly than other herbicides (e.g. glyphosate, auxins (dicamba), dinitroanilines).

Herbicide resistance could b ecome a limiting factor in cr op production if the resistant weed population cannot be controlled with othe r herbicides or cultu ral practices. This generally has not been the case for any herbic ide and weed combination. For most crops, there are multiple herbicide options for grow ers to use. However, good management practices to delay the development of herbic ide resistance have been identified, are being actively promoted by the public and privat e sectors, and are being implemented by

growers.

Monsanto considers product st ewardship to be a fundament al component of customer service and business practices. Stewardship of the glyphosate molecu le to preserve its usefulness for growers is an important aspect of Monsanto’s stewardship commitment. Although herbicide resistance may eventually oc cur in weed species when a herbicide is widely used, resistance can be delayed, contained and mana ged through research, education and good management practices. Monsan to will invest in research to identify best management practices, monitoring for resi stance, and in dealer/grower education and training programs to provide information on best practices to ma nage glyphosate weed resistance in canola. These are the key el ements of Monsanto’s approach to providing stewardship of glyphosate relative to a ll uses including use in the MON 88302 canola production system. This document provides an overview of resistance to glyphosate and Monsanto’s approach to the development of best management practices to mitigate glyphosate resistance.Monsanto Company 11-CA-233U 452 of 500 J.2. The Herbicide Glyphosate

Glyphosate (N-phosphonomethyl-glycine) (C AS Registry #: 1071-83-6), the active ingredient in the Roundup family of nons elective, foliar-applie d, post-emergent agricultural herbicides, is among the world’s most widely used herbicidal active ingredients. Glyphosate is highly effectiv e against the major ity of economically significant annual and perennial grasses and broadleaf weeds. Currently, glyphosate is

labeled for control of more than 300 weed species world- wide. Glyphosate kills plant cells by inhibition of 5-enolpyruvylshikimate- 3-phosphate synthase (EPSPS), an enzyme

involved in the shikimic acid pathway for aromatic amino aci d biosynthesis in plants and microorganisms (Franz et al., 1997; Steinr ücken and Amrhein, 1980). Glyphosate is the only known herbicide with this mode of act ion (Franz et al., 1997). The relevant aromatic amino acid pathway is not presen t in mammalian metabolic systems (Cole, 1985; Franz et al., 1997). A comprehensive hum an safety evaluation and risk assessment concluded that glyphosate has low toxicity to mammals, is not a carcinogen, does not adversely affect reproduction and developm ent and does not bioaccumulate in mammals (Williams et al., 2000). An ecotoxicological ri sk assessment concluded that the use of glyphosate does not pose an unreasonable risk of adverse effects to nontarget species, such as birds and fish, when used according to label directions (Giesy et al., 2000). Glyphosate has favorable envi ronmental characteristics, including a low potential to move through the soil to reach ground water and is degraded over tim e by soil microbes. Because it binds tightly to so il, glyphosate’s bioavailability is reduced immediately after application, which is why glyphosate has no re sidual soil activity. Similar conclusions were reached in an indepe ndent assessment of risk to human health and to the environment that was conducted as a part of the Environmental Impact Statement for Glyphosate Tolerant Alfalf a (USDA-APHIS, 2010).

J.3. Herbicide Use in Canola Syst ems and Herbicide-resistant Weeds

Weed control in canola productio n fields is critical for obtaining optimized yields.

Because failure to control w eeds within the crop can resu lt in decreased yields and reduced crop quality, a program for weed cont rol is essential to ensure a grower can optimize yields. In using MON 88302 weed co ntrol will be obtained through the use of glyphosate plus other herbicides. Control of w eeds in a crop is essential because weeds compete with the crop for the same limited resources in the fiel d, including sunlight, water and nutrients. Weed competition can be a major limiting factor in canola production leading to significant yield reductions (CCC, 2006). Certain perennial weeds, such as Canada thistle, are known to be pa rticularly important to control in canola production. For example, studies have demonstr ated that only 10 Canada thistle plants per square meter have resulted in 10% yiel d loss while 40 plants pe r square meter have resulted in over 50% yield loss (CCC, 2006). Glyphosate is highly effective against the majority of annual and perennial grasses an d broad-leaf weeds including difficult-tocontrol weeds such as Canada thistle (NDSU, 2005; Padgett e et al., 1996).

With any herbicide use, howev er, comes the potential for the selection of weeds resistant to that herbicide. Within a weed species, individuals may possess an inherent ability to

withstand the effects of a particular herbicide. Repeated use of that herbicide will exposeMonsanto Company 11-CA-233U 453 of 500 the weed population to a "selec tion pressure," which may le ad to an increase in the number of surviving resistant individuals in the population (Jasieni uk et al., 1996). In other words, plants susceptible to the applied herbicide will die, while those few having some type of natural resistan ce may survive and reproduce.

A resistant weed must demonstrate two crite ria as defined by the Weed Science Society of America website at www.wssa.net: (1) th e ability to survive application rates of herbicide product that once were effective in control ling it and; (2) resist ance is heritable. Herbicide-resistant weeds are neither a new phenomena nor is resistance unique to glyphosate. Growers have been managing herb icide-resistant weeds for decades with the

use of alternative herbicides and/or cultural methods such as tillage or crop rotation that are combined to provide a diverse weed management program.

J.4. Characteristics of Herbicides and Herbicide Use Influencing Resistance

While the incidence of weed resistance is often associated with repeat ed applications of a herbicide product, the actual onset of resist ance within a population depends very much on the specific herbicide chemistry in question, as well as the inherent presence of gene(s) that confer the ability of a plant to be resistant to a particular chemical within a specific weed species and even a specific pop ulation of that species (Sammons et al., 2007). Some herbicide products are much more prone to develop herbicide resistance than others (Heap, 2009). Considering th e substantial worldw ide glyphosate-treated acreage, and the total number of weeds that glyphosate can control, glyphosate, a member of the glycine herbicide chemical family, has been used extensively for over three decades with relatively few cases of resistance development, particularly when compared to many other herbicides (e.g., ALS inhibitors, triazines, and ACCase inhibitors). The graph in Figure J-1 illustrates the instances of weed resistance to various herbicide groups. The different slopes observed are la rgely due to the factors described above, which relate to chemistry and function, in add ition to levels of exposure in the field. The summary below describes herbicide-specific factors determined to be important in the process of selecting for individuals that ar e inherently resistant to a herbicide.Monsanto Company 11-CA-233U 454 of 500 Figure J-1. Weed Resistance to Vari ous Herbicide Chemical FamiliesMonsanto Company 11-CA-233U 455 of 500 J.5.1. Mechanisms of Resistance

The application of a herbicide to a weed does not, itself, cause a mutation in later generations of the plant. Rath er, over time, the repeated application of herbicide selects for those few biotypes that are less suscepti ble to the herbicide and they increase in frequency within a population. To date, th e three known mechanisms by which a weed species develops resistant to a herbicide have been identified as 1) target site alteration (target site); 2) enhanced metabolism of th e herbicides (metabolism); and 3) reduced absorption and/or translocation of the herbicide such that the herbicide does not get to the

site of action within th e plant cell (exclusion) (S ammons et al., 2007).

Herbicide resistance via target site is th e most common resistance mechanism among the

various herbicide classes. It has been found that a target site mechanism is the most common mechanism for ALS inhibitors, ACCase inhibitors, and triazines, but is less common for glyphosate. One ty pe of target site alte ration involves amino acid substitution(s) in the enzyme that prevents the binding of the herbic ide without impacting enzyme activity, and the plant is able to grow normally without any fitness penalty. For ALS inhibitors, the level of resistance conf erred by a target site mechanism has been found to be as high as 3,400 X (Ferguson et al ., 2001). (Note: X is the labeled or recommended rate for a herbicide on a particul ar weed species.) For glyphosate, species found to exhibit a target site mechanism can demonstrate low levels of resistance (2-3X) due to the fact that glyphosate is a true tr ansition state inhibitor (Sammons et al., 2007; Schönbrunn et al., 2001) differe ntiating glyphosate from AL S inhibitors and ACCase

inhibitors. The transition state is an unsta ble state during an enzyme reaction half-way between the substrate and product. Transition state inhibitors are very effective enzyme inhibitors. In addition, multip le alterations of the same en zyme have been found for ALS inhibitors (Tranel and Wright, 2002). This ma y explain the apparent high frequency of

resistance and the short time in which resist ance developed to herbicides in these two

classes of chemistries. On ly one altered site in the targeted plant EPSPS enzyme impacting glyphosate insensitivity has been f ound for glyphosate (Baerson et al., 2002). Another type of a target site resistance m echanism recently discovered for glyphosate, is an over amplification of the EPSPS gene wh ich results in an overproduction of the EPSPS enzyme (Gaines et al., 2010). This mechanism was discovered in Palmer pigweed.

The second general type of he rbicide resistance mechanism, metabolism, has not been

found to be a resistance mechanism associated with glyphosate in any of the weed species studied thus far. However, legumes ha ve been shown to degrade glyphosate and therefore this type of resistance mechanism ma y be active in some species (Gaines et al., 2010).

Herbicide resistance as a result of exclus ion mechanisms is the glyphosate resistant mechanism among the majority of the weed sp ecies studied to date . This resistance

mechanism has also been found to be associat ed with 2,4D and paraquat. Within this category, there are two types of translocation alterations th at have been observed for glyphosate; (a) restricted move ment of glyphosate from leaf cells into the meristematic cells of the plant (Shaner, 2009) and (b) decreased delivery of glyphosate to theMonsanto Company 11-CA-233U 456 of 500 chloroplast (Sammons et al., 2007). The le vel of glyphosate resist ance conferred with this mechanism is higher (6-8X) than for sp ecies exhibiting amino acid substitution type target site mutations (2-3X).

In some species, the experimental evidence su ggests that more than one mechanism of glyphosate resistance may occur within the sa me plant to protect the plant from the phytotoxic effects of glyphosate (Yu et al., 2 007). This implies that multiple genes (polygenic resistance) are necessary and thus the selection of plants with multiple genes

needed to confer resistance would be e xpected to occur at a low frequency.

In summary, the overall low o ccurrence of glyphosate resistance may be in part explained by: (1) the nature of the targ et site inhibition by glyphosate relative to other herbicides; (2) the lack of metabolism as a mechanism of selectivity for weed resistance, and (3) evidence of multiple mechanisms being necessary for resistance; thus, resistance is polygenic and difficult to assemble and maintain. Recommendations to manage glyphosate resistance are not dependent upon th e type of resistant mechanism operating within a species or popu lation of a species.

J.5.2. Use of Recommended Rate

The interaction between herbicide applica tion rate and resistance for post-emergence herbicides, such as glyphos ate, is dependent upon the na ture of the plant gene(s) conferring resistance to the chemical. In ge neral, herbicide rate has more effect on selecting for resistant individua ls in a population if the resist ant gene is semi-dominant or recessive as compared to the resistant gene being dominant. Likewise, herbicide rates

would have more of an effect on the onset of resistance if commercially significant resistance required the additive effect of multiple genes (i.e. quantitative or polygenic resistance). Low rates would tend to allow certain biotypes to survive and mate with other biotypes of the same or an alternate resistant gene. The offspring of this mating may then be able to survive a full rate.

Less-than-recommended or suboptimal rates have been implicated as or speculated to be

the causal factor in herbicide resistance for several different weed species, including chlortoluron-resistant blackgr ass, diclofop-resistant ryegrass and dicamba-resistant kochia (Beckie, 2006). It has been demonstrat ed that, in three gene rations of a ryegrass biotype sprayed at sublethal rates of diclofop-methyl or glyphosate, a high level of resistance to diclofop-methyl and a moderate level of re sistance to glyphosate evolved (Busi et al., 2009). The conclusion of this research was that growers should avoid lowering the application rate of herbicides.

J.6. Weeds Resistant to Glyphosate

As with any other herbicide, the use of glyphosate may lead to the development of glyphosate-resistant weed species. A list of glyphosate resistant weeds is provided below in Table J-1. However, th e potential for the development of a glyphosate-resistant weed needs to be considered in the following context: 1) if a glyphosate-based weed control system was not available, other herbicide(s) wi th equal or greater potential for resistanceMonsanto Company 11-CA-233U 457 of 500 would be used to control weed s and 2) other herbicides and cultural practices can be used to manage the glyphosate resistant sp ecies (Gustafson, 2008; Neve, 2008).

Through March 2011, biotypes of twenty-one we ed species resistant to glyphosate have been identified and confirmed worldwide (WSS A, 2011a). Twelve species resistant to glyphosate have been confirmed in the U.S., three of which were identified outside of Roundup Ready cropping systems. The speed of spread and geographical distribution of the resistant species has varied. Some speci es with resistant biotypes, such as common ragweed ( Ambrosia artemisfolia ), have been found in a limite d number of sites across the mid-west, whereas marestail ( Conyza canadensis ) has been found in many states in the northeast, mid-west and the south. The re productive biology of the particular weed species involved appears to be a factor contribut ing to the spread of resistant biotypes. In the above examples, marestail produces a large number of wind-dispersed seeds, which contributes to rapid spread (Weaver, 2001), while ragweed seeds do not have features that allow for such easy di stribution by the wind.

Table J-1. U.S. Glyphosate Resistant Weeds through March 2011

Weeds identified outside of Roundup Ready Systems Rigid ryegrass ( Lolium rigidum ) Annual bluegrass ( Poa annua ) Hairy fleabane ( Conyza bonariensis )

Weeds identified in Roundup Ready Systems Horseweed ( Conyza canadensis ) Common ragweed ( Ambrosia artemisiifolia ) Giant ragweed ( Ambrosia trifida ) Palmer amaranth ( Amaranthus palmeri ) Common waterhemp ( Amaranthus rudis ) Italian ryegrass ( Lolium multiflorum ) Johnson grass ( Sorghum halepense ) Kochia ( Kochia scoparia ) Goosegrass ( Eleusine indica )

Some weed species, such as Equisetum arvensis (field horseweed), are tolerant, as opposed to resistant, to glyphosat e. That is, they have the inherent ability to survive herbicide treatments as opposed to resistan ce induced by such techniques as genetic engineering or selection of variants produced by tissue culture or mutagenesis (WSSA, 2011b). Some species are more difficult to control with glyphosat e than others (e.g. lambsquarters ( Chenopodium album ) and morninglory ( Ipomea sp.)) and require more care to make sure the correct amount of glyphosa te is applied at the right growth stage. For these difficult-to-control weeds, environmental conditions can affect herbicide performance more than for weeds that are ea sier to control, and therefore it is more

critical that the correct rate be applied at the right growth stage when making applications to weeds in the difficult-to-control category. Weed control situati ons involving tolerant or difficult-to-control species are of ten confused with resistance.Monsanto Company 11-CA-233U 458 of 500 J.7. Use of Glyphosate for In-crop Weed Management

Monsanto has developed plants through biotec hnology to be tolerant to glyphosate. The development, approval and cultivation of these Roundup Ready crop s have facilitated additional uses of glyphosate in crops wher e such uses were not previously possible given the non-selective nature of glyphosate. This development has provided growers with an additional weed management option and benefits relative to existing weed management options. The glyphosate tolera nce in Roundup Ready crops has no effect per se on the control of weeds. From a weed resistan ce standpoint, the us e of glyphosate with glyphosate-tolerant canola is no different than the use of a selective herbicide in a conventional canola crop.

The most often cited benefits of glyphosate as an in-crop weed management option are simplicity, flexibility of application timing, weed spectrum, crop safety and environmental safety (Dill, 2005). The abili ty to use glyphosate in-crop has allowed farmers to change their farming practices in some cases. For example, planting of Roundup Ready cotton, corn and soybean have resulted in an increa se in no-tillage practices (Dill et al., 2008).

Since Monsanto commercialized the firs t Roundup Ready canola varieties in 1998, growers have enthusiastically adopte d the technology. The Roundup Ready canola system, (i.e., planting Roundup Ready canol a and applying glyphos ate in-crop), has become the standard weed control program in U.S. canola production. In addition, weed control in a Roundup Ready canola system likely will involve not only glyphosate-based

herbicides but also other herbicides and weed management practices to effectively manage weeds, thus increasing crop yield and reducing development of resistant weed populations. State Universi ties/Cooperative Extension Services (CES) publish information on best weed management practic es in Roundup Ready crops to address both of these objectives (see Table J-2). In a ddition Monsanto and other companies selling glyphosate products provide information on thes e same best management practices as detailed later in this Appendix.

J.8. Weed Resistance Management Strategies for Glyphosate

As part of Monsanto’s stewardship of Roundup agricultural herbicides and Roundup Ready crop systems, the company has conducte d investigations and worked extensively

with academics and other herbicide manufact urers to understand the best practices to manage resistance. These investigations have demonstrated that one of the major factors that can contribute to the development of resistant weed populati ons is weed control management practices such as the application of herbicides at rates below those indicated on the EPA-approved label for the weed species (Beckie, 2006), and sole reliance on a particular herbicide for weed control without the use of other herbicides or cultural control methods (i.e. pre-plant and in-cr op tillage) over an extended period of time (Beckie, 2006; Peters on et al., 2007).

As detailed in the Petition and Appendix I, the purpose of MON 88302 is to provide growers with improved weed control through gr eater flexibility of glyphosate herbicideMonsanto Company 11-CA-233U 459 of 500 application. By virtue of enhanced CP4 EPSPS expression in male reproductive tissues, MON 88302 provides tolerance to glyphosate du ring the sensitive reproductive stages of growth, and enables the application of glyphosat e at higher rates and at later stages of development than is possible wi th the current product.

Monsanto will communicate to all canola growers recommended weed resistance management practices. Monsanto will provid e instructions to canola growers regarding reporting any incidence of re peated non-performance of R oundup agricultural herbicides on a particular weed, and Monsanto will invest igate cases of unsatisfactory weed control to determine the cause of poor performance. In cases wh ere resistance is confirmed, Monsanto will provide recommendations for al ternative control methods for farmers (see Table J-2). These recommendations are made available through Monsanto supplemental labels, the Monsanto Technology Use Gu ide (TUG), Monsanto and University publications and internet sites for growers, cons ultants, retailers and distributors. In all cases of glyphosate-resistant weeds in the U.S. and glob ally, there are alternative

herbicides and cultural methods available to fa rmers to effectively control these species. Some examples of these recommendations from University/CES personnel are found in Table J-2. It is important to note that there are many alternative options in each situation.

The weed resistance management recommenda tions that will be made for the use of glyphosate in conjunction with varieties containing the second generation glyphosatetolerant MON 88302 event will not differ fr om recommendations being made for commercial varieties containing event RT 73 (Roundup Ready canola varieties). These

recommendations are consistent with th e Herbicide Resistance Action Committee’s guidelines for prevention and management of herbicide resistance (HRAC, 2011). These guidelines recommend an integrated approach to weed resistance management including crop management (i.e. row spacing, etc), cultura l techniques and herbicides. The EPA is the U.S. federal regulatory agency that admi nisters the federal law governing pesticide sale and use (FIFRA). The U.S. EPA enc ourages pesticide manu facturers to provide growers with information regarding a herbic ide’s mode of action to aid growers in planning herbicide use practices and to foster the adoption of effective weed-resistance management practices as specified by the U.S. EPA in PR Notice 2001-5. In that document the U.S. EPA states that “this appr oach to resistance ma nagement is sound and would be highly beneficial to pesticide manu facturers and pesticide users” (U.S. EPA, 2001). The U.S. EPA approves al l pesticide label use instruc tions based on the agency’s evaluation of supporting data supplied by the pe sticide registrant or manufacturer. After the U.S. EPA approves a pesticide label, it is a violation of federal law to use the pesticide for a use or in a manner not in accordance with the la bel directions.

Monsanto incorporates the U.S. EPA’s guide lines for pesticide resistance management labeling on its glyphosate-based agricultural herbicide labels, and will do so on the label for products to be applied over the top of varieties developed c ontaining MON 88302 (An example of the current Roundup Weathe rMAX product label is available at http://www.cdms.net/LabelsMsds/LMDefault.aspx?manuf=23&t ). The U.S. EPAapproved labels for Roundup branded he rbicide weed-resistant management recommendations are designed to minimi ze the potential for the development of glyphosate-resistant weeds. By approving a label for a glyphosate-based agriculturalMonsanto Company 11-CA-233U 460 of 500 herbicide, the U.S. EPA has concluded th at the product will not cause unreasonable

adverse effects to the environment or human health when used in accordance with the

label’s directions.

The weed resistance management guideline s on the labels of Roundup agricultural

herbicides include recommendati ons that are well-documented in the scientific literature as being appropriate and effective for weed control, and that mitigate weed resistance. Significant research has been conducted to id entify the appropriate application rate of glyphosate required to control a particular w eed at various growth stages under various agronomic and environmental conditions. These rates are based on over 35 years of ongoing research at Monsanto to evalua te the efficacy of Roundup agricultural herbicides. Studies have included efficacy of weed control for a broad spectrum of weeds and under a wide range of conditions. A key element of effective weed control and weed resistance management, therefore, is using the correct rate of glyphosate at the right time for the weed species and the size of the weed (i.e., using a lethal dose which avoids the need for subsequent applications). This important strategy is well-supported by field research studies at several universi ties (Westra et al., 2008) . Additionally, it is accepted in the weed science community that the use of multiple herbicide modes of action via tank mixtures, use of herbicides w ith different modes of action in a rotational crop, or using multiple herbicides in seque nce within a crop will reduce the risk of developing weed resistance (Beckie, 2006; Gressel and Segel, 1990). Tank-mixing involves mixing two or more herbicides in the spray tank immediately prior to application. To provide growers with th e tools needed to minimize resistant weed development, Monsanto will continue to i nvestigate and recommend appropriate residual and post-emergence herbicide products that have a different mode of action from glyphosate. As an example, the herbicide clopyralid (tradename Stinger) is a postemergence herbicide that will help control fl ushes of annual broadleaf seed which could slow the selection and potential spread of glyphosate-resistant weeds in Roundup Ready systems. The general concept that Monsanto promotes for management of resistance has been referred to by several authors as applying “diversity” across cropping/fallow seasons to manage weed resistance (B eckie, 2006; Powles, 2008). Crop rotation and management of the fallow period and cover cr ops can be important considerations in managing resistance.Monsanto Company 11-CA-233U 461 of 500 Table J-2. Management Recommendations for Control of Glyphosate Resistant Weeds in Canola and Rotational Crops

Glyphosate

Resistant

Weed Crop Recommendations for alternative herbicides to manage glyphosate resistant

weeds 1 Reference 2

Palmer

amaranth Canola PPI: trifluralin, ethalflurafin Post: glufosinate(LL canola) ND

Maize PP: 2,4-D, paraquat Pre: alachlor, alachlor+atrazine Post: 2,4-D, dicamba, atrazine AR and GA

Soybean PP: flumioxazin, metribuzin/chlorimuron Pre: flumioxazin, metolachlor/metribuzin Post: fomesafen, metolachlor/fomesafen AR and GA

Wheat/Barley PP: paraquat, 2,4-D, dicamba

Pre: saflufenacil Post: fluroxypyr/florasulam, pyrasolfotole/bromoxynil, 2,4-D ND, GA,

Product

Labels

Waterhemp Canola PPI: trifluralin, ethalflurafin Post: glufosinate(LL canola) ND

Maize PP: 2,4-D, atrazine Pre: acetochlor, acetochlor+atrazine, topramezone, tembotrione Post: mesotrione, atrazine, 2,4-D+atrazine OH/IN

Soybean PP: metribuzin/chlorimuron Pre: metolachlor/metribuzin, sulfentrazone,

flumioxazin Post: lactofen, fomesafen, acifluorfen OH/IN

Wheat/Barley PP: paraquat, 2,4-D, dicamba

Pre: saflufenacil Post: fluroxypyr/florasulam, pyrasolfotole/bromoxynil, 2,4-D ND

Common ragweed Canola Post: clopyralid, gl ufosinate(LL canola) ND

Maize PP: 2,4-D, dicamba, atrazine Pre: acetochlor, atrazine, rimsulfuron/isoxaflutole, saflufenacil Post: 2,4-D, dicamba, atrazine,

mesotrione/atrazine OH/IN

Soybean PP: 2,4D, chlorimuron,

metribuzin/chlorimuron Pre: sulfentrazone/chloransulam, chloransulam/flumioxazin, Post: cloransulam, fomesafen OH/IN

Wheat/Barley PP: 2,4-D, dicamba, paraquat

Pre: saflufenacil Post: 2,4-D, dicamba, bromoxynil NDMonsanto Company 11-CA-233U 462 of 500 Table J-2. Management Recommendations for Contro l of Glyphosate Resistant Weeds in Canola and Rotational Crops (continued)

Glyphosate

Resistant

Weed Crop Recommendations for alternative herbicides to manage glyphosate resistant

weeds Reference 2

Giant ragweed Canola Post: clopyralid, gl ufosinate(LL canola) OH/IN

Maize PP: 2,4-D, dicamba Pre: atrazine, saflufenacil, Post: 2,4-D, dicamba, clopyralid,

mesotrione/atrazine OH/IN

Soybean PP: 2,4-D Pre: metribuzin/chlorimuron, cloransulam, sulfentrazone/chlorimuron,

flumioxazin/cloransulam Post: fomesafen, lactofen OH/IN

Wheat/Barley PP: 2,4-D, dicamba, bromoxynil

Pre: saflufenacil Post: 2,4-D, dicamba, bromoxynil OH/IN,

Product

Labels

Marestail Canola PP: 2,4-D, dicamba Post: glufosinate(LL canola) ND, NE

Maize PP: 2,4-D+dicamba, atrazine+2,4-D Pre: mesotrione, saflufenacil,

rimsulfuron/isoxaflutole Post: 2,4-D+dicamba, clopyralid OH/IN, ND

Soybean PP: 2,4D, 2,4-D+metribuzin/chlorimuron Pre: sulfentrazone/chloransulam, saflufenacil, flumioxazin, metribuzin, saflufenacil Post: cloransulam, fomesafen OH/IN, ND

Wheat/Barley PP: 2,4-D, dicamba+MCPA

Pre: saflufenacil Post: 2,4-D, pyrasolfotole/bromoxynil, dicamba/fluroxypyr ND

Kochia Canola Pre: ethalflurafin Post: glufosinate(LL canola) ND

Maize PP: dicamba, bromoxynil, atrazine Pre: atrazine, saflufenacil, sulfentrazone Post: dicamba, bromoxynil, fluroxpyr ND and NE

Soybean PP: flumioxazin Pre: sulfentrazone, clomazone, saflufenacil,

flumioxazin/chlorimuron Post: chlorimuron, fomesafen, imazethapyr ND and NE

Wheat/Barley PP: dicamba, bromoxynil, paraquat

Pre: saflufenacil Post: dicamba, bromoxynil, tribenuron ND

1 Application Timings: PP = Preplant burndown before planting; Pre = pre-emergence; Post = post-emergence; Postdirected = applied post-emergence directed at the base of the crop; PPI = Pre-plant incorporated.

2 References: AR: (University of Arka nsas, 2011); GA: (University of Georgia, 2010); TN: (Univers ity of Tennessee, 2010); OH/IN: (OSU, 2011) ; NE: (University of Nebraska-Lincoln, 2011); ND: (Zollinger, 2010).Monsanto Company 11-CA-233U 463 of 500 J.9. Monsanto Weed Performance Evaluation and Weed Resistance Management

Plan

Monsanto and/or Monsanto seed company licen sees are directly in a position to be aware of the performance of glyphosate in all fields through its extensive presence in the markets where Roundup Ready canola is grow n, through its relati onship with farm advisors, and its relatio nship with key University/CES pe rsonnel. This will allow the timely recognition of performance issues that could arise related to weed resistance or other means. Monsanto field employees a nd hired consultants ar e trained and provided processes for responding to product performa nce inquiries. As warranted, individual performance issues that could be related to potential resistan ce are promptly handled. In addition, performance inquires are periodically re viewed for trends that could indicate the need for follow up action on a broad scale. If broad scale actions in the areas where canola is produced are needed, Monsanto and/or licensees will alert the canola growers of the need for any prescribed action.

In general, when resistance is confirmed, the scientific and grower communities are notified and a weed resistance mitigation plan is implemented by Monsanto in cooperation with the University/CES. The m itigation plan is designed to manage the resistant biotype through e ffective and economical weed management recommendations implemented by the grower. The scope and level of intensity of the mitigation plan may vary depending on a combination of the following factors: 1) biology and field characteristics of the weed (seed shed, seed dormancy, etc.); 2) importance of the weed in the agricultural system; 3) resistance status of the weed to other herbicides with alternate modes of action; and 4) availability of a lternative control options. These factors are analyzed by Monsanto and University/CES personnel in combination with economic and practical management considerations to develop a tailored mitigation strategy. The plan considers what is technically appropriate for the particular weed and incorporates practical management strategies that can be implemented by the grower.

After a mitigation plan is developed, Monsan to communicates the plan to the grower community through the use of supplemental labeling (labeling which includes newly approved uses, use directions or other instructions which have been added since the last EPA-approved Master label), informational fact sheets, retailer training programs, agriculture media and/or other means, as appropriate.

In addition to the grower inquiry initiated process, Monsanto, alone and in cooperation with University/CES, conducts field studi es to understand the potential for weed

resistance and weed shifts as the resu lt of various weed management programs implemented in a Roundup Ready canola system . These studies allow researchers to better track specific factors that can influence the development of resistance to specific

weeds.Monsanto Company 11-CA-233U 464 of 500 J.10. Summary

Development of weed resistance is a complex process. No single agronomic practice will mitigate resistance for all herbic ides or all weeds. As a re sult, weed resistance needs to be managed on a case-by-case basis and tailored for the particular herbicide and weed in order to meet grower needs. Using good weed management principles, built upon achieving high levels of cont rol through proper applicati on rate, choice of cultural practices, and appropriate companion weed c ontrol tools will allow Roundup agricultural

herbicides to continue to be used effec tively. In cases where weed populations have developed resistance to glyphos ate, effective management options are available and experience has shown that growers continue to find value in using glyphosate in their weed control programs.

The key principles for effective stewards hip of glyphosate use, including Roundup Ready crops, include: 1) basing weed management and weed resistance management practices on local needs and using the tools necessary to optimize crop yield; 2) using proper rate and timing of application; 3) not relying solely on one herbicide weed control option across a cropping system; 4) responding rapidl y to instances of unsatisfactory weed control; and 5) providing up to date weed management and weed resistance management training.Monsanto Company 11-CA-233U 465 of 500 References for Appendix J

Baerson, S.R., D.J. Rodriguez, M. Tra n, Y. Feng, N.A. Biest and G.M. Dill. 2002. Glyphosate-resistant goosegrass. Identification of a mutation in the target enzyme 5- enolpyruvylshikimate-3-phosphate synthase. Plant Physiology 129: 1265-1275.

Beckie, H.J. 2006. Herbicide-resistant weeds: Management tactics and practices. Weed Technology 20: 793-814.

Busi, R., S.M. Velayudhan and S.B. Powles. 2009. Use of below label herbicide rates can

lead to evolution of herbicide resistant w eeds. IBG Workshop, Weed Science Society of America, Lawrence, Kansas. http://wssa.net/Meetings/WSSAAbstracts/ abstractsearch.php [Accessed June 24, 2009].

CCC. 2006. How many weeds are too many. Canola Council of Canada, Winnipeg, Manitoba. https://canola-coun cil.merchantsecure.com/canol a_resources/product30.aspx [Accessed December 16, 2010].

Cole, D.J. 1985. Mode of action of glyphosate - A literature analysis . Pages 48-74 in The Herbicide Glyphosate. E. Grossbard and D. At kinson (eds.). Butterworth's & Co., Boston,

Massachusetts.

Dill, G.M. 2005. Glyphosate-resistant crops: Hist ory, status and future. Pest Management

Science 61: 219-224.

Dill, G.M., C.A. CaJacob and S.R. Padgette. 2008. Glyphosate-resistant crops: Adoption,

use and future considerations . Pest Management Science 64: 326-331.

Ferguson, G.M., A.S. Hamill and F.J. Tardif. 2001. ALS inhibitor resistance in populations of Powell amaranth a nd redroot pigweed. Weed Science 49: 448-453.

Franz, J.E., M.K. Mao and J.A. Sikorski. 1997. General properties of glyphosate and glyphosate salts. Pages 27-65 in Glyphosate: A Unique Globa l Herbicide. American Chemical Society, Washington, D.C.

Gaines, T.A., W. Zhang, D. Wang, B. Bukun, S. T. Chisholm, D.L. Shaner, S.J. Nissen, W.L. Patzoldt, P.J. Tranel, A.S. Culpepper, T.L. Grey, T.M. Webster, W.K. .Vencill, R.D. Sammons, J. Jiang, C. Preston, J.E. Leach and P. Westra. 2010. Gene amplification confers glyphosate resistance in Amaranthus palmeri . Proceedings of the National Academy of Sciences of the United States of America 107: 1029-1034.

Giesy, J.P., S. Dobson and K.R. Solomon. 2000. Ecotoxicological risk assessment for Roundup ® herbicide. Reviews of Environm ental Contamination and Toxicology 167: 35-

120.

Gressel, J. and L.A. Segel. 1990. Modeling th e effectiveness of he rbicide rotations and mixtures as strategies to delay or preclude resistance. Weed Technology 4: 186-198.Monsanto Company 11-CA-233U 466 of 500 Gustafson, D.I. 2008. Sustainable use of glyphos ate in North American cropping systems. Pest Management Science 64: 409-416.

Heap, I. 2009. International survey of herbicide resistant weeds. Weed Science Society of America, Corvallis, Oregon. http://www.w eedscience.org/in.asp [Accessed June 23, 2010].

HRAC. 2011. Weed resistance management: Pr oposed HRAC stewardship guidelines for

herbicide labels. Herbicide Resistance Ac tion Committee, St. Louis, Missouri. http://www.hracglobal.com/Publications/HRA CStewardshipGuidelinesforHerbicideLabel s/tabid/604/Default.aspx [Accessed March 28, 2011].

Jasieniuk, M., A.L. Brule-Babel and I.N. Morrison. 1996. The evolution and genetics of

herbicide resistance in weeds. Weed Science 44: 176-193.

NDSU. 2005. Canola production field guide. A-1280, North Da kota State University Extension Service, Fargo, North Dakota.

Neve, P. 2008. Simulation modelling to understand the evolution and management of glyphosate resistance in weeds. Pest Management Science 64: 392-401.

OSU. 2011. Weed control guide for Ohio field crops. Bulletin 789. The Ohio State University Extension Research, Columbus, Ohio. http://ohioline.osu.edu/b789/ [Accessed March 28, 2011].

Padgette, S.R., D.B. Re, G.F. Barry, D.E. Eichholtz, X. Delannay, R.L. Fuchs, G.M. Kishore and R.T. Fraley. 1996. New weed control opportunities: Development of soybeans with a Roundup Ready TM gene. Pages 53-84 in Herbicide-Resistant Crops: Agricultural, Environmental, Economic, Regul atory and Technical Aspects. S.O. Duke (ed.). CRC Press, Inc., Boca Raton, Florida.

Peterson, D., B. Olson, K. Al-Kha tib, R. Currie, J.A. Dille, J. Falk, P. Geier, D. Regehr, P. Stahlman and C. Thompson. 2007. Glyphosat e stewardship. MF-2767, Kansas State University Agricultural Experiment Stat ion and Cooperative Extension Service, Manhattan, Kansas.

Powles, S.B. 2008. Evolved glyphosate-resistant weeds around the world: Lessons to be learnt. Pest Management Science 64: 360-365.

Sammons, R.D., D.C. Heeri ng, N. Dinicola, H. Glick and G.A. Elmore. 2007. Sustainability and stewardship of glyphos ate and glyphosate-resistant crops. Weed Technology 21: 347-354.

Schönbrunn, E., S. Eschenburg, W.A. Shuttlewort h, J.V. Schloss, N. Amrhein, J.N.S.

Evans and W. Kabsch. 2001. Interaction of the herbicide glyphosat e with its targetMonsanto Company 11-CA-233U 467 of 500 enzyme 5-enolpyruvylshikimate 3-phosphate synt hase in atomic detail. Proceedings of the National Academy of Sciences of the United States of America 98: 1376-1380.

Shaner, D.L. 2009. Role of translocation as a mechanism of resistance to glyphosate.

Weed Science 57: 118-123.

Steinrücken, H.C. and N. Amrhein. 1980. The herbicide glyphosate is a potent inhibitor of 5-enolpyruvylshikimic-acid-3-phosphate synthase. Biochemica l and Biophysical

Research Communications 94: 1207-1212.

Tranel, P.J. and T.R. Wright. 2002. Resistan ce of weeds to ALS-inhibiting herbicides:

What have we learned? Weed Science 50: 700-712.

U.S. EPA. 2001. Pesticide registration (P R) notice 2001-5, guidance for pesticide registrants on pesticide resi stance management labeling. U.S. Environmental Protection Agency, Washington, D.C. http://www. epa.gov/opppmsd1/PR_Notices/pr2001-5.pdf [Accessed May 12, 2010].

University of Arkansas. 2011. Recommended chemicals for weed and brush control. University of Arkansas, Cooperative Exte nsion Service, Divisi on of Agriculture, Fayetteville, Arkansas.

University of Georgia. 2010. Corn production gu ide. University of Georgia, College of Agricultural & Environmental Sciences, Athens, Georgia. http://www.caes.uga.edu/commodities/fieldc rops/gagrains/2008ComProductionGuide.ht ml [Accessed December 9, 2010].

University of Nebraska-Lincoln. 2011. Weed control guide results. University of Nebraska-Lincoln Agronomy & Horticu lture Department, Lincoln, Nebraska. http://weedscience.unl.edu/cornburn downfs.cfm [Accessed March 21, 2011].

University of Tennessee. 2010. 2010 weed cont rol manual for Tennessee. University of Tennessee, Institute of Agriculture, Knoxville, Tennessee.

USDA-APHIS. 2010. Glyphosate-tole rant alfalfa events J101 and J163: Request for nonregulated status. U.S. Department of Agri culture, Animal and Pl ant Health Inspection Service, Washington, D.C.

Weaver, S.E. 2001. The biology of Canadian weeds. 115. Conyza canadensis . Canadian

Journal of Plant Science 81: 867-875.

Westra, P., R.G. Wilson, S.D. Miller, P.W. Stahlman, G.W. Wick s, P.L. Chapman, J. Withrow, D. Legg, C. Alford and T.A. Gain es. 2008. Weed population dynamics after six years under Glyphosate- and conve ntional herbicide-based weed control strategies. Crop

Science 48: 1170-1177.Monsanto Company 11-CA-233U 468 of 500 Williams, G.M., R. Kroes and I.C. Munro. 2000. Safety evaluation and risk assessment of the herbicide Roundup and its active ingredient, glyphosate, for humans. Regulatory Toxicology and Pharmacology 31: 117-165.

WSSA. 2011. Resistance and tolerance defini tions. Weed Science Society of Ameica, Lawrence, Kansas. http://www.wssa.net/Weed s/Resistance/definitions.htm [Accessed March 28, 2011].

Yu, Q., A. Cairns and S. Powles. 2007. Glyphosate, paraquat and ACCase multiple

herbicide resistance evolved in a Lolium rigidum biotype. Planta 225: 499-513.

Zollinger, R. 2010. North Dakota weed contro l guide. North Dakota State University Extension Service, Fargo, North Dakota. http://www.ag.ndsu.edu/weeds/weed-controlguides/nd-weed-control-guide -1/nd-weed-control-guide/ [Accessed May 12, 2010].Monsanto Company 11-CA-233U 469 of 500 Appendix K: Potential Impact of Glyphosate on Human Health and the

Environment

K.1. Overview

Glyphosate is a herbicide approved for use (registered) by the U.S. Environmental Protection Agency (EPA) for the control of weed s that would interfere with the growth of many food and non-food crops, including biotechnology-derive d crops, as well as for control of weeds growing in non-crop areas. Glyphosate rece ived a label for use in cropping systems, and has had food and feed to lerances for residues in the U.S. since 1975. In 2001, the U.S. U.S. identified glyphos ate as the most widely used conventional agricultural herbicide in the U. S. (Kiely et al., 2004).

A comprehensive database on glyphosate has been evaluated by the EPA to support all currently approved uses. The EPA has repeatedly stated that it has a high level of confidence in the quality of the existing st udies and the reliability of the toxicity endpoints that are the basis for human health and environmental risk assessments ((U.S. EPA, 2006a; 2006b). In establishing food and feed tolerances to support the use of glyphosate on animal feed and forage crops, the U.S.EPA concluded, “that there is a reasonable certainty that no harm will result to the general population, and to infants and children from aggregate exposure to gl yphosate residues” (U.S. EPA, 2006d).

The following discussion provides an overview of the regulatory and risk assessment processes applicable to glyphos ate and all other ag ricultural use pesticides. Glyphosate has been approved by the EPA for a large numbe r of food and feed us es, including uses associated with glyphosate-tolerant crops. Over 180 food and feed tolerances (40 CFR § 180.364) have been established for glyphosate in support of these uses. A complete listing of all U.S. glyphosate tolerances is provided in Attachment 2.

K.1.1. Pesticide Registration and Tolerance Setting

The Federal Insecticide, Fungicide, and Rode nticide Act (FIFRA) requires that before sale or distribution of a pesticide in th e U.S., a person or company must obtain a registration, or license, from the U.S. EPA. Before registering a new pesticide or a new use for a previously registered pesticide, the U. S. EPA must first ensure that the pesticide, when used according to its label directions, will not cause unreasonable adverse effects

on the environment. In order to address th is standard, the U.S. EPA must evaluate potential risks to humans and the environmen t, and may require applicants to submit

more than 100 different scientific studies and tests conducted accord ing to the U.S. EPA guidelines. According to the U.S. EPA, glyphosate is one of more than over 1000 active ingredients currently registered as pesticides, which are formulated into many thousands of pesticide products that are av ailable in the marketplace.

The process of registering a pesticide is a scientific, legal and administrative procedure through which the U.S. EPA examines the ingredie nts of the pesticide; the particular site or crop on which it is to be used; the amount, frequency, method and timing of application and other conditions of its use; and storage and dis posal practices. InMonsanto Company 11-CA-233U 470 of 500 evaluating a pesticide registration application, the U.S. EPA assesses a wide variety of potential human health and environmental eff ects associated with use of the product.

The data required by the EPA are used to eval uate whether a pesticid e has the potential to cause adverse effects on humans, wildlife, fi sh and plants (incl uding endangered species and non-target organisms that the pesticide is not intended to act against). This includes potential human health and safety risks rang ing from short-term toxicity to long-term effects such as cancer and reproductive system disorders. The registration applicant must also supply data addressing the pesticide’s potential impact on surf ace water or ground water (which can result from leaching or runoff, for example).

The U.S. EPA also must approve the language that appears on each pesticide label. A pesticide product can only be used legally a ccording to the directions for use on the labeling accompanying it at the time of sale . Following these directions carefully and precisely is necessary to ensure safe use as defined by FIFRA.

The initial registration of a pe sticide is not the only opportun ity an agency like the U.S. EPA has to evaluate its safety. The 1988 am endments to FIFRA authorized the EPA to conduct a re-registration program of pesticides first regist ered before November 1, 1984. The goal of the re-registration program was to ensure that these pesticides met current scientific and regulatory standards and could be declared "eligible " for re-registration.

The results of the U.S. EPA's reviews ar e summarized in Re-registration Eligibility Decision (RED) documents. In 1993, the U.S. EPA produced a 291-page RED on glyphosate (U.S. EPA, 1993b), de scribing/listing the data on which it made a decision to re-register all existing uses of the pesticide, based on the pesticide having met the no

unreasonable adverse effects standard descri bed in FIFRA. As mandated by the Food Quality Protection Act of 1996, the U.S. EPA initiated the Registration Review program to periodically re-evaluate all registered pestic ides to ensure that as changes in science, public policy, and pesticide use practices occu r, products in the marketplace can still be used safely. The Registration Review pr ocess for glyphosate started in 2009 and is expected to be completed in 2015. During th e Registration Review process, the U.S. EPA will be requesting additional data for gl yphosate due to recent changes in FIFRA data requirements and will be updating the risk assessments for all currently registered glyphosate uses.

The EPA also sets tolerances (m aximum pesticide r esidue l evels or MRLs) for the amount of the pesticide that can legally remain in or on foods and feeds. The U.S. EPA undertakes this analysis under the authority of the Federa l Food, Drug, and Cosmetic Act (FFDCA). Under the FFDCA, the U.S. EPA must find that such tolera nces will be safe, meaning that there is a reasonable certaint y that no harm will result from aggregate exposure to the pesticide chemical residue and other potential exposure routes. This finding must be made and the appropriate tole rance established before a pesticide can be registered for use on the particular food or f eed crop in question. Several factors must be

addressed before a tolerance can be established, including:Monsanto Company 11-CA-233U 471 of 500  the aggregate exposure from the pes ticide (now includi ng occupational

exposure 41

, exposure through diet, from using pesticides in and around the home, and from drinking water);

 the cumulative effects from exposure to di fferent pesticides th at produce similar effects in the human body;

 whether there is increased exposure to in fants and children, or other potentially high exposure subpopulations; and

 whether the pesticide produces an effect in humans similar to an effect produced by a naturally occurring estrogen or produces other endocrine-disr uption effects.

K.1.2. Pesticide Risk Assessment

The process the U.S. EPA uses for evaluating the health impacts of a pesticide, under either FIFRA or the FFDCA, is called a ri sk assessment. The EPA uses the National Research Council’s four-step process for human health risk assessment, which involves hazard identification, dose-response asse ssment, exposure assessment and risk

characterization. Each of thes e steps is discussed below:

Hazard Identification

The first step in the risk assessment process is to identify potential health effects, or hazards that may occur from different type s of pesticide exposure. The U.S. EPA considers the full spectrum of a pesticide’ s potential health effects. Hazards are identified through a battery of studies that examine the poten tial toxicity of the pesticide in various tests including, where appropria te, tests with laboratory animals.

Generally, for human health risk assessments , many toxicity studies are conducted, based on EPA guidelines, by pesticide companies in independent laboratories following the Good Laboratory Practice (GLP) standards, and evaluated for acceptability by U.S. EPA scientists. The U.S. EPA evaluates pesticides for a wide range of effects, from eye and

skin irritation to cancer and birth defects. The U.S. EPA may also consult the public

literature or other sources of informa tion on any aspect of the chemical.

Dose-response assessment

The next step of the risk assessment consider s the levels at which the pesticide produces adverse effects. Dose-response assessment i nvolves considering the dose levels at which adverse effects were observed in test animals, and using these dose levels to calculate an equal dose in humans.

41 Historically, issues associated with potential occupa tional exposure for each new use are were considered separately under FIFRA’s unreasonable risk standard ; however, under a recently announced revised policy, the EPA has stated that it intends to begin to include occupational exposure into the aggregate assessment (74 FR 65121, EPA–HQ– OPP–2009-0889; Decem ber 9, 2009).Monsanto Company 11-CA-233U 472 of 500 Exposure Assessment

Step three of the process i nvolves an exposure assessment. People can be exposed to pesticides in three ways: 1) inhaling pe sticides (inhalation e xposure); 2) absorbing pesticides through the skin (der mal exposure); and 3) ingesting pesticides (oral exposure). Depending on the situation, pesticides could enter the body by any one or all of these routes. Typical sources of pesticide expos ure include agricultu ral (food); home and personal use pesticides; pesticides applied to lands that make their way into the drinking water; or occupational exposure for agricultu ral workers or pesticide applicators.

Risk Characterization

Risk characterization is the final step in asse ssing human health risks from pesticides. It is the process of combining the hazard, dose-response and exposure assessments to describe the overall risk from the use of a pe sticide. It explains the assumptions used in assessing exposure as well as the uncertain ties that are built into the dose-response assessment. The strength of the overall database is considered, and broad conclusions are

made. The U.S. EPA’s role is to evaluate both toxicity and exposure and to determine

the risk associated with use of the pesticide.

The risk to human health from pesticide exposure depends on both the toxicity of the pesticide and the likelih ood of people coming into contact w ith it (exposure). At least

some exposure and some toxicity are required to result in a risk. For example, if the pesticide is found to have a high level of toxicity, but people are not exposed to the pesticide, there is no risk. Likewise, if there is ample exposure but the pesticide is essentially nontoxic, there is no risk. However, usually when pesticides are used, there is some toxicity and exposure, whic h results in a potential risk.

The U.S. EPA recognizes that effects of expos ure to all pesticides including glyphosate vary between animals of differe nt species (interspecies extrapolation) and from person to person (intraspecies variability). To account for this variability, a 100-fold uncertainty factor is built into the risk assessment (10X fo r interspecies extrapolation and 10X for intraspecies variability). This uncertainty fact or creates an additional margin of safety for protecting people who may be e xposed to the pesticides.

Once the risk assessment proce ss for a pesticide is complete, the U.S. EPA uses this information to determine if (when used according to label directions) there is a reasonable certainty that the pesticide will not harm a person’s health and will not cause

unreasonable adverse effects on the environment.

Using the conclusions of a risk assessment, th e U.S. EPA can then make a more informed decision regarding whether to approve a pest icide chemical or use, as proposed, or whether additional protective measures ar e necessary to limit occupational or nonoccupational exposure to a pesticide. For example, the U.S. EPA may prohibit a pesticide from being used on certain crops because consuming that commodity treated with the pesticide may result in an unacceptabl e risk to consumers. Another example of protective measures is requiring workers to wear personal protect ive equipment (PPE)Monsanto Company 11-CA-233U 473 of 500 such as a respirator or chemical resistant gl oves, or not a llowing workers to enter treated crop fields until a specific period of time has elapsed.

The U.S. EPA is responsible under the Federa l Insecticide, Fungicide and Rodenticide Act (FIFRA) to evaluate pesticides to ensure that they will not have unreasonable adverse effects on humans, the environment and non-targ et species when used according to label

directions. Tier 1 “worst-cas e” or appropriately-ref ined higher tier risk assessments assist the U.S. EPA in determining whether or not risks would be below the U.S. EPA’s levels

of concern. Pesticides must be registered or exempted by the EPA's Office of Pesticide Programs before they may be sold or distributed in the U.S.

K.2. Potential Impact of Glyphosate on Human Health

Glyphosate presently has 186 esta blished food and feed tole rances in the U.S. (see Attachment 2). Each time the U.S. EPA review s an application to add a new food or feed use to the glyphosate label the Agency is required by FFDCA to conduct an aggregate risk assessment, considering all sources of hum an exposure to the pesticide, and find that aggregate exposure to the pestic ide will be safe as defined by the statute and regulations. As noted above, historically, i ssues associated with potenti al occupational exposure were considered separately under FIFRA’s unreasonable risk standard. However, under a recently announced revised policy, the U.S. EPA has stated that it intends to begin to include occupational exposur e into the aggregate asse ssment (74 FR 65121, EPA–HQ– OPP–2009-0889; December 9, 2009).

Over the course of these numerous revi ews the toxicology of glyphosate has been extensively studied. Multiple comprehensiv e toxicological studies in animals have demonstrated that glyphosate does not cause cancer, birth defects, mutagenic effects, nervous system effects or reproductive problems (EC, 2002; U.S. EPA, 1993b; WHOFAO, 2004). In fact, after a thorough review of all availa ble toxicology data, the EPA concluded that glyphosate should be classi fied in Group E carcinogen (evidence of noncarcinogenicity in Humans), the most favorab le category possible (U.S. EPA, 1993a).

K.2.1. Glyphosate Safety Evaluations

Despite this extensive safety data, glyphosate safety is reviewed with every new use for which registration is sought, including, wher e necessary, uses associated with glyphosatetolerant crops developed through biotechnolo gy. As discussed above, prior to the approval of any new use of an existing register ed pesticide, U.S. EPA must consider the potential human health effects from the a ggregate (total combin ed) human exposure to that pesticide, combining the potential exposur e from the proposed new use with all other existing exposures to the pesticide. Diet ary exposure is considered, which addresses pesticide residues that may remain on food fr om crops on which the pesticide is applied (pre- or post-emergence), as well as any resi due that could be found in drinking water as a result of pesticide use. Non- dietary exposure is also includ ed in this assessment, which includes exposure to the pesticide through reside ntial use, such as on lawns or in flower beds, as well as exposure in a recreational cont ext, such as from a golf course or sportsMonsanto Company 11-CA-233U 474 of 500 field. Based on these data, the U.S. EPA mu st be able to make a determination of reasonable certainty of no harm to human health as required by the FFDCA.

The U.S. EPA does not conduct an acute diet ary risk assessment for glyphosate because no acute human health concerns have ever been determined from toxicological studies conducted with glyphosate. A ccordingly, the U.S. EPA does not expect glyphosate to pose an acute risk (U.S. EPA, 2006a; 2006b). Th e U.S. EPA assesses ch ronic dietary risk for glyphosate using “worst-case” (Tier 1) exposure assumpti ons that include tolerance level residues and 100% crop treated. For food, this estimat e assumes that glyphosate is used on 100 percent of all the crops on which the pesticide is currently approved for use. It further assumes that the resulting pest icide residues found on all harvested food crops are at the level of the legally established to lerance (i.e., the maximum allowable pesticide residue level). For water, the U.S. EPA assu mes that glyphosate is used to control weeds in water bodies by direct application to the water at the maximum application rate, without taking into account de gradation in the water body or partitioning to sediment

within the water column (U.S. EPA, 2006a; 2006b).

Applying this highly conservative, theoretica l maximum exposure estimate, the U.S. EPA

determines how much of the established Refe rence Dose (RfD) would be utilized by all currently approved produc t uses. The chronic RfD (cRfD) is an estimate of the amount of daily pesticide exposure to the human popul ation that can occur over a lifetime with a

reasonable certaint y of no harm to human health 42 . For glyphosate, the chronic RfD is 1.75 mg per kg body weight per day (mg/kg bw/day) (U.S. EPA, 2006a; 2006b). The

U.S. EPA will utilize the Chr onic Population Adjusted Dose (cPAD, the cRfD with any FQPA uncertainty factors appl ied) from aggregated expos ures and from the exposure assessment to determine if these exposures ex ceed the U.S. EPA level of concern (i.e., 100 percent of the cPAD).

If the aggregate risk assessment shows that utilization of the cP AD does not exceed the U.S. EPA level of concern, then the U.S. EPA will conclude that the new use does not pose an unreasonable risk to human health. The U.S. EPA will then establish or revise, as needed, any food or animal feed crop tolerances to allow for the presence of glyphosate residue on that crop. However, under a recently announced policy aimed at increasing transparency to th e general and regulated public, the U.S. EPA may choose to publish the risk assessment and proposed regul atory decision on the Office of Pesticides website and ask for public comment; this posting occurs prior to the establishment of the tolerances (http://www.epa.gov/pesticides/regul ating/registration-status.html). The U.S. EPA publishes the new tolerances in the Federa l Register, along with a final summary of the risk assessment and approves pesticide labe ling for the new use. In issuing the final tolerance rule, the U.S. EPA considers and discusses any comments received in response to the original notice regarding the U.S. EPA’ s intention to establish tolerances that was published in the Federal Register and any co mments received in the transparency policy

notice.

42 RfD is the current terminology used by EPA; however earlier EPA risk assessment terminology used the term Allowable Daily Intake (ADI). RfD and ADI are synonymous.Monsanto Company 11-CA-233U 475 of 500 Despite the large number of approved food and feed uses of glyphosate, including uses associated with glyphosate-toler ant crops, a large margin of sa fety exists for glyphosate. While use of glyphosate has increased in th e decade since the intr oduction of glyphosatetolerant crops, the associated risk to human health as a result of the increased human exposure to glyphosate remains low, due to the low mammalian toxicity of glyphosate and the relatively low dietar y exposure associated with the herbicide’s approved uses.

Prior to the first approval of a glyphosat e-tolerant crop (soybean ) in 1996, theoretical maximum dietary exposure for all registered conventional uses of glyphosate utilized approximately 2.9% of the glyphosate RfD fo r the most highly exposed subpopulation of non-nursing infants less than one year old (U .S. EPA, 1993a). A more recent U.S. EPA risk estimate determined chronic dietary e xposure estimates for f ood and drinking water to glyphosate to be below the U.S. EPA le vel of concern for both the general U.S. population and population subgroups; 2% and 9% of the chronic population adjusted dose (cPAD) for the general population and non-nur sing infants (most exposed subpopulation) respectively (U.S. EPA, 2006b). The combin ed short/intermediate-term estimated exposure for dietary and non-dietary exposures with all current registered uses of glyphosate utilizes only 11% of the gl yphosate cPAD for the most sensitive subpopulation of non-nursing infants less than one year old (U.S. EPA, 2006b). The utilization of the glyphosat e cPAD, which is well below 100 percent, has allowed the

U.S. EPA to continue to make the conclusi on of reasonable certainty of no harm to human health for each glyphosate use, includ ing new glyphosate-tolerant crop uses.

These figures are supported by the data prov ided in the tables below. Table K-1

summarizes the established food and feed to lerances supporting the use of glyphosate in the conventional crops of alfalfa, cotton, su gar beet and soybean prior to the first glyphosate-tolerant crop in 1996. A summary of the regulator y approvals, including new or modified food and feed tolerances, and associated dietary exposure assessments for approved glyphosate-tolerant crops is provided in Table K-2. Tabl e K-3 summarizes the

most recent chronic and short/intermedia te-term aggregate risk assessments for glyphosate.Monsanto Company 11-CA-233U 476 of 500 Table K-1. Established Glyphosate Tolerances in Selected Crops Prior to Glyphosate-tolerant Crops (1993)

Crop Established Food/Feed

Tolerances Publication % of Reference Dose (RfD) 1

Soybean • Seed – 20 ppm

• forage & hay – 15 ppm

• hulls – 100 ppm Glyphosate Reregistration Eligibility

Decision

Document Sept. 1993 (U.S. EPA, 1993a) General Population - 1.2 Non-nursing infants <1 year

old - 2.9

Alfalfa 200 ppm

Cotton forage, hay, & seed – 15

ppm

Sugar beet Roots – 0.2 ppm

Maize 0.1 ppm seed

0.2 ppm forage/fodder

Canola none

1 % of Reference Dose based on all established tolerances in 1993.Monsanto Company 11-CA-233U 477 of 500 Table K-2. Summary of EPA Approvals for Glyphosate Use in Glyphosate-tolerant Crops

Commercial

Introduction

Year Required Changes in

Food/Feed Tolerances Federal Register

Publication Establishing New

or Modified

Tolerance Dietary Exposure Only

(Food + Water)

Roundup Ready soybean 1996 • Increase soybean forage to 100 ppm.

• Increase soybean hay to 200 ppm.

• Establish new tolerance for aspirated grain fractions at 50

ppm. 61 FR 15192

Petition No.

4F4369
Apr. 1996
(U.S. EPA, 1996b) General Population –

1% of RfD Non-nursing infants <1 year old- 2.5% of RfD Children 1-6 yrs – 2.5%

of RfD

Roundup Ready

cotton 1997 Establish new tolerance for gin byproduct at 100

ppm. 61 FR 7729

Petition No.

5F4493

Feb. 1996
(U.S. EPA, 1996a) General Population –

1% of RfD Non-nursing infants <1 year old - 2.4% of RfD Children 1-6 yrs – 2.3%

of RfD

Roundup Ready corn 1998 Establish new tolerance

for maize

 forage at 1 ppm

 grain at 1 ppm

 stover at 100 ppm. 62 FR 17723

Petition No.

5F4555
Apr. 1997
(U.S. EPA, 1997) General Population –

1% of RfD Non-nursing infants < 1 year old – 3% of RfD Children 1-6 yrs – 3% of

RfD

Establish new tolerance for maize forage at 3 ppm Sept 27 2000

65 FR 57957
Sep. 2000

U.S. EPA, 2000 #53040} General Population -

1.5% of RfD All infants <1 year old –

3.1% of RfD Children 1-6 yrs – 3.2%

of RfD

Roundup Ready corn

2 2004 Increased tolerance for maize forage to 6 ppm. 68 FR 36472

Jun. 2003
(U.S. EPA, 2003) Change in forage

tolerance did not affect estimated dietary exposure from animal products; therefore no dietary risk assessment

was conducted.

Roundup Ready

canola 1999 Establish new tolerances

for canola.

• seed at 10 ppm

• meal at 15 ppm 64 FR 18360

Petition No.

2E4118
Apr. 1999
(U.S. EPA, 1999) General Population -

1.5% of RfD Non-nursing infants <1 year old - 3.3 % of RfD Children 1-6 yrs – 3.2%

of RfD Roundup Ready sugar

beet 2008 Establish new tolerances for sugar beet.

• roots at 10 ppm

• tops at 10 ppm

• pulp (dried) at 25 ppmMonsanto Company 11-CA-233U 478 of 500 Table K-2. Summary of EPA Approvals fo r Glyphosate Use in Glyphosate-tolerant Crops (Continued)

Commercial

Introduction

Year Required Changes in

Food/Feed Tolerances Federal Register

Publication Establishing New

or Modified

Tolerance Dietary Exposure Only

(Food + Water)

Roundup Ready Flex

cotton 2006 • Increase tolerance for gin byproducts to 175

ppm.

• Increase tolerance for

undelinted cottonseed to 35 ppm. 69 FR 65081

Petition No.

3F6570

Nov. 2004
(U.S. EPA, 2004c) General Population -

2.2% of cPAD All infants < 1 year old -

-3.9% of cPAD Children 1-2 years -

5.4% of cPAD

Roundup Ready

alfalfa 2006 Establish new tolerances for alfalfa seed at 0.5 ppm. 70 FR 7861

Petition No.

2F6487

Feb. 2005
(U.S. EPA, 2005) Dietary exposure insignificant, did not

conduct new risk

assessment. Deferred to

assessment conducted

for flex cotton as published in 69 FR

65081.

Maize Increased tolerance for maize grain to 5 ppm. 73 FR 52607
Sept. 2008
(U.S. EPA, 2008a) Tolerance adjusted to

harmonize with

CODEX. Based on

conventional and glyphosate-tolerant uses

in maizeMonsanto Company 11-CA-233U 479 of 500 Table K-3. Aggregate Exposu re Assessment for Glyphosate

Population Subgroup Acute Aggregate 2 RfD (mg/kg/day) 2 Chronic Aggregate 1,2 Short/Intermediate Term Aggregate ,2,3

Exposure (mg/kg/day) %

cPAD Exposure (mg/kg/day) % RfD

General U.S. population

Not applicable 1.75 0.041 2 - -

All infants (<1 year) 0.127 7 0.157 9

Non-nursing infants (<1 year) 0.158 9 0.188 11

Children 1-2

years 0.095 5 0.125 7

Children 3-5

years 0.088 5 0.118 7

Children 6-12

years 0.059 3 0.089 5

Youth 13-19

years 0.037 2 - -

Adults 20-49

years 0.033 2 0.063 4

Adults 50+

years 0.028 2 - -

Females 13- 49 years 0.031 2 - -

1 These aggregate exposure assessments were performed by the U.S. EPA prior to the issuance of latest

guidance ( http://www.epa.gov/pesticides/h ealth/revisedRAmethods.pdf , December 2009), and thus do not include occupational exposure. As such, the ch ronic aggregate exposure estimated in this fashion was the same as chronic dietary exposure because chronic non-dietary expos ure was not expected based upon the registered non-crop uses of glyphosate.

2 U.S.EPA (2006b).

3 Calculated from values given in U.S.EPA (2006b).Monsanto Company 11-CA-233U 480 of 500 K.2.2. Glyphosate Safety Evaluation for Applicator and Bystander Exposure

Another potential impact of the use of gl yphosate on human health that the U.S. EPA

considers in its human health analysis is applicator and bys tander exposure resulting from increased glyphosate use. Based on the toxi city of glyphosate and its registered uses, including use on glyphosate-tolerant crops, the U.S. EPA has concluded that occupational exposures (short-term dermal and inhalation) to glyphosate are not of concern because no short-term dermal or inhalation toxicity e ndpoints have been id entified for glyphosate (U.S. EPA, 2006a; 2006b).

Additional evidence to support th e U.S. EPA conclusion can be found in the Farm Family Exposure Study (Acquavella et al., 2004), a bi omonitoring study of pe sticide applicators conducted by independent investigators. Th is biomonitoring study determined that the highest estimated systemic dose of glyphosate for applicators as the result of routine labeled applications of registered glyphosat e-based agricultural herbicides to crops, including glyphosate-tolerant crops, was a pproximately 400 times lower than the RfD established for glyphosate. Furthermore, investigators determined that 40% of applicators did not have detectable exposure on the day of application, and 90% of the applicators had an estimated systemic dose of glyphosate less than 0.06% the RfD (Acquavella et al., 2004).

The biomonitoring study also found little eviden ce of detectable exposure to individuals

on the farm who were not activ ely involved in or located in the immediate vicinity of labeled applications of glyphosate-based agricu ltural herbicides to crops. Considering the similarity of the use pattern and appli cation rates of the glyphos ate products in this study compared to those registered for use on glyphosate-tolera nt crops, bystander exposure attributed to the use of glyphosate on glyphosate-tole rant crops is expected to be negligible.

K.3. Potential Impact of Glyphosate on the Environment

Potential environmental effects are carefully co nsidered as a part of the FIFRA pesticide registration process. Prior to the approval of a new pesticide or a new use (including a change in pesticide applicati on rates and/or timing) and befo re re-registering an existing pesticide, the U.S. EPA must consider the pot ential for environmental effects and make a determination that no unreasonable adverse effects to the environment will be caused by the new pesticide, new use or continued use.

To make this determination, the EPA requires a comprehensive set of environmental fate and ecotoxicology data on the pesticide’s active ingredient (40 CFR § 158). The U.S. EPA uses these data to assess the pesticide’s potential environmental risk (exposure/hazard). The required data include both short and long-term hazard data on representative organisms that are used to pr edict hazards to terrestrial animals (birds, nontarget insects, and mammals), aquatic anim als (freshwater fish and invertebrates, estuarine and marine organisms) and nontarge t plants (terrestrial and aquatic).Monsanto Company 11-CA-233U 481 of 500 The U.S. EPA re-evaluated the environmental safety of glyphosate in 1993 as part of the FIFRA-required re-registration of all pesticides. At the e nd of this evaluation, the EPA concluded that all registered uses of glyphosat e were eligible for re -registration, including terrestrial (i.e., land-based) applications up to 6 lbs glyphosat e acid equivalents (a.e.) per acre on crops, and 8 lbs glyphosate a.e pe r acre for certain limited uses.

Since the re-registration evaluation in 1993, th e U.S. EPA has reviewed and approved a significant number of new glyphosate uses: on conventiona l crops such as legume vegetables and sunflower/safflower seed, glyphosat e-tolerant crops such as alfalfa, maize, cotton, canola, sugar beet and soybean and in non-crop areas. In each case, the EPA concluded that the new use, including a ny incremental environmental exposure to glyphosate caused by that new use, did not pose an unreasonable risk to the environment, and approved pesticide labe ling for the new use.

The studies and data collected by Monsanto, both for the initi al U.S. EPA registration and re-registration of glyphosate, as well as data developed by inde pendent academics, present a well-established safety profile fo r glyphosate. The following sections provide greater detail regarding some of th e key findings from these studies.

K.3.1. Persistence of Glyphosate in the Soil

Persistence of agricultural chemicals in the soil is widely regard ed as an undesirable

environmental characteristic. Glyphosate has been shown to degrade over time from most agricultural ecosystems across a wide ra nge of soil and climatic conditions, with a median soil half-life (the time it takes for half of the glyphosate to di ssipate in the soil) of 13.9 days (U.S. EPA, 1993a). The potential for glyphosate to accumulate in soil following repeated applications has been stud ied both in the laborat ory and the field.

A laboratory study was conducted on two soil samp les, with each sample receiving up to three sequential applicat ions of 5 pounds glyphosat e a.e. per acre over a 6-week period, at

two-week intervals. The concentration of glyphosate in soil 24 weeks following application had declined to 1-5% of the concentration i mmediately after application, regardless of whether it was the firs t, second or third application.

Glyphosate degradation in the soil following mu ltiple glyphosate applications was also

shown under field conditions. Soil was colle cted from pesticide efficacy and tolerance trials in orchards and vineyards that receiv ed repeated applications of glyphosate over a one- to six-year period, at cumulative rate s of 6 to 120 pounds glyphosate a.e. per acre. These soil samples did not show any accumula tion of glyphosate residues, even at the exaggerated rate of three seque ntial applications of eight pounds glyphosa te a.e. per acre
within a three-month interval for five out of six sequential years. Glyphosate degradation continued after multiple applications, and less than 10 percent of the total applied glyphosate remained in the soil one year after the last glyphos ate application.

Similar to some of the other glyphosate tole rant crops, a typical agronomic (annual) use pattern for glyphosate on MON 88302 could include a pre-emergence burn down application of up to 3.7 pounds of glyphosate acid equivalent (a.e.) per acre. This couldMonsanto Company 11-CA-233U 482 of 500 be followed by one to two post-emergence in-cro p applications for weed control of up to a total of 1.6 pounds glyphosate a.e. per acre. The total amount of glyphosate that could be applied per season (pre-emergence thr ough first flower) cannot exceed 5.3 pounds glyphosate a.e. per acre. Thus , the maximum labeled rates a nd typical use patterns of glyphosate on MON 88302 are well within the rates and frequencies used in the soil persistence studies described in this section. As a result, glyphosate is not expected to accumulate in soil when MON 88302 is appl ied according to label directions.

K.3.2. Persistence of Surfactant in the Soil

Herbicide products approved for application to emerged weed s normally are applied with surfactants. Glyphosate products are formul ated with surfactan ts to increase the permeability of the cuticle wax of the weed foliage, resulting in increased foliar uptake of glyphosate. In other words, the surfactant acts to alter the physical properties of the plant’s natural protective wax coating, allowing the plant to better absorb the glyphosate, thereby improving the efficacy of the herbicide.

One common surfactant used in formulated glyphosate products is polyethoxylated alkyl amine (POEA). When degradati on of POEA was investigated in three types of soil (silt loam, silty clay loam and sandy loam), microbi al degradation was determined to be the primary degradation route, with minimal de gradation occurring unde r sterile conditions. Approximately 25-30% of applied 14 C-POEA was mineralized to 14 CO 2 within seven weeks. The estimated degradation half-life for parent POEA was less than one week and possibly as short as one to two days. B ecause limited data are available for POEA dissipation, a conservative estim ate of half-life values for POEA in soil would be 7-14 days (Giesy et al., 2000). Glyphosate a nd the POEA surfactan t have similar soil dissipation rates and the same primary route of dissipation, i.e., microbial degradation. Therefore, it is reasonable to assume that the POEA surfactant will behave similarly to glyphosate in field soil, and an in crease in residual soil con centrations (accumulation) of

the POEA surfactant is not anticipated as a result of increased use of glyphosate associated with the planting of MON 88302.

K.3.3. Surface Water and Groundwater

Glyphosate binds strongly to agricultural soils and has a low potential to move offsite to

surface water or leach to groundwater (U.S. EPA, 1993a). The U.S. EPA has used computer models to estimate worst-case glyphosate levels in surface water based on presently approved use patterns. Relying on toxicological data fr om acute and chronic tests on fish and other aquatic organisms, th e EPA has determined that “the potential for environmental effects of glyphosate in surf ace water is minimal” (U.S. EPA, 2002).Monsanto Company 11-CA-233U 483 of 500 K.3.4. Wildlife

Animals

As a part of the re-regist ration evaluation under FIFRA, the U.S. EPA conducted an ecological assessment for glyphosate. This assessment compared the results from toxicity tests using glyphosat e, conducted with various plan t and animal species to a

conservative estimate of the c oncentration of glyphosate to which an organism might be exposed in the environment. This estimate, called the Es timated Exposure Concentration (EEC), is a point estimate for exposur e that does not take into account normal environmental dilution or dissipation, or the frequency of exposure to the pesticide by wildlife. In the Re-registr ation Eligibility Decision (R ED) for glyphosate (U.S. EPA, 1993a), the exposure estimates were determ ined assuming an application rate of

5.0625 lb a.e. 43 per acre, which exceeds the maximum labeled use rate for a single application for agricultural purposes. When the EECs were calculated for aquatic plants and animals, the direct application of this rate to water was assumed. Based on this assessment, the U.S. EPA concluded that effects to birds, mammals, fish and

invertebrates are minimal based on av ailable data (U.S. EPA, 1993a).

Glyphosate is classified as pr actically non-toxic to honey bees (which are us ed to assess effects on nontarget insects in general) and as practically non-toxic to slightly toxic to birds, freshwater fish, marine and estuarin e species, aquatic invertebrates and mammals (U.S. EPA, 1993a). Glyphosate has a low octanol-water co efficient, indicating that it has a tendency to remain in the water phase rather than move from the water phase into fatty substances; therefore, it is not expected to accumulate in fish or other animal tissues.

The glyphosate end-use products used in agricult ure contain a surfactan t to facilitate the uptake of glyphosate into the plant (Ash ton and Crafts, 1981). Depending on the surfactant used, the to xicity of the end-use product ma y range from practically nontoxic to moderately toxic to fish and aquatic inve rtebrates (U.S. EPA, 1993a ). For this reason, the 1993 Glyphosate RED stated that some fo rmulated end-use products of glyphosate

needed to be labeled as “Toxic to fish” since some glyphosate products are applied directly to aquatic environments. Due to the associated hazard to fish and other aquatic organisms, glyphosate end-use products that are labeled for applic ations to aquatic environments generally do not contain surfactant, or contain a surfactant approved for direct application to aquatic environments.

Plants

Glyphosate is a non-selective he rbicide with post-emergence activity on essentially all annual and perennial plants. As such, e xposure to glyphosate co uld put aquatic and terrestrial nontarget plants as well as threaten ed or endangered plants at risk (U.S. EPA, 1993a). Nontarget plants may potentially be at risk from applicati ons of glyphosate as a result of spray drift. As discussed earlier, glyp hosate binds tightly to agricultural soils

43 Although the RED refers to the maximum application ra te as a.i., it can be determined from the EEC’s obtained that the maximum rate in acid equivalents was actually used in the calculation of the EEC’sMonsanto Company 11-CA-233U 484 of 500 and has low potential to move offsite dissolv ed in water. More over, glyphosate is not taken up from agricultural soils by plants. Th erefore, risks to nont arget plants are only attributed to the spray drift of the pesticid e. Pesticide labels include specific risk management measures to manage spray dr ift, including mandato ry requirements for aerial applications.

During the re-registration process in 1993, add itional data on terrestrial nontarget plants were requested by the U.S. EPA. These additional data have been utilized in conjunction with an exposure assessment to further unde rstand the potential ri sk to nontarget and threatened and endangered plants from the use of glyphosate he rbicides in agriculture. Using the methodology described in a recent EPA effects determination for glyphosate (U.S. EPA, 2008a), it can be determ ined that there is minimal risk to terrestrial plants that are not listed as threatened or endangere d at the maximum glyphos ate single application rate for ground applications (3.75 lbs a.e. glyphos ate per acre), and at rates just below the maximum single aerial application rate (1.48 lbs a.e. glyphosate per acre compared to 1.55 lbs a.e. glyphosate per acre). Nonliste d plants, therefore, are unlikely to be significantly impacted by glyphosate applic ations to MON 88302. The potential impact to threatened and endangered plant species ha s been further considered in an analysis conducted by Monsanto (Mor tensen et al., 2008).

K.3.5. Endangered and Threatened Species

The U.S. EPA Endangered Species Protection Program web site, http://www.epa.gov/espp/, describes the U.S. EPA assessment process for endangered species. The essential elements of that pro cess, generally taken from the web site, are

summarized below.

The Endangered Species Act (ESA) was intended to protect and promote the recovery of animals and plants that are in danger of becoming extinct. All federal agencies are required under the ESA to ensure that their regulatory actions, including the U.S. EPA’s registration of pesticides in the U.S., are not likely to jeopardize the continued existence of threatened or endangered species (“list ed” species) or destroy or adversely modify

their critical habitat.

The U.S. EPA's Endangered Species Protecti on Program (ESPP) which helps promote the recovery of listed speci es is designed to determine whether pesticide use in a certain geographic area may affect any listed species.

When registering a pesticide or reassessing th e potential ecological risks from use of a currently registered pesticide, the U.S. EPA extensively evaluates environmental fate and ecological effects data to determine how a pesticide will move through and break down in the environment. Risks to birds, fish, inve rtebrates, mammals and plants are routinely

assessed and used in U.S. EPA’s determinati ons of whether a pesticide may be licensed

for use in the U.S.

The U.S. EPA’s core pesticide risk assessm ent and regulatory pr ocesses ensure that protections are in place fo r all populations of nontarget species. Because endangeredMonsanto Company 11-CA-233U 485 of 500 species may need specific protection, the U.S. EPA has developed risk assessment procedures described in the Overview of the Ecological Risk A ssessment Process (U.S. EPA, 2004d) to determine whether individuals of a listed species have the potential to be harmed by a pesticide, and if s o, what specific protections ma y be appropriate. The U.S. EPA’s conclusion regarding the potential risks a pesticide ma y pose to a listed species and any designated critical habitat for the species, after conducting a thorough ecological risk assessment, results in an "effects determination."

An evaluation of the effects of glyphosate us e on all types of threatened and endangered species was conducted by Monsanto. This ev aluation generally followed the procedures described in the Overview of the Ecologi cal Risk Assessment Process (U.S. EPA, 2004d), as summarized in Figure K-1.Monsanto Company 11-CA-233U 486 of 500 Figure K-1. Tier I Endangered Species Analysis

Risk quotients (RQ’s) were calculated as th e quotient of the Estimated Environmental Concentration (EEC) and the relevant toxicity endpoint for the most sensitive species for a given taxon (class of species). For acu te studies of a few days duration, the concentration calculated to result in 50% mortality (LC 50 ) or 50% designated effect (EC 50 ) on the test species was utilized in th e RQ calculation. For chronic studies, representing a significant porti on of the species life-cycle, the highest concentration at which no effects were observed (No Observed Effect Concentration, NOEC) was used in the RQ calculation.

Toxicity values (effects endpoints) for most categories of species were taken from a recent U.S. EPA assessment for a new glyphosate use (U.S. EPA, 2006c), or from the U.S. EPA guideline studies c onducted by Monsanto if these e ndpoints were lower. Studies from the literature were considered when the study design was appropriate for the assessment being made and where suffici ent information regarding glyphosate or

formulation test concentrations was availa ble. Exposure estimates were based on standard U.S. EPA methods for calculating exposure (U.S. EPA, 2004d). For aquatic organisms, the model GENEEC2 (U.S. EP A, 2004d), which calculates high-end

estimates of surface water concentrations of pesticides in a generic farm pond, was utilized. When formulation toxicity was c onsidered, default drift values and the U.S. EPA standard pond 44 were utilized for estimation of aquatic exposure. For terrestrial

44 A water body with a depth of 2 m and a volume of 20,000 liters. Exposure Estimate:

Worst Case

Estimated Environmental Concentration (EEC) Using Standard EPA Models Or Analysis Methods Toxicity Endpoint (TE):

Lowest Acute Or Chronic Value Meeting EPA Quality Standards

RQ ≥ Level Of Concern (LOC)?

Further Analysis Determine Risk Quotient (RQ = EEC/TE)

Yes For Each Taxon

Risk To Taxon Is Expected To Be Low NoMonsanto Company 11-CA-233U 487 of 500 animals, the T-REX model (U.S. EPA, 2008c) wa s utilized to calcula te estimated dietary exposure and risk. For terrestri al and semi-aquatic plants, only the drift component of the Terrplant model (U.S. EPA, 2004d) was used to determine exposure levels (the runoff component was disregarded). Runoff was not c onsidered to contribut e to exposure, since glyphosate binds very tightly to agricultural soils and does no t have herbicidal properties when bound to soil (U.S. EPA, 2006c).

Risk quotients calculated using toxicity endpo ints and estimated exposure concentrations determined as described above were compared with the U.S. EPA established Levels of Concern (U.S. EPA, 2004d). If the RQ exceeded the Endangered Species Level of Concern, a conclusion was reached that a threatened or endangered species may potentially be affected by the glyphosate use. Because initial RQ calculations are made with worst case exposure estimates, furthe r refinement of the estimated exposure concentration or of toxicity endpoints may subs equently indicate the RQ is not as high as initially calculated. If the RQ is less than the Endangered Species Level of Concern it can be concluded that the species will not be affected by glyphosate use.

The conclusion from this evaluation, submitted to the USDA and the U.S. EPA, is that threatened or endangered terrestrial or semi-aquatic plant species are not at risk 45 from ground applications of glyphosate at rates less than 3.5 lb glyphosate a.e. per acre, or from aerial applications at rates less than 0.70 lb a.e. per acre. However, potential effects to these species cannot be excluded when rates exceed these levels. All in-crop applications to MON 88302 will be 1.6 lb a.e. per acre or less, which are not predicted to affect threatened and endangered plants if a pplications are made us ing ground equipment.

Rates that exceed 3.5 lb a.e. per acre, if used by growers, are generally for control of perennial species prior to crop emer gence or prior to harvest.

The same evaluation determined that other ta xa (including birds, ma mmals, insects, fish, amphibians, aquatic invertebrates and non-vascul ar aquatic plants) w ould not be affected by the use of glyphosate herbicides in crop production. Furthermore, this evaluation

determined that these other taxa were not at risk from indirect effects resulting from habitat alteration from the use of glyphosate, since non-endangered terrestrial or semiaquatic plants were not c onsidered to be at risk of direct effects.

Based on Monsanto’s determination that threatened and endangered plant species potentially could be affected by certain us es of glyphosate in crop production (e.g., aerial applications at the maximum aerial rate), a more detailed evaluation of the locations of threatened and endangered plant species rela tive to areas of crop production has been undertaken. The first crop to be assessed wa s alfalfa (Honegger et al., 2008), but canola, maize, cotton, soybeans and sugar beets have now also been evaluated. The evaluation process was divided into three phases, as outlined below.

 First, the co-occurrence of observations of threatened or endangered plant species and the presence of alfalfa, canola, maize, cotton, soybeans or sugar beet

45 Risk to threatened or endangered plant species is only assessed outside of agricultural production areas.Monsanto Company 11-CA-233U 488 of 500 production was determined at the county level. This analysis (Phase 1)

considered the 3028 counties in which at l east one of these six crops are grown, which comprise 96% of the 3141 counties and equivalent areas 46 in the 50 states of the U.S. Species were reviewed for a pplicable exclusions at the county-level, which indicated, for some species, that glyphosate use in th ese crops posed no risk of adverse effects to these species.

 Next, for threatened or endangered plan t species where effects could not be excluded at the county leve l, the possible exposure to glyphosate was assessed at the sub-county level (Phase 2) in the sa me counties considered in Phase 1. This analysis used information available at the sub-county level for threatened and endangered plant species locations and for la nd use. Land uses considered in this analysis are those classified as Pasture/Hay and Cultivated Crops. 47

 Finally, in sub-county areas where, unde r certain application conditions, the potential for threatened and endangered pl ant species to be affected by exposure to glyphosate could not be excluded, areas have been defined so that grower practices can be implemented to limit glyphosate exposure (Phase 3). Measures to limit glyphosate exposure in these ar eas have been proposed. These measures include: 1) limiting ground application rate s to less than 3.5 lb glyphosate a.e. per acre in areas identified for potential use limitation when the potential habitat for the threatened or endangered species is present and 2) for aerial applications greater than 0.7 lb a.e./acre, implementing an unsprayed buffer between the potential habitat for the listed species a nd the application area. Proposed buffer distances are based on app lication rate, droplet si ze and wind direction.

This analysis was initially completed for th e U.S. counties in which alfalfa, canola, maize, cotton, soybeans and sugar beets we re grown based on the 2002 Ag Census (USDA-NASS, 2002) and listed species in formation available through early 2008.

Of the 3028 U.S. counties where alfalfa, ca nola, maize, cotton, soybeans and sugar beets were produced, 11% of counties (334 counties) required the definiti on of potential areas

for use limitations. In the other 2694 counties, either th ere were no threatened or endangered plant species present, or the sp ecies present were ei ther excluded from concern (based on habitat or pr oximity information), had existing protections, or were not in proximity to potential areas of production of the si x crops evaluated. This analysis is being updated based on the 2007 Ag Census (USDA-NASS, 2009) and updated listed species information.

The Roundup Ready crop analysis considered the land classifications where agricultural crop production may occur (in counties with reported farms producing any of the six crops) in the assessment of proximity to observations of threatened or endangered

46 Equivalent areas include independent cities th at are not within the boundaries of a county.

47 Land use was based on the National La nd Cover Database (2001) for the continental U.S. and on the NOAA (National Oceanic and Atmospheric Ad ministration) Coastal Services Ce nter land cover data for Hawaii.Monsanto Company 11-CA-233U 489 of 500 species. Thus, the identification of potential use limitation areas al so applies to other crops in those counties.

K.3.6. Potential Effects on Endangered Anim al Species Identified by the U.S. EPA or in Litigation

As previously discussed, no indirect effect s on threatened or endangered animal species are predicted, since no significant direct effects due to pe sticide drift onto nonendangered plant species are predicted. In the Glyphosate RED (U.S. EPA, 1993a), the U.S. EPA suggested that glyphosate may have ef fects on the habitat of the Houston Toad. After the issuance of the 1993 Glyphosate RED, Monsanto conducted a vegetative vigor study. When relevant effects data from that study are considered, it can be determined that the amount of glyphosate per unit area pred icted to drift away from the site of an agricultural application is less than the amount per unit area observed to have a 25% effect on plant dry weight or growth of the most sensitive of ten species tested in the study. Thus, the habitat of the toad is not lik ely to be significantly affected by glyphosate drift, and hence the toad is not likely to be at risk from the agricultural use of glyphosate.

The EPA evaluated the effect of glyphosat e on the California Red-legged Frog in response to a consent agreement reached in a lawsuit filed by the Center for Biological Diversity 48 . In the California red-legged frog effects determination (U.S. EPA, 2008b), the U.S. EPA considered glyphosate rates up to 7.95 lb a.e. per acre. Even at this high rate, the U.S. EPA concluded th at there would be no direct effect to th e aquatic phase of the California red-legged frog (CRLF) from application of glyphosat e or any glyphosate

formulation or salts 49 . The U.S. EPA also concluded th at there are no direct effects to the terrestrial-phase of the CRLF at rates of 3.85 lb a.e. per acre and below (with the exception of one formulation that is not registered for use in MON 88302). 50 Since the maximum in-crop application rate for MON 88302 is 1.6 lb a.e. per acre, glyphosate

formulations and rates used in MON 88302 do not pose any potent ial risk of direct effects to the aquatic or terrestri al-phase of the CRLF.

With respect to indirect effects on prey, th e U.S. EPA concluded that no effect on the following prey items: algae, aquatic invert ebrates, aquatic-phase frogs or fish, or terrestrial-phase frogs would occur at glyphosate rates of 3.84 lb a.e. per acre. 51 The potential for some effects on terrestrial inve rtebrate and small mammal prey items were identified but only at rates above 7.5 lb a.e. per acre and 3.75 lb a.e. per acre, respectively. 52 No chronic effects on mammals were predicted at rates of 3.75 lb a.e. per

48 Center For Biological Diversity v. Leavitt

, 2005 WL 2277030 (N.D.Cal., September 19, 2005).

49 (U.S. EPA, 2008a)Page 11, “The acute and chronic LOC’s for freshwater fish and aqua tic-phase amphibians are not exceeded for either glyphosate, its salts or its formulations”.

50 Ibid., Page 14, Table 1.3.

51 Ibid., Page 15, Table 1.4.

52 Ibid., Page 156, ibid. For terrestrial inve rtebrate prey items that we re not listed (i.e. not thre atened or endangered), no effects were predicted at any rate up to 7.95 lb a.e. per acre for large in vertebrates, and the Level of Concern was only exceeded for small invertebrates at application rates of 7.5 lb a.e. per acre and above. Levels of Concern were exceeded at lower rates for listed (threatened or endangered) terrestrial invertebrates, but listed species would not be anticipated to be a significant portion of the CRLF diet. Listed small inverteb rates in areas adjacent to a MON 88302 field, would not be at risk from spray drift e xposure from glyphosate applications to MON 88302.Monsanto Company 11-CA-233U 490 of 500 acre and below. 53 Therefore, no effects on CRLF prey were identified at the maximum single application rate for MON 88302. Simila rly, no effects on terrestrial plant habitat are identified for ground applicatio ns at 1.6 lb a.e. per acre. 54

Based on the CRLF effects determination conduct ed by the U.S. EPA, it is not expected that the glyphosate rates applied to MON 88302 would have any direct or indirect impact

on the CRLF.

The EPA also has evaluated the potential eff ect of glyphosate on salmon in eleven areas in California and Southern Oregon 55 in response to the cons ent agreement reached in

another lawsuit 56 . The conclusion of the U.S. EPA’s risk assessment was that for all uses of glyphosate with application rates of 5 lb ac tive ingredient (a.i.) per acre or below, the Agency determined that glyphosat e will have no effect on the s ubject listed species. (U.S. EPA, 2004a; 2004b). All glyphosate use rates for agricultural uses are 5 lb a.i. per acre (3.7 lb glyphosate a.e. pe r acre) or below, so no risk to salmon is anticipated from these

uses.

K.3.7. Other Potential Environmental Impact s Associated with Glyphosate Use in Glyphosate-tolerant Crops

As discussed more fully below, the potential im pacts to soil attributable to the change in production (cultivation) practices associated with the deregul ation of glyphosate-tolerant crops have been assessed. The adoption of glyphosate-tolerant crop s and the ability to use glyphosate-based agricultural herbicides is not expected to significantly change agricultural practices, except to enable th e adoption of no-till s eeding practices.

No-Till Practices: No-till production is the practice of establishing an agricultural seed bed and controlling weeds without mechanically tilling the soil. In stead, the only tillage

of the soil is done at the tim e of planting, with the crop bei ng seeded direc tly into the previous year’s crop residue. Among other environmen tal benefits, no-till production reduces soil erosion and the use of petroleum- based fuels for tractors. The practice has

been shown to minimize surface water runoff and soil erosion and to improve soil quality by increasing the soil organic matter that helps bind soil nutrients and prevent their loss to runoff, erosion and leaching (Leep et al., 2003).

No-till agriculture can provide benefits to water bodies, as well. No-till practices reduce soil erosion to surface water bodies, decreasing the amount of sediment in rivers and

53 Ibid., Page 12. The potential for chroni c effects on small mammals were only identified at rates of 3.84 lb a.e. and above (Risk Quotient (RQ) greater than the LOC). Since the next lower rate consider ed in the CRLF evaluation is 3.75 lb a.e.per acre, it can be concluded that the RQ did not exceed the LOC for these rates, and, therefore, no effects would be anticipated from an applica tion rate of 1.6 lb a.e.per acre)

54 Ibid., Page 136-138. Risk quotients < 1 are below the Level of Concern. Table 5.6 and 5.7 indicate RQ’s for ground applications of 1.54 lb a.e.per acre ar e well below the LOC of 1. Since th e maximum in-crop application rate for MON 88302 is 1.6 lb a.e.per acre no risk of indirect effects to CRLF because of effects on plants in the habitat are predicted from glyphosate use on MON 88302.

55 These areas are call Evolutionarily Significant Units ba sed on the salmonid populations present in these areas.

56 Washington Toxics Coalition v. Environmental Protection Agency , 413 F.3d 1024 (9th Cir. 2005).Monsanto Company 11-CA-233U 491 of 500 streams. Sedimentation increases the turbidity (cloudiness) of surface water bodies, reducing light penetration, impairing photos ynthesis and altering oxygen levels, which cause a reduction of food sources for some aquatic organisms. Sediment can also cover spawning beds and impact fish populati ons. Phosphorus (a major component of fertilizer) bound to soil particle s can be transferred to rivers and lakes via soil erosion, giving rise to high levels of phosphorus in surface waters, which may lead to algae blooms that can impact desirable fish populations (Hill and Mannering, 1995).

Soil Microorganisms : Results of standardized tests with glyphosate formulations performed for submission to regulatory ag encies indicate no long-term effects on microorganisms in soil even at rates that exce ed maximum use rates (up to five times the labeled rate). In addition, independent researchers have reviewed numerous laboratory and field studies, investigatin g the effects of glyphosate on so il bacteria and fungi (Felsot, 2000; Giesy et al., 2000). Although some laboratory test s have shown effects on nitrogen-fixing bacteria (M oorman et al., 1992; Santos a nd Flores, 1995) and soil fungi (Busse et al., 2001; Estok et al., 1989), effects ar e typically observed only under artificial laboratory conditions and at glyphosate concentrations well above normal field application rates. Several researchers have co ncluded that it is difficult to extrapolate results from the laboratory to the natural soil environment (Busse et al., 2001; Estok et al., 1989; Wan et al., 1998; Wardle and Parkinson, 1990b).

In studying microorganisms from soil in pine pl antations, (Busse et al., 2001) noted: “Our findings suggest that artificial media assa ys are of limited relevance in predicting glyphosate toxicity to soil organi sms and that field rate applic ations of glyphosate should

have little or no effect on soil microbial co mmunities in ponderosa pine plantations.” Long-term studies following repeated applicati ons of Roundup agricult ural herbicides in the field at labeled use rates for multiple appl ications in one year (Olson and Lindwall, 1991) or over 15 years (Biederbeck et al., 1997; Hart and Brookes, 1996) have shown no long-term adverse effects on soil microbes. Investigations by Hane y et al. . (2002; 2000)

related to the increased use of glyphosate-tolerant crops indicate that glyphosate was degraded over time by soil microbes, even at high application rates, without adversely impacting the soil microbial community. In ad dition, results from fiel d studies that have evaluated the fungal co mponent of the soil microbial comm unity indicate that glyphosate

treatment had no deleterious effects on bene ficial soil fungi (Araujo et al., 2003; Biederbeck et al., 1997; Bu sse et al., 2001; Wardle an d Parkinson, 1990a; 1990b). Moreover, the history of safe use and yi eld data obtained for nearly 10 years of glyphosate-tolerant crop production, combined with in-crop applications of glyphosatebased agricultural herbicides, reinforce the findings that soil mi crobes and microbiallymediated processes are not adversely impacted by field-rate applications of glyphosate.

The Potential for Glyphosate Metal Chelation to Affect Soil Fertility: Plants are dependent on the uptake of a number of different metal cations from the soil for optimal growth. Glyphosate is known to chelate, or tig htly bind, to several di- and trivalent metal

cations such as Fe 3+, Cu 2+ , Mn 2+ , Al 3+ and Ca 2+ that are needed by plants (Glass, 1984; Madsen et al., 1978). Cations that chelate glyphosate have been shown to reduce the efficacy of glyphosate when present in suffi cient amounts in the tank mix spray solution (Bernards et al., 2005). In the spray soluti on, there is a simple interaction betweenMonsanto Company 11-CA-233U 492 of 500 glyphosate and metal cations, which reduces the herbicidal activ ity of glyphosate. However, in the soil environment, the interactions between metals and chelators are much more complex (Parker et al., 2005). Glyphosate can interact w ith metals that are present

on the surface of soil particles, as well as with disso lved metal ions in the water soil solution. In addition to glyphosate, many other potential ligands or chelators are present

in soil that can also interact with metals. As a result, there is a complex multi-component equilibrium between glyphosate, other ligands or chelators, and numerous metals present in soil. Glyphosate is only one factor in this system. Numerous compositional analysis studies have demonstrated a lack of any si gnificant immobilization of mineral nutrients by glyphosate in soil that results in reduced uptake by plants. These studies have shown that glyphosate-tolerant crops that have been sprayed with glyphosate do not have decreased micronutrient levels compared to untreated controls (McCann et al., 2006; Obert et al., 2004; Ridl ey et al., 2002).

Transport through the Soil – Surfactant: Available data also suggest that the POEA surfactant used in Roundup agricultural herbic ides binds strongly to soil (estimated soil organic carbon-water pa rtition coefficient (K

oc ) values range from 2500 to 9600 57 ) and undergoes microbial degradation with an estima ted half-life of less than 14 days (Marvel et al., 1974). POEA is rapidly partitioned (half-life of 13 to 18 hours) from water to sediment in a water / sediment study (Wang et al., 2005). The rapid partitioning of the POEA surfactant to soil / sediment combined with the high K

oc values indicates that the surfactant will be tightly bound to the soil. The Groundwater Ubiquity Score (GUS) is an index that indicates the potential for com pounds to leach from soil into groundwater

based on their ha lf-life and K

oc (Gustafson, 1989). Using an estimated half-life of 14 days and a K

oc of 2500 as conservative estimates of the rate of degradation and binding to soil, the GUS index for the POEA surfactan t is 0.69. According to the GUS movement ranking, this GUS index indicates that POEA has a very low potential to leach to groundwater.

57 Estimated from the partition ratio between water and ster ile soil as reported in the POEA soil degradation study

(Marvel et al., 1974).Monsanto Company 11-CA-233U 493 of 500 Attachment 2. Appendix K. U.S. Gly phosate Tolerances for Food & Feed Commodities (40 CFR § 180.364)

Commodity Parts/million Commodity Parts/million

Acerola 0.2 Durian 0.2

Alfalfa, seed 0.5 Egg 0.05

Almond, hulls 25 Epazote 1.3

Aloe vera 0.5 Feijoa 0.2

Ambarella 0.2 Fig 0.2

Animal feed, nongrass, group 18 400 Fish 0.25

Artichoke, globe 0.2 Flax, meal 8.0

Asparagus 0.5 Flax, seed 4.0

Atemoya 0.2 Fruit, citrus, group 10 0.5

Avocado 0.2 Fruit, pome, group 11 0.2

Bamboo, shoots 0.2 Fruit, stone, group 12 0.2

Banana 0.2 Galangal, roots 0.2

Barley, bran 30 Ginger, white, flower 0.2

Barley, grain 20 Goat, kidney 4.0

Beet, sugar, dried pulp 25 Goat, liver 0.5

Beet, sugar, roots 10 Gourd, buffalo, seed 0.1

Beet, sugar, tops 10 Governor's plum 0.2

Berry group 13 0.2 Gow kee, leaves 0.2

Betelnut 1.0 Grain, as pirated fractions 100

Biriba 0.2 Grain, cereal, forage, fodder and straw, group 16, except maize forage 100

Blimbe 0.2 Grain, cereal, group 15, except barley, commercial maize, grain sorghum, oat

and wheat 0.1

Borage, seed 0.1 Grape 0.2

Breadfruit 0.2 Grass, forage, fodder and hay, group 17 300

Cacao bean 0.2 Guava 0.2

Cactus, fruit 0.5 Herbs subgroup 19A 0.2

Cactus, pads 0.5 Hog, kidney 4.0

Canistel 0.2 Hog, liver 0.5

Canola, meal 15 Hop, dried cones 7.0

Canola, seed 10 Horse, kidney 4.0

Cattle, kidney 4.0 Horse, liver 0.5

Cattle, liver 0.5 Ilama 0.2

Chaya 1.0 Imbe 0.2

Cherimoya 0.2 Imbu 0.2

Citrus, dried pulp 1.5 Jackfruit 0.2

Coconut 0.1 Jaboticaba 0.2

Coffee, bean 1.0 Jojoba, seed 0.1

Maize, field, forage 6.0 Juneberry 0.2

Maize, field, grain 1.0 Kava, roots 0.2

Cotton, gin byproducts 175 Kenaf, forage 200

Cotton, undelinted seed 35 Kiwifruit 0.2

Cranberry 0.2 Lesquerella, seed 0.1

Crambe, seed 0.1 Leucaena, forage 200

Custard apple 0.2 Lingonberry 0.2Monsanto Company 11-CA-233U 494 of 500 Commodity Parts/million Commodity Parts/million

Date 0.2 Longan 0.2

Dokudami 2.0 Lychee 0.2

Mamey apple 0.2 Sapote, black 0.2

Mango 0.2 Sapote, mamey 0.2

Mangosteen 0.2 Sapote, white 0.2

Marmaladebox 0.2 Sesame, seed 0.1

Meadowfoam, seed 0.1 Sheep, kidney 4.0

Mioga, flower 0.2 Sheep, liver 0.5

Mustard, seed 0.1 Shellfish 3.0

Noni 0.20 Sorghum, grain, grain 15

Nut, pine 1.0 Soursop 0.2

Nut, tree, group 14 1.0 Soybean, forage 100

Oat, grain 20 Soybean, hay 200

Okra 0.5 Soybean, hulls 100

Olive 0.2 Soybean, seed 20

Oregano, Mexican, leaves 2.0 Spanish lime 0.2

Palm heart 0.2 Spearmint, tops 200

Palm heart, leaves 0.2 Spice subgroup 19B 7.0

Palm, oil 0.1 Star apple 0.2

Papaya 0.2 Starfruit 0.2

Papaya, mountain 0.2 Stevia, dried leaves 1.0

Passionfruit 0.2 Strawberry 0.2

Pawpaw 0.2 Sugar apple 0.2

Pea, dry 8.0 Sugarcane, cane 2.0

Peanut 0.1 Sugarcane, molasses 30

Peanut, hay 0.5 Sunflower 85

Pepper leaf, fresh leaves 0.2 Sunflower, seed 0.1

Peppermint, tops 200 Surinam cherry 0.2

Perilla, tops 1.8 Tamarind 0.2

Persimmon 0.2 Tea, dried 1.0

Pineapple 0.1 Tea, instant 7.0

Pistachio 1.0 Teff, grain 5.0

Pomegranate 0.2 Ti, leaves 0.2

Poultry, meat 0.1 Ti, roots 0.2

Poultry, meat byproducts 1.0 Ugli fruit 0.5

Pulasan 0.2 Vegetable, leafy, brassica, group 5 0.2

Quinoa, grain 5.0 Vegetable, bulb, group 3 0.2

Rambutan 0.2 Vegetable, cucurbit, group 9 0.5

Rapeseed, meal 15 Vegetable, foliage of legume, except soybean, subgroup 7A 0.2

Rapeseed, seed 10 Vegetable, fruiting, group 8 0.1

Rose apple 0.2 Vegetable, leafy, except brassica, group 4 0.2

Safflower 85 Vegetable, leaves of root and tuber, group 2, except sugar beet 0.2

Safflower, seed 0.1 Vegetable, legume, group 6, except soybean 5.0

Salal 0.2 Vegetable, legume, group 6 except soybean and pea,dry 5.0Monsanto Company 11-CA-233U 495 of 500 Commodity Parts/million Commodity Parts/million

Sapodilla 0.2 Vegetable, root and tuber, group 1, except sugar beet 0.2

Wasabi, roots 0.2 Wheat, grain 5.0

Water spinach, tops 0.2 Wheat, middlings 20

Watercress, upland 0.2 Wheat, shorts 20

Wax jambu 0.2 Yacon, tuber 0.2

Wheat, bran 20Monsanto Company 11-CA-233U 496 of 500 References for Appendix K

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