Petition for the Determination of Nonregulated 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 Principal 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 advance 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 any information based on appropriate legal 

grounds, e.g., responsiveness, confidentiality, and/or competitive 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 (including website posting) without Monsanto's 

prior notice and consent. 

©2011 Monsanto Company. All Rights Reserved. 

This document is protected under copyright law. 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 Company. 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 30f500 
Monsanto Company 11-CA-233U 4 of 500 

EXECUTIVE SUMMARY 

The Animal and Plant Health Inspection Service (APHIS) of the United States (U.S.) 

Department of Agriculture (USDA) has responsibility 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 pests 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 article does not present a plant pest risk and no 

longer should be regulated. If APHIS determines that the regulated article does not 

present a plant pest risk, the petition is granted, thereby allowing unrestricted introduction 

of the article. 

Monsanto Company is submitting this request to APHIS for a determination of 

nonregulated status for the new biotechnology-derived canola product, 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 nonregulated status under 7 CFR Part 340. 

Product Description

Weed competition can be a major limiting factor in canola production leading to 

significant yield reductions. Monsanto Company has developed a second-generation 

glyphosate-tolerant canola product, MON 88302, designed to provide growers with 

improved weed control through greater flexibility 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 CP4 EPSPS protein confers 

tolerance to the 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 (i.e., pollen), compared to the promoter used to drive 

CP4 EPSPS production in the first-generation product, Roundup Ready canola (RT73). 

Enhanced CP4 EPSPS expression in the male reproductive tissues of MON 88302 allows 

the greater flexibility of glyphosate 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 herbicide 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, that accumulate glyphosate and lack sufficient 

CP4 EPSPS expression are considered to be at risk for 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 higher rates and at later stages of development than is 

possible with the current Roundup Ready canola product. 

 

®Roundup and Roundup Ready are registered trademarks of Monsanto Technology, LLC 
Monsanto Company 11-CA-233U 5 of 500 

Use of MON 88302 will enable growers to apply Roundup herbicide at higher rates and 

at later stages of development than is possible with the current product facilitating: 1) 

better control of tough-to-kill weeds; 2) an increased opportunity to control weeds if 

glyphosate application is delayed due to weather or equipment failure; 3) an enhanced 

ability to tailor labeled glyphosate applications to the 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. 

Data and Information Presented Confirms the 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 compositionally comparable to conventional canola, 

with the exception of the introduced 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 confirmed based on multiple, well established 

lines of evidence: 

 Canola is a familiar crop that has a history of safe consumption, and serves as an 

appropriate basis of comparison for MON 88302. 

 A detailed molecular characterization of the introduced DNA demonstrated a 

single, intact copy of the transgenic insert in a single locus within the canola 

genome. 

 The CP4 EPSPS protein in MON 88302 is identical to the CP4 EPSPS protein 

produced in several other commercially available crops that have been reviewed 

by USDA and previously deregulated (e.g., Roundup Ready soybean, Roundup 

Ready 2 Yield soybean, 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 

biotechnology-derived crops has 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 extensively 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 phenotypic 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 effects on these organisms, similar to 

conventional canola. 

 Evaluation of MON 88302 using intended and current cultivation and 

management practices for canola concluded that deregulation of MON 88302 will 

not significantly impact canola agronomic 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 feed to the global economy 

and whose biology is well understood and documented. 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 will 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 and 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 Dakota. 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 undisturbed 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 species including low genetic diversity, lack of long-distance 

seed dispersal mechanisms, limited population 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 recipient for the DNA insertion to create 

MON 88302 was Ebony, a non-transgenic conventional 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 genetic backgrounds with the exception of the 

cp4 epsps expression cassette. 
Monsanto Company 11-CA-233U 7 of 500 

Molecular Characterization Verified the Integrity and Stability of the Inserted DNA 

in MON 88302

MON 88302 was developed through Agrobacterium-mediated transformation of 

hypocotyls from canola variety Ebony utilizing plasmid vector PV-BNHT2672. 

PV-BNHT2672 contains one T-DNA that is delineated 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 and 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 blot analyses determined that MON 88302 

contains one copy of the T-DNA at a single 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 demonstrated that the insert in MON 88302 has been 

maintained over multiple generations of breeding, thereby confirming the stability of the 

insert. Further, results from segregation analyses 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 genetic 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 protein safety studies. The 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 protein supports the conclusion 

that the CP4 EPSPS protein does not pose a significant allergenic risk to humans or 

animals. In addition, the 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 animal pathogenicity or allergenicity. Bioinformatics analysis 

determined that CP4 EPSPS protein does not share amino acid sequence similarities with 

known allergens, gliadins, glutenins, or protein toxins. The CP4 EPSPS protein is rapidly 

digested in simulated digestive fluids and demonstrates no oral toxicity in mice at the 

level tested. Hence, the consumption of the CP4 EPSPS protein from MON 88302 or its 

progeny is considered safe for 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 had 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 nutrients, toxicants, and antinutrients present in seed from this new product. 

Safety assessments of biotechnology-derived 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 88302 to the conventional control variety 

(Ebony) and commercial reference varieties demonstrated that MON 88302 is 

compositionally equivalent to conventional canola. The background genetics of the 

conventional control were similar to that of MON 88302, but did not contain the 

cp4 epsps expression cassette. The commercial reference 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. The samples utilized for compositional analysis 

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

Nutrients assessed in this analysis included proximates (ash, carbohydrates by 

calculation, moisture, protein, and total fat), fibers (acid detergent fiber [ADF], neutral 

detergent fiber [NDF], and total dietary fiber [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 acid and glucosinolates (alkyl glucosinolates 

[including 3-butenyl, 4-pentenyl, 2-hydroxy-3-butenyl, and 2-hydroxy-4-pentenyl 

glucosinolates], indolyl glucosinolates [including 3-indolylmethyl and 4-hydroxy-3-

indoylmethyl], and total glucosinolates). The anti-nutrients assessed in seed included 

phytic acid and sinapine (as sinapic acid). 

Combined-site analyses were conducted to determine statistically significant differences 

(α = 0.05) between MON 88302 and the conventional control seed samples. Statistical 

results from the combined-site data were evaluated using considerations relevant to the 

safety and nutritional quality of MON 88302 when compared to the conventional 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 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 statistically significant (α = 0.05) combined-site 

component differences at individual sites, and 4) an assessment of the differences within 
Monsanto Company 11-CA-233U 9 of 500 

the context of natural variability of commercial canola composition 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 MON 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 combined-site analysis, the magnitudes of the differences 

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

interval established from the commercial reference varieties grown concurrently. The 

mean values for the significantly different components were within the 99% tolerance 

interval, differences were not observed across all individual sites, and with the exception 

of alkyl glucosinolates for which a range of values was not available, mean values were 

within the natural variability of canola components as reported 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 Plant Pest Potential or Environmental 

Interactions

Plant pest potential of a biotechnology-derived 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 conventional plant hybrid or variety whose biological 

properties and plant pest potential are well known. Familiarity considers the biology of 

the plant, the introduced trait, the receiving environment, and the interactions among 

these factors. This provides a basis for comparative risk assessment between a 

biotechnology-derived plant and the conventional control. Thus, the phenotypic, 

agronomic, and environmental interaction assessment of MON 88302 included the 

genetically similar conventional control as a comparator. This evaluation used a weight 

of evidence approach and considered statistical differences between MON 88302 and the 

conventional control with respect to reproducibility, magnitude, and directionality. The 

observations were taken on plants not treated 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 variability for canola, and provided a context 

from which to further evaluate any statistical differences. Characteristics assessed 

included: seed dormancy and germination, pollen morphology, and plant phenotypic 

observations and environmental interaction evaluations conducted in the field. 

Commercial references grown concurrently were used to establish a range of natural 

variability for each assessed characteristic in canola. The phenotypic, agronomic, and 

environmental interaction assessment demonstrated that MON 88302 is comparable to 
Monsanto 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 characterization indicated that MON 88302 seed had no 

changes in the dormancy or germination characteristics that could 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 differences in general pollen morphology were 

observed between MON 88302 and the conventional control, demonstrating that the 

introduction of the glyphosate-tolerance trait 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 conducted at eight field sites in the U.S. and 

nine field sites in Canada. These 17 field sites provided a diverse range of environmental 

and agronomic conditions representative of commercial canola production areas in North 

America. Assessments included 12 phenotypic and agronomic characteristics, as well as 

observations for plant responses to abiotic stressors and plant-disease and plant-arthropod 

interactions. The observed phenotypic characteristics were comparable between 

MON 88302 and the conventional control. Across sites, data show no statistically 

significant differences between MON 88302 and the conventional control for early stand 

count, seedling vigor, seed maturity, lodging, plant height, 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 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 natural 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 had 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 difference in seed moisture is unlikely to be biologically 

meaningful in terms of increased weediness potential. 

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

observed between MON 88302 and the conventional control for 130 out of 131 

comparisons involving 9 assessed abiotic stressors. 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 frost damage to MON 88302 was within 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 frost damage observations among the sites. 

Thus, the single difference for frost damage was not considered biologically meaningful 
Monsanto Company 11-CA-233U 11 of 500 

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 diseases or for any of the 165 comparisons for 

any of the 13 assessed arthropods among all 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 beneficial arthropod abundance, no statistically 

significant differences were detected between MON 88302 and the conventional control 

for any of the 51 comparisons, including 36 arthropod pest comparisons and 15 beneficial 

arthropod comparisons, among the observations at the four sites. 

In summary, the phenotypic, agronomic, and environmental interaction data were 

evaluated to characterize MON 88302, and to assess 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 approach, considered the reproducibility, 

magnitude, and direction of detected differences (trends) between MON 88302 and the 

conventional control and comparison to the range of the commercial reference varieties. 

Results from the phenotypic, agronomic, and environmental interactions assessment 

indicated that MON 88302 does not possess weedy characteristics, increased 

susceptibility or tolerance to specific abiotic 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 pest risk assessment. Since MON 88302 does not possess 

pesticidal activity, all organisms that interact 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 CP4 EPSPS protein found in MON 88302 did not 

unexpectedly alter plant-arthropod interactions, including beneficial arthropods, or alter 

disease susceptibility compared to the conventional control. 

The safety of the CP4 EPSPS protein present 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 presence of functionally identical EPSPS 

proteins in plants and microbes in the environment make it unlikely that the presence of 

CP4 EPSPS protein in MON 88302 will have a significant impact on the receiving 
Monsanto Company 11-CA-233U 12 of 500 

environment, including soil function and water quality. Data from the 2009 North 

American phenotypic and agronomic study including observational data on 

environmental interactions such as plant-disease interaction, arthropod damage and 

arthropod abundance, were collected at select sites for MON 88302 and the conventional 

control. These results 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. 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. 

Brassica napus is predominantly self-pollinating although 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 over greater distances at low frequency. Brassica 

napus produces a large amount of pollen that can remain viable up to four to five days 

under field conditions. This, coupled 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 reports of hybridization under field conditions 

with B. napus as the pollen donor with six species including Brassica rapa, Brassica 

juncea, Brassica oleracea, Hirschfeldia incana, Raphanus raphanistrum, and Sinapis 

arvensis. In all cases these hybrids had decreased environmental fitness evidenced by a 

variety of characteristics including decreased pollen viability, 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 are 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 considered weedy, and 

are 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. 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 Significantly 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 likely to be used in common 

rotations on land previously used for agricultural purposes. Certified seed production 

will continue to use well-established industry practices to deliver high quality seed with 
Monsanto Company 11-CA-233U 13 of 500 

MON 88302 to growers. Cultivation of MON 88302 is not expected to differ from 

current canola cultivation using glyphosate-tolerant 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 authorized. Due 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. Therefore, no significant impacts on current 

cultivation and management practices for canola 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 practices 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, Monsanto Company requests a 

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

between MON 88302 and conventional Brassica species or deregulated biotechnologyderived crop be granted nonregulated status under 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 Determination 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 Foreign 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 THE 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 Determine the Presence or Absence of 

PV-BNHT2672 Backbone Sequences in MON 88302 .................................................51
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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 Genetic Insert in MON 88302 ...............................................63

IV.G. Genetic Modification Characterization 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 Equivalence of CP4 EPSPS Protein from 

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

V.C. Expression Levels of CP4 EPSPS Protein in MON 88302 .................................70

V.D. Assessment of Potential Allergenicity of the CP4 EPSPS Protein ......................72

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

V.E.1. The Donor Organism Has 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 MON 88302 is Not Acutely Toxic .......................74

V.F. CP4 EPSPS Protein Characterization 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, AGRONOMIC, AND ENVIRONMENTAL 

INTERACTIONS ASSESSMENT ...............................................................................106
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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 Phenotypic, 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 Glyphosate-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. ENVIRONMENTAL CONSEQUENCES ...........................................................167

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

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

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

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

IX.B 3. Phenotypic and Agronomic and Environmental Interaction 

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

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

IX.D. Potential for Pollen Mediated Gene Flow and Introgression ...........................180
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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 Information 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 CONSEQUENCES 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, and 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 ...................................................................................................................469
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LIST OF TABLES 

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

Table IV-1. Summary Chart of the Expected DNA Segments Based on 

Hybridizing Probes and Restriction Enzymes Used in MON 88302 Analysis ...........45

Table IV-2. Summary of Genetic 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 Protein 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 of Combined-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 Characteristics of MON 88302 and Conventional 

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

Table VII-3. Field Phenotypic Evaluation 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 Characteristics 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. .............................................................................................148
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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 ...........................................................................................................152

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

Table VIII-9. Grass Weed Species Responses 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 ............................................................................................................175

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

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

Table IX-3. Summary of Published Literature on Unassisted Hybridization 

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

Table A-1. USDA Notifications Approved 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 Site MBNW Canola Seed Anti-nutrient 

Content for MON 88302 vs. the Conventional Control ..............................................268
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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 Site 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 Site MNCA Canola Seed Anti-nutrient 

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

Table E-9. Statistical Summary of Site 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 Site SKSA Canola Seed Anti-nutrient 

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

Table F-1. Starting Seed of MON 88302, Control and Commercial Canola 

Reference Varieties Used in Dormancy Assessment ...................................................309

Table F-2. Dormancy and Germination Characteristics of MON 88302 and 

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

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

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

Table G-2. Field and Planting Information ................................................................315

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

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

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

Table G-5. Abiotic Stress Response Evaluations of MON 88302 and 

Conventional Control Using an Observational Severity Scale ..................................329

Table G-6. Disease Damage Evaluations of MON 88302 and Conventional 

Control Using an Observational Severity Scale ..........................................................330

Table G-7. Arthropod-Related Damage Evaluations of MON 88302 and 

Conventional Control Using an Observational Severity Scale ..................................331

Table G-8. Individual Site Analysis: Quantitative 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 Arthropods in Cone Beat Sheet Samples 

Collected from MON 88302, Conventional Control and the Commercial 

Reference Varieties ........................................................................................................333
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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 Potential 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 Winter Canola in the Great Plains .................380

Table I-5. Most Common Weeds in Spring 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 Common Weeds in Top Five U.S. CanolaProducing States in 2010 ...............................................................................................386

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

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

Table I-10. Cross Pollination Potential 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 Tolerances 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 ....................................479
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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 Analysis 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 Generating 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 Coverage 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 ................................................358
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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 Various Herbicide Chemical Families ...................454

Figure K-1. Tier I Endangered Species Analysis .......................................................486
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ABBREVIATIONS AND DEFINITIONS1

~ Approximately 

a.e. Acid Equivalent 

a.i. Active Ingredient 

AA Amino Acid 

ADF Acid Detergent Fiber 

AOSA Association of Official 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 Blocks Substitution Matrix, used to score similarities between 

pairs of distantly related protein 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-phosphate 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 Evaluation Model-Food Commodity Intake 

Database 

DTT Dithiothreitol 

dw Dry Weight 

E. coli Escherichia coli

EDTA Ethylenediaminetetraacetic Acid 

ELISA Enzyme-linked Immunosorbent 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 Organization 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-ethanesulfonic acid)] 

ILSI International Life Sciences Institute 

kDa Kilodalton 

LB Laemmli buffer 

LOD Limit of Detection 

LOQ Limit of Quantitation 

MALDI-TOF-MS Matrix Assisted Laser Desorption Ionization - Time of Flight 

Mass Spectrometry 

MMT Million metric tons 

MOE Margin of Exposure 

MW Molecular Weight 

MWCO Molecular Weight Cutoff 

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 Phosphoenolpyruvate 

Pi Inorganic phosphate 

PPA Plant Protection Act 

ppm Parts Per Million 

PRESS Predicted Residual Sum of Squares 

PVDF Polyvinylidene Difluoride 

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-Polyacrylamide Gel Electrophoresis 

SE Standard Error 
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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 Potential 
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I. RATIONALE FOR THE DEVELOPMENT OF MON 88302 

I.A. Basis for the Request for a Determination of Nonregulated Status under 

7 CFR § 340.6 

The Animal and Plant Health Inspection Service (APHIS) of the United States (U.S.) 

Department of Agriculture (USDA) has responsibility, 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 pests 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 article does not present a plant pest risk and no 

longer should be regulated. If APHIS determines that the regulated article does not 

present a plant pest risk, the petition is granted, thereby allowing unrestricted introduction 

of the article. 

Monsanto Company is submitting this request to APHIS for a determination of 

nonregulated status for the new biotechnology-derived canola product, 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 nonregulated status under 7 CFR Part 340. 

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

Monsanto Company has developed a second-generation glyphosate-tolerant canola 

product, MON 88302, designed to provide growers with improved weed control through 

greater flexibility for glyphosate herbicide application. 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 particularly 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 unreasonable 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 the 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 agricultural 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 systemic herbicide and is translocated in the 

plant generally from a strong source tissue (e.g., leaf) to rapidly developing, or sink tissue 
Monsanto Company 11-CA-233U 28 of 500 

(Devine et al., 1993). Sink tissues, such as pollen, that accumulate glyphosate and lack 

sufficient CP4 EPSPS expression are considered to be at risk for 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 (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 possible with the current product 

facilitating: 1) better control of tough-to-kill weeds; 2) an increased opportunity to 

control weeds if glyphosate application is delayed due to weather or equipment failure; 3) 

an enhanced ability to tailor labeled glyphosate 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 Regulation of Biotechnology, the responsibility 

for regulatory oversight of biotechnology-derived crops falls 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). Deregulation of MON 88302 by USDA constitutes only one 

component of the overall regulatory 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 the 1992 FDA policy statement concerning 

regulation of products derived from new plant varieties, including those developed 

through biotechnology (U.S. FDA, 1992). In compliance with this policy, Monsanto has 

initiated a consultation with the FDA (BNF No. 127) on the 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 pesticide 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. Environmental Protection Agency (U.S. EPA) in 

February 2011 for EPA Registration Numbers 524-537 (Roundup WeatherMAX®

Herbicide) and 524-549 (Roundup PowerMAX® Herbicide), that propose to modify the 

current use pattern of glyphosate 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 canola development, this use of glyphosate does 

not present any new environmental exposure scenarios not previously evaluated for use 
Monsanto Company 11-CA-233U 29 of 500 

on other Roundup Ready crops which have already been deemed acceptable by the U.S. 

EPA. 

I.C.3. Submissions to Foreign Government Agencies 

To support commercial introduction of MON 88302 in the U.S., regulatory submissions 

will be made to countries that import significant quantities of canola and canola products 

from the U.S. This results in submissions 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 Agriculture, Forestry, and Fisheries and the 

Ministry of Health, Labor, and Welfare; the Canadian Food Inspection Agency and 

Health Canada; the Intersectoral Commission for Biosafety of Genetically Modified 

Organisms, Mexico; the European Food Safety Authority, Food Standards Australia New 

Zealand, the Korea Food and Drug Administration, and the Rural Development 

Administration of Korea, as well as to regulatory authorities in other canola importing 

countries with functioning regulatory systems. As appropriate, notifications will be made 

to countries that import significant quantities of canola and canola products and do not 

have a formal regulatory review process for biotechnology-derived crops. 
Monsanto Company 11-CA-233U 30 of 500 

II. THE BIOLOGY OF CANOLA 

Brassica napus L. is a versatile crop that provides both food and feed to the global 

economy, and whose biology is well understood and documented. 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 document, B. napus will 

be referred to as oilseed rape and the term canola will be used to denote B. napus

varieties that produce low (< 2%) erucic acid oil and have levels of glucosinolates below 

the accepted standard of 30 μmoles/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 relatives 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 Technology Regulator) website (OGTR, 2008). 

Information on the taxonomy of B. napus can be found in the U.S. Department of 

Agriculture Natural Resources Conservation Service PLANTS database (USDA-NRCS, 

2010b). 

To support the evaluation of the plant pest 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 practices for canola in 

Section VIII; volunteer management of canola in Section VIII; 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 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 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 and 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 (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, goitrogenic (suppresses thyroid function), reproductive, and 

palatability problems (Fenwick et al., 1989). However, in the 1960s intensive breeding 

programs resulted in the development and introduction of low erucic acid or canola 

(Canadian oil, low acid) varieties 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 the 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 outcross 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 canola varieties. Spring canola, 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 Plains. 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, glufosinate and imazamox herbicides for weed control in canola 

fields are widely available. 

Canola oil is currently the world’s third largest source of vegetable oil (15%) after 

soybean oil (28%) and palm oil (32%) (ASA, 2010; USDA-ERS, 2010). Canola oil 

appeals to health conscious consumers because it has the lowest level of saturated fat of 

all edible oils, the second-highest level of monounsaturated fat, and is free of artificial 

trans-fatty acids (Brown et al., 2008; USDA-ERS, 2010). Canola seed is also processed 

into canola meal which is used as high protein 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 meal was 30.8 million metric tons (MMT) in 

2008/2009 compared to 151.6 MMT for soybean meal (USDA-ERS, 2010). Canola meal 
Monsanto 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 recipient for the DNA insertion to create 

MON 88302 was Ebony, a non-transgenic conventional spring canola variety registered 

with the Canadian Food Inspection Agency in 1994 by Monsanto Company (CFIA, 

2010). Ebony originated from a cross of varieties (Bienvenu × Alto) × Cesar. Selection 

criteria for the non-transgenic variety included yield, oil and protein content, and 

tolerance to the fungus Leptosphaeria maculans, commonly known as blackleg (CFIA, 

1994). Ebony was used to produce the glyphosate-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 comparator (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, commercial conventional canola varieties 

(referred to in this consultation document as commercial reference varieties) were used to 

establish ranges of natural variability or responses representative of commercial canola 

varieties. The commercial reference varieties used at each location were selected based 

on their availability and agronomic fit for the geographic region. 
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III. DESCRIPTION OF THE GENETIC MODIFICATION 

MON 88302 was developed through Agrobacterium-mediated transformation of 

hypocotyls from Ebony canola variety utilizing plasmid vector PV-BNHT2672. This 

section describes the plasmid vector, the donor gene, and the regulatory elements 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, transfer 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 the 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 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 and is derived from 

CTP2 target sequence of the Arabidopsis thaliana shkG gene. 

The backbone sequence of PV-BNHT2672, located outside of the T-DNA, contains two 

origins of replication for maintenance 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 described by Radke et al. (1992), utilizing 

PV-BNHT2672 (Figure III-1). In summary, hypocotyl segments were excised from 

Ebony seedlings grown in the dark. After co-culturing with Agrobacterium carrying the 

vector, the hypocotyl segments were placed on callus growth medium that contained 

carbenicillin, ticarcillin disodium 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 untransformed cells and plant growth regulators 

conducive to shoot regeneration. Rooted R0 plants with normal phenotypic 

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

The R0 plants generated through the Agrobacterium-mediated transformation were 

transferred to soil for growth and then self pollinated to produce R1 seed. R0 and R1

plants were evaluated for tolerance to glyphosate 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 R1 plant was self-pollinated to give rise to R2
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plants. Homozygous R2 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 characteristics and its comprehensive molecular 

profile. Regulatory studies on MON 88302 were initiated to further characterize the 

genetic insertion and the expressed protein, and to establish the food, feed, and 

environmental safety relative to conventional canola. The major steps involved in the 

development of MON 88302 are depicted 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 polypeptide of 455 amino acids (Figure III-3) 

(Padgette et al., 1996). The cp4 epsps coding sequence is the 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 endogenous plant EPSPS enzymes, but has a much reduced 

affinity for glyphosate, the active ingredient in Roundup agricultural herbicides, relative 

to endogenous plant EPSPS (Barry et al., 2001; Padgette et al., 1996). 
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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 the plasmid vector) are shown on the exterior of the map. The 

probes used in the Southern analyses are shown on the interior of the map and listed in 

the table. PV-BNHT2672 contains a single T-DNA. 

 

PV-BNHT2672

9664 bp

Ase I 4026

Ase I 8525

BamHI 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

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

Transformed Ebony, a conventional canola variety, hypocotyl 

segments with the vector PV-BNHT2672 in Agrobacterium tumefaciens

Selected transformants and generated rooted shoots from the 

transformed hypocotyls segments

Identified MON 88302 as lead candidate and further evaluated its 

progeny in the laboratory and field for insert integrity, glyphosate 

tolerance and agronomic performance

Assembled Agrobacterium binary plasmid vector PV-BNHT2672 and

transferred to Agrobacterium tumefaciens strain ABI

Evaluated the transformed plants for tolerance to glyphosate and 

screened the transformed plants for the presence of T-DNA (cp4 epsps

expression cassette) and absence of the T-DNA backbone (Ori V)

Selected homozygous plants using a quantitative polymerase chain

reaction method
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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 promoter 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-1α (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 carboxylase small subunit (rbcS2) E9 gene (Coruzzi et al., 

1984) and is present to direct polyadenylation of the cp4 epsps transcript. The 

chloroplast transit peptide CTP2 directs transport 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 Left 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 that 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 plasmid 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 exist 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 origin 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 transposon Tn7 that confers spectinomycin and 

streptomycin resistance (Fling et al., 1985) in E. coli and Agrobacterium during 

molecular cloning. As these elements are outside the border regions, they are not 

expected to be transferred into the canola genome. The absence of the backbone 

sequence in MON 88302 has been confirmed by Southern blot analyses (see 

Section IV.B). 
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Table III-1. Summary of Genetic Elements in PV-BNHT2672 

Genetic Element Location 

in Plasmid Function (Reference) 

T-DNA 

B1-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 

P2-FMV/Tsf1 428-1467 

Chimeric promoter consisting of the promoter 

of the Tsf1 gene from the Arabidopsis thaliana

encoding elongation factor EF-1α (Axelos et 

al., 1989) and enhancer sequences from the 

35S promoter from the figwort mosaic virus 

(Richins et al., 1987) 

L3-Tsf1 1468-1513 

5' untranslated leader (exon 1) from the 

Arabidopsis thaliana Tsf1 gene encoding 

elongation factor EF-1α (Axelos et al., 1989) 

I

4

-Tsf1 1514-2135 

Intron from the Arabidopsis thaliana Tsf1 gene 

encoding elongation factor EF-1α (Axelos et 

al., 1989) 

Intervening Sequence 2136-2144 Sequence used in DNA cloning 

TS5-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 

CS6-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 

T7-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) 
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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 

OR8- oriV 4997-5393 

Origin of replication from 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 replication 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 Replication 

 
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 1 MAQVSRICNG VQNPSLISNL SKSSQRKSPL SVSLKTQQHP RAYPISSSWG

 51 LKKSGMTLIG SELRPLKVMS SVSTACMLHG 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 Protein 

The 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. 
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IV. CHARACTERIZATION OF THE GENETIC MODIFICATION 

Characterization of the DNA insert in MON 88302 was conducted by Southern blot, PCR 

and DNA sequence analyses. The 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 entire canola genome for the presence of T-DNA 

and the absence of the backbone sequences 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 confirm that only the expected 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 transformation;. 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 the insert were as expected across multiple 

generations. Taken together, the characterization of the genetic modification 

demonstrates that a single copy of the T-DNA was 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 number and insertion sites of the 

integrated DNA as well as the presence or absence of plasmid vector backbone 

sequences. The Southern blot strategy was 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 presence of the insert as 

well as confirm the absence of any plasmid 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 sensitivity 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 fully 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 cleaves 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 containing a portion of the insert with the adjacent 

3' flanking DNA generated by another set of the enzyme(s). This two-set-enzyme design 

ensures that the entire insert is identified in a predictable hybridization pattern. This 

strategy also maximizes the possibility of detecting an insertion 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 insertion sites of the T-DNA, and the presence or 

absence of the plasmid vector backbone sequences, duplicated samples that consisted of 
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equal amounts of digested DNA were run on the agarose gel. One set of samples was run 

for a longer period of time (long run) than the second set (short run). The long run allows 

for greater resolution of large molecular weight DNA, whereas the short run allows for 

retaining the small molecular weight DNA on the gel. The molecular weight markers on 

the left of the figures were used to estimate the sizes of the bands present in the long run 

lanes of the Southern blots, and the molecular weight markers on the right of the figures 

were used to estimate the sizes of bands present in the short run lanes of the Southern 

blots (Figure IV-2 through Figure IV-6). Southern blot analyses determined that a single 

copy of the T-DNA was inserted at a single locus of the canola genome, and no 

additional genetic elements, including backbone 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 sequences in MON 88302, confirmed the predicted 

organization of the genetic elements within the insert, and determined the sequences 

flanking the insert. In addition, DNA sequence analyses confirmed that each genetic 

element in the insert is intact and the sequence of the insert is identical to the 

corresponding sequence in PV-BNHT2672. Furthermore, genomic organization at the 

MON 88302 insertion site was determined by comparing the 5' and 3' flanking sequences 

of the insert to the sequence of the insertion 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 (Figure 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 adjacent genomic DNA that assesses not only the 

stability of the insert, but also the stability of the DNA directly adjacent to the insert. 

Segregation analysis was conducted to determine 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 insert 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 insert, 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 through 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. The 
Monsanto Company 11-CA-233U 43 of 500 

generations used in the generational stability analysis are depicted 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 the segregation analysis are presented in 

Table IV-3. Materials and methods used for the characterization of the insert in 

MON 88302 are found in Appendix B. 
Monsanto Company 11-CA-233U 

Figure IV-1. Schematic Representation of the Insert and Flanking DNA in MON 88302 A linear map of the insert and DNA flanking the insert in MON 88302 is shown. Right-angled arrows ind

integrated T-DNA and the beginning of the flanking DNA. Identified on the linear map are genetic elements wias the sites of the restriction enzymes used in the Southern analyses with positions relative to the first base pair represented in this map. The relative sizes and locations of the T-DNA probes and the expected sizes of res

indicated in the lower portion of the scheme. This schematic diagram is not drawn to scale. Locations of 

T-DNA probes are approximate. Probes are also shown in Figure III-1. 

~3.8 kb ~1.4 kb

Ase I

>1.8 kb ~4.3 kb

SalI and Sca I

Probe 1

Probe 2

Probe 3

B-Right Border Regionr1

P-FMV/Tsf1

L-Tsf1

I-Tsf1

TS-CTP2

CS-cp4 epsps

T-E9

B-Left Border Regionr1

Ase I 757 Ase I 4552 Sal I 6093 Sca I 1833

Ase I 5975

5' Flanking DNA 3' Flanking DNA

1 6174 
Monsanto Company 11-CA-233U 

Table IV-1. Summary Chart of the Expected DNA Segments Based on Hybridizing Probes and RestrictiMON 88302 Analysis 1 probe template spikes were used as positive hybridization controls in Southern blot analyses when multiple probes were hybrid

simultaneously. 

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 Backbone Figure Number IV-2 IV-3 IV-4 IV-5 IVProbe Used 1, 3

2

4

5

Probing Target Digestion Enzyme Expected Band Sizes on Each Southern Plasmid PV-BNHT2672 Bam HI and Sca I ~2.5 kb ~7.2 kb ~2.5 kb ~7.2 kb ~7.2 kb ~7.Probe Template Spikes1 N/A ~2.3 kb ~1.3 kb ~~2 ~~2 ~~2 ~MON 88302 

Ase I ~3.8 kb ~1.4 kb ~3.8 kb No band No band No Sal I and Sca I >1.8 kb ~4.3 kb ~4.3 kb No band No band No 
Monsanto Company 11-CA-233U 46 of 500 

Table IV-2. Summary of Genetic 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 

B1-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 

P2-FMV/Tsf1 953-1992 

Chimeric promoter consisting of the promoter of the 

Tsf1 gene from the Arabidopsis thaliana encoding 

elongation factor EF-1α (Axelos et al., 1989) and 

enhancer sequences from the 35S promoter from the 

figwort mosaic virus (Richins et al., 1987) 

L3-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-1α (Axelos et al., 1989) 

Intervening Sequence 2661-2669 Sequence used in DNA cloning 

TS5-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 

CS6-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 

T7-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 indicate 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 Determine 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 probes that span the T-DNA (Figure IV-1). 

Each restriction digest is expected to produce a specific banding pattern on the Southern 

blots (Table IV-1). Any additional copies and/or integration sites would be detected as 

additional bands on the blots. 

The restriction enzyme Ase I cleaves once within the inserted 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 segments 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 site 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-DNA 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 digested with the restriction enzyme Ase I and 

spiked with either probe templates and/or digested PV-BNHT2672 DNA served as 

positive hybridization controls. The positive hybridization control was spiked at 

approximately 0.1 and 1 genome equivalents to demonstrate sufficient sensitivity of the 

Southern blot. Conventional control genomic DNA digested with the appropriate 

restriction enzymes was used as a negative control. 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 genomic DNA digested with Ase I (Figure IV-2, Lane 1 and 

Lane 5) or the combination of restriction enzymes Sal I and Sca I (Figure IV-2, Lane 3 

and Lane 7) and simultaneously hybridized with Probe 1 and Probe 3 (Figures III-1 and 

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 two bands at ~7.2 kb and ~2.5 kb 

(Figure IV-2, Lane 10), as expected. Conventional 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 probes 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 Figure IV-2 indicate that the sequences 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 and 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 Lane 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 combination 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 indicate that the sequence covered by Probe 2 

resides at a single detectable locus 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 simultaneously hybridized with two 32P-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 the size of the DNA, in 

kilobase pairs, 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 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 RUN
Monsanto 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 32P-labeled probe that spans a portion of the T-DNA 

sequence (Figure III-1, Probe 2). Each lane contains approximately 10 μg of digested 

genomic DNA. Arrows denote the size of the DNA, in kilobase pairs, 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 RUN
Monsanto Company 11-CA-233U 51 of 500 

IV.B. Southern Blot Analysis to Determine the Presence or Absence of 

PV-BNHT2672 Backbone Sequences 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, and Probe 6). If backbone sequences are 

present in MON 88302, then probing with backbone probes should result in hybridizing 

bands. Conventional control genomic DNA digested with the restriction enzyme Ase I 

and spiked with digested PV-BNHT2672 DNA served as positive hybridization controls. 

The positive hybridization control was spiked at approximately 0.1 and 1 genome 

equivalents to demonstrate sufficient sensitivity of the Southern blot. Conventional 

control genomic DNA digested with the appropriate restriction enzymes was used as a 

negative control. The results of these analyses 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) produced 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 hybridized 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 indicate that MON 88302 contains 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) produced 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 hybridized 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 indicate that MON 88302 contains 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) produced 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 hybridized 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 indicate that MON 88302 contains 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 Sequences in MON 88302: Probe 4 

The blot was hybridized with a 32P-labeled probe that spans a portion of the plasmid 

vector backbone sequences (Figure III-1, Probe 4). Each lane contains approximately 

10 μg of digested genomic DNA. Arrows denote 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 SHORTRUN
Monsanto Company 11-CA-233U 54 of 500 

Figure IV-5. Southern Blot Analysis to Determine the Presence or Absence of the 

PV-BNHT2672 Backbone Sequences in MON 88302: Probe 5 

The blot was hybridized with a 32P-labeled probe that spans a portion of the plasmid 

vector backbone sequences (Figure III-1, Probe 5). Each lane contains approximately 

10 μg of digested genomic DNA. Arrows denote 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 RUN
Monsanto 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 32P-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/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 RUN
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IV.C. Organization and Sequence of the Insert and Adjacent DNA in MON 88302 

The organization and sequence of the elements within the MON 88302 insert was 

confirmed by DNA sequence analysis. PCR primers were designed with the intent to 

amplify two overlapping DNA amplicons 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 analyses. 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-BNHT2672 as described in Table III-1. 

 
Monsanto Company 11-CA-233U 57 of 500 

Figure IV-7. Overlapping PCR Analysis across the Insert in MON 88302 

PCR was performed on both conventional 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 appears at the bottom of the figure. Arrows 

on the agarose gel photograph denote 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 kb

B-Right Border Regionr1

P-FMV/Tsf1

L-Tsf1

I-Tsf1

TS-CTP2

CS-cp4 epsps

T-E9

B-Left Border Regionr1
Monsanto 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 performed on genomic DNA extracted from 

MON 88302 and the conventional control to examine the MON 88302 insertion site. The 

PCR was performed with a forward primer specific to the genomic DNA sequence 

flanking the 5' end of the insert paired with a reverse primer specific to the genomic DNA 

sequence flanking the 3' end of the insert (Figure IV-8). The amplified PCR product 

from the conventional control was subjected to DNA sequence analysis. Alignments 

between the conventional control 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 genomic organization of the insertion site in 

MON 88302. From these alignment analyses, a 9 base pair insertion 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 presumably resulted from double-stranded break 

repair mechanisms in the plant during the Agrobacterium-mediated transformation 

process (Salomon and Puchta, 1998). A single 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 difference was most likely caused by a single 

nucleotide polymorphism (SNP) segregating in the canola population (Trick et al., 2009). 

 
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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 ́ Flank B-Right Border Regionr1 P-FMV/Tsf1 L-Tsf1 I-Tsf1 TS-CTP2 CS-cp4 epsps T-E9 B-Left Border Regionr1
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IV.E. Southern Blot Analysis to Examine Insert Stability in Multiple Generations of 

MON 88302 

In order to demonstrate the stability of the insert in MON 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 generations tested are indicated in the legend of 

Figure IV-9. The R3 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 R3 generation. Genomic DNA, isolated from each of the selected 

generations of MON 88302, was digested with the restriction enzyme Ase I and 

simultaneously hybridized with Probe 1 and Probe 3 (Figures III-1 and IV-1), which was 

designed to detect both fragments 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 digested with restriction enzyme Ase I and 

simultaneously hybridized with Probe 1 and Probe 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), produced the expected bands at ~2.5 kb 

and ~7.2 kb (Figure IV-10, Lane 8). Conventional control genomic DNA digested with 

Ase I and spiked with probe templates of Probe 1 and Probe 3 produced the expected 

bands at ~2.3 kb and ~1.3 kb (Figure IV-10, Lane 9 and Lane 10). Detection of the 

positive controls indicates that the probes hybridized to their target 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 produce 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, Lane 4, Lane 5, and Lane 6), of MON 88302 

are consistent with the one produced from the fully characterized generation R3

(Figure IV-2, Lane 2 and Lane 6, and Figure 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 

R0 corresponds to the transformed canola plant. All generations were self pollinated. 

 designates self-pollination. The R3 generation was used for the molecular 

characterization and commercial development of MON 88302. The R2, R3, R4, R5a, and 

R5b (bolded in the breeding tree) generations of MON 88302 were used for analyzing the 

stability of the insert across generations. R5b was propagated independently of R5a 

beginning with the R3 generation. 

R0- Originally transformed plant ; -self pollination 

R0

(transformation)

R1

R2

R3

R4

R5b R5a

R4b



Commercial Development









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

Generations of MON 88302: Probes 1 and 3 

The blot was simultaneously hybridized with two 32P-labeled probes that span a portion 

of the T-DNA sequence (Figure III-1, Probe 1 and Probe 3). Each lane contains ~10 μg 

of digested genomic DNA. Arrows denote the size of the DNA, in kilobase pairs, 

obtained from 1Kb DNA Extension Ladder on the ethidium bromide stained gel. Lane 

designations are as follows: 

Lane

1 Conventional control (Ase I) 

2 R2 generation of MON 88302 (Ase I) 

3 R3 generation of MON 88302 (Ase I) 

4 R4 generation of MON 88302 (Ase I) 

5 R5a generation of MON 88302 (Ase I) 

6 R5b 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

0.5
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IV.F. Inheritance of the Genetic Insert in MON 88302 

During development of MON 88302, segregation data were recorded to assess the 

inheritance and stability of the coding sequence present in MON 88302. Chi-square (χ

2

) 

analysis was performed over several generations to confirm the segregation and stability 

of the MON 88302 insert. The χ

2 analysis is based on testing the observed segregation 

ratio to the expected segregation ratio according to Mendelian principles. 

The MON 88302 breeding path for generating segregation data is described in 

Figure IV-11. The transformed R0 plant was self-pollinated to generate R1 seed. From 

the R1 segregating population, an individual 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 R1

plant was self-pollinated to give rise to R2 plants that were self-pollinated to produce R3

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 R3 plant, which was confirmed by Endpoint TaqMan 

PCR assay, was crossed to a Monsanto proprietary canola inbred, which does not contain 

the MON 88302 insert, via traditional breeding techniques to produce hemizygous F1

seed. The resulting F1 plant, was shown to contain a single copy of the cp4 epsps gene by 

real-time TaqMan PCR, and was then self-pollinated to produce F2 seed. A cp4 epsps

hemizygous F2 plant from the F2 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 F3

population. A cp4 epsps hemizygous F3 plant from the F3 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 F4 population. The copy number of the cp4 epsps gene in 

the F2, F3, and F4 populations was then assessed using a real-time TaqMan PCR assay. 

A χ

2

analysis was performed on each of the F2, F3, and F4 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 statistical significance was predetermined to 

be 5% (α = 0.05). 

The results of the χ

2 analysis of the MON 88302 segregating progeny are presented in 

Table IV-3. The χ

2 value in the F2, F3, and F4 populations indicated no statistically 

significant difference between the observed and expected 1:2:1 segregation ratio 
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(homozygous positive: hemizygous: homozygous negative) of cp4 epsps coding 

sequence. These results support the conclusion that the cp4 epsps expression cassette in 

MON 88302 resides at a single locus within the canola genome and is inherited according 

to Mendelian principles of inheritance. These results are also consistent with the 

molecular characterization data indicating that MON 88302 contains a single, intact copy 

of the cp4 epsps expression cassette inserted at a single locus in the canola genome. 
Monsanto Company 11-CA-233U 

Figure IV-11. Breeding Path for Generating Segregation Data for MON 88302 An individual hemizygous plant from each of the F1, F2, and F3 populations was self-pollinated to produce the 

generation. Chi-square analyses were conducted on segregation data from the F2, F3, and F4 populations. 

Transformed R0 Plant

R1



R2R3





(homozygous)

cross with a Monsanto proprietary canola inbred 

which does not contain the MON 88302 insert

F1 (hemizygous)



F2 (expected segregation ratio 

(homozygous positive : hemizygous : homozygous negativ



F3

(homozygous positive : hemizygous : homozygous negati



F4

(homozygous positive : hemizygous : homozygous negati

(expected segregation ratio 

(expected segregation ratio 
Monsanto Company 11-CA-233U 

Table IV-3. Segregation of the cp4 epsps Gene During the Development of MON 88302 

1:2:1 Segregation

Generation Total Plants* 

Observed # 

Plants 

Homozygous 

Positive 

Observed # 

Plants 

Hemizygous 

Observed # 

Plants 

Homozygous 

Negative 

Expected # 

Plants

Homozygous 

Positive

Expected # 

Plants 

Hemizygous

Expected # 

Plants

Homozygous 

Negative 

F2 220 51 122 47 55.00 110.00 55.00 F3 166 39 94 33 41.50 83.00 41.50 F4 198 53 97 48 49.50 99.00 49.50 

*Plants were evaluated for the copy number of the cp4 epsps gene using a real-time TaqMan PCR assay. 
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IV.G. Genetic Modification Characterization Conclusion 

Molecular characterization of MON 88302 by Southern 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, confirmed the predicted organization of the 

genetic elements within the insert, determined 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 deletion 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 nucleotide difference between the conventional 

control sequence and the known DNA sequence flanking the 3' end of the MON 88302 

insert was also identified. This single nucleotide difference was most likely caused by 

single nucleotide polymorphism (SNP) segregating in the canola population (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 analysis and establishes the genetic behavior of 

the T-DNA in MON 88302 at a single chromosomal locus. 
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V. CHARACTERIZATION AND SAFETY ASSESSMENT OF THE 

CP4 EPSPS PROTEIN PRODUCED IN MON 88302 

Characterization of the introduced protein in a biotechnology-derived 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 expression of the CP4 EPSPS protein. 

This section summarizes: 1) the identity 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 allergenicity 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 conclusion that MON 88302 is safe for the 

human or animal consumption based on several lines of evidence summarized below. 

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

The enzyme, 5-enolpyruvylshikimate-3-phosphate 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 shikimic acid pathway and EPSPS enzymes are 

ubiquitous to plants and microorganisms, but absent in mammals, fish, birds, reptiles, and 

insects (Alibhai and Stallings, 2001). EPSPS proteins have been isolated from both plant 

and microbial sources and their properties have been extensively studied (Harrison et al., 

1996; Haslam, 1993; Klee et al., 1987; Schönbrunn et al., 2001; Steinrücken and 

Amrhein, 1984). The plant and microbial enzymes are mono-functional with a molecular 

weight of 44-51 kDa (Franz et al., 1997; Kishore et al., 1988). EPSPS enzymes catalyze 

the transfer of the enolpyruvyl group from phosphoenolpyruvate (PEP) to the 5-hydroxyl 

of shikimate-3-phosphate (S3P), thereby yielding inorganic phosphate and 5-

enolpyruvylshikimate-3-phosphate (EPSP) (Alibhai and Stallings, 2001). Shikimic acid 

is a substrate for the biosynthesis of the aromatic amino acids (phenylalanine, tryptophan 

and tyrosine) and other aromatic molecules necessary for plant growth. 

The EPSPS transgene in MON 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 (Padgette et al., 1996). The CP4 EPSPS protein 

is similar and functionally identical to endogenous plant EPSPS enzymes, but has a much 

reduced affinity for glyphosate, the active ingredient in Roundup agricultural herbicides, 

relative to endogenous plant EPSPS (Padgette et al., 1996). In conventional plants, 

glyphosate blocks the biosynthesis of EPSP, thereby depriving plants of essential amino 

acids (Haslam, 1993; Steinrücken and Amrhein, 1980). In Roundup Ready plants, which 

are tolerant to Roundup agricultural herbicides, requirements for aromatic amino acids 

and other metabolites are met by the continued action of the 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 Ready 2 Yield soybeans, as well as the 
Monsanto 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 Equivalence of CP4 EPSPS Protein from MON 88302 

The safety assessment of crops derived through biotechnology includes characterization 

of the physicochemical and functional properties of the protein(s) produced from the 

inserted DNA, and confirmation of the safety of the protein(s). The safety of E. coliproduced CP4 EPSPS protein has been assessed 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 in MON 88302, the equivalence of the plantand E. coli-produced protein was established. The MON 88302-produced CP4 EPSPS 

protein was characterized and the equivalence 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 analytical tests, 

including: 1) N-terminal sequence analysis of the MON 88302-produced CP4 EPSPS 

protein to establish identity; 2) matrix-assisted 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 establish identity; 3) western 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 weight between MON 88302-produced protein and 

the E. coli-produced protein; 5) glycosylation analysis of the MON 88302-produced 

CP4 EPSPS protein to establish the equivalence between the MON 88302-produced and 

E. coli-produced CP4 EPSPS proteins; and 6) CP4 EPSPS enzymatic activity analysis to 

demonstrate functional equivalence between MON 88302-produced and the E. coliproduced protein. The details of the materials, 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 identity of the MON 88302-produced CP4 EPSPS 

protein and established the equivalence of the plant produced protein to the E. coliproduced 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. The 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 expected 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 CP4 EPSPS protein. Furthermore, the 

immunoreactive properties and electrophoretic 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 molecular weight, glycosylation status, and 

functional activity of the MON 88302-produced CP4 EPSPS protein and E. coli-produced 

CP4 EPSPS protein were also all found to be equivalent. Taken together, these data 
Monsanto 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 tissues of MON 88302 relevant to the risk 

assessment were determined by a validated 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.: Power 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 (OSL-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 and 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. The 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 collections are detailed in Appendix D. The mean 

CP4 EPSPS protein levels were highest in leaf (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). 
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Table V-1. Summary of CP4 EPSPS Protein Levels in Canola Tissues from 

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

Tissue1 Development 

Stage2

Days 

After 

Planting 

(DAP)

CP4 EPSPS

Mean (SD) 

Range

(μg/g fw)3

CP4 EPSPS

Mean (SD) 

Range

(μg/g dw)4

LOQ/LOD5

(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 collected. 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 (μg) 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 all 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 (μg) of 

protein per gram (g) of tissue on a dry weight basis (dw). The dry weight values were calculated by 

dividing the μg/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 Allergenicity of the CP4 EPSPS Protein 

The allergenic potential of an introduced protein is assessed by comparing the 

biochemical characteristics of the introduced protein to biochemical characteristics of 

known allergens (Codex Alimentarius, 2009). A protein is not likely to be associated 

with allergenicity if: 1) the protein is from 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 based 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, therefore, is highly 

unlikely to contain immunologically cross-reactive allergenic epitopes. 

4) Finally, in vitro digestive fate experiments conducted with the CP4 EPSPS protein 

demonstrate that the protein is rapidly digested in simulated gastric fluid (SGF) 

and in simulated intestinal fluid (SIF). 

Taken together, these data support the conclusion that the CP4 EPSPS protein does not 

pose a significant allergenic risk 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 feed 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 commonly allergenic (FAO-WHO, 1991). Agrobacterium sp. 

strain CP4 has been previously reviewed as a part of the safety 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 beet (1998), and Roundup Ready Flex cotton 

(2005). Further, the Environmental Protection Agency has established an exemption 

from the requirement of a tolerance for residues 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 consumption (Harrison et al., 1996; Padgette et al., 

1996). The similarity of the CP4 EPSPS protein to EPSPS proteins in a variety of foods 

supports extensive human consumption of the family of EPSPS proteins and the lack of 

health concerns. The ubiquitous presence of homologous EPSPS enzymes in food crops 

and common microorganisms establishes that 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 pathway that is integral to aromatic amino 

acid biosynthesis in plants and microorganisms (Levin and Sprinson, 1964; Steinrücken 

and Amrhein, 1980). Therefore, this enzyme and its activity are found widely in food 

and feed derived from plant and microbial sources. Genes for numerous EPSPS proteins 

have been cloned (Padgette et al., 1996) and the catalytic domains of this group of 

proteins are conserved. Bacterial EPSPS proteins have been well characterized with 

respect to their three dimensional X-ray crystal structures (Stallings et al., 1991) and 

detailed kinetic and chemical mechanisms (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 acid sliding window search. An amino acid 

sequence is considered to have allergenic potential 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). Rapid degradation of the fulllength CP4 EPSPS protein in SGF and SIF reduces 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 mice 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 currently produced from canola is refined, 

bleached, and deodorized (RBD) oil. Because RBD oil contains negligible amounts of 

protein (Martín-Hernández et al., 2008), oil produced from MON 88302 will contain 

negligible levels of CP4 EPSPS protein. Therefore 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 assessed (Harrison et al., 1996) and 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 protein exposure to animals from consumption of MON 88302 

in feeds was evaluated by calculating an estimate of daily dietary intake (DDI). The 

highest percentage of CP4 EPSPS protein (g/kg bw) per total protein consumed was in 

the dairy cow, 0.0141% (g/g) of the total 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 percentages 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 relation to the total protein consumed. 

V.F. CP4 EPSPS Protein Characterization and Safety Conclusion 

The data and information provided in this section address the questions important to the 

food and feed safety of the CP4 EPSPS protein in MON 88302 including its potential 

allergenicity and toxicity. To summarize, the physicochemical characteristics of the 

CP4 EPSPS protein were determined and shown to be equivalent to those of an E. coliproduced CP4 EPSPS protein. The 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 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 animal 
Monsanto Company 11-CA-233U 75 of 500 

pathogenicity, or allergenicity. The CP4 EPSPS protein lacks structural similarity to 

allergens, toxins or other proteins known to have adverse effects on mammals. The 

CP4 EPSPS protein is rapidly digested in simulated digestive 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 ASSESSMENT 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 Ready 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 88302 would affect nutritionally important 

nutrients, toxicants, or anti-nutrients present in seed from this new product. 

Safety assessments of biotechnology-derived 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 principles 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 crop varieties, nine countries 

and eleven growing seasons concluded that incorporation of biotechnology-derived 

agronomic traits has had little impact on natural variation in crop composition. Most 

compositional variation is attributable to growing region, agronomic practices, and 

genetic background (Harrigan et al., 2010). Compositional quality, therefore, implies a 

very broad range of endogenous levels of individual constituents. Numerous scientific 

publications have further documented the extensive variability in the concentrations of 

crop nutrients, anti-nutrients, and secondary metabolites that reflect the influence of 

environmental and genetic factors as well as extensive conventional breeding efforts to 

improve nutrition, agronomics, and yield (Harrigan et al., 2010; Mailer 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 biotechnology-derived and conventional crops 

provides an “equal or increased assurance of the safety of foods derived from genetically 

modified plants” (OECD, 1998). OECD consensus documents on compositional 

considerations for new crop varieties emphasize quantitative measurements 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 commodities 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 potential differences between the assessed crop and 

its comparator in the context of the well-documented variation in the concentrations of 

crop nutrients, toxicants, and anti-nutrients. 
Monsanto Company 11-CA-233U 77 of 500 

This section provides analyses of concentrations of key nutrients, toxicants, and antinutrients of MON 88302 compared with equivalent analyses of a conventional 

counterpart grown and harvested 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 variability for each analyte, defined by a 99% 

tolerance interval. The production of materials 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 sites were replicated with an adequate number 

of plant samples, and the methods of analysis were sufficiently sensitive and specific to 

detect variations in the components measured and to allow statistically rigorous analyses. 

The information provided in this section also addresses the relevant factors in Codex 

Plant Guidelines, Section 4, paragraphs 44 and 45 for compositional analyses (Codex 

Alimentarius, 2009). 

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

Compositional analysis comparing MON 88302 to the conventional control variety 

(Ebony) and commercial reference varieties demonstrated that MON 88302 is 

compositionally equivalent to conventional canola. Seed samples 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 consensus 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 variability of each compositional component 

analyzed. The samples utilized for compositional analysis were obtained from two U.S. 

sites [Wilkin County, Minnesota (MNCA) 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 four replicates per site. MON 88302, the 

conventional control, and commercial reference varieties were treated with maintenance 

pesticides as necessary throughout the growing 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 equivalents per acre (1800 g a.e./ha). 

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 conventional control and to the composition of commercial 

reference varieties. Nutrients assessed in this analysis included proximates (ash, 

carbohydrates by calculation, moisture, protein, and total fat), fibers (acid detergent fiber 

[ADF], neutral detergent fiber [NDF], total dietary fiber [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 
Monsanto Company 11-CA-233U 78 of 500 

zinc) in seed. The toxicants assessed in seed included erucic acid and glucosinolates 

(alkyl glucosinolates [including 3-butenyl, 4-pentenyl, 2-hydroxy-3-butenyl, and 2-

hydroxy-4-pentenyl glucosinolates], indolyl glucosinolates [including 3-indolylmethyl 

and 4-hydroxy-3-indoylmethyl], and total glucosinolates [sum of alkyl and indolyl]). The 

anti-nutrients assessed in seed included phytic 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-nutrient 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 toxicant (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 were 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 comparisons to the conventional control were conducted 

for each compositional component. One comparison was based on compositional data 

combined across all five field sites (combined-site analysis) and five separate 

comparisons were conducted on data from each of the individual field sites. Statistically 

significant differences were identified 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 component to define the natural variability 

of each component in canola varieties that have 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 using considerations relevant to the safety 

and nutritional quality of MON 88302 when compared to the conventional control, which 

is the conventional counterpart that has a history of safe consumption. 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 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 statistically significant (α = 0.05) combined-site component 

differences at individual 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 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. 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 MON 88302 and the conventional 

control, discussed in the context of natural variability in composition of commercial 
Monsanto Company 11-CA-233U 79 of 500 

canola. Results of the comparison indicate that the composition of the seed of 

MON 88302 is equivalent to that of the conventional 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 types of fiber (ADF and NDF), 18 amino acids 

(alanine, arginine, aspartic acid, cystine, glutamic acid, glycine, histidine, isoleucine, 

leucine, lysine, methionine, phenylalanine, proline, serine, threonine, 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 (16:1 palmitoleic acid, 18:0 stearic acid, 18:1 

oleic acid, 18:2 linoleic acid, 18:3 linolenic acid, 20:0 arachidic acid, and 22:0 behenic 

acid) (Tables VI-1 and VI-2). 

1) The statistically significant differences in nutrients were evaluated using 

considerations relevant to the nutritional quality of MON 88302 when compared 

to the conventional control: eight combined-site nutrient significant differences 

(α = 0.05) between MON 88302 and the conventional 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 behenic 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 individual sites were between 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 components found to be significantly different 

(α = 0.05) from the combined-site analysis of MON 88302 were within the 99% 

tolerance interval established from the commercial reference varieties grown 

concurrently and were, therefore, within the range of natural variability of that 

component in commercial canola varieties that have a history of safe consumption 

(Table VI-1). 
Monsanto Company 11-CA-233U 80 of 500 

3) Assessment of the reproducibility of the combined-site differences at the five 

individual sites demonstrated no significant 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 differences for 22:0 behenic acid 

at two sites (Table VI-1). The magnitudes 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 determined by the 99% tolerance interval 

established by the concurrently 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 significant differences fell within the 99% tolerance 

interval established from the commercial reference varieties grown concurrently 

and were, therefore, within the range of natural variability of that component in 

commercial canola varieties that have a history 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 components 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 established by the concurrently grown commercial 

reference varieties that have a history of safe consumption. Of these significant 

differences, only 18:0 stearic acid, 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 and corresponding individual site analyses, were 

within the natural variability of commercial canola defined by the 99% tolerance interval 

established by the concurrently grown commercial reference varieties, and were within 

the published literature ranges (there is no published reference data for TDF) for these 

components in conventional canola. Therefore, 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 contains toxicants including erucic acid and 

glucosinolates, and anti-nutrients, including phytic acid and sinapine. Erucic acid has 

been shown to have cardiopathic 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 chemical groups, alkyl and indolyl, with alkyl 

being the most common (CCC, 2009). Upon enzymatic hydrolysis with myrosinase, 

certain glucosinolates form compounds that can depress growth and thyroid function 
Monsanto Company 11-CA-233U 81 of 500 

(Bell, 1984). The standard for glucosinolates in canola seed is <18 μmoles/g 

(Szmigielska et al., 2000). Phytic acid is present in canola seed. Phytic acid chelates 

mineral nutrients, including calcium, magnesium, potassium, iron, and zinc, rendering 

them biologically unavailable to monogastric animals consuming the seed (Liener, 2000). 

Sinapine is the choline ester of sinapic acid, the primary phenolic component in canola 

seed. Sinapine imparts a bitter taste and reduces palatability of the seed (OECD, 2001). 

Sinapine levels were determined based on quantitation of the hydrolysis product, sinapic 

acid. 

MON 88302 levels of 22:1 erucic acid were below the level of detection (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 anti-nutrient in MON 88302. The following considerations show 

that this difference is not a meaningful concern 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 determined by the 99% tolerance interval 

established by the concurrently grown commercial 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 value was, therefore, within the range of 

natural variability for alkyl glucosinolates in commercial canola varieties that 

have a history of safe consumption (Tables VI-1 and VI-3). 

3) The combined-site difference for alkyl glucosinolates was not consistently 

observed at all individual sites. A significant difference for alkyl glucosinolates 

was observed at one of the individual sites. However, the mean value for alkyl 

glucosinolates in MON 88302 at this individual 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 conventional mean value, and not consistently 

observed at the individual sites. The mean alkyl glucosinolates value for MON 88302 

was within the natural variability of commercial canola defined by the 99% tolerance 

interval established from the concurrently grown commercial reference varieties that have 
Monsanto 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, and E-12), within the standard for canola. 

Thus, an evaluation of anti-nutrient components in seed supports the conclusion that 

MON 88302 is as safe as and compositionally equivalent to conventional canola. 
Monsanto Company 11-CA-233U 

Table VI-1. Summary of Differences (p < 0.05) for the Comparison of Canola Seed Component Levels foConventional Control 

 Mean Difference 

(MON 88302 minus Control)

Analytical Component (Units)1

MON 883022

Mean3

Control4

Mean

Mean Difference 

(% of Control)

Significance

(p-Value)

MON 8830

Range

Statistical Differences Observed in Combined-Site AnalysisSeed Fiber (% dw)Total Dietary Fiber 20.90 18.37 13.81 0.004 16.91 - 27.8Seed Fatty Acid (% Total FA)16:1 Palmitoleic 0.22 0.24 -7.56 0.008 0.20 - 0.26 18:0 Stearic 1.68 1.98 -15.06 <0.001 1.54 - 1.87 18:1 Oleic 62.82 65.79 -4.52 <0.001 60.51 - 65.218:2 Linoleic 19.26 17.67 8.98 <0.001 17.78 - 20.618:3 Linolenic 9.58 7.98 20.01 <0.001 8.71 - 11.220:0 Arachidic 0.54 0.60 -10.68 <0.001 0.50 - 0.57 22:0 Behenic 0.27 0.28 -6.01 0.016 0.24 - 0.29 
Monsanto Company 11-CA-233U 

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

 Mean Difference 

(MON 88302 minus Control)

Analytical Component (Units)1

MON 883022

Mean3

Control4

Mean

Mean Difference 

(% of Control)

Significance

(p-Value)

MON 8830

Range

Statistical Differences Observed in Combined-Site AnalysisSeed Anti-nutrientAlkyl Glucosinolate (μmole/g dw) 3.68 5.08 -27.59 0.035 1.19 - 5.87 Statistical Differences Observed in More than One Individual SiteSeed Fatty Acid (% Total FA)18:0 Stearic Site MBNW 1.73 1.97 -12.23 0.028 1.64 - 1.87 18:0 Stearic Site MBPL 1.58 1.87 -15.64 <0.001 1.55 - 1.59 18:0 Stearic Site MNCA 1.67 1.86 -10.01 0.022 1.65 - 1.71 18:0 Stearic Site NDVA 1.77 2.11 -16.06 0.004 1.71 - 1.84 18:0 Stearic Site SKSA 1.66 2.08 -20.14 0.001 1.54 - 1.72 18:1 Oleic Site MBNW 63.40 65.71 -3.51 0.004 62.94 - 64.0
Monsanto Company 11-CA-233U 

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

 Mean Difference 

(MON 88302 minus Control)

Analytical Component (Units)1

MON 883022

Mean3

Control4

Mean

Mean Difference 

(% of Control)

Significance

(p-Value)

MON 8830

Range

Statistical Differences Observed in More than One Individual SiteSeed Fatty Acid (% Total FA)18:1 Oleic Site MBPL 62.06 64.30 -3.48 <0.001 61.82 - 62.318:1 Oleic Site MNCA 61.67 64.86 -4.92 0.005 61.70 - 61.818:1 Oleic Site NDVA 65.14 68.38 -4.74 0.003 64.90 - 65.218:1 Oleic Site SKSA 61.91 65.69 -5.75 0.001 60.51 - 62.218:2 Linoleic Site MBNW 19.27 17.89 7.71 0.011 18.82 - 19.618:2 Linoleic Site MBPL 20.43 19.18 6.50 <0.001 20.13 - 20.618:2 Linoleic Site MNCA 20.20 18.35 10.07 0.001 20.00 - 20.318:2 Linoleic Site NDVA 17.86 15.71 13.67 0.009 17.78 - 18.0
Monsanto Company 11-CA-233U 

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

 Mean Difference 

(MON 88302 minus Control)

Analytical Component (Units)1

MON 883022

Mean3

Control4

Mean

Mean Difference 

(% of Control)

Significance

(p-Value)

MON 8830

Range

Statistical Differences Observed in More than One Individual SiteSeed Fatty Acid (% Total FA)18:2 Linoleic Site SKSA 18.49 17.22 7.36 0.019 18.08 - 19.4Seed Vitamin (mg/100g dw)Vitamin E (a-tocopherol) Site MBNW 13.06 9.36 39.51 0.004 12.22 - 13.4Vitamin E (

α-tocopherol) Site MBPL 11.50 7.63 50.83 <0.001 10.70 - 12.2

Vitamin E (

α-tocopherol) Site MNCA 13.39 10.82 23.73 0.006 12.58 - 14.6

Vitamin E (

α-tocopherol) Site NDVA 15.89 9.43 68.39 0.010 15.23 - 16.5

Vitamin E (

α-tocopherol) Site SKSA 1.49 6.91 -78.47 0.019 1.30 - 1.66 

Seed Anti-nutrientSinapic Acid (% dw) Site MBNW 1.02 0.92 10.34 0.001 0.99 - 1.06 
Monsanto Company 11-CA-233U 

Table VI-1. Summary of Differences (p<0.05) for the Comparison of Canola Seed Component Levels foConventional Control (continued) 

 Mean Difference 

(MON 88302 minus Control)

Analytical Component (Units)1

MON 883022

Mean3

Control4

Mean

Mean Difference 

(% of Control)

Significance

(p-Value)

MON 8830

Range

Statistical Differences Observed in More than One Individual SiteSeed Anti-nutrientSinapic Acid (% dw) Site MBPL 0.97 0.86 12.04 <0.001 0.95 - 0.99 Sinapic Acid (% dw) Site MNCA 1.06 0.96 10.66 0.001 1.02 - 1.08 Sinapic Acid (% dw) Site NDVA 1.02 0.83 23.56 0.001 1.00 - 1.04 Sinapic Acid (% dw) Site SKSA 0.22 0.81 -73.12 0.001 0.16 - 0.28 Seed Fatty Acid (% Total FA)16:1 Palmitoleic Site MBNW 0.21 0.23 -9.71 0.015 0.20 - 0.21 16:1 Palmitoleic Site MBPL 0.23 0.25 -10.10 0.008 0.22 - 0.23 16:1 Palmitoleic Site MNCA 0.21 0.24 -10.88 0.001 0.21 - 0.21 16:1 Palmitoleic Site NDVA 0.20 0.22 -11.05 0.036 0.20 - 0.20 
Monsanto Company 11-CA-233U 

Table VI-1. Summary of Differences (p<0.05) for the Comparison of Canola Seed Component Levels for MConventional Control (continued) 

 Mean Difference 

(MON 88302 minus Control)

Analytical Component (Units)1

MON 883022

Mean3

Control4

Mean

Mean Difference 

(% of Control)

Significance

(p-Value)

MON 8830

Range

Statistical Differences Observed in More than One Individual SiteSeed Fatty Acid (% Total FA)18:3 Linolenic Site MBNW 9.19 8.12 13.27 0.004 8.88 - 9.42 18:3 Linolenic Site MBPL 9.28 7.74 19.89 <0.001 9.12 - 9.43 18:3 Linolenic Site NDVA 8.82 7.31 20.67 <0.001 8.71 - 8.94 18:3 Linolenic Site SKSA 10.78 8.38 28.69 <0.001 10.39 - 11.2Seed Fatty Acid (% Total FA)20:0 Arachidic Site MBPL 0.53 0.60 -11.73 <0.001 0.52 - 0.54 20:0 Arachidic Site NDVA 0.57 0.65 -12.58 <0.001 0.56 - 0.57 20:0 Arachidic Site SKSA 0.54 0.62 -13.28 <0.001 0.52 - 0.55 
Monsanto Company 11-CA-233U 

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

 Mean Difference 

(MON 88302 minus Control)

Analytical Component (Units)1

MON 883022

Mean3

Control4

Mean

Mean Difference 

(% of Control)

Significance

(p-Value)

MON 88

Range

Statistical Differences Observed in More than One Individual SiteSeed MineralCopper (mg/kg dw) Site MBNW 3.72 3.41 9.28 0.013 3.61 - 3Copper (mg/kg dw) Site MBPL 3.47 3.97 -12.50 0.016 3.35 - 3Copper (mg/kg dw) Site MNCA 4.40 4.11 6.91 0.027 4.16 - 4Seed Fatty Acid (% Total FA)22:0 Behenic Site MBPL 0.27 0.30 -13.00 <0.001 0.26 - 022:0 Behenic Site NDVA 0.27 0.30 -9.83 0.007 0.27 - 0Seed MineralIron (mg/kg dw) Site MBPL 44.13 51.01 -13.48 0.001 42.80 - 4Iron (mg/kg dw) Site MNCA 42.57 50.64 -15.93 0.007 40.56 - 44
Monsanto Company 11-CA-233U 

Table VI-1. Summary of Differences (p<0.05) for the Comparison of Canola Seed Component Levels for MConventional Control (continued) 

 Mean Difference 

(MON 88302 minus Control)

Analytical Component (Units)1

MON 883022

Mean3

Control4

Mean

Mean Difference 

(% of Control)

Significance

(p-Value)

MON 8830

Range

Statistical Differences Observed in More than One Individual SiteSeed MineralPotassium (g/100g dw) Site MBPL 0.70 0.77 -8.91 0.023 0.63 - 0.76 Potassium (g/100g dw) Site SKSA 0.82 0.71 15.32 <0.001 0.77 - 0.90 Zinc (mg/kg dw) Site MBPL 31.25 33.88 -7.76 0.024 30.45 - 32.0Zinc (mg/kg dw) Site SKSA 41.58 33.10 25.61 0.010 39.33 - 45.4Statistical Differences Observed in One Individual SiteSeed Proximate (% dw)Carbohydrates Site MNCA 27.31 25.99 5.07 0.035 26.27 - 27.9Moisture (% fw) Site MNCA 5.52 6.69 -17.46 <0.001 5.37 - 5.61 Protein Site SKSA 23.82 22.14 7.58 0.038 23.62 - 24.5
Monsanto Company 11-CA-233U 

Table VI-1. Summary of Differences (p<0.05) for the Comparison of Canola Seed Component Levels for MConventional Control (continued) 

 Mean Difference 

(MON 88302 minus Control)

Analytical Component (Units)1

MON 883022

Mean3

Control4

Mean

Mean Difference 

(% of Control)

Significance

(p-Value)

MON 8830

Range

Statistical Differences Observed in One Individual SiteSeed Proximate (% dw)Total Fat Site NDVA 48.04 45.17 6.35 0.014 47.20 - 48.8Seed Fiber (% dw)Acid Detergent Fiber Site MBPL 16.75 14.19 18.00 0.005 15.17 - 18.1Neutral Detergent Fiber Site MBPL 19.45 16.87 15.31 0.017 18.35 - 20.0Seed Amino Acid (% dw)Tyrosine Site MBPL 0.72 0.71 2.46 0.028 0.72 - 0.73 Valine Site MNCA 1.15 1.24 -7.32 0.048 1.13 - 1.15 Seed Fatty Acid (% Total FA)16:0 Palmitic Site SKSA 4.51 4.07 10.90 <0.001 4.46 - 4.57 20:1 Eicosenoic Site SKSA 1.24 1.13 9.55 0.005 1.22 - 1.26 
Monsanto Company 11-CA-233U 

Table VI-1. Summary of Differences (p<0.05) for the Comparison of Canola Seed Component Levels for MConventional Control (continued) 

 Mean Difference 

(MON 88302 minus Control)

Analytical Component (Units)1

MON 883022

Mean3

Control4

Mean

Mean Difference 

(% of Control)

Significance

(p-Value)

MON 8830

Range

Statistical Differences Observed in One Individual SiteSeed Fatty Acid (% Total FA)24:0 Lignoceric Site MBPL 0.16 0.19 -12.24 0.029 0.16 - 0.17 24:1 Nervonic Site MBPL 0.13 0.16 -20.37 0.031 0.12 - 0.13 Seed Anti-nutrientAlkyl Glucosinolate (μmole/g dw) Site SKSA

1.61 5.82 -72.32 0.005 1.19 - 2.17 

Indolyl Glucosinolate (μmole/g dw) Site SKSA

0.86 3.30 -73.88 0.001 0.49 - 1.31 

Total Glucosinolate (μmole/g dw) Site SKSA

2.53 9.22 -72.58 0.002 1.73 - 3.51 

1dw = 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 population of commercial conventional reference varieties. to zero. 
Monsanto Company 11-CA-233U 

Table VI-2. Statistical Summary of Combined-site Canola Seed Nutrients for MON 88302 vs. Convention

 Difference (MON 88302 minus Control)

Analytical Component (Units)1

MON 883022 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% CI3

Lower, Upper

Significan

(p-Value

Proximate (% dw)Ash 3.96 (0.18) 3.90 (0.18) 0.055 (0.095) -0.14, 0.25 0.565 

(3.31 - 4.45) (3.20 - 5.10) (-0.21 - 0.64) 

Carbohydrates 25.96 (0.68) 26.13 (0.68) -0.17 (0.54) -1.42, 1.09 0.765 

(21.83 - 28.81) (23.91 - 28.73) (-4.18 - 1.94) 

Moisture (% fw) 5.35 (0.34) 5.45 (0.34) -0.10 (0.24) -0.65, 0.45 0.688 

(3.90 - 6.08) (4.41 - 6.98) (-1.53 - 0.87) 

Protein 23.04 (0.70) 23.14 (0.69) -0.10 (0.52) -1.32, 1.11 0.847 

(19.68 - 25.98) (20.29 - 27.02) (-2.29 - 2.50) 

Total Fat 47.06 (0.83) 46.82 (0.83) 0.24 (0.52) -1.00, 1.48 0.659 

(43.96 - 49.26) (43.65 - 50.24) (-2.28 - 4.10) 

Fiber (% dw)Acid Detergent Fiber 15.32 (1.36) 14.47 (1.36) 0.84 (0.41) -0.14, 1.83 0.082 

(9.19 - 20.24) (8.94 - 18.71) (-2.71 - 3.57) 
Monsanto Company 11-CA-233U 

Table VI-2. Statistical Summary of Combined-Site Seed Nutrient Content for MON 88302 vs. the C(continued) 

 Difference (MON 88302 minus Control)

Analytical Component (Units)1

MON 883022 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% CI3

Lower, Upper

Significan

(p-Value

Fiber (% dw)Neutral Detergent Fiber 17.43 (1.38) 16.70 (1.38) 0.74 (0.57) -0.58, 2.05 0.231 

(9.48 - 21.36) (11.56 - 19.58) (-2.74 - 4.43) 

Total Dietary Fiber 20.90 (0.79) 18.37 (0.78) 2.54 (0.84) 0.85, 4.23 0.004 

(16.91 - 27.81) (14.58 - 23.00) (-0.49 - 9.96) 

Amino Acid (% dw)Alanine 1.02 (0.025) 1.04 (0.025) -0.015 (0.022) -0.066, 0.035 0.502 

(0.88 - 1.15) (0.93 - 1.19) (-0.12 - 0.069) 

Arginine 1.45 (0.054) 1.51 (0.054) -0.063 (0.032) -0.13, 0.00082 0.052 

(1.23 - 1.72) (1.29 - 1.77) (-0.27 - 0.15) 

Aspartic Acid 1.65 (0.067) 1.71 (0.067) -0.055 (0.043) -0.16, 0.045 0.238 

(1.40 - 1.93) (1.46 - 1.97) (-0.37 - 0.12) 

Cystine 0.57 (0.027) 0.58 (0.027) -0.0044 (0.015) -0.040, 0.031 0.781 

(0.48 - 0.73) (0.49 - 0.79) (-0.054 - 0.053) 
Monsanto Company 11-CA-233U 

Table VI-2. Statistical Summary of Combined-Site Seed Nutrient Content for MON 88302 vs. the C(continued) 

 Difference (MON 88302 minus Control)

Analytical Component (Units)1

MON 883022 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% CI3

Lower, Upper

Significan

(p-Value

Amino Acid (% dw)Glutamic Acid 4.06 (0.18) 4.24 (0.17) -0.19 (0.10) -0.43, 0.049 0.103 

(3.37 - 5.06) (3.64 - 5.26) (-0.68 - 0.36) 

Glycine 1.14 (0.040) 1.19 (0.040) -0.041 (0.025) -0.10, 0.018 0.142 

(1.02 - 1.32) (1.01 - 1.38) (-0.18 - 0.044) 

Histidine 0.63 (0.023) 0.65 (0.023) -0.015 (0.011) -0.038, 0.0074 0.181 

(0.55 - 0.77) (0.57 - 0.78) (-0.065 - 0.044) 

Isoleucine 0.93 (0.028) 0.96 (0.028) -0.024 (0.021) -0.074, 0.026 0.299 

(0.81 - 1.08) (0.82 - 1.12) (-0.13 - 0.041) 

Leucine 1.64 (0.049) 1.68 (0.049) -0.042 (0.039) -0.13, 0.048 0.308 

(1.40 - 1.90) (1.46 - 1.95) (-0.25 - 0.086) 

Lysine 1.39 (0.041) 1.41 (0.041) -0.019 (0.023) -0.064, 0.027 0.410 

(1.22 - 1.63) (1.25 - 1.65) (-0.12 - 0.086) 
Monsanto Company 11-CA-233U 

Table VI-2. Statistical Summary of Combined-Site Seed Nutrient Content for MON 88302 vs. the Conven(continued)

 Difference (MON 88302 minus Control)

Analytical Component (Units)1

MON 883022 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% CI3

Lower, Upper

Significan

(p-Value

Amino Acid (% dw)Methionine 0.46 (0.015) 0.46 (0.015) -0.0018 (0.0089) -0.022, 0.019 0.847 

(0.40 - 0.54) (0.40 - 0.56) (-0.038 - 0.034) 

Phenylalanine 0.98 (0.029) 1.00 (0.028) -0.024 (0.024) -0.079, 0.031 0.348 

(0.84 - 1.11) (0.87 - 1.15) (-0.17 - 0.044) 

Proline 1.40 (0.054) 1.42 (0.054) -0.028 (0.027) -0.093, 0.036 0.335 

(1.20 - 1.71) (1.20 - 1.73) (-0.16 - 0.17) 

Serine 1.02 (0.030) 1.05 (0.030) -0.035 (0.019) -0.080, 0.0095 0.105 

(0.87 - 1.14) (0.94 - 1.18) (-0.17 - 0.052) 

Threonine 0.98 (0.030) 1.00 (0.030) -0.025 (0.018) -0.066, 0.016 0.192 

(0.86 - 1.11) (0.88 - 1.12) (-0.12 - 0.065) 

Tryptophan 0.23 (0.010) 0.24 (0.010) -0.013 (0.0093) -0.032, 0.0059 0.172 

(0.17 - 0.26) (0.19 - 0.31) (-0.063 - 0.036) 
Monsanto Company 11-CA-233U 

Table VI-2. Statistical Summary of Combined-Site Seed Nutrient Content for MON 88302 vs. the Conven(continued)

 Difference (MON 88302 minus Control)

Analytical Component (Units)1

MON 883022 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% CI3

Lower, Upper l

Significan

(p-Value

Amino Acid (% dw)Tyrosine 0.67 (0.019) 0.69 (0.019) -0.017 (0.013) -0.048, 0.015 0.249 

(0.59 - 0.75) (0.61 - 0.77) (-0.11 - 0.028) 

Valine 1.20 (0.035) 1.22 (0.035) -0.025 (0.025) -0.084, 0.034 0.352 

(1.04 - 1.37) (1.05 - 1.41) (-0.16 - 0.054) 

Fatty Acid (% Total FA)16:0 Palmitic 4.23 (0.078) 4.10 (0.077) 0.13 (0.067) -0.027, 0.28 0.094 

(3.95 - 4.57) (3.94 - 4.41) (-0.22 - 0.48) 

16:1 Palmitoleic 0.22 (0.0081) 0.24 (0.0081) -0.018 (0.0053) -0.030, -0.0059 0.008 

(0.20 - 0.26) (0.22 - 0.26) (-0.039 - 0.0074) 

18:0 Stearic 1.68 (0.044) 1.98 (0.044) -0.30 (0.031) -0.37, -0.23 <0.001 

(1.54 - 1.87) (1.78 - 2.19) (-0.48 - -0.059) 

18:1 Oleic 62.82 (0.62) 65.79 (0.62) -2.97 (0.31) -3.69, -2.26 <0.001 

(60.51 - 65.20) (63.72 - 68.44) (-4.30 - -1.52) 
Monsanto Company 11-CA-233U 

Table VI-2. Statistical Summary of Combined-Site Seed Nutrient Content for MON 88302 vs. the Convent(continued)

 Difference (MON 88302 minus Control)

Analytical Component (Units)1

MON 883022 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% CI3

Lower, Upper

Significanc

(p-Value)

Fatty Acid (% Total FA)18:2 Linoleic 19.26 (0.51) 17.67 (0.51) 1.59 (0.17) 1.20, 1.97 <0.001 

(17.78 - 20.66) (15.72 - 19.29) (0.40 - 2.42) 

18:3 Linolenic 9.58 (0.27) 7.98 (0.27) 1.60 (0.21) 1.12, 2.07 <0.001 

(8.71 - 11.23) (7.19 - 8.99) (0.76 - 2.64) 

20:0 Arachidic 0.54 (0.011) 0.60 (0.011) -0.064 (0.0074) -0.081, -0.047 <0.001 

(0.50 - 0.57) (0.54 - 0.65) (-0.091 - -0.0032) 

20:1 Eicosenoic 1.13 (0.024) 1.09 (0.024) 0.036 (0.017) -0.0034, 0.076 0.068 

(1.06 - 1.26) (1.00 - 1.18) (-0.042 - 0.14) 

22:0 Behenic 0.27 (0.0072) 0.28 (0.0072) -0.017 (0.0056) -0.030, -0.0041 0.016 

(0.24 - 0.29) (0.24 - 0.31) (-0.047 - 0.016) 

24:0 Lignoceric 0.16 (0.016) 0.16 (0.015) 0.0038 (0.017) -0.030, 0.038 0.823 

(0.049 - 0.23) (0.045 - 0.22) (-0.14 - 0.11) 
Monsanto Company 11-CA-233U 

Table VI-2. Statistical Summary of Combined-Site Seed Nutrient Content for MON 88302 vs. the Conven(continued)

 Difference (MON 88302 minus Control)

Analytical Component (Units)1

MON 883022 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% CI3

Lower, Upper

Significan

(p-Value

Fatty Acid (% Total FA)24:1 Nervonic 0.12 (0.015) 0.11 (0.015) 0.013 (0.014) -0.020, 0.047 0.377 

(0.046 - 0.20) (0.045 - 0.17) (-0.072 - 0.081) 

MineralCalcium (g/100g dw) 0.41 (0.030) 0.40 (0.030) 0.015 (0.012) -0.0089, 0.039 0.210 

(0.30 - 0.51) (0.28 - 0.49) (-0.068 - 0.081) 

Copper (mg/kg dw) 3.78 (0.17) 3.65 (0.17) 0.14 (0.14) -0.19, 0.46 0.361 

(3.27 - 4.57) (2.96 - 4.18) (-0.83 - 0.57) 

Iron (mg/kg dw) 48.73 (4.28) 54.01 (4.24) -5.28 (2.89) -11.85, 1.30 0.102 

(40.55 - 69.61) (41.65 - 77.74) (-20.41 - 14.87) 

Magnesium (g/100g dw) 0.37 (0.014) 0.36 (0.014) 0.0048 (0.0070) -0.011, 0.021 0.508 

(0.31 - 0.42) (0.31 - 0.42) (-0.032 - 0.043) 

Manganese (mg/kg dw) 41.44 (2.02) 40.34 (1.99) 1.10 (1.83) -2.62, 4.82 0.551 

(35.28 - 51.55) (33.12 - 50.97) (-8.36 - 12.63) 
Monsanto Company 11-CA-233U 

Table VI-2. Statistical Summary of Combined-Site Seed Nutrient Content for MON 88302 vs. the Conven(continued)

 Difference (MON 88302 minus Control)

Analytical Component (Units)1

MON 883022 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% CI3

Lower, Upper

Significan

(p-Value

MineralPhosphorus (g/100g dw) 0.72 (0.042) 0.72 (0.041) -0.0090 (0.022) -0.055, 0.037 0.692 

(0.56 - 0.87) (0.56 - 0.93) (-0.095 - 0.16) 

Potassium (g/100g dw) 0.64 (0.053) 0.64 (0.052) 0.0016 (0.025) -0.056, 0.060 0.951 

(0.48 - 0.90) (0.53 - 0.81) (-0.097 - 0.14) 

Zinc (mg/kg dw) 35.58 (1.78) 33.01 (1.76) 2.57 (1.83) -1.66, 6.80 0.198 

(29.81 - 45.56) (28.46 - 40.66) (-4.50 - 11.44) 

Vitamin (mg/100g dw)Vitamin E (

α-tocopherol) 11.06 (2.08) 8.85 (2.08) 2.21 (1.66) -1.61, 6.03 0.218 

(1.30 - 16.55) (3.33 - 11.77) (-6.92 - 8.09) 

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 population of commercial conventional reference varietiesset to zero. 
Monsanto Company 11-CA-233U 

Table VI-3. Statistical Summary of Combined-site Canola Seed Anti-nutrients for MON 88302 vs. Conven

 Difference (MON 88302 minus Control)

Analytical Component (Units)1

MON 883022 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% CI3

Lower, Upper

Significan

(p-Value

Anti-nutrientAlkyl Glucosinolate (μmole/g dw) 3.68 (0.43) 5.08 (0.42) -1.40 (0.59) -2.69, -0.11 0.035 

(1.19 - 5.87) (2.45 - 8.28) (-6.11 - 1.43) 

Indolyl Glucosinolate (μmole/g dw) 3.50 (0.51) 3.89 (0.50) -0.39 (0.45) -1.42, 0.64 0.408 

(0.49 - 5.76) (1.83 - 5.89) (-3.05 - 2.83) 

Phytic Acid (% dw) 1.95 (0.18) 2.11 (0.18) -0.16 (0.083) -0.33, 0.010 0.064 

(1.20 - 2.58) (1.46 - 2.77) (-0.67 - 0.68) 

Sinapic Acid (% dw) 0.86 (0.12) 0.88 (0.12) -0.023 (0.11) -0.27, 0.22 0.837 

(0.16 - 1.08) (0.65 - 0.97) (-0.76 - 0.21) 

Total Glucosinolate (μmole/g dw) 7.35 (0.87) 9.08 (0.86) -1.73 (1.01) -4.06, 0.61 0.127 

(1.73 - 11.42) (4.38 - 12.72) (-9.21 - 3.58) 

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 population of commercial conventional reference varieties set to zero. 
Monsanto Company 11-CA-233U 102 of 500 

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

Component1 Literature Range2

 

Proximates (% dw)

Ash 4.067 – 5.917a

Carbohydrates N

Moisture (% fw) 3.177 – 8.045 a; 7.4 – 10.0b

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

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

 

Fiber (% dw)

Acid Detergent Fiber (ADF) 11.934 – 26.799 a

;11.6f; 12.4g; 22.2h

Neutral Detergent Fiber (NDF) 18.653 – 34.720a

; 17.8f; 16.49g; 31.3h

Total Dietary Fiber N

 

Amino Acids (% dw)

Alanine 0.93 – 0.96b ; 1.15 – 1.38e

Arginine 1.13 – 1.21 b; 2.23 – 2.46e

Aspartic acid 1.54 – 1.59e

Cystine/Cysteine 0.52 – 0.54 b

Glutamic acid 4.60 – 4.71e

Glycine 1.04 – 1.06 b; 2.20 – 2.22e

Histidine 0.51 – 0.66 b; 0.80 – 0.82e

Isoleucine 0.80 – 0.86 b; 0.96 – 1.03e

Leucine 1.35 – 1.47 b; 1.83 – 1.99e

Lysine 1.03 – 1.19 b; 1.67 – 1.85e

Methionine 0.42 – 0.44 b

Phenylalanine 0.75 – 0.82 b; 0.90 – 1.03e

Proline 1.19 – 1.33 b; 3.36 – 3.74e

Serine 0.90 – 0.94 b; 1.44 – 1.55e

Threonine 0.87 – 0.94 b; 1.28 – 1.30e

Tryptophan 0.23 – 0.27b;

Tyrosine 0.51 – 0.59 b; 0.81 – 0.92e

Valine 1.02 – 1.13 b; 1.45 – 1.55e

 

Vitamins (mg/kg dw)

Vitamin E (α-tocopherol) 71.1 – 108.4i

 

 
Monsanto Company 11-CA-233U 103 of 500 

Table VI-4. Literature Ranges for Components in Canola Seed (continued) 

Component1 Literature Range2

Minerals 

Calcium (% dw) 0.29 – 0.48b

; 0.348 – 0.729a

Copper (mg/kg dw) 7b

; 1.388 – 5.492a

Iron (mg/kg dw) NDb

; 0.0 – 965.6a

Magnesium (% dw) 0.29 – 0.31b

; 0.272 – 0.402a

Manganese (mg/kg dw) NDb

; 33.813 – 64.757a

Phosphorus (% dw) 0.48 – 0.85b

; 0.581 – 0.895a

Potassium (% dw) 0.83 – 0.91b

; 0.681 – 1.016a

Sodium (% dw) 0.05b

; 0.003 – 0.030a

Zinc (mg/kg dw) 62b

; 0 – 126.953a

 

Fatty Acids (% total)

16:0 Palmitic 3.3 – 6.0b

16:1 Palmitoleic 0.1 – 0.6b

18:0 Stearic 1.1 – 2.5 b

18:1 Oleic 52.0 – 66.9b

18:2 Linoleic 16.1 – 24.8b

18:3 Linolenic 6.4 – 14.1b

20:0 Arachidic 0.2 – 0.8b

20:1 Eicosenoic 0.1 – 3.4b

20:2 Eicosadienoic 0.0 – 0.1b

20:3 Eicosatrienoic N 

20:4 Arachidonic N 

22:0 Behenic 0.0 – 0.5b

22:1 Erucic 0.0 – 2.0b

24:0 Lignoceric 0.0 – 0.2b

24:1 Nervonic 0.0 – 0.04b

 

Anti-nutrients 

Total Glucosinolates (μmol/g) 6 – 29b

; 7.8 – 26.8c; 18 – 57j

Phytic Acid (% dw) 2.0 – 5.0b

Sinapine (% dw) 0.6 – 1.8b

 

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 anti-nutrient levels in MON 88302 and the 

conventional control were conducted to assess 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 compositional analysis, conducted in accordance with 

OECD guidelines, included measurement of nutrient, toxicant, and anti-nutrient 

components in a genetically similar conventional control variety, Ebony and also in 

commercial conventional reference varieties that have a history of safe consumption to 

establish the natural range of variability. MON 88302, the conventional control, and 

commercial reference varieties were treated with conventional weed control programs. In 

addition, MON 88302 plots were treated with glyphosate 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 using 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 analysis were small (4.52% to 20.01%), when 

considered relative to the natural variability determined by the 99% tolerance 

interval established by the concurrently grown commercial reference varieties that 

have a history of safe consumption. The relative magnitude of difference for the 

anti-nutrient alkyl glucosinolate that was statistically significant in the combined 

site analysis was small (27.59%) when considered relative to the natural 

variability determined by the 99% tolerance interval established by the 

concurrently grown commercial reference varieties 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 varieties grown concurrently. Therefore, the 

differences were within the range of natural variability of those components in 

commercial canola varieties that have a history of safe consumption (Tables VI-2 

and VI-3). 

3) Assessment of the reproducibility 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% tolerance interval established from the 

concurrently grown commercial reference varieties. Therefore, these components 
Monsanto Company 11-CA-233U 105 of 500 

were within the range of natural variability in commercial canola that have a 

history of safe consumption. 

4) With the exception of TDF and alkyl glucosinolates, all 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 relevant 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 MON 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 and therefore the food and feed safety and 

nutritional quality of this product is comparable 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 components of MON 88302 have been performed 

and have demonstrated that MON 88302 is compositionally 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 originating from conventional canola. 
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VII. PHENOTYPIC, AGRONOMIC, AND ENVIRONMENTAL 

INTERACTIONS ASSESSMENT 

This section provides an assessment of the phenotypic, agronomic, and environmental 

interaction characteristics of MON 88302 compared to the conventional control Ebony. 

The data support a conclusion that MON 88302 is similar to conventional canola with the 

exception of the glyphosate-tolerance trait and, therefore, is no more likely to pose a plant 

pest risk or have a significant environmental impact. These conclusions are based on the 

results of multiple evaluations reported here. 

Phenotypic, agronomic, and environmental interaction characteristics of MON 88302 

were evaluated in a comparative manner to assess plant pest potential. These assessments 

included evaluation of seed germination characteristics, plant growth and development 

characteristics, observations for plant responses to abiotic stress, plant-disease and plantarthropod interactions, and pollen characteristics. Results from the phenotypic, 

agronomic, and environmental interaction assessment demonstrate that MON 88302 does 

not possess a) increased weediness characteristics; b) increased susceptibility or tolerance 

to specific abiotic stress, diseases, or arthropods; or c) characteristics that would confer a 

plant pest risk or a significant environmental impact compared to the conventional 

control. 

VII.A. Characteristics Measured for Assessment 

In the phenotypic, agronomic, and environmental interactions assessment of 

MON 88302, data were collected to evaluate specific aspects of altered plant pest 

potential. A detailed description of the regulated article phenotype is requested as part of 

the petition for determination of nonregulated status in 7 CFR § 340. 6 including 

differences from the unmodified recipient organism that would “substantiate 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 detailed phenotypic, agronomic, and environmental interaction 

description of MON 88302. A subset of these data were included in an evaluation of 

specific characteristics related to altered plant pest potential. 

As part of the phenotype description, the plant characterization of MON 88302 

encompassed five general categories: 1) germination, dormancy, and emergence; 2) 

vegetative growth; 3) reproductive growth (including pollen characteristics); 4) seed 

retention on the plant and lodging; and 5) plant response to abiotic stress and 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, 
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(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 represent a range of genetic 

backgrounds and phenotypic characteristics and have been grown in the canola 

production regions of Canada and the U.S. The commercial reference varieties reflect a 

range of values for the agronomic, phenotypic and environmental interaction 

characteristics assessed within the crop and therefore can provide context for interpreting 

experimental results. 
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Table VII-1. Phenotypic, Agronomic and Environmental Interaction Characteristics 

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

Data 

category 

Characteristics 

measured 

(associated section 

where discussed) Evaluation timing 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 

characteristics2

Abnormal 

germinated 

(VII.C.1) 

Day 7 (15/25 °C) Percentage of seed producing seedlings 

that could not be classified as normal 

germinated2

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 test2

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 when 

subsequently moved to the AOSArecommended temperature (15/25°C) they 

did germinate2

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 test2

) 

Early stand count 

(VII.C.2.1) Seedling (G.S. 12 – 14)1 Number of emerged plants estimated by counting 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 viable 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% moisture 
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Table VII-1. Phenotypic, Agronomic and Environmental Interaction Characteristics 

Evaluated in U.S. and Canadian Field Trials 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). 
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VII.B. Interpretation of Phenotypic and Environmental Interaction Data 

Plant pest risk assessments for biotechnology-derived crops are comparative assessments. 

Familiarity provides a basis from which the 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 interaction of these factors, and provides a basis 

for comparative environmental risk assessment between a biotechnology-derived plant 

and its conventional counterpart. 

Expert knowledge and experience with conventionally bred canola was the basis for 

selecting appropriate endpoints and estimating 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, agronomic, and environmental interaction 

characteristics. An overview of the characteristics assessed is presented in Table VII-1. 

A subset of the data relating to well-understood 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 potential of MON 88302, an element of Animal and 

Plant Health Inspection Service’s (APHIS) plant 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 collected, 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 unexpected plant response. No unexpected observations or issues 

were identified. 

VII.B.1. Interpretation of Detected Differences Criteria 

Comparative plant characterization data between a biotechnology-derived crop and the 

conventional control are interpreted in the context of contributions to increased plant pest 

potential as assessed by APHIS. Under the framework of familiarity, characteristics for 

which no differences are detected support a conclusion 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 plant pest potential of the biotechnology-derived 

crop. Ultimately, a weight of evidence approach considering all characteristics and 

studies was used for the final risk assessment of differences and their significance. 

Figure VII-1 illustrates the step-wise assessment process employed in detail: 
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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 “yes” 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 Statistically Significant Differences 

Data on each measured characteristic are statistically analyzed, where appropriate, within 

each individual site and in a combined-site analysis, in which data are pooled among 

sites. Differences detected in the individual 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 detected 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 analyses 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 1 
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difference for a characteristic is detected, then the mean value for the biotechnologyderived crop is assessed relative to the commercial reference varieties. 

Step 4 - Evaluate Differences in the Context of the Crop 

If the mean value of the biotechnology-derived crop is outside the variation of the 

commercial reference varieties (e.g., reference range), the mean value of the 

biotechnology-derived crop for the characteristic is assessed relative to known values 

common for the crop (e.g., published values). 

Step 5 - Plant Pest Potential 

If the mean value of the biotechnology-derived 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 plant pest potential. 

Step 6 - Conduct Risk Assessment on Identified Hazard 

If an adverse effect (hazard) is identified, a risk assessment on the difference is 

conducted. The risk assessment considers contributions to enhanced plant pest potential 

of the crop itself, the impact of differences detected in other measured characteristics, and 

potential for, and effects of trait transfer to feral populations of the crop or a sexually 

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

Environmental Interaction Characteristics of MON 88302 

This section provides the results of comparative assessments conducted in replicated 

laboratory and/or multi-site field experiments to provide a detailed phenotypic, 

agronomic, and environmental interaction description of MON 88302. The 

characteristics of MON 88302 evaluated in these assessments included: seed dormancy 

and germination characteristics (Section VII.C.1), plant phenotypic, agronomic, and 

environmental interaction characteristics under 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 weediness 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 dormancy is an important characteristic that is 

often associated with plants that are considered weeds (Anderson, 1996; Lingenfelter and 

Hartwig, 2007). Canola seed dormancy is known to occur under field conditions and can 

contribute to seed persistence in soil (Gulden et al., 2004) and Section IX.C). 

Standardized germination assays are routinely used to measure the germination 

characteristics of canola seed. The Association of Official Seed Analysts (AOSA), an 

internationally recognized seed testing organization, recommends a temperature regime 

of 15/25 °C for testing the germination characteristics of canola seed (AOSA, 2009a). 

Comparative assessments of seed dormancy 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 commercial canola varieties. The seed lots for MON 88302, 

conventional control, and the commercial reference 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 the experimental materials and methods are 

presented in Table VII-1 and Appendix F. 

No statistically significant differences (α = 0.05) were detected between MON 88302 and 

the control at any of the temperature regimes for normal and abnormal germination and 

dead seed (Table VII-2). No statistically significant differences (α = 0.05) were detected 

between MON 88302 and the control at the 5 oC temperature regime for viable nondormant seed (defined in Table VII-1; Table 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. 
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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 control (0.0% vs. 0.3%). A reduction in the 

number of dormant seed would not increase plant weediness since non-dormant seed 

would be more likely to germinate reducing the 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 conventional control was 

small and there were no observable trends in the mean difference in this category across 

the different temperature regimes. 

The biological characteristics 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 increased plant weediness or pest potential of 

MON 88302 compared to the conventional control. 
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Table VII-2. Germination Characteristics of MON 88302 and Conventional Control 

Temperature Germination1 Mean% (S.E.)2 

Regime (°C) Category MON 88302 Control Reference Range3

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

Characteristics 

Phenotypic, agronomic, and environmental interactions characteristics were evaluated 

under field conditions as part of the plant characterization assessment of MON 88302. 

These data were developed to provide USDA-APHIS with a detailed description of 

MON 88302 relative to the conventional control and commercial reference varieties. 

According to 7 CFR § 340.6, as part of the petition to seek deregulation, a petitioner must 

submit “a detailed description of the phenotype of the regulated article.” This 

information is being provided to assess whether there are phenotypic differences between 

MON 88302 and the conventional control that may impact its pest potential. Certain 

growth, reproduction, and pre-harvest seed loss characteristics (e.g., lodging, pod 

shattering) were used to assess whether there is an increase in weediness of MON 88302, 

an element of APHIS’s plant pest risk determination. 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 the U.S. and nine field 

sites in Canada during 2009 to evaluate phenotypic, agronomic, and environmental 

interaction characteristics (Table VII-3). These 17 field sites provided a diverse range of 

environmental and agronomic conditions representative of commercial canola production 

areas in North America. The sites were planted 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 trait altered the phenotypic and 

agronomic characteristics or the environmental 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 environmental interaction 

characteristics and the designated developmental stages when evaluations occurred are 

listed in Table VII-1. The methods and detailed 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 trait did not alter MON 88302 compared to the 

conventional control in terms of weediness potential. 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 result 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 characteristics 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 significant differences were detected (α = 0.05) between 

MON 88302 and the conventional control for early stand count, seedling vigor, seed 

maturity, lodging, plant height, visual rating for pod shattering, quantitative pod 
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shattering, seed quality, yield, and final stand count (Table VII-4). Two statistically 

significant differences were detected between 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 natural 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 

had 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 

difference in seed moisture is unlikely to be biologically meaningful in terms of increased 

weediness potential. 

The plant phenotypic and agronomic characteristics and environmental interactions 

evaluated in this study were used to characterize the plant and its interactions 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 unexpected changes indicative of increased plant 

pest potential or adverse environmental impact of MON 88302 compared to conventional 

canola. 
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Table VII-3. Field Phenotypic Evaluation 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 

Table VII-4. Combined-Site Comparison of MON 83302 to Conventional Control During 2009 for PhenotCharacteristics 

MON 88302 Control Re

Phenotypic Characteristic (units) Mean (S.E.) Mean (S.E.) Minimu

Early stand count (# plants per linear meter)2 18.2 (0.8) 19.2 (0.7) 12Seedling vigor (1-9 scale)3 5.1(0.2) 4.5 (0.2) 1

Days to first flowering (Days after planting)4 61.1 (1.5)* 56.2 (1.4) 45

Seed maturity (Days after planting)5 102.6 (1.4) 101.3 (1.4) 84Lodging (1-9 scale)6 1.7 (0.3) 1.8 (0.2) 1Plant height (inches)7 45.4 (0.9) 44.3 (0.8) 29Visual rating for pod shattering (0-9 visual scale)8 1.2 (0.1) 1.4 (0.1) 1Quantitative pod shattering (seeds per ft.2

)

9 64.7 (22.7) 132.5 (52.2) 13

Seed moisture (%)10 13.2 (0.5)* 11.7 (0.4) 7

Seed quality (%)11 3.3 (0.5) 2.8 (0.5) 0

Yield (bushels per acre)12 43.4 (2.2) 44.5 (2.2) 11

Final stand count (# plants per linear meter)13 18.3 (0.7) 18.1 (0.6) 12

Note: The experimental design was a randomized complete block with four replicates. S.E. = Standard Error. The number of plotanalysis 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. 

2 Early 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 contr4Days to first flowering was determined from the number of days after planting when 50% of the plants in a plot had one or more flo

for 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 

to 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 the conv7Plant height data plot number (n)=60 for MON 88302 and the conventional control. 
Monsanto Company 11-CA-233U 

8Visual pod shattering was estimated with a rated on a 1 - 9 scale, where 1 = 0 to 10% shatter, and each subsequent value on th

increments up to 9 = greater than 80% shatter. Plot number (n)=56 for MON 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

the 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 from a 100 seed subsample from each plot. 

MON 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 interaction 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 phenotypic and agronomic characteristics of 

MON 88302, data were also collected on plant response to abiotic stress (drought, wind, 

nutrient deficiency, etc.), disease damage, 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 potential and provide an indication of potential effects of 

MON 88302 on non-target organisms (NTOs) as well as threatened 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 showed that the glyphosatetolerance trait did not unexpectedly alter the assessed environmental interactions of 

MON 88302 compared to the conventional control. The lack of significant biologicallymeaningful differences in plant response to abiotic stress, disease damage, arthropod 

damage, and pest and beneficial arthropod abundance support the conclusion that the 

introduction of the glyphosate-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 observations of plant response to abiotic stress, 

disease damage, and arthropod-related damage were performed four times during the 

growing season at all 17 sites. At four of the 17 sites (MBMI, MNPY, NDBI and SKSA) 

quantitative assessments were conducted four times for arthropod abundance, twice for 

flea beetle damage and once for seedpod weevil damage during the growing season. 

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

observed between MON 88302 and the conventional control for 130 out of 131 

comparisons involving 9 assessed abiotic stressors (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 frost damage to MON 88302 was within 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 frost damage 

observations among the sites. Thus, the difference in frost damage rating during the 

single observation was not indicative of a consistent 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 diseases or for any of the 165 comparisons for 
Monsanto 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 quantitative 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 beneficial arthropod abundance, no statistically 

significant differences (α = 0.05) were detected between MON 88302 and the 

conventional control for any of the 51 comparisons, including 36 arthropod pest 

comparisons and 15 beneficial arthropod comparisons, 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-tolerance trait did not 

unexpectedly alter the assessed environmental interactions of MON 88302 compared to 

the conventional control. The lack of significant biological differences in plant responses 

to abiotic stress, disease damage, arthropod-related damage, and pest and beneficial 

arthropod abundance support the conclusion that the introduction of the glyphosatetolerance trait in MON 88302 is unlikely to result in increased plant pest potential or an 

adverse environmental impact as compared to conventional canola. 
Monsanto Company 11-CA-233U 123 of 500 

Table VII-5. Summary of Qualitative and Quantitative Environmental Interaction 

Assessments During 2009 

Assessments1 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 

stress2 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 88302 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 

Table VII-6. Combined-Site Analysis: Quantitative Assessment of Flea Beetle and Seedpod Weevil Dam

Compared to the Conventional Control in 2009 Field Trials1

 Mean (S.E) 

Arthropod Pest Damage assessment MON 88302 ControFlea beetles3(Chrysomelidae) 

Mean (S.E.) damage of 10 plants per plot (0-10 rating scale) – Observation 1 1.18 (0.24) 1.13 (0.2

Mean (S.E.) damage of 10 plants per plot (0-10 rating scale) – Observation 2 1.22 (0.19) 1.23 (0.2

Seedpod weevil4

(Ceutorhynchus obstrictus) 

Mean (S.E.) number of holes in pods from 10 plants per plot 0.03 (0.02) 0.05 (0.0

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. The number of plot

analysis 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 tw

crop emergence at all four sites. The second assessment was conducted approximately 1 week later at the MBMI, MNPY, and N

assessed quantitatively using a 0 - 10 rating scale where 0 = no damage and 10 = 100% of leaf area damaged. The first flea beetle asse

be conducted due to a severe rain storm (n = 12 plots). 

4Damage assessments for seedpod weevil were 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 impact weediness of the receiving species, 

the USDA-APHIS considers the potential for gene flow and introgression of the 

biotechnology derived trait into other canola varieties and wild relatives. An assessment 

of pollen morphology and viability provides information to characterize the plant and are 

also pertinent to assessments of gene flow and, therefore, were assessed for MON 88302. 

In addition, characterization of pollen 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 compared to the conventional control. 

The purpose of this evaluation was to assess 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 five replicates with 1 plant of each entry 

(MON 88302, conventional control and commercial reference varieties) per replicate. 

Once the plants started flowering a total of three newly opened flowers were collected 

from each plant. Pollen was extracted, combined among flowers collected from the same 

plot, and stained with Alexander’s stain (Alexander, 1980). Pollen viability was 

evaluated for each sample, and pollen grain diameter was measured for ten representative 

viable pollen grains per replicate. General morphology of the pollen was observed for 

each of the five replicates of MON 88302, the conventional control, and the four 

commercial reference varieties (see Appendix H). 

No statistically significant differences were detected (α = 0.05) between MON 88302 and 

the conventional control for percent viable pollen or pollen grain diameter (Table VII-7). 

Furthermore, no visual differences in general pollen morphology were observed between 

MON 88302 and the conventional control. These results demonstrate that the 

introduction of the glyphosate-tolerance trait 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 description of MON 88302 

compared to the conventional control. The result supports an overall conclusion that 

MON 88302 is comparable to conventional canola 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 Range2

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, Agronomic, and Environmental Interactions 

Evaluation 

An extensive and robust set of information and data were used to assess whether the 

introduction of the glyphosate-tolerance trait altered the plant pest potential of 

MON 88302 compared to the conventional control. Phenotypic, agronomic, and 

environmental interaction characteristics of MON 88302 were evaluated and compared to 

those of the conventional control and considered within the variation among commercial 

reference varieties. These assessments included five general data categories: 1) 

germination, dormancy, and emergence; 2) vegetative growth; 3) reproductive growth 

(including pollen characteristics); 4) seed retention on the plant and lodging; and 5) plant 

response to abiotic stress and interactions with diseases and arthropods. Results from the 

phenotypic, agronomic, and environmental interaction assessment demonstrate that 

MON 88302 does not possess: a) increased weediness characteristics; b) increased 

susceptibility or tolerance to specific abiotic 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. AGRONOMIC PRACTICES 

VIII.A. Introduction 

As part of the plant pest assessment required 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 cultivation of MON 88302. Discussions include canola 

production, canola seed production, plant growth and development, general management 

practices during the season, management of weeds, insects and diseases, canola rotational 

crops, and volunteer canola management. Information presented in Section VII.C.2 

demonstrated that MON 88302 is no more susceptible to diseases or pests than 

conventional canola. Additionally data presented in Section VII.C show that, with the 

exception of tolerance to the herbicide glyphosate, MON 88302 is phenotypically 

equivalent to conventional canola. Thus, there 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 are 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 preparation, good genetics, proper planting dates and 

density and good integrated pest management practices are important to optimizing the 

yield potential and economic 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 left 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 weed control, particularly 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-emergence 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 pests and diseases can also be considered 
Monsanto Company 11-CA-233U 128 of 500 

problematic in canola fields and can be managed 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 have 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 years (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. Researchers began investigating other market 

opportunities for oilseed rape. The high erucic acid content of the industrial oil was not 

desirable for human consumption. In addition, the meal derived from oilseed rape had 

relatively high concentrations of glucosinolates which reduced the animal feed value of 

the meal. In the 1960s, through intensive breeding programs, Canadian scientists made 

two important genetic modifications to oilseed rape which lead to the first double-low 

(low-erucic acid and low glucosinolate) variety (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 Rapeseed Association of Canada) chose the 

word “canola” (Canadian oil, low acid) to become 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. Food 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 glucosinolates 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 Alimentarius, 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 the 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 and is the most valuable component of canola 

seed. It is the world’s third largest source of vegetable oil with 15% of world 

vegetable oil consumption after soybean oil at 28% and palm oil at 32% (ASA, 2010; 

USDA-ERS, 2010). Canola oil appeals to health conscious consumers because it 
Monsanto Company 11-CA-233U 129 of 500 

contains a low level (7%) of saturated fatty acids which have been 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 appreciable amount (approximately 

10%) of alpha-linolenic acid relative to other oils (CCC, 2010) that are essential to 

human health and must be supplied in the diet. Canola oil has well established heart 

health benefits. The FDA has confirmed GRAS 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 canola 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 metric tons in 2008/2009 compared to 151.6 

million metric tons for soybean meal (USDA-ERS, 2010). 

The European Union, Canada, and China are the largest producers of oilseed rape with 

19.0, 12.64, and 12.10 million metric tons, respectively. The U.S. produced only about 

0.66 million metric tons (1.1%) of the world’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 recorded in 2009 and the 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 ranged 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 (USDA-NASS, 2011a), canola was planted on 

approximately 1.5M acres in the U.S. in 2010 producing 2.45 billion 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, soybean 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 types, 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 requirements 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 rotations 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 VIII-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 production 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 with 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 areas in recent years and canola acreage has 

declined accordingly. Oregon produced the 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 – 20101

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 

Planted1

Acres 

Harvested1

Average 

Yield1 

(pounds/acre) 

Total Production1 

(thousands pounds) 

Value2 

(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 States3 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 producing a canola crop. 

The key decisions on input costs include choosing what seed or canola varieties to plant, 

amounts of fertilizer to apply 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 Management Specialists (Table VIII-3). 

The total direct and overhead 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 the 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 – 

20091

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 acres
Monsanto 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. agriculture. 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 varieties that were distributed to 

growers for use. Seed was saved by growers and later sold to neighbors; however, the 

desirable traits of the varieties often were lost through random genetic changes and 

contamination with other crop and 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 identified as a major factor in crop yields. 

States in the U.S. developed seed laws and 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 official certifying agencies. 

Regulations first adopted in 1969 under the Federal Seed Act recognize land history, field 

isolation, and varietal purity standards for foundation, registered, and certified seed. 

Under international agreements such as the Organization for Economic Co-Operation and 

Development (OECD) scheme, the U.S. and 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 America, Australia, and 

New Zealand. 

Canola seed is broadly separated 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 organization 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 and identity. Certified seed 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 canola production is produced predominately to 

meet or exceed certified seed standards. This section of the petition 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 measures to assure the quality 

and genetic purity of seed varieties for commercial planting. The need for such systems 

arose from the recognition that the quality of improved seed varieties quickly 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. There are special land requirements, seed stock 

eligibility requirements, field inspections and seed labeling standards for seed 

certification. Seed certification services are available through various state agencies 

affiliated with the AOSCA. Large seed producers implement their own seed quality 

assurance programs. However, large seed producers often will utilize the services of 
Monsanto Company 11-CA-233U 135 of 500 

state certifying agencies as a third party source to perform certain field inspections and 

audits. 

The U.S. canola production for all purposes has varied from 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 acre, 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 required for distribution excess, seed returns, 

replants, and potential increases in canola acreage. Approximately 5,000 acres of 

commercial seed production would supply sufficient seed to plant the entire U.S. canola 

acreage with the various production and distribution allowances mentioned above 

(Bateman, Monsanto - Personal Communication, 3/17/11). 

Canola seed production involves a three stage process. The first stage or pre-foundation 

seed stage occurs in isolated tents for complete pollination control. Individual inbred 

plants are grown and tested to ensure the plants 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. The 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 produced in Southern Alberta, Canada and 

Northwestern U.S. These are preferred locations for seed production because of ideal 

heat units, accessibility to irrigation, and distance from commercial canola fields 

(Monsanto Company, 2009). To meet the demand for hybrid seed and to minimize 

production risks, most seed companies have off-season seed production locations in the 

Southwestern U.S. and Chile. 

Canola is a relatively easy crop to manipulate genetically and many new varieties with 

new quality and agronomic characteristics are available from universities and private 

seed companies (NDSU, 2005). Canola seed varieties are developed from three different 

breeding techniques, namely open-pollinated, synthetic hybrids, and hybrids (NDSU, 

2005). Over 95% of the acreage in the U.S. is planted with hybrid canola varieties 

(Bonnetta, Monsanto Company – Personal Communication, 8/22/2010). Hybrid canola 

varieties generally have higher yield potentials, but also have higher seed production 

costs. Hybrid canola varieties are produced from a cross between two genetically 

different inbred parent lines providing hybrid vigor (heterosis). 

Producing hybrid canola requires using a pollination control system to prevent unwanted 

self-pollination. Three primary pollination control methods used in North America: 1) 

the biotechnology derived male sterility system; 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 hybrid seed production (Mariani et al., 1992). The MSL-system (Male 

Sterility Lembke) is a private system owned 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 
Monsanto 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) and 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 (Buzza, 1995). The first step in the hybrid 

seed production process involves foundation seed crossing block where female parent 

Line A is pollinated by the genetically identical Line B to produce sufficient quantities of 

Line A seed. The second step involves pollination of Line A with restorer Line R to 

produce fully fertile F1 hybrid seed (Thomas, 2003b). 

Special planting methods are utilized in the production of hybrid canola seed involving 

planting alternating strips of male 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 International, Bayer CropScience, Croplan 

Genetics, and Dow AgroSciences. Seed companies in turn contract acreage with growers 

to produce the required amount of canola seed. Seed companies identify top canola 

growers to produce the seed and also monitor 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 seed as well as monitor and inspect all the 

processes at the plant. 

The entire seed production process for the majority of the seed companies and tollers is 

certified by the International Organization for Standardization (ISO) and includes internal 

and external audits (ISO, 2009). ISO standards ensure desirable characteristics of seeds 

and services, such as quality, safety, reliability, 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 requirements and applicable seed regulatory 

requirements, and must aim to enhance customer satisfaction. 

The field operations and management practices 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 germination rates, and high genetic purity. General 

guidelines specific for seed production are discussed below. 

A seed production field should not have been planted with canola the preceding five 

years in order to avoid volunteer 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 emergence 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 weed seed in the harvested canola seed. 

Roguing should be performed on all seed production fields to remove volunteer canola 

and off-type plants prior to or during flowering. Fields should be scouted frequently for 
Monsanto Company 11-CA-233U 137 of 500 

insect pests and insecticides should be 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 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 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, swathing should 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 begin when the seed moisture drops below 

10% and no green pods are visible (Boyles et al., 2009). Harvesting 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 because they can increase seed damage. 

Field inspections are vital to ensure canola seed meets seed certification requirements, 

ISO certification standards, regulatory standards, and trait licensing agreement standards. 

Field inspections are conducted on seed production fields throughout the growing season 

to evaluate variety purity, ensure canola plants are developing properly, 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 screening, cleaning, and bagging process. Specific methods are 

used to assure the genetic purity and identity 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 quantified 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 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 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%. State 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 crops 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. There is a second type of winter canola that is 

planted in the fall and overwinters, but does not require vernalization 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 vernalization 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 agronomic practices and 

can be affected by different insect pests and diseases. 

Crop rotation, tillage system, seed or variety selection(s), and soil fertility are 

management issues which require production decisions well in advance of planting the 

canola crop. Many of the decisions in this area are made immediately after harvest of the 

previous crop or sooner, particularly with 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 rotation is generally a long term decision, but the 

rotation sequence can be modified to take advantage of a particular economic or market 

opportunity. Canola is commonly grown following small grain cereal crops (winter or 

spring wheat, barley, maize or sorghum) (Brown et al., 2008). Growers are advised not 

to plant canola following canola to avoid a build-up of soil-borne diseases like Sclerotinia 

stem rot, blackleg, or club root. Ideally, canola 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 et al., 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 planting canola in any field to avoid severe crop injury and/or yield 

loss due to carryover herbicide residues. For example, bromoxynil has only a 30-day 

plant back restriction for canola, but imazethapyr has a restriction of 40 months for most 

varieties. In addition to the plant back restriction, 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 legume crops in the Pacific Northwest and crop 

injury to canola has been reported six years after an imazethapyr application (Brown et 

al., 2008). 
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Canola can be grown with conventional tillage, conservation tillage, 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 systems include reduced soil erosion, reduced 

fuel and labor costs, soil moisture conservation, and improved soil structure and organic 

matter content (Brown et al., 2008). 

Primary and secondary pre-plant tillage helps remove crop residue, control weeds and 

volunteer plants from the previous crop and is an important consideration prior to 

adopting no-till or direct-seeding systems for canola. Regardless of the tillage system 

utilized by the grower, the tillage system must facilitate good stand establishment and 

emergence. Good seed-to-soil contact is essential for good seed germination and uniform 

emergence that are critical to obtaining maximum 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 for erosion control and 

moisture conservation. However, no-till planting or direct seeding 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 tillage systems (Brown et al., 

2008). Winter canola is not recommended when planting into excessive amounts of fresh 

straw, or when soil temperatures are lower than average in the fall months, because it can 

result in poor emergence. Seed yields with direct-seeded winter canola have been 

significantly lower than with conventional tillage systems (Brown et al., 2008). Spring 

canola is better suited to conservation tillage due to better soil moisture conservation and 

availability. Spring canola has fewer problems dealing with heavy straw residue and, 

residue from the previous crop has usually decomposed to some degree during the winter 

months prior to planting spring canola. In addition, cooler soil temperature can be 

advantageous to spring canola. 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). The 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 canola. Nitrogen, phosphorus, potassium, sulfur, and boron, 

depending on the region, are the most limiting nutrients for successful canola production 

(Brown et al., 2008). Soil sampling and testing is the first step in assessing soil nutrient 

levels and the requirements for supplemental fertilizers. The availability of soil nutrients 

is dependent on soil acidity or the pH level and must be included in this assessment. The 
Monsanto 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 nutrients 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 previous crop credit for nitrogen (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 medium-textured soils (Franzen and Lukach, 

2007). 

Canola has a moderate requirement for phosphorus 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 (Brown et al., 2008). A 

starter fertilizer rate of 20-30 lbs. of P2O5 per acre is generally sufficient for most soil test 

levels unless the grower intends to build up phosphorus levels in the soil (Franzen and 

Lukach, 2007). Potassium requirements are also based on soil tests. Many soils contain 

sufficient levels of potassium requiring no potassium 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 (Brown et al., 2008). Sulfur deficiencies result in yellowing 

between leaf veins, cupped leaves and stunting (Franzen and Lukach, 2007). In addition, 

in the presence of sulfur deficiency flowering 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 applications (Franzen and Lukach, 2007). Since 

soil tests tend to overestimate available sulfate 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 show less than 0.5 ppm boron (Brown et al., 

2008). Canola has not shown yield responses to applications of micronutrients such as 

chlorine, copper, iron, manganese, molybdenum 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 growing a successful crop (NDSU, 2005). The 
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performance of a given variety may differ from year to year and location to location due 

to changing environmental conditions. Therefore, 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 several factors to consider in choosing canola 

varieties, namely: yield, maturity, plant height and lodging, disease tolerance, and 

seedling vigor (NDSU, 2005). 

Planting and Early Development

An understanding of the growth stages of canola is important for the proper timing of 

certain management practices such as herbicide and insecticide applications. In addition, 

the impact of certain weather conditions and diseases on canola yield 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, Stage 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 process 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 economically beneficial for protecting seedlings 

from seed rot, damping off, seedling blight, and early season root rot caused by Pythium,

Rhizoctonia, and Fusarium. The fungicide seed treatments are also effective protection 

against seed-borne blackleg and 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 treatments registered for canola contain multiple 

fungicides for broad spectrum disease protection plus an insecticide for control of flea 

beetles. For example, Prosper 400 contains the fungicides thiram, carboxin, and 

metalaxyl plus the insecticide clothianidin. 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. The 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 near 
Monsanto Company 11-CA-233U 142 of 500 

freezing temperatures will undergo a gradual hardening process that 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 dependent 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 crop can result in lower soil temperature and 

higher soil moisture which could delay planting (Brown et al., 2008). However, water 

infiltration improves after utilizing no-tillage or direct-seeded systems for several years. 

Frequently, that allows growers to enter fields sooner, plant earlier and establish the crop 

earlier. For optimum seed germination, seeding 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 height, maturity, and final yield 

(Brown et al., 2008). Spring canola should be planted in April to early May in North 

Dakota to maximize yields (NDSU, 2005). Spring 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 susceptible to heat and drought stress. 

Since winter canola is planted in the fall, planting dates should be selected to provide 

time for plants to develop sufficient growth before the onset of winter to minimize winter 

damage (Brown et al., 2008). Generally, winter canola should be planted six weeks 

before the first killing frost in most states (Boyles et al., 2009; Brown 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 winter. 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 makes them more susceptible to winter kill. 

On the other hand, planting too late will result in seedlings that are too small that have 

insufficient reserves to survive winter conditions 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 planting can be in early August to the second week of 

September (Brown et al., 2008). Recommended planting dates for winter canola in the 

Great Plains vary according to latitude. For example, optimum planting dates for Kansas 

and Oklahoma are August 26 through September 25 and August 20 to September 21 

respectively (Brown et al., 2008). 

Canola is adapted to a wide range of plant population densities. The optimum seeding 

rate for spring canola is 600,000 pure live seeds (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 square foot. Similar seeding rates are recommending in winter 
Monsanto Company 11-CA-233U 143 of 500 

canola. The seeding rate for winter canola should result in 10 to 16 established seedlings 

per square foot (Brown et al., 2008). Significant yield differences do not occur unless 

populations at harvest are less than one or greater than 15 plants per square foot (Boyles 

et al., 2009). Low plant populations of canola will not compete as effectively with 

weeds, and can produce lower yields although canola plants in low planting densities 

have the ability to compensate (Brown et al., 2008). Individual 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 rates to compensate for poor 

seedling emergence. Direct-seeding or no-tilling 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 conditions (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 being planted. Canola seeds 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). Generally, 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 seed germination percentage which is vital 

information in determining the actual seeding rate. Seeding rates will generally be 4 to 8 

pounds per acre (NDSU, 2005). 

Most commercial grain drills used 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 shattering before harvest (Boyles et al., 

2009). Canola should be seeded to a depth that allows the seed to be covered and must 

be planted into moist soil. The best germination 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 crusted 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 weeds and becomes more 

competitive as it approaches the late rosette and bolting stage (NDSU, 2005). Planting 

canola into a weed-free seedbed is essential and can be achieved with tillage, herbicides, 

or a combination of both methods. Seedbed 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 progresses. Effective weed management in 

canola relies heavily on selective post-emergent herbicides. The list of registered 

herbicides in the U.S. is relatively small compared to other major crops. Sethoxydim and 

clethodim are post-emergent in-crop herbicides for control of annual grasses while 

quizalofop will control annual grass and quackgrass (perennial). Clopyralid will control 

several broadleaf species in canola. Herbicide-tolerant canola varieties are also available 
Monsanto Company 11-CA-233U 144 of 500 

which allow the use of broadspectrum post-emergent herbicides such as glyphosate, 

glufosinate, and imazamox. Early canopy closure 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 per day and increasing yield potential. Bud 

formation is triggered as the days lengthen and temperatures rise. The plant reaches its 

maximum leaf area index in the late bud stage. The vegetative stages (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 temperatures together with moisture stress 

during this stage can severely reduce yield potential. 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 flowers, 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 winter canola is impacted by the same insect 

pests, but the degree of damage may be different. 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 insecticides. 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, Sclerotinia stem rot and Alternaria black spot are 

seed-borne diseases which can be effectively 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 applications for control these diseases are not 

economical under most situations, although foliar applications of boscalid and 

azoxystrobin fungicides are effective on Sclerotinia stem rot and Alternaria black spot 

respectively as needed. 

Harvest 

Spring canola is potentially at risk for frost damage in the fall. It is important to have 

stands of canola that ripen early and uniformly since the amount of fall frost damage 
Monsanto 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% moisture while seeds at 35% moisture will 

escape damage (NDSU, 2005). Frost fixes the 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 plant reach physiological maturity and complete filling at 

about 40% moisture. They then 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 change in three to five days or less. Seeds 

lose moisture at the rate of about 2% to 3% or more each day depending 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, and hail can result in yield losses of greater than 50% when 

the crop is ripe and ready to harvest (Boyles et al., 2009). Therefore, if possible, canola 

should be harvested immediately upon ripening 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 with a short growing season (Boyles et al., 

2009). The direct combining method does not require additional equipment (swather) for 

growers. There has been considerable research 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 Dakota (NDSU, 2007). However, canola is more 

vulnerable to seed and shatter loss when direct 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 beneficial for spring canola if a hard frost is 

expected. It is important to harvest at the optimum stage of ripening using the swathing 

and combining method to reduce green seed problems and seed shatter losses (NDSU, 

2007). The quantity of green seed in the harvested crop affects quality 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 (Brown et al., 2008). 

Swathing can hasten the loss of green seed by about 2 days compared to seeds in standing 

crops (Brown et al., 2008). Direct combining 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 and harvest period (Boyles et al., 2009). 

However, both methods have been used successfully for harvesting canola in this region. 

Swathing is generally recommended if harvest cannot be completed in a timely manner 

with direct combining. 

Pushing, a new procedure being considered for harvesting canola, is reportedly a faster 

and less expensive alternative to swathing (Boyles 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 the 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 immature 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 potential for pod shatter, growers 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 averages 30% to 35%, about 30% 

to 40% of the seeds in pods on the main stem 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 particularly when humidity is low (Brown et 

al., 2008). These weather conditions can result in higher shatter 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 canola (80 % seed color change) results in fluffy cut rows of 

canola that are more susceptible to blowing and increased shattering. Seed moisture 

content and green seed counts should 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 cool, dry conditions. The optimum storage conditions for 

canola are 55°F and 7% seed moisture and the storage life of canola 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 levels that cause yield reductions and often a 

corresponding reduction in the oil content of canola. Insect pests can 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 especially vulnerable to 

chewing insects. A severe infestation of flea beetles can completely destroy a stand of 

canola seedlings. Late season insect pests can typically cause yield 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. However, some pests negatively impact one variety 
Monsanto Company 11-CA-233U 147 of 500 

more than the other. Crop rotation, volunteer and wild mustard control, and managing 

crop residues are all important cultural practices for insect control. 

The insect pests of spring and winter canola varieties in the U.S. are listed in Table VIII4. Flea beetle is a serious insect pest in spring 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 injury 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 advisable in spring canola to protect the seedlings 

(NDSU, 2005; 2007). Seed treatments usually provide 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 when 25% defoliation occurs on the cotyledons and true leaves 

(NDSU, 2005). Minimum or no-tillage systems generally have lower infestations of flea 

beetles (Weiss et al., 2009). In addition, planting early can allow establishment of the 

canola prior to beetle emergence. 

Insect pests causing serious damage to winter canola include flea beetles, 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 seedling 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 controlling aphid populations. However, these insects 

alone will not prevent aphid populations from reaching damaging levels. An insecticide 

seed treatment (clothianidin, 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 subsequent 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 Minnesota (Weiss et al., 2009). Plant 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 seeds (Weiss et al., 

2009). A foliar insecticide (bifenthrin or lambda-cyhalothrin) is recommended when 

three to four weevils are collected in a sampling sweep during 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 configurata 

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 levels 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 used 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 application of insecticides. Most insecticides are non-selective and may 

damage non-pest insects. Bee keepers frequently set their hives close to flowering winter 
Monsanto Company 11-CA-233U 149 of 500 

canola fields because canola is an early and very rich source of pollen for bees (Brown et 

al., 2008). Therefore, growers need to exercise special care to protect 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 soilborne, seedborne, or airborne, 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 introduced by planting infected 

seeds (Brown et al., 2008). Once introduced into an area, it will remain as long as canola 

is grown there. The virulent or aggressive strains can infect 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 and 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 disease (NDSU, 2005). Several management 

practices are available to control or minimize this disease (NDSU, 2005). The most 

important practice is planting disease-free, certified seed where blackleg has not been 

introduced. Blackleg has yet to be introduced in the Pacific Northwest region and 

growers are advised to plant locally produced seed to prevent the introduction of the 

disease (Brown et al., 2008). A fungicide seed treatment should be used such as Helix 

Liteor Helix XTra which contains fungicides and an insecticide for flea beetle control. 

Growers should plant blackleg resistant 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 residue and prevent the release of blackleg 

ascospores to nearby fields. Foliar applications 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 canola in North Dakota and Minnesota (NDSU, 2005). This 

disease has caused estimated losses up to 13% in North Dakota and Minnesota and yield 

reductions approaching 50% have been reported on seriously infected fields (NDSU, 

2005). Canola primarily is susceptible during all bloom stages and shortly thereafter 

(NDSU, 2007). Wet weather immediately preceding and at flowering favors 

development of the Sclerotinia fungus. The first step in minimizing this disease is to 

plant canola in a four-year rotation with non-susceptible crops (Brown et al., 2008). The 

Sclerotinia fungus may survive four to six years in the soil (NDSU, 2005). Field peas and 
Monsanto Company 11-CA-233U 150 of 500 

flax are much less susceptible to the Sclerotinia 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 soybeans are some of the most susceptible host 

crops and should be avoided in the rotation. Many broadleaf weeds, including 

lambsquarters, Canada thistle, ragweed and marshelder are also susceptible host plants. 

All canola varieties are susceptible to Sclerotinia fungus, but some varieties are less 

susceptible than others and will perform better in moderate to severe disease pressure 

(NDSU, 2005). Foliar applications of a fungicide (bosclid or azoxystrobin) provide 

effective suppression of Sclerotinia stem rot. Since fungicides are expensive, 

applications should be limited to fields where: 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 expected, and 4) Sclerotinia has been a problem in 

recent years (NDSU, 2005). Foliar 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 yields 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 weeds that emerge before or 

with the crop such as wild oats result in greater yield loss than weeds that emerge after 

the crop (CCC, 2006). Canola in the seedling stage is a poor competitor with weeds 

(NDSU, 2005). However, once established and 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 are very competitive with canola early in the 

growing season. Canada thistle is three or four times more competitive with canola than 

wild oats (CCC, 2006). Ten Canada thistle plants per square meter resulted in 10% yield 

loss while forty plants per square meter resulted in over 50% yield loss (CCC, 2006). A 

wild oat density of thirty plants per square meter caused between 15 and 20% yield loss 

(CCC, 2006). However, an average infestation 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 which 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 emergence). 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 the 
Monsanto Company 11-CA-233U 151 of 500 

dominate method of herbicide application. The goal in timing post-emergent applications 

is to delay application for as long as practical to capture the most weed flushes without 

incurring yield loss. Frequently the crop canopy 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 determined 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 different locations with three different herbicide-tolerant 

canola systems generally confirmed these findings (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 when removing weeds as early 

as the one- to two-leaf stage of canola in certain conditions. Although late-emerging 

weeds are not always competitive with the crop, controlling these weeds may be 

beneficial to reduce contaminants such as chaff, stems, weed seeds etc. that could lower 

the grade of the canola seed and prevent weeds from producing seed and infesting 

subsequent crops (Harker 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 and 7. Common weeds are those species which 

can be found abundantly infesting a significant 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 Dakota1

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 Plains1

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 Spring and Winter Canola in the Pacific 

Northwest1

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 tillage can effectively reduce 

the competitive ability of weeds by burying the plants, disturbing or weakening the root 
Monsanto 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 limited compared to the number available for 

use in other crops such as maize, soybean, and small grain cereals. Sethoxydim, 

clethodim, quizalofop and imazamox are post-emergence in-crop herbicides for control 

of annual grasses and broadleaf weeds in conventional canola varieties (NDSU, 2005). 

In addition, along with pre-plant incorporated herbicides ethalfluralin and trifluralin, 

glyphosate and glufosinate herbicides can be applied in conventional 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 20081

 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 control. The herbicide-tolerance traits enable 

the use of certain herbicides in canola that previously would not provide 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, glufosinate, and imazamox, respectively. In 2008 

herbicide-tolerant canola varieties were planted on approximately 97% of the canola 

acreage in North Dakota (Zollinger et al., 2009). Glyphosate-tolerant canola was 

commercially introduced in 1999. Roundup Ready canola is tolerant to post-emergence 

applications of glyphosate. Glyphosate was applied 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 canola was introduced in 1995 and is tolerant to 

post-emergence applications of glufosinate. Glufosinate is the second most commonly 

applied herbicide used in canola (38% of acres) (Table VIII-8). Clearfield varieties are 

tolerant to imazamox herbicide. Roundup Ready and Liberty Link are biotechnology 

derived traits while Clearfield was developed through traditional breeding techniques. 

Tables VIII-9 and 10 provide a summary of the 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 and 
Monsanto 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 the annual and biennial broadleaf weeds with 

the exception of Canada thistle, buckwheat, mallow, ragweed and wormwood. The 

ratings for glyphosate are more variable on broadleaf weeds ranging from excellent to 

poor-excellent for all annual and biennial weeds and good to excellent 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 herbicide-tolerant canola varieties include 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, respectively (Zollinger et al., 2009). The acreage planted with 

Clearfield varieties is only 1.9%. The superior weed control ratings presented for 

glyphosate and glufosinate in Tables VIII-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 control 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 important 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 indicate glyphosate agricultural herbicides can be applied postemergence on glyphosate-tolerant canola from emergence to the 6-leaf stage of 

development in spring canola varieties. To prevent early weed competition and 

maximize yield potential, the first application of glyphosate to glyphosate-tolerant canola 

should be made at the two- to three-leaf stage. 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 acreage (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 second application is in-crop to control weeds 

emerging later. 

.
Monsanto Company 11-CA-233U 

Table VIII-9. Grass Weed Species Responses and Persistence in Soil to Herbicides Applied in Canola Pro

Product 

Barnyardgrass 

Field Sandbur 

Foxtail, Green 

Foxtail, Yellow 

Volunteer Cereals 

Wild Oat 

Wild Proso Millet 

Quackgrass 

Herbicide 

Persistence 

Preplant Incorporated Ethalfluralin E G E E G F P-F N S Trifluralin E G E E N P-F P-F N S PostemergenceClopyralid N N N N N N N N S Imaxamox E F-G E G-E G-E E G-E F N Glyphosate E E E E E G-E E E N Glufosinate E G E G F-G G-E E P N Quizalofop E E E G-E E G-E E G-E N Sethoxydim E E E E E G-E E F N Clethodim E E E E E E E G N 1Source: (NDSU, 2005). Weed control ratings based on the following scale: E = Excellent = 90 to 99% control, G = Good = 80 to 90% 

80% control, P = Poor = 40 to 65 % control, and N = None = no control. Herbicide persistence ratings are for residues present 12 moOften, S = Seldom, and N = None. 
Monsanto Company 11-CA-233U 

Table VIII-10. Broadleaf Weed Species Responses to Herbicides Applied in Canola Production1

Product 

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 

Preplant Incorporated OnlyEthalfluralin P-F P P F-G E N - F-G N N P P E P P P Trifluralin P-F N P F G-E N - F-G N N N N E N P P PostemergenceClopyralid F-G E N N P-F F P P E N E E P E G-E G-E GImaxamox P G-E E E F E P P G-E E E E E - P G-E Glyphosate F-G E G-E F-E P-E E F-G E G-E G-E F-G F-G E E E F-E GGlufosinate E E G-E E F-G E E E E E E E E G-E E E Quizalofop N N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

Sethoxydim N N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

Clethodim N N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

1Source: (NDSU, 2005). Weed control ratings based on the following scale: E = Excellent = 90 to 99% control, G = Good = 80 to 90% 

80% control, P = Poor = 40 to 65 % control, and N = None = no control. ‘-‘ = No data available 
Monsanto Company 11-CA-233U 157 of 500 

VIII.G. Second Generation Glyphosate-tolerant Canola – MON 88302 

Monsanto Company has developed a second-generation glyphosate-tolerant canola 

product, MON 88302, designed to provide growers with improved weed control through 

greater flexibility of 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 CP4 EPSPS protein confers tolerance to the 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-generation product, Roundup Ready canola (RT73). 

Glyphosate is a systemic herbicide and is translocated 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 for 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 rates than is possible with the 

current product. 

It is anticipated that canola production practices with MON 88302 will remain the same 

as they are for the first-generation Roundup Ready canola system. This includes the use 

of rotational crops, tillage systems, planting and harvesting operations and equipment. In 

addition to having similar agronomic practices, 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 facilitated 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 after the 4-leaf stage can result 

in crop injury. As an alternative, two sequential 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 application of glyphosate cannot exceed 0.78 lbs. a.e. per acre. 

MON 88302 provides greater crop tolerance which 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 glyphosate label is amended by the EPA, 

MON 88302 will permit two in-crop sequential glyphosate applications up to 0.77 lbs. 

a.e. per acre each in spring canola or one application prior to the 6-leaf growth stage up to 

1.55 lbs of glyphosate a.e. per acre. 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 application 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. Recommended application rates for glyphosate in 

winter canola will likely remain unchanged 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 greater flexibility to 

utilize the appropriate glyphosate rate for difficult to control weed species when making 

sequential application of glyphosate 

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 particularly 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; 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 practice of crop rotation is a key management tool for 

growers. One of the main purposes of growing canola in rotation 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 resulted in 10% to 20% yield increases in wheat 

compared to continuous wheat (Brown et al., 2008). Incorporating canola in small grain 

rotations has also decreased disease incidence, increased quality of cereal crops and 

provides better herbicide options for controlling certain weeds such as downy brome, 

jointed goat grass, wild oat, Italian ryegrass and 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 still must make cropping decisions based on both the agronomic 

and economic returns of various cropping systems. 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 buildup of soilborne 

diseases like Sclerotinia stem rot, blackleg 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 after other crops like legumes (pea, lentil 

chickpea, soybean, field and dry been) or sunflower that are susceptible to Sclerotinia, 
Monsanto Company 11-CA-233U 159 of 500 

Rhizoctonia and Fusarium root rots. Canola is most commonly grown following small 

grain cereals such as winter or spring wheat, barley, maize 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 description and quantification of the rotational 

cropping practices immediately following canola, by state, that account for 99+% of the 

total canola acreage. These data are 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 - Personal 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 added 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 with wheat depending on economic 

conditions. A typical 3-year rotation sequence 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) (Peeper, Oklahoma State University – Personal 

Communication, 8/3/2010). Soybean or sorghum 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 sequences 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 dry bean, field bean, flax, 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., (Table 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 crop in the U.S. Roundup Ready soybean is 

the only Roundup Ready crop rotated with canola and it has been deregulated by the 

USDA. For the purposes of this assessment, the adoption rates used for Roundup Ready 
Monsanto Company 11-CA-233U 160 of 500 

soybean in 2010 were obtained from the USDA-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 

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

 A B C D E F G H I 

State 

Total 

Canola 

Acres1

Major Crops 

That Follow 

Canola In 

Rotation 

Total Acreage 

of Rotation 

Crop in States1

Percent of 

Rotational 

Crop Rotated 

From Canola2

Rotational 

Crop Acres 

Following 

Canola3

Percent 

Rotational 

Crop of 

Total 

Canola4

Percent 

Roundup 

Ready 

Rotational 

Crop 

Option5

Acreage of 

Roundup 

Ready 

Rotational 

Crop 

Option6

United 

States 

811 Barley 

Dry Bean 

Field Pea 

Flax 

Oats 

Sorghum 

Soybean 

Sunflower 

Wheat 

3567 

654 863 317 

3404 6633 

77451 

2030 

59133

Total: 154,369 

3.2 1.1 

0.02 

2.2 0.2 

0.02 0.01 

0.3 1.1 

113 

7 

0.2 

7 7 1 8 7 

659 

Total: 809 

13.9 

0.9 

0.02 

0.9 0.9 0.1 1.0 0.9 

81.3 

NA NA NA NA NA NA 

93% 

NA NA 

7.4 

Total: 7.4 

ID 15 Wheat 1,310 

Total: 1,310

1.1 15 

Total: 15

100 NA 0

Total: 0 1

MN 13 Barley 

Dry Bean 

Flax 

Oats 

Soybean 

Sunflower 

Wheat 

95 68 

3 

250 

7200 

45 

1655 

Total: 9,316

2.1 0.2 3.3 

0.04 

0.001 

0.2 0.6 

2 

0.1 0.1 0.1 0.1 0.1 

10

Total: 13

15 

1 1 1 1 1 

80 

NA NA NA NA 

93% 

NA NA 

0.1

Total: 0.1 

MT 6.5 Barley 

Field Pea 

Wheat 

870 240 

5520 

Total: 6,630

0.1 

0.04 

0.1 

1 

0.1 

5 

Total: 6.5

15 

2 

83 

NA NA NA 

Total: 0 
Monsanto Company 11-CA-233U 

Table VIII-11. Rotational Practices in the U.S. Following Canola Production (continued) 

A B C D E F G H I 

State 

Total 

Canola 

Acres1

Major Crops 

That Follow 

Canola In 

Rotation 

Total 

Acreage of 

Rotation 

Crop in 

States1

Percent of 

Rotational 

Crop Rotated 

From 

Canola2

Rotational 

Crop Acres 

Following 

Canola3

Percent 

Rotational 

Crop of 

Total 

Canola4

Percent 

Roundup 

Ready 

Rotational 

Crop 

Option5

Acreage of 

Roundup 

Ready 

Rotational 

Crop 

Option6

P

P

R

ND 730 Barley 

Dry Bean 

Flax 

Oats 

Soybean 

Sunflower 

Wheat 

1210 

533 295 350 

3900 

770 

8680 

Total: 15,738

8.3 1.3 2.4 

2 

0.2 0.9 6.7 

110 

7 7 7 7 7 

584 

Total: 730

15 

1 1 1 1 1 

80 

NA NA NA NA 

94% 

NA NA 

6.6 

Total: 6.6 

OK 42 Sorghum 

Soybean 

Wheat 

250 405 

5700 

Total: 

6355

0.4 0.3 0.7 

1 1 

40 

Total: 42

2.5 2.5 

95 

NA 

90% 

NA 

0.9 

Total: 0.9 

OR 4.9 Wheat 890 

Total: 890

0.6 4.9 

Total: 4.9

100 NA 0 

Total: 0 

The United States summary was developed by compiling the data from all states. All acreages are expressed as 1000s of acres. Unlike the indColumn G for the US were obtained by dividing Column F by Column B. NA denotes not applicable. 1 Acreage planted of the specific crops is based on 2009 planting data from the USDA-NASS, Crop Production - 2009 Summary, January 2010 (2010b2 Column E obtained by dividing Column F by Column D and multiplying by 100. 3 Column F obtained by multiplying Column B by Column G. 4 Rotational crop percentages based on estimates from personal communications with individual state canola Extension Crop Production Specialists 

University, 8/2/2010; Peeper, Oklahoma State University, 8/3/2010; Davis, University of Idaho, 7/29/2010; Wysocki, Oregon State University, 7/29/2

5 Roundup Ready rotational crop adoption rates for soybean are based on 2010 planting data taken from USDA-NASS, Acreage Report, June 2010 (206 Column I obtained by multiplying Column F by Column H except as noted above for U.S. 7 Column J obtained by dividing Column B by Column D Total and multiplying by 100. 8 Column 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 subsequent rotational crop. Canola seeds can remain in a field 

after canola harvest as a result of pods shattering 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 (NDSU, 2005). Although 

canola can volunteer for several years following a canola 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 after summer rains and can be 

controlled by tillage. A Canadian study indicates that the incidence of fields with canola 

volunteers in the subsequent rotational 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 volunteers with tillage prior to planting. With 

no-till small grains, growers should add a labeled herbicide (2,4-D) to the glyphosate preemergence application for control of glyphosate-resistant canola 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 volunteer glyphosate-tolerant canola in the various rotational 

crops, and their effectiveness. 

No changes are anticipated from the introduction of MON 88302 on current volunteer 

management practices in the cultivation 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 Crops1

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 

Finesse2

 0.3 oz G-E 

Amber2

 0.56 oz G-E 

Agility2 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. Monsanto considers product stewardship to be a 

fundamental component of customer service 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 resistance. Detailed information regarding 

herbicide resistance is presented in Appendix J. 

VIII.K. Stewardship of MON 88302 

Monsanto Company develops effective products and technologies and is committed to 

assuring that its products and technologies are safe and environmentally responsible. 

Monsanto demonstrates this commitment by implementing product stewardship processes 

throughout the lifecycle of a product and by participation in the Excellence Through 

StewardshipSM (ETS) Program. These policies and practices include rigorous field 

compliance and quality management systems and verification through auditing. 

Monsanto’s Stewardship Principles are also articulated in Technology Use Guides that 

are distributed annually to growers who utilize Monsanto branded traits. 

As an integral action of fulfilling this stewardship commitment, Monsanto will seek 

biotechnology regulatory approvals for MON 88302 in all key canola import countries 

with a functioning regulatory system 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 from the U.S. for the development of formal 

biotechnology approval processes. If new functioning regulatory processes are 

developed, Monsanto will make appropriate and timely regulatory submissions. 

Monsanto is committed to utilizing the best industry practices on seed quality assurance 

and control to ensure the purity and integrity of MON 88302 seed. As with all of 

Monsanto’s products, before commercializing MON 88302 in any country, a 

MON 88302 detection method will be made available to canola producers, processors 

and buyers. 

Glyphosate is a non-selective herbicide registered with the U.S. EPA for the postemergence control of annual and perennial grasses and broadleaf weeds in agriculture. 

Glyphosate has a long history of effective use 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 higher application rates and later timing for applications. 

Although the amended labeling increases the rate of application and widens the 

application period relative to canola development, this use of glyphosate does not present 

any new environmental exposure scenarios. Glyphosate residue levels for this product 
Monsanto Company 11-CA-233U 166 of 500 

fall within the existing U.S. and Codex Maximum Residue Level of 20 ppm (Codex, 

2011). 

As with all U.S. EPA registered herbicides for 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 drift. Potential impacts from drift have been 

mitigated through specific label use restrictions for glyphosate when applied with aerial 

equipment. Good management practices for application of all pesticides are also 

promoted by state university extension services (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 agricultural areas that are dedicated to crop 

production. MON 88302 is likely to be used in common rotations on land previously 

used for agricultural purposes. Certified seed production will continue to use wellestablished industry practices to deliver high quality seed containing MON 88302 to 

growers. Cultivation of MON 88302 is not expected to differ from current canola 

cultivation using first-generation glyphosate-tolerant 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 authorized for the first-generation product. 

MON 88302 is similar to conventional canola in its agronomic, phenotypic, 

environmental, and compositional characteristics 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 and management practices for canola 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 practices or 

weed management practices, other than intended weed 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 agronomic practices as well as the environmental impact 

of the introduced CP4 EPSPS protein. USDA-APHIS has responsibility, 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 does not present a plant pest risk, the petition 

is granted, thereby allowing unrestricted introduction of the article. 

According to the PPA, the definition of “plant pest” includes living organisms that can 

directly or indirectly injure, damage, or cause disease in any plant or plant product (7 

U.S.C. § 7702[14]). 

The regulatory endpoint under the PPA for biotechnology-derived crop products is not 

zero risk, but rather a determination that deregulation 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 action and changes to plant metabolism; 2) 

composition; 3) expression and characteristics 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 practices; 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 expressed in MON 88302; 3) safety and mode 

of action of the CP4 EPSPS protein; 4) compositional equivalence of MON 88302 seed to 

conventional canola; 5) phenotypic and agronomic characteristics demonstrating no 

increased plant pest potential including disease and pest susceptibilities; 6) negligible risk 

to non target organisms (NTOs) and threatened or endangered species (TES); 7) 

familiarity with canola as a cultivated crop; and 8) no greater likelihood to significantly 

impact agronomic practices, including land use, cultivation practices, or the management 

of weeds, diseases, and insects compared to commercially grown canola. 

Using the assessment above, the data and analysis 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 engineered plants (73 FR 600008). 

Because the data presented in this petition 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 “noxious 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 Genetic 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 Left 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 and is derived from CTP2 target sequence of the Arabidopsis thaliana

shkG gene (Figure III-1; Table III-1). The characterization 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 elements, including backbone sequences, from 

PV-BNHT2672 were detected in MON 88302 (Figures 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 conventional 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 adjacent 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 during plant transformation; these changes 

presumably resulted from double-stranded break repair mechanisms in the plant during 

the Agrobacterium-mediated transformation process (Salomon and Puchta, 1998). 

Finally, Southern blot analysis demonstrated that the DNA fingerprint of the T-DNA 

insert in MON 88302 has been maintained through multiple generations of breeding, 

thereby confirming the stability of the 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 Ready canola, which confers tolerance to 

glyphosate. In most plants, glyphosate binds to the endogenous EPSPS enzyme and 

blocks the biosynthesis of EPSP thereby depriving the plant of essential amino acids 
Monsanto 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 glyphosate (Padgette et al., 1996). 

IX.B.1.3. Expressed Protein Safety 

Several Roundup Ready crops that produce the CP4 EPSPS protein have been 

deregulated by the USDA and been reviewed during the biotechnology consultation 

process by the FDA. The CP4 EPSPS protein expressed in MON 88302 is identical to 

the CP4 EPSPS proteins in other Roundup Ready crops including Roundup Ready 

soybean, Roundup Ready 2 Yield soybean, 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 extensively assessed (Harrison et al., 1996). 

Results from the protein characterization 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 previously to demonstrate the safety of the CP4 EPSPS protein 

produced in other Roundup Ready crops. The Environmental Protection Agency (EPA) 

also reviewed the safety of the CP4 EPSPS protein and has established a tolerance 

exemption for the protein and the genetic material 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 included rapid digestion in simulated 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 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 comparable 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 conventional control and to the composition of 

commercial reference varieties (Section VI). The background genetics of the 

conventional control (Ebony) were similar to that of MON 88302, but did not contain the 

cp4 epsps expression cassette. Seed was harvested 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 trial. The commercial reference varieties were 

used to calculate a 99% tolerance interval for each compositional component to define 
Monsanto 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 included proximates (ash, carbohydrates by 

calculation, moisture, protein, and total fat), fibers (acid detergent fiber [ADF], neutral 

detergent fiber [NDF], and total dietary fiber [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 acid and glucosinolates (alkyl glucosinolates 

[including 3-butenyl, 4-pentenyl, 2-hydroxy-3-butenyl, and 2-hydroxy-4-pentenyl 

glucosinolates], indolyl glucosinolates [including 3-indolylmethyl and 4-hydroxy-3-

indoylmethyl], and total glucosinolates). 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 different, were within the natural variability of 

canola composition as expressed in the 99% tolerance interval. Collectively, the 

compositional analyses data support the conclusion that MON 88302 is as safe as and 

compositionally equivalent to conventional canola. 

IX.B 3. Phenotypic and Agronomic and Environmental Interaction Characteristics 

An extensive and robust set of information and data were used to assess whether the 

introduction of the glyphosate-tolerance trait altered the plant pest potential of 

MON 88302 compared to the conventional control. Phenotypic, agronomic, and 

environmental interaction characteristics of MON 88302 were evaluated and compared to 

those of the conventional control and considered within the variation among commercial 

reference varieties. These assessments included five general data categories: 1) 

germination, dormancy, and emergence; 2) vegetative growth; 3) reproductive growth 

(including pollen characteristics); 4) seed retention on the plant and lodging; and 5) plant 

response to abiotic stress and interactions with diseases and arthropods. Results from the 

phenotypic, agronomic, and environmental interaction assessment demonstrate that 

MON 88302 does not possess: a) increased weediness characteristics; b) increased 

susceptibility or tolerance to specific abiotic stresses, diseases 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 likely 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 vary with species and their genetic basis 

tends to be complex. Seed dormancy is an important characteristic that is often 

associated with plants that are considered weeds (Anderson, 1996; Lingenfelter and 

Hartwig, 2007). There were no changes in the dormancy or germination characteristics 

that are indicative of increased plant weediness or pest potential 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 lodging 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 significant differences were 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-site 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 terms 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 conventional control and their response to 

abiotic stressors, such as drought, wind, nutrient deficiency, etc. (Section VII.C.2.). The 

lack of significant biological differences in plant responses to abiotic stress support the 

conclusion that the introduction of the glyphosate-tolerance trait is unlikely to result in 

increased plant pest potential or an adverse 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 potential for gene flow to, 

and introgression of, the biotechnology-derived trait into other canola varieties and wild 

relatives (Section VII.C.3.). Pollen morphology and viability evaluations demonstrated 

no statistically significant differences (α = 0.05) between MON 88302 and the 

conventional control. Taken together, these comparative assessments indicate that 

MON 88302 is not fundamentally different from conventional canola or more or less 

likely to have increased weediness or plant pest potential. 

IX.B.3.5. Interactions with Non-target Organisms Including Threatened and 

Endangered Species 

Evaluation of MON 88302 for potential adverse impacts on non target organisms (NTOs) 

is a component of the plant pest risk assessment. The safety of CP4 EPSPS proteins 

present in biotechnology-derived crops has been extensively assessed (Harrison et al., 

1996; ILSI-CERA, 2010). A mouse gavage study demonstrated no acute oral toxicity 

(Harrison et al., 1996), and consequently the low potential for impact to terrestrial 

vertebrate NTOs including threatened and endangered vertebrate species. Impacts of 

deregulation of MON 88302 to NTOs and threatened and endangered species are 
Monsanto 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 interaction, arthropod damage and arthropod 

abundance, were collected at select sites for MON 88302 and the conventional control 

(Section VII.C.2.; Appendix G). These results 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 between CP4 EPSPS protein expression and any 

increased tendency for persistence or spread in the environment, alterations in 

reproductive biology affecting gene flow, or negative impacts on other organisms in the 

environment (ILSI-CERA, 2010). 

MON 88302 produces the identical CP4 EPSPS protein as is found in commercial 

Roundup Ready crop products including Roundup Ready 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 Amrhein, 1984). The CP4 EPSPS protein is an 

enzyme in the shikimate metabolic pathway in plants and microorganisms and is 

important in the production of aromatic amino acids that are essential to growth and 

development. The mode of action of this family 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, fish, birds and reptiles (Alibhai and Stallings, 

2001; Haslam, 1993). This explains the selective 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, fish, terrestrial invertebrates or aquatic 

invertebrates. APHIS further concluded that potential impacts to non-target plants may 

occur from drift resulting from the aerial application of glyphosate, but that these 

potential impacts have been mitigated through specific label use restrictions for 

glyphosate when applied with aerial equipment. Good management practices for 

application of all pesticides are also promoted by state university 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 acres are planted with glyphosate-tolerant 

canola and are routinely treated 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 glyphosate 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 opportunistic species, that is, a species 

adapted to take advantage of temporary conditions such as disturbed areas (CFIA, 1994), 

not as an environmentally hazardous colonizing (EC, 2000) or invasive species in 

undisturbed natural ecosystems (Crawley et al., 2001). Although B. napus has some 

characteristics typical of weedy species such as a high reproductive capacity, rapid 

growth and multiple pollination mechanisms (self, wind, insect), it also has many 

characteristics typical of domesticated species including low genetic diversity, lack of 

long-distance seed dispersal mechanisms, limited 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 present 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 transportation 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, populations of canola outside agricultural fields 

do not effectively compete with perennial vegetation, and usually persist only for a few 

years in the absence of ongoing seed introductions into areas from spillage during 

handling and transport or processing (Baker and Preston, 2008; Crawley and Brown, 

1995; 2004; Crawley et al., 1993; Knispel et al., 2008). The persistence 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 mowing, the use of broadleaf herbicides, animal 

predation, diseases and environmental conditions (Crawley and Brown, 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 seed in soil declines over time (Gulden et al., 

2003; Gulden et al., 2004; Hails et al., 1997). Biotechnology-derived canola seed has not 

been demonstrated to persist longer than conventional canola seed (Gruber et al., 2004; 

Hails et al., 1997). However, canola seed can persist in the soil for several years 

becoming secondarily dormant, and then germinate as volunteers (Gruber et al., 2004). 

Canola that has germinated and emerged unintentionally in a subsequent 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 applications or by mechanical means (Boyles et al., 

2009; EC, 2000). 

In comparative studies between MON 88302 and the conventional control, phenotypic, 

agronomic and environmental interaction data were evaluated (Section VII.C.2.) for 
Monsanto Company 11-CA-233U 174 of 500 

changes that would impact the plant pest potential, and, in particular, plant weediness 

potential. Results of these evaluations show that there is no biologically significant 

difference between MON 88302 and the conventional control for traits potentially 

associated with weediness such as germination, dormancy, reproductive growth and 

pollen characteristics, response to abiotic stress and disease or pest susceptibilities. 

Furthermore, comparative field observations 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. Collectively, these findings support the conclusion that 

MON 88302 has no increased weed potential compared to conventional canola and it is 

no more likely to become a weed than conventional canola. 
Monsanto Company 11-CA-233U 

Table IX-1. Summary of Literature on the Persistence of Canola Outside of Cultivated Fields Reference Methods Results (Nishizawa et al., 2009) 

Three years of monitoring for canola on roadsides along routes for transportation of canola seed from the shipping port of Kashima (1 of 11 shipping ports for canola seed in Japan). Plants of 

B. juncea were 

also observed, and tested for the presence of glyphosate- and glufosinate-tolerance proteins and genes. 

The number of plants found each year; along a 20 km section of roadway v2005, 4066 plants in 2006 and 278 plants in 2007. Brassica napus popula

roadsides or sidewalks, with some found in vacant lots and flower beds. MB. napus but a few 

B. juncea plants were also observed. In all three years 

plants were found, with the percentage of canola plants with either the glytolerance trait varying from 0 to 1.8%. The authors concluded that no masterm persistence of canola populations had occurred and that environmentaspillage along transportation routes would be negligible. 

(Baker and Preston, 2008) 

Monitoring of canola seed persistence in managed fields in Australia for up to 3.5 years after the crop was grown over 3 geographical regions from a total of 66 fields. Canola seeds were isolated and counted from soil samples taken from commercial fields. 

The overall mean number of seeds recovered per meter squared that germicollected 6 months after harvest and for seed collected 1.5 and 2.5 years afwas 1.4% and 1.5%, respectively. No seed collected 3.5 years after the las

Seed viability was not tested in the study. 

(Knispel et al., 2008) 

Observations of canola plant densities along field edges and roadways in Manitoba Canada from 2004 to 2006. Sampling of seed from 16 canola populations and testing for herbicide tolerance. Soil sampling and testing for canola seed viability and dormancy. 

Many canola plants along roadways died from mowing and most did not s25% of the populations had plants that set seed over 2 years, and only one 3 years of the study. Herbicide-tolerant canola plants were detected at a hpopulations sampled, and evidence of pollen-mediated gene flow of herbicdetected. Soil sampling from along roadways indicated that the seedbanks 

germinable seeds per square meter) and had little dormancy. The authors populations outside of cultivated areas are highly transient. 
Monsanto Company 11-CA-233U 

Table 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 along roadsides and field margins in France in a large-scale study over 4 years. Regression modeling was used to estimate the contribution of ecological processes involved in canola presence and persistence. 

The presence of 35–40 % of the ruderal canola populations was associated 

from neighboring fields. Approximately 15 % of the populations were attrduring transportation. The presence of no more than 10 % of the populatio

produced by ruderal canola populations in the previous year. The remaini40 %, were attributed to recruitment from seed that had been in the seed b

(Aono et al., 2006) 

Screening of seed collected from canola (B. napus), B. juncea and 

B. rapa plants from 95 sites from 7 port areas in Japan for herbicide tolerance. 

No biotechnology-derived seeds were found in B. juncea and B. rapa sampfrom canola plants and tolerance to glyphosate and glufosinate was found. of two individual plants had both the glyphosate-tolerance and glufosinate

(Yoshimura et al., 2006) 

Monitoring of sites in Saskatchewan, where canola is grown, and in Vancouver, British Columbia, where most canola destined for export is transported by rail. Observations at both areas of railway and along side roads for Brassica species were conducted Plants were tested for glyphosate and glufosinate herbicide-tolerance proteins. 

The glyphosate and glufosinate tolerance traits were found in approximatesampled in Saskatchewan and British Columbia. In Saskatchewan and Vacultivated (163 samples) and weedy relatives of canola (223 samples) werand glufosinate tolerance traits. These relatives included wild mustard (Si

mustard (B. juncea), dog mustard (Erucastrum gallicum) and bird’s rape (B

samples only a single B. rapa  B. napus hybrid with the glyphosate-tolera

British Columbia. 
Monsanto Company 11-CA-233U 

Table 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 by shattering at harvest and persistence in soil of glyphosate-, glufosinateand imidazolinone-tolerant and conventional canola varieties at 5 sites over 4 years. 

No difference between glyphosate-tolerant, glufosinate-tolerant, imidazoliconventional canola varieties for seed shattering at harvest and seed persisof canola seeds of all varieties declined rapidly in the soil in the first few mmean loss of 60 %. The number of canola seeds in the soil declined by ap

for the remainder of the experiment. 

(Saji et al., 2005) 

Collection of seeds from canola, B. rapa and 

B. juncea found 

growing near ports, along roadsides and along riverbanks. Seeds were grown and tested with either of glufosinate or glyphosate herbicide, and survivors were tested for presence of both herbicidetolerance proteins and genes. 

Seeds were collected from 7500 feral B. napus, 300 B. rapa and 5800 B. ju

locations. No herbicide-tolerance genes or proteins were detected in B. rapB. napus plants found along riverbanks. 

(Crawley and Brown, 2004) 

Survey of B. napus populations in 

100-m quadrants along roadside in the U.K. for 10 years (1993–2002) 

Seed spillage from trucks was a significant contributor to the observed 

B. n

the roadside. Plants were mostly observed within 1 m of the roadside and in deep shade. In the years following road works B. napus population denhigher in 9 of 12 cases. Overall the populations were not self-replacing. Cextinct after 1 to 4 years. The most frequent pattern was for quadrants to hto 2 years out of 10 with only 1 % of quadrants surveyed having one or moyears. 

(Gruber et al., 2004) 

Study of seed viability after burial in two separate years in the UK, with evaluation of seed viability of conventional and transgenic seeds after burial. 

No differences between biotechnology-derived and the near isogenic counseed survival in soil. Mean canola seed viability of buried seed was 66 % second years of the study, respectively. 
Monsanto Company 11-CA-233U 

Table 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 studies of canola secondary dormancy and seed persistence in soil. 

In the field experiments, canola viability after 4 weeks in soil ranged from seedling recruitment from seed in the shallow soil seedbank was low, rangThe authors conclude that germination may have contributed to loss of seeseedbank but because seedlings were not recruited from the deep seedbank germination at these deeper depths must have been lethal. 

(Gulden et al., 2004) 

Seeds were planted at two sites in Canada and persistence was measured each spring for 3 years. 

Different canola varieties persisted for different lengths of time. After 1, 2 maximum persistence was 44, 1.4 and 0.2 % of the original buried seeds, rvarieties. In 2001, canola genotypes with high potential for dormancy tengreater persistence than medium dormancy potential canola genotypes, indmortality in high potential dormancy varieties canola. 

(Norris and Sweet, 2002) 

Monitoring along field margins of a glufosinate-tolerant canola trial in the UK which had not previously been cropped to biotechnologyderived canola. 

Only one persistent population of feral B. napus was found at any site in anpersisted for 3 years. In the third year of monitoring all of the plants in the late in the season, and no plants were found in the fourth year of monitorinoilseed rape were rare in this study, and where populations were found in ogenerally persist to following years. Individual plants growing in roadsidesurvived to maturity and set seed. Many factors were involved in the mortmain ones being competition with perennial weeds (especially grasses), prbirds and insects) and susceptibility to diseases of canola. 

(Crawley et al., 2001) 

Conventional and biotechnologyderived canola planted at 12 habitats and monitored for 10 years. 

No difference in recruitment between conventional and biotechnology-derconventional or biotechnology-derived canola increased in abundance. Bicanola did not persist longer than conventional canola. Establishment of sbiotechnology-derived canola than conventional at 6 of the 12 sites, and w

at any site. Survival of biotechnology-derived canola relative to conventioof 12 cases and higher in 2 of 12 cases. All populations were completely e
Monsanto Company 11-CA-233U 

Table IX-1. Summary of Literature on the Persistence of Canola Outside of Cultivated Fields (continued)

Reference Methods Results 

(Hails et al., 1997) 

Seed burial study for 2 years at 12 four habitats (sites) in each of 3 different geographic sites in the UK (12 total habitats). The source of canola seed was the variety Westar for the conventional and biotechnology-derived lines (one antibiotic-tolerant, one herbicidetolerant and a third with both traits). A wild relative, Sinapis arvensis(charlock or wild mustard), was also included. 

After burial in soil only 1.5 % of the canola seeds persisted after the first ysecond year at all sites. The survival rate of biotechnology-derived seeds w

conventional seeds at both burial depths at all 12 habitats. However, when examined this trend was only detectable at 5 habitats and no detectable difhabitats. The weed S. arvensis persisted significantly longer than any of th

habitats and sites. 

(Crawley and Brown, 1995) 

Monitoring for canola plants in road verges adjacent to the M25 highway (UK). 

Canola plants were not seen in dense grass. Populations established by setransportation in the first year of the study were extinct within 4 years. In local canola populations were typically extinct within 2–4 years. In the yedisturbance canola densities were often high. 

(Crawley et al., 1993) 

Experiment to examine whether biotechnology-derived herbicidetolerance affects the likelihood of 

B. napus becoming invasive of 

natural habitats. 

No evidence of invasiveness of canola in undisturbed natural habitats. No or persistence between biotechnology-derived and conventional varieties. measured were declining and local extinction in all habitats was likely. Th

there is no evidence that B. napus is invasive of undisturbed natural habita
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 biology/volume, plant 

phenology, overlap of flowering times, proximity of the pollen source and sink, ambient 

conditions such as temperature and humidity and field architecture. Because pollen 

mediated gene flow is a natural biological process, it does not constitute an 

environmental risk in and of itself. 

Introgression is a process whereby 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 biotechnology-derived plant, 

and the likelihood that the presence of the transgenes and their subsequent transfer to 

recipient plants and plant populations will result in increased plant pest potential. The 

potential for gene flow and introgression from deregulation 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 although 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 travels 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 production 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 (Rantio-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 

0.14φ

0.07φ

0.08φ

0.07φ

0.04φ

0.02φ

(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 from 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 information on the potential for hybridization (cross 

pollination) and introgression from B. napus to related species. Reports of unassisted 

(without human intervention or assistance) field hybridization of B. napus to related 

species provide biologically relevant information 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 field 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 observed 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 will 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 control 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 reviewed. There are reports of hybridization 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 (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 hybrids 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). Hybridization frequencies between B. napus and B. rapa in 

neighboring fields or inter-planted in large plots vary from 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 rate of hybridization in 

commercial fields (13.6%) vs. natural 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 evidence for this were the 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). 

F1 hybrids between B. napus and B. rapa had reduced pollen viability with an average 

viability of approximately 50% (Warwick et al., 2003). F1 hybrids between B. napus and 

B. rapa produced approximately half as many germinable seeds as their respective 

parents, and had very low (< 2%) hybrid seedling survival rates (Scott and Wilkinson, 

1998). Additionally, unlike the B. rapa parent, under field conditions, seeds of F1 hybrids 

between these two species lacked seed dormancy (Jørgensen et al., 1998) which may 

limit F1 hybrid persistence. In a field study backcrosses with B. rapa as the female parent 

with F1 and BC1 hybrids occurred at very low frequencies 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, F1

hybrids, F2 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). F1 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 simulated herbivory and interspecific competition 

(Sutherland et al., 2006). 

Introgression, i.e., the stable 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 the glyphosatetolerance trait declined drastically from the first to fifth year of monitoring, 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 conclusion that hybridization between B. napus and B. rapa is 

possible under field conditions, but resulting progeny have decreased fitness. In the rare 

occurrence that the glyphosate-tolerance trait introgressed and persistence of hybrids 

between MON 88302 and B. rapa were to occur, these plants could be controlled by 
Monsanto 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 areas, and may be found in areas 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 chromosomes (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 third of pollination due to insect pollination 

(Duke, 1997). As reviewed in Devos (2009), in field plots with interplanted B. napus and 

B. juncea interspecific hybridization frequencies were low ranging from 0.3% to 3%. F1 

hybrids between B. juncea and B. napus had reduced male and female fertility, very low 

pollen viability ranging from 0% to 28% and low seed set (Bing et al., 1996; Frello et al., 

1995). The majority of studies of hybridization between B. napus and B. juncea have 

shown some directional effects such that F1 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 field experiments with 

neighboring B. juncea and B. napus plants to assess the potential 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%). Hybrid seeds formed when B. napus was the female parent were 

often deformed (e.g., without an embryo) or they started 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 reduced male and female fertility significantly 

decreasing the likelihood of introgression. 

Brassica oleracea 

Brassica oleracea is distributed primarily in northeastern and midwestern states, and may 

be found in areas where canola is grown (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 limited sexual compatibility under open 

pollination conditions (Ford et al., 2006). Brassica oleracea is predominately a selfpollinating species (Devos et al., 2009) limiting its potential for hybridization with 

B. napus. In field plots with inter-planted B. napus and B. oleracea, formation of F1

hybrid seed was reported when B. napus was the male parent, but the 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 samples from two sites (Ford et 
Monsanto 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 conditions 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 reproductive success makes introgression highly 

unlikely. Further, no hybrid plants were obtained when B. napus was the male parent 

(Kerlan et al., 1992). Therefore, the likelihood of gene flow from B. napus to B. nigra

under field conditions is extremely low. 

Brassica carinata

No field studies of the hybridization potential between B. napus and B. carinata have 

been reported (FitzJohn et al., 2007). Manual pollination results in only 0.08 seeds 

produced per pollination (Getinet 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 members 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 are 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 vegetable 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 pollination) and introgression 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 these 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 F1 hybrids produced was 

approximately 10-6 relative to the H. incana parent (Lefol et al., 1996b). In a two year 

field study evaluating the potential for introgression of a herbicide-tolerance gene from 

B. napus to H. incana, from 0.17% to 0.79% hybrid seed was produced (Darmency and 

Fleury, 2000). F1 hybrid plants obtained from both the 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 seeds 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 irregular (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 F1 hybrid seed produced from field studies, the survival and 

emergence after burial in soil under field conditions was assessed. Prior to burial in soil 

the F1 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 F1 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 phenomenon (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 experimental 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 frequency of crossing 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 Devos et al. (2009), seed dormancy of hybrids 

of B. napus and R. raphanistrum was within the range of their 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 than 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 reduction in chromosome number. By the fourth 

backcross generation, the hybrids had a chromosome number close to R. raphanistrum 

and the percentage of herbicide tolerant 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 number 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 hybrids 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 except 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 developed using hand pollination demonstrated 

poor pollen viability and fertility and 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 presence 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). Thus, 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 potential between B. napus and E. vesicaria

have been reported. Using protoplast fusion, hybrids can be produced (Fahleson et al., 

1988). F1 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 been reported in the literature. 

Other Species in the Family Brassicaceae

Under field conditions hybrids have 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; Norris and Sweet, 2002). Cross 

pollination of B. napus with any of these species is highly unlikely. 
Monsanto Company 11-CA-233U 

Table IX-3. Summary of Published Literature on Unassisted Hybridization under Field Conditions with Parent Species F1 Hybrids F2 or Backcross (BC) Hybrids Comments B. rapa 160 hybrids N/A 

Brassica napus and B. rapa cross readily whe

the male or female parent. Self-sterile B. rap

produced many hybrid progeny when compatpollen from other B. rapa plants was not avaifew hybrids when B. rapa pollen was availab

B. rapa 18 putative F1 hybrids. 

All F1s were self-sterile but 

backcrosses with both parents yielded some seed. 

Seed formed on recipients from the crosses wand the progeny visually assessed for morphovariants indicative of putative hybrids. 

B. rapa 

Some populations did not have any hybrids. In one population, flowering near a winter B. napuscrop, identified 33 hybrids among the 952 plants examined. 

N/A 

B. rapa plants with well developed seed pods collected from six Danish localities and plantgreenhouse. Seeds were harvested from each individual plant at maturity. Potential hybridB. napus were selected based on morphology subset characterized with isozymes. 

B. rapa 9% to 93% depending on experimental design. 

N/A but pollen fertility of the F1

hybrids under field conditions ranged form 12 to 54% (pollen fertility of the B. rapa and B. napus parents were 89%-99%, and 99%-100%, respectively) 

When B. rapa was the female parent F1 seed 

germination was 55% with a seed dormancy btreatment. The frequency of hybrids when B. 

planted at low density (93%) among B. napus 

was much higher than when the two were plaratio (13%). 

B. rapa More than 4000 seeds from 32 hybrids. 

F1 hybrids crossed with B. rapa had 

pronounced seed dormancy. BC1

crossed with B. rapa resulted in an average 6.4 viable BC2 offspring 

plants per pollination. 

From 44 hybrids selected with B. rapa-like m

some had 20 chromosomes (like B. rapa) and 

than 90% pollen fertility. 
Monsanto Company 11-CA-233U 

Table IX-3. Summary of Published Literature on Unassisted Hybridization under Field Conditions with 

Parent (continued) 

Species

F1 Hybrids

F2 or Backcross (BC) Hybrids Comments 

B. rapa 

0.09% and 1.5% F1 hybrid 

seed of the total harvested seed. 

BC1 failed under field conditions 

when B. rapa was the female. 

Field trials produced F1 seed. B. rapa was so

field with interspecific F1 hybrids, but backcrbetween the hybrid and B. rapa as the female observed. Under field conditions, backcrosseB. rapa as the female parent were attempted bproduce seed. F1 hybrids produced approxim

seeds per pod, whereas, the original parents h23. The seeds of the F1 hybrids showed almo

of dormancy, which the authors suggest could interspecific gene flow because germination uunfavorable conditions (e.g., with effective wmanagement) may occur. 

B. rapa 

1 F1 hybrid from 505 

(0.2%) plants screened from sympatric locations. 

N/A 

Remote sensing was used to find potential aresympatry between 

B. napus and populations o

in England. Two 

B. rapa populations sympatr

B. napus were found. No 

B. rapa plants were 

in any cultivated fields in the survey, indicatinegligible scope for transgene movement into B. rapa from B. napus x B. rapa hybrids form

agricultural fields.

B. rapa 

F1 hybrid frequency with 

different B. napus lines ranged from 0.7% to 16.9%. 

Backcross seed could only be produced with half of the B. napuslines in the greenhouse. 

Field experiments with 1200:1 ratio of B. napB. rapa. 

B. rapa 

F1 hybrid frequency was 

7% and 13.6% in field experiments and commercial fields, respectively. 

F1 hybrids had ~54% (range of two 

populations was 20-77% and 10-86%) pollen viability. 

From field experiments a total of 32,154 seedscreened and from the commercial fields 956seedlings were screened. 
Monsanto Company 11-CA-233U 

Table IX-3. Summary of Published Literature on Unassisted Hybridization under Field Conditions with 

Parent (continued) 

Species

F1 Hybrids

F2 or Backcross (BC) Hybrids Comments 

B. rapa 

F1 hybrid frequency with 

different B. napus lines ranged from 4% to 22% with an overall mean of 10%. 

When B. rapa was the female parent backcross frequencies from two locations were 0.088% and 0.06%. 

A 600:1 ratio of B. napus to B. rapa with sev

different transformed lines of the former. Scr12,388 B. rapa seedlings. 

B. juncea 

Mean F1 hybrid frequency 

of 3% under field conditions. 

Two F1 hybrids with ~24% and 28% 

pollen fertility were used in backcrosses to B. juncea. The backcrosses in the greenhouse produced 148 seeds with 75% germination compared to 90-100% for the initial cross. 

F1 hybrid seed germination was 93% and poll

ranged from 0% to 31%. BC1 pollen fertility 

from 24% to 90%, but note the study only usehighest pollen fertility F1 hybrids for these cr

B. juncea 

3 F1 hybrids from 990 

B. juncea plants as the 

female parent. 

N/A 

Two experiments consisting of (1) five-row p61 cm inter-row spacing with a 50:50 mixture cultivating species sown in the center row, or row plots with 25 cm inter-row spacing, 6 m female plants transplanted at low density inte

B. juncea 

2.3%, 1.1% and 0.3% hybrids in co-cultivation plots with 1:3, 1:10 and 1:15 ratio of B. napus to 

B. juncea plants, respectively. 

BC1 with B. juncea as the female 

parent. Results from backcrosses reported in Frello (

B. oleracea 

0 F1 hybrids from one 

B. oleracea population sympatric with a B. napusfield. 

N/A 

Remote sensing was used to identify potentiasympatry between B. napus production fields populations of B. oleracea in England. Every recruited plant in the population was screened hybrid status using flow cytometry and molecanalyses. 
Monsanto Company 11-CA-233U 

Table IX-3. Summary of Published Literature on Unassisted Hybridization under Field Conditions with 

Parent (continued) 

Species

F1 Hybrids

F2 or Backcross (BC) Hybrids Comments 

B. oleracea 1 triploid F1 hybrid,. 

Twelve tentative introgessants were identified with B. napus specific markers from 842 (1.4%). Of these nine were diploid and two near triploid hybrids 

A single F1 hybrid from 842 samples did not sufficient statistical power to provide an accuestimate of hybridization frequency. The 12 introgressed genotypes with crop-specific matriploids and nine diploids) out of 842 providfrequency of 1.4%. 

B. nigra 0 F1 hybrids from 198 B. nigra plants. N/A Male parents were sown in four-row plots, 6 m 

with female plants transplanted at low density row. 

B. nigra No hybrids. N/A 

No hybrids were observed form B. nigra plan

surrounded by B. napus plants in cages with bas pollinators 

H. incana 

0 F1 in experiment with 

cages from 36, 900 seeds screened and 16 F1 hybrids from 1061 in open field (~1/plant) 

N/A 

An experiment with cages and bees with B. nH. incana plants produced no hybrids. F1 hybrids produced almost no pollen and the grains were aborted. On average F1 hybrids p

40% fewer flowers, 20x fewer pods/flower, 5seeds/pod and 2x105 fewer seeds/plant than H

Of the 168 F1 hybrids only 32 seeds were col

which only 5 germinated. The seeds that did 3 were completely sterile, 1 produced 5 viabland 1 produced 57 viable seeds. 

H. incana Mean of 0.6 F1 hybrid seeds per plant. 

Backcrossing interspecific hybrids to H. incana over five generations showed that introgression was not successful. 

Seeds output in each backcross generation deand no viable seeds were produced by the 5th

generation. 
Monsanto Company 11-CA-233U 

Table IX-3. Summary of Published Literature on Unassisted Hybridization under Field Conditions with 

Parent (continued) 

Species

F1 Hybrids

F2 or Backcross (BC) Hybrids Comments 

R. raphanistrum 2 F1 seeds from 147,671 total seeds. 

Seed production of F1 hybrids and 

their F2 descendants was up to 0.4% 

and 2%, respectively, of that of R. raphanistrum. 

Male-fertile B. napus in cages with different proportions of R. raphanistrum and bees did n

produce hybrids. Two hybrids produced undconditions without cages. 

R. raphanistrum 

1 F1 hybrid from 189,084 

seedlings obtained from seeds harvested from R. raphanistrum plants. 

N/A The 1 F1 hybrid obtained in the study had pol

fertility of 6.5%. 

R. raphanistrum 

0 F1 hybrids from 25,000 

seeds harvested from R. raphanistrum plants. 

N/A 

Two hybrids were obtained when B. napus w

female parent from 52-million seeds screened (frequency of 4 x 10-8). 

R. raphanistrum 1 F1 hybrid from 32,821 seedlings screened. N/A F1 hybrid had <1% pollen viability. R. raphanistrum 0 F1 hybrids from 19,274 seeds harvested. N/A Ratio of 6001 of B. napus to R. raphanistrumS. arvensis 1 putative F1 hybrid. N/A 

Blowflies were added to cages to encourage pmovement. The chromosome number and ferthe single hybrid was not confirmed in this pu

S. arvensis 0 F1 hybrids from 61 

S. arvensis plants. N/A 

Male parents were sown in four-row plots, 6 m with female plants transplanted at low density row. 

S. arvensis 0 F1 hybrid seeds from 2.9 million seeds harvested. Artificial hybrids grown in the presence of S. 

or hand-crossed, produced a few aborted seed

S. arvensis 1 F1 hybrid. 

The F1 hybrid did not have viable 

pollen. The F1 hybrid produced no 

seed. 

The authors concluded that the risk of gene trfrom B. napus to S. arvensis is minimal. 

S. arvensis 0 F1 hybrids. N/A Screened approximately 43,000 seedlings of S

and no hybrids were detected. 
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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 confirming 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 from any other herbicidetolerant canola currently available. Furthermore, MON 88302 produces the identical 

CP4 EPSPS protein as is found in commercial Roundup Ready crop products including 

Roundup Ready canola (RT73) and has been shown to have no meaningful toxicity to 

humans and to other NTOs under 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 previously used for agricultural purposes. Certified seed 

production will continue to use well-established industry practices to deliver high quality 

seed containing MON 88302 to growers. Cultivation of MON 88302 is not expected to 

differ from current canola cultivation using glyphosate-tolerant canola, with the 

exception of an opportunity to use glyphosate during an expanded period of application 

and at rates higher than those currently 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. Therefore, no significant impacts on current 

cultivation and management practices for canola 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 practices or weed management practices, other 

than intended weed control benefits. 

IX.F. Summary of Plant Pest Assessments 

Plant pests, as defined in the Plant Protection Act, are living organisms that can directly 

or indirectly injure, cause damage to or cause disease to any plant or plant product (7 

U.S.C. § 7702[14]). Data presented in Sections 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 potential. Monsanto is not aware of any study 

results or observations associated with MON 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 compared to conventional canola, followed by a 

risk assessment on detected differences. The plant pest risk assessment in this petition 

was based on the following lines of evidence: 1) insertion of a single functional copy of 
Monsanto Company 11-CA-233U 195 of 500 

the cp4 epsps expression cassette; 2) characterization of the CP4 EPSPS protein 

expressed in MON 88302; 3) safety of the CP4 EPSPS protein; 4) compositional 

equivalence of MON 88302 seed as compared to a conventional control; 5) phenotypic 

and agronomic characteristics demonstrating no increased plant pest potential; 6) 

negligible risk to NTO and threatened or endangered 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 highly unlikely to be a plant 

pest. Thus, the results support a conclusion of no increased weediness potential of 

MON 88302 compared to conventional canola. In addition, APHIS has proposed to 

amend 7 CFR § 340 to include its noxious weed authority. MON 88302 would not be 

considered a “noxious weed” as defined by the Plant Protection Act because the data in 

this petition show that it has no potential to cause direct injury or damage (physical harm) 

to any protected interest. Therefore, Monsanto Company requests a determination from 

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

and other commercial canola be granted non-regulated status under 7 CFR § 340. 
Monsanto Company 11-CA-233U 196 of 500 

X. ADVERSE CONSEQUENCES OF INTRODUCTION 

Monsanto is not aware of any study results or observations associated with MON 88302 

or the CP4 EPSPS protein indicating that there would be an adverse environmental 

consequence from the introduction of MON 88302. 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 firstgeneration product, Roundup Ready canola (RT73). Enhanced CP4 EPSPS expression in 

the male reproductive tissues of MON 88302 allows the greater flexibility of glyphosate 

herbicide applications possible 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. Sink tissues, such as pollen, that accumulate glyphosate and 

lack sufficient CP4 EPSPS expression are considered to be at risk for glyphosate injury. 

MON 88302 plants can be sprayed with higher rates 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 development 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 nonregulated status by APHIS, and have been widely planted 

in the U.S. and globally. As demonstrated by field results and laboratory tests, the only 

phenotypic difference between MON 88302 and conventional canola is glyphosate 

tolerance. 

The data and information presented in this 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 molecular analyses, which confirmed the 

insertion of a single functional copy of the CP4 EPSPS expression cassette at a single 

locus within the canola genome. Additionally, protein expression analyses demonstrate 

the CP4 EPSPS protein is expressed in vegetative and male reproductive tissues. The 

CP4 EPSPS protein produced by MON 88302 is similar and functionally identical to 

endogenous plant EPSPS enzymes, but has a much reduced affinity for glyphosate, 

relative to endogenous plant EPSPS. The amino acid sequence of the CP4 EPSPS protein 

expressed in MON 88302 is identical to the amino acid sequence of the recombinant E. 

coli-produced CP4 EPSPS protein standard previously utilized. Analyses of key 

nutrients, anti-nutrients and toxicants of MON 88302 seed demonstrate that MON 88302 

is compositionally equivalent to conventional canola. The 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 unchanged compared to conventional canola. 

There is no indication that MON 88302 would have an adverse impact on beneficial or 

non-target organisms, including threatened or endangered species. Therefore, based on 

the lack of increased pest potential or adverse environmental consequences compared to 
Monsanto 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 adversely impact cultivation practices or the 

management of weeds, diseases and insects in canola production systems. Growers 

familiar with the Roundup Ready canola system would continue to employ the same crop 

rotational practices, weed control practices and/or volunteer control measures currently in 

place for the first-generation Roundup Ready canola product. 
Monsanto Company 11-CA-233U 198 of 500 

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transportation routes and port of Vancouver in western Canada. Environmental Biosafety 

Research 5: 67-75. 

Zambryski, P., A. Depicker, K. Kruger and H.M. Goodman. 1982. Tumor induction by 

Agrobacterium tumefaciens: Analysis of the boundaries of T-DNA. Journal of Molecular 

and Applied Genetics 1: 361-370. 

Zollinger, R., M. McMullen, J. Knodel, J. Gray, D. Jantzi, G. Kimmet, K. Hagemeister 

and C. Schmitt. 2009. Statewide pesticide usage for North Dakota. Page 23 in Pesticide 

Use and Pest Management Practices in North Dakota 2008. North Dakota State 

University Extension Service, North Dakota Agricultural Experiment Station, Fargo, 

North Dakota. 
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APPENDICES 
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Appendix A: USDA Notifications 

Field trials of MON 88302 have been conducted in the U.S. since 2005. The protocols 

for these trials include field 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, observational 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 preparation. A list of trials conducted under USDA notifications and 

the status of the final reports for these trials are provided in Table A-1. 

Table A-1. USDA Notifications 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 Progress
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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 R3

generation of MON 88302 and the conventional control (Ebony). The leaf tissue was 

harvested from a greenhouse production in 2009. For generational stability analysis, 

genomic DNA was extracted from leaf tissue of the R2, R3, R4, R5a and R5b generations of 

MON 88302. The leaf tissue was harvested 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 hybridization controls. As additional 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 Southern 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 Southern Blot and PCR Analyses 

Genomic DNA was isolated from MON 88302 leaf 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 transferred 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, and 0.4% w/v sodium 

bisulfate) and 300 μg of RNase A were added 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 incubation, the samples were allowed to come 

to room temperature and 20 ml of phenol/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 was repeated at least once followed by a 

chloroform extraction. The aqueous phase was 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 centrifugation at 4,000 x g for 5 min at 2-8 °C and the 70% 

ethanol was discarded. After being air-dried, the DNA pellet was suspended in 
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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 (Roche, 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 digested 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 ~500 microliter (μl) with ~50 units of each 

restriction enzyme. Digests conducted with the combination of restriction enzymes Sal I 

and Sca I also included 1X BSA (New England Biolabs, Inc.) in the reaction. For the 

purpose of running positive hybridization controls, ~10 μg of genomic DNA extracted 

from the conventional control was digested 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, individual 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. The long run allows for greater resolution of large molecular 

weight DNA, whereas the short run allows for 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 generations of MON 88302 and the conventional 

control along with the positive hybridization controls were loaded on the agarose gel in a 

single run format. 

B.7. DNA Probe Preparation for Southern Blot Analyses 

Probe templates were prepared by PCR amplification using the PV-BNHT2672 DNA as a 

template. The PCR products were separated on an agarose gel by electrophoresis and 

purified from the gel using QIAquick Gel 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 possible, probes possessing similar melting 

temperature (Tm) were combined in the same Southern blot hybridization. 

Approximately 25 ng of each probe template were radiolabeled with either [α32P] 

deoxycytidine triphosphate (dCTP) (6000 Ci/mmol) or [α32P] deoxyadenosine 
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triphosphate (dATP) (6000 Ci/mmol) using 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 the conventional control was digested and 

evaluated using Southern blot analyses (Southern, 1975). The PV-BNHT2672 DNA, 

previously digested with the combination of restriction 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 probe 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 separated by agarose gel electrophoresis and 

transferred onto a nylon membrane. Southern blots were hybridized and washed at 

55 °C, 60 °C, or 65 °C, depending on the calculated melting temperature (Tm) of the 

probes that were used. Table A-1 lists the radiolabeling conditions and hybridization 

temperatures of the probes used in this study. Multiple exposures of each blot were then 

generated using Kodak Biomax MS film (Eastman Kodak, Rochester, NY) in conjunction 

with one Kodak Biomax MS intensifying screen in a -80 °C freezer. 
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Table B-1. Hybridization Conditions of Utilized Probes 

Probe DNA Probe

Element Sequence 

Spanned by DNA 

Probe

Probe labeled 

with dNTP 

(

32P)

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 60
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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 according 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 containing a final concentration of 1.5 mM 

MgSO4 (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 included in the reaction. The amplification of 

Product A was performed under the 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 amplification of Product B was performed under the 

following cycling conditions: 1 cycle at 94 oC for 2 minutes; 35 cycles at 94 oC for 30 

seconds, 60 oC for 15 seconds, 72 oC for 2 minutes; 1 cycle at 72 oC for 5 minutes. 

Aliquots of each PCR product were separated on 1.0% (w/v) agarose gels and visualized 

by ethidium staining to verify that the products were of the expected size. Prior to 

sequencing, each verified PCR product was purified with the QIAquick PCR Purification 

Kit (Qiagen, Inc., Valencia, CA) according to manufacturer’s instruction and gel 

quantified according to SOP BR-ME-1222-01. The purified PCR products were 

sequenced using multiple primers, including primers used for PCR amplification. All 

sequencing was performed by the Monsanto Genomics Sequencing Center using BigDye 

terminator chemistry (Applied Biosystems, Foster City, CA). 

Numerous sequencing reactions performed on the overlapping PCR products were 

compiled and a consensus sequence was generated. This consensus sequence was 

aligned with the sequence of plasmid vector PV-BNHT2672 to determine the integrity 

and genomic organization of the integrated DNA and the junctions of the 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 organization 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. 
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The PCR analyses were performed according to SOP BR-ME-0486-01. The PCR 

reactions were conducted using 30 ng of genomic DNA template in a 50 μl reaction 

volume containing a final concentration of 2 mM MgSO4, 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) agarose 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 conventional 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 Sequencing Center using BigDye terminator 

chemistry. 

Numerous sequencing reactions performed on the PCR product were compiled and a 

consensus sequence was generated. This consensus 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. 

 
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References for Appendix B 

Southern, E.M. 1975. Detection of specific sequences among DNA fragments separated 

by gel electrophoresis. Journal of Molecular Biology 98: 503-517. 
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Appendix C: Materials, Methods, and Results for Characterization of 

CP4 EPSPS Protein Produced 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-produced CP4 EPSPS protein was stored 

in a -80 oC freezer in a buffer solution containing 50 mM Tris-HCl, pH 7.5, 50 mM KCl, 

2 mM dithiothreitol (DTT), 1 mM benzamidine-HCl, and 25% glycerol. 

The E. coli-produced CP4 EPSPS protein (lot 10000739) was used as the reference 

substance. The CP4 EPSPS protein reference substance was 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 previously characterized. 

C.2. Description of Assay Controls 

Protein MW standards (Precision Plus Protein Standards Dual color; Bio-Rad, Hercules, 

CA) were used to calibrate some SDS-PAGE gels and verify protein transfer to 

polyvinylidene difluoride (PVDF) and nitrocellulose membranes. Broad Range SDSPAGE molecular weight standards (Bio-Rad, Hercules, CA) were used to generate a 

standard curve for the apparent 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 protein 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 calibrate the MALDI-TOF mass 

spectrometer for tryptic mass and a bovine serum albumin (BSA) standard (NIST, 

Gaithersburg, MD) was used to calibrate the 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 applicable, 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 chromatography, and cellulose phosphate affinity 

chromatography. The purification procedure 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 crushed seed was further ground using a Magic 

Bullet grinder. The ground seed was then defatted by extraction with heated hexane 
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(~50 °C) followed by vacuum filtration. This was repeated three times at a ground seed 

(g) to hexane volume (ml) ratio of approximately 1:5. The defatted ground seed was 

allowed to dry overnight at room temperature in a fume hood. The following day the 

defatted ground seed was mixed with extraction 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 addition 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 liters) was again collected and 592 g of 

ammonium sulfate was added to bring the solution to 70% ammonium 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 DTT, 10% (v/v) glycerol,1.5 M ammonium 

sulfate]. The sample was loaded onto a 460 ml column (5 cm x 23 cm) of Phenyl 

Sepharose Fast Flow (GE Healthcare, Piscataway, NJ) equilibrated with PS(A) buffer. 

Proteins were eluted with a linear salt gradient 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 analysis 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 eluted with a linear salt gradient that 

increased from 0 M to 0.4 M KCl in QS(A) buffer over 2.1 liters. Fractions containing 

CP4 EPSPS, identified by immunoblot analysis, were pooled 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 total of 18 h at ~4 °C 

using a dialysis tubing (Spectrum Laboratories, Inc. Rancho Dominguez, CA; MWCO: 

3.5 kDa). 

Of the 350 ml recovered after dialysis, approximately 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-equilibrated 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 adjusted 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 SDS-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 sample 
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was then loaded onto a freshly prepared 20 ml column (2.6 x 3.7 cm) of cellulose 

phosphate P11 cation exchange (Whatman, Kent, UK) pre-equilibrated with freshly 

prepared CP(A) buffer. After an initial wash 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) buffer. The bound CP4 EPSPS protein was eluted 

with freshly prepared CP(D) buffer. Fractions containing CP4 EPSPS protein, based on 

SDS-PAGE analysis and confirmed by immunoblot, 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 centrifugation 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 containing 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 concentrator (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 automated Edman degradation chemistry, was used 

to confirm the identity of the MON 88302-produced CP4 EPSPS. 

MON 88302-produced CP4 EPSPS was separated by SDS-PAGE and transferred to a 

PVDF membrane. The blot was stained using Coomassie Blue R-250. The major band at 

~44 kDa containing the test protein was excised 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 Microgradient 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 observed during analysis. 

 
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C.4.2. Results of the N-terminal 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 EPSPS protein were compared to the 

expected sequence. The single letter IUPAC-IUB amino acid code is M, methionine; L, 

leucine; H, histidine; G, glycine; A, alanine, 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 bicarbonate 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 bicarbonate 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/ml). After 1 h, excess liquid 

was removed and the gel was incubated at ~37 °C for 16 h in 40 μl of 10% acetonitrile in 
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25 mM ammonium bicarbonate. Gel bands were sonicated for 5 min to further elute 

proteolytic fragments. The resulting extracts 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% acetonitrile, 0.1% trifluoroacetic acid, 0.1% β-octylglucopyranoside solution and sonicated for 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 (sinapinic 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 reflector 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 generate a theoretical trypsin digest of the 

CP4 EPSPS protein sequence. Those experimental 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 coverage was considered 

acceptable if  40% of the protein sequence was identified by matching experimental 

masses observed for the tryptic peptide fragments 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 fragments produced from tryptic digestion of the 

MON 88302-produced CP4 EPSPS protein. The ability to identify a protein using this 

method is dependent upon matching a sufficient number of observed tryptic peptide 

fragment masses with predicted tryptic peptide fragment masses. In general, protein 

identification made by peptide mapping is considered 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 produced by tryptic digestion of the CP4 EPSPS protein. The identified 

masses were used to assemble a coverage map of the entire CP4 EPSPS protein 

(Figure C-1). The experimentally determined 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 

Table C-2. Summary of the Tryptic Masses Identified for the MON 88302-produced CP4 EPSPS Using 

Spectrometry 

1Only 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 ex3Position 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-hyd

Ave = experimental mass average (for large peptides the monoisotopic mass is poorly resolved, therefore the mass average value 

-Cyano

-Cyano DHB DHB Sinapinic acid Sinapinic acid Expected Mass1 Diff2 Position3 Sequence

Extract 1 Extract 2 Extract 1 Extract 2 Extract 1 Extract 2

389.28 389.25 0.03 225-227 TIR474.32 474.27 0.05 228-231 LEGR

506.29 506.22 0.07 354-357 ESDR 

529.36 529.30 0.06 24-28 IPGDK

599.43 599.51 599.41 599.33 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 VLNPLR

790.55 790.48 0.07 306-312 VRSSTLK790.55 790.41 0.14 139-145 EMGVQVK805.54 805.43 0.11 447-453 IELSDTK

835.54 835.58 835.53 835.39 0.15 62-69 AMQAMGA863.61 863.68 863.60 863.46 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 LSAVANGL

930.66 930.51 0.15 169-177 VPMASAQV

948.68 948.74 948.68 948.75 948.52 0.16 161-168 TPTPITYR991.72 991.71 991.55 0.17 14-23 KSSGLSGTV

1115.75 1115.83 1115.77 1115.86 1115.69 1115.57 0.18 295-305 LAGGEDVAD

1357.94 1358.01 1357.97 1358.05 1357.89 1357.71 0.23 146-157 SEDGDRLPVT1359.88 1359.96 1359.91 1360.00 1359.81 1359.87 1359.72 0.16 354-366 ESDRLSAVANG1359.88 1359.96 1359.91 1360.00 1359.81 1359.87 1359.64 0.24 34-46 SFMFGGLASG1559.11 1559.18 1559.13 1559.01 1558.83 0.28 47-61 ITGLLEGEDVIN1647.10 1647.24 1647.16 1647.24 1646.84 0.26 389-405 GLGNASGAAVAT1764.10 1764.26 1764.16 1764.06 1763.81 0.29 367-382 LNGVDCDEGETS1994.31 1994.43 1994.35 1994.55 1994.21 1994.35 1993.97 0.34 206-224 MLQGFGANLTVETD2183.54 2183.67 2183.57 2183.80 2183.45 2183.53 2183.17 0.37 275-294 TGLILTLQEMGADI2367.73 2367.87 2367.77 2367.85 2367.65 2367.8 2367.33 0.40 178-200 SAVLLAGLNTPGITT2450.65 2450.83 2450.80 2450.51 2450.6 2450.23 0.42 24-46 IPGDKSISHRSFMFGG2450.65 2450.83 2450.80 2450.51 2450.6 2450.22 0.43 105-127 LTMGLVGVYDFDSTF

3247.10 (Ave) 3247.05 (Ave) 3246.89 (Ave) 3246.97 (Ave) 3246.54 (Ave) 0.56 73-104 EGDTWIIDGVGNGGLLAPEA3251.94 (Ave) 3252.18 (Ave) 3252.06 (Ave) 3253.42 (Ave) 3252.58 (Ave) 3252.04 (Ave) 3251.75 (Ave) 0.19 321-351 APSMIDEYPILAVAAAFAEG

4191.34 (Ave) 4191.48 (Ave) 4191.89 (Ave) 4191.63 (Ave) 4190.89 (Ave) 0.37 234-274 LTGQVIDVPGDPSSTAFPLVAALLV
Monsanto 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 regions 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 covered by the identified peptides. 

C.6. Western Blot Analysis-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 gel. 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 verify 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 (Invitrogen, Carlsbad, CA) was performed for 

90 min at a constant voltage of 30 V. After 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 DTKAA
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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 antibody (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 horseradish peroxidase (HRP)-conjugated rabbit 

anti-goat IgG (Thermo, Rockford, IL) at a dilution of 1:10,000 in 5% NFDM in PBST for 

1 h at room temperature. Excess HRP-conjugate was removed using three 10 min washes 

with PBST. All washes were performed at room temperature. Immunoreactive bands 

were visualized using the ECL detection system (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 SRX-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 One 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 quantified 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 the CP4 EPSPS bands were within 35% of one another. 

 
Monsanto Company 11-CA-233U 234 of 500 

C.6.2. Results of CP4 EPSPS Protein Immunoreactivity Equivalence 

Western blot analysis was conducted using goat anti-CP4 EPSPS polyclonal antibody to 

1) confirm the identity of the CP4 EPSPS protein isolated from the seed of MON 88302 

and 2) to determine the relative immunoreactivity of the MON 88302- and the E. coliproduced 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 the 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 difference (24.1%) are also shown in 

Table C-3. The acceptance criterion for equivalence of immunoreactivity (±35%) of the 

MON 88302-produced CP4 EPSPS bands and E. coli-produced CP4 EPSPS bands was 

met. Thus, the western blot analysis established 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 CP4 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 -

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

coli-produced EPSPS Proteins 

Sample Gel 

lane

Amount 

(ng)

Contour 

Qty

(OD × 

mm2

)

Average 

Contour 

Qty1

Percent 

Difference2 

(%)

Average 

Difference3 

(%)

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-produced CP4 EPSPS protein and an E. coli-produced 

CP4 EPSPS protein was mixed with 5X LB and diluted with H2O to a final total protein 

concentration of 0.2 μg/μl. Bio-Rad broad range Molecular Weight Standards (Hercules, 

CA) were diluted to a final total protein concentration of 0.9 g/l. The 

MON 88302-produced CP4 EPSPS protein was analyzed 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 samples 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 solution of 40% (v/v) methanol and 7% (v/v) acetic acid for 

~30 min, stained for ~16 h with Brilliant Blue G-Colloidal stain (Sigma-Aldrich, St. 

Louis, MO). Gels were destained for 30 to 45 sec with a solution 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 were quantified using Quantity One software. 

The purity of the MON 88302-produced CP4 EPSPS protein was reported as the percent 
Monsanto Company 11-CA-233U 237 of 500 

of the total of all quantified bands in a lane. Apparent MW and purity were reported as 

an average of all six lanes containing the MON 88302-produced CP4 EPSPS 

C.7.2. Results of CP4 EPSPS Protein 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) migrated 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. coliproduced CP4 EPSPS protein as reported on its Certificate of Analysis was 43.8 kDa 

(Table C-4). The apparent molecular weights of the MON 88302- and E. coli-produced 

CP4 EPSPS proteins were considered equivalent if they were within 10% of one another. 

Because the experimentally determined apparent 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 EPSPS 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 Comparison Between the MON 88302- and E. coliproduced CP4 EPSPS Proteins 

Molecular Weight 

of MON 88302-Produced 

CP4 EPSPS Protein 

Molecular Weight of 

E. coli-Produced 

CP4 EPSPS Protein1

% 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 reported on its Certificate of 

Analysis. 

 
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Figure C-3. Molecular Weight and Purity 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 polyacrylamide gradient gel and then stained with 

Brilliant Blue G-Colloidal stain. Approximate 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 - 
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C.8. Glycosylation Analysis 

C.8.1. Methods 

Glycosylation analysis was used to determine whether the MON 88302-produced 

CP4 EPSPS was post-translationally modified with covalently bound carbohydrate 

moieties. Aliquots of the MON 88302-produced CP4 EPSPS protein, the E. coliproduced 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-glycine 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 for 75 min. Electrotransfer to a 0.45 μm PVDF 

membrane (Invitrogen, Carlsbad, CA) was performed for 35 min at a constant voltage of 

100 V. 

Carbohydrate detection was performed directly on the PVDF membrane at room 

temperature using the Amersham ECL glycoprotein Detection Module (GE, Healthcare, 

Piscataway, NJ). With this module, carbohydrate moieties of proteins are oxidized with 

sodium metaperiodate and are then biotinylated with biotin-X-hydrazide. The 

biotinylated proteins can be detected on the blot by addition of streptavidin conjugated to 

HRP for luminol-based detection using ECL reagents (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 the 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 the supplied Quantity One software (version 

4.4.0). 

C.8.2. Results of Glycosylation Analysis 

Some eukaryotic proteins are post-translationally modified by the addition of 

carbohydrate moieties (Rademacher et al., 1988). To test whether the CP4 EPSPS 

protein was glycosylated when expressed 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). Transferrin, a glycosylated protein, was used 

as a positive control in the assay. To assess equivalence of the MON 88302- and E. coliproduced 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 positive 
Monsanto Company 11-CA-233U 240 of 500 

control was clearly detected at expected molecular weight (~76 kDa) and the band 

intensity increased with increasing concentration (Figure C-4, Panel A, lanes 2-5). In 

contrast, signals were not observed in the lanes containing the MON 88302- or E. coliproduced protein at the expected molecular weight for the CP4 EPSPS protein (Figure C4 panel A, lanes 6-9). To confirm that sufficient MON 88302- and E. coli-produced 

CP4 EPSPS proteins were present for glycosylation analysis, a second membrane (with 

identical loadings and transfer times) was stained 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 glycosylated 
Monsanto Company 11-CA-233U 241 of 500 

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

Protein 

Aliquots of the transferrin (positive control), E. coli-produced CP4 EPSPS 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 blot was stained with Coomassie Blue R250 

to confirm the presence of proteins. The signal was captured using a Bio-Rad GS-800 

with Quantity One software (version 4.4.0). Approximate molecular weights (kDa) 

correspond to the Precision Plus, dual color markers (used to verify transfer and MW) in 

Lane 1. Arrows indicate the band corresponding 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 - 
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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 concentration 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 without 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 green, 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 treated with the phosphate assay reagent and 

33% (w/v) sodium citrate. The absorbance 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 released 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 using 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 assay that measures formation of inorganic 

phosphate (Pi) from the EPSPS-catalyzed reaction 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 minute at 25 °C. The MON 88302- and E. coliproduced CP4 EPSPS proteins were considered to have equivalent functional activity if 

the specific activities were within 2-fold of one another. 

The experimentally determined specific activities for the MON 88302- and E. coliproduced CP4 EPSPS proteins are presented in Table C-5. The specific 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. Because the specific activity of the MON 88302-

produced CP4 EPSPS protein falls within the preset acceptance criterion (Table C-5), the 

MON 88302-produced CP4 EPSPS protein was considered to have equivalent functional 

activity to that of the E. coli-produced CP4 EPSPS protein. 
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Table C-5. CP4 EPSPS Functional Activity Assay 

MON 88302-produced 

CP4 EPSPS Protein1 

(U/mg) 

E. coli-produced 

CP4 EPSPS Protein1 

(U/mg) 

Previously set acceptance 

limits2 

(U/mg) 

4.93 ± 0.36 2.79 ± 0.26 1.40 – 5.58 

1

Value refers to mean and standard deviation calculated 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) 
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References for Appendix C 

Aebersold, R. 1993. Mass spectrometry 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. Hammond, 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-tolerant soybean, 5-enolpyruvylshikimate-3-

phosphate synthase from Agrobacterium sp. strain CP4, is rapidly digested 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 sequenator. 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 Protein in MON 88302 

D.1. Materials 

Seed, forage, over-season leaf (OSL-1-4), and root (Root-1-2) tissue samples from 

MON 88302 were harvested from three field sites in the U.S. and three field sites in 

Canada during the 2009 growing season from starting seed lot 11225246. An 

E. coli-produced CP4 EPSPS protein (lot 10000739) was used as the analytical reference 

standard. 

D.2. Characterization of the Materials 

The identity of MON 88302 was confirmed by verifying the chain of custody 

documentation prior to analysis. To further confirm the identities 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 unexpectedly during PCR verification, were not 

analyzed in this study. 

D.3. Field Design and Tissue Collection 

Field trials were initiated during the 2009 planting season 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 Saskatoon, Saskatchewan, Canada (SKSA). 

These field sites were representative of canola producing regions 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 replicated 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 impacted 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 management 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 sample from site NDVA. The processed tissue 

samples were stored in a -80 °C freezer. 
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CP4 EPSPS protein was extracted from the tissue samples as described in Table D-1. 

CP4 EPSPS protein was extracted from all tissues samples using a Harbil Mixer with the 

appropriate amount of Tris-borate buffer with L-ascorbic acid (1× TBA) [0.1 M Tris, 

0.1 M Na2B4O7 • 10H2O, 0.005 M • 6H2O MgCl2, 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 Scientific, Pittsburgh, PA). The extracts were aliquotted and stored 

frozen in a -80 °C freezer until ELISA analysis. 
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Table D-1. Protein Extraction Methods for Tissue Samples 

Sample Type Tissue-to-Buffer Ratio Extraction Buffer 

Leaf2 1:100 1× TBA

Root3 1:100 1× TBA

Forage 1:100 1× TBA

Seed 1:100 1× TBA 1

Over- season leaf (OSL-1, OSL-2, OSL-3, and OSL-4). 2

Root (Root-1 and Root-2). 
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D.5. CP4 EPSPS Antibodies 

Mouse monoclonal antibody clone 39B6.1 (IgG2a isotype, kappa light chain; lot 

10002190) specific for the CP4 EPSPS protein was purified from mouse ascites fluid 

using Protein-A Sepharose affinity 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 Biosolutions (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 Na2CO3, 

35 mM NaHCO3, and 150 mM NaCl, pH 9.6) to a final concentration of 2.0 μg/ml, and 

immobilized onto 96-well microtiter plates followed by incubation in a 4 °C refrigerator 

for ≥8 hours. Prior to each step in the assay, plates were washed with 1× PBST. 

CP4 EPSPS protein standard or sample extract was added at 100 μl per well and 

incubated for 1 hour at 37 °C. The captured CP4 EPSPS protein was detected by the 

addition of 100 μl per well of anti-CP4 EPSPS HRP conjugate. Plates were developed by 

adding 100 μl per well of 3,3',5,5' tetramethyl-benzidine (TMB; Kirkegaard & Perry, 

Gaithersburg, MD). The enzymatic reaction was terminated by the addition of 100 μl per 

well of 6 M H3PO4. Quantification of the CP4 EPSPS protein was accomplished by 

interpolation from a CP4 EPSPS protein standard curve that ranged from 

0.456-14.6 ng/ml. 

D.7. Moisture Analysis 

Tissue moisture content was determined using an IR-200 Moisture Analyzer (Denver 

Instrument Company, Arvada, CO). A homogeneous tissue-specific site pool (TSSP) 

was prepared consisting of samples of a given tissue type grown at a given site. The 

average percent moisture for each TSSP was calculated from triplicate analyses. A TSSP 

Dry Weight Conversion Factor (DWCF) 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 fresh 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 spectrophotometer, 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 using Molecular Devices SOFTmax PRO GxP 

version 5.0.1. Absorbance readings and protein standard concentrations were fitted with 

a four-parameter logistic curve. Following the interpolation from the standard curve, the 

amount of protein (ng/ml) in the tissue was converted 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. The sample means, standard deviations, and 

ranges were also calculated by Microsoft Excel 2007. 

Any MON 88302 sample extracts that resulted 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. 
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Appendix E: Materials, Methods, and Individual-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 varieties were seven conventional canola varieties 

(Table E-1). 

Table E-1. Commercial Reference Canola Varieties 

Material Name Seed Lot Number Field Sites1

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 the chain of custody documentation prior to 

analysis. To further confirm the identities 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 control and commercial reference varieties 

were collected from replicated plots at each of two U.S. sites [Wilkin County, Minnesota 

(MNCA); and McHenry County, North Dakota (NDVA)] and three Canadian sites 

[Portage la Prairie, Manitoba (MBPL); Newton, Manitoba (MBNW); and Saskatoon, 

Saskatchewan (SKSA)]. Seeds were planted in a randomized 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 conditions for their respective geographic 

regions. Seed samples were harvested from all plots and shipped at ambient temperature 

from the field sites to Monsanto 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 Covance 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 calculation, 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, manganese, phosphorus, potassium, sodium, and 

zinc) in the grain. The toxicants assessed in grain included erucic acid and glucosinolates 

(alkyl glucosinolates, indolyl glucosinolates, and total glucosinolates). 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 acetone wash prior to 

analysis. The fibrous residue that is primarily cellulose, lignin, and insoluble protein 

complexes remained in the Ankom filter bag, and were determined gravimetrically. 

(Komarek et al., 1994; USDA, 1970). The results are reported on fresh weight basis. 

The limit of quantitation was 0.100%. 
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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 glutamic 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. The sulfur-containing amino 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 acid 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 Monohydrate, 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 furnace at 550 °C and ignited. The nonvolatile 

matter remaining was quantified gravimetrically and calculated to determine percent ash 

(AOAC, 2005b). The limit of quantitation was 0.100%. 
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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 fresh 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 thimble containing sodium sulfate and dried to 

remove excess moisture. Pentane was dripped through the sample to remove the fat. The 

extract was then evaporated, dried, and weighed (AOAC, 2005c). The results are 

reported on fresh weight basis. The 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 methanol. The resulting methyl esters of the 

fatty acids were extracted with heptane containing an internal standard. The methyl 

esters of the fatty acids were analyzed by gas chromatography 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 Linolenate, used as 100%, Lot Number U-63M08-T 

 Nu Chek Prep Methyl Tridecanoate, used 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, used as 100%, Lot Number N-24M-S8-T 

 Nu Chek Prep Methyl Docosapentaenoate, used as 100%, Lot Number U-101MD4-T 

 Nu Chek Prep Methyl Docosahexaenoate, used as 100%, Lot Number U-84MJA15-U 
Monsanto Company 11-CA-233U 254 of 500 

 Nu Chek Prep Methyl Eicosapentaenoate, used as 100%, Lot Number U-99MS22-T 

 Nu Chek Prep Methyl Nervonate, used as 100%, Lot Number U-88M-MA31-U 

 Cayman Chemicals Steariodonic Acid Methyl Ester, 100%, Lot Number 0407775 

*Overall purity of the sum of the mixture of components was used as 100% 

E.4.7. Glucosinolates 

Glucosinolates were extracted using 70% methanol at 75 °C. They were then purified 

and enzymatically desulfatated on ion-exchange 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 standards. Peak identification was made based on retention 

times determined by comparing the chromatograms of internal standard(s) and three BCR 

certified oilseed rape controls (ISO, 1992). The results are reported on fresh weight 

basis. The limit of quantitation 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 dryness, 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, measured on the inductively coupled plasma 

spectrometer, with the emission 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 Standards 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 weight 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 and insoluble protein fraction was left in the 

filter bag and determined gravimetrically (AACC, 1998; Komarek et al., 1994; USDA, 

1970). The results are reported on fresh weight basis. The 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-performance 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 basis. 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 0.400 
Monsanto 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 sulfuric acid containing a catalyst mixture. The acid digest was 

made alkaline. The ammonia was distilled and then titrated with a previously 

standardized acid. The percent nitrogen was calculated and converted to equivalent 

protein using the factor 6.25 (AOAC, 2005g; AOCS, 1998). The results are reported on 

fresh weight basis. The limit of quantitation was 0.100%. 

E.4.13. Sinapic Acid 

The ground sample was extracted with methanol followed by alkaline hydrolysis and 

buffering prior to injection on an analytical high-performance liquid chromatography 

(HPLC) system for quantification of sinapic 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 products and moisture. Protein content was 

determined for one of the duplicates; ash content was determined for the other. The total 

dietary fiber in the sample was calculated using protein and ash values (AOAC, 2005h). 

The results were reported on fresh weight basis. 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 and 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 weight basis. The limit of quantitation was 

0.500 mg/100g. 

Reference Standard: 

USP, α-Tocopherol, 98.9%, Lot Number N0F068 
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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 (Chesterfield, 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 Statistical Analysis of Composition Data 

Component From (X) To Formula1

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/(104d)

Calcium, Magnesium, Phosphorus, 

Potassium, Sodium ppm fw g/100g dw X/(104

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)Xj/X, for each FAj

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 greater than the assay limit of 

quantitation (LOQ). Components with more than 50% of observations below the assay 

LOQ were excluded from summaries 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 lauric acid, 14:0 myristic acid, 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 docosapentaenoic acid, 22:6 docosahexaenoic acid, and 

sodium. 

If less than 50% of the observations for a component were below the LOQ, individual 

analyses that were below the LOQ were assigned 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 

Residual Sum of Squares) calculation. A PRESS residual is the difference between any 

value and its value predicted from a statistical 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 commercial 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 total fat value from one commercial reference 

at the MBNW site were extreme data points that were outside the  6 studentized PRESS 

residual range and were removed from the statistical analysis. 
Monsanto Company 11-CA-233U 259 of 500 

All canola components were statistically analyzed using a mixed model analysis of 

variance. The five replicated field sites were analyzed individually and as a combined 

data set. Individual replicated site analyses used model (1). 

(1) Yij = U + Ti + Bj + eij, 

where Yij = unique individual observation, U = overall mean, Ti = substance effect, 

Bj = random block effect, and eij = residual error. 

Combined-site analyses used model (2). 

(2) Yijk = U + Ti + Lj + B(L)jk + LTij + eijk, 

where Yijk = unique individual observation, U = overall mean, Ti = substance effect, 

Lj = random site effect, B(L)jk = random block within site effect, LTij = random site by 

substance interaction effect, and eijk = 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 specified proportion, p, of an entire 

sampled population for the parameter measured. The calculated tolerance intervals are 

expected to contain, with 95% confidence, 99% of the quantities expressed in the 

population of conventional canola. Each tolerance interval estimate 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 generate 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 

Table E-3. Statistical Summary of Site MBNW Canola Seed Nutrient Content for MON 88302 vs. the Con

 Difference (MON 88302 minus Control)

Analytical Component (Units)1

MON 883022 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence 

Interval3

Significan

(p-Value

Proximate (% dw)Ash 3.98 (0.087) 3.84 (0.10) 0.14 (0.13) -0.28, 0.56 0.367 

(3.72 - 4.10) (3.66 - 4.03) (0.076 - 0.41) 

Carbohydrates 27.18 (0.29) 26.02 (0.33) 1.16 (0.40) -0.13, 2.45 0.063 

(26.75 - 28.02) (25.81 - 26.35) (1.08 - 1.67) 

Moisture (% fw) 5.26 (0.16) 4.90 (0.18) 0.36 (0.24) -0.39, 1.12 0.225 

(4.99 - 5.56) (4.69 - 5.13) (0.12 - 0.87) 

Protein 21.00 (0.62) 20.78 (0.71) 0.22 (0.94) -2.78, 3.22 0.830 

(19.68 - 22.64) (20.29 - 21.61) (-0.86 - 0.49) 

Total Fat 47.84 (0.47) 49.35 (0.54) -1.51 (0.72) -3.79, 0.77 0.125 

(46.87 - 49.26) (48.89 - 49.93) (-2.28 - -0.61) 

Fiber (% dw)Acid Detergent Fiber 16.26 (0.63) 14.93 (0.71) 1.32 (0.81) -1.25, 3.90 0.199 

(15.05 - 17.66) (13.64 - 16.34) (0.019 - 3.09) 
Monsanto Company 11-CA-233U 

Table 

E -3. Statistical Summary of Site MBNW Canola Seed Nutrient Content for MON 88302 vs. the Conv

(continued) 

 Difference (MON 88302 minus Control)

Analytical Component (Units)1

MON 883022 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence 

Interval3

Significance

(p-Value)

Fiber (% dw)Neutral Detergent Fiber 19.08 (0.75) 17.16 (0.87) 1.92 (1.07) -1.50, 5.33 0.171 

(17.16 - 21.36) (16.68 - 17.45) (-0.29 - 3.97) 

Total Dietary Fiber 22.93 (1.65) 19.30 (1.87) 3.63 (2.10) -3.05, 10.32 0.181 

(19.17 - 27.81) (15.21 - 22.29) (-0.29 - 7.36) 

Amino Acid (% dw)Alanine 0.95 (0.026) 0.95 (0.030) 0.0018 (0.040) -0.13, 0.13 0.967 

(0.88 - 1.02) (0.93 - 0.98) (-0.047 - 0.011) 

Arginine 1.35 (0.042) 1.37 (0.049) -0.019 (0.065) -0.23, 0.19 0.784 

(1.23 - 1.44) (1.36 - 1.38) (-0.12 - 0.025) 

Aspartic Acid 1.60 (0.057) 1.58 (0.066) 0.018 (0.087) -0.26, 0.30 0.846 

(1.44 - 1.72) (1.55 - 1.64) (-0.10 - 0.090) 

Cystine 0.52 (0.020) 0.51 (0.023) 0.0048 (0.031) -0.093, 0.10 0.886 

(0.48 - 0.59) (0.50 - 0.54) (-0.043 - 0.0090) 
Monsanto Company 11-CA-233U 

Table E-3. Statistical Summary of Site MBNW Canola Seed Nutrient Content for MON 88302 vs. the Conventi(continued) 

 Difference (MON 88302 minus Control)

Analytical Component (Units)1

MON 883022 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence 

Interval3

Significa

(p-Valu

Amino Acid (% dw)Glutamic Acid 3.68 (0.12) 3.71 (0.14) -0.030 (0.19) -0.64, 0.58 0.886 

(3.37 - 4.02) (3.64 - 3.84) (-0.27 - 0.043) 

Glycine 1.09 (0.028) 1.09 (0.032) 0.0020 (0.043) -0.13, 0.14 0.965 

(1.02 - 1.16) (1.06 - 1.12) (-0.046 - 0.014) 

Histidine 0.59 (0.017) 0.58 (0.019) 0.0052 (0.026) -0.076, 0.087 0.851 

(0.55 - 0.64) (0.57 - 0.60) (-0.023 - 0.0092) 

Isoleucine 0.87 (0.028) 0.86 (0.032) 0.010 (0.042) -0.12, 0.14 0.820 

(0.81 - 0.94) (0.82 - 0.90) (-0.029 - 0.0069) 

Leucine 1.51 (0.044) 1.51 (0.051) 0.00056 (0.067) -0.21, 0.21 0.993 

(1.40 - 1.62) (1.48 - 1.56) (-0.082 - 0.026) 

Lysine 1.31 (0.034) 1.28 (0.040) 0.033 (0.052) -0.13, 0.20 0.573 

(1.22 - 1.41) (1.25 - 1.32) (-0.030 - 0.057) 
Monsanto Company 11-CA-233U 

Table E-3. Statistical Summary of Site MBNW Canola Seed Nutrient Content for MON 88302 vs. the ConvenControl (continued) 

 Difference (MON 88302 minus Control)

Analytical Component (Units)1

MON 88302 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence 

Interval3

Significanc

(p-Value)

Amino Acid (% dw)Methionine 0.41 (0.012) 0.42 (0.014) -0.0029 (0.019) -0.063, 0.057 0.887 

(0.40 - 0.45) (0.40 - 0.44) (-0.038 - 0.0034) 

Phenylalanine 0.91 (0.025) 0.91 (0.029) 0.0043 (0.038) -0.12, 0.12 0.916 

(0.84 - 0.97) (0.90 - 0.93) (-0.053 - 0.019) 

Proline 1.27 (0.038) 1.24 (0.044) 0.029 (0.058) -0.16, 0.21 0.659 

(1.20 - 1.35) (1.20 - 1.29) (-0.030 - 0.036) 

Serine 0.96 (0.029) 0.95 (0.033) 0.0032 (0.044) -0.14, 0.14 0.945 

(0.87 - 1.03) (0.94 - 0.97) (-0.077 - 0.051) 

Threonine 0.94 (0.022) 0.94 (0.025) 0.0053 (0.034) -0.10, 0.11 0.884 

(0.88 - 0.98) (0.92 - 0.96) (-0.044 - 0.058) 

Tryptophan 0.21 (0.017) 0.21 (0.020) -0.0040 (0.026) -0.088, 0.080 0.889 

(0.17 - 0.26) (0.19 - 0.25) (-0.037 - -

0.0025)
Monsanto Company 11-CA-233U 

Table E-3. Statistical Summary of Site MBNW Canola Seed Nutrient Content for MON 88302 vs. the ConvenControl (continued) 

 Difference (MON 88302 minus Control)

Analytical Component (Units)1

MON 883022 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence 

Interval3

Significanc

(p-Value)

Amino Acid (% dw)Tyrosine 0.64 (0.015) 0.63 (0.017) 0.0046 (0.022) -0.066, 0.075 0.850 

(0.59 - 0.66) (0.63 - 0.64) (-0.037 - 0.016) 

Valine 1.12 (0.036) 1.10 (0.041) 0.021 (0.054) -0.15, 0.19 0.719 

(1.04 - 1.21) (1.05 - 1.15) (-0.032 - 0.014) 

Fatty Acid (% Total FA)16:0 Palmitic 4.10 (0.029) 4.00 (0.033) 0.10 (0.044) -0.039, 0.24 0.105 

(4.02 - 4.16) (3.97 - 4.06) (0.0047 - 0.18) 

16:1 Palmitoleic 0.21 (0.0031) 0.23 (0.0036) -0.022 (0.0044) -0.036, -0.0081 0.015 

(0.20 - 0.21) (0.22 - 0.23) (-0.028 - -0.015) 

18:0 Stearic 1.73 (0.039) 1.97 (0.045) -0.24 (0.060) -0.43, -0.049 0.028 

(1.64 - 1.87) (1.93 - 2.01) (-0.35 - -0.059) 

18:1 Oleic 63.40 (0.19) 65.71 (0.22) -2.30 (0.29) -3.24, -1.37 0.004 

(62.94 - 64.03) (65.55 - 65.93) (-3.00 - -1.52) 
Monsanto Company 11-CA-233U 

Table E-3. Statistical Summary of Site MBNW Canola Seed Nutrient Content for MON 88302 vs. the ConvenControl (continued) 

 Difference (MON 88302 minus Control)

Analytical Component (Units)1

MON 883022 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence 

Interval3

Significan

(p-Valu

Fatty Acid (% Total FA)18:2 Linoleic 19.27 (0.16) 17.89 (0.19) 1.38 (0.25) 0.59, 2.17 0.011 

(18.82 - 19.66) (17.70 - 18.17) (0.65 - 1.96) 

18:3 Linolenic 9.19 (0.091) 8.12 (0.10) 1.08 (0.14) 0.64, 1.52 0.004 

(8.88 - 9.42) (7.98 - 8.25) (0.76 - 1.43) 

20:0 Arachidic 0.52 (0.010) 0.56 (0.012) -0.042 (0.015) -0.091, 0.0064 0.069 

(0.50 - 0.54) (0.54 - 0.58) (-0.081 - -0.0032) 

20:1 Eicosenoic 1.08 (0.021) 1.03 (0.024) 0.055 (0.032) -0.046, 0.16 0.180 

(1.06 - 1.15) (1.00 - 1.08) (-0.016 - 0.064) 

22:0 Behenic 0.25 (0.0058) 0.26 (0.0067) -0.010 (0.0089) -0.038, 0.018 0.337 

(0.24 - 0.26) (0.24 - 0.27) (-0.030 - 0.00017) 

24:0 Lignoceric 0.15 (0.0053) 0.15 (0.0061) 0.0020 (0.0081) -0.024, 0.028 0.824 

(0.14 - 0.16) (0.15 - 0.15) (-0.0041 - 0.0063) 
Monsanto Company 11-CA-233U 

Table E-3. Statistical Summary of Site MBNW Canola Seed Nutrient Content for MON 88302 vs. the ConvenControl (continued) 

 Difference (MON 88302 minus Control)

Analytical Component (Units)1

MON 883022 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence 

Interval3

Significan

(p-Value

Fatty Acid (% Total FA)24:1 Nervonic 0.096 (0.027) 0.090 (0.031) 0.0065 (0.041) -0.13, 0.14 0.884 

(0.046 - 0.12) (0.046 - 0.12) (-0.069 - 0.072) 

MineralCalcium (g/100g dw) 0.48 (0.010) 0.44 (0.012) 0.038 (0.016) -0.012, 0.089 0.095 

(0.45 - 0.51) (0.43 - 0.46) (0.017 - 0.081) 

Copper (mg/kg dw) 3.72 (0.040) 3.41 (0.046) 0.32 (0.061) 0.12, 0.51 0.013 

(3.61 - 3.83) (3.36 - 3.44) (0.22 - 0.40) 

Iron (mg/kg dw) 42.22 (1.46) 46.51 (1.66) -4.30 (1.90) -10.33, 1.74 0.108 

(40.55 - 43.60) (41.65 - 51.30) (-8.99 - -1.10) 

Magnesium (g/100g dw) 0.34 (0.010) 0.33 (0.012) 0.0078 (0.016) -0.042, 0.057 0.651 

(0.31 - 0.35) (0.31 - 0.35) (0.0036 - 0.039) 

Manganese (mg/kg dw) 39.62 (1.70) 39.54 (1.96) 0.078 (2.60) -8.18, 8.34 0.977 

(35.28 - 43.84) (37.35 - 41.11) (-3.95 - 6.49) 
Monsanto Company 11-CA-233U 

Table E-3. Statistical Summary of Site MBNW Canola Seed Nutrient Content for MON 88302 vs the Convent(continued) 

 Difference (MON 88302 minus Control)

Analytical Component (Units)1

MON 883022 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence 

Interval3

Significan

(p-Value

MineralPhosphorus (g/100g dw) 0.72 (0.041) 0.72 (0.047) 0.0064 (0.062) -0.19, 0.20 0.925 

(0.60 - 0.78) (0.61 - 0.79) (-0.034 - 0.16) 

Potassium (g/100g dw) 0.56 (0.0096) 0.56 (0.011) -0.0054 (0.015) -0.052, 0.041 0.734 

(0.54 - 0.57) (0.54 - 0.58) (-0.0098 - 0.021) 

Zinc (mg/kg dw) 34.91 (1.09) 30.24 (1.26) 4.66 (1.66) -0.63, 9.95 0.067 

(32.40 - 37.15) (28.46 - 32.84) (-0.44 - 7.72) 

Vitamin (mg/100g dw)Vitamin E (

-tocopherol) 13.06 (0.31) 9.36 (0.36) 3.70 (0.48) 2.17, 5.23 0.004 

(12.22 - 13.47) (8.89 - 10.15) (3.07 - 4.46) 

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 genetically similar, conventional control. 5With 95% confidence, interval contains 99% of the values expressed in the population of commercial conventional references. Negatzero. 
Monsanto Company 11-CA-233U 

Table E-4. Statistical Summary of Site MBNW Canola Seed Anti-nutrient Content for MON 88302 vs. the Control 

 Difference (MON 88302 minus Control)

Analytical Component (Units)1

MON 883022 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence 

Interval3

Significan

(p-Value

Anti-nutrientAlkyl Glucosinolate (μmole/g dw) 5.19 (0.28) 5.43 (0.31) -0.23 (0.28) -1.13, 0.67 0.472 

(4.47 - 5.87) (4.85 - 6.16) (-0.38 - 0.091) 

Indolyl Glucosinolate (μmole/g dw) 4.23 (0.57) 4.01 (0.65) 0.22 (0.86) -2.53, 2.97 0.817 

(2.92 - 5.75) (2.92 - 5.31) (-1.32 - 2.83) 

Phytic Acid (% dw) 2.06 (0.17) 2.27 (0.20) -0.21 (0.26) -1.05, 0.63 0.489 

(1.73 - 2.46) (1.77 - 2.56) (-0.67 - 0.68) 

Sinapic Acid (% dw) 1.02 (0.014) 0.92 (0.014) 0.095 (0.0093) 0.066, 0.12 0.001 

(0.99 - 1.06) (0.92 - 0.94) (0.076 - 0.11) 

Total Glucosinolate (μmole/g dw) 9.60 (0.77) 9.61 (0.88) -0.014 (1.05) -3.35, 3.32 0.990 

(7.60 - 11.42) (8.44 - 11.56) (-1.54 - 2.98) 

1dw = dry weight. 2 MON 88302 treated with glyphosate 3Mean (S.E.) = least-square mean (standard error); CI = confidence interval. 4Control refers to the genetically similar, conventional control. 5With 95% confidence, interval contains 99% of the values expressed in the population of commercial conventional references. Negatzero. 
Monsanto Company 11-CA-233U 

Table E-5. Statistical Summary of Site MBPL Canola Seed Nutrient Content for MON 88302 vs. the C

 Difference (MON 88302 minus Control)

Analytical Component (Units)1

MON 883022 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence 

Interval3

Significan

(p-Value

Proximate (% dw)Ash 4.15 (0.070) 4.28 (0.080) -0.14 (0.089) -0.38, 0.11 0.201 

(3.99 - 4.38) (4.17 - 4.38) (-0.21 - 0.020) 

Carbohydrates 27.51 (0.48) 28.11 (0.56) -0.59 (0.74) -2.64, 1.45 0.466 

(26.55 - 28.81) (26.87 - 28.73) (-2.18 - 1.94) 

Moisture (% fw) 5.68 (0.12) 5.24 (0.14) 0.44 (0.19) -0.078, 0.95 0.077 

(5.45 - 5.93) (4.93 - 5.47) (0.12 - 0.67) 

Protein 23.70 (0.26) 23.46 (0.30) 0.23 (0.40) -0.87, 1.33 0.590 

(23.17 - 24.33) (23.03 - 24.12) (-0.95 - 1.30) 

Total Fat 44.66 (0.36) 44.20 (0.41) 0.46 (0.49) -0.91, 1.83 0.405 

(43.96 - 45.72) (43.65 - 44.85) (-0.84 - 1.11) 

Fiber (% dw)Acid Detergent Fiber 16.75 (0.70) 14.19 (0.73) 2.55 (0.46) 1.28, 3.83 0.005 

(15.17 - 18.19) (12.59 - 16.16) (2.03 - 3.57) 
Monsanto Company 11-CA-233U 

Table E-5. Statistical Summary of Site MBPL Canola Seed Nutrient Content for MON 88302 vs. the ConvenControl (continued) 

 Difference (MON 88302 minus Control)

Analytical Component (Units)1

MON 883022 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence 

Interval3

Significan

(p-Value

Fiber (% dw)Neutral Detergent Fiber 19.45 (0.50) 16.87 (0.57) 2.58 (0.66) 0.74, 4.43 0.017 

(18.35 - 20.02) (15.44 - 18.06) (1.50 - 4.43) 

Total Dietary Fiber 22.61 (1.30) 17.91 (1.50) 4.70 (1.99) -0.82, 10.23 0.077 

(18.67 - 24.98) (14.58 - 20.42) (3.52 - 9.96) 

Amino Acid (% dw)Alanine 1.06 (0.013) 1.04 (0.015) 0.017 (0.020) -0.040, 0.073 0.460 

(1.04 - 1.08) (1.02 - 1.09) (-0.049 - 0.059) 

Arginine 1.57 (0.033) 1.54 (0.039) 0.031 (0.051) -0.11, 0.17 0.578 

(1.51 - 1.64) (1.48 - 1.65) (-0.14 - 0.15) 

Aspartic Acid 1.84 (0.024) 1.79 (0.027) 0.047 (0.029) -0.034, 0.13 0.179 

(1.81 - 1.89) (1.73 - 1.85) (-0.023 - 0.086) 

Cystine 0.55 (0.016) 0.57 (0.019) -0.013 (0.025) -0.082, 0.056 0.625 

(0.50 - 0.59) (0.53 - 0.60) (-0.054 - 0.052) 
Monsanto Company 11-CA-233U 

Table E-5. Statistical Summary of Site MBPL Canola Seed Nutrient Content for MON 88302 vs. the ConventControl (continued) 

 Difference (MON 88302 minus Control)

Analytical Component (Units)1

MON 883022 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence 

Interval3

Significan

(p-Value

Amino Acid (% dw)Glutamic Acid 4.26 (0.084) 4.24 (0.097) 0.017 (0.13) -0.34, 0.37 0.903 

(4.15 - 4.41) (4.05 - 4.53) (-0.37 - 0.36) 

Glycine 1.23 (0.013) 1.21 (0.015) 0.013 (0.020) -0.043, 0.070 0.542 

(1.21 - 1.24) (1.19 - 1.26) (-0.047 - 0.044) 

Histidine 0.65 (0.0098) 0.65 (0.011) 0.0039 (0.015) -0.037, 0.045 0.806 

(0.63 - 0.67) (0.62 - 0.68) (-0.041 - 0.044) 

Isoleucine 0.99 (0.011) 0.98 (0.012) 0.0090 (0.016) -0.036, 0.054 0.609 

(0.95 - 1.01) (0.96 - 1.00) (-0.050 - 0.034) 

Leucine 1.73 (0.021) 1.71 (0.024) 0.027 (0.032) -0.063, 0.12 0.448 

(1.70 - 1.76) (1.66 - 1.78) (-0.076 - 0.086) 

Lysine 1.41 (0.019) 1.40 (0.022) 0.0064 (0.029) -0.073, 0.086 0.833 

(1.37 - 1.45) (1.36 - 1.45) (-0.067 - 0.086) 
Monsanto Company 11-CA-233U 

Table E-5. Statistical Summary of Site MBPL Canola Seed Nutrient Content for MON 88302 vs. the Conventi(continued) 

 Difference (MON 88302 minus Control)

Analytical Component (Units)1

MON 883022 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence 

Interval3

Significa

(p-Valu

Amino Acid (% dw)Methionine 0.46 (0.0083) 0.47 (0.0095) -0.0073 (0.013) -0.042, 0.028 0.593 

(0.43 - 0.48) (0.45 - 0.49) (-0.024 - 0.030) 

Phenylalanine 1.05 (0.011) 1.03 (0.013) 0.014 (0.017) -0.032, 0.060 0.443 

(1.04 - 1.05) (1.01 - 1.07) (-0.032 - 0.039) 

Proline 1.39 (0.033) 1.36 (0.038) 0.021 (0.050) -0.12, 0.16 0.696 

(1.32 - 1.47) (1.30 - 1.45) (-0.12 - 0.17) 

Serine 1.08 (0.016) 1.07 (0.018) 0.012 (0.018) -0.039, 0.063 0.559 

(1.05 - 1.09) (1.04 - 1.12) (-0.037 - 0.052) 

Threonine 1.06 (0.013) 1.02 (0.015) 0.031 (0.016) -0.013, 0.075 0.119 

(1.04 - 1.06) (0.99 - 1.07) (-0.0054 - 0.065) 

Tryptophan 0.25 (0.0075) 0.26 (0.0087) -0.011 (0.011) -0.043, 0.021 0.388 

(0.24 - 0.25) (0.25 - 0.27) (-0.014 - -0.0041) 
Monsanto Company 11-CA-233U 

Table E-5. Statistical Summary of Site MBPL Canola Seed Nutrient Content for MON 88302 vs. the Convent(continued) 

 Difference (MON 88302 minus Control)

Analytical Component (Units)1

MON 883022 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence 

Interval3

Significan

(p-Value

Amino Acid (% dw)Tyrosine 0.72 (0.0065) 0.71 (0.0069) 0.017 (0.0052) 0.0030, 0.032 0.028 

(0.72 - 0.73) (0.69 - 0.73) (0.0078 - 0.028) 

Valine 1.26 (0.014) 1.25 (0.016) 0.016 (0.021) -0.041, 0.074 0.474 

(1.21 - 1.29) (1.23 - 1.27) (-0.058 - 0.054) 

Fatty Acid (% Total FA)16:0 Palmitic 4.25 (0.031) 4.34 (0.035) -0.090 (0.047) -0.22, 0.040 0.126 

(4.20 - 4.29) (4.23 - 4.41) (-0.22 - 0.030) 

16:1 Palmitoleic 0.23 (0.0035) 0.25 (0.0040) -0.026 (0.0053) -0.040, -0.011 0.008 

(0.22 - 0.23) (0.24 - 0.26) (-0.031 - -0.015) 

18:0 Stearic 1.58 (0.023) 1.87 (0.026) -0.29 (0.028) -0.37, -0.22 <0.001 

(1.55 - 1.59) (1.79 - 1.93) (-0.34 - -0.22) 

18:1 Oleic 62.06 (0.11) 64.30 (0.13) -2.24 (0.13) -2.59, -1.88 <0.001 

(61.82 - 62.35) (64.19 - 64.56) (-2.40 - -1.84) 
Monsanto Company 11-CA-233U 

Table E-5. Statistical Summary of Site MBPL Canola Seed Nutrient Content for MON 88302 vs. the Conventi(continued) 

 Difference (MON 88302 minus Control)

Analytical Component (Units)1

MON 883022 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence 

Interval3

Significan

(p-Value

Fatty Acid (% Total FA)18:2 Linoleic 20.43 (0.088) 19.18 (0.099) 1.25 (0.11) 0.95, 1.55 <0.001 

(20.13 - 20.66) (19.01 - 19.26) (0.92 - 1.41) 

18:3 Linolenic 9.28 (0.085) 7.74 (0.091) 1.54 (0.073) 1.34, 1.74 <0.001 

(9.12 - 9.43) (7.52 - 7.92) (1.35 - 1.67) 

20:0 Arachidic 0.53 (0.0046) 0.60 (0.0049) -0.070 (0.0042) -0.082, -0.059 <0.001 

(0.52 - 0.54) (0.59 - 0.61) (-0.079 - -0.063) 

20:1 Eicosenoic 1.09 (0.0059) 1.08 (0.0068) 0.011 (0.0089) -0.014, 0.035 0.298 

(1.08 - 1.10) (1.06 - 1.09) (-0.013 - 0.042) 

22:0 Behenic 0.27 (0.0027) 0.30 (0.0032) -0.040 (0.0039) -0.050, -0.029 <0.001 

(0.26 - 0.27) (0.29 - 0.31) (-0.047 - -0.030) 

24:0 Lignoceric 0.16 (0.0046) 0.19 (0.0054) -0.023 (0.0068) -0.042, -0.0038 0.029 

(0.16 - 0.17) (0.18 - 0.19) (-0.024 - -0.022) 
Monsanto Company 11-CA-233U 

Table E-5. Statistical Summary of Site MBPL Canola Seed Nutrient content for MON 88302 vs. the Conventio(continued) 

 Difference (MON 88302 minus Control)

Analytical Component (Units)1

MON 883022 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence 

Interval3

Significan

(p-Value

Fatty Acid (% Total FA)24:1 Nervonic 0.13 (0.0066) 0.16 (0.0076) -0.033 (0.010) -0.061, -0.0047 0.031 

(0.12 - 0.13) (0.15 - 0.17) (-0.052 - -0.014) 

MineralCalcium (g/100g dw) 0.36 (0.0051) 0.34 (0.0059) 0.021 (0.0078) -0.00057, 0.043 0.053 

(0.35 - 0.37) (0.32 - 0.34) (0.0015 - 0.035) 

Copper (mg/kg dw) 3.47 (0.082) 3.97 (0.094) -0.50 (0.12) -0.84, -0.15 0.016 

(3.35 - 3.56) (3.68 - 4.18) (-0.83 - -0.23) 

Iron (mg/kg dw) 44.13 (0.64) 51.01 (0.73) -6.87 (0.90) -9.38, -4.37 0.001 

(42.80 - 45.09) (49.75 - 52.89) (-9.20 - -4.80) 

Magnesium (g/100g dw) 0.41 (0.0070) 0.41 (0.0081) -0.0029 (0.011) -0.033, 0.027 0.797 

(0.39 - 0.42) (0.40 - 0.42) (-0.022 - 0.021) 

Manganese (mg/kg dw) 39.33 (0.90) 37.78 (1.03) 1.55 (1.37) -2.25, 5.34 0.321 

(37.24 - 41.46) (36.29 - 39.99) (-1.82 - 4.38) 
Monsanto Company 11-CA-233U 

Table E-5. Statistical Summary of Site MBPL Canola Seed Nutrient Content for MON 88302 vs. the Conventio(continued) 

 Difference (MON 88302 minus Control)

Analytical Component (Units)1

MON 883022 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence 

Interval3

Significan

(p-Valu

MineralPhosphorus (g/100g dw) 0.78 (0.0092) 0.81 (0.011) -0.031 (0.014) -0.069, 0.0077 0.090 

(0.75 - 0.80) (0.80 - 0.82) (-0.033 - -0.0099) 

Potassium (g/100g dw) 0.70 (0.025) 0.77 (0.027) -0.068 (0.019) -0.12, -0.015 0.023 

(0.63 - 0.76) (0.77 - 0.81) (-0.097 - -0.017) 

Zinc (mg/kg dw) 31.25 (0.49) 33.88 (0.56) -2.63 (0.75) -4.70, -0.56 0.024 

(30.45 - 32.05) (32.82 - 35.76) (-4.50 - -1.02) 

Vitamin (mg/100g dw)Vitamin E (

-tocopherol) 11.50 (0.24) 7.63 (0.27) 3.88 (0.36) 2.87, 4.89 <0.001 

(10.70 - 12.20) (7.50 - 7.72) (3.20 - 4.23) 

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 genetically similar, conventional control. 5With 95% confidence, interval contains 99% of the values expressed in the population of commercial conventional references. Negati
Monsanto Company 11-CA-233U 

Table E-6. Statistical Summary of Site MBPL Canola Seed Anti-nutrient Content for MON 88302 vControl 

 Difference (MON 88302 minus Control)

Analytical Component (Units)1

MON 883022 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence 

Interval3

Significan

(p-Value

Anti-nutrientAlkyl Glucosinolate (μmole/g dw) 2.98 (0.64) 5.03 (0.74) -2.05 (0.98) -4.76, 0.66 0.103 

(1.91 - 4.03) (3.06 - 6.50) (-2.68 - -1.15) 

Indolyl Glucosinolate (μmole/g dw) 3.90 (0.69) 4.29 (0.79) -0.39 (0.93) -2.98, 2.20 0.697 

(1.67 - 5.76) (3.26 - 5.89) (-1.59 - 0.96) 

Phytic Acid (% dw) 2.36 (0.066) 2.39 (0.076) -0.027 (0.10) -0.31, 0.25 0.803 

(2.19 - 2.58) (2.35 - 2.41) (-0.15 - 0.17) 

Sinapic Acid (% dw) 0.97 (0.0080) 0.86 (0.0090) 0.10 (0.010) 0.076, 0.13 <0.001 

(0.95 - 0.99) (0.86 - 0.86) (0.085 - 0.12) 

Total Glucosinolate (μmole/g dw) 7.01 (1.30) 9.40 (1.49) -2.39 (1.83) -7.45, 2.68 0.261 

(3.66 - 9.77) (6.42 - 12.59) (-2.82 - -0.44) 

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 genetically similar, conventional control. 5With 95% confidence, interval contains 99% of the values expressed in the population of commercial conventional references. Negat
Monsanto Company 11-CA-233U 

Table E-7. Statistical Summary of Site MNCA Canola Seed Nutrient Content for MON 88302 vs. the Con

 Difference (MON 88302 minus Control)

Analytical Component (Units)1

MON 883022 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence 

Interval3

Significan

(p-Value

Proximate (% dw)Ash 4.35 (0.23) 4.18 (0.20) 0.17 (0.30) -0.60, 0.94 0.591 

(4.20 - 4.45) (3.76 - 5.10) (0.33 - 0.64) 

Carbohydrates 27.31 (0.35) 25.99 (0.30) 1.32 (0.46) 0.13, 2.51 0.035 

(26.27 - 27.90) (25.57 - 26.55) (-0.29 - 1.92) 

Moisture (% fw) 5.52 (0.13) 6.69 (0.11) -1.17 (0.17) -1.60, -0.74 <0.001 

(5.37 - 5.61) (6.33 - 6.98) (-1.53 - -0.72) 

Protein 22.00 (0.70) 23.23 (0.61) -1.23 (0.81) -3.30, 0.85 0.189 

(21.51 - 22.03) (21.50 - 24.27) (-2.29 - 0.53) 

Total Fat 46.04 (0.72) 46.59 (0.69) -0.55 (0.46) -1.72, 0.62 0.280 

(45.76 - 47.55) (45.26 - 48.05) (-0.78 - -0.21) 

Fiber (% dw)Acid Detergent Fiber 17.89 (0.87) 17.66 (0.75) 0.23 (1.15) -2.73, 3.20 0.847 

(15.99 - 20.24) (16.11 - 18.71) (-2.71 - 2.09) 
Monsanto Company 11-CA-233U 

Table E-7. Statistical Summary of Site MNCA Canola Seed Nutrient Content for MON 88302 vs. the Convent(continued) 

 Difference (MON 88302 minus Control)

Analytical Component (Units)1

MON 883022 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence 

Interval3

Significan

(p-Value

Fiber (% dw)Neutral Detergent Fiber 19.55 (0.82) 18.78 (0.71) 0.76 (1.08) -2.01, 3.54 0.511 

(17.90 - 21.19) (17.83 - 19.58) (-1.56 - 3.36) 

Total Dietary Fiber 20.18 (1.55) 19.75 (1.35) 0.44 (2.06) -4.85, 5.72 0.839 

(16.91 - 22.24) (17.40 - 23.00) (-0.49 - 3.79) 

Amino Acid (% dw)Alanine 0.98 (0.027) 1.05 (0.023) -0.070 (0.032) -0.15, 0.014 0.084 

(0.97 - 0.98) (0.98 - 1.10) (-0.12 - -0.014) 

Arginine 1.40 (0.054) 1.53 (0.048) -0.13 (0.063) -0.29, 0.029 0.089 

(1.38 - 1.39) (1.40 - 1.65) (-0.27 - -0.010) 

Aspartic Acid 1.59 (0.067) 1.79 (0.058) -0.20 (0.082) -0.41, 0.0098 0.057 

(1.57 - 1.60) (1.61 - 1.97) (-0.37 - -0.045) 

Cystine 0.57 (0.025) 0.55 (0.022) 0.015 (0.031) -0.064, 0.094 0.650 

(0.53 - 0.58) (0.52 - 0.61) (0.012 - 0.035) 
Monsanto Company 11-CA-233U 

Table E-7. Statistical Summary of Site MNCA Canola Seed Nutrient Content for MON 88302 vs. the Conventi(continued) 

 Difference (MON 88302 minus Control)

Analytical Component (Units)1

MON 883022 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence 

Interval3

Significan

(p-Valu

Amino Acid (% dw)Glutamic Acid 3.89 (0.15) 4.25 (0.13) -0.36 (0.17) -0.80, 0.083 0.091 

(3.84 - 3.87) (3.98 - 4.45) (-0.59 - -0.11) 

Glycine 1.12 (0.034) 1.22 (0.030) -0.099 (0.044) -0.21, 0.014 0.073 

(1.11 - 1.12) (1.13 - 1.30) (-0.18 - -0.0061) 

Histidine 0.62 (0.018) 0.65 (0.016) -0.031 (0.022) -0.087, 0.025 0.214 

(0.61 - 0.62) (0.61 - 0.67) (-0.058 - -0.00063) 

Isoleucine 0.90 (0.027) 0.97 (0.024) -0.076 (0.031) -0.16, 0.0044 0.059 

(0.88 - 0.90) (0.89 - 1.03) (-0.13 - -0.0068) 

Leucine 1.56 (0.052) 1.70 (0.045) -0.15 (0.064) -0.31, 0.015 0.066 

(1.55 - 1.55) (1.58 - 1.80) (-0.25 - -0.034) 

Lysine 1.37 (0.032) 1.40 (0.029) -0.033 (0.036) -0.13, 0.059 0.397 

(1.35 - 1.36) (1.35 - 1.44) (-0.055 - -0.0095) 
Monsanto Company 11-CA-233U 

Table E-7. Statistical Summary of Site MNCA Canola Seed Nutrient Content for MON 88302 vs. the Convent(continued) 

 Difference (MON 88302 minus Control)

Analytical Component (Units)1

MON 883022 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence 

Interval3

Significan

(p-Value

Amino Acid (% dw)Methionine 0.44 (0.020) 0.44 (0.017) 0.0058 (0.024) -0.056, 0.067 0.819 

(0.43 - 0.45) (0.42 - 0.48) (0.011 - 0.019) 

Phenylalanine 0.93 (0.030) 1.02 (0.026) -0.089 (0.036) -0.18, 0.0043 0.057 

(0.92 - 0.93) (0.94 - 1.08) (-0.17 - -0.011) 

Proline 1.36 (0.045) 1.40 (0.040) -0.040 (0.053) -0.18, 0.096 0.479 

(1.29 - 1.39) (1.30 - 1.46) (-0.16 - 0.076) 

Serine 0.98 (0.038) 1.08 (0.033) -0.095 (0.050) -0.22, 0.033 0.114 

(0.98 - 0.99) (1.02 - 1.16) (-0.17 - -0.037) 

Threonine 0.95 (0.027) 1.02 (0.024) -0.073 (0.031) -0.15, 0.0074 0.067 

(0.94 - 0.94) (0.97 - 1.06) (-0.12 - -0.027) 

Tryptophan 0.22 (0.020) 0.24 (0.017) -0.022 (0.026) -0.089, 0.044 0.426 

(0.18 - 0.24) (0.22 - 0.27) (-0.063 - 0.024) 
Monsanto Company 11-CA-233U 

Table E-7. statistical Summary of Site MNCA Canola Seed Nutrient Content for MON 88302 vs. the Convent(continued) 

 Difference (MON 88302 minus Control)

Analytical Component (Units)1

MON 883022 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence 

Interval3

Significan

(p-Value

Amino Acid (% dw)Tyrosine 0.65 (0.019) 0.71 (0.017) -0.061 (0.024) -0.12, 0.0017 0.054 

(0.64 - 0.65) (0.66 - 0.75) (-0.11 - -0.0055) 

Valine 1.15 (0.031) 1.24 (0.027) -0.091 (0.035) -0.18, -0.00082 0.048 

(1.13 - 1.15) (1.15 - 1.31) (-0.16 - -0.017) 

Fatty Acid (% Total FA)16:0 Palmitic 4.27 (0.051) 4.14 (0.045) 0.13 (0.061) -0.029, 0.28 0.090 

(4.27 - 4.28) (4.07 - 4.19) (0.087 - 0.21) 

16:1 Palmitoleic 0.21 (0.0030) 0.24 (0.0026) -0.026 (0.0039) -0.036, -0.016 0.001 

(0.21 - 0.21) (0.23 - 0.25) (-0.039 - -0.020) 

18:0 Stearic 1.67 (0.044) 1.86 (0.038) -0.19 (0.057) -0.33, -0.039 0.022 

(1.65 - 1.71) (1.78 - 1.92) (-0.26 - -0.074) 

18:1 Oleic 61.67 (0.59) 64.86 (0.52) -3.19 (0.69) -4.98, -1.41 0.005 

(61.70 - 61.87) (63.72 - 65.52) (-3.81 - -3.11) 
Monsanto Company 11-CA-233U 

Table E-7. Statistical Summary of Site MNCA Canola Seed Nutrient Content for MON 88302 vs. the Convent(continued) 

 Difference (MON 88302 minus Control)

Analytical Component (Units)1

MON 883022 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence 

Interval3

Significan

(p-Value

Fatty Acid (% Total FA)18:2 Linoleic 20.20 (0.24) 18.35 (0.21) 1.85 (0.30) 1.07, 2.63 0.001 

(20.00 - 20.32) (17.90 - 19.29) (1.86 - 2.42) 

18:3 Linolenic 9.79 (0.58) 8.40 (0.50) 1.39 (0.75) -0.54, 3.32 0.122 

(9.76 - 9.79) (8.16 - 8.64) (1.31 - 1.60) 

20:0 Arachidic 0.53 (0.013) 0.57 (0.012) -0.042 (0.018) -0.088, 0.0027 0.060 

(0.52 - 0.54) (0.55 - 0.60) (-0.081 - -0.019) 

20:1 Eicosenoic 1.08 (0.012) 1.07 (0.011) 0.016 (0.016) -0.025, 0.058 0.354 

(1.06 - 1.09) (1.05 - 1.09) (-0.024 - 0.041) 

22:0 Behenic 0.27 (0.0056) 0.27 (0.0049) 0.0022 (0.0074) -0.017, 0.021 0.776 

(0.27 - 0.28) (0.26 - 0.29) (-0.021 - 0.016) 

24:0 Lignoceric 0.11 (0.033) 0.14 (0.029) -0.032 (0.044) -0.15, 0.081 0.499 

(0.049 - 0.16) (0.049 - 0.19) (-0.14 - 0.069) 
Monsanto Company 11-CA-233U 

Table E-7. Statistical Summary of Site MNCA Canola Seed Nutrient Content for MON 88302 vs. the Convent(continued) 

 Difference (MON 88302 minus Control)

Analytical Component (Units)1

MON 883022 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence 

Interval3

Significan

(p-Value

Fatty Acid (% Total FA)24:1 Nervonic 0.10 (0.025) 0.099 (0.021) 0.0042 (0.032) -0.079, 0.088 0.902 

(0.049 - 0.15) (0.049 - 0.12) (-0.072 - 0.062) 

MineralCalcium (g/100g dw) 0.47 (0.016) 0.45 (0.014) 0.018 (0.022) -0.038, 0.074 0.438 

(0.46 - 0.47) (0.42 - 0.49) (-0.0037 - 0.053) 

Copper (mg/kg dw) 4.40 (0.074) 4.11 (0.064) 0.28 (0.093) 0.046, 0.52 0.027 

(4.16 - 4.57) (4.06 - 4.18) (0.056 - 0.39) 

Iron (mg/kg dw) 42.57 (1.72) 50.64 (1.53) -8.07 (1.87) -12.89, -3.25 0.007 

(40.56 - 44.18) (46.23 - 54.03) (-12.92 - -4.82) 

Magnesium (g/100g dw) 0.38 (0.0074) 0.37 (0.0067) 0.014 (0.0076) -0.0049, 0.034 0.113 

(0.36 - 0.40) (0.36 - 0.38) (0.0074 - 0.018) 

Manganese (mg/kg dw) 38.70 (2.68) 40.94 (2.32) -2.24 (3.54) -11.34, 6.86 0.554 

(37.83 - 39.93) (33.70 - 46.19) (-8.36 - -2.11) 
Monsanto Company 11-CA-233U 

Table E-7. Statistical Summary of Site MNCA Canola Seed Nutrient Content for MON 88302 vs. the Convent(continued) 

 Difference (MON 88302 minus Control)

Analytical Component (Units)1

MON 883022 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence 

Interval3

Significan

(p-Value

MineralPhosphorus (g/100g dw) 0.81 (0.039) 0.79 (0.034) 0.022 (0.044) -0.090, 0.13 0.633 

(0.74 - 0.87) (0.72 - 0.93) (-0.011 - 0.098) 

Potassium (g/100g dw) 0.65 (0.027) 0.64 (0.023) 0.012 (0.035) -0.079, 0.10 0.746 

(0.58 - 0.70) (0.60 - 0.72) (-0.020 - 0.098) 

Zinc (mg/kg dw) 39.18 (2.73) 35.29 (2.41) 3.90 (3.15) -4.21, 12.00 0.271 

(35.19 - 45.56) (32.63 - 36.66) (-1.44 - 10.33) 

Vitamin (mg/100g dw)Vitamin E (

-tocopherol) 13.39 (0.48) 10.82 (0.42) 2.57 (0.57) 1.11, 4.03 0.006 

(12.58 - 14.62) (10.15 - 11.77) (2.18 - 3.99) 

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 genetically similar, conventional control. 5With 95% confidence, interval contains 99% of the values expressed in the population of commercial conventional references. Negat
Monsanto Company 11-CA-233U 

Table E-8. Statistical Summary of Site MNCA Canola Seed Anti-nutrient Content for MON 88302 vControl 

 Difference (MON 88302 minus Control)

Analytical Component (Units)1

MON 883022 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence 

Interval3

Significan

(p-Value

Anti-nutrientAlkyl Glucosinolate (μmole/g dw) 4.64 (0.62) 4.88 (0.54) -0.24 (0.79) -2.28, 1.80 0.775 

(4.35 - 4.88) (2.92 - 6.16) (-1.48 - 1.43) 

Indolyl Glucosinolate (μmole/g dw) 4.17 (0.52) 4.35 (0.45) -0.19 (0.69) -1.96, 1.59 0.798 

(3.79 - 4.47) (3.28 - 5.66) (-1.42 - 0.13) 

Phytic Acid (% dw) 2.28 (0.12) 2.36 (0.10) -0.078 (0.15) -0.47, 0.31 0.630 

(2.14 - 2.47) (2.15 - 2.77) (-0.011 - 0.12) 

Sinapic Acid (% dw) 1.06 (0.015) 0.96 (0.014) 0.10 (0.015) 0.063, 0.14 0.001 

(1.02 - 1.08) (0.94 - 0.97) (0.082 - 0.12) 

Total Glucosinolate (μmole/g dw) 9.08 (0.69) 9.42 (0.61) -0.33 (0.81) -2.41, 1.74 0.696 

(8.35 - 9.36) (7.15 - 10.65) (-1.29 - 1.19) 

1dw = dry weight. 2 MON 88302 treated with glyphosate. 3Mean (S.E.) = least-square mean (standard error); CI = confidence interval. 4Control refers to the genetically similar, conventional control. 5With 95% confidence, interval contains 99% of the values expressed in the population of commercial conventional references. Negat
Monsanto Company 11-CA-233U 

Table E-9. Statistical Summary of Site NDVA Canola Seed Nutrient Content for MON 88302 vs. the C

 Difference (MON 88302 minus Control)

Analytical Component (Units)1

MON 883022 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence 

Interval3

Significan

(p-Value

Proximate (% dw)Ash 3.31 (0.31) 3.25 (0.26) 0.063 (0.40) -1.22, 1.35 0.886 

(3.31 - 3.31) (3.20 - 3.35) (0.11 - 0.11) 

Carbohydrates 24.01 (0.95) 25.49 (0.78) -1.48 (1.22) -5.37, 2.42 0.314 

(21.83 - 26.20) (24.69 - 26.01) (-4.18 - 0.45) 

Moisture (% fw) 5.72 (0.23) 5.61 (0.20) 0.12 (0.22) -0.58, 0.82 0.633 

(5.72 - 6.08) (5.24 - 6.18) (-0.10 - 0.090) 

Protein 24.66 (0.74) 26.12 (0.61) -1.46 (0.96) -4.52, 1.60 0.226 

(23.33 - 25.98) (25.33 - 27.02) (-1.99 - -0.028) 

Total Fat 48.04 (0.44) 45.17 (0.36) 2.87 (0.57) 1.07, 4.67 0.014 

(47.20 - 48.87) (44.77 - 45.78) (1.42 - 4.10) 

Fiber (% dw)Acid Detergent Fiber 15.20 (0.37) 15.22 (0.31) -0.016 (0.39) -1.26, 1.23 0.969 

(14.53 - 15.86) (14.88 - 15.35) (-0.62 - 0.52) 
Monsanto Company 11-CA-233U 

Table E-9. Statistical Summary of Site NDVA Canola Seed Nutrient Content for MON 88302 vs. the Conventi(continued) 

 Difference (MON 88302 minus Control)

Analytical Component (Units)1

MON 883022 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence 

Interval3

Significan

(p-Value

Fiber (% dw)Neutral Detergent Fiber 17.32 (0.81) 17.74 (0.66) -0.42 (1.05) -3.76, 2.93 0.718 

(15.91 - 18.74) (17.16 - 18.65) (-2.74 - 1.58) 

Total Dietary Fiber 18.71 (1.39) 17.17 (1.15) 1.54 (1.65) -3.70, 6.79 0.418 

(17.08 - 21.08) (14.88 - 19.61) (-0.30 - 1.47) 

Amino Acid (% dw)Alanine 1.08 (0.042) 1.14 (0.034) -0.061 (0.054) -0.23, 0.11 0.344 

(1.01 - 1.15) (1.11 - 1.19) (-0.10 - 0.031) 

Arginine 1.60 (0.076) 1.72 (0.062) -0.12 (0.098) -0.43, 0.19 0.316 

(1.47 - 1.72) (1.68 - 1.77) (-0.21 - 0.030) 

Aspartic Acid 1.79 (0.067) 1.83 (0.054) -0.040 (0.086) -0.31, 0.23 0.675 

(1.65 - 1.93) (1.79 - 1.89) (-0.14 - 0.12) 

Cystine 0.70 (0.040) 0.71 (0.033) -0.018 (0.046) -0.16, 0.13 0.715 

(0.64 - 0.73) (0.66 - 0.79) (-0.028 - 0.037) 
Monsanto Company 11-CA-233U 

Table E-9. Statistical Summary of Site NDVA Canola Seed Nutrient Content for MON 88302 vs. the Conventi(continued) 

 Difference (MON 88302 minus Control)

Analytical Component (Units)1

MON 883022 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence 

Interval3

Significan

(p-Value

Amino Acid (% dw)Glutamic Acid 4.66 (0.25) 5.04 (0.20) -0.38 (0.32) -1.41, 0.64 0.319 

(4.25 - 5.06) (4.92 - 5.26) (-0.68 - 0.13) 

Glycine 1.24 (0.048) 1.33 (0.039) -0.094 (0.062) -0.29, 0.10 0.229 

(1.16 - 1.32) (1.30 - 1.38) (-0.15 - 0.0093) 

Histidine 0.72 (0.034) 0.75 (0.027) -0.032 (0.043) -0.17, 0.11 0.509 

(0.67 - 0.77) (0.73 - 0.78) (-0.065 - 0.032) 

Isoleucine 1.01 (0.041) 1.07 (0.034) -0.067 (0.053) -0.24, 0.10 0.299 

(0.94 - 1.08) (1.04 - 1.12) (-0.099 - 0.0095) 

Leucine 1.77 (0.073) 1.88 (0.059) -0.11 (0.094) -0.40, 0.19 0.336 

(1.64 - 1.90) (1.83 - 1.95) (-0.19 - 0.051) 

Lysine 1.55 (0.066) 1.60 (0.054) -0.054 (0.085) -0.32, 0.22 0.566 

(1.46 - 1.63) (1.58 - 1.65) (-0.12 - 0.052) 
Monsanto Company 11-CA-233U 

Table E-9. Statistical Summary of Site NDVA Canola Seed Nutrient Content for MON 88302 vs. the Conventi(continued) 

 Difference (MON 88302 minus Control)

Analytical Component (Units)1

MON 883022 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence 

Interval3

Significan

(p-Value

Amino Acid (% dw)Methionine 0.52 (0.019) 0.53 (0.016) -0.0059 (0.020) -0.068, 0.056 0.782 

(0.49 - 0.54) (0.50 - 0.56) (-0.013 - 0.0079) 

Phenylalanine 1.04 (0.038) 1.11 (0.031) -0.066 (0.049) -0.22, 0.091 0.272 

(0.97 - 1.11) (1.08 - 1.15) (-0.12 - 0.031) 

Proline 1.59 (0.065) 1.68 (0.053) -0.086 (0.083) -0.35, 0.18 0.377 

(1.46 - 1.71) (1.62 - 1.73) (-0.16 - 0.041) 

Serine 1.09 (0.036) 1.16 (0.029) -0.064 (0.046) -0.21, 0.083 0.258 

(1.05 - 1.14) (1.13 - 1.18) (-0.12 - 0.0094) 

Threonine 1.05 (0.035) 1.09 (0.029) -0.037 (0.045) -0.18, 0.11 0.470 

(0.99 - 1.11) (1.06 - 1.12) (-0.065 - 0.020) 

Tryptophan 0.26 (0.023) 0.26 (0.019) 0.0021 (0.025) -0.079, 0.083 0.938 

(0.25 - 0.26) (0.21 - 0.31) (0.0061 - 0.036) 
Monsanto Company 11-CA-233U 

Table E-9. Statistical Summary of Site NDVA Canola Seed Nutrient Content for MON 88302 vs. the Conventi(continued) 

 Difference (MON 88302 minus Control)

Analytical Component (Units)1

MON 883022 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence 

Interval3

Significan

(p-Value

Amino Acid (% dw)Tyrosine 0.71 (0.023) 0.74 (0.019) -0.031 (0.029) -0.13, 0.062 0.364 

(0.67 - 0.75) (0.72 - 0.77) (-0.060 - 0.025) 

Valine 1.29 (0.050) 1.36 (0.041) -0.073 (0.064) -0.28, 0.13 0.340 

(1.21 - 1.37) (1.32 - 1.41) (-0.12 - 0.020) 

Fatty Acid (% Total FA)16:0 Palmitic 3.98 (0.065) 3.95 (0.053) 0.036 (0.084) -0.23, 0.30 0.699 

(3.95 - 4.02) (3.94 - 3.96) (-0.016 - 0.083) 

16:1 Palmitoleic 0.20 (0.0057) 0.22 (0.0046) -0.025 (0.0068) -0.047, -0.0031 0.036 

(0.20 - 0.20) (0.22 - 0.23) (-0.029 - -0.023) 

18:0 Stearic 1.77 (0.033) 2.11 (0.027) -0.34 (0.042) -0.47, -0.20 0.004 

(1.71 - 1.84) (2.10 - 2.12) (-0.41 - -0.28) 

18:1 Oleic 65.14 (0.35) 68.38 (0.29) -3.24 (0.37) -4.43, -2.05 0.003 

(64.90 - 65.20) (68.11 - 68.44) (-3.24 - -3.21) 
Monsanto Company 11-CA-233U 

Table E-9. Statistical Summary of Site NDVA Canola Seed Nutrient Content for MON 88302 vs. the Conventi(continued) 

 Difference (MON 88302 minus Control)

Analytical Component (Units)1

MON 883022 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence 

Interval3

Significan

(p-Value

Fatty Acid (% Total FA)18:2 Linoleic 17.86 (0.31) 15.71 (0.25) 2.15 (0.36) 1.00, 3.29 0.009 

(17.78 - 18.02) (15.72 - 15.77) (2.06 - 2.26) 

18:3 Linolenic 8.82 (0.070) 7.31 (0.057) 1.51 (0.091) 1.22, 1.80 <0.001 

(8.71 - 8.94) (7.19 - 7.40) (1.35 - 1.54) 

20:0 Arachidic 0.57 (0.0047) 0.65 (0.0046) -0.082 (0.0023) -0.089, -0.074 <0.001 

(0.56 - 0.57) (0.64 - 0.65) (-0.085 - -0.077) 

20:1 Eicosenoic 1.15 (0.011) 1.16 (0.0088) -0.0098 (0.014) -0.054, 0.035 0.531 

(1.13 - 1.17) (1.15 - 1.18) (-0.042 - 0.020) 

22:0 Behenic 0.27 (0.0036) 0.30 (0.0029) -0.029 (0.0044) -0.043, -0.015 0.007 

(0.27 - 0.27) (0.30 - 0.30) (-0.031 - -0.027) 

24:0 Lignoceric 0.17 (0.053) 0.16 (0.043) 0.012 (0.068) -0.20, 0.23 0.873 

(0.16 - 0.18) (0.049 - 0.22) (-0.045 - 0.11) 
Monsanto Company 11-CA-233U 

Table E-9. Statistical Summary of Site NDVA Canola Seed Nutrient Content for MON 88302 vs. the Conventi(continued) 

 Difference (MON 88302 minus Control)

Analytical Component (Units)1

MON 883022 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence 

Interval3

Significan

(p-Value

Fatty Acid (% Total FA)24:1 Nervonic 0.12 (0.028) 0.090 (0.023) 0.031 (0.037) -0.086, 0.15 0.460 

(0.11 - 0.13) (0.049 - 0.12) (-0.0061 - 0.081) 

MineralCalcium (g/100g dw) 0.34 (0.049) 0.31 (0.040) 0.030 (0.063) -0.17, 0.23 0.663 

(0.30 - 0.37) (0.28 - 0.34) (-0.038 - 0.068) 

Copper (mg/kg dw) 3.80 (0.092) 3.53 (0.076) 0.28 (0.11) -0.084, 0.64 0.092 

(3.72 - 3.89) (3.33 - 3.67) (0.16 - 0.22) 

Iron (mg/kg dw) 51.55 (4.68) 63.44 (3.82) -11.89 (6.04) -31.12, 7.34 0.143 

(46.78 - 56.32) (60.79 - 67.18) (-20.41 - -6.03) 

Magnesium (g/100g dw) 0.36 (0.012) 0.35 (0.0099) 0.0074 (0.016) -0.043, 0.058 0.668 

(0.34 - 0.37) (0.33 - 0.37) (-0.012 - 0.043) 

Manganese (mg/kg dw) 50.88 (6.56) 50.07 (5.45) 0.81 (7.57) -23.29, 24.90 0.921 

(46.85 - 51.55) (47.11 - 50.97) (-0.26 - 0.58) 
Monsanto Company 11-CA-233U 

Table E-9. Statistical Summary of Site NDVA Canola Seed Nutrient Content for MON 88302 vs. the Conventi(continued) 

 Difference (MON 88302 minus Control)

Analytical Component (Units)1

MON 883022 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence 

Interval3

Significan

(p-Value

MineralPhosphorus (g/100g dw) 0.57 (0.077) 0.57 (0.063) 0.0026 (0.094) -0.30, 0.30 0.979 

(0.56 - 0.59) (0.56 - 0.58) (0.0027 - 0.014) 

Potassium (g/100g dw) 0.48 (0.037) 0.54 (0.030) -0.053 (0.048) -0.20, 0.099 0.346 

(0.48 - 0.49) (0.53 - 0.54) (-0.053 - -0.048) 

Zinc (mg/kg dw) 30.46 (1.73) 32.41 (1.43) -1.94 (2.07) -8.52, 4.63 0.416 

(29.81 - 31.08) (28.89 - 34.23) (-3.15 - 0.93) 

Vitamin (mg/100g dw)Vitamin E (

-tocopherol) 15.89 (0.88) 9.43 (0.72) 6.45 (1.14) 2.82, 10.08 0.010 

(15.23 - 16.55) (8.46 - 10.20) (5.03 - 8.09) 

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 genetically similar, conventional control. 5With 95% confidence, interval contains 99% of the values expressed in the population of commercial conventional references. Negat
Monsanto Company 11-CA-233U 

Table E-10. Statistical Summary of Site NDVA Canola Seed Anti-nutrient Content for MON 88302 vControl 

 Difference (MON 88302 minus Control)

Analytical Component (Units)1

MON 883022 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence 

Interval3

Significan

(p-Value

Anti-nutrientAlkyl Glucosinolate (μmole/g dw) 3.90 (0.65) 4.22 (0.54) -0.32 (0.76) -2.73, 2.10 0.705 

(3.74 - 3.88) (2.45 - 5.22) (-1.34 - 1.29) 

Indolyl Glucosinolate (μmole/g dw) 4.51 (0.57) 3.40 (0.49) 1.11 (0.58) -0.74, 2.96 0.152 

(3.99 - 4.48) (1.83 - 4.23) (0.25 - 2.16) 

Phytic Acid (% dw) 1.58 (0.097) 1.59 (0.088) -0.0050 (0.082) -0.27, 0.26 0.954 

(1.41 - 1.57) (1.46 - 1.68) (-0.052 - -0.012) 

Sinapic Acid (% dw) 1.02 (0.027) 0.83 (0.025) 0.19 (0.018) 0.14, 0.25 0.001 

(1.00 - 1.04) (0.83 - 0.88) (0.18 - 0.21) 

Total Glucosinolate (μmole/g dw) 8.59 (1.20) 7.72 (1.00) 0.87 (1.33) -3.37, 5.10 0.560 

(7.96 - 8.57) (4.38 - 9.61) (-1.04 - 3.58) 

1dw = dry weight. 2 MON 88302 treated with glyphosate. 3Mean (S.E.) = least-square mean (standard error); CI = confidence interval. 4Control refers to the genetically similar, conventional control. 5With 95% confidence, interval contains 99% of the values expressed in the population of commercial conventional references. Negat
Monsanto Company 11-CA-233U 

Table E-11. Statistical Summary of Site SKSA Canola Seed Nutrient Content for MON 88302 vs. the Con

 Difference (MON 88302 minus Control)

Analytical Component (Units)1

MON 883022 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence 

Interval3

Significan

(p-Value

Proximate (% dw)Ash 3.95 (0.18) 4.01 (0.17) -0.058 (0.10) -0.34, 0.22 0.594 

(3.68 - 4.44) (3.70 - 4.33) (-0.20 - 0.12) 

Carbohydrates 23.46 (0.51) 25.05 (0.44) -1.59 (0.59) -3.23, 0.056 0.055 

(23.11 - 23.84) (23.91 - 26.81) (-2.97 - -0.39) 

Moisture (% fw) 4.36 (0.21) 4.69 (0.18) -0.33 (0.28) -1.12, 0.45 0.306 

(3.90 - 4.82) (4.41 - 4.88) (-0.75 - 0.010) 

Protein 23.82 (0.55) 22.14 (0.50) 1.68 (0.55) 0.15, 3.21 0.038 

(23.62 - 24.58) (21.03 - 24.16) (0.42 - 2.50) 

Total Fat 48.83 (0.65) 48.81 (0.59) 0.022 (0.64) -1.76, 1.80 0.974 

(47.91 - 49.22) (46.96 - 50.24) (-1.02 - 0.95) 

Fiber (% dw)Acid Detergent Fiber 10.40 (0.63) 9.85 (0.61) 0.54 (0.35) -0.42, 1.51 0.193 

(9.19 - 11.50) (8.94 - 10.78) (-0.011 - 1.18) 
Monsanto Company 11-CA-233U 

Table E-11. Statistical Summary of Site SKSA Canola Seed Nutrient Content for MON 88302 vs. the Convent(continued) 

 Difference (MON 88302 minus Control)

Analytical Component (Units)1

MON 883022 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence 

Interval3

Significan

(p-Value

Fiber (% dw)Neutral Detergent Fiber 11.61 (0.73) 12.59 (0.67) -0.98 (0.63) -2.72, 0.77 0.194 

(9.48 - 12.75) (11.56 - 13.91) (-2.08 - 0.013) 

Total Dietary Fiber 18.68 (0.54) 17.21 (0.48) 1.48 (0.56) -0.075, 3.03 0.057 

(17.17 - 19.24) (16.57 - 17.89) (0.60 - 2.52) 

Amino Acid (% dw)Alanine 1.05 (0.033) 1.01 (0.028) 0.040 (0.043) -0.080, 0.16 0.405 

(1.02 - 1.07) (0.93 - 1.10) (-0.031 - 0.069) 

Arginine 1.33 (0.052) 1.39 (0.045) -0.062 (0.069) -0.26, 0.13 0.420 

(1.27 - 1.38) (1.29 - 1.55) (-0.18 - -0.018) 

Aspartic Acid 1.45 (0.037) 1.53 (0.032) -0.085 (0.049) -0.22, 0.052 0.159 

(1.40 - 1.50) (1.46 - 1.65) (-0.15 - -0.084) 

Cystine 0.55 (0.022) 0.55 (0.019) -0.0025 (0.025) -0.072, 0.067 0.923 

(0.53 - 0.57) (0.49 - 0.62) (-0.050 - 0.053) 
Monsanto Company 11-CA-233U 

Table E-11. Statistical Summary of Site SKSA Canola Seed Nutrient Content for MON 88302 vs. the Convent(continued) 

 Difference (MON 88302 minus Control)

Analytical Component (Units)1

MON 883022 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence 

Interval3

Significan

(p-Value

Amino Acid (% dw)Glutamic Acid 3.84 (0.14) 3.98 (0.12) -0.14 (0.18) -0.64, 0.36 0.493 

(3.66 - 3.97) (3.65 - 4.39) (-0.50 - 0.094) 

Glycine 1.05 (0.023) 1.08 (0.020) -0.026 (0.030) -0.11, 0.058 0.436 

(1.02 - 1.07) (1.01 - 1.15) (-0.084 - -0.016) 

Histidine 0.59 (0.015) 0.61 (0.013) -0.016 (0.019) -0.070, 0.037 0.443 

(0.57 - 0.60) (0.57 - 0.65) (-0.058 - 0.0030) 

Isoleucine 0.91 (0.021) 0.90 (0.018) 0.011 (0.028) -0.067, 0.088 0.716 

(0.89 - 0.93) (0.84 - 0.96) (-0.049 - 0.041) 

Leucine 1.61 (0.045) 1.58 (0.039) 0.029 (0.059) -0.13, 0.19 0.644 

(1.55 - 1.65) (1.46 - 1.71) (-0.073 - 0.085) 

Lysine 1.33 (0.037) 1.37 (0.032) -0.034 (0.050) -0.17, 0.10 0.527 

(1.27 - 1.37) (1.30 - 1.48) (-0.12 - 0.024) 
Monsanto Company 11-CA-233U 

Table E-11. Statistical Summary of Site SKSA Canola Seed Nutrient Content for MON 88302 vs. the Conventi(continued) 

 Difference (MON 88302 minus Control)

Analytical Component (Units)1

MON 883022 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence 

Interval3

Significan

(p-Valu

Amino Acid (% dw)Methionine 0.45 (0.015) 0.44 (0.013) 0.0082 (0.016) -0.037, 0.054 0.643 

(0.44 - 0.47) (0.40 - 0.49) (-0.022 - 0.034) 

Phenylalanine 0.96 (0.023) 0.94 (0.020) 0.026 (0.031) -0.059, 0.11 0.443 

(0.93 - 0.98) (0.87 - 1.01) (-0.028 - 0.044) 

Proline 1.39 (0.035) 1.44 (0.030) -0.050 (0.046) -0.18, 0.077 0.335 

(1.34 - 1.42) (1.35 - 1.52) (-0.13 - 0.0026) 

Serine 0.97 (0.025) 0.99 (0.022) -0.022 (0.033) -0.11, 0.070 0.546 

(0.93 - 0.99) (0.94 - 1.06) (-0.067 - -0.0013) 

Threonine 0.89 (0.022) 0.93 (0.019) -0.038 (0.029) -0.12, 0.043 0.263 

(0.86 - 0.92) (0.88 - 0.99) (-0.072 - -0.031) 

Tryptophan 0.20 (0.0091) 0.22 (0.0082) -0.018 (0.0093) -0.043, 0.0084 0.134 

(0.19 - 0.22) (0.21 - 0.24) (-0.035 - 0.0041) 
Monsanto Company 11-CA-233U 

Table E-11. Statistical Summary of Site SKSA Canola Seed Nutrient Content for MON 88302 vs. the C(continued) 

 Difference (MON 88302 minus Control)

Analytical Component (Units)1

MON 883022 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence 

Interval3

Significan

(p-Value

Amino Acid (% dw)Tyrosine 0.64 (0.014) 0.64 (0.012) -0.0084 (0.018) -0.060, 0.043 0.672 

(0.61 - 0.65) (0.61 - 0.68) (-0.037 - 0.0079) 

Valine 1.16 (0.025) 1.15 (0.022) 0.0090 (0.034) -0.085, 0.10 0.801 

(1.13 - 1.19) (1.08 - 1.23) (-0.073 - 0.046) 

Fatty Acid (% Total FA)16:0 Palmitic 4.51 (0.047) 4.07 (0.042) 0.44 (0.049) 0.31, 0.58 <0.001 

(4.46 - 4.57) (4.05 - 4.10) (0.41 - 0.48) 

16:1 Palmitoleic 0.26 (0.0047) 0.25 (0.0043) 0.0062 (0.0044) -0.0061, 0.019 0.235 

(0.25 - 0.26) (0.24 - 0.25) (0.0043 - 0.0074) 

18:0 Stearic 1.66 (0.055) 2.08 (0.049) -0.42 (0.057) -0.58, -0.26 0.001 

(1.54 - 1.72) (1.91 - 2.19) (-0.48 - -0.35) 

18:1 Oleic 61.91 (0.55) 65.69 (0.51) -3.78 (0.47) -5.08, -2.48 0.001 

(60.51 - 62.29) (64.73 - 66.86) (-4.30 - -2.59) 
Monsanto Company 11-CA-233U 

Table E-11. Statistical Summary of Site SKSA Canola Seed Nutrient Content for MON 88302 vs. the Convent(continued) 

 Difference (MON 88302 minus Control)

Analytical Component (Units)1

MON 883022 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence 

Interval3

Significan

(p-Value

Fatty Acid (% Total FA)18:2 Linoleic 18.49 (0.37) 17.22 (0.34) 1.27 (0.34) 0.33, 2.20 0.019 

(18.08 - 19.48) (16.64 - 18.01) (0.40 - 1.90) 

18:3 Linolenic 10.78 (0.23) 8.38 (0.20) 2.40 (0.25) 1.72, 3.09 <0.001 

(10.39 - 11.23) (7.94 - 8.99) (2.08 - 2.64) 

20:0 Arachidic 0.54 (0.0079) 0.62 (0.0069) -0.082 (0.0090) -0.11, -0.057 <0.001 

(0.52 - 0.55) (0.59 - 0.63) (-0.091 - -0.070) 

20:1 Eicosenoic 1.24 (0.015) 1.13 (0.013) 0.11 (0.020) 0.052, 0.16 0.005 

(1.22 - 1.26) (1.10 - 1.17) (0.050 - 0.14) 

22:0 Behenic 0.28 (0.0031) 0.29 (0.0027) -0.0088 (0.0037) -0.019, 0.0014 0.073 

(0.28 - 0.29) (0.28 - 0.30) (-0.014 - 0.0025) 

24:0 Lignoceric 0.20 (0.029) 0.15 (0.026) 0.050 (0.030) -0.032, 0.13 0.163 

(0.20 - 0.23) (0.045 - 0.22) (-0.016 - 0.062) 
Monsanto Company 11-CA-233U 

Table E-11. Statistical Summary of Site SKSA Canola Seed Nutrient Content for MON 88302 vs. the Conventio(continued) 

 Difference (MON 88302 minus Control)

Analytical Component (Units)1

MON 883022 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence 

Interval3

Significa

(p-Valu

Fatty Acid (% Total FA)24:1 Nervonic 0.17 (0.020) 0.12 (0.018) 0.055 (0.021) -0.0021, 0.11 0.055 

(0.16 - 0.20) (0.045 - 0.16) (0.034 - 0.055) 

MineralCalcium (g/100g dw) 0.41 (0.015) 0.44 (0.013) -0.031 (0.020) -0.086, 0.025 0.199 

(0.40 - 0.42) (0.40 - 0.47) (-0.068 - 0.0021) 

Copper (mg/kg dw) 3.52 (0.16) 3.23 (0.15) 0.29 (0.18) -0.21, 0.78 0.181 

(3.27 - 3.85) (2.96 - 3.48) (-0.21 - 0.57) 

Iron (mg/kg dw) 63.21 (8.79) 59.66 (7.61) 3.56 (11.63) -28.72, 35.84 0.774 

(55.62 - 69.61) (50.11 - 77.74) (-13.33 - 14.87) 

Magnesium (g/100g dw) 0.36 (0.017) 0.36 (0.016) -0.0044 (0.014) -0.042, 0.033 0.764 

(0.31 - 0.39) (0.34 - 0.39) (-0.032 - 0.026) 

Manganese (mg/kg dw) 41.77 (2.44) 34.73 (2.12) 7.04 (3.03) -1.37, 15.45 0.080 

(38.56 - 47.24) (33.12 - 37.61) (0.95 - 12.63) 
Monsanto Company 11-CA-233U 

Table E-11. Statistical Summary of Site SKSA Canola Seed Nutrient Content for MON 88302 vs. the Convent(continued) 

 Difference (MON 88302 minus Control)

Analytical Component (Units)1

MON 883022 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence 

Interval3

Significan

(p-Value

MineralPhosphorus (g/100g dw) 0.68 (0.051) 0.74 (0.048) -0.057 (0.043) -0.18, 0.062 0.254 

(0.57 - 0.80) (0.67 - 0.78) (-0.095 - 0.040) 

Potassium (g/100g dw) 0.82 (0.030) 0.71 (0.030) 0.11 (0.012) 0.077, 0.14 <0.001 

(0.77 - 0.90) (0.67 - 0.80) (0.084 - 0.14) 

Zinc (mg/kg dw) 41.58 (2.06) 33.10 (1.90) 8.48 (1.84) 3.36, 13.60 0.010 

(39.33 - 45.49) (29.75 - 40.66) (4.84 - 11.44) 

Vitamin (mg/100g dw)Vitamin E (

-tocopherol) 1.49 (1.08) 6.91 (0.94) -5.43 (1.43) -9.40, -1.45 0.019 

(1.30 - 1.66) (3.33 - 9.22) (-6.92 - -1.67) 

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 genetically similar, conventional control. 5With 95% confidence, interval contains 99% of the values expressed in the population of commercial conventional references. Negat
Monsanto Company 11-CA-233U 

Table E-12. Statistical Summary of Site SKSA Canola Seed Anti-nutrient Content for MON 88302 vControl 

 Difference (MON 88302 minus Control)

Analytical Component (Units)1

MON 883022 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence 

Interval3

Significan

(p-Value

Anti-nutrientAlkyl Glucosinolate (μmole/g dw) 1.61 (0.71) 5.82 (0.63) -4.21 (0.78) -6.36, -2.06 0.005 

(1.19 - 2.17) (4.87 - 8.28) (-6.11 - -3.28) 

Indolyl Glucosinolate (μmole/g dw) 0.86 (0.34) 3.30 (0.32) -2.44 (0.29) -3.24, -1.64 0.001 

(0.49 - 1.31) (2.68 - 4.35) (-3.05 - -2.19) 

Phytic Acid (% dw) 1.58 (0.20) 1.95 (0.18) -0.37 (0.24) -1.03, 0.29 0.191 

(1.20 - 1.91) (1.69 - 2.20) (-0.62 - 0.22) 

Sinapic Acid (% dw) 0.22 (0.059) 0.81 (0.051) -0.60 (0.075) -0.80, -0.39 0.001 

(0.16 - 0.28) (0.65 - 0.95) (-0.76 - -0.49) 

Total Glucosinolate (μmole/g dw) 2.53 (1.02) 9.22 (0.93) -6.69 (1.02) -9.51, -3.86 0.002 

(1.73 - 3.51) (7.85 - 12.72) (-9.21 - -5.78) 

1dw = dry weight. 2 MON 88302 treated with glyphosate. 3Mean (S.E.) = least-square mean (standard error); CI = confidence interval. 4Control refers to the genetically similar, conventional control. 5With 95% confidence, interval contains 99% of the values expressed in the population of commercial conventional references. Nzero.
Monsanto Company 11-CA-233U 305 of 500 

References for Appendix E 

AACC. 1998. Official method 32-20. American Association of Cereal Chemists, St. Paul, 

Minnesota. 

AOAC. 2005a. AOAC official method 982.30. Association of Analytical Communities 

International, Gaithersburg, Maryland. 

AOAC. 2005b. AOAC official method 923.03. Association of Analytical Communities 

International, Gaithersburg, Maryland. 

AOAC. 2005c. AOAC official method 960.39 and 948.22. Association of Analytical 

Communities International, Gaithersburg, Maryland. 

AOAC. 2005d. AOAC official 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 official 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 official 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 and 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 gluconsinolates content. Part 1: Method using 

high-performance liquid chromatography. International Standards Organization, Geneva, 

Switzerland. 

Komarek, A.R., J.B. Robertson and P.J. Van Soest. 1994. Improved efficiency of ADF 

analysis using a filter bag procedure. Journal of Animal Science 72 114. 

Lehrfeld, J. 1989. High-performance liquid chromatography analysis of phytic acid on a 

pH-stable macroporous polymer column. Cereal Chemistry 66: 510-515. 

Lehrfeld, J. 1994. HPLC separation and quantitation of phytic acid and some inositol 

phosphates in foods: Problems and solutions. Journal of Agricultural and Food Chemistry

42: 2726-2731. 

McMurray, C.H., W.J. Blanchflower and D.A. Rice. 1980. Influence 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. Schreurs. 1985. Vitamin E composition of some 

seed oils as determined by high-performance liquid chromatography with fluorometric 

detection. Journal of Food Science 50: 121-124. 

USDA. 1970. Forage and fiber analyses (apparatus, reagents, procedures, and some 

applications). Agricultural 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 and Results for Seed Dormancy and 

Germination Assessment of MON 88302 

F.1. Materials 

Starting seed of MON 88302, the conventional 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 MON 88302 was confirmed by event-specific 

polymerase chain reaction analyses. 

F.3. Germination Testing Facility and Experimental Methods 

Seed dormancy and germination evaluations were conducted at BioDiagnostics, Inc. in 

River Falls, WI. The principal investigator is qualified to conduct seed dormancy and 

germination testing consistent with the standards established by the Association of 

Official Seed Analysts (AOSA), a seed trade association (AOSA, 2009a; AOSA, 2009b). 

Seed lots of MON 88302, the conventional control and four commercial reference 

varieties were tested under six different temperature 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 light and the alternating temperatures regimes 

were maintained for 16 h at the lower temperature 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 XTra (insecticide 20.7% thiamethoxam and 

fungicides 1.25% difenoconazole, 0.40% mefenoxam 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 seeds per entry were placed in a germination tray on moist 

blotters (one entry per germination tray) using a vacuum planting system. Four replicates 

of each entry of MON 88302, conventional control 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 characteristic 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). AOSA only provides guidelines (AOSA, 2009a) for testing seed 

under a recommended temperature (15 °C/25 °C). Additional temperature regimes were 
Monsanto 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, and 5/25 °C) were assessed 7 and 14 days 

after planting (DAP) and categorized as either germinated, dead, viable non-dormant or 

dormant. For the additional temperature regimes, no distinction was made between 

normal and abnormal germinated seed. Any seedling 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 categorized as “normal germinated” under the AOSArecommended temperature conditions may not be so at other temperatures. 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) germinated 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 seed were defined the same as in the AOSA 

temperature regime. 

F.4. Statistical Analysis 

Analysis of variance (ANOVA) was conducted according to a randomized complete 

block design with four replicates using SAS (version 9). MON 88302 was compared to 

the conventional control for germination characteristics. The level of statistical 

significance was predetermined to be 5% (α = 0.05). MON 88302 was not statistically 

compared to the commercial reference varieties and no comparisons were made across 

temperature regimes. The minimum and maximum mean values (reference range) were 

determined from the commercial reference varieties. 
Monsanto Company 11-CA-233U 309 of 500 

Table F-1. Starting Seed of MON 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 11263707 
Monsanto Company 11-CA-233U 

Table F-2. Dormancy and Germination Characteristics of MON 88302 and the Conventional Control See

Temperature Regimes and Seed Characteristics Evaluated

Evaluation AOSA 2, 3 Additional Temperatures 4 

Timing 1 15/25 °C 5, 15, 25, 30, 5/25 °C followed by 

15/25 °C 

7 DAP 5 Normal germinated 

Abnormal germinated 

Dea

d

Germinated Dead 

14 DAP 5 Dormant 4

Dead 

Germinated Dead 

21 DAP 5

No Data Collected 

Viable non-dormant Dormant Dea

d 1 Seed in the AOSA recommended (15/25 °C) temperature regime were evaluated 7 and 14 days after planting, while seed in the additi

were evaluated on Days 7, 14 and 21 days after planting. Ungerminated seeds in the AOSA temperature regime on day 14 were subj

to determine viability. Ungerminated seeds in the additional temperature regimes on day 14 were moved to the AOSA recommend

regime for 7 days (21 days after planting) and then subject to tetrazolium test (Tz) to determine viability. 

2 The Association of Official Seed Analysts. 3 AOSA Category definitions: Normal germinated seed: Seedlings that exhibited normal developmental characteristics and possesse

Abnormal germinated seed: Seedlings that could not be classified as normal germinated (i.e., insufficient root and shoot development, 

deep cracks or lesions or exhibited mechanical damage). Dead seed: Seeds that had visibly deteriorated and had become soft to t

seeds that when tested with Tz had a negative result. Dormant seed: Ungerminated seeds that when tested with Tz had a positive resul

4 Non-AOSA category definitions: Germinated: Any seedling with a radical of 1 mm or more was classified as germinated. 

Ungerminated seeds in the additional temperature regimes at day 14 that when moved to the AOSA temperature regime germinated. D

visibly deteriorated and had become soft to the touch or ungerminated seeds that when tested with Tz had a negative result. Dormant 

that when tested with Tz had a positive result. 

5 DAP; Days after planting. 
Monsanto Company 11-CA-233U 311 of 500 

References for Appendix F 

AOSA. 2009a. AOSA Rules for testing seeds. Volume 1. Principles and Procedures. 

Association of Official Seed Analysts, Lincoln, Nebraska. 

AOSA. 2009b. AOSA Rules for testing seeds. Volume 4. Seedling Evaluation. 

Association of Official Seed Analysts, Lincoln, Nebraska. 
Monsanto Company 11-CA-233U 312 of 500 

Appendix G: Materials, Methods and Individual Site Results from Phenotypic, 

Agronomic and Environmental Interaction Assessment of MON 88302 under 

Field Conditions 

G.1. Materials 

The materials for phenotypic assessment included: 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 starting seed lots, the presence or absence 

of the glyphosate-tolerance trait was confirmed 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 nine sites in Canada during 2009 

(Section VII, Table VII-3). These 17 locations provided a diverse range of 

environmental and agronomic conditions representative of commercial canola production 

areas in North America. The researchers at each field site were familiar with the growth, 

production and evaluation of canola 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 occurred. 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 ft2

 over an area for each plot of 551 to 754 ft2. At the remaining sites, the 

seeding rate was approximately 12 to 18 seeds per ft2 over an area for each plot of 85 to 

131 ft2

 (Table G-2). 

At sites without arthropod collection, each plot was planted with a single pass of the 

seeder drill. One additional seeder pass was 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 qualitative environmental interactions data were 

collected from the middle of the seeder pass in each plot. 

At sites with arthropod collection, each plot was planted with four passes of the seeder 

drill. Two additional seeder passes were planted at the 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 replicate 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 collection of more robust arthropod abundance data. 

The four seeder passes in each plot were dedicated to specific types of data collection. 

Seeder pass 1 was used as a buffer. Seeder pass 2 was designated for the collection of the 
Monsanto Company 11-CA-233U 313 of 500 

phenotypic and qualitative environmental interactions 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 

Material1 Material 

Type Phenotype Monsanto 

Seed Lot # Sites2

MON 88302 Test Glyphosate-tolerant 11225246 All sites 

Ebony Control Conventional 11225243 All sites 

Hyola 401 Reference Conventional 10001850 MNCA, NDBI3

, SKME 

46A65 Reference Conventional 11220680 

ABLE, MBBR, MNCA, MNPY3, 

MNRO, NDBI3

, NDCL, SKRA, 

SKSA3

, SKWA 

71-45 RR Reference Glyphosate-tolerant4

11225379 

MBBR, MBMI3

, MNRO, NDBI3, 

SKRA, SKSA3

In Vigor 5630 Reference Glufosinate-tolerant4

11225748 MBMI3

, NDBI3, SKWA 

71-30 CL Reference Conventional 10001852 ABLE, IARL, MNPY3, NDBO, SKSA3

71-40 CL Reference Conventional 11261966 SKRA 

Hyola 357 Magnum Reference Glyphosate-tolerant4 10001727 IARL, MBBR, MNPY3, NDBO 

In Vigor 5550 Reference Glufosinate-tolerant4

 10001855 MNPY3, NDCL, SKME 

In Vigor 5020 Reference Glufosinate-tolerant4 11225944 ABLE, MBNW, SKSA3

In Vigor 5030 Reference Glufosinate-tolerant4 11225943 SKRA, SKRO 

Python 1651H Reference Conventional 11225749 MBBR, NDBO, SKWA 

Q2 Reference Conventional 11220679 MBMI3

, MBNW, NDVA, SKRO, 

SKME 

45H73 Reference Conventional 10008476 MBMI3, MBNW, NDCL, SKRO 

Cropland 601 Reference Conventional 10001849 IARL 

In Vigor 8440 Reference Glufosinate-tolerant4 11225747 IARL 

50 Calibur Reference Glyphosate-tolerant4 11225744 NDCL 

72-55 RR Reference Glyphosate-tolerant4 11225751 NDBO, SKME 

HiLite 618 Reference Conventional 10001851 MNRO, NDVA 

52-41 RR Reference Glyphosate-tolerant4 11225752 NDVA, SKRO 

45H28 Reference Glyphosate-tolerant4

 11225746 MNCA 

30 Calibur Reference Glyphosate-tolerant4

 11225745 MNCA 

34-65 RR Reference Glyphosate-tolerant4

 10001423 MNRO, SKWA 

30-42 RR Reference Glufosinate-tolerant4 11225750 ABLE 

45H26 Reference Glyphosate-tolerant4

 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 = Otter 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; SKRO = Wallace, SK; SKSA = Rosthern, SK; SKWA = 

Viscount, SK. 

3

Sites with arthropod collections. 

4

Commercially available herbicide-tolerant canola varieties. 
Monsanto Company 11-CA-233U 

Table G-2. Field and Planting Information Site1

Planting Date2 

Planting 

rate 

(seeds/ft.2

)

Planting 

depth 

(inches) 

Plot 

area 

(ft.2

) ‡

Seeder passes/ 

plot Soil series, organic matter, pH 

ABLE 05/15/09 14.0 0.6 108 1 Morinville clay loam, 10%, 6.3 IARL 05/15/09 14.0 1.0 114 1 Barnes clay loam,.3%, 7.1 MBBR 05/21/09 18.0 0.4 85 1 Newdale clay loam, 5.5%, 7-8.4 MBMI 05/28/09 12.0 0.5 754 4 Ryerson clay loam, 5.4%, 7.3 MBNW 05/26/09 18.0 0.4 85 1 Read Rome clay loam, 5.2%, 7.2-7.9 MNCA 05/30/09 12.0 0.5 130 1 Sisseton Heimdal loam, 3%, 7 MNPY 05/21/09 12.0 0.8 751 4 Estherville loam, 3.4%, 6.4 MNRO 05/24/09 12.0 0.5 130 1 Aazdahl clay loam, 4-6%, 6.6-7.3 NDBI 05/22/09 14.0 0.8 665 4 Gardena silt loam, 4.5%, 6.8 NDBO 05/20/09 12.0 0.5 131 1 Svea-Barnes loam, 3.3%, 6.9 NDCL 05/15/09 14.0 1.0 114 1 Barnes clay loam , 4.5%, 7.1 NDVA 05/19/09 12.0 0.5 131 1 Williams loam, 2.4%, 5.4 SKME 05/28/09 15.0 0.8 106 1 Melfort clay loam, 9.1%, 6.5 SKRA 05/17/09 15.0 0.8 106 1 Weyburn loam, 3.3%, 7.5 SKRO 05/19/09 15.0 0.5 103 1 Canora silty clay loam, 6.5%, 7.7 SKSA 05/27/09 17.0 0.5 551 4 Hamlin loam , 2.19%, 6.35 SKWA 05/22/09 15.0 0.8 106 1 Weyburn loam, 4.6%, 6.7 1Site codes are as follows: ABCA = Camrose, AB; ABFS = Sturgeon, AB; ABLE = Leduc, AB; IARL = Brookings, SD; MBBR = E

Tail, MN; MBMI = Whitewater, MB; MBNW = Portage le Prairie, MB; MNPY = Stearns, MN; MNRO = Wilkins, MN; NDBI = G

McHenry, ND; NDCL = Brookings, SD; NDGF = Grand Forks, ND; NDVA = McHenry, ND; SKME = Flett’s Springs, SK; SKRA = = 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 characteristics measured and the designated developmental stages 

where observations occurred are listed in Table VII-1. 

G.6. Environmental Interaction Observations 

Environmental interactions (i.e., interactions between the crop plants and their receiving 

environment) were used to characterize MON 88302 by evaluating plant response to 

abiotic stress, disease damage, arthropod-related 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 evaluated at all 17 sites for differences in 

plant response to abiotic stressors, disease damage and arthropod-related damage. Three 

abiotic stressors, three diseases and three 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 stressors, 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 often varied between observations at a site and between 

sites. 

Abiotic stressor and disease damage observations were collected from a single seeder 

pass of each plot (sites without arthropod collection) and from seeder pass 2 of each plot 

(sites with arthropod collection) using a continuous 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 week 

later. At each assessment time, flea beetle damage was assessed by examining 10 nonsystematically selected plants from the middle of seeder pass 3 of each plot using a 0 - 10 

rating scale adapted from (Palaniswamy and 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 the middle of seeder pass 3 of each plot at the 

ripening stage. Seedpod weevil damage was assessed by counting the number of exit 

holes in each pod. 

G.8. Arthropod Abundance 

Pest and beneficial arthropods were collected at the MBMI, MNPY, NDBI and SKSA 

sites four times during the growing season 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 plants were folded in half), to collect the 

dislodged insects into the container. Arthropods were collected from five nonsystematically selected plants from the middle 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 Company, St. Louis, MO and Department of 

Agricultural, Food and Nutritional Science at University of Alberta, respectively for 

arthropod identification and enumeration. 

A maximum of the five most abundant pest and five most abundant beneficial arthropods 

were determined from four non-systematically 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 often 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 Interactions Evaluation Criteria 

For the qualitative assessments of abiotic stress response, disease damage, and arthropod 

damage MON 88302 and the conventional control were considered different in 

susceptibility or tolerance to an abiotic stressor, 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 control across all four replicates. These data 

are categorical and were not subjected to statistical 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 representative of commercially-released canola 

varieties. Arthropod abundance, flea beetle damage and seedpod weevil damage data 

were quantitatively evaluated and subjected to statistical analysis (Section G.11.). 

G.10. Data Assessment 

Experienced scientists familiar with the experimental design and evaluation criteria were 

involved in all components of data collection, summarization, and analysis. Study 

personnel assessed that measurements were taken properly, data were consistent with 

expectations based on experience 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 during the study that would impact the study 

objectives were noted. Data were then subjected to statistical analysis as indicated 

below. 

G.11. Statistical Analysis 

Analysis of variance (ANOVA) was conducted 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 site (individual site 

analysis) and across sites (combined-site analysis). 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 for each characteristic from the 24 unique 

commercial reference varieties that were included in this study. Data excluded from the 

study and the reasons for their exclusion are listed in Table G-3. Exclusion of these data 

did not adversely affect the quality of the study. 

An analysis of variance (ANOVA) was conducted according to a randomized complete 

block design using SAS (Version 9) for the flea beetle damage, seedpod weevil damage 

and the arthropod abundance. The level of statistical significance 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 damage and seedpod weevil damage data were 

pooled across sites (combined-site analysis) for a statistical comparison between 

MON 88302 and the conventional control. Minimum and maximum mean values were 
Monsanto Company 11-CA-233U 319 of 500 

calculated for flea beetle damage and seedpod weevil damage from 10 commercial 

reference varieties that were included at the MBMI, MNPY, NDBI and SKSA sites. The 

reference range for the arthropod 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. Exclusion of these data did not adversely affect the 

quality of the study. 

G.12. Individual Field Site Plant Growth, 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 rating) and NDBO (5.3 vs. 3.5 rating). 

MON 88302 reached days to first flowering later than the conventional 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). MON 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 conventional 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 lower 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 conventional 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 conventional control at the MBNW site (13.6 vs. 19.8 

plants/linear meter). 

The statistical differences detected in the individual site analyses for early stand count, 

seedling vigor, seed maturity, lodging, plant height, visual pod shattering, quantitative 

pod shattering, seed quality, yield and final stand counts were not detected in the 

combined-site analysis. Thus, the differences detected for these phenotypic 

characteristics are not indicative of a consistent response associated with the trait and are 

unlikely to be biologically meaningful in terms of increased weediness potential of 

MON 88302 compared to the conventional control (Figure VII-1, step 2, “no” answer). 
Monsanto Company 11-CA-233U 320 of 500 

While a statistical difference was detected for days to first flowering and seed moisture in 

both the individual site analysis 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 commercial reference varieties (Figure VII-1, step 3, 

“no” answer). Therefore, the difference in days to first flowering and seed moisture are 

unlikely to be biologically meaningful in terms of increased weediness potential. 

In the individual site analysis of arthropod damage among all 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 differences. 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 difference was not indicative of a consistent plant 

response associated with the trait and is unlikely to be biologically meaningful in terms of 

an adverse environmental impact of MON 88302 compared to conventional canola 

(Figure VII-1, step 2, “no” answer). 
Monsanto Company 11-CA-233U 

Table G-3 Data Missing or Excluded from Analysis Site1 Substance Rep Characteristic Reason for exclusion ABLE 46A65 3 Seed maturity Protocol revision to increase the precisiABLE MON 88302 1 & 2 Seed maturity Protocol revision to increase the precisiIARL All All Days to first flowering Data was not recorded correctly. IARL All All Visual pod shattering Data assessment added to the protoc

harvest was completed. 

IARL All All Abiotic stressors, disease and arthropod damage 

ratings 

Data not recorded. 

MBBR All All Seed maturity, lodging, plant height, visual pod 

shattering, seed moisture, seed quality, yield and final stand count

A hail storm destroyed the plants after f

MBBR All All Seedling vigor Data were not recorded or was recorded MBBR Ebony 2 All The results of the seeding quality te

control plot may have contained plants o

MBNW All All Seedling vigor Data were not recorded or were recordeMNCA All All Seedling vigor Data were not recorded or were recordeMNCA All All Abiotic stressors and 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 not recorded or were recordeMNRO All All Abiotic stressors and 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 protoc

harvest was completed. 

NDCL ALL ALL Abiotic stressors, disease and arthropod damage 

ratings 

Data not recorded. 
Monsanto Company 11-CA-233U 

Table G-3. Data Missing or Excluded from Analysis (continued)

Site1 Substance Rep Characteristic Reason for exclusion 

NDCL All All Days to first flowering Data was not recorded correctly. NDCL MON 88302 

& Ebony 

1 Seed maturity Protocol revision to increase the precisi

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 damage 

ratings 

First two of the four total ratings were n

SKSA All All Seedling vigor and seed moisture Data was not recorded correctly. SKSA All All Quantitative pod shattering Snow storm prevented the collection of SKSA All All First arthropod collection and second flea beetle 

assessment 

Weather prevented the collection of arof 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

MBMI=Whitewater, MB; MBNW=Portage le Prairie, MB; MNPY=Stearns, MN; MNRO=Wilkins, MN; NDBI=Grand Forks, ND; ND

NDCL=Brookings, SD; NDGF=Grand Forks, ND; NDVA=McHenry, ND; SKME=Flett’s Springs, SK; SKRA=Corman Park, SK; SKRSKSA=Rosthern, SK; SKWA=Viscount, SK. 
Monsanto Company 11-CA-233U 

Table G-4. Individual Site Phenotypic Comparison of MON 88302 to Conventional Control 

Phenotypic Characteristic (units)

Early stand count2

(number of plants per linear meter)

Seedling vigor3

(1-9 scale)

Days to f

(days a

MON 88302 Control MON 88302 Control MON 88302 

Site 1 Mean (S.E.) Mean (S.E.) Mean (S.E.) Mean (S.E.) Mean (S.E.) 

ABLE 12.8 (1.9) 18.3 (1.6) 6.3 (0.5) 5.5 (0.3) 61.0 (0.0) * 

IARL 21.7 (1.9) 21.5 (2.4) 6.0 (0.0) 6.0 (0.0)

–

MBBR 7.1 (1.1) 6.8 (0.6)

–

– 68.0 (0.0) * 

MBMI 17.8 (1.4) 19.1 (0.6) 4.0 (0.0)* 3.0 (0.0) 57.0 (0.0)* 

MBNW 14.8 (1.6) 20.1 (1.7)

–

– 62.0 (0.0) * 

MNCA 23.7 (1.2) 20.8 (2.2)

–

– 63.3 (0.8)* 

MNPY 26.8 (1.1) 24.5 (0.5) 6.0 (0.0)* 5.0 (0.0) 100.0 (0.0)* 

MNRO 22.6 (0.8) 20.2 (0.9)

–

– 52.0 (0.0) * 

NDBI 19.9 (1.5) 17.9 (1.7) 7.0 (0.0) 6.3 (0.5) 58.3 (0.5)* 

NDBO 17.8 (2.1) 16.6 (0.8) 5.3 (0.8)* 3.5 (0.3) 53.0 (0.0)* 

NDCL 23.0 (0.5) 24.9 (0.5) 7.0 (0.0) † 6.0 (0.0)

–

NDVA 14.3 (1.3) 13.2 (1.5) 6.5 (0.5) 5.8 (0.6) 54.0 (0.0)* 

SKME 23.6 (1.2)* 29.0 (1.7) 3.3 (0.5) 3.3 (0.3) 56.0 (0.0)* 

SKRA 22.0 (0.9) 21.8 (1.9) 3.3 (0.3) 3.0 (0.0) 54.0 (0.0) SKRO 8.8 (1.9) 10.6 (1.9) 3.0 (0.0) 3.5 (0.3) 59.0 (0.0)* 

SKSA 10.5 (1.1) 14.3 (0.8)

–

– 53.8 (0.3)* 

SKWA 23.0 (3.2) 23.3 (2.3) 3.5 (0.3) 3.0 (0.0) 61.0 (0.0)* 
Monsanto Company 11-CA-233U 

Table 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)

Plan

(

MON 88302 Control MON 88302 Control MON 88302 

Site 1 Mean (S.E.) Mean (S.E.) Mean (S.E.) Mean (S.E.) Mean (S.E.) ABLE 108.0 (0.0)† 108.0 (0.0) 1.0 (0.0)† 1.0 (0.0) 36.3 (0.8) IARL 90.3 (1.4) 88.8 (0.8) 1.0 (0.0)† 1.0 (0.0) 41.8 (1.0)* 

MBBR

–

–

–

–

–

MBMI 101.0 (0.0)† 101.0 (0.0) 1.0 (0.0) 2.0 (0.0) 52.5 (1.3) 

MBNW 105.0 (0.0) 104.0 (1.0) 1.0 (0.0) 1.8 (0.5) 54.5 (0.9) MNCA 101.0 (0.0)† 101.0 (0.0) 1.0 (0.0)† 1.0 (0.0) 51.3 (0.5)* MNPY 118.0 (0.0) 118.0 (0.0) 9.0 (0.0)† 9.0 (0.0)

–

MNRO 91.0 (0.0)† 86.0 (0.0) 1.0 (0.0)* 1.5 (0.3) 55.8 (0.5)* 

NDBI 104.0 (0.0) 101.8 (0.8) 3.3 (0.5)* 2.0 (0.4) 55.8 (1.8)* NDBO

–

– 1.0 (0.0) 1.0 (0.0) 42.8 (0.9) 

NDCL 92.3 (1.7) 92.3 (1.7) 1.0 (0.0) 1.5 (0.3) 36.3 (2.2) NDVA

–

– 1.3 (0.3) 1.0 (0.0) 45.0 (0.6) 

SKME

–

– 1.0 (0.0) 1.5 (0.3) 43.3 (0.5)* 

SKRA

–

– 1.0 (0.0)† 1.0 (0.0) 38.0 (0.8) 

SKRO 112.0 (0.0)† 112.0 (0.0) 1.0 (0.0) 1.0 (0.0) 39.0 (0.6) SKSA 106.5 (2.3)* 99.5 (1.9) 2.3 (0.3) 1.8 (0.3) 42.5 (0.5) SKWA

–

– 1.0 (0.0)† 1.0 (0.0) 47.0 (1.5) 
Monsanto Company 11-CA-233U 

Table 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 

MON 88302 Control MON 88302 Control MON 88302 

Site 1 Mean (S.E.) Mean (S.E.) Mean (S.E.) Mean (S.E.) Mean (S.E.) ABLE 1.0 (0.0)† 1.0 (0.0)

–

– 8.6 (0.1) 

IARL

–

–

–

– 14.9 (0.6)* 

MBBR

–

–

–

–

–

MBMI 1.0 (0.0) 1.0 (0.0) 155.9 (38.6)* 365.0 (53.7) 14.5 (0.4)* 

MBNW 1.0 (0.0) 1.0 (0.0)

–

– 11.8 (0.4)* 

MNCA 4.0 (0.4)* 4.8 (0.5)

–

– 13.2 (0.2)* 

MNPY 1.0 (0.0)† 1.0 (0.0) 18.4 (0.8) 12.6 (0.9) 16.0 (0.4)* MNRO 1.3 (0.3) 1.8 (0.3)

–

– 18.1 (0.1)* 

NDBI 1.0 (0.0) 1.0 (0.0) 19.9 (4.8) 20.0 (6.5) 10.3 (0.2) NDBO 1.0 (0.0) 1.0 (0.0)

–

– 24.2 (0.7)* 

NDCL

–

–

–

– 9.1 (0.4)* 

NDVA 1.0 (0.0) 1.5 (0.3)

–

– 14.4 (0.4)* 

SKME 1.0 (0.0)† 1.0 (0.0)

–

– 13.0 (0.4)* 

SKRA 1.0 (0.0)† 1.0 (0.0)

–

– 7.5 (0.2) 

SKRO 1.0 (0.0)† 1.0 (0.0)

–

– 15.1 (0.5)* 

SKSA 1.0 (0.0) 1.0 (0.0)

–

–

–

SKWA 1.0 (0.0)† 1.0 (0.0)

–

– 12.7 (0.2) 
Monsanto Company 11-CA-233U 

Table G-4. Individual Site Phenotypic Comparison of MON 88302 to Conventional Control (continued) 

Phenotypic Characteristic (units)

Seed quality11

(%)

Yield12

(bu per ac)

Final stand c

(number of stubs pe

MON 88302 Control MON 88302 Control MON 88302 

Site 1 Mean (S.E.) Mean (S.E.) Mean (S.E.) Mean (S.E.) Mean (S.E.) ABLE 0.8 (0.5) 0.5 (0.3) 29.5 (1.2) 26.6 (1.3) 13.8 (2.2) IARL 6.5 (1.6)* 1.8 (0.6) 21.0 (1.8) 26.9 (2.4) 21.7 (1.9) 

MBBR

–

–

–

–

–

MBMI 1.0 (1.0) 3.3 (1.7) 62.1 (3.5) 66.9 (2.3) 17.8 (1.4) 

MBNW 0.5 (0.3) 1.5 (0.6) 72.3 (4.9) 66.2 (4.0) 13.6 (1.1)* MNCA 3.3 (0.9) 3.0 (0.4) 19.4 (0.4)* 15.5 (0.7) 19.3 (0.6) MNPY 0.5 (0.3)* 0.0 (0.0) 24.7 (0.2) 23.8 (0.3) 26.7 (1.0) MNRO 11.3 (0.9)* 6.8 (1.4) 50.1 (1.2)* 43.6 (1.0) 20.6 (0.8) NDBI 0.3 (0.3) 0.3 (0.3) 64.5 (3.2) 67.9 (4.7) 19.1 (2.6) NDBO 8.5 (3.1) 4.5 (1.0) 29.6 (1.5) 35.1 (1.0) 17.4 (1.5) 
Monsanto Company 11-CA-233U 

Table G-4. Individual Site Phenotypic Comparison of MON 88302 to Conventional Control (continued) 

Phenotypic Characteristic (units)

Seed quality11

(%)

Yield12

(bu per ac)

Final stan

(number of stubs 

MON 88302 Control MON 88302 Control MON 88302 

Site 1 Mean (S.E.) Mean (S.E.) Mean (S.E.) Mean (S.E.) Mean (S.E.) NDCL 3.5 (0.6) 7.5 (3.1) 23.4 (2.1) 30.3 (3.5) 23.0 (0.5) NDVA 9.3 (0.9) 10.3 (2.1) 40.4 (2.0) 38.4 (2.8) 16.7 (1.0) 

SKME 1.3 (0.5) 0.0 (0.0) 45.3 (2.0) 52.7 (1.6) 20.9 (1.5) SKRA 0.5 (0.3) 0.8 (0.5) 47.4 (1.5) 47.7 (2.1) 22.4 (1.3) SKRO 3.8 (0.8) 2.5 (0.5) 58.1 (4.3) 66.6 (4.8) 9.1 (2.0) 

SKSA 0.5 (0.3) 0.0 (0.0) 39.4 (1.0) 41.2 (3.6) 10.5 (0.9) 

SKWA 1.0 (0.4) 2.5 (1.6) 60.3 (2.1) 62.6 (0.9) 19.7 (0.7) 

Note: The experimental design was a randomized complete block with four replicates. S.E. = Standard Error. The number of plotanalysis 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. 1 Site codes are as follows: ABCA=Camrose, AB; ABFS=Sturgeon, AB; ABLE=Leduc, AB; IARL=Brookings, SD; MBBR=Elton

MN; MBMI=Whitewater, MB; MBNW=Portage le Prairie, MB; MNPY=Stearns, MN; MNRO=Wilkins, MN; NDBI=Grand Forks, N

NDCL=Brookings, SD; NDGF=Grand Forks, ND; NDVA=McHenry, ND; SKME=Flett’s Springs, SK; SKRA=Corman Park, SSKSA=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 num

and 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 more flowe

data 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=3 for the conventional control. 
Monsanto Company 11-CA-233U 

5Seed maturity was determined as the number of days after planting when 30% or more of the seed in the lower third of the main ra

green to black/brown/tan color. Seed maturity data at ABLE plot number (n)=2 for MON 88302 and n=4 for the conventional cont(n)=3 for MON 88302 and the conventional control. 

6Rated on a 0 - 9 scale, where 0 = completely upright 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 th

increments up to 9 = greater than 80% shatter. 9Collection trays placed within the crop canopy and seed losses from shattering counted once per week for three weeks. This data wa

MBMI, 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=4 for the conventional control. 

11Seed quality was determined at harvest by counting the percentage of green seeds from a 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 MO

conventional 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 Evaluations 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 development 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 330 of 500 

Table G-6. Disease Damage Evaluations 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. 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 development 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 Damage 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 worms1 (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 development 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 caterpillars 
Monsanto Company 11-CA-233U 332 of 500 

Table G-8. Individual Site Analysis: Quantitative Assessment of Flea Beetle and 

Seedpod Weevil Damage to MON 88302 Compared to the Conventional Control in 

2009 Field Trials 

Pest Damage assessment Site1 MON 88302 Control 

 

Flea beetles2 

(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 weevil3

(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 rounding 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 emergence 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 

Table G-9. Abundance of Pest Arthropods in Cone Beat Sheet Samples Collected from MON 88302, C

and the Commercial Reference Varieties 

 Pest Arthropod2 Aphids 

(Aphididae) 

Bertha armyworms 

(Mamestra configurata) 

Diamondback mo

(Plutella xylos

Col. Site1 MON 88302 Mean (S.E.) Control Mean (S.E.) Reference Range3 MON 88302 Mean (S.E.) Control Mean (S.E.) Reference Range3 MON 88302 Mean (S.E.) Control Mean (S.E

1 MBMI 

−

−

−

−

−

− 1.00 (0.00) 0.50 (0.2

 MNPY 1.00 (1.00) 0.25 (0.25) 0.25 – 1.25 

−

−

−

−

−

 NDBI 

−

−

−

−

−

−

−

−

 SKSA 

−

−

−

−

−

−

−

−

2 MBMI 

−

−

−

−

−

− 0.25 (0.25) 0.50 (0.2

 MNPY 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 (0.2

 MNPY 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 (0.7

 MNPY 0.25 (0.25) 0.25 (0.25) 0.00 – 0.25 

−

−

− 0.25 (0.25) 0.25 (0.2

 NDBI 

−

−

−

−

−

−

−

−

 SKSA 

−

−

−

−

−

− 0.00 (0.00) 0.75 (0.4
Monsanto Company 11-CA-233U 

Table G-9. Abundance of Pest Arthropods in Cone Beat Sheet Samples Collected from MON 88302, C

and the Commercial Reference Varieties (continued) 

 Pest Arthropod2  Flea beetles 

(Chrysomelidae) 

Lygus bugs 

(Miridae) 

T

(Th

Col. Site1 MON 88302 Mean (S.E.) Control Mean (S.E.) Reference Range3 MON 88302 Mean (S.E.) Control Mean (S.E.) Reference Range3 MON 88302 Mean (S.E.) C

Mea

1 MBMI 

−

−

−

−

−

− 0.50 (0.50) 0.25 

 MNPY 

−

−

− 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 

 SKSA 

−

−

−

−

−

−

−

2 MBMI 

−

−

−

−

−

− 0.75 (0.75) 0.00 

 MNPY 

−

−

−

−

−

−

−

 NDBI 

−

−

−

−

−

− 0.25 (0.25) 0.75 

 SKSA 

−

−

− 0.00 (0.00) 0.00 (0.00) 0.00 – 0.00 8.25 (5.12) 5.00 

3 MBMI 0.25 (0.25) 0.00 (0.00) 0.00 – 0.25 

−

−

− 4.25 (2.46) 0.25 

 MNPY 

−

−

− 0.25 (0.25) 1.25 (0.63) 0.50 – 2.25 1.00 (0.71) 0.00 

 NDBI 0.50 (0.29) 2.00 (0.82) 0.00 – 0.75 

−

−

− 4.00 (1.68) 2.75 

 SKSA 

−

−

−

−

−

− 0.50 (0.50) 3.25 

4 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 

 MNPY 

−

−

− 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 

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 to first flowering stage and the three subsequent collections at approximately two week inte

arthropod 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 

Table G-10. Abundance of Beneficial Arthropods in Cone Beat Sheet Samples Collected from MON 

Control and the Commercial Reference Varieties 

 Beneficial Arthropod2 

Chironomid midge Lacewings (chrysopidae) Ladybir

(Cocci

Col. Site1 MON 88302 Mean (S.E.) Control Mean (S.E.) Reference Range3 MON 88302 Mean (S.E.) Control Mean (S.E.) Reference Range3 MON 88302 Mean (S.E.) Co

Mean 

1 MBMI 

−

−

−

−

−

−

−

 MNPY 

−

−

−

−

−

−

−

 NDBI 

−

−

−

−

−

−

−

 SKSA 

−

−

−

−

−

−

−

2 MBMI 

−

−

−

−

−

−

−

 MNPY 

−

−

−

−

−

−

−

 NDBI 

−

−

−

−

−

−

−

 SKSA 

−

−

−

−

−

− 0.00 (0.00) 0.00 

3 MBMI 

−

−

−

−

−

−

−

 MNPY 

−

−

−

−

−

− 0.00 (0.00) 0.00 

 NDBI 

−

−

−

−

−

−

−

 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 

Table G-10. Abundance of Beneficial Arthropods in Cone Beat Sheet Samples Collected from MON 

Control and the Commercial Reference Varieties (continued) 

 Beneficial Arthropod2  Micro-parasitic hymenoptera Macro-parasitic hymenoptera Oriu

Col. Site1 MON 88302 Mean (S.E.) Control Mean (S.E.) Reference Range3 MON 88302 Mean (S.E.) Control Mean (S.E.) Reference Range3 MON 88302 Mean (S.E.) Co

Mean 

1 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 

 SKSA 

−

−

−

−

−

−

−

3 MBMI 0.75 (0.75) 0.25 (0.25) 0.00 – 0.50 

−

−

−

−

 MNPY 

−

−

−

−

−

− 2.00 (1.08) 1.75 

 NDBI 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 

 NDBI 

−

−

−

−

−

−

−

 SKSA 0.00 (0.00) 0.25 (0.25) 0.00 – 0.25 

−

−

−

−
Monsanto Company 11-CA-233U 

Table G-10. Abundance of Beneficial Arthropods in Cone Beat Sheet Samples Collected from MON 

Control and the Commercial Reference Varieties (continued) 

 Beneficial Arthropod2

Spiders 

(Araneae) 

Sphecid wasps 

(Sphecidae) 

Col. Site1 MON 88302 Mean (S.E.) Control Mean (S.E.) Reference Range3 MON 88302 Mean (S.E.) Control Mean (S.E.) Reference Range3

1 MBMI 

−

−

−

−

−

−

 MNPY 

−

−

− 0.00 (0.00) 0.00 (0.00) 0.00 – 0.25 

 NDBI 

−

−

−

−

−

−

 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 to first flowering stage and the three subsequent collections at approximately two week inte

arthropod 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 Dakota State University 

Extension Service, Fargo, North Dakota. 

Palaniswamy, P. and R.J. Lamb. 1992. Screening for antixenosis resistance to flea 

beetles, Phyllotreta cruciferae (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 Methods for Pollen Morphology and Viability 

Assessment 

H.1. Plant Production 

MON 88302, a conventional control, and four commercial reference varieties 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% fludioxonil) at the recommended rate. 

Canola was seeded into 4” pots with Metro-Mix 200 containing 14 oz/yds3 Micromax 

soil medium. Three seeds of MON 88302, the 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 plant per pot when all of the plants had 

approximately one true leaf. Pots were placed 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 glyphosate herbicide. 

H.2. Flower Collection 

A total of three newly opened flowers were collected 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 solution (Alexander, 1980). The stain solution 

was prepared on the day anthers were collected with 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 stored for at least 24 h at approximately 4 °C 

prior to preparation 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 cross-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 viability, 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 running 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 imaging 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 solution, viable pollen 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 counted from a minimum of 75 pollen grains 

from a random field of view under the microscope. Dense clusters of pollen or pollen 

grains adhering to flower parts were not counted because they may not have absorbed the 

stain solution uniformly. 

H.4.2. Pollen Diameter 

Micrographs (400X resolution) of ten representative 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 values 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 micrographs that were used for pollen diameter 

measurements. 

H.5. Statistical Analysis 

Analysis of variance (ANOVA) was conducted 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 pollen and pollen grain diameter. MON 88302 

was not statistically different when compared to the commercial reference varieties. A 

reference range for each measured characteristic 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 11220680 
Monsanto Company 11-CA-233U 342 of 500 

References for Appendix H 

Alexander, M.P. 1980. A versatile 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-effective solution for broad spectrum weed control. 

The current Roundup Ready canola product RT73 has restrictions in application rates and timing 

which make the ability to control some important annual and perennial 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 significant 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 application up to first flower instead of 

the 6-leaf growth stage and provides tolerance that allows for 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-tolerant canola, which incorporates the higher application rates 

and later timing of application. Under the proposed expanded glyphosate use pattern with 

MON 88302, glyphosate residue levels fall within the U.S. EPA’s existing tolerance 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 been prepared to support an anticipated APHIS 

environmental assessment, evaluates two alternatives: the “deregulation in whole” alternative 

and the “no action” alternative. Under the deregulation in whole alternative, MON 88302 and 

any progeny derived from crosses between MON 88302 and conventional Brassica species, and 

crosses of MON 88302 with other biotechnology-derived 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 remain a regulated article and 

plantings could be conducted under APHIS notification or permit. Interstate movements and 

field trials of MON 88302 have been conducted 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 was unsuited for food or feed use because of the 

high erucic acid and high glucosinolate content. 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 primarily 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 the 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 late 
Monsanto Company 11-CA-233U 344 of 500 

spring or early summer. The remaining 4% of U.S. production is both winter and spring canola 

grown primarily in Idaho, Montana, Oregon, Colorado, Kansas and Washington. Winter canola 

has higher yield potential than spring canola, but can be planted 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 short 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. since 1999. In 2008 approximately 95% 

of the North Dakota canola crop was produced using 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 grown for sale for crop production for 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 Washington, Oregon, Idaho, North Dakota and Colorado. 

Specialty and Organic Canola Production: Specialty canola includes high erucic acid varieties 

used in industrial processes, organic, and varieties 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 conducted in 2008, there were approximately 232 acres of organic canola 

grown in the U.S., all grown in four states, none of which are major canola production states. As 

noted above, the vast majority of canola currently produced in the U.S. contains a herbicidetolerant trait. 

Other Brassica Production: Because of potential for cross 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 available information on acreage and areas of 

production does not differentiate areas of organic and non-organic production. 

The primary Brassica vegetable crops in the U.S. are B. napus (rutabaga and Siberian kale), 

Brassica oleracea (cabbage, broccoli, cauliflower, Brussels 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 harvested before they produce seed, 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 western Washington; and the Columbia Basin in 

eastern Washington. To maintain the desired vegetable 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 broccoli seed is grown primarily in 

California and Arizona. According to the 2007 Census 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 addition to canola include high erucic acid or 

industrial oilseed rape, mustard seed and biofumigant mustard seed. High erucic acid oilseed 

rape is grown on very limited acres in Washington, Oregon, and Idaho. These states all regulate 
Monsanto Company 11-CA-233U 345 of 500 

where canola and oilseed rape can be grown, to prevent 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 production typically 

involves the extensive use of agronomic inputs and cultivation practices to maximize 

productivity and grower profitability. 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 planted with glyphosate- and glufosinate-tolerant 

varieties and application of in-crop herbicides plus tillage practices are used to control weeds. In 

combination with appropriate agronomic practices, herbicide-tolerant canola volunteers can be 

controlled in the subsequent rotational crop with a variety of herbicides. 

While some of these weeds have developed resistance to other herbicides in canola production 

states, no glyphosate-resistant population of these weed species has been reported in the major 

U.S. canola production area in North Dakota/Minnesota. 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 rotational 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 managed agricultural settings and can interact with the environment 

directly, via pollen movement to sexually compatible plants and potential hybridization/gene 

introgression, or via persistence 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 another) cross pollination can occur at a rate of 

approximately 30%. Most canola pollen (98%) travels 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 possible, 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 minimizing cross pollination, hybridization and seed 

formation. Additionally, cross pollination between canola and vegetable seed crops, whether 

organic or non-organic, is expected to be non-existent or negligible given that there are strict 

controls on canola production in the major Brassica vegetable seed production areas (Oregon 

and Washington). In other Brassica vegetable seed production areas (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 potential for cross pollination to native species. 
Monsanto Company 11-CA-233U 346 of 500 

Cross Pollination Potential – Naturalized or Weedy Brassica2: There are several Brassica weed 

species in the U.S. and Canada, including several 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 very 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 the 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 temporary conditions such as disturbed areas. It is not considered 

to be a colonizing or invasive species 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 edges of roadways and other transportation 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 soil not yet colonized by 

grasses and other more competitive plants. 

Public Health, Worker Safety 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 limited use as a forage crop. Pesticide use on canola including 

glyphosate is regulated by the U.S. EPA, which 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 canola production since the introduction 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 might be affected by agricultural operations. 

Canola can interact with the environment directly, 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 draining the canola field are part of the affected 

environment. Plants on adjacent land may be affected by fertilizer runoff, water runoff, and/or 

herbicide drift. Threatened or endangered animal or plant species that may inhabit the 

surrounding area are also a part of the affected environment. Significant 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 increased in recent years, and may continue 

to increase based on U.S. demand exceeding U.S. production. Recent increases in canola 

production have resulted primarily from canola replacing wheat in wheat monocultures or 

cropping systems in which wheat is the primary crop, 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 currently 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 significant increases in conservation tillage 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 metric 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 Potential 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 Growers 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 and 

environmental 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 control 
Monsanto 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 production 
Monsanto Company 11-CA-233U 351 of 500 

I.2. Background and Rationale 

Monsanto Company has developed a second-generation glyphosate-tolerant canola 

product, MON 88302, designed to provide growers with improved weed control through 

greater flexibility and tolerance to higher rates of 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 CP4 EPSPS protein confers 

tolerance to the herbicide glyphosate, the active ingredient in the family of Roundup 

agricultural herbicides. 

I.3. Purpose and Need for Action 

I.3.1. Regulatory Authority 

“Protecting American agriculture" is the basic charge of the U.S. Department of 

Agriculture's (USDA) Animal and Plant Health 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 that all methods of agricultural production 

(conventional, organic, or the use of genetically engineered varieties) 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 known as the Coordinated Framework for the Regulation of 

Biotechnology. This document specifies three Federal 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 biotechnology-derived organisms under the Plant 

Protection Act of 2000 (PPA).3

 U.S. FDA regulates biotechnology-derived food products 

under the authority of the Federal Food, Drug, and Cosmetic Act (FFDCA).4

 The U.S. 

FDA policy statement concerning regulation of products derived from new plant 

varieties, including those genetically engineered, was published in the Federal Register 

on May 29, 19925

. Under this policy, U.S. FDA uses a consultation process to ensure 

that human food and animal feed safety, nutrition 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 protectants 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- 23005 
Monsanto 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 herbicides under FIFRA, and establishes 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 environment. 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 tolerance has been established. 

Residue tolerances for pesticides are established by U.S. EPA under the FFDCA; the U.S. 

FDA enforces the tolerances 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-based regulatory framework that allows for 

the safe development and use of biotechnology-derived organisms. APHIS regulations7

, 

which were promulgated pursuant to authority granted by the PPA, regulate the 

introduction (importation, interstate movement, or release into the environment) of 

certain biotechnology-derived organisms and products. A biotechnology-derived 

organism is considered a regulated article if the donor organism, recipient organism, 

vector, or vector agent used in engineering the 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 plant 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 information 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 the unmodified organism. A biotechnology-derived 

organism is no longer subject to the regulatory requirements of 7 CFR part 340 when 

APHIS determines that it is unlikely 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 produces the same 5-enolpyruvylshikimate-3-

 

6

 7 USC § 136a et seq.; 15 USC § 2601 et seq.

7 7 CFR part 340
Monsanto 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 higher rates of glyphosate application with 

MON 88302 result from an increased level of glyphosate tolerance in the male 

reproductive tissues of MON 88302, which is achieved through use of improved 

promoter sequences that regulate expression 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-derived organisms under the provisions of the 

PPA. APHIS must respond to petitioners that request a determination of the regulated 

status of biotechnology-derived organisms, including genetically engineered crop plants 

such as MON 88302. If a petition for nonregulated 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 notifications of these field trials acknowledged by APHIS are 

listed in the petition in Appendix A. These field tests allow for evaluation in agricultural 

settings under confinement measures designed to minimize the likelihood of the test 

crop’s persistence in the environment after completion 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 were evaluated to 

characterize MON 88302, and to assess 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 approach, considered the reproducibility, 

magnitude, and direction of detected differences (trends) between MON 88302 and the 

conventional control, and comparison to the range of the commercial reference varieties. 

Results from the phenotypic, agronomic, and environmental interactions assessment 

indicated that MON 88302 does not possess weedy characteristics, increased 

susceptibility or tolerance to specific abiotic 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 plant 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 potential environmental effects of its 

actions/decisions and reasonable alternatives to those actions, consistent with NEPA 

regulations9

 and the USDA and APHIS NEPA implementing regulations and 

 

8 42 USC §4321 et seq. 9

 40 CFR Parts 1500-1508 
Monsanto 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 consultation 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 March 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 timing 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 from 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 still 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 made oilseed rape oil and meal derived from 

B. napus unattractive for human consumption and as an animal feed, respectively, 

particularly in western countries. High erucic acid oilseed rape 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 (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, goitrogenic (suppresses thyroid function), 

reproductive, and palatability problems (Fenwick, et al., 1989). However, in the 1960s 

intensive breeding programs resulted in the development and introduction of low erucic 

acid or canola (Canadian oil, low acid) varieties 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 

 

10 7 CFR § 1b and 7 CFR part 372 

11 40 CFR § 180.364
Monsanto 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 varieties in the U.S. (Boyles, et al., 2009). 

In North America canola is grown primarily in prairie areas that have high quality soil, 

but where shorter, drier growing seasons make maize and soybean production less 

attractive (Figure I-1). As shown in Figure I-1, the major canola production region is 

primarily in Canada and extends into North Dakota. 

I.4.1.1. Canola Types 

There are generally three types of canola crops 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. There is a second type of winter canola that is 

planted in the fall and overwinters, but does not require vernalization 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 spring and is harvested in late summer of that same year. 

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 varieties may require different agronomic practices and can be affected by 

different insect pests 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-council.org/gallery/726/canola_growing_region_map.aspx 
Monsanto 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 Columbia Basin (Hang, et al., 2009). Winter 

and spring canola varieties require slightly different agronomic practices and can be 

affected by different insect pests and diseases (Brown et al., 2008). Winter canola has a 

higher yield potential than spring canola (Boyles 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 North 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 grown in Canada was herbicide-tolerant 

(USDA-NASS, 2011c; Zollinger, et al., 2009; CCC, 2010b). An additional 2% of the 

North Dakota crop in 2008 was tolerant to the herbicide imazamox. The imazamox 

tolerance was developed through conventional breeding techniques. 

In North Dakota in 2008, 56% of the canola 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 largest source of vegetable oil 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 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 

appreciable amount (approximately 10%) of alpha-linolenic acid, 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-FAS, 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 distinguish canola but report all rapeseed.
Monsanto Company 11-CA-233U 358 of 500 

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, 2011b). 

In 2010 canola was planted on approximately 1.45M acres in the United States producing 

1.1 million metric tons of canola, with a value of approximately $487 million (USDANASS, 2011a; USDA-NASS, 2011b). 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 dramatically 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 throughout 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 generally 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 states, canola production occurred on 

3 or fewer farms and acreage was not reported (Alaska, Arkansas, Illinois, Indiana, Iowa, 

New York, North Carolina, South Dakota, Utah, Vermont, Virginia and Wyoming). 

Harvested acres were reported for the following states (harvested acres in parentheses): 

Washington (10,449), Kansas (3,362), Wisconsin (1,996), Colorado (1,757), Maine 

(1,364), Texas (486), Michigan (152), Pennsylvania (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). Based on these constraints, most of the 

canola seed produced for sale for crop production 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 production 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 eastern 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 North Dakota (and possibly northwestern Minnesota) 

in the Red River Valley, and the San Luis Valley in south central Colorado (Bateman, 

Monsanto Company, Personal Communication, 2011; Bewley 2008). 

To meet the demand for seed and to minimize production risks, most seed companies 

have off-season seed production locations in the southwestern U.S. (Imperial Valley, 

California and Yuma Valley, Arizona) 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 production 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, distribution 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 strict 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, and germination. All certified seed must pass 

field inspection and be sampled and pass laboratory testing before it can be labeled and 

sold as certified seed. In the U.S. the state 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 Official 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) breeder; 2) foundation; and 3) certified. Breeder 

seed is seed directly controlled by the originating or sponsoring plant breeding 

organization 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 and identity. Certified seed is the progeny of breeder or foundation seed, 

and is the class recommended for commercial 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 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%. 

State seed certification standards and isolation distances vary slightly from state to state 

and can be more restrictive than the seed standards 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 field history restriction is utilized to limit the 

spontaneous germination and sprouting of seeds left in the seedbank from previous 

canola or oilseed rape cultivation. All volunteer plants and noxious weeds must be 

eradicated and definite boundaries to the field set before field use. 
Monsanto Company 11-CA-233U 362 of 500 

Note that Table I-2 does not include 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. The next stage or foundation seed stage occurs 

in highly isolated open fields. During this stage seed lines are further tested to ensure the 

desired genetic characteristics and purity are maintained under field conditions. 

I.4.2.3. Production of Open-Pollinated and Hybrid Seed 

Producing hybrid canola requires using a pollination control system to prevent unwanted 

self-pollination. Three primary pollination control methods used in North America: 1) 

the biotechnology derived male sterility system; 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 hybrid seed production (Mariani, et al., 1992). The MSL-system (Male 

Sterility Lembke) is a private system owned 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) and 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 (Buzza, 1995). The first step in the hybrid 

seed production process involves foundation seed crossing block where female parent 

Line A is pollinated by the genetically identical Line B to produce sufficient quantities of 

Line A seed. The second step involves pollination of Line A with restorer Line R to 

produce fully fertile F1 hybrid seed (Thomas, 2003). 
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Table I-2. Certified Canola Seed Standards 

Category Characteristic 

Coloradoa

(Hybrid/Nonhybrid Varieties) Idahob

Minnesotac

(Hybrid/Non-hybrid 

Varieties) 

North 

Dakota

Field Isolation 

Distances 

Fields of any Variety of the Same Kind 

(feet) 2,640 / 660 660 2,640 / 660 660 

Fields of any Variety from Another 

Kind (feet) NA / 660 330 NA / NA 660 

Field 

Standards 

Years Field Has Not Produced Oilseed 

rape, Mustard and/or Canola 5 / 3 3 5 / 3 3 

Ratio of Plants - other varieties 1.5:10,000 / 1:500 NA 1.5:10,000 / 1:500 1:500 

Plants of other Brassica & Inseparable 

Other Crops 1:10,000 / 1:10,000 NA 1:10,000 / 1:10,000 1:500 

Seed Standards 

Pure Seed (Min) 99% / 99% 99% 99% / 99% 99% 

Inert Matter (Max) 1% / 1% 1% 1% / 1% 1% 

Other Crop Seeds (Max) 0.25% / 0.25% 40/50 kg 0.25% / 0.25% 0.25% 

Weed Seeds (Max) 33/kg / 33/kg 400/50 kg 0.10% / 0.10% 33/kg 

Objectionable Weed Seeds(Max) 2 11/kg / 11/kg 40/50 kg 31/kg / 31/kg 11/kg 

Sclerotia bodies (Max) 15/kg / 15/kg None 15/kg / 15/kg 15/kg 

Germination Rate (Min) 85% / 85% 85% 85% / 85% 85% 

a Colorado Seed Growers Association (CSGA, 2010). b Idaho Crop Improvement Association (ICIA, 2006). c Minnesota Crop Improvement Association (MCIA, 2010). d North Dakota State Seed Department (NDLB, 2008). e Oregon 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 International, Bayer CropScience, Croplan 

Genetics, and Dow AgroSciences. Seed companies contract with growers to produce the 

specified amount of canola seed. Seed companies 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 facility processes. 

For most of the major seed companies, the entire seed production process is International 

Organization for Standardization (ISO) certified and; therefore, includes internal and 

external audits (ISO, 2009). The ISO standards represent an international consensus on 

good management practices with the aim of ensuring that the organization can 

consistently deliver excellent product or services. 

While field operations and management practices 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 volunteer canola plants and to ensure genetic purity. All seed 

stock is 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 emergence and uneven stands. Every effort must 

be made to eliminate weeds in a seed field through the use of herbicides, 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 frequently 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 fields 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, swathing 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 drops below 10% and no green pods are visible 

(Boyles et al., 2009). Harvesting equipment must be adjusted to minimize or avoid seed 

damage and must be cleaned before entering the seed fields to minimize genetic 

contamination. Certain handling equipment, such 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, regulatory standards and trait licensing agreement standards. 

Field inspections are conducted on seed production fields throughout the growing season 
Monsanto Company 11-CA-233U 365 of 500 

to evaluate variety purity, ensure canola plants are developing properly 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 screening, cleaning and bagging process. Specific methods are 

used to assure the genetic purity and identity 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 quantified 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) Extension Office, for example, has conducted variety trials for 

many years, and publishes the results each year. The 2010 trials included 58 varieties: 49 

glyphosate-tolerant varieties (representing 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 selection: 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, which 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, including planting, harvesting, transporting, processing, and 

marketing in order to preserve their value. While standard seed production practices 

preserve variety identity through planting, segregation in the remainder of the production 

process (including the need for special cleaning of harvesting and transportation 

equipment and custom crushes) adds incremental costs that, for the production to be 

worthwhile, must be recovered in a price premium for the end product (Phillips and 

Smyth, 2003). 

In some cases, and particularly for organic production, specialty production is required 

which utilizes oil processing facilities other than the very large, regional facilities where 

commodity canola is processed. Reproducing the 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 using a screw-type mechanical press (expeller). 

Expellers are available in a range of sizes suitable for commercial production to backyard 

use. 
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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 Marketing Service (AMS), requires organic 

production operations to have distinct, defined boundaries and buffer zones to prevent 

unintended contact with prohibited 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 organic production system plan 

approved by an accredited certifying agent. This plan enables the production operation to 

achieve and document compliance with the National Organic Standards, including the 

prohibition on the use of excluded methods.14 Excluded methods include a variety of 

methods used to genetically engineer organisms or influence their growth and 

development by means that are not possible under 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 agent of the materials 

and practices used to produce or handle an organic agricultural product. This oversight 

includes an annual review of the certified operation’s organic system 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 necessarily constitute a violation of the 

National Organic Standards. The unintentional presence of the products of excluded 

methods will not affect the status of an organic product or operation when the operation 

has not used excluded methods and has taken reasonable steps to avoid contact with the 

products of excluded methods as detailed in an approved organic system plan. Organic 

certification indicates that organic production and handling processes have been 

followed, not that the product itself is “free” from any particular substance. As USDA 

AMS has recently re-iterated in a policy memorandum, organic certification is process 

based. The NOP regulations do not allow the use of excluded methods such as 

biotechnology; however, the inadvertent presence of products of biotechnology “does not 

constitute a use because there was no intent on the part of the certified operator to use 

excluded methods” (USDA-AMS Organic, 2011). 

Many crop management practices are similar to those used by non-organic canola 

growers, except that organic growers may not use synthetic fertilizers or pesticides. 

Because synthetic herbicides are not allowed, organic growers use cover crops and/or 

tillage to control weeds (Myers, 2002). 

Organic producers use production practices designed to prevent commingling of their 

crop with neighboring crops treated with herbicides and other pesticides (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. 
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These well established practices include isolation 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 production 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 (USDA-NASS, 2008). To avoid reporting 

data for individual farms, the only state-specific 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 four organic canola farms in those states 

reported in 2008. The census reported 5 farms growing canola in Michigan and 56 in 

Washington, the other two states with organic canola production in 2008 (USDA-NASS 

2009). 

There is little specific information available about organic canola production 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. However, only the larger organic crops (flax 

and sunflowers) are differentiated; canola is grouped in the “Other” 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 reported 12,487 acres of “Unclassified/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 Richter, 2011). California safflower acreage 

for that period ranged from zero to 4,875 acres. 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 would 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 has a 3,500-acre farm in Windsor, North Dakota, 

that he has operated as organic since 1980 (Leopold Center for Sustained Agriculture 

 

16 The organic survey was part of the 2007 census, but was done in 2008. Data were collected by mail, with a overall 

response rate of 87%. The mailing list 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 that canola had been a “very good 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 growing canola (Kirschenmann, 2009). The other 

grower has a 640-acre farm in the Red River Valley in eastern North Dakota and grows 

organic wheat, clover, sunflowers, rye, barley, 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 production is the development 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 traditional or commodity 

canola oil (CanolaInfo, 2007). High-oleic canola oil is sold most commonly to food 

companies and food service operations (CanolaInfo, 2007). In the 2010 NDSU variety 

trials, five of the 58 varieties tested were high-oleic (Kandel, 2010). 

Canola products labeled “GMO-Free” have no detectable presence of genetically 

modified material, and may be specialty products, 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 that are grown in the U.S. 

Because of potential concerns with canola related to cross pollination and spread of 

disease among related species, this section focuses 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, Brussels sprouts, collards, 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). Also 

grown are radish, daikon, and 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 concern for cross pollination with canola. 

Brassica species represent approximately 6.8% of the total vegetable 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 the 
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harvested acreage and head cabbage (cabbage) 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 state in 2007 was Arizona (11,869 acres or 9% 

harvested), then Oregon with 1,410 acres or 1% harvested. All other states had less than 

1,000 acres harvested (USDA-NASS, 2009). The distribution of cauliflower production 

is similar to that of broccoli, with 82% of harvested acres in California, and with Arizona 

and Oregon with the next highest production. California is also the 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 Brussels sprouts, Chinese cabbage 

and kale. Georgia is the leading producer of collards and Michigan leads in turnips. 

I.4.5.2. Brassica Vegetable Seed Production 

Because of the wealth of literature and 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 three distinct areas: the Willamette Valley 

in Oregon; Skagit County in western Washington; and the Columbia Basin in eastern 

Washington (Bateman, Monsanto Company, Personal Communication, 2011). These 

areas have strict regulations or prohibitions on growing canola. Brassica vegetable seed 

crops are also produced in California and Arizona, and broccoli seed is grown primarily 

in California and Arizona. According to the 2007 Census of Agriculture, canola was 

grown on 16 acres in California and 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 grade 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 operators, 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 implemented regulations on where canola can and 

cannot be grown (Appendix Section I.4.6). 

Mustard seed was harvested on approximately 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, Chinese cabbage, mustard cabbage, collards, kale, mustard 

greens, turnips, and turnip greens, with harvested acreages ranging from 66 acres (mustard cabbage) to 11,480 acres 

(Chinese cabbage). Other vegetable crops in the Brassicaceae family include daikon (624 acres), horseradish (3,692 

acres), radishes (14,599 acres) and watercress (679 acres). 
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Washington and Idaho together accounting for 98% of the acreage (61%, 25%, 9% and 

3%, respectively). In North Dakota mustard is grown in rotation with small grains, but is 

not recommended for close rotation with canola because of similarity of disease 

susceptibility (NDSU, 2007b). The most common type grown is yellow mustard 

(S. alba), which is used as condiment mustard. 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 manure crop (crop is grown then 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 same 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 restrictions (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. Part of the reason for these restrictions is to 

prevent cross pollination between canola and industrial oilseed rape. There is little 

industrial oilseed rape grown in these states today; however, in the 1980s and early 1990s 

industrial oilseed rape acreages 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 vegetable seed production 

areas. 

I.4.6.1. Oregon Restrictions 

Since 1989, the Oregon Department of Agriculture has had the authority under Oregon 

law to establish control areas for the production of oilseed rape (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 preventing 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 acid levels and canola with low erucic acid content used for 

edible oils. 
Monsanto Company 11-CA-233U 371 of 500 

conflicts between specialty seed production and canola [oilseed rape] grown for biodiesel 

or edible oil.” The Department of Agriculture made small modifications to the 

regulations in 2009 and will be reviewing them 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 (described below) to 

help prevent blackleg’s introduction into Oregon. Long distance spread of blackleg is 

usually by infected seed (NDSU, 2004). 

The regulations establish a “general production 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 restrictions apply to all oilseed rape grown within 

the general production area: 

• All oilseed rape seed that “trades within 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 same plot more often than two years (nonconsecutively) in five. 

• All unbagged loads of oilseed rape transported through protected districts “must be in 

enclosed bins or in containers 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 District includes parts of Crook, Jefferson and 

Deschutes counties. The Northeast Oregon Protected District includes part or all of 

Baker, Union and Wallowa counties. The Malheur Protected District includes a 3 mile 

strip of northern Malheur County adjacent to the Idaho border (ODA, 2011). Within all 

the protected districts except the Northeast District, oilseed rape production for seed or 

oil is prohibited except under special permit. Oilseed rape production for seed or oil is 

allowed in the Northeast District without a permit. The same restrictions applicable to 

the general production area apply to production in the protected districts, except that 

oilseed rape can be grown on the same plot of land only once every four years. The 

following additional restrictions apply to all protected districts:23 

• Oilseed rape “must be isolated from other crops with which it will cross pollinate, 

by a distance of not less than three miles,” except that in the Northeast District, 

the isolation distance is two miles. 

• The locations of oilseed rape fields must 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) 
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• Forage and cover crop oilseed rape shall not be allowed to flower. 

• Any volunteer or uncontrolled oilseed rape “in and around production fields must 

be prevented from flowering by the producer.” 

• Violators may be fined or subjected to crop destruction.24

I.4.6.2. Washington Restrictions 

Since 2007, Washington state law allows the State 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 Agriculture has established two Brassica seed production 

districts. Seed Production District 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 participation in the Washington State University 

Extension Center pinning process. Within both districts, seed must be transported in 

“covered containers from which the seed cannot 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 seed or oil is prohibited 

unless grown under the conditions of a Brassica production agreement.28 A production 

agreement must be developed through the director of the State Department of Agriculture 

and an advisory committee and the agreement must contain “terms and conditions that 

are necessary and sufficient to mitigate reasonably 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 distance 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 fuel 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 16-326-050 
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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 (District 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 cultivated fields “shall be destroyed before 

flowering” by “the person responsible 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 production is to produce a crop with high yield, high oil 

content, low green seed content, low weed seed content, and little seed left in the field, all 

while managing input costs. This section discusses those practices that are within a 

grower’s control that help achieve this goal. Agronomic practices 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 choosing 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 compiled 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 expenses 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 was $55.27 per acre. For more detailed information see Table VIII-3 

in the Petition. 

I.4.7.2. Tillage 

Canola can be grown with conventional tillage, conservation tillage, 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 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 §150 
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soil erosion, reduced fuel and labor costs, conservation of soil moisture, improvement of 

soil structure, reduction of soil compaction 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 tillage systems (Brown et al., 

2008). Winter canola is not recommended when planting into excessive amounts of fresh 

straw, or when soil temperatures are lower than average in the fall months. This can 

result in poor emergence. Seed yields with direct-seeded winter canola have been 

significantly lower than with conventional tillage systems (Brown et al., 2008). Spring 

canola is better suited to conservation tillage due to better soil moisture conservation and 

availability. Spring canola has fewer problems dealing with heavy straw residue and, 

residue from the previous crop has usually decomposed to some degree during the winter 

months prior to planting spring canola. In addition, cooler soil temperatures can be 

advantageous to spring canola. 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, 2010). The 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. 

I.4.7.3. Planting 

Canola is typically seeded in 6 or 7-inch rows 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 damaged 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 canola. Nitrogen, phosphorus, potassium, sulfur, and boron, 

depending on the region, are the most limiting nutrients for successful canola production 

(Brown et al., 2008). Soil sampling and testing is the first step in assessing soil nutrient 

levels and the requirements for supplemental fertilizers. The availability of soil nutrients 

is dependent on soil acidity or the pH level and 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 nutrients 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 previous crop credit for nitrogen. 
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Research studies have shown that nitrogen 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 phosphorus 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 (Brown et al., 2008). A 

starter fertilizer rate of 20-30 pounds of P2O5 per acre is generally sufficient for most soil 

test levels unless the grower intends to build up phosphorus levels in the soil (Franzen 

and Lukach, 2007). Potassium requirements are also based on soil tests. Many soils 

contain sufficient levels of potassium requiring no potassium 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 (Brown et al., 2008). Sulfur deficiencies result in yellowing 

between leaf veins, cupped leaves and stunting (Franzen and Lukach, 2007). In addition, 

in the presence of sulfur deficiency flowering 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 applications (Franzen and Lukach, 2007). Since 

soil tests tend to overestimate available sulfate 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 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 show less than 0.5 ppm boron (Brown et al., 

2008). Canola has not shown yield responses to applications of micronutrients such as 

chlorine, copper, iron, manganese, molybdenum or zinc (Brown et al., 2008). 

I.4.7.5. Harvesting 

The grower’s challenge is to harvest canola during the very brief optimum stage when 

most seeds are mature but the pods are not so dry that they will shatter. The brevity of 

this optimum stage is one of the factors limiting canola production (USDA-ERS, 2010b). 

Harvesting too early results in higher green seed 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 is 
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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 growing season (Boyles et 

al., 2009). Direct combining is generally recommended for winter canola in the southern 

Great Plains region because dry-down is accelerated by high air temperatures during seed 

ripening (Boyles et al., 2009). 

Canola seed is sensitive to heating in storage 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 small grains, but is less tolerant of drought 

conditions. However, according to the USDA 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 reductions and often a 

corresponding reduction in the oil content of canola. Some aphids act as vectors for plant 

viruses (Brown et al., 2008). Canola seedlings are especially vulnerable to chewing 

insects. A severe infestation of flea beetles can completely destroy a stand of canola 

seedlings. A firm seedbed and adequate fertilizer 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 uncontrolled (Brown, et al., 2004). 

Damage from insect pests is more severe during periods of stress, especially drought 

stress. Spring and winter canola types are impacted 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 insect 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 protect 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 early can allow establishment of the canola 

prior to beetle emergence. 

Insect pests causing serious damage 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., 2009). Aphids have become the most 

important insect pest of canola in this region. 

Crop rotation, controlling volunteer canola and wild mustard plants and managing crop 

residue are all important cultural practices for insect control. 

Insect Pests and Herbicide-Tolerant Canola: In a three-year study (2000-2002) in 

southwestern Canada researchers compared the results of insect damage in glyphosate-
Monsanto Company 11-CA-233U 377 of 500 

tolerant, glufosinate-tolerant and conventional canola. Insect 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 canola 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). Diseases are managed primarily through use 

of genetics, seed treatment, chemical application and crop rotation. 

Blackleg: Blackleg a fungal disease caused by Leptosphearia maculans, first detected in 

Saskatchewan in 1975, is a serious disease in the major North American canola 

production areas (SMA, 2009; NDSU, 2004). The 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 Canadian provinces. It is less common in 

Minnesota (Lamey, et al., 2003). Highly aggressive strains are present in Saskatchewan, 

Manitoba, and in some parts of North Dakota (NDSU, 2004). Blackleg 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 planting in North Dakota have been bred for 

resistance to blackleg (Kandel, 2010). Canola 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 migration from adjacent fields and from infected 

seeds, a study showed that most infection comes 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 susceptible 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 mustard, 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 facilitate fungal infection (Markell, et al., 2009). SSR 

occurs in canola, dry beans, soybeans, peas, lentils and chickpeas, but does not occur in 
Monsanto 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 minimal (Boyles et al., 2009). Crop rotation is 

important in managing the disease, and a preferred rotation would have canola planted 

after two or more nonsusceptible crops (Boyles et al., 2009; Markell 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: Scientific evidence indicates that at recommended 

use rates, glyphosate-based herbicides applied in conventional or glyphosate-tolerant 

cropping systems do not result in increased susceptibility 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 years 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 equal to or greater than that of conventional 

varieties. 

Scientists from Purdue University Extension recently published an article assessing 

whether glyphosate use has impacted crop production and disease development 

(Camberato, et al., 2011). Prior research from Purdue University (and research published 

by others) had previously found that plants sprayed with non-glyphosate herbicides were 

more susceptible to biological and physiological disorders, suggesting that plants already 

weakened by the herbicide application are more susceptible to infection. However, 

research found that glyphosate-tolerant soybeans and wheat are no more susceptible to 

soil-borne fungal diseases than conventional glyphosate-sensitive varieties, regardless of 

whether or not glyphosate is applied (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 (Camberato, 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 yields 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. 
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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 yield losses, wild mustard is a serious seed 

contaminant in canola and can cause price discounts or rejection in the market. 

Extension advisors recommend special attention to controlling this weed prior to planting 

in canola fields (NDSU, 2007a). 

Research studies have shown that Canada thistle 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 loss, 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 meter. 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). 
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I.4.7.9.2. Problem Weeds in Canola – Minor Production Areas 

Much less information specific to the impacts of weeds on canola is available for minor 

production areas. Boyles et al. (2009) identified the weeds 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 the 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). Components of IWM in canola production can 

include the following (Harker, 2011; NDSU, 2007a): 
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Appropriate Tillage: 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 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 close to the seed row can damage the crop 

(Harker, 2011). High seeding rates can also 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 (Blackshaw, 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 Company, Personal Communication, 2010). 

Early Weed Removal: Early weed removal is important to attain high canola yield 

(Harker, et al., 2008; Harker, 2011). 

Crop Diversity and Rotation: Because different crop species and their associated 

management practices present different challenges for weeds, crop diversity and rotation 

can help prevent creating advantages for any given species (Harker, 2011). 

Combining Optimal Agronomics: Researchers have demonstrated optimized weed 

control associated with applying multiple optimal agronomic practices such as early 

seeding, higher seeding rates, spring-applied fertilizer and timely 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 injury to kill the plants. (NDSU, 2007a). 

As discussed in Petition Section VIII, the great majority of the canola grown in North 

America is herbicide-tolerant. In North 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 acres in North Dakota in 2008 

(Zollinger et al., 2009). The number of registered herbicides 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 quizalofop are post-emergence in-crop herbicides 

for control of annual grasses and broadleaf weeds in conventional canola varieties 

(NDSU, 2005). In addition, glyphosate herbicide can be applied 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 applications 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 the 
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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 all but one annual grass (wild oat – good to 

excellent). Glufosinate and imazamox are rated excellent or good-excellent on annual 

grasses except volunteer cereals and field sandbur, respectively, where they are rated fair 

to good. Glyphosate is the only herbicide in canola that is rated excellent on quackgrass, 

a perennial grass species. Glufosinate 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 and biennial weeds and good to excellent 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, excellent for wild mustard, but poor for 

Canada thistle. Of the herbicides, other than glyphosate, only clopyralid is effective on 

Canada thistle, but it provides no control for wild oat or wild mustard. None of the 

herbicides, other than glyphosate and glufosinate, control wild mustard. 
Monsanto Company 11-CA-233U 

Table 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 

Preplant Incorporated Only Ethalfluralin E E G F P-F F-G E N E G-E Trifluralin E E N P-F P-F F G-E N E G Postemergence

Clopyralid N N N N F-G N P-F N P P-F Imazamox E G-E G-E E P E F E E G-E Glyphosate E E E G-E F-G F-E P-E G-E E G Glufosinate E G F-G G-E E E F-G E E G-E Quizalofop E G-E E G-E N N N N N N Sethoxydim E E E G-E N N N N N N Clethodim E E E E N N N N N N 

1Source: NDSU, 2005. Weed control ratings based on the following scale: E = Excellent = 90 to 99% control, G = Good = 80 to 90% control, F = F

Poor = 40 to 65% control, and N = None = no control. 
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I.4.7.10. Canola Volunteers 

Volunteer canola is defined as a plant that has germinated and emerged unintentionally in the 

production of a subsequent rotational crop. Canola 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). Although canola can volunteer for several years 

following a canola crop (Beckie and Warwick, 2010), problems with volunteer canola in 

rotations are not common (Boyles et al., 2009). Beckie and Warwick (2010), documented “the 

longest persistence of oilseed rape volunteers in Canada” (7 years) 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 three-year study in which 

wheat was planted for three years (2001, 2002 and 2003) following canola in 2000, researchers 

found average canola densities of 6.2, 0.7, and 0.0 plants per square meter in the first, second, 

and third year of the wheat crops. Researchers concluded that preventing seed production in new 

canola volunteers in 2001 reduced canola densities in subsequent years (2002 and 2003) below 

those required to mitigate weed-crop competition influences in most crops (Harker, et al., 2006). 

Data from Canada suggest that presence of a herbicide tolerance trait is not a major factor 

influencing volunteer canola abundance (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 herbicide tolerant canola was widely used. Based 

on estimated crop yield loss, the economic impact of volunteer wheat and oilseed rape is ranked 

third and eighth, respectively, among weedy 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 the spring immediately following a sown canola crop 

and that an effective management strategy to limit long-term volunteers may 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 germinates after summer rains and can be controlled 

by tillage before planting the next rotational crop. 

Many of the same management practices for weed management discussed in Petition Section 

VIII.F are applicable to control 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. Burndown 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). 
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However, volunteer canola can be controlled through herbicide applications and with appropriate 

rotational crop management. Table VIII-11 in the Petition provides a detailed description of the 

rotational cropping practices immediately following canola by state. Table VIII-12 in the Petition 

provides a listing of herbicides that control volunteer glyphosate-tolerant canola in the various 

rotational crops and their 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) theorized that annual, repeated use of any herbicide could lead 

to shifts in weed species composition within a crop-weed community. Similarly, Bandeen, et al. 

(1982) suggested that a normal variability in response to herbicides exists among plant species 

and tolerance can increase with repeated 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 genetic traits. Biotypes possess certain traits or characteristics not 

common to the entire population. Herbicides that suppress or kill weeds exert selection pressure 

on weed populations. When a herbicide is applied, surviving plants, those that had reduced 

sensitivity to the herbicide, have a competitive reproductive advantage with progeny more likely 

to possess the same or superior herbicide resistance. With repeated application of the same 

herbicide and no other herbicide or weed control practice, the resistant biotypes can become the 

dominant biotype in that weed community. As of May 2011, 358 herbicide resistant 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 herbicides that inhibit acetolactate synthase (ALS) 

account for 31% of the herbicide resistant biotypes (Heap, 2011b; Heap, 2011a). 

For as long as herbicide resistance has been a known phenomenon, public sector 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 population. 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 biotype that is resistant to a herbicide being used, the grower needs 

to utilize an alternate method of weed control. Management practices that can be used to retard 

the development of resistance, such as those routinely used by canola growers, 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 Canola 
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Herbicide resistance in weeds causing yield losses in the major U.S. canola production area are 

summarized in I-8 along with the herbicide mode of action (MOA). 

Table I-8. Herbicide Resistant 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 identified in soybean fields in two counties in 

eastern central North Dakota (Stachler and Christoffers, 2010). These counties are not major 

production counties for canola (USDA-NASS, 2007). In addition, 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 common in spring canola production fields in North 

Dakota (Table I-3). 

Glyphosate-resistant Italian 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-resistant 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-tolerant technology, which has been shown to provide 

superior weed management, yield and environmental performance (see Appendix Section 

I.4.7.9.3.). In addition, the Herbicide Resistance Action Committee’s provides guidelines for 

prevention and management of herbicide resistance (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 available for winter canola, winter 

canola is grown in rotation primarily with wheat, which utilizes herbicides with alternate modes 

of action. 
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Glyphosate-resistant weeds may occur in canola fields. 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 volunteers (Appendix Section I.4.7.10). 

I.4.7.13. Crop Rotation 

One of the main purposes of growing canola in rotation 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 blackleg and Sclerotinia stem rot. Some of the 

other purposes of rotations are to improve or maintain soil fertility, reduce erosion, reduce the 

build-up of pests, reduce risk of weather-related crop damage, improve weed control and 

increase net profits through yield increases (Peel, 1998). Compared 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). Incorporating canola in small grain rotations has also 

decreased disease incidence and increased quality of cereal crops. Including canola in a small 

grain cereal rotation provides broader, more effective herbicide options for controlling certain 

weeds such as downy brome, jointed 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 description 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 quantitative data for the various crops that are rotated 

with canola, and also shows the percent of glyphosate-tolerant crops that 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, researchers 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 Agricultural 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 flaked 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 with 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 removal 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. 
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Canola oil has well established heart health benefits 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 including dairy and grain 

products, fruit and vegetable juices, salad dressings, 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. FDA, 2010) and the first commercial 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 dairy 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, canola meal has slightly less digestible energy 

value. Therefore, animals with requirements for intermediate energy levels such as dairy cattle 

and laying chickens perform well 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 family 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 potential 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 environment including other canola plants, other 

Brassica crops, canola seed production 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. Introgression is a multi-generation 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 Table IX-3 summarizing the 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 although 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 
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by insects, such as honey bees (Thompson, et al., 1999) although wind is also responsible for 

some pollen movement. Most (98.8%) of pollen travels 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). 

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 possibility 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 information on the potential for hybridization (cross 

pollination) and introgression from B. napus to related species. Reports of unassisted (e.g. 

without human intervention or assistance) field hybridization of B. napus to related species 

provide biologically relevant information that can be useful in assessing the potential for 

hybridization and gene introgression under field conditions. 

There are reports of hybridization 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 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, 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-planted in large plots vary from 

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 et al. (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 mean 

hybridization frequency was 7% in natural environmental field trials and 13.6% in the 

commercial fields. The first-generation hybrids between B. napus and B. rapa had reduced 

pollen viability, produced fewer germinable seeds, and had very low (<2%) hybrid seedling 

survival rates (Scott and Wilkinson, 1998; Warwick et al., 2003). Additionally, unlike the 

B. rapa parent, under field conditions, seeds of F1 hybrids between these two species lacked seed 

dormancy (Jørgensen, et al., 1998).
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The presence of a herbicide-tolerance trait (glufosinate tolerance) 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). 

Collectively, these data support the conclusion that hybridization between B. napus and B. rapa

is possible under field conditions, but resulting progeny demonstrate decreased fitness and 

likelihood of gene introgression 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 derived 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 conditions (Bing et al., 1996; 

Frello, et al., 1995; Jørgensen et al., 1998). As reviewed in Devos (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 from 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 likelihood of introgression. 

Brassica oleracea 

Brassica oleracea is distributed primarily in northeastern 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 conditions (Ford, et al., 2006). Hybridization 

between these two species under field conditions appears to be relatively rare and introgression 

under field conditions is unlikely to occur (Ford 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, cauliflower, 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 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. 

Closely Related Non-Brassica Species

In addition to those reported for B. rapa, B. juncea, B. oleracea, there are reports of 

hybridization under non-cultivation field conditions with B. napus as the pollen donor and 
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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 characteristics including decreased pollen viability, seed production, 

seedling survival, etc. when compared to parental varieties. Based on field 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 Gene Flow between Canola Crops 

Gene flow may occur between canola crops planted close together. Many studies have been 

performed and are discussed in Petition Section 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 cross 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 populations, variety (genotype), presence and 

activity of insect vectors, environmental conditions (temperature, wind speed and direction, 

humidity, etc.) and topography. Information on pollen movement is useful for managing pollen 

flow during canola breeding, seed production and for identity preservation. 
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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 

0.14φ

0.07φ

0.08φ

0.07φ

0.04φ

0.02φ

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 from publication. 

Since the development of biotechnology-derived canola researchers often use the biotechnologyderived trait as a marker in their cross pollination 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 cross pollination does occur at short distances, 
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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 distance 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 varieties that can be found in certified seed (Table I-2). North 

Dakota has required isolation distance of 660 feet from other varieties of canola and a standard 

of 1:500 (0.2%) for the ratio of seed of other canola varieties (Table I-2). These standards are 

intended to ensure that the seed produced meets 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 producer wishes to produce seed with more stringent purity standards 

than those specified by AOSCA and the individual 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 considered weedy, and are generally 

harvested prior to flowering, preventing cross pollination, hybridization and seed formation. 

Biofumigant Mustard

As discussed in Appendix Section I.4.4.3, biofumigant mustard (S. alba and B. juncea) is used as 

a green manure crop. As discussed in Appendix Section I.4.9.1.2, the potential 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 would 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) 
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As discussed in Appendix Section I.4.5.3, mustard seed is grown on approximately 48,000 acres, 

in the same general areas where canola is grown. As discussed in that section, because of 

similarity of disease susceptibility, it is not recommended for rotation 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 Petition Section IX.D.4., the potential for gene flow between B. 

napus and B. juncea is very low, and the potential 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 probability of a rotation immediately following canola, cross 

pollination between S. alba mustard and canola is highly unlikely. Based on the very small 

acreage, the low potential for introgression and the low probability of close rotation with canola, 

cross pollination between 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 appear to be variations of Siberian kale. 

Therefore, the greatest possibility for cross pollination with B. napus canola in vegetable seed 

production would be with rutabaga and Siberian kale. Rutabaga seed is produced on 

approximately 80 acres in western Washington 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 rutabaga seed production area 

is within that part of western Washington where there are strict limitations on growing canola 

that are intended to prevent any cross pollination with related Brassica species (Appendix 

Section I.4.6). We were unable to find any information 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 (Attachment 1). Based on USDA census data, no 

canola is grown in Arizona; therefore, there is no potential for cross pollination from canola with 

these seed crops. 

For the other Brassica and related vegetables, Table I-10 summarizes the potential for 

hybridization and introgression with canola, based on the information presented in Petition 

Section IX and summarized in Appendix Section I.4.9.1. 
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Table I-10. Cross Pollination Potential for Canola 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 almost entirely in areas with 

prohibitions or strict limitations on canola production and transport (eastern Washington, western 

Washington, the Willamette Valley in Oregon and Treasure Valley in Idaho), or in areas where 

canola production is either negligible or non-existent (California and Arizona; Appendix Section 

I.4.5; Attachment 1). 

Aside from these regulatory and geographic features 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 extremely low potential for introgression with B. napus. 

In greenhouse and field experiments, (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 evaluated 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 field during peak flowering of both species (Quinn 

2010). 

The Willamette Valley Specialty Seed Association (WVSSA) seed production isolation 

guidelines for vegetable seed production are established to minimize the potential for cross 

pollination of B. napus canola with related vegetable species (WVSSA, 2008). 

I.4.9.3. Potential for Gene Flow from Canola to Species in the 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) shows 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 other related weed species on this list. 

Introgression, i.e., the stable 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 the glyphosate-tolerance trait declined drastically 

from the first to fifth year of monitoring, 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 glyphosate-tolerant B. napus confirming the presence of the herbicide 

tolerance trait over time (Warwick et al., 2008). 

Warwick et al. (2008) noted that they had no data to suggest that the presence of a herbicidetolerant trait in a weedy species poses a specific 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 boundary 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 weeds have been found in the literature or in 

information from university extension offices and industry sources other than that reported by 

Warwick et al. (2008). To date, herbicide-tolerant canola has proved 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 North Dakota, as it is in the Canadian 

provinces. The NDSU recommends that conventional canola not be planted on fields with heavy 

infestations of wild mustard. However, glyphosate, glufosinate and imazamox tolerant canola 
Monsanto Company 11-CA-233U 397 of 500 

can all be planted on heavily infested wild mustard 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 (Appendix Section I.4.7.10). 

When it occurs outside an agricultural field, the 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 whether herbicide-tolerance has affected the 

survivability of ruderal canola. A literature review 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 disturbed areas along roadways and railroads where 

canola has been transported (Aono, et al., 2006; Crawley and Brown, 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 adjacent 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 concluded 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. The most frequent pattern observed 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; 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 mowing, the use of 

broadleaf herbicides, animal predation, diseases and environmental conditions (Crawley and 

Brown, 1995; Knispel et al., 2008; Norris and Sweet, 2002; Yoshimura, et al., 2006). 

Herbicide-Tolerant Traits and Ruderal Canola 

Frequency of occurrence of biotechnology-derived plants in ruderal areas was similar to the 

proportion of the canola area planted with herbicide-tolerant canola varieties in recent preceding 

years (Yoshimura et al., 2006). Biotechnology-derived 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 seed in soil declines over time (Gulden et al., 

2003; Gulden, et al., 2004; Hails, et al., 1997) and biotechnology-derived canola seed has not 

been demonstrated to persist longer than conventional canola seed (Gruber, et al., 2004; Hails et 

al., 1997). Populations of glyphosate-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 mechanical 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 canola is canola oil, which has 

well established heart health benefits. According to the OECD (2001) canola oil contains one 

key toxicant, erucic acid. A key toxicant is a potentially significant toxic 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 erucic acid has been affirmed as GRAS by U.S. 

FDA. The Codex Standard for Named Vegetable Oils (Codex Alimentarius, 2005) also specifies 

that canola oil cannot contain more than 2% erucic acid. In addition the U.S. FDA has no 

objections to the recent GRAS notification for canola protein isolate. 

Canola oil derived from the current glyphosate-tolerant canola RT73 has been shown to be 

compositionally equivalent to canola oil derived from conventional canola (Nickson and 

Hammond, 2002). As noted by the National Research Council (NRC), unexpected and 

unintended compositional changes arise with all forms of genetic modification, including both 

conventional breeding and genetic 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 nutritional quality of biotechnology-derived foods have generally 

concluded that there are no significant nutritional differences 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 food, for protection of human and animal health. 

The U.S. EPA also regulates other pesticides, including insecticides and fungicides, that are used 

on canola and establishes application rates that are deemed not to have an unreasonable adverse 

effect. The herbicides, insecticides and fungicides typically used on canola are discussed in 

Petition Sections VIII.E and VIII.F. This discussion focuses on glyphosate, as MON 88302 is 

tolerant to glyphosate. Glyphosate has been widely used in canola fields since 1999 when the 

first generation Roundup Ready canola RT73 was launched. 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 (registered) 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 control of weeds growing in non-crop areas. Currently over 70% of 

canola acres in the major U.S. canola producing region are treated with glyphosate as a 

component of a weed control regimen either in-crop or pre-plant. A more detailed discussion on 
Monsanto Company 11-CA-233U 399 of 500 

the potential impact of glyphosate on human health and the environment may be found in 

Appendix K. 

According to the Reregistration Eligibility Decision (RED) document for glyphosate (U.S. EPA, 

1993), glyphosate is of relatively low oral and dermal acute toxicity. For this reason, glyphosate 

has been assigned to Toxicity Categories III and IV for these effects (i.e., Toxicity Category I 

indicates the highest degree of acute 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 are considered sufficient (U.S. EPA, 1993). 

Expert toxicological reviews the World Health Organization (WHO-FAO, 2004) are in 

agreement that glyphosate does not pose any human 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 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 

endpoints have been identified for glyphosate (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 biomonitoring study of pesticide applicators 

conducted by independent investigators. This biomonitoring study determined that the highest 

estimated systemic dose of glyphosate for applicators as the result of routine labeled applications 

of registered glyphosate-based agricultural herbicides to crops, including glyphosate-tolerant 

crops, was approximately 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 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 agricultural herbicides to crops. Considering the similarity of 

the use pattern and application rates of the glyphosate products in this study compared to those 

registered for use on glyphosate-tolerant crops, bystander exposure attributed to the use of 

glyphosate on glyphosate-tolerant crops is expected to be negligible. Therefore, the use of 

currently registered products containing glyphosate in accordance with the labeling will not pose 

unreasonable risks and adverse effects to humans or the environment. In general, the herbicidal 

activity of glyphosate is due primarily 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 carcinogen; it has been classified by the U.S. EPA as a Group E 

carcinogen (evidence of non-carcinogenicity 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 glucosinolates, and canola meal has a history of safe use 
Monsanto Company 11-CA-233U 400 of 500 

as an animal feed source. Although 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 key toxicant in canola meal, glucosinolates. Glucosinolates are 

organic compounds that contain both 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. The low levels of glucosinolates in canola as 

compared to other oilseed rape make the meal safe for use as animal feed. The standard for 

glucosinolates in canola is 30 μmoles/g (OECD, 2001). 

Canola meal from biotechnology-derived canola has been shown to be as safe and nutritious for 

animals as canola meal from conventional canola (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-biotechnology-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 Communities Including Threatened and Endangered 

Species 

This section discusses animal and plant communities that may be affected 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 animal and plant communities as they relate to 

current, widespread glyphosate application on canola fields. 

As a part of the reregistration evaluation under 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 determined 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 calculate the estimated environmental concentrations (EECs) for 

aquatic plants and animals. Based 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 agricultural fields) plus adjacent areas extending beyond the intended 

plantings that might be affected by agricultural operations. Mammals and birds, including 

migratory mammals and birds, may seasonally consume seeds from the planted fields, and 

invertebrates can feed on the plant and surrounding vegetation during 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 canola fields, and the raptors, snakes and other 

animals that may prey on them are part of the affected environment. Deer may also browse in 

canola fields on the forage. Fish and other aquatic organisms in streams draining agricultural 

fields are also part of the affected environment. 

Glyphosate is practically nontoxic to slightly toxic to birds, freshwater 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 substances; 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) areas as well as adjacent areas extending beyond the 

intended plantings that might be affected by agricultural operations. Plants, extraneous to the 

crop, which grow in planted fields can be considered weeds and are dealt with in a separate 

section in this document. Plants not growing in a field amongst the canola would include those 

in ditches, hedge rows, fence rows, wind breaks, yards, etc. 

Glyphosate is a non-selective herbicide with post-emergence activity 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, glyphosate binds 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. Therefore, any potential effects to non-target plants 

from glyphosate use are only attributed to spray drift. During the re-registration process in 1993, 

additional 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 understand the 

potential risk to nontarget and threatened and endangered plants from the use of glyphosate 

herbicides in agriculture. Using the methodology 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. glyphosate per acre), and at rates 

just below the maximum single aerial application 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, nutrient content, and moisture capacity, 2) typical 

physico-chemical factors such as temperature, pH, and redox potential, and 3) soil management 

practices. Agricultural practices such as fertilization and cultivation may also have profound 

effects on soil microbial populations, species composition, colonization, and associated 

biochemical processes (Buckley and Schmidt, 2001; Buckley and Schmidt, 2003). 

Consequently, significant variation in microbial populations is expected within and among 
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agricultural fields. A recent study performed on the effects of glyphosate-tolerant canola on soil 

microbial biomass, functional diversity and enzyme activity (Lupwayi, et al., 2007) found that 

effects on soil microorganisms were minor and inconsistent over a wide range of growing 

conditions and crop management. 

The effects of glyphosate and glyphosate-based formulations on soil microorganisms have been 

extensively investigated (Cerdeira and Duke, 2006; Sullivan and Sullivan, 2000). Results of 

standardized tests with glyphosate formulations performed 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 researchers have reviewed 

numerous laboratory and field studies, investigating the effects of glyphosate on soil bacteria and 

fungi (Felsot, 2000; Giesy, et al., 2000) and have concluded that glyphosate has a “remarkable 

level of safety for virtually all organisms tested”. In a recent greenhouse investigation, Arango 

(2009) evaluated the effects of multiple glyphosate applications on the rhizosphere bacterial 

community associated with glyphosate-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 glyphosate-tolerant soybean. Although some laboratory 

tests have shown effects on nitrogen-fixing bacteria (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 concentrations 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 et al., 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 over 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, Powell, et al. (2009) assessed effects of glyphosate 

applications on soil food web properties and crop litter decomposition in a glyphosate-tolerant 

soybean and maize rotation. The researchers concluded that: “Permanent responses in soil biota 

were not observed, suggesting a high level of resilience 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 community was similar when glyphosate or conventional 

herbicides were applied to crop rotations of glyphosate-tolerant soybean and maize cultivars. 

Moreover, the history of safe use and yield data obtained for nearly 15 years of glyphosatetolerant crop production, combined with in-crop applications of glyphosate-based agricultural 

herbicides, reinforce the findings that soil microbes 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 threatened or endangered under the Endangered Species 
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Act may be part of the affected 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 affected 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 use 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 Alfalfa 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 impacts 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 glyphosate tolerant alfalfa. The conclusion from the APHIS review 

is that threatened or endangered terrestrial or semi-aquatic plant 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 called Pre-Serve that describes location-specific 

mitigation measures that must be implemented when glyphosate is applied at a ground 

application rate of 3.5 lbs a.e. glyphosate 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 their aerial application rate is 0.7 lb a.e. glyphosate per acre or 

more (Monsanto Company, 2010). Only a very small percentage of glyphosate applications will 

require implementation of management practices. This is because the vast majority of U.S. 

cropland is not in close proximity to threatened and endangered plant species. 

I.4.14. Physical Environment 

I.4.14.1. Land Use 

The total U.S. acreage of canola increased more 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 increased rapidly following commercial introduction 

in 1999 and has remained between 50 and 70% of planted canola acres since 2001. Fluctuations 

in total canola acreage before and after glyphosate-tolerant canola was commercialized 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-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 pre-plant burn down and in-crop applications. 

Glufosinate-tolerant canola was introduced in 1995 and is tolerant to post-emergence 

applications of glufosinate. Glufosinate is the second most commonly applied herbicide used in 

canola (38% of acres) in 2008. 

As canola cropland has increased, 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 cropping 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 Canada and some European Union 

countries, where canola production is higher and soybeans are not grown on significant acreage. 

In the U.S., soybean oil is currently substantially less expensive than canola oil and the USDA 

ERS has concluded that this reduces 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 nitrogen fertilizer. These agricultural activities 

individually have potentially adverse environmental 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 emission of carbon dioxide, nitrogen 

oxide, sulfur oxide, and particulate matter. Agricultural practices have the potential to directly 

and indirectly impact air quality and to contribute emissions which could lead to climate change. 

Tillage contributes to the release of greenhouse 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 equipment (e.g., irrigation pumps and tractors) 

include carbon monoxide, nitrogen oxides, particulate matter, and sulfur oxides. Nitrous oxide 

may also be released following the 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 agriculture 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 discussed in Appendix Section I.4.7.2. The USDA 

Economic Research Service defines conservation tillage as cultural operations that maintain at 
Monsanto Company 11-CA-233U 405 of 500 

least 30% cover of the soil surface by plant residue at the time of planting (Anderson and 

Magleby, 1997). Conservation tillage can encompass a range of management practices, from notill to ridge- and strip-till cultivation to minimum tillage systems that restrict equipment traffic to 

dedicated zones. Special tillage field equipment can often perform the equivalent functions of 

several standard implements, reducing the necessity 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 residues on the surface of the planted field. 

Implementing conservation tillage practices can lead to both economic and production quality 

benefits, as well as having positive environmental impacts (www.nrcs.usda.gov). Use of 

conservation tillage compared to use of conventional 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 surfaces 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 production 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 data, the soil component of runoff is a much more 

important contributor to surface water impacts than is the pesticide component. 

Tillage causes widespread soil disturbance. Thus, erosion, topsoil loss and the resulting 

sedimentation and turbidity in streams 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 leading 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 conservation 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 those 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 leading canola-producing states, almost all are 
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previously used, highly persistent chemicals that are no longer registered for use in the U.S. 

(EPA 2008a). 

I.4.15. Economics 

The affected trade economic environment is defined as those countries with which the U.S. 

engages in canola feed, seed and food trade. The affected domestic environment is defined as 

any land in the U.S. that is currently producing crops that could incorporate a canola rotation, as 

well as land that could be converted from inactive 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-share 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 (Figure I-2). The U.S. 

shortfall is made up with imports primarily from Canada. In 2010 canola was planted on 

approximately 1.45M acres in the United States producing 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, 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 (USDANASS, 2011b). 

Canola has been able to benefit from the same price subsidies as other commodity crops, 

strengthening the profit potential of this crop. In most side-by-side tests, canola has provided a 

greater net profit than wheat, though ease of marketing and variability in yield are off-setting 

factors. 

Researchers have found that growers have realized substantial economic benefits with the 

adoption of herbicide-tolerant canola. A survey of canola growers in western Canada revealed 

that herbicide-tolerant technology generated from $1.063 – 1.192 billion in annual direct and 

indirect benefits over the 2005-2007 period partly attributed to lower input costs and partly 

attributed to better weed control (Smyth et al., 2010). 

I.5. Alternatives 

The decision-making process of deregulation is governed by 7 CFR § 340.6 (d)(3)(i) which 

states that APHIS may approve the petition in whole or in part, resulting in three possible 

outcomes from Monsanto’s petition, described below. 

 

38 The USDA FAS reports do not distinguish 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 whole” alternative, MON 88302 would no longer be a regulated 

article under 7 CFR Part 340 and would be widely 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” alternative, 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, currently deregulated herbicide-tolerant canola events including 

glyphosate-tolerant RT73 would continue to be available 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” alternative 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 information 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 MON 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 the 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 part 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 are subject to regulation in 

many countries. In order that canola seed harvested in the U.S. may be freely traded, Monsanto 
Monsanto 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 international trade (Petition Section VIII.K.). Monsanto adheres to 

the BIO Product Launch Policy39 including: 1) conducting a market and trade assessment, 2) 

securing regulatory approvals in key export countries prior to full commercial launch, 3) 

following generally accepted best seed management practices to prevent unintended low level 

presence of the event in seed, 4) providing reliable detection methods to growers, processors and 

buyers prior to commercialization, and 5) communicating to stakeholders the company’s product 

launch stewardship policies. These 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 alternative. The majority of commercial canola 

grown in the U.S. is already glyphosate-tolerant, and company stewardship policies described 

above ensure there will be no disruption of trade from the introduction of new biotechnologyderived products. The difference between the no action and the deregulation in whole alternative 

is expected to be the gradual replacement of the current Roundup Ready canola RT73 event with 

MON 88302. As described in Petition Sections VI. and VII., MON 88302 is compositionally 

and phenotypically equivalent to conventional canola. MON 88302 allows for a wider period of 

glyphosate application than RT73 at rates comparable to those already used for maize and 

soybean. The change in the biotechnology-derived trait, and the resulting allowable increase in 

glyphosate application, is not expected to have any impact on canola production and use. 

Therefore, the no action alternative and the approval 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 part 340. Under this alternative, 

seed production practices and geographies where 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, and canola seed production practices associated with herbicidetolerant varieties is not expected to change. 

Deregulation in Whole Alternative: Certified seed production is a carefully managed process 

(Petition Section VIII.B.) for maintaining high quality seed stocks, an essential basis for U.S. 

agriculture. Seed producers have learned to account 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 preserve the purity of canola seed developed for 

commodity and specialty uses. The Association of Seed Certifying Agencies (AOSCA) is 

dedicated to assisting companies in the production, identification, distribution and promotion of 

certified classes of seed. AOSCA establishes minimum standards for quality and identity. Its 

 

39 BIO’s Product Launch guidelines can be found at: 

http://www.excellencethroughstewardship.org/facts/documents/Guide%20for%20Product%20Launch%20Stewardsh

ip.pdf. 
Monsanto Company 11-CA-233U 409 of 500 

goal is to standardize certification regulations and procedures internationally so companies 

compete with one set of standards. The association cooperates with the Organization for 

Economic Cooperation and Development (OECD) and other international organizations to 

develop standards, regulations, procedures, and policies to expedite movement of seed and 

encourage international commerce in improved seed products. 

The difference between the no action and the deregulation in whole alternative is expected to be 

the gradual replacement of the current Roundup 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 breeders 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 management in seed 

production operations relative to practices used to produce existing glyphosate-tolerant varieties. 

Impacts on weed management from the introduction of MON 88302 are discussed in Appendix 

Section I.6.5. The change in the biotechnology-derived trait, and the potential changes in weed 

management, is not expected to have any impact on canola seed production. Therefore, the no 

action alternative and the approval in whole alternative would not differ in their impact to canola 

seed production. 

I.6.3. Specialty Canola 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 part 340. Under this alternative, 

specialty canola production and other Brassica production, including Brassica vegetable 

production, is expected to continue as described in Appendix Sections I.4.3., I.4.4. and I.4.5. 

Specialty canola growers would continue to manage their production 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 production 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 products that use herbicides or 

other pesticide treatments, as well as biotechnology-derived canola. These well established 

practices to avoid “excluded methods” will continue 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 biotechnology-derived trait to 

MON 88302 will have no impact on organic canola production practices 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, cauliflower, Brussels sprouts, collard, kale, kohlrabi), B. juncea
Monsanto 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 grown primarily in states such as California, 

Arizona, New York, Florida, Georgia, North Carolina, Wisconsin, Texas and Michigan where 

canola production is minimal. In states 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 process that maps production sites and ensures 

adherence to appropriate isolation distances. 

The difference between the no action and the deregulation in whole alternative is expected to be 

the gradual replacement of the current Roundup Ready canola RT73 event with MON 88302. 

The adoption of biotechnology-derived trait MON 88302 will have no impact on the current 

processes employed for planting and identity preservation in specialty canola, including organic 

canola or canola with modified oil composition or Brassica vegetables. Therefore, the no action 

alternative and the approval in whole alternative would not differ in their impact to specialty 

canola or Brassica vegetable production. 

I.6.4. Agronomic Practices – Tillage, Irrigation, Crop Rotations, Insect and Disease 

Management 

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 part 340 and would not be 

widely grown. Under this alternative, the agronomic practices for tillage, irrigation, crop 

rotation, insect pest management and disease management 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 ecological characteristics (Petition Sections VII, VIII and IX), and 

has the same levels of resistance to insects and diseases as conventional 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 deregulation in whole alternative is expected to be 

the gradual replacement of the current Roundup Ready canola RT73 event with MON 88302. 

The adoption of biotechnology-derived trait MON 88302 will have no impact on the current 

agronomic practices of tillage, irrigation, crop rotations and insect and disease management. 

Therefore, the no action alternative and the approval 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 canola 
Monsanto Company 11-CA-233U 411 of 500 

RT73 and associated glyphosate use on approximately the same percentage of acres as currently. 

Other weed control practices would continue to be utilized at current rates. Based on U.S. EPA 

approved labels for Roundup agricultural herbicides, 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 glyphosate 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 gradual 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 submitted 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 grower 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 that control of problematic weeds such as dandelion and Canada 

thistle would be improved. In addition, the increased 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 weed 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). Additionally 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-derived 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 alternative and the approval in whole alternative 

would not differ significantly in their impact 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 Appendix 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 replacement of the current Roundup Ready canola RT73 

event with MON 88302. As described in Petition Section VII. , MON 88302 is equivalent to 

conventional canola in terms of seed germination, dormancy and emergence. In addition no 

differences were detected between MON 88302 and the conventional control for early stand 

count, seedling vigor, pod shattering, seed quality, yield and final stand count. Based on the 

assessed characteristics, the results of this study demonstrate that there were no unexpected 

changes indicative of increased plant pest potential or adverse environmental impact of 

MON 88302 compared to conventional canola. Therefore, 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 practices, growers may control volunteer MON 88302 

canola with herbicides other than glyphosate and cultural practices just as they now control 

Roundup Ready canola RT73 volunteer canola with herbicides 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 articles and would not be widely grown. For the reasons described in 

Appendix Section I.4.7.11. and Appendix J, weeds that are glyphosate resistant are not currently 

an issue in canola production. With appropriate integrated weed management practices, this 

condition would be expected to continue. Under the no action alternative, 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 replacement of the current Roundup Ready canola RT73 

event with MON 88302. Canola, including glyphosate-tolerant canola, is produced in a crop 

rotation system that has relatively low susceptibility for development of weeds with herbicide 

resistance. As discussed in Appendix Section I.4.7.13, canola is typically grown in a three- to 

four-year rotation with small grains (wheat, barley and oats) that 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 glyphosate 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 Agricultural Commodities 

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 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 products like MON 88302 undergo a 

voluntary food and feed consultation process with the U.S. FDA prior to release on the market. 

Monsanto has already initiated this process and 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 deregulation in whole alternative is expected to be 

the gradual replacement of the current Roundup Ready canola RT73 event with MON 88302. 

Compositional assessments (Petition Section VI) conducted on MON 88302 seed support a 

conclusion that the composition of the seed is equivalent to that of the conventional control. The 

genetic modification in MON 88302, has no impact on the composition, and therefore on the 

food and feed safety or nutritional quality of this product compared to conventional canola. 

Residue tolerances for glyphosate 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 canola commodities. Therefore, the no action 

alternative and the approval in whole alternative would not differ significantly in their impact to 

canola weed management 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 articles and would not be widely grown. As such, any plantings of 

MON 88302 would take place under APHIS notification 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 Section I.4.9. As discussed in that section, there are 

reports of unassisted (e.g. without human intervention) 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 environmental fitness evidenced by a variety of characteristics including 

decreased pollen viability, seed production and seedling survival, when compared to parental 

varieties. 

As a result of state and federal regulatory requirements, seed certification requirements, seed 

grower agreements, geography, and/or biological constraints 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 continue to use biotechnology-derived, herbicide-tolerant canola having 

the same potential for cross pollination with sexually compatible species as conventional canola. 

Deregulation in Whole: The difference between the no action and the deregulation in whole 

alternative is expected to be the gradual 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 and is not expected to have a greater potential for cross 

pollination with related species than conventional 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 Alternative, MON 88302 and its progeny would 

continue to be regulated articles and would not be widely grown. Most ruderal canola results 

from seed loss during transportation and is found 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 year or two, and are usually found within a few 

feet of a roadway in disturbed soil not yet supplanted by grasses 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 detail 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 replacement of the current Roundup Ready canola RT73 

event with MON 88302. In studies biotechnology-derived canola populations have not been 

found to be more invasive or more persistent than conventional canola populations, and 

biotechnology-derived canola seed has not been demonstrated to persist longer than conventional 

canola seed. The introduction of MON 88302 is not expected to impact canola survival or 

control as compared to conventional canola. Thus, the no action alternative and the approval in 

whole alternative would not differ 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 regulated articles and would not be widely grown. Under this alternative, 

potential affects to public health relating to the production of canola are expected to remain 

unchanged and similar to those described in Appendix Section I.4.11. Canola is expected to 

continue to be grown at approximately 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. Biotechnology-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 the 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 enforces the tolerances set by the 

U.S. EPA. The U.S. EPA also reviews the proposed 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 deregulation in whole alternative is expected to be 

the gradual replacement of the current Roundup Ready canola RT73 event with MON 88302. As 
Monsanto Company 11-CA-233U 415 of 500 

summarized in Petition Section V.F, the CP4 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 structurally 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 EPSPS protein present in 

biotechnology-derived crops has been extensively evaluated (Harrison et al., 1996; ILSI-CERA, 

2010). The U.S. EPA has also reviewed the safety of the CP4 EPSPS protein 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 § 174.523). This exemption was based on a safety 

assessment that included rapid digestion in simulated 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 effects since the introduction 

of other Roundup Ready crops in 1996. 

Roundup Ready canola RT73 is planted on greater than 50% of U.S. canola acreage (USDANASS, 2008). Consequently glyphosate is the most widely used herbicide in canola. The 

toxicology of glyphosate has been 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 aspects, 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 composition 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 that has a history of safe use. 

Workers may be exposed to higher levels of glyphosate with MON 88302 because of the 

proposed higher glyphosate application rates relative to currently registered use patterns on 

canola. However, recall that the proposed rates of glyphosate applications are comparable to 

rates already used in maize and soybean (2 pounds a.e. per acre of glyphosate in-crop). The 

affected environment for pesticide use and human health is discussed in Appendix Section 

I.4.11.2. That section describes the U.S. EPA’s conclusions and the conclusions of other studies 

regarding worker exposure, and incidental exposure to non-workers who may be in the vicinity 

where glyphosate is being applied. Currently the 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 Alternative, 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 fractions as a feed source is expected to continue as described in 

Appendix Section I.4.12. Under the no action alternative, farmers will likely continue to plant 

Roundup Ready canola RT73 as a feed source at approximately the same rate as currently, 
Monsanto Company 11-CA-233U 416 of 500 

continue to apply glyphosate at label rates to control 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 gradual replacement of the current Roundup Ready canola 

RT73 event with MON 88302. As described in Petition Section V, the CP4 EPSPS protein 

expressed in MON 88302, as in other Roundup Ready 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) (Harrison et al., 1996). Furthermore, the 

composition of the seed produced by MON 88302 is unchanged compared to conventional 

canola. This information indicates that there would be no negative 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 feed derived from MON 88302 is as safe and nutritious as feed 

derived from conventional canola. 

I.6.13. Animal, Plant and Microbial Communities 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 articles and would not be widely grown. 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 grown 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 gradual 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 significant health 

risk to animals. The safety of CP4 EPSPS protein present in biotechnology-derived crops has 

been extensively evaluated (Harrison et al., 1996) and reviewed by the U.S. EPA which 

established a tolerance exemption for the protein and the genetic material necessary for its 

production in or on all raw agricultural commodities (40 CFR § 174.523). A history of safe use 

is supported by the lack of any documented reports of adverse effects since 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 on 
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MON 88302. Similarly, it is expected that there would 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 feeding damage were observed (Petition Section VII.) 

indicating similar insect susceptibility for MON 88302 compared to conventional canola. 

Additionally, in a quantitative assessment of pest and beneficial arthropod abundance, no 

statistically significant differences were detected between MON 88302 and the conventional 

control. As MON 88302-containing canola exhibits no toxic effects on animals or pollinators of 

other plants in or around fields cultivated with MON 88302, it is unlikely insects and animals 

will be significantly affected. 

 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. 

This maximum rate is similar to rates currently allowed for soybean and maize 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 environmental 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 Section I.4.13. and the maximum application rate of 

glyphosate at 1.55 pounds a.e. per acre, glyphosate use associated with MON 88302 is not 

expected to have any impacts on animal communities in or adjacent to canola fields. Thus, the 

no action alternative and the approval 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 articles and would not be widely grown. Under the no action 

alternative, the effect of canola on plant communities is expected to continue as described in 

Appendix Section I.4.13. Canola and glyphosate-tolerant canola are expected 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 occur 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; however these impacts can be minimized through good 

management practices such as decreasing spray pressure, lowering boom height, increasing 

nozzle size, avoiding making applications during 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 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 Section I.4.9. As discussed in that section, there are 

reports of unassisted (e.g. without human intervention) 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 environmental fitness evidenced by a variety of characteristics including 

decreased pollen viability, seed production and seedling survival, when compared to parental 

varieties. 

There is also the potential for ruderal canola populations 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 usually found in disturbed soil not 

yet supplanted by grasses and other more aggressive plants. In studies biotechnology-derived 

canola populations have not been found to be more invasive or more 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 deregulation in whole alternative is expected to be 

the gradual replacement of the current Roundup Ready canola RT73 event with MON 88302. 

MON 88302 does not exhibit characteristics associated 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 for cross pollination with closely related species or increased 

potential for the development of ruderal populations over conventional canola or the current 

Roundup Ready canola RT73. 

As discussed in Appendix Section I.4.11.2, glyphosate is a non-selective herbicide with postemergence activity on essentially all annual and perennial plants. As such, exposure to 

glyphosate could affect aquatic and 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, binds 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. Therefore, any potential glyphosate effects to nontarget plants are 

only due to exposure via spray drift. As described in Appendix Section I.4.13., using 

methodology from a recent U.S. EPA effects determination, 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 addition, glyphosate labels include specific risk 

management measures to manage spray drift, including mandatory requirements for aerial 

applications. 

Therefore, the no action alternative and the approval in whole alternative would not differ 

significantly in their impact on plant communities. 
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I.6.13.3. Soil Microorganisms 

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 the 

effect of canola on soil microorganisms is expected to continue as described 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 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 gradual 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 CP4 EPSPS protein pose any concern to soil 

microorganisms (ILSI-CERA, 2010). Therefore, MON 88302 is not expected to have any 

impacts on soil microorganisms. 

The effects of glyphosate on soil microorganisms have been extensively investigated (Giesy et 

al., 2000; Sullivan and Sullivan, 2000). Long-term studies following repeated applications of 

glyphosate in the field for six (Olson and Lindwall, 1991) or over ten years (Biederbeck, et al., 

1997; Hart and Brookes, 1996) have shown no detectable adverse effects on soil microbes. 

Investigations by Haney (2002; 2000) related to the increased use of glyphosate-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 regulated articles and would not be widely grown. Under this alternative, the 

potential impact of canola on threatened or endangered (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 proposed for listing, or a host of any TE species 

because there are no listed species or species 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 the use of glyphosate applied to canola have been 

found, and none would be expected, based on the application 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 gradual replacement of the current Roundup Ready canola 

RT73 event with MON 88302. 

As with the no action alternative, 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 the environment and is not commonly 

known for human or animal pathogenicity or allergenicity. The CP4 EPSPS protein lacks 

structural similarity to allergens, toxins or other proteins known to have adverse effects on 

mammals. The CP4 EPSPS protein is rapidly digested in simulated digestive 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. 

As the action agency for pesticide registrations, the U.S. EPA has the responsibility to conduct 

an assessment of effects of a registration action on threatened and endangered 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 endangered species. Some of the elements of that 

process, generally taken from the 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 extensive exposure and ecological effects data to 

determine how a pesticide will move through and break down in the environment. Risks to 

birds, fish, invertebrates, mammals 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 and regulatory processes ensure 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 develop label use restrictions for the pesticide. 

These label restrictions carry the weight of law 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 whether individuals of a listed species have 

the potential to be harmed by a pesticide, 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 species, after conducting a thorough ecological risk 

assessment, results in an "effects determination" in accordance with Section 7 (a)(2) of the 

Endangered Species Act. 

As a part of the U.S. EPA’s threatened and 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 acre on fish, amphibians, aquatic invertebrates, 

aquatic plants, birds, mammals, and terrestrial invertebrates. This assessment determined that at 

the maximum application rate for in-crop applications of glyphosate to MON 88302 (1.55 lbs 

a.e. glyphosate per acre) there would be no effects of glyphosate use on the following taxa of 

threatened and endangered species: fish, amphibians, birds, and mammals. The U.S. EPA 

assessment was uncertain of the effects on terrestrial invertebrates, citing the potential to affect 

small insects at all application rates and large insects at the higher application rates. The U.S. 

EPA considered these potential effects as part of their review process and label use restrictions 
Monsanto Company 11-CA-233U 421 of 500 

imposed under authority of FIFRA. To mitigate potential adverse effects to threatened and 

endangered species, the U.S. EPA has imposed specific 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, non-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 possibility of exposure and adverse impacts to TE species from 

glyphosate application to MON 88302; the U.S. EPA has considered potential impacts to TE 

species as part of their registration and labeling process for glyphosate; and adherence to the U.S. 

EPA label use restrictions by the pesticide applicator will ensure that the use of glyphosate will 

not adversely affect threatened and endangered species or critical habitat. Based on these factors 

and the legal requirements for pesticide applicators to follow the U.S. EPA label use restrictions, 

the use of the U.S. EPA registered glyphosate for MON 88302 production 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 are expected to continue as described in Appendix Section 

I.4.14. Under the no action alternative, growers 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 applications as currently. 

Deregulation in Whole Alternative: The difference between the no action and the deregulation in 

whole alternative is expected to be the gradual replacement of the current Roundup Ready canola 

RT73 event with MON 88302. 

Glyphosate-tolerant canola has been deregulated and grown in the U.S. since 1999. Roundup 

Ready canola RT73 currently occupies greater than 50% of total canola commercial acres. The 

acreage of glyphosate-tolerant canola increased rapidly following commercial introduction and 

has remained between 50 and 70% of planted canola acres since 2001. Fluctuations in total 

canola acreage before and after glyphosate-tolerant canola was commercialized (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-tolerant trait. 

Despite the rapid adoption of herbicide-tolerant canola products in the past decade, there has 

been no significant impact on total cropland acreage in the U.S. For example, from 2001 to 

2010, the total annual commercial canola acres planted 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 dictated by many factors, the 
Monsanto 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 availability or adoption of biotechnology-derived 

traits. Thus, the introduction of MON 88302 is not anticipated to facilitate production of canola 

in areas where it is not currently grown or have significant 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 articles and would not be widely grown. Potential impacts on air quality 

and climate change associated with canola production are not expected to change under the no 

action alternative (Appendix Section I.4.14.). Under the no action 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 gradual 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 MON 88302 is not anticipated to result in significant 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 articles 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 Appendix Section I.4.14. Under the no action 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 impacts on surface and groundwater. 

Deregulation in Whole Alternative: Water quality could be impacted either directly by 

MON 88302 via plant material impacts on water resources, or indirectly 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 the previous crop residue covering the soil when 

planting another crop has been increasingly employed 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 water 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 action 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 EPSPS protein present in other glyphosate-
Monsanto Company 11-CA-233U 423 of 500 

tolerant crops has been extensively evaluated, 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 practices 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 tolerance trait as in MON 88302. 

Therefore, it is unlikely that the presence of CP4 EPSPS protein in MON 88302 will have a 

significant impact on water quality. 

The difference between the no action and the deregulation in whole alternative is expected to be 

the gradual replacement of the current Roundup 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 allowed 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 environment” (U.S. EPA, 1993). Glyphosate is rapidly 

adsorbed and tightly complexed by soil particles 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 other 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; Cerdeira 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 articles and would not be widely grown. Economic conditions 

associated with growing canola, which are not expected to change under the no action 

alternative, are discussed in Appendix Section I.4.15. Under the no action alternative, growers 

will likely continue to use Roundup Ready canola 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 other organic crops when biotechnology-derived 

canola was introduced in the U.S. Another organic grower considered 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 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, 2011b). Although canola production in the U.S. has increased dramatically 

since the 1980s, it has always been far short of demand (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 canola acres have not grown substantially (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 alternative is expected to be the gradual replacement 

of the current Roundup Ready canola RT73 event with MON 88302. It is not expected that the 

introduction of MON 88302 will result 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 growers would be able to apply higher rates of 

glyphosate for a greater period of time during canola’s life cycle. Thus the costs of weed 

management may rise incrementally. However, researchers have found that growers have 

realized substantial economic benefits with the adoption of herbicide-tolerant canola. A survey 

of canola growers in western Canada revealed that herbicide-tolerant technology generated from 

$1.063 – 1.192 billion in annual direct and indirect benefits over the 2005-2007 period partly 

attributed to lower input costs and partly attributed to better weed control (Smyth et al., 2010). 

Therefore it is unlikely the deregulation 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 environment 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. Cumulative 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 timeframe. The cumulative impact analysis follows 

CEQ’s guidance: Considering Cumulative Effects under the National Environmental Policy Act 

(CEQ, 1997). The steps associated with the analysis include: 

 Specify the class of actions for which 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 past, present, and reasonably foreseeable future 

actions within the affected environment. 

 

40 40 CFR 1508.7
Monsanto 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 articles and would not be widely grown. For MON 88302 the only 

impacts are those associated with the increased application rate of glyphosate. Under the no 

action alternative, weed management 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 acres as currently. Other 

weed control practices would continue to be utilized at current rates. Based on U.S. EPA 

approved labels for Roundup agricultural herbicides, 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 expected to change significantly. 

 

Deregulation in Whole Alternative: The impact of the deregulation in whole alternative results 

from the option to use increased application rates 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 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 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 for herbicides or pesticides that persist 

in the environment and thus can continue to accumulate with repeated applications. 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 tended to 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 today, its effects, both positive and negative, would 

soon be negligible. Therefore, the temporal cumulative effects of increased 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 effects 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 could potentially be impacted by drift. Cumulative effects would 

occur only when the area of influence for some other glyphosate application intersected with the 

area of influence for a MON 88302 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 approximately 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 

Glyphosate 

per acre1

RR 

Adoption2

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 total 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 Washington 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 produced in three areas of Washington 

and Oregon: in western Washington in the lower-lying areas around the Puget Sound between 

the Olympic Mountains and the Cascades; in the Columbia Basin of eastern Washington; and in 

Oregon’s Willamette Valley, located between the Coastal Range and the Cascades. 

The most comprehensive information is available for Washington State. Based on the discussion 

below, the total estimated Washington acreage of Brassica and related vegetable seed production 

is approximately 4,500 acres, of which approximately 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 data 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 Washington, 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 production 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 acres in eastern Washington and 2,160 acres in 

western Washington for an overall total maximum of 4,473 acres) (Thomas, 1997). 

McMoran (2010) and du Toit (2007) both report that Washington produces 75% of the U.S. 

cabbage seed crop. Thomas et al., (1997,) reported that Washington produced 100% of the U.S. 

Brussels sprouts and kale seed crop, 75% of the U.S. cauliflower seed 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 crops 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 production; most of the remaining acreage is in 

grains, hay and forage (OSU 2011). While there 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 vegetable and flower seed production (OSU, 2011). 

According to the Willamette Valley Specialty 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) western Washington and western Oregon combined produce nearly 
Monsanto Company 11-CA-233U 

Attachment Table 1. Washington State Brassica and Related Vegetable Seed Production West of Cascades (Western) Colu

(East

 Acres 

Skagit 

Island 

Jefferson 

Snohomish 

Clallam 

Lewis 

San Juan 

Whatcom 

Grant 

Cabbage (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; or B. 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 REPORTS 3,750-4,500 

Source: WSU, 2010. 
Monsanto Company 11-CA-233U 429 of 500 

all (~90%) of European cabbage, Brussels sprouts, rutabaga and turnip seed, and a 

substantial portion (20-30%) of radish, 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, cabbage, cauliflower, kale, kohlrabi, mustard 

and radish. 

 Region 4 – southeast, bordering Nevada and Arizona; includes Imperial Valley – 

broccoli, cabbage, cauliflower, collard greens, 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, but there is no acreage information. 

California and Arizona are the only states for which specific mention 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 specify the seed types (University 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 Idaho’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 

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Appendix J: Herbicide Resistance

J.1. Introduction 

Herbicides are pesticides intended to prevent or kill weeds that can compete with a crop 

for nutrients, water and in some cases, sunlight. By killing weeds, herbicides allow 

planted crops to grow and thrive, thereby increasing crop yield and allowing these crops 

to be grown on fewer acres, protecting habitat and wildlife from unnecessary expansion 

of cropland production wherever possible. 

Plant populations can develop resistance to a herbicide 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 the herbicide in the absence of other 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) exhibiting resistance more quickly than other 

herbicides (e.g. glyphosate, auxins (dicamba), dinitroanilines). 

Herbicide resistance could become a limiting factor in crop production if the resistant 

weed population cannot be controlled with other herbicides or cultural practices. This 

generally has not been the case for any herbicide and weed combination. For most crops, 

there are multiple herbicide options for growers to use. However, good management 

practices to delay the development of herbicide resistance have been identified, are being 

actively promoted by the public and private sectors, and are being implemented by 

growers. 

Monsanto considers product stewardship to be a fundamental component of customer 

service and business practices. Stewardship of the glyphosate molecule to preserve its 

usefulness for growers is an important aspect of Monsanto’s stewardship commitment. 

Although herbicide resistance may eventually occur in weed species when a herbicide is 

widely used, resistance can be delayed, contained and managed through research, 

education and good management practices. Monsanto will invest in research to identify 

best management practices, monitoring for resistance, and in dealer/grower education and 

training programs to provide information on best practices to manage glyphosate weed 

resistance in canola. These are the key elements of Monsanto’s approach to providing 

stewardship of glyphosate relative to all 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. 
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J.2. The Herbicide Glyphosate

Glyphosate (N-phosphonomethyl-glycine) (CAS Registry #: 1071-83-6), the active 

ingredient in the Roundup family of nonselective, foliar-applied, post-emergent 

agricultural herbicides, is among the world’s most widely used herbicidal active 

ingredients. Glyphosate is highly effective against the majority 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 acid biosynthesis in plants and 

microorganisms (Franz et al., 1997; Steinrücken and Amrhein, 1980). Glyphosate is the 

only known herbicide with this mode of action (Franz et al., 1997). The relevant 

aromatic amino acid pathway is not present in mammalian metabolic systems (Cole, 

1985; Franz et al., 1997). A comprehensive human safety evaluation and risk assessment 

concluded that glyphosate has low toxicity to mammals, is not a carcinogen, does not 

adversely affect reproduction and development and does not bioaccumulate in mammals 

(Williams et al., 2000). An ecotoxicological risk 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 environmental characteristics, including a low potential to 

move through the soil to reach ground water and is degraded over time by soil microbes. 

Because it binds tightly to soil, glyphosate’s bioavailability is reduced immediately after 

application, which is why glyphosate has no residual soil activity. Similar conclusions 

were reached in an independent assessment of risk to human health and to the 

environment that was conducted as a part of the Environmental Impact Statement for 

Glyphosate Tolerant Alfalfa (USDA-APHIS, 2010). 

J.3. Herbicide Use in Canola Systems and Herbicide-resistant Weeds 

Weed control in canola production fields is critical for obtaining optimized yields. 

Because failure to control weeds within the crop can result in decreased yields and 

reduced crop quality, a program for weed control is essential to ensure a grower can 

optimize yields. In using MON 88302 weed control will be obtained through the use of 

glyphosate plus other herbicides. Control of weeds in a crop is essential because weeds 

compete with the crop for the same limited resources in the field, 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 particularly 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 difficult-tocontrol weeds such as Canada thistle (NDSU, 2005; Padgette et al., 1996). 

With any herbicide use, however, 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 expose 
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the weed population to a "selection pressure," which may lead to an increase in the 

number of surviving resistant individuals in the population (Jasieniuk et al., 1996). In 

other words, plants susceptible to the applied herbicide will die, while those few having 

some type of natural resistance may survive and reproduce. 

A resistant weed must demonstrate two criteria as defined by the Weed Science Society 

of America website at www.wssa.net: (1) the ability to survive application rates of 

herbicide product that once were effective in controlling it and; (2) resistance is heritable. 

Herbicide-resistant weeds are neither a new phenomena nor is resistance unique to 

glyphosate. Growers have been managing herbicide-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 repeated applications of a 

herbicide product, the actual onset of resistance 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 population of that species (Sammons et al., 

2007). Some herbicide products are much more prone to develop herbicide resistance 

than others (Heap, 2009). Considering the substantial worldwide 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 largely due to the factors described above, 

which relate to chemistry and function, in addition 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 are inherently resistant to a herbicide. 
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Figure J-1. Weed Resistance to Various Herbicide Chemical Families 
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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. Rather, over time, the repeated application of herbicide selects 

for those few biotypes that are less susceptible to the herbicide and they increase in 

frequency within a population. To date, the 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 the 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 the plant cell (exclusion) (Sammons et al., 2007). 

Herbicide resistance via target site is the 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 type of target site alteration involves amino acid 

substitution(s) in the enzyme that prevents the binding of the herbicide without impacting 

enzyme activity, and the plant is able to grow normally without any fitness penalty. For 

ALS inhibitors, the level of resistance conferred 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 particular 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 transition state inhibitor (Sammons et al., 2007; 

Schönbrunn et al., 2001) differentiating glyphosate from ALS inhibitors and ACCase 

inhibitors. The transition state is an unstable state during an enzyme reaction half-way 

between the substrate and product. Transition state inhibitors are very effective enzyme 

inhibitors. In addition, multiple alterations of the same enzyme have been found for ALS 

inhibitors (Tranel and Wright, 2002). This may explain the apparent high frequency of 

resistance and the short time in which resistance developed to herbicides in these two 

classes of chemistries. Only one altered site in the targeted plant EPSPS enzyme 

impacting glyphosate insensitivity has been found for glyphosate (Baerson et al., 2002). 

Another type of a target site resistance mechanism recently discovered for glyphosate, is 

an over amplification of the EPSPS gene which results in an overproduction of the 

EPSPS enzyme (Gaines et al., 2010). This mechanism was discovered in Palmer 

pigweed. 

The second general type of herbicide 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 have been shown to degrade glyphosate and 

therefore this type of resistance mechanism may be active in some species (Gaines et al., 

2010). 

Herbicide resistance as a result of exclusion mechanisms is the glyphosate resistant 

mechanism among the majority of the weed species studied to date. This resistance 

mechanism has also been found to be associated with 2,4D and paraquat. Within this 

category, there are two types of translocation alterations that have been observed for 

glyphosate; (a) restricted movement of glyphosate from leaf cells into the meristematic 

cells of the plant (Shaner, 2009) and (b) decreased delivery of glyphosate to the 
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chloroplast (Sammons et al., 2007). The level of glyphosate resistance conferred with 

this mechanism is higher (6-8X) than for species exhibiting amino acid substitution type 

target site mutations (2-3X). 

In some species, the experimental evidence suggests that more than one mechanism of 

glyphosate resistance may occur within the same plant to protect the plant from the 

phytotoxic effects of glyphosate (Yu et al., 2007). This implies that multiple genes 

(polygenic resistance) are necessary and thus the selection of plants with multiple genes 

needed to confer resistance would be expected to occur at a low frequency. 

In summary, the overall low occurrence of glyphosate resistance may be in part explained 

by: (1) the nature of the target 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 the type of resistant mechanism operating 

within a species or population of a species. 

J.5.2. Use of Recommended Rate 

The interaction between herbicide application rate and resistance for post-emergence 

herbicides, such as glyphosate, is dependent upon the nature of the plant gene(s) 

conferring resistance to the chemical. In general, herbicide rate has more effect on 

selecting for resistant individuals in a population if the resistant 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 blackgrass, diclofop-resistant ryegrass and dicamba-resistant 

kochia (Beckie, 2006). It has been demonstrated that, in three generations 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 resistance 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, the 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) with equal or greater potential for resistance 
Monsanto Company 11-CA-233U 457 of 500 

would be used to control weeds and 2) other herbicides and cultural practices can be used 

to manage the glyphosate resistant species (Gustafson, 2008; Neve, 2008). 

Through March 2011, biotypes of twenty-one weed species resistant to glyphosate have 

been identified and confirmed worldwide (WSSA, 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 species with resistant biotypes, such as common 

ragweed (Ambrosia artemisfolia), have been found in a limited 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 reproductive biology of the particular weed 

species involved appears to be a factor contributing 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 distribution 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 glyphosate. That is, they have the inherent ability to survive 

herbicide treatments as opposed to resistance 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 glyphosate than others (e.g. 

lambsquarters (Chenopodium album) and morninglory (Ipomea sp.)) and require more 

care to make sure the correct amount of glyphosate 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 easier 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 situations involving tolerant 

or difficult-to-control species are often confused with resistance. 
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J.7. Use of Glyphosate for In-crop Weed Management 

Monsanto has developed plants through biotechnology to be tolerant to glyphosate. The 

development, approval and cultivation of these Roundup Ready crops have facilitated 

additional uses of glyphosate in crops where 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 tolerance in Roundup Ready crops has no effect 

per se on the control of weeds. From a weed resistance standpoint, the use 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 ability 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 increase in no-tillage 

practices (Dill et al., 2008). 

Since Monsanto commercialized the first Roundup Ready canola varieties in 1998, 

growers have enthusiastically adopted the technology. The Roundup Ready canola 

system, (i.e., planting Roundup Ready canola and applying glyphosate 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 Universities/Cooperative Extension Services (CES) publish 

information on best weed management practices in Roundup Ready crops to address both 

of these objectives (see Table J-2). In addition Monsanto and other companies selling 

glyphosate products provide information on these 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 conducted investigations and worked extensively 

with academics and other herbicide manufacturers 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 populations 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-crop tillage) over an extended period of time 

(Beckie, 2006; Peterson 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 greater flexibility of glyphosate herbicide 
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application. 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 

development than is possible with the current product. 

Monsanto will communicate to all canola growers recommended weed resistance 

management practices. Monsanto will provide instructions to canola growers regarding 

reporting any incidence of repeated non-performance of Roundup agricultural herbicides 

on a particular weed, and Monsanto will investigate cases of unsatisfactory weed control 

to determine the cause of poor performance. In cases where resistance is confirmed, 

Monsanto will provide recommendations for alternative control methods for farmers (see 

Table J-2). These recommendations are made available through Monsanto supplemental 

labels, the Monsanto Technology Use Guide (TUG), Monsanto and University 

publications and internet sites for growers, consultants, retailers and distributors. In all 

cases of glyphosate-resistant weeds in the U.S. and globally, there are alternative 

herbicides and cultural methods available to farmers 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 recommendations that will be made for the use of 

glyphosate in conjunction with varieties containing the second generation glyphosatetolerant MON 88302 event will not differ from recommendations being made for 

commercial varieties containing event RT73 (Roundup Ready canola varieties). These 

recommendations are consistent with the 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), cultural techniques and herbicides. The EPA is 

the U.S. federal regulatory agency that administers the federal law governing pesticide 

sale and use (FIFRA). The U.S. EPA encourages pesticide manufacturers to provide 

growers with information regarding a herbicide’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 approach to resistance management is sound and 

would be highly beneficial to pesticide manufacturers and pesticide users” (U.S. EPA, 

2001). The U.S. EPA approves all pesticide label use instructions based on the agency’s 

evaluation of supporting data supplied by the pesticide 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 label directions. 

Monsanto incorporates the U.S. EPA’s guidelines 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 containing MON 88302 (An 

example of the current Roundup WeatherMAX product label is available at 

http://www.cdms.net/LabelsMsds/LMDefault.aspx?manuf=23&t). The U.S. EPAapproved labels for Roundup branded herbicide weed-resistant management 

recommendations are designed to minimize the potential for the development of 

glyphosate-resistant weeds. By approving a label for a glyphosate-based agricultural 
Monsanto Company 11-CA-233U 460 of 500 

herbicide, the U.S. EPA has concluded that 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 guidelines on the labels of Roundup agricultural 

herbicides include recommendations 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 identify the appropriate application rate of 

glyphosate required to control a particular weed at various growth stages under various 

agronomic and environmental conditions. These rates are based on over 35 years of 

ongoing research at Monsanto to evaluate 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 universities (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 with different modes of action in a rotational 

crop, or using multiple herbicides in sequence 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 the tools needed to minimize resistant weed 

development, Monsanto will continue to investigate 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 flushes 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 (Beckie, 2006; Powles, 2008). Crop rotation and 

management of the fallow period and cover crops 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 

weeds1

Reference2

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, glufosinate(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

ND
Monsanto Company 11-CA-233U 462 of 500 

Table J-2. Management Recommendations for Control of Glyphosate Resistant Weeds in 

Canola and Rotational Crops (continued) 

Glyphosate 

Resistant 

Weed

Crop Recommendations for alternative 

herbicides to manage glyphosate resistant 

weeds

Reference2

Giant 

ragweed

Canola Post: clopyralid, glufosinate(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 Arkansas, 2011); GA: (University of Georgia, 2010); TN: (University 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 licensees 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 grown, through its relationship with farm 

advisors, and its relationship with key University/CES personnel. This will allow the 

timely recognition of performance issues that could arise related to weed resistance or 

other means. Monsanto field employees and hired consultants are trained and provided 

processes for responding to product performance inquiries. As warranted, individual 

performance issues that could be related to potential resistance are promptly handled. In 

addition, performance inquires are periodically reviewed 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 mitigation plan is designed to manage the 

resistant biotype through effective 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 alternative 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, Monsanto 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 studies to understand the potential for weed 

resistance and weed shifts as the result 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 herbicides or all weeds. As a result, 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 control through proper application rate, choice of cultural 

practices, and appropriate companion weed control tools will allow Roundup agricultural 

herbicides to continue to be used effectively. In cases where weed populations have 

developed resistance to glyphosate, 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 stewardship 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 rapidly 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. Tran, 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 weeds. 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-council.merchantsecure.com/canola_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. Atkinson (eds.). Butterworth's & Co., Boston, 

Massachusetts. 

Dill, G.M. 2005. Glyphosate-resistant crops: History, 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 and 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 Global 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 Environmental Contamination and Toxicology 167: 35-

120. 

Gressel, J. and L.A. Segel. 1990. Modeling the effectiveness of herbicide rotations and 

mixtures as strategies to delay or preclude resistance. Weed Technology 4: 186-198. 
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Gustafson, D.I. 2008. Sustainable use of glyphosate 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.weedscience.org/in.asp [Accessed June 23, 

2010]. 

HRAC. 2011. Weed resistance management: Proposed HRAC stewardship guidelines for 

herbicide labels. Herbicide Resistance Action Committee, St. Louis, Missouri. 

http://www.hracglobal.com/Publications/HRACStewardshipGuidelinesforHerbicideLabel

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 Dakota 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 ReadyTM gene. Pages 53-84 in Herbicide-Resistant Crops: 

Agricultural, Environmental, Economic, Regulatory and Technical Aspects. S.O. Duke 

(ed.). CRC Press, Inc., Boca Raton, Florida. 

Peterson, D., B. Olson, K. Al-Khatib, R. Currie, J.A. Dille, J. Falk, P. Geier, D. Regehr, 

P. Stahlman and C. Thompson. 2007. Glyphosate stewardship. MF-2767, Kansas State 

University Agricultural Experiment Station 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. Heering, N. Dinicola, H. Glick and G.A. Elmore. 2007. 

Sustainability and stewardship of glyphosate and glyphosate-resistant crops. Weed 

Technology 21: 347-354. 

Schönbrunn, E., S. Eschenburg, W.A. Shuttleworth, J.V. Schloss, N. Amrhein, J.N.S. 

Evans and W. Kabsch. 2001. Interaction of the herbicide glyphosate with its target 
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enzyme 5-enolpyruvylshikimate 3-phosphate synthase 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. Biochemical and Biophysical 

Research Communications 94: 1207-1212. 

Tranel, P.J. and T.R. Wright. 2002. Resistance of weeds to ALS-inhibiting herbicides: 

What have we learned? Weed Science 50: 700-712. 

U.S. EPA. 2001. Pesticide registration (PR) notice 2001-5, guidance for pesticide 

registrants on pesticide resistance 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 Extension Service, Division of Agriculture, 

Fayetteville, Arkansas. 

University of Georgia. 2010. Corn production guide. University of Georgia, College of 

Agricultural & Environmental Sciences, Athens, Georgia. 

http://www.caes.uga.edu/commodities/fieldcrops/gagrains/2008ComProductionGuide.ht

ml [Accessed December 9, 2010]. 

University of Nebraska-Lincoln. 2011. Weed control guide results. University of 

Nebraska-Lincoln Agronomy & Horticulture Department, Lincoln, Nebraska. 

http://weedscience.unl.edu/cornburndownfs.cfm [Accessed March 21, 2011]. 

University of Tennessee. 2010. 2010 weed control manual for Tennessee. University of 

Tennessee, Institute of Agriculture, Knoxville, Tennessee. 

USDA-APHIS. 2010. Glyphosate-tolerant alfalfa events J101 and J163: Request for 

nonregulated status. U.S. Department of Agriculture, Animal and Plant 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. Wicks, P.L. Chapman, J. 

Withrow, D. Legg, C. Alford and T.A. Gaines. 2008. Weed population dynamics after six 

years under Glyphosate- and conventional herbicide-based weed control strategies. Crop 

Science 48: 1170-1177. 
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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 definitions. Weed Science Society of Ameica, 

Lawrence, Kansas. http://www.wssa.net/Weeds/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 control guide. North Dakota State University 

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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 weeds that would interfere with the growth of 

many food and non-food crops, including biotechnology-derived crops, as well as for 

control of weeds growing in non-crop areas. Glyphosate received a label for use in 

cropping systems, and has had food and feed tolerances for residues in the U.S. since 

1975. In 2001, the U.S. U.S. identified glyphosate 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 studies 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 glyphosate residues” (U.S. EPA, 2006d). 

The following discussion provides an overview of the regulatory and risk assessment 

processes applicable to glyphosate and all other agricultural use pesticides. Glyphosate 

has been approved by the EPA for a large number of food and feed uses, 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 Rodenticide Act (FIFRA) requires that before 

sale or distribution of a pesticide in the 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 this standard, the U.S. EPA must evaluate 

potential risks to humans and the environment, and may require applicants to submit 

more than 100 different scientific studies and tests conducted according 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 available in the marketplace. 

The process of registering a pesticide is a scientific, legal and administrative procedure 

through which the U.S. EPA examines the ingredients 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 disposal practices. In 
Monsanto 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 effects associated with use of the product. 

The data required by the EPA are used to evaluate whether a pesticide has the potential to 

cause adverse effects on humans, wildlife, fish and plants (including endangered species 

and non-target organisms that the pesticide is not intended to act against). This includes 

potential human health and safety risks ranging 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 surface 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 according 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 pesticide is not the only opportunity an agency like the U.S. 

EPA has to evaluate its safety. The 1988 amendments to FIFRA authorized the EPA to 

conduct a re-registration program of pesticides first registered 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 are 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), describing/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 described 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 pesticides to ensure that as changes in science, 

public policy, and pesticide use practices occur, products in the marketplace can still be 

used safely. The Registration Review process for glyphosate started in 2009 and is 

expected to be completed in 2015. During the Registration Review process, the U.S. 

EPA will be requesting additional data for glyphosate 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 (maximum pesticide residue levels 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 Federal Food, Drug, and Cosmetic Act 

(FFDCA). Under the FFDCA, the U.S. EPA must find that such tolerances will be safe, 

meaning that there is a reasonable certainty 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 tolerance established before a pesticide can be 

registered for use on the particular food or feed 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 pesticide (now including occupational 

exposure41, exposure through diet, from using pesticides in and around the home, 

and from drinking water); 

 the cumulative effects from exposure to different pesticides that produce similar 

effects in the human body; 

 whether there is increased exposure to infants 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-disruption 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 risk assessment. The EPA uses the National 

Research Council’s four-step process for human health risk assessment, which involves 

hazard identification, dose-response assessment, exposure assessment and risk 

characterization. Each of these 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 types 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 potential toxicity of the pesticide 

in various tests including, where appropriate, 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 information on any aspect of the chemical. 

Dose-response assessment 

The next step of the risk assessment considers the levels at which the pesticide produces 

adverse effects. Dose-response assessment involves 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 occupational 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; December 9, 2009). 
Monsanto Company 11-CA-233U 472 of 500 

Exposure Assessment 

Step three of the process involves an exposure assessment. People can be exposed to 

pesticides in three ways: 1) inhaling pesticides (inhalation exposure); 2) absorbing 

pesticides through the skin (dermal 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 exposure include agricultural (food); home and 

personal use pesticides; pesticides applied to lands that make their way into the drinking 

water; or occupational exposure for agricultural workers or pesticide applicators. 

Risk Characterization

Risk characterization is the final step in assessing 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 pesticide. It explains the assumptions used in 

assessing exposure as well as the uncertainties 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 likelihood of people coming into contact with 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, which results in a potential risk. 

The U.S. EPA recognizes that effects of exposure to all pesticides including glyphosate 

vary between animals of different 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 for interspecies extrapolation and 10X for 

intraspecies variability). This uncertainty factor creates an additional margin of safety for 

protecting people who may be exposed to the pesticides. 

Once the risk assessment process 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, the U.S. EPA can then make a more informed 

decision regarding whether to approve a pesticide chemical or use, as proposed, or 

whether additional protective measures are 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 unacceptable risk to consumers. Another example of 

protective measures is requiring workers to wear personal protective equipment (PPE) 
Monsanto Company 11-CA-233U 473 of 500 

such as a respirator or chemical resistant gloves, or not allowing workers to enter treated 

crop fields until a specific period of time has elapsed. 

The U.S. EPA is responsible under the Federal 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-target species when used according to label 

directions. Tier 1 “worst-case” or appropriately-refined 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 established food and feed tolerances in the U.S. (see 

Attachment 2). Each time the U.S. EPA reviews 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 human exposure to the pesticide, and find that 

aggregate exposure to the pesticide will be safe as defined by the statute and regulations. 

As noted above, historically, issues associated with potential 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 exposure into the aggregate assessment (74 FR 65121, EPA–HQ–

OPP–2009-0889; December 9, 2009). 

Over the course of these numerous reviews the toxicology of glyphosate has been 

extensively studied. Multiple comprehensive 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 available toxicology data, the EPA 

concluded that glyphosate should be classified in Group E carcinogen (evidence of noncarcinogenicity in Humans), the most favorable 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, where necessary, uses associated with glyphosatetolerant crops developed through biotechnology. As discussed above, prior to the 

approval of any new use of an existing registered pesticide, U.S. EPA must consider the 

potential human health effects from the aggregate (total combined) human exposure to 

that pesticide, combining the potential exposure from the proposed new use with all other 

existing exposures to the pesticide. Dietary exposure is considered, which addresses 

pesticide residues that may remain on food from crops on which the pesticide is applied 

(pre- or post-emergence), as well as any residue that could be found in drinking water as 

a result of pesticide use. Non-dietary exposure is also included in this assessment, which 

includes exposure to the pesticide through residential use, such as on lawns or in flower 

beds, as well as exposure in a recreational context, such as from a golf course or sports 
Monsanto Company 11-CA-233U 474 of 500 

field. Based on these data, the U.S. EPA must 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 dietary risk assessment for glyphosate because 

no acute human health concerns have ever been determined from toxicological studies 

conducted with glyphosate. Accordingly, the U.S. EPA does not expect glyphosate to 

pose an acute risk (U.S. EPA, 2006a; 2006b). The U.S. EPA assesses chronic dietary risk 

for glyphosate using “worst-case” (Tier 1) exposure assumptions that include tolerance 

level residues and 100% crop treated. For food, this estimate 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 pesticide residues found on all harvested food crops 

are at the level of the legally established tolerance (i.e., the maximum allowable pesticide 

residue level). For water, the U.S. EPA assumes 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 degradation in the water body or partitioning to sediment 

within the water column (U.S. EPA, 2006a; 2006b). 

Applying this highly conservative, theoretical maximum exposure estimate, the U.S. EPA 

determines how much of the established Reference Dose (RfD) would be utilized by all 

currently approved product uses. The chronic RfD (cRfD) is an estimate of the amount 

of daily pesticide exposure to the human population that can occur over a lifetime with a 

reasonable certainty of no harm to human health42 . 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 Chronic Population Adjusted Dose (cPAD, the cRfD with any 

FQPA uncertainty factors applied) from aggregated exposures and from the exposure 

assessment to determine if these exposures exceed the U.S. EPA level of concern (i.e., 

100 percent of the cPAD). 

If the aggregate risk assessment shows that utilization of the cPAD 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 the general and regulated public, the U.S. EPA may choose to 

publish the risk assessment and proposed regulatory 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/regulating/registration-status.html). The U.S. 

EPA publishes the new tolerances in the Federal Register, along with a final summary of 

the risk assessment and approves pesticide labeling 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 comments received in the transparency policy 

notice. 

 

42RfD 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-tolerant crops, a large margin of safety exists for glyphosate. 

While use of glyphosate has increased in the decade since the introduction 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 dietary exposure associated with the herbicide’s approved uses. 

Prior to the first approval of a glyphosate-tolerant crop (soybean) in 1996, theoretical 

maximum dietary exposure for all registered conventional uses of glyphosate utilized 

approximately 2.9% of the glyphosate RfD for 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 exposure estimates for food and drinking water 

to glyphosate to be below the U.S. EPA level 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-nursing infants (most exposed subpopulation) 

respectively (U.S. EPA, 2006b). The combined short/intermediate-term estimated 

exposure for dietary and non-dietary exposures with all current registered uses of 

glyphosate utilizes only 11% of the glyphosate cPAD for the most sensitive 

subpopulation of non-nursing infants less than one year old (U.S. EPA, 2006b). The 

utilization of the glyphosate cPAD, which is well below 100 percent, has allowed the 

U.S. EPA to continue to make the conclusion of reasonable certainty of no harm to 

human health for each glyphosate use, including new glyphosate-tolerant crop uses. 

These figures are supported by the data provided in the tables below. Table K-1 

summarizes the established food and feed tolerances supporting the use of glyphosate in 

the conventional crops of alfalfa, cotton, sugar beet and soybean prior to the first 

glyphosate-tolerant crop in 1996. A summary of the regulatory 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. Table K-3 summarizes the 

most recent chronic and short/intermediate-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% 

Roundup of RfD 

Ready sugar 

beet

2008 Establish new tolerances 

for sugar beet. 

• roots at 10 ppm 

• tops at 10 ppm 

• pulp (dried) at 25 ppm 
Monsanto Company 11-CA-233U 478 of 500 

Table K-2. Summary of EPA Approvals for 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 maize 
Monsanto Company 11-CA-233U 479 of 500 

Table K-3. Aggregate Exposure Assessment for Glyphosate 

Population 

Subgroup 

Acute 

Aggregate2

RfD 

(mg/kg/day) 2

Chronic 

Aggregate1,2 

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/health/revisedRAmethods.pdf, December 2009), and thus do 

not include occupational exposure. As such, the chronic aggregate exposure estimated in this fashion 

was the same as chronic dietary exposure because chronic non-dietary exposure was not expected 

based upon the registered non-crop uses of glyphosate. 2

U.S.EPA (2006b). 

3Calculated 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 glyphosate on human health that the U.S. EPA 

considers in its human health analysis is applicator and bystander exposure resulting from 

increased glyphosate use. Based on the toxicity 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 endpoints have been identified for glyphosate 

(U.S. EPA, 2006a; 2006b). 

Additional evidence to support the U.S. EPA conclusion can be found in the Farm Family 

Exposure Study (Acquavella et al., 2004), a biomonitoring study of pesticide applicators 

conducted by independent investigators. This biomonitoring study determined that the 

highest estimated systemic dose of glyphosate for applicators as the result of routine 

labeled applications of registered glyphosate-based agricultural herbicides to crops, 

including glyphosate-tolerant crops, was approximately 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 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 agricultural herbicides to crops. Considering 

the similarity of the use pattern and application rates of the glyphosate products in this 

study compared to those registered for use on glyphosate-tolerant crops, bystander 

exposure attributed to the use of glyphosate on glyphosate-tolerant crops is expected to 

be negligible. 

K.3. Potential Impact of Glyphosate on the Environment 

Potential environmental effects are carefully considered 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 application rates and/or timing) and before re-registering an existing 

pesticide, the U.S. EPA must consider the potential 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 predict hazards to terrestrial animals (birds, 

nontarget insects, and mammals), aquatic animals (freshwater fish and invertebrates, 

estuarine and marine organisms) and nontarget 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 end of this evaluation, the EPA 

concluded that all registered uses of glyphosate were eligible for re-registration, including 

terrestrial (i.e., land-based) applications up to 6 lbs glyphosate acid equivalents (a.e.) per 

acre on crops, and 8 lbs glyphosate a.e per acre for certain limited uses. 

Since the re-registration evaluation in 1993, the U.S. EPA has reviewed and approved a 

significant number of new glyphosate uses: on conventional crops such as legume 

vegetables and sunflower/safflower seed, glyphosate-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 any incremental environmental exposure to 

glyphosate caused by that new use, did not pose an unreasonable risk to the environment, 

and approved pesticide labeling for the new use. 

The studies and data collected by Monsanto, both for the initial U.S. EPA registration and 

re-registration of glyphosate, as well as data developed by independent academics, 

present a well-established safety profile for glyphosate. The following sections provide 

greater detail regarding some of the key findings from these studies. 

K.3.1. Persistence of Glyphosate in the Soil 

Persistence of agricultural chemicals in the soil is widely regarded as an undesirable 

environmental characteristic. Glyphosate has been shown to degrade over time from 

most agricultural ecosystems across a wide range of soil and climatic conditions, with a 

median soil half-life (the time it takes for half of the glyphosate to dissipate in the soil) of 

13.9 days (U.S. EPA, 1993a). The potential for glyphosate to accumulate in soil 

following repeated applications has been studied both in the laboratory and the field. 

A laboratory study was conducted on two soil samples, with each sample receiving up to 

three sequential applications of 5 pounds glyphosate 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 immediately after application, 

regardless of whether it was the first, second or third application. 

Glyphosate degradation in the soil following multiple glyphosate applications was also 

shown under field conditions. Soil was collected from pesticide efficacy and tolerance 

trials in orchards and vineyards that received repeated applications of glyphosate over a 

one- to six-year period, at cumulative rates of 6 to 120 pounds glyphosate a.e. per acre. 

These soil samples did not show any accumulation of glyphosate residues, even at the 

exaggerated rate of three sequential applications of eight pounds glyphosate 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 glyphosate application. 

Similar to some of the other glyphosate tolerant 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 could 
Monsanto Company 11-CA-233U 482 of 500 

be followed by one to two post-emergence in-crop 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 through first flower) cannot exceed 5.3 pounds 

glyphosate a.e. per acre. Thus, the maximum labeled rates and 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 applied according to label directions. 

K.3.2. Persistence of Surfactant in the Soil 

Herbicide products approved for application to emerged weeds normally are applied with 

surfactants. Glyphosate products are formulated with surfactants 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 degradation of POEA was investigated in three types of soil (silt 

loam, silty clay loam and sandy loam), microbial degradation was determined to be the 

primary degradation route, with minimal degradation occurring under sterile conditions. 

Approximately 25-30% of applied 14C-POEA was mineralized to 14CO2 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. Because limited data are available for POEA 

dissipation, a conservative estimate of half-life values for POEA in soil would be 7-14 

days (Giesy et al., 2000). Glyphosate and the POEA surfactant 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 increase in residual soil concentrations (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 from acute and chronic 

tests on fish and other aquatic organisms, the EPA has determined that “the potential for 

environmental effects of glyphosate in surface 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-registration evaluation under FIFRA, the U.S. EPA conducted an 

ecological assessment for glyphosate. This assessment compared the results from 

toxicity tests using glyphosate, conducted with various plant and animal species to a 

conservative estimate of the concentration of glyphosate to which an organism might be 

exposed in the environment. This estimate, called the Estimated Exposure Concentration 

(EEC), is a point estimate for exposure that does not take into account normal 

environmental dilution or dissipation, or the frequency of exposure to the pesticide by 

wildlife. In the Re-registration Eligibility Decision (RED) for glyphosate (U.S. EPA, 

1993a), the exposure estimates were determined 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 available data (U.S. EPA, 1993a). 

Glyphosate is classified as practically non-toxic to honey bees (which are used to assess 

effects on nontarget insects in general) and as practically non-toxic to slightly toxic to 

birds, freshwater fish, marine and estuarine species, aquatic invertebrates and mammals 

(U.S. EPA, 1993a). 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 

substances; therefore, it is not expected to accumulate in fish or other animal tissues. 

The glyphosate end-use products used in agriculture contain a surfactant to facilitate the 

uptake of glyphosate into the plant (Ashton and Crafts, 1981). Depending on the 

surfactant used, the toxicity of the end-use product may range from practically nontoxic 

to moderately toxic to fish and aquatic invertebrates (U.S. EPA, 1993a). For this reason, 

the 1993 Glyphosate RED stated that some formulated 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 applications 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 herbicide with post-emergence activity on essentially all 

annual and perennial plants. As such, exposure to glyphosate could put aquatic and 

terrestrial nontarget plants as well as threatened or endangered plants at risk (U.S. EPA, 

1993a). Nontarget plants may potentially be at risk from applications of glyphosate as a 

result of spray drift. As discussed earlier, glyphosate binds tightly to agricultural soils 

 

43Although the RED refers to the maximum application rate 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’s 
Monsanto Company 11-CA-233U 484 of 500 

and has low potential to move offsite dissolved in water. Moreover, glyphosate is not 

taken up from agricultural soils by plants. Therefore, risks to nontarget plants are only 

attributed to the spray drift of the pesticide. Pesticide labels include specific risk 

management measures to manage spray drift, including mandatory requirements for 

aerial applications. 

During the re-registration process in 1993, additional 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 understand the potential risk to nontarget and 

threatened and endangered plants from the use of glyphosate herbicides in agriculture. 

Using the methodology described in a recent EPA effects determination for glyphosate 

(U.S. EPA, 2008a), 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 lbs a.e. glyphosate 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). Nonlisted plants, therefore, are unlikely to be 

significantly impacted by glyphosate applications to MON 88302. The potential impact 

to threatened and endangered plant species has been further considered in an analysis 

conducted by Monsanto (Mortensen 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 process, 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 (“listed” species) or destroy or adversely modify 

their critical habitat. 

The U.S. EPA's Endangered Species Protection Program (ESPP) which helps promote the 

recovery of listed species is designed to determine whether pesticide use in a certain 

geographic area may affect any listed species. 

When registering a pesticide or reassessing the 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, invertebrates, mammals and plants are routinely 

assessed and used in 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 and regulatory processes ensure that 

protections are in place for all populations of nontarget species. Because endangered 
Monsanto 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 Assessment Process (U.S. 

EPA, 2004d) to determine whether individuals of a listed species have the potential to be 

harmed by a pesticide, 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 species, after conducting a thorough ecological 

risk assessment, results in an "effects determination." 

An evaluation of the effects of glyphosate use on all types of threatened and endangered 

species was conducted by Monsanto. This evaluation generally followed the procedures 

described in the Overview of the Ecological 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 the 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 acute studies of a few days duration, the 

concentration calculated to result in 50% mortality (LC50) or 50% designated effect 

(EC50) on the test species was utilized in the RQ calculation. For chronic studies, 

representing a significant portion 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 conducted by Monsanto if these endpoints were lower. 

Studies from the literature were considered when the study design was appropriate for the 

assessment being made and where sufficient information regarding glyphosate or 

formulation test concentrations was available. 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. EPA, 2004d), which calculates high-end 

estimates of surface water concentrations of pesticides in a generic farm pond, was 

utilized. When formulation toxicity was considered, default drift values and the U.S. 

EPA standard pond44 were utilized for estimation of aquatic exposure. For terrestrial 

 

44A 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 

No 
Monsanto Company 11-CA-233U 487 of 500 

animals, the T-REX model (U.S. EPA, 2008c) was utilized to calculate estimated dietary 

exposure and risk. For terrestrial 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 considered to contribute to exposure, since 

glyphosate binds very tightly to agricultural soils and does not have herbicidal properties 

when bound to soil (U.S. EPA, 2006c). 

Risk quotients calculated using toxicity endpoints 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, further refinement of the estimated exposure 

concentration or of toxicity endpoints may subsequently 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 risk45 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 applications are made using 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 emergence or prior to harvest. 

The same evaluation determined that other taxa (including birds, mammals, insects, fish, 

amphibians, aquatic invertebrates and non-vascular aquatic plants) would 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 considered to be at risk of direct effects. 

Based on Monsanto’s determination that threatened and endangered plant species 

potentially could be affected by certain uses 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 relative to areas of crop production has been 

undertaken. The first crop to be assessed was 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 least one of these six crops are grown, 

which comprise 96% of the 3141 counties and equivalent areas46 in the 50 states 

of the U.S. Species were reviewed for applicable exclusions at the county-level, 

which indicated, for some species, that glyphosate use in these crops posed no 

risk of adverse effects to these species. 

 Next, for threatened or endangered plant species where effects could not be 

excluded at the county level, the possible exposure to glyphosate was assessed at 

the sub-county level (Phase 2) in the same counties considered in Phase 1. This 

analysis used information available at the sub-county level for threatened and 

endangered plant species locations and for land use. Land uses considered in this 

analysis are those classified as Pasture/Hay and Cultivated Crops.47

 Finally, in sub-county areas where, under certain application conditions, the 

potential for threatened and endangered plant 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 areas have been proposed. These measures 

include: 1) limiting ground application rates 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 and the application area. Proposed buffer 

distances are based on application rate, droplet size and wind direction. 

This analysis was initially completed for the U.S. counties in which alfalfa, canola, 

maize, cotton, soybeans and sugar beets were grown based on the 2002 Ag Census 

(USDA-NASS, 2002) and listed species information available through early 2008. 

Of the 3028 U.S. counties where alfalfa, canola, maize, cotton, soybeans and sugar beets 

were produced, 11% of counties (334 counties) required the definition of potential areas 

for use limitations. In the other 2694 counties, either there were no threatened or 

endangered plant species present, or the species present were either excluded from 

concern (based on habitat or proximity information), had existing protections, or were not 

in proximity to potential areas of production of the six 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 that are not within the boundaries of a county. 47Land use was based on the National Land Cover Database (2001) for the continental U.S. and on the NOAA 

(National Oceanic and Atmospheric Administration) Coastal Services Center land cover data for Hawaii.
Monsanto Company 11-CA-233U 489 of 500 

species. Thus, the identification of potential use limitation areas also applies to other 

crops in those counties. 

K.3.6. Potential Effects on Endangered Animal Species Identified by the U.S. EPA 

or in Litigation 

As previously discussed, no indirect effects on threatened or endangered animal species 

are predicted, since no significant direct effects due to pesticide drift onto nonendangered plant species are predicted. In the Glyphosate RED (U.S. EPA, 1993a), the 

U.S. EPA suggested that glyphosate may have effects 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 predicted 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 likely 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 glyphosate on the California Red-legged Frog in 

response to a consent agreement reached in a lawsuit filed by the Center for Biological 

Diversity48 . 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 that there would be no direct effect to the aquatic phase of 

the California red-legged frog (CRLF) from application of glyphosate or any glyphosate 

formulation or salts49 . The U.S. EPA also concluded that 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 potential risk of direct effects 

to the aquatic or terrestrial-phase of the CRLF. 

With respect to indirect effects on prey, the U.S. EPA concluded that no effect on the 

following prey items: algae, aquatic invertebrates, 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 invertebrate 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 aquatic-phase amphibians are not 

exceeded for either glyphosate, its salts or its formulations”. 50Ibid., Page 14, Table 1.3. 

51Ibid., Page 15, Table 1.4. 

52Ibid., Page 156, ibid. For terrestrial invertebrate prey items that were not listed (i.e. not threatened or endangered), no 

effects were predicted at any rate up to 7.95 lb a.e. per acre for large invertebrates, 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 invertebrates in areas adjacent to a MON 88302 

field, would not be at risk from spray drift exposure 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. Similarly, no effects on terrestrial plant habitat 

are identified for ground applications at 1.6 lb a.e. per acre. 54 

Based on the CRLF effects determination conducted 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 effect of glyphosate on salmon in eleven areas 

in California and Southern Oregon55 in response to the consent agreement reached in 

another lawsuit56. The conclusion of the U.S. EPA’s risk assessment was that for all uses 

of glyphosate with application rates of 5 lb active ingredient (a.i.) per acre or below, the 

Agency determined that glyphosate will have no effect on the subject 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. per acre) or below, so no risk to salmon is anticipated from these 

uses. 

K.3.7. Other Potential Environmental Impacts Associated with Glyphosate Use in 

Glyphosate-tolerant Crops 

As discussed more fully below, the potential impacts to soil attributable to the change in 

production (cultivation) practices associated with the deregulation of glyphosate-tolerant 

crops have been assessed. The adoption of glyphosate-tolerant crops and the ability to 

use glyphosate-based agricultural herbicides is not expected to significantly change 

agricultural practices, except to enable the adoption of no-till seeding practices. 

No-Till Practices: No-till production is the practice of establishing an agricultural seed 

bed and controlling weeds without mechanically tilling the soil. Instead, the only tillage 

of the soil is done at the time of planting, with the crop being seeded directly into the 

previous year’s crop residue. Among other environmental 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 

 

53Ibid., Page 12. The potential for chronic 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 considered 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 application rate of 1.6 lb a.e.per acre) 54Ibid., 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 are well below the LOC of 1. Since the 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 based 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 photosynthesis and altering oxygen levels, which 

cause a reduction of food sources for some aquatic organisms. Sediment can also cover 

spawning beds and impact fish populations. Phosphorus (a major component of 

fertilizer) bound to soil particles 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 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 researchers have reviewed numerous laboratory 

and field studies, investigating the effects of glyphosate on soil bacteria and fungi (Felsot, 

2000; Giesy et al., 2000). Although some laboratory tests have shown effects on 

nitrogen-fixing bacteria (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 concentrations 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 et al., 2001; Estok et 

al., 1989; Wan et al., 1998; Wardle and Parkinson, 1990b). 

In studying microorganisms from soil in pine plantations, (Busse et al., 2001) noted: “Our 

findings suggest that artificial media assays are of limited relevance in predicting 

glyphosate toxicity to soil organisms and that field rate applications of glyphosate should 

have little or no effect on soil microbial communities in ponderosa pine plantations.” 

Long-term studies following repeated applications of Roundup agricultural herbicides in 

the field at labeled use rates for multiple applications 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 Haney 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 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; 1990b). 

Moreover, the history of safe use and yield 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 microbes 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 tightly bind, to several di- and trivalent metal 

cations such as Fe3+, Cu2+, Mn2+, Al3+ and Ca2+ 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 sufficient amounts in the tank mix spray solution 

(Bernards et al., 2005). In the spray solution, there is a simple interaction between 
Monsanto Company 11-CA-233U 492 of 500 

glyphosate and metal cations, which reduces the herbicidal activity of glyphosate. 

However, in the soil environment, the interactions between metals and chelators are much 

more complex (Parker et al., 2005). Glyphosate can interact with metals that are present 

on the surface of soil particles, as well as with dissolved 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 significant 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; Ridley et al., 2002). 

Transport through the Soil – Surfactant: Available data also suggest that the POEA 

surfactant used in Roundup agricultural herbicides binds strongly to soil (estimated soil 

organic carbon-water partition coefficient (Koc) values range from 2500 to 960057) and 

undergoes microbial degradation with an estimated 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 Koc 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 compounds to leach from soil into groundwater 

based on their half-life and Koc (Gustafson, 1989). Using an estimated half-life of 14 

days and a Koc of 2500 as conservative estimates of the rate of degradation and binding to 

soil, the GUS index for the POEA surfactant 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 sterile 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. Glyphosate 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, aspirated 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 0.2 
Monsanto 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 5.0 
Monsanto 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 20 
Monsanto Company 11-CA-233U 496 of 500 

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