Monsanto Company 10-SY-210U 1 of 721 

### Petition for the Determination of Nonregul ated Status for Dicamba-Tolerant Soybean

MON 87708

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

make a determination that the article s hould not be regulated under 7 CFR Part 340

July 6, 2010

(Revised on October 10, 2011, March 1, 2012 and March 26, 2012)

OECD Unique Identifier: MON-877Ø8-9

Monsanto Petition Number: 10-SY-210U

Submitted by:

Rhonda M. Mannion, B.S. Monsanto Company 800 North Lindbergh Blvd. St. Louis, MO 63167 Phone: (314) 694-8785

Fax: (314) 694-3080

E-mail: rhonda.m.mannion@monsanto.com

Prepared by: L.A. Burzio, Ph.D., B.A. Comstock, B.S., A. Deffenbaugh, B.S., K.S. Giddings, Ph.D., R.L. Girault M.S., G.G. Harrigan, Ph.D., J.L. Honegger, Ph.D., D.L. Kendrick, M.S., R.M. Mannion B.S., M.C. McCann, M.S., B. Morrison, M.S., K.E. Niemeyer B.S., G.J. Rogan, M.S., R.W. Schneider, B.S., J.K. Soteres Ph.D., C. Wang, Ph.D., G. Watson, Ph.D., J. Wright, Ph.D.



### Contributors and/or Pr incipal Investigators: C. Arnevik, M.S., J. Berry, M.S., M. Bleeke, Ph.D., C.R. Brown, M.S., K. Carr, M.S., D. Carson, Ph.D.,
C.M. Dalton, B.A., A.J. Evans, M.P.H., R.A. H eeren, B.S., S. Hill, M.S., K.M. Huizinga, Ph.D., J. Kronenberg, Ph.D., T.C. Laufer, M.S., K.D. Lawry, M.S., V. Leopold, M.S., F. Lloyd, M.S., S.L. Phillips, M.S., J.F. Rice, Ph.D., Z. Song, M.S., R.S. Thoma, M.S., Q. Tian, Ph.D. M. Miller, Ph.D., M.
Starke, Ph.D., J. Whitsel, M.S.Monsanto Company 10-SY-210U 2 of 721 

### 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 § 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 th e 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 upo n 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 in formation (including website posting) without Monsanto's prior no tice and consent.

©2012 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 Mo nsanto 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 subm itting 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.Monsanto Company 10-SY-210U 4 of 721 

### EXECUTIVE SUMMARY

The Animal and Plant Health Inspection Serv ice (APHIS) of the United States (U.S.) Department of Agriculture (US DA) has responsibility, under the Pl ant 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 pe st risk and should no longer be regulated. If APHIS determines that the regulated article does not present a pl ant pest risk, the petition is granted, thereby allowing unrestricted intr oduction of the article.

Monsanto Company is submitting this request to APHIS for a determination of nonregulated status in whole for the new biotechnology-de rived soybean product, MON 87708, any progeny

derived from crosses between MON 87708 and c onventional soybean, and any progeny derived

from crosses of MON 87708 with other biotech nology-derived soybean that has been granted nonregulated status under 7 CFR Part 340.

Product Description

Monsanto Company has developed biotechnology- derived soybean MON 87708 that is tolerant to dicamba (3,6-dichloro-2-methoxybenzoic acid ) herbicide. MON 87708 offers growers an expanded use of dicamba in soybean production from the current preplant and preharvest labeled

uses. The tolerance of MON 87708 to dicamba facilitates a wider window of application in soybean, allowing preemergence application up to the day of crop emergence and in-crop postemergence applications through the early re productive (R1/R2) growth stage. Dicamba provides effective control of ove r 95 annual and biennial weed species, and suppression of over 100 perennial broadleaf and woody plant species. Dicamba is efficacious on broadleaf weeds

that are hard-to-control with glyphosate, such as common la mbsquarters, hemp sesbania, morning glory species, nightshade, Pennsylvania smartweed, prickly sida, velvetleaf, waterhemp and wild buckwheat. Hard-to-control weeds genera lly require a higher rate and/or application at a smaller growth stage in order to consistently achieve commercially acceptable control. Refer to the Roundup WeatherMax labe l (U.S. EPA Reg. No. 524-537) for a listing of these weeds.

Additionally, dicamba provides e ffective control of herbicide-resistant broadleaf weeds, including glyphosate-resistant weeds such as ma restail, common ragweed, giant ragweed, palmer pigweed, and waterhemp. Herbicide resistant weed s are those listed on the International Survey

of Resistant Weeds website (www.weedscience.org).

MON 87708 will be combined with MON 89788 (Roundup Ready 2 Yield ® 

### soybean) utilizing traditional breeding techniques. Dicamba is an effective broadleaf herbicide and the potential use of dicamba and glyphosate herbicides at the same time in mixtures for weed control will provide growers greater applicatio n flexibility prior to planting as well as in-crop for greater consistency of control in both conventional and c onservation tillage situations. Use of dicamba, in addition to glyphosate and the other herbicide options currently labeled for use on soybean, provides more options to implement diversified weed management programs to control a broad

® Roundup Ready 2 Yield and Roundup Ready are regi stered trademarks of Monsanto Technology LLC.Monsanto Company 10-SY-210U 5 of 721 

### spectrum of grass and broadleaf weed species. Successful adoption of the dicamba tolerance trait, into the Roundup Ready® soybean system, w ill provide: 1) growers with an opportunity for an efficient, effective weed management syst em; 2) an option to delay or prevent further resistance to glyphosate and other critically important soybean herbicides, in particular, herbicides in the ALS and PPO class of chemistry; 3) excellent crop safet y, and 4) continue to provide soybean growers with e ffective weed control systems n ecessary for production yields to meet the growing needs of the food, feed, and i ndustrial markets. The combination of dicamba and glyphosate tolerance in soybean s will also provide the basis for delaying or preventing the

evolution of further weed resist ance to glyphosate, dicamba, and herbicides in ge neral, because of the ability to use these two modes of action in mixtures and sequences.

MON 87708 contains a gene from Stenotrophomonas maltophilia that expresses a monooxygenase enzyme that rapidly demethylates dicam ba rendering it inactive, thereby conferring tolerance to dicamba. The demethylation of dicamba produces 3,6-dichlorosalicylic acid (DCSA), a known soybean, soil, and livestock metabol ite whose safety has be en evaluated by the Environmental Protection Agency (U.S. EPA). DC SA, in addition to dicamba, is included in the current 10 ppm pesticide residue to lerance for soybean seed that supports the ex isting uses of dicamba on commercial soybean (40 CFR § 180.227). Even with the expanded use of dicamba on MON 87708, compared to commercial soybean uses, the rapid metabolism of dicamba results in residues in dicamba-treated MON 87708 seed, including the DCSA metabolite, that are well below the established 10 ppm tolerance, and th erefore no modification to the existing soybean

seed tolerance is needed. Cons equently, only approval for the expanded use pattern of dicamba on MON 87708 has been requested of EPA.

Data and Information Presented Confirm the Lack of Plant Pest Potential of MON 87708 Compared to Conventional Soybean

The data and information presented in this petition demonstrate MON 87708 is agronomically, phenotypically, and compositionally comparable to conventional soybean with the exception of its tolerance to dicamba. Moreover, the data presented demonstrate MON 87708 is unlikely to pose an increased plant pest risk, including weediness or adverse environmental impact, compared to conventional soybean. The food, feed, and environmenta l safety of MON 87708 was confirmed based on multiple, well-established lines of evidence:

1. Soybean is a familiar crop that does not possess any of the attributes commonly associated with weeds and has a history of safe consumption.

2. A detailed molecular characterization of the inserted DNA demonstrated a single, intact copy of the T-DNA insert in a single locus within the soybean genome.

3. Data confirmed that the dicamba m ono-oxygenase (DMO) in MON 87708 (MON 87708 DMO) is unlikely to be a t oxin or allergen based on extensive information collected.

4. A compositional assessment of seed a nd forage confirmed that MON 87708 is compositionally equivalent to conventional soybean.Monsanto Company 10-SY-210U 6 of 721 

### 5. An extensive evaluation on phenotypic and ag ronomic characteristic s and environmental

interactions of MON 87708 dem onstrated no increas ed plant pest potential compared to conventional soybean.

6. An assessment of potential impact on non-ta rget organisms (NTOs) and endangered species indicated that, under normal agricu ltural conditions, MON 87708 is unlikely to have adverse effects on these organism s compared to conventional soybean.

7. Evaluation of MON 87708 usi ng current cultivation and management practices for soybean concluded that deregulation of MON 87708 will not significantly impact soybean agronomic practices or land use, with the exception of the expanded window of dicamba application.

Soybean is a Familiar Crop Lacking Weedy Characteristics

There is a longstanding history of safe use and consumption of conventional soybean and processed products. Soybean is grown as a commerc ial crop in over 35 countries. Domestication occurred as early as 1000 B.C. and is now the mo st widely grown oilseed crop in the world, with approximately 211 million metric tons of harvested seed produced in 2008, which represented

56% of world oilseed seed production that year.

The commercial soybean species in the U.S. ( Glycine max L. Merr.) does not exhibit weedy characteristics, does not invade es tablished ecosystems, and does not outcross to weedy relatives. Soybean is not listed as a weed in major weed references, nor is it present on the lists of noxious weed species distributed by the federal governme nt (7 CFR Part 360). During 2004 to 2008, U.S. growers planted between 64.7 and 75.7 million acres of soybean. Soybean does not possess any of the attributes commonly associated with weeds, such as long persistence of the seed in the soil, ability to disperse, invade, or become a dominant species in ne w or diverse landscapes, or the ability to compete well with nati ve vegetation. However, due to a pronounced lack of dormancy it is known that soybean seed can germinate qui ckly under adequate temperature and moisture conditions, and can potentially grow as a volunteer plant. However, a volunteer soybean plant likely would be killed by frost during the autumn or winter of the year it germinated. Furthermore, if a volunteer plant were to survive, it would not compete well with the succeeding crop, and would be controlled readily via mechanical or other chemical means. Twenty commonly used agricultural herbicides , representing eight modes-of-action ( i.e. , ALS-inhibitor, chloroacetamide, EPSPS, PPO inhibitor, PSI di sruption, PSII inhibitor, synthetic auxin, and tubulin inhibitor classes) were tested as potenti al substrates for MON 87708 DMO. None of the

herbicides tested were found to effect the tolerance of M ON 87708 at commercial application rates, therefore, herbicides effective for control of volunteer soybean can s till be used to control MON 87708 volunteers. Finally, since wild populations of Glycine species are not known to exist in the U.S., there is no potential for M ON 87708 to outcross to wild or weedy relatives.

Conventional Soybean A3525 is an Appropriate Comparator to MON 87708

Soybean variety A3525 is the near isogenic line to MON 87708 and was used as the conventional soybean comparator to support the safety assessment of MON 87708. MON 87708 and the near isogenic conventional soybean co ntrol A3525 have similar genetic backgroundsMonsanto Company 10-SY-210U 7 of 721 

### with the exception of the dmo expression cassette, thus, the effect of the dmo expression cassette and the expressed MON 87708 DMO could be as sessed in an unbiased manner.

Molecular Characterization V erifies the Integrity and Stab ility of the Inserted DNA in

MON 87708

MON 87708 was developed through Agrobacterium -mediated transformation of conventional soybean A3525 meristem tissue with the 2T-DNA plasmid vector PV-GMHT4355. PV-GMHT4355 contains two separate T-DNAs th at are each delineated by Left and Right Border sequences. The first T-DNA, designated as T-DNA I, contains the dmo expression cassette regulated by the peanut chlorotic streak virus ( PC1SV ) promoter and the pea E9 3 ′ nontranslated region. The second T-DNA, designated as T-DNA II, contains the cp4 epsps expression cassette under the regulati on of the figwort mosaic virus ( FMV ) promoter and the pea

E9 3 ′ non-translated region. Duri ng transformation, both T-DNAs were inserted into the soybean genome, where T-DNA II, containing the cp4 epsps expression cassette, functioned as a marker gene for the selection of transforme d plantlets. Subsequently, conventional selfpollination breeding methods and segregation we re used to isolate a plant containing the dmo expression cassette but not containing the cp4 epsps expression cassette, resulting in the production of marker-free, dicam ba-tolerant soybean MON 87708.

Molecular characterization by Southern blot an alyses determined that MON 87708 contains one copy of the T-DNA I at a single in tegration locus and all expression elements are present. These

data also demonstrated that MON 87708 does not contain detectable backbone sequences from the plasmid vector or T-DNA II sequences. Th e complete DNA sequence of the insert and adjacent genomic DNA sequence in MON 87708 confirmed the integrity of the inserted dmo expression cassette within the insert ed sequences and identified the 5 ′ and 3 ′ insert-to-genomic DNA junctions. Furthermore, Southern blot anal ysis demonstrated that the insert in MON 87708 has been maintained through at least five ge nerations of breeding, thereby confirming the stability of the insert over multiple generations.

Data Confirm MON 87708 DMO Safety

MON 87708 contains a dmo expression cassette that results in two forms of the DMO protein; referred to as DMO and DMO+27 (S ection V.A.). The active form of these proteins, necessary to confer dicamba tolerance, is a trimer co mprised of three DMO monomers. In MON 87708, the trimer can be comprised of DMO, DMO+27, or a combination of bot h. Therefore, this document will refer to both forms of the protei n and all forms of the trimer as MON 87708

DMO.

A multistep approach was used to char acterize MON 87708 DMO. This detailed

characterization and assessment confirmed that MON 87708 DMO is safe for human and animal consumption. The assessment involved: 1) characterization of the physicochemical and functional properties of MON 87708 DMO; 2) quantification of MON 87708 DMO levels in plant tissues; 3) comparison of the amino acid sequence of MON 87708 DMO to known allergens, gliadins, glutenins, toxins, and othe r biologically active pr oteins known to have adverse effects on mammals; 4) evaluation of the digestibility of MON 87708 DMO in simulated gastric and intestinal fluids; 5) endogenous and exogenous substr ate specificity of DMO; 6)Monsanto Company 10-SY-210U 8 of 721 

### documentation of the history of safe consumpti on of mono-oxygenases (the class of enzymes to which MON 87708 DMO belongs); and 7) investig ation of the potential mammalian toxicity through an oral gavage assay.

DMO was found to be specific to dicamba when tested using structurally similar endogenous substrates and exogenous herbicid e substrates representing a wi de range of modes-of-action. MON 87708 DMO has no relevant amino acid seque nce similarities with known allergens, gliadins, glutenins, toxins, and other biologically active proteins that may have adverse effects on mammals. MON 87708 DMO was rapidly degraded in simulated gastric and intestinal fluids and a high dose of this protein in a mouse acute or al toxicity evaluation demonstrated that it is not acutely toxic, and does not cause any adve rse effect. The safety assessment supports the conclusion that exposure to MON 87708 DMO poses no meaningful risk to the environment, or

human and animal health.

MON 87708 is Compositionally Equivalent to Conventional Soybean

Detailed compositional analyses in accordance with OECD guideline s were conducted to assess whether levels of key nutrients and anti-nutrients in MON 87708 were comparable to levels present in the aforementioned near isogenic c onventional soybean cont rol A3525 and several commercial reference soybean varieties. Seed a nd forage were harvested from five individual sites in which MON 87708 (both treated with dicam ba herbicide at the V2-V3 growth stage and not treated with dicamba herbicide), the conv entional control, and a range of commercial reference varieties were grown c oncurrently in the same field tr ial. The commercial reference varieties used to establish a range of natural vari ability for the key nutrients and anti-nutrients in commercial soybean varieties have a history of safe consumption. Nutr ients assessed in this analysis included proximates (as h, carbohydrates by calculation, moistu re, protein, and fat), fiber, amino acids (18 components), fatty acids (FA, C8-C22), and vitamin E ( α -tochopherol) in seed, and proximates (ash, carbohydrates by calculation, moisture, protein, and fat) and fiber in forage.

The anti-nutrients assessed in seed included ra ffinose, stachyose, lecti n, phytic acid, trypsin inhibitors, and isoflavones (daid zein, genistein, and glycitein).

The combined-site analysis was conducted to determine statistica lly significant differences (5% level of significance) between MON 87708 and th e near isogenic conventional control A3525.

The results from the combined-site data were revi ewed using considerations relevant to food and feed safety and nutritional quality. These consider ations included assessment s of: 1) the relative magnitudes of the difference in the mean values of nutrient and anti- nutrient components of MON 87708 and the conventional control, 2) whether the MON 87708 component mean value was within the range of natural variability of that component as repr esented by the 99% tolerance

interval of the commercial refe rence varieties grown concurrently in the same field trial, 3) analyses of the reproducibility of the sta tistically significant combined-site component differences at individual sites, and 4) assessing the differences within the context of natural variability of commercial soybean composition published in the scientific literature and in the International Life Sciences Institute (ILSI) Crop Composition Database.

Assessment of the analytical results confirmed th at the differences observed in the combined-site analysis were not meaningful to food and f eed safety or the nutritional quality of MON 87708 soybean. In addition, the levels of assesse d components in MON 87708 were compositionallyMonsanto Company 10-SY-210U 9 of 721 

### equivalent to the conventional control and with in the range of variability of the commercial reference varieties that were grown conc urrently in the same field trial.

MON 87708 Does Not Change Soybean Plant Pest Potential or Environmental Interactions

Assessing the plant pest potential of a biot echnology-derived crop includes the concept of familiarity that the USDA recognizes as an impo rtant consideration. Familiarity is based upon the fact that the new biotechnology-derived plant is developed from a conventional plant variety whose biological properties and plant pest potential are well know n. Familiarity considers the biology of the plant, the introduced trait, the re ceiving environment, and the interactions among these factors that provides a basis for comp arative risk assessment between a biotechnologyderived plant and the conventional control. Fo llowing this concept, the phenotypic, agronomic,

and environmental interacti on assessment of MON 87708 included the near isogenic conventional soybean control A3525 and the commer cial reference varieties. Characteristics assessed included: seed dormancy and ge rmination, pollen morphology, and symbiont

interactions conducted in the laboratory and greenhouse; and plant phenotypic and agronomic

evaluations and environmental interaction observa tions conducted in the field. The commercial soybean reference varieties grown concurrently were used to establish a range of natural variability for each assessed characteristic in soybean. The phenotypic, agronomic, and

environmental interaction assessment demonstr ated that MON 87708 is equivalent to the conventional control. Thus, MON 87708 is unlik ely to have a changed plant pest potential compared to conventional soybean.

Seed dormancy and germination characteriza tion demonstrated that MON 87708 seed had germination characteristics similar to seed of the conventional control. In particular, the lack of hard seed, a well-accepted characteristic of w eediness affecting seed germination, supports a

conclusion of no increased weediness of M ON 87708 when compared to the conventional control. For pollen characteristics and symbi ont interactions, there were no statistically significant differences (5% level of signi ficance) observed between MON 87708 and the conventional control for any of the parameters measured, including pollen viability and diameter, nodule number and dry weight, shoot total nitrogen, and shoot and root dry weight. Collectively, these results support the conclusion that MON 87708 is not likely to exhib it increased plant pest potential compared to conventional soybean.

The field evaluation of phenotypic, agronomic, and environmental in teraction characteristics of MON 87708 also support the conclu sion that MON 87708 is not likely to have an increased plant pest potential compared to conventional soyb ean. The evaluations were conducted at 18 replicated field sites across North American soybean production regions. These assessments included plant growth and development charact eristics, as well as observations for plant responses to abiotic stressors and plant-diseas e and plant-arthropod interactions. The observed phenotypic characteristics were similar between MON 87708 and the conventional control.

In a combined-site analysis, data show no sta tistically significant differences (5% level of significance) between MON 87708 and the conventi onal control for early stand count, seedling vigor, days to 50% flowering, lodging, pod shatteri ng, final stand count, seed moisture, seed test weight, or yield. Two statisti cally significant differences we re detected between MON 87708Monsanto Company 10-SY-210U 10 of 721 

### and the conventional control for plant height and 100 seed weight. MON 87708 was slightly

taller and had a lower 10 0 seed weight than the conventional control. Ho wever, both differences were small in magnitude. Additionally, MON 87708 and the conventional control were within the same range of plant growth stages for 131 out of the 132 growth stage observations among the sites. Except for the differences in plant height, 100 seed weight, a nd a single growth stage

observation at one site , all values for MON 87708 fell within the range of the commercial reference varieties grown concurrently. None of these differences were considered biologically meaningful in terms of increased plant pest potential of MON 87708 compared to conventional soybean.

In an individual-site assessment of abiotic stre ss response and diseas e damage, no differences

were observed between MON 87708 and the conventional control for 193 out of 194 comparisons for the assessed abio tic stressors or for any of th e 215 comparisons for the assessed diseases among all observations at the 18 sites. One difference was observed between

MON 87708 and the conventional control for wind damage during a single observation at one site. The damage rating for MON 87708 (slight damage) was outside the range of the commercial reference varieties (no damage); how ever, the difference wa s not observed during any of the other 29 wind damage observations among the sites. Thus, the slight difference in wind damage rating was not indicative of a c onsistent plant response associated with

MON 87708 and is not considered biologically meaningful in te rms of increased plant pest potential or an altered environmental imp act from MON 87708 compared to conventional soybean.

In an assessment of arthropod-re lated damage, no statistically significant differences (5% level of significance) were detected between MON 87 708 and the conventional control for 89 out of 95 comparisons for the assessed arthropods. Lack of variability in the data precluded statistical comparisons between MON 87708 and the conventi onal control for 121 additional comparisons; however, the means for MON 87708 and the conventi onal control were the same value for these comparisons, indicating no biological differences. For each of th e six statistically significant

differences between MON 87708 and the conventional control, the severity of arthropod-related damage to MON 87708 was within or slightly outside the range of th e commercial reference

varieties. The differences between MON 87708 and the conventional c ontrol were small in magnitude and were not consistent across observ ations or sites. Thus, the differences in arthropod-related damage are not indicative of a consistent pl ant response associated with MON 87708 and are not considered biologically m eaningful in terms of increased plant pest potential or an altered environmental imp act from MON 87708 compared to conventional soybean.

In an assessment of pest and beneficial ar thropod abundance, no statistically significant differences (5% level of significance) were de tected between MON 87708 and the conventional control for 142 out of 151 compar isons (including 74 arthropod pe st and 77 beneficial arthropod comparisons) among the multiple collections conducte d during the season at four sites. For the nine detected differences in arthropod abundance, seven were arthropod pests (green cloverworm, Japanese beetles, and sti nk bugs) and two were benefici al arthropods (spiders and Nabis spp). The differences detected in pest and benefici al arthropod abundance were small in magnitude

and were not consistent with other collection time s at the individual sites or across the sites. Consequently, it is concluded that the differences in pest and beneficial arthropod abundance areMonsanto Company 10-SY-210U 11 of 721 

### not indicative of a cons istent plant response a ssociated with MON 87708 and are not biologically meaningful in terms of increased plant pest po tential or an altered environmental impact from MON 87708 compared to conventional soybean.

Field evaluations of phenotypic, agronomic, and environmental interaction characteristics of

MON 87708 treated with dicamba herbicide were also conducted. Data were collected from field trials conducted at eight sites within the U.S. soybean producing regions. These assessments included plant growth and developmen t characteristics, as well as observations for plant responses to abiotic stressors, plant-di sease and plant-arthropod interactions. The phenotypic, agronomic, and enviro nmental interaction assessment demonstrated that treated MON 87708 is equivalent to the conventional cont rol. Thus, MON 87708 is unlikely to have an altered plant pest potential comp ared to conventional soybean.

The observed phenotypic characteristics were si milar between the dicamba-treated MON 87708

and the conventional control. In a combined-s ite assessment, no statistically significant

differences were detected be tween treated MON 87708 and the c onventional control for early stand count, seedling vigor, days to 50% flower ing, plant height, lodging, pod shattering, final stand count, seed moisture, or yield. One st atistically significant di fference was detected between treated MON 87708 and the control, for 100 seed weight. The difference in 100 seed weight was relatively small in magnitude and the mean 100 seed weight of treated MON 87708 was slightly below the reference range. It is unlikely that this small difference in 100 seed weight would contribute to increased weed pot ential of MON 87708 when treated with dicamba compared to conventional soybean. Addition ally, treated MON 87708 and the control were within the same range of plant growth stages for all growth stage observations among the sites.

None of these differences were considered biol ogically meaningful in terms of increased plant pest potential of treated MON 87708 compared to conventional soybean.

In an assessment of plant response to abiotic stressors and disease damage, no differences were

observed between treated MON 87708 and the c onventional control for 181 of 182 comparisons among all observations at the eight sites. One difference was observed between treated

MON 87708 and the control for white mold duri ng a single observation (s light vs. none). The damage rating for treated MON 87708 was outsid e of the reference range (no damage was observed in the references). This difference was not observed in any of the other two white mold

evaluations across the sites and is not considered biologically meaningful in terms of increased plant pest potential or an altered environmen tal impact from treated MON 87708 compared to conventional soybean.

In an assessment of arthropod-re lated damage, there were no statistically significant differences

detected between treated MON 87708 and the cont rol for 56 out of 59 comparisons. Lack of variability in the data precl uded statistical co mparisons between treated MON 87708 and the conventional control for 34 additional comparisons. The mean damage ratings for bean leaf beetle and grasshopper damage was outside the reference range however the response was not

consistent across observations or sites. Thus , the results are not c onsidered biologically meaningful in terms of adve rse environmental impacts of tr eated MON 87708 compared to the conventional soybean.Monsanto Company 10-SY-210U 12 of 721 

### In summary, the phenotypic, agronomic, and envir onmental interaction data were collected to provide a detailed characteriza tion of MON 87708 and to assess whether the introduction of the

dicamba tolerance trait in MON 87708 and the associ ated application of di camba herbicide alters the plant pest potential compared to conventi onal soybean. The analysis considered the comparisons of MON 87708 to the conventional control, the reproducibility, magnitude, and direction of detected differences (trends), an d comparison to the range of the commercial reference varieties. Results from the phenotyp ic, agronomic, and environmental interactions

assessment indicated that MON 87708 does not possess weedy characteristics, increased susceptibility or tolerance to specific ab iotic stress, diseases, or arth ropods, or char acteristics that would confer a plant pest risk or a significant environmental im pact compared to conventional soybean.

MON 87708 Will Not Adversely Affect NTOs or Threatened and Endangered Species

Evaluation of the impacts of a biotechnology-de rived crop on Non-Target Organisms (NTOs) and threatened and endangered species is a compone nt of the plant pest risk assessment. Since MON 87708 does not possess pesticid al activity, all organisms that interact with MON 87708 are

considered to be NTOs. The environmental asse ssment demonstrated that the presence of the

dicamba tolerance trait in MON 87708 and the asso ciated application of dicamba did not alter plant-arthropod interactions, incl uding beneficial arthropods, or alter disease susceptibility compared to the conventional control.

The biochemical information and experimental data for evaluation of MON 87708 included molecular characterization, MON 87708 DMO safety assessments, the history of environmental exposure to mono-oxygenases (the class of enzymes to which MON 87708 DMO belongs),

information from the environmental interactio n assessment, demonstration of compositional equivalence to conventional soybean, and demonstration of agronomic and phenotypic equivalence to conventional soybean. Taken toge ther, these data support the conclusion that

MON 87708 has no reasonable mechanism for harm to NTOs, or to pose an additional risk to threatened and endangered species compared to the cultivation of conventional soybean.

The potential for outcrossing and gene introgr ession from MON 87708 to sexually-compatible species in the U.S. is unl ikely since no known wild Glycine species related to cultivated soybean are known to be present in North America. Furthermore, should cross-pollination occur, MON 87708 and its progeny are not expected to exhibit a significant environmental impact because, as described above, evaluations have sh own that the presence of the dicamba tolerance trait is not likely to enhance weediness or plant-pest potential. Therefore, the environmental consequence of pollen transf er from MON 87708 to other Glycine species is considered negligible.

Deregulation of MON 87708 Will Not Significan tly Impact Soybean Agronomic Practices

or Land Use

Soybean fields are typically hi ghly managed agricultu ral areas that are dedicated to crop production for many years. Cultivation of MO N 87708 would not be expe cted to differ from typical soybean cultivation, w ith the sole exception of an expanded window of dicamba applications due to the pres ence of the dicamba toleranc e trait in MON 87708. MON 87708Monsanto Company 10-SY-210U 13 of 721 

### likely would be used in common rotations on land currently used for agricultural purposes. As demonstrated, MON 87708 is similar to conven tional soybean in its agronomic, phenotypic, ecological, and compositional characteristics and has comparable levels of resistance to insects and diseases as compared to commercial soybe an. Therefore, the in troduction of MON 87708 into the Roundup Ready soybean syst em is not expected to have a significant impact on current cultivation and management practices for so ybean. The adoption of MON 87708 into the Roundup Ready soybean system will provide grower s with another herbicide mode-of-action and the means to control broadleaf weeds, including hard-to-control and herbic ide-resistant broadleaf weeds, and will help preserve conservation t illage practices by providing growers with an additional weed management tool. Based on thes e considerations, there is no apparent potential for significant impacts on agronomic practices or land use, with the exception of the expanded application window of dicamba.

Conclusion

Based on the data and information presented in this petition, it is concluded that MON 87708 is not likely to be a plant pest. Therefore, M onsanto Company requests a determination from APHIS that MON 87708 and any progeny deri ved from crosses between MON 87708 and conventional soybean or previously deregulat ed biotechnology-derive d soybean, be granted nonregulated status under 7 CFR Part 340.Monsanto Company 10-SY-210U 14 of 721 

### TABLE OF CONTENTS

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

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

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

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

LIST OF TABLES .............................................................................................................19

LIST OF FIGURES ...........................................................................................................26

ABBREVIATION AND DEFINITIONS ..........................................................................28

I. RATIONALE FOR THE DE VELOPMENT OF MON 87708 .....................................32

I.A. Basis for the Request for a Determ ination of Nonregulated Status under 7 CFR § 340.6 ...............................................................................................................32

I.B. Rationale for the Development of Dicamba-Tolerant Soybean

MON 87708 ...................................................................................................................32

I.C. Submissions to Other Regulatory Agencies ..........................................................34

I.C.1. Submission to FDA .....................................................................................34

I.C.2. Submission to EPA .....................................................................................35

I.C.3. Submissions to Fore ign Government Agencies ..........................................35

II. THE BIOLOGY OF SOYBEAN .................................................................................36

II.A. Soybean as a Crop ................................................................................................36

II.B. Characteristics of the Recipient Plant ..................................................................36

II.C. Soybean as a Test System in Product Safety Assessment ...................................37

III. DESCRIPTION OF THE GENETIC MODIFICATION ...........................................38

III.A. The Plasmid Vector PV-GMHT4355 .................................................................38

III.B. Description of the Transformation System .........................................................43

III.C. The dmo Coding Sequence and MON 87708 DMO (T-DNA I) ........................45

III.D. The cp4 epsps Coding Sequence and the CP4 EPSPS Protein (T-DNA II) .................................................................................................................... 45

III.E. Regulatory Sequences .........................................................................................45

III.F. T-DNA Borders ...................................................................................................46

III.G. Genetic Elements Outside of the T-DNA Borders .............................................46

IV. CHARACTERIZATION OF TH E GENETIC MODIFICATION .............................47

IV.A. Insert and Copy Number of T-DNA I in MON 87708 .......................................52

IV.A.1. Probe 8 .....................................................................................................53

IV.A.2. Probe 9 .....................................................................................................53Monsanto Company 10-SY-210U 15 of 721 

### IV.A.3. Probe 10 ...................................................................................................54

IV.B. Southern Blot Analysis to De termine the Presence or Absence of T-DNA II Sequences in MON 87708 ............................................................................58

IV.B.1. Probe 4 .....................................................................................................58

IV.B.2. Probe 5 .....................................................................................................58

IV.B.3. Probe 6 .....................................................................................................59

IV.C. Southern Blot Analysis to De termine the Presence or Absence of PV-GMHT4355 Backbone Sequences in MON 87708 ................................................63

IV.C.1. Plasmid Vector Backbone Probes 1, 2, 3, and 7 ......................................63

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

MON 87708 ...................................................................................................................65

IV.E. PCR and DNA Sequence Analyses to Examine the MON 87708

Insertion Site ................................................................................................................ ..65

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

Generations of MON 87708 ..........................................................................................65

IV.F.1. Probe 9 ......................................................................................................66

IV.G. Inheritance of the Genetic Insert in MON 87708 ...............................................69

IV.H. Genetic Modification Ch aracterization Conclusion ...........................................73

V. CHARACTERIZATION AND SAFETY ASSESSMENT OF THE

MON 87708 DMO .............................................................................................................74

V.A. Function of DMO and MON 87708 DMO ..........................................................74

V.A.1. Formation of MON 87708 DMO ..............................................................75

V.A.2. Specificity of MON 87708 DMO .............................................................75

V.B. Characterization of MON 87708 DMO ...............................................................77

V.C. Expression Levels of MON 87708 DMO ............................................................78

V.D. Assessment of Potentia l Allergenicity of MON 87708 DMO .............................81

V.E. Safety Assessment Summary of MON 87708 DMO ...........................................81

V.F. MON 87708 DMO Characteriza tion and Safety Conclusion ...............................84

VI. COMPOSITIONAL A SSESSMENT OF MON 87708 ..............................................86

VI.A. Compositional Equivalence of MON 87708 Seed and Forage to Conventional Soybean ...................................................................................................86

VI.A.1. Composition of Soybean Seed and Forage (Treated) ..............................88

VI.A.2. Composition of Soybean Seed and Forage (Untreated)..........................93

VI.B. Compositional Assessment Conclusion ...........................................................144

VII. PHENOTYPIC, AGRONOM IC, AND ENVIRONMENTAL

INTERACTIONS ASSESSMENT ..................................................................................146Monsanto Company 10-SY-210U 16 of 721 

### VII.A. Characteristics Measured for Assessment ......................................................146

VII.B. Interpretation of Phenotypic and Environmental Interaction Data .................150

VII.B.1. Interpretation of Detected Differences Criteria ....................................150

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

Environmental Interaction Characteristics of MON 87708 ........................................153

VII.C.1. Seed Dormancy and Germination Characteristics ................................153

VII.C.2. Phenotypic, Agronomic, and Environmental Interaction

Characteristics Evaluated under Field Conditions ...............................................156

VII.C.2.1. Phenotypic, Agronomic, and Environmental Interaction

Characteristics for Untreated MON 87708 Evaluated under 2008 Field

Conditions ............................................................................................................156

VII.C.2.2. Phenotypic, Agronomic, and Environmental Interaction

Characteristics for Dicamba-Tr eated Evaluated under 2009 Field

Conditions ............................................................................................................157

VII.C.2.3. Field Phenotypic and Agronomic Characteristics .............................158

VII.C.2.4. Environmental In teraction Characteristics ........................................162

VII.C.3. Pollen Characteristics ...........................................................................166

VII.C.4. Symbiont Interactions ...........................................................................167

VII.D. Phenotypic, Agronomic, and Environmental Interactions Assessment

Conclusion ...................................................................................................................1 69

VIII. U.S. AGRONOMIC PRACTICES ........................................................................170

VIII.A. Introduction ...................................................................................................170

VIII.B. Overview of U.S. Soybean Production ..........................................................171

VIII.B.1. Soybean Production .............................................................................171

VIII.B.2. Soybean Seed Production ....................................................................176

VIII.C. Production Management Considerations .......................................................179

VIII.C.1. Pre-Season ...........................................................................................179

VIII.C.2. Planting and Early Season ...................................................................180

VIII.C.3. Mid to Late Season ..............................................................................182

VIII.C.4. Harvest Season ....................................................................................183

VIII.D. Management of Insects ..................................................................................183

VIII.E. Management of Diseases and Other Pests .....................................................184

VIII.F. Weed Management .........................................................................................186

VIII.F.1. Methods of Weed Control in Soybean .................................................189

VIII.G. Dicamba Herbicide Use in the U.S. ...............................................................197

VIII.G.1. Dicamba Application Timing for Labeled Crops ................................200Monsanto Company 10-SY-210U 17 of 721 

### VIII.G.2. Distribution of Dicamba Use in the U.S. ............................................202

VIII.G.3. Potential Imp acts to Adjacent Crops ...................................................207

VIII.H. Dicamba-Tolerant Soybean MON 87708 ......................................................208

VIII.H.1. Potential Impact of Dicamba Application Timing to

MON 87708 .........................................................................................................217

VIII.H.2. Impact on Dicamba Use in U.S. Soybean Production Following Integration of MON 87708 into th e Roundup Ready Soybean System ...............221

VIII.H.3. Conclusions on Dicamba-Tolerant Soybean MON 87708 ..................224

VIII.I. Crop Rotation Practices in Soybean ................................................................224

VIII.J. Soybean Volunteer Management ....................................................................238

VIII.K. Weed Resistance to Dicamba Herbicide........................................................241

VIII.L. Stewardship of MON 87708 ..........................................................................241

VIII.M. Impact of the Introduction of MON 87708 on Agricultural Practices ..........243

IX. ENVIRONMENTAL CONSEQUENCES ...............................................................245

IX.A. Introduction ......................................................................................................245

IX.B. Plant Pest Assessment of the MON 87708 Insert and Expressed Protein ........246

IX.B.1. Characteristics of the Geneti c Insert and the Expressed Protein ...........246

IX.B.2. Compositional Characteristics ...............................................................247

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

Characteristics ......................................................................................................248

IX.C. Weediness Potential of MON 87708 ................................................................253

IX.D. Potential for Pollen Mediated Gene Flow ........................................................254

IX.D.1. Hybridization with Cultivated Soybean .................................................255

IX.D.2. Hybridization with Wild Annual Species within Subgenus Soja ..........255

IX.D.3. Hybridization with th e Wild Perennial Species of Glycine Subgenus ..............................................................................................................256

IX.D.4. Transfer of Genetic Informa tion to Species with Which Soybean Cannot Interbreed (Horizontal Gene Flow) .........................................................256

IX.E. Potential Impact on Soybean Agronomic Practices ..........................................257

IX.F. Summary of Plant Pest Assessments ................................................................257

X. ADVERSE CONSEQUENC ES OF INTRODUCTION ...........................................260

REFERENCES ................................................................................................................261

APPENDICES .................................................................................................................282

Appendix A: USDA Notifications ..................................................................................283

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

MON 87708 ..................................................................................................................... 287Monsanto Company 10-SY-210U 18 of 721 

### Appendix C: Materials, Methods, and Results for the Characterization of

MON 87708 DMO and Substrat e Specificity of DMO ...................................................294

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

MON 87708 DMO ...........................................................................................................323

Appendix E: Materials, Methods, and I ndividual-Site Results for Compositional Analysis of MON 87708 Soybean Seed and Forage .......................................................326

Appendix F: Materials, Methods, and I ndividual Site Results for the Seed Dormancy and Germination Assessment of MON 87708 ...............................................437

Appendix G: Materials, Methods, an d Individual-Site Results from the Phenotypic, Agronomic, and Environm ental Interaction Assessment of

MON 87708 under Field Conditions ...............................................................................443

Appendix H: Materials and Methods for Pollen Morphology and Viability

Assessment .................................................................................................................... ...486

Appendix I: Materials and Met hods for Symbiont Assessment .....................................489

Appendix J: Petitioner’ s Environmental Report .............................................................492

Appendix K: Herbicide Resistance .................................................................................588

Appendix L: Comparative Analysis of Dicamba and Alternative Soybean

Herbicides .................................................................................................................... ....606

Appendix M: Potential Impact of Dicamba on Human Health and the

Environment ................................................................................................................... ..676

Appendix N: Overview of Evaluation of Potential Exposure and Biological Effects on Endangered Species for Dicamba Use in MON 87708 ..................................703Monsanto Company 10-SY-210U 19 of 721 

### LIST OF TABLES

Table III-1. Summary of Genetic Elements in the Plasmid Vector

PV-GMHT4355 .................................................................................................................40

Table IV-1. Summary Chart of the Expected DNA Segments Based on Hybridizing Probes and Restriction En zymes Used in MON 87708 Analysis ..................50

Table IV-2. Summary of Gene tic Elements in MON 87708 ............................................51

Table IV-3. Segregation of the dmo Expression Cassette During the Development

of MON 87708 .................................................................................................................. .72

Table V-1. Herbicides Applied to MON 87708 and Conventional Control .....................77

Table V-2. Tissues Collected a nd Analyzed for MON 87708 DMO ...............................79

Table V-3. Summary of th e Levels of MON 87708 DMO in Leaf, Root, Forage,

and Seed from MON 87708 Grown in 2008 U.S. Field Trials ..........................................80

Table VI-1. Summary of Differences ( α =0.05) for the Comparison of Soybean Component Levels for MON 87708 (Tre ated) vs. Conventional Control .........................98

Table VI-2. Statistical Summary of Co mbined-Site Soybean Seed Nutrients for MON 87708 (Treated) vs. Conventional Control ............................................................112

Table VI-3. Statistical Summary of Co mbined-Site Soybean Seed Anti-Nutrients for MON 87708 (Treated) vs. Conventional Control ......................................................118

Table VI-4. Statistical Summary of Comb ined-Site Soybean Forage Nutrients for MON 87708 (Treated) vs. Conventional Control ............................................................120

Table VI-5. Summary of Differences ( α =0.05) for the Comparison of Soybean C omponent Levels for MON 87708 (Untr eated) vs. Conventional Control ......................122

Table VI-6. Statistical Summary of CombinedSite Soybean Seed Nutrients for MON 87708 (Untreated) vs. Conventional

Control ....................................................................................................................... ......132

Table VI-7. Statisti cal Summary of Combined-Site Soybean Seed AntiNutrients for MON 87708 (Untreated) vs. Conventional Control ...................................138

Table VI-8. Statistical Summary of CombinedSite Soybean Forage Nutrients for M ON 87708 (Untreated) Conventional Control ......140

Table VI-9. Literature and ILSI Data base Ranges for Components in Soybean Seed and Forage ............................................................................................................... 142

Table VII-1. Phenotypic, Agronomi c, and Environmen tal Interaction

Characteristics Evaluated in U.S. Fiel d Trials, Laboratory, or Greenhouse Tests ..........148

Table VII-2. Combined-Site Comparison of MON 87708 to Conventional Control for Seed Dormancy and Germination Characteristics ........................................155

Table VII-3. 2008 Field Phenotypic Evaluation Site s for Untreated MON 87708 ........157Monsanto Company 10-SY-210U 20 of 721 

### Table VII-4. 2009 Field Phenotypic Evaluation Site s for Dicamba Treated

MON 87708 ..................................................................................................................... 158

Table VII-5. Combined-Site Comparison of Untreated MON 87708 to Conventional Control during 2008 for Phe notypic and Agronomic Characteristics .......159

Table VII-6. Combined-Site Compar ison of Dicamba Treated MON 87708 to Conventional Control during 2009 for Phe notypic and Agronomic Characteristics .......161

Table VII-7. Pollen Characteristics of MON 87708 Compared to Conventional

Control ....................................................................................................................... ......167

Table VII-8. Symbiont Interaction Assessment of MON 87708 and Conventional

Control ....................................................................................................................... ......169

Table VIII-1. Soybean Production in the U.S., 1999 – 2008 1 

### ........................................173

Table VIII-2. U.S. Soybean Pr oduction by Region and State in 2008 1 

### ..........................174

Table VIII-3. U.S. Soybean Pr oduction Costs and Returns in 2008 1 

### .............................175

Table VIII-4. Common Weeds in Soyb ean Production: Midwest Region ....................188

Table VIII-5. Common Weeds in Soybean Production: Southeast Region ...................189

Table VIII-6. Common Weeds in Soybean Production: Eastern Coastal Region .........189

Table VIII-7. Herbicide Use in Soyb ean in the U.S. from 1995 through 2001 1 

### .............192

Table VIII-8. Agricultural Chemical App lications Registered for Soybean Use in AR, IA, IL, IN, KS, KY, LA, MI, MN, MS, MO, NE, NC, ND , OH, SD, TN, VA,

and WI in 2006 1 

### ...............................................................................................................193

Table VIII-9. Crop Tolerance and Common Grass Weed Responses to Herbicides Applied in Soybean Production .......................................................................................195

Table VIII-10. Common Br oadleaf Weed Responses to Herbicides Applied in Soybean Production .........................................................................................................196

Table VIII-11. Dicamba Use in All Labeled Crops from 1990 to 2008 1 

### ........................198

Table VIII-12. Dicamba-Treated Acres and Amounts Applied to Labeled Crops

and Uses in 2008 1 

### .............................................................................................................199

Table VIII-13. Dicamba A pplications – Average Number and Rates to Labeled Crops 1



### .............................................................................................................................. .199

Table VIII-14. Dicamba-Treated Acres (000) by Application Timing and Crop in

2008 1



### .............................................................................................................................. ..201

Table VIII-15. Dicamba-Treated Ac res by State and Labeled Crop in 2008 1 

### ................203

Table VIII-17. Common Br oadleaf Weed Responses to Preplant Burndown

Herbicides .................................................................................................................... ....213

Table VIII-18. Common Br oadleaf Weed Responses to Dicamba Compared to Labeled Postemergence Herbicides in Soybean Production ............................................214

Table VIII-19. Summary of Comparative Analysis of Dicamba and Alternative

Herbicides .................................................................................................................... ....215Monsanto Company 10-SY-210U 21 of 721 

### Table VIII-20. Dates and Days to Reach Various Growth Stages in Corn and Soybean in Central Illinois ...............................................................................................218

Table VIII-21. Dates and Days to Reach Various Growth Stages in Corn and Soybean in Western Tennessee ........................................................................................220

Table VIII-22. Average Daily Temperat ures in Major Soybean Producing States 1 

### .......220

Table VIII-23. Average Relative Hu midity (1971-2000) in Major Soybean Producing States 1 

### .............................................................................................................221

Table VIII-24. Rotational Practices in the U.S. Following Soybean Production ...........227

Table VIII-25. Rotational Practices Following Soybean Production in the Midwest Region ...............................................................................................................2 28

Table VIII-26. Rotational Practices Following Soybean Production in the Southeast Region .............................................................................................................2 33

Table VIII-27. Rotational Practices Fo llowing Soybean Production in the East Coastal Region ................................................................................................................ .236

Table VIII-28. Ratings for Postemerge nce Control of Volunteer Soybean in Labeled Rotational Crops 1 

### ...............................................................................................240

Table IX-1. Summary of Published Lite rature on Soybean Cross Pollination ...............259

Table A-1. USDA Notifications Approve d for MON 87708 and Status of Trials

Conducted under These Notifications and Permits ..........................................................284

Table B-1. Hybridization Conditions of Utilized Probes ................................................289

Table C-1. Molecular We ight and Purity of th e MON 87708-Produced DMO

Proteins ...................................................................................................................... ......302

Table C-2. Summary of the Tryptic Masses Identif ied for the MON 87708 DMO Protein Using MALDI-TOF MS ......................................................................................306

Table C-3. Summary of the Tryptic Masses Identified for the MON 87708 DMO+27 Protein Using MALDI-TOF MS .....................................................................307

Table C-4. MON 87708 DMO F unctional Activity Assay .............................................312

Table C-5. Herbicides Tested in Ex ogenous Specificity Herbicide Tolerance

Greenhouse Trials ............................................................................................................3 13

Table C-6. Compounds Used in Specificity In Vitro Experiments .................................314

Table C-7. Herbicide Toleran ce Trials Injury Ratings ...................................................317

Table E-1. Commercial Reference Varieties ..................................................................326

Table E-2. Re-Expression Formulas for Sta tistical Analysis of Composition Data .......332

Table E-3. Component with Observations Below the Assay Limit of Quantitation Not Excluded from Statistical Analysis ...........................................................................332

Table E-4. Statistical Summary of S ite IARL Soybean Seed Nutrients for MON 87708 (Treated) vs. Conventional Control ............................................................334Monsanto Company 10-SY-210U 22 of 721 

### Table E-5. Statistical Summary of Site IARL Soybean Seed Anti-nutrients for MON 87708 (Treated) vs. Conventional Control ............................................................340

Table E-6. Statistical Summary of Site IARL Soybean Forage Nutrients for MON 87708 (Treated) vs. Conventional Control ............................................................342

Table E-7. Statistical Summary of S ite ILCY Soybean Seed Nutrients for MON 87708 (Treated) vs. Conventional Control ............................................................344

Table E-8. Statistical Summary of Site ILCY Soybean Seed Anti-nutrients for MON 87708 (Treated) vs. Conventional Control ............................................................350

Table E-9. Statistical Summary of Site ILCY Soybean Forage Nutrients for MON 87708 (Treated) vs. Conventional Control ............................................................352

Table E-10. Statistical Summary of S ite ILWY Soybean Seed Nutrients for MON 87708 (Treated) vs. Conventional Control ...........................................................354

Table E-11. Statistical Summary of Site ILWY Soybean Seed Anti-nutrients for MON 87708 (Treated) vs. Conventional Control ............................................................360

Table E-12. Statistical Summary of Site ILWY Soybean Forage Nutrients for MON 87708 (Treated) vs. Conventional Control ............................................................362

Table E-13. Statistical Summary of S ite INRC Soybean Seed Nutrients for MON 87708 (Treated) vs. Conventional Control ............................................................364

Table E-14. Statistical Summary of Site INRC Soybean Seed Anti-nutrients for MON 87708 (Treated) vs. Conventional Control ............................................................370

Table E-15. Statistical Summary of S ite INRC Soybean Forage Nutrients for MON 87708 (Treated) vs. Conventional Control ............................................................372

Table E-16. Statistical Summary of S ite PAHM Soybean Seed Nutrients for MON 87708 (Treated) vs. Conventional Control ............................................................374

Table E-17. Statistical Summary of Site PAHM Soybean Seed Anti-nutrients for MON 87708 (Treated) vs. Conventional Control ............................................................380

Table E-18. Statistical Summary of Site PAHM Soybean Forage Nutrients for MON 87708 (Treated) vs. Conventional Control ............................................................382

Table E-19. Statistical Summary of S ite IARL Soybean Seed Nutrients for MON 87708 (Untreated) vs. Conventional Control ........................................................384

Table E-20. Statistical Su mmary of Site IARL Soybean Seed Anti-Nutrients for MON 87708 (Untreated) vs. Conventional Control ........................................................390

Table E-21. Statistical Su mmary of Site IARL Soybean Forage Nutrients for MON 87708 (Untreated) vs. Conventional Control ........................................................392

Table E-22. Statistical Summary of Site ILCY Soybean Seed Nutrients for MON 87708 (Untreated) vs. Conventional Control ........................................................394

Table E-23. Statistical Su mmary of Site ILCY Soybean Seed Anti-Nutrients for MON 87708 (Untreated) vs. Conventional Control ........................................................400

Table E-24. Statistical Su mmary of Site ILCY Soybean Forage Nutrients for MON 87708 (Untreated) vs. Conventional Control ........................................................402Monsanto Company 10-SY-210U 23 of 721 

### Table E-25. Statistical Summary of Site ILWY Soybean Seed Nutrients for MON 87708 (Untreated) vs. Conventional Control ........................................................404

Table E-26. Statistical Su mmary of Site ILWY Soybean Seed Anti-Nutrients for MON 87708 (Untreated) vs. Conventional Control ........................................................410

Table E-27. Statistical Su mmary of Site ILWY Soybean Forage Nutrients for MON 87708 (Untreated) vs. Conventional Control ........................................................412

Table E-28. Statistical Summary of Site INRC Soybean Seed Nutrients for MON 87708 (Untreated) vs. Conventional Control ........................................................414

Table E-29. Statistical Su mmary of Site INRC Soybean Seed Anti-Nutrients for MON 87708 (Untreated) vs. Conventional Control ........................................................420

Table E-30. Statistical Summary of Site INRC Soybean Forage Nutrients for MON 87708 (Untreated) vs. Conventional Control ........................................................422

Table E-31. Statistical Summary of Site PAHM Soybean Seed Nutrients for MON 87708 (Untreated) vs. Conventional Control ........................................................424

Table E-32. Statistical Su mmary of Site PAHM Soybean Seed Anti-Nutrients for MON 87708 (Untreated) vs. Conventional Control ........................................................430

Table E-33. Statistical Su mmary of Site PAHM Soybean Forage Nutrients for MON 87708 (Untreated) vs. Conventional Control ........................................................432

Table F-1. Starting Seed of MON 87708, Conventional Control, and Commercial Reference Varieties Used in the Dormancy and Germination Assessment .....................437

Table F-2. Dormancy and Germina tion Characteristics of MON 87708 and

Conventional Control Seed Produced at each of Three Field Sites .................................440

Table G-1. Starting Seed for the Phe notypic, Agronomic, and Environmental

Interaction Assessment for 2008 Field Trials ..................................................................445

Table G-2. Starting Seed for the Phe notypic, Agronomic, and Environmental

Interaction Assessment for 2009 Field Trials ..................................................................446

Table G-3. Field and Planting In formation for 2008 Field Trials...................................448

Table G-4. Field and Planting In formation for 2009 Field Trials...................................450

Table G-5. 2008 Individual-Site Ph enotypic Comparison of Untreated

MON 87708 to Conventional Control .............................................................................457

Table G-6. 2009 Individual-Site Phenot ypic Comparison of Dicamba Treated

MON 87708 to Conventional Control .............................................................................460

Table G-7. Growth Stage Monitoring of Untreated MON 87708, Conventional Control, and the Commercial Referen ce Varieties from 2008 Field Trials .....................463

Table G-8. Growth Stage Monito ring of Dicamba Treated MON 87708, Conventional Control, and the Commercial Reference Varieties from 2009 Field

Trials ........................................................................................................................ ........467

Table G-9. Abiotic Stress Response Ev aluations of Untr eated MON 87708 and

Conventional Control from 2008 Field Tria ls Using an Observational Severity

Scale ......................................................................................................................... ........469Monsanto Company 10-SY-210U 24 of 721 

### Table G-10. Disease Damage Eval uations of Untreated MON 87708 and

Conventional Control from 2008 Field Tria ls Using an Observational Severity

Scale ......................................................................................................................... ........470

Table G-11. Arthropod–Related Damage Evaluations of Untreated MON 87708

and Conventional Control from 2008 Field Tr ials Using an Observational Severity

Scale ......................................................................................................................... ........471

Table G-12. Abundance of Pest Arthropods in Beat Sheet Samples Collected from Untreated MON 87708, Conventional Control, and the Commercial

Reference Varieties fr om 2008 Field Trials .....................................................................473

Table G-13. Abundance of Beneficial Arthropods in Beat Sheet Samples Collected from Untreated MON 87708, Conventional Control, and the

Commercial Reference Variet ies from 2008 Field Trials ................................................477

Table G-14. Abiotic Stress Response Evaluations of Dicamba-Treated

MON 87708 and Conventional Control fr om 2009 Field Trials Using an Observational Severity Scale ...........................................................................................481

Table G-15. Disease Damage Evaluati ons of Dicamba-Treated MON 87708 and

Conventional Control from 2009 Field Tria ls Using an Observational Severity

Scale ......................................................................................................................... ........482

Table G-16. Arthropod–Related Damage Evaluations of Dicamba-Treated

MON 87708 and Conventional Control fr om 2009 Field Trials Using an Observational Severity Scale ...........................................................................................483

Table I-1. Starting Seed of MON 87708, Conventional Control, and Commercial Reference Varieties Used in the Symbiont Assessment ..................................................489

Table J-1. Summary of Dicamba Uses on Soybean ........................................................493

Table J-2. 2008 Soybean Productivity by Region ..........................................................496

Table J-3. Known Weed Resistance in the Southern U.S. 1 

### ............................................508

Table J-4. Known Weed Resi stance in the Midwest U.S. 1 

### ............................................509

Table J-5. Deregulated or Submitted Bi otechnology-derived Soybean Products ...........514

Table J-6. Organic and Conventi onal Soybean Seed Sources .........................................544

Table K-1. Management Recommendations for Control of Dicamba- and Other Synthetic Auxin-Resistant Weeds....................................................................................596

Table L-1. Dicamba Acid Ac ute Toxicity Study Findings .............................................611

Table L-2. Dicamba Acid Reproductive, Developmental, Mutagenic, and Neurotoxicologic Findings...............................................................................................612

Table L-3. Dicamba Acid Subchron ic, Chronic and Cancer Findings ...........................613

Table L-4. Summary of Toxicological Findings fr om Testing of DCSA .......................615

Table L-5. Dicamba Acid Ecotoxicity Findings on Mammals, Birds and Fish (U.S. EPA. 2005a) ...........................................................................................................61 6Monsanto Company 10-SY-210U 25 of 721 

### Table L-6. Dicamba Acid Ecotoxicity Findings on Aquatic and Terrestrial Invertebrates (U.S. EPA. 2005a) ......................................................................................617

Table L-7. Dicamba Acid Ecotoxicity Resu lts on Aquatic and Terrestrial Plants (U.S. EPA. 2005a) ...........................................................................................................61 7

Table L-8. Dicamba, Diglycolamine ( DGA) Salt Ecotoxicity Findings (U.S. EPA. 2005a) ................................................................................................................... ..618

Table L-9. Environmental Fate and Physical Proper ties of Dicamba Acid ....................619

Table L-10. Ten Most Widely Used Alte rnative Herbicides in U.S. Soybean

Production .................................................................................................................... ....623

Table L-11. Alternative Regist ered Soybean Herbicides ...............................................624

Table L-12. Active Ingredients Contai ned in Alternative Herbicides ............................629

Table L-13. Human Health Risk Parame ters for Alterna tive Herbicides .......................632

Table L-14. Aquatic Toxicity Parameters for Fish and Aquatic Invertebrates for

Alternative Herbicides .....................................................................................................637

Table L-15. Aquatic Toxicity Paramete rs for Aquatic Plants for Alternate Herbicide Active Ingredients ...........................................................................................641

Table L-16. Herbicide Efficacy Compar ison: Herbicide Resistant Weeds and Hard-to-Control Weeds in Soybean .................................................................................646

Table L-17. Summary of Co mparative Analysis of Dicamba and Alternative

Herbicides .................................................................................................................... ....653

Table L-18. Planting Restrictions (months ) for Alternative He rbicide Products ...........659

Table M-1. Summary of Di etary Exposure and Risk for Dicamba: Food and

Water ......................................................................................................................... .......682

Table M-2. Aggregate (Short-term) Exposure Assessment for Dicamba .......................683

Table M-3. Residues of Dicamba, DC SA, 5-Hydroxydicamba and DCGA in Dicamba-Tolerant Soybean Forage, Hay and Seed .........................................................684

Table M-4. Dicamba Concentrations (μ g/L) in Groundwater in Major Soybean Growing States ................................................................................................................ .688

Table M-5. Dicamba Concentrations (μg/ L) in Surface Water in Major Soybean Growing States ................................................................................................................ .690

Table N-1. EPA Levels of Concern fo r Threatened and Endangered Species ...............705

Table N-2. Comparison of Default Ke naga Residues and Dicamba-specific

Residues in Food-Items....................................................................................................707

Table N-3. Risk Quotients for Chronic Exposure a for Mammalian Species Based on Dicamba-Specific Residue Values and DT 50 

### . .............................................................709

Table N-4. Risk Quotients for Acut e Exposure to Birds Using Measured

Dicamba Residue and Residue Decline Values ...............................................................710Monsanto Company 10-SY-210U 26 of 721 

### LIST OF FIGURES

Figure III-1. Circular Map of Plasmi d Vector PV-GMHT4355 Showing Probes 1-

10............................................................................................................................. ...........39

Figure III-2. Schematic of the Development of MON 87708 ...........................................44

Figure III-3. Deduced Amino Acid Sequence of the RbcS Targeting Sequence

and MON 87708 DMO ......................................................................................................45

Figure IV-1. Schematic Representa tion of the Insert and DNA Flanking Sequences in MON 87708 .................................................................................................49

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

T-DNA I in MON 87708: Probe 8 ....................................................................................55

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

T-DNA I in MON 87708: Probe 9 ....................................................................................56

Figure IV-4. Southern Blot Analysis to Determine Insert and Copy Number of

T-DNA I in MON 87708: Probe 10 ..................................................................................57

Figure IV-5. Southern Blot Analysis to Detect the Presence or Absence of T-DNA II Sequences in MON 87708: Probe 4 .................................................................60

Figure IV-6. Southern Blot Analysis to Detect the Presence or Absence of T-DNA II Sequences in MON 87708: Probe 5 .................................................................61

Figure IV-7. Southern Blot Analysis to Detect the Presence or Absence of T-DNA II Sequences in MON 87708: Probe 6 .................................................................62

Figure IV-8. Southern Blot Analysis to Determine the Presence or Absence of PV-GMHT4355 Backbone Sequences in MON 87708: Probes 1, 2, 3, and 7 .................64

Figure IV-9. Breeding History of MON 87708 ................................................................67

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

Generations of MON 87708: Probe 9 ...............................................................................68

Figure IV-11. Breeding Path for Gene rating Segregation Data for MON 87708 .............71

Figure V-1. Three Components of the DMO Oxygenase System ....................................74

Figure V-2. Dicamba and Set of Poten tial Endogenous Substrates Tested in in

vitro Experiments with DMO ............................................................................................76

Figure VII-1. Schematic Diagram of Agronomic and Phenotypic Data Interpretation Methods .....................................................................................................151

Figure VIII-1. Mean Annual Dicamba Use for Agricultural Uses in the U.S. During 1999-2004 1 

### ...........................................................................................................206

Figure VIII-2. Planted Soybean Acreage by County in the U.S. in 2009 1 

### .....................206

Figure B-1. Overlapping PCR Analysis Across the Insert in MON 87708 ....................292

Figure B-2. PCR Amplification of the MON 87708 Insertion Site ................................293Monsanto Company 10-SY-210U 27 of 721 

### Figure C-1. Molecular Weight and Purity Analysis of MON 87708 DMO

Proteins ...................................................................................................................... ......303

Figure C-2. Immunoblot Analysis of MON 87708 DMO Proteins ................................304

Figure C-3. MALDI-TOF MS Coverage Map of the MON 87708 DMO Protein .........308

Figure C-4. MALDI-TOF MS Coverage Map of the MON 87708 DMO+27

Protein ....................................................................................................................... .......308

Figure C-5. N-Terminal Sequen ce of the MON 87708 DMO Protein ...........................310

Figure C-6. N-Terminal Sequence of the MON 87708 DMO+27 Protein .....................310

Figure C-7. Glycosylation Analysis of the MON 87708 DMO Proteins ........................311

Figure C-8. UPLC separation of dicamba (DCB) and DCSA ........................................319

Figure C-9. E. coli -produced DMO Conversion of Dicamba (DCB) to DCSA and 2,4-D to 2,4-Dichlorophenol (2,4-DCP) ..........................................................................320

Figure C-10. E. coli -produced DMO Conversion of Endogenous Substrates ................321

Figure K-1. Weed Resistance to Various Herbicide Families 1 

### .......................................592

Figure L-1. Structure of Dicamba ...................................................................................609

Figure L-2. Structure of DCSA .......................................................................................614

Figure M-1. Ground Water Sampling Site s for Dicamba (Top: Detects only; Bottom: detects and non-detects) .....................................................................................689

Figure M-2. Surface Water Sampling Sites for Dicamba (Top: Detects only; Bottom: detects and non-detects) .....................................................................................691

Figure N-1. Identification of Taxa Ex ceeding Endangered Species Levels of

Concern ....................................................................................................................... .....708

Figure N-2. County-Leve l Analysis Process ..................................................................712

Figure N-3. Counties with Soybean Farms and Adjacent Counties ...............................713

Figure N-4. Counties with Soybean Farm s (or Adjacent Counties) with Listed Species in the Taxa of Interest .........................................................................................715Monsanto Company 10-SY-210U 28 of 721 

### ABBREVIATION AND DEFINITIONS 

~ approximately

aadA Bacterial promoter and coding sequence for an aminoglycosidemodifying enzyme, 3’(9)-O-nucleotidyltransferase from the transposon Tn7 that confers spectinomycin and streptomycin

resistance AAPSE American Association of Pesticide Safety Educators ADF acid detergent fiber ALS acetolactate synthase AOSA Association of Official Seed Analysts aPAD acute population adjusted dose APHIS Animal and Plant Health Inspection Service B. japonicum Bradyrhizobium japonicum BIO Biotechnology Industry Organization bp base pair BRS Biotechnology Regulatory Services

BSA bovine serum albumin bu/A bushels per acre CEQ Council on Environmental Quality CES Cooperative Extension Service CFR Code of Federal Regulations CHT ceramic hydroxyapatite column cp4 epsps codon modified coding sequence of the aroA gene from Agrobacterium
species strain CP4 encoding CP4 EPSPS

CP4 EPSPS 5-Enolpyruvylshikimate-3-phosphate synthase protein cPAD chronic population adjusted dose CTAB Hexadecyltrimethylammonium bromide

CTP2 Sequences encoding the chloroplast transit peptide region of Arabidopsis thaliana EPSPS used to direct proteins into chloroplasts

CV column volume

Da Dalton DAP days after planting dATP Deoxyadenosine triphosphate DCSA 3,6-dichlorosalicylic acid also known as 3,6-dichloro-2-hydroxybenzoic acid DCGA 3,6-dichlorogentisic acid dCTP Deoxycytidine triphosphate DDI daily dietary intake DEEM dietary exposure evaluation model DGA Diglycolamine DHB dihydroxybenzoic acid dicamba 3,6-dichloro-2-methoxybenzoic acid

dmo Coding sequence of the dicamba mono-oxygenase gene from Stenotrophomonas maltophilia encoding DMO DMO full-length dicamba mono-oxygenase protein DMO+27 full-length dicamba mono-oxygenase protein with an additional 24

Note: Standard abbreviations, e.g ., units of measure, are used accor ding to the format described in

‘Instructions to Authors’ in the Journal of Biological Chemistry

.Monsanto Company 10-SY-210U 29 of 721 amino acids from the Rubisco sm all subunit and 3 amino acids from an intervening sequence

DnaK 5 ′ non-translated leader sequence from the Petunia hybrid Hsp70 gene that is involved in regulating gene expression dNTP deoxynucleoside triphosphate

DTT dithiothreitol DWCF dry weight conversion factor dwt dry weight of tissue

E9 3 ′ non-translated region of the pea RbcS2 gene that functions to direct polyadenylation of the mRNA

E. coli Escherichia coli EEC estimated environmental exposure concentration

EFED Environmental Fate and Effects Division ELISA enzyme-linked immunosorbent assay EPA U.S. Environmental Protection Agency

ERS Economic Research Service ETS Excellence Through Stewardship

Exo exonuclease I FA fatty acid FASTA algorithm used to find local high scoring alignments between a pair of protein or nucleotide sequences FDA U.S. Food and Drug Administration FFDCA Federal Food, Dr ug, and Cosmetic Act FIFRA Federal Insecticide, F ungicide, and Rodenticide Act FLt full-length transcript

FMV Promoter for the figwort mosaic virus 35S RNA FQPA Food Quality Protection Act fwt fresh weight of tissue GLP good laboratory practice H.U. hemagglutinating unit HAL health advisory level

HED Health Effects Division HPPD 4-hydroxyphenylpy ruvate dioxygenase HRP horseradish peroxidase

IAA indole acetic acid

ILSI International Life Sciences Institute IPM integrated pest management

kb kilobase

kDa kiloDalton LB loading buffer lb/bu pounds per bushel

LC lethal concentration

LD lethal dose Left Border DNA region from Agrobacterium tumefaciens containing the left border sequence used for transfer of the T-DNA LOAEL lowest dosing level that produced an observable effect

LOC level of concern

LOD limit of detection

LOEC lowest observed effect concentration LOQ limit of quantitation MALDI-TOF MS matrix-assisted laser desorption/ionization time-of-flight massMonsanto Company 10-SY-210U 30 of 721 spectrometry
MMT million metric tons MOE margin of exposure MON 87708 DMO The active form of DMO, a trimer comprised of three monmers. The DMO trimer can be comprised of DMO, DMO+27 or a combination

of both forms. MSTA Monsanto Technology Stewardship Agreement MW molecular weight MWCO molecular weight cut off N/A not applicable

NADH nicotinamide adenine dinucleotide NASS National Agricultural Statistics Service, a branch of the USDA NAWQA National Water-Quality Assessment NDF neutral detergent fiber NEPA National Environmental Policy Act

NFDM non-fat dried milk

NOAEL no observable adverse effect level

NOEC no observable effect concentration NTOs non-target organisms OECD Organization of Economic Co-Operation and Development OPP Office of Pesticide Programs ORETF Outdoor Residential Exposure Task Force

ori

- pBR322 Origin of replication from pBR322 necessary for maintenance of plasmid in E. coli

ori V Origin of replication from the broad host range plasmid RK2 necessary for maintenance of plasmid in Agrobacterium

OSL over-season leaf PAD population adjusted dose PBS phosphate buffered saline solution PBST phosphate buffered saline solution containing 0.05% (v/v) Tween-20

PC1SV Promoter for the full length transcript of peanut chlorotic streak virus that directs transcription in plant cells PCR polymerase chain reaction

pea Pisum sativum PHED Pesticide Handler Exposure Database RED Reregistration Eligibility Decision

RfD reference dose PMSF phenylmethylsulfonyl fluoride

PPA Plant Protection Act PPO protoporphyrinogen oxidase PRZM/EXAMS Pesticide Root Zone Model/Exposure Analysis Modeling System PVDF polyvinylidene difluoride

RbcS Sequences encoding the transit peptide region of the ribulose-1,5- bisphosphate carboxylase oxygenase gene from Pisum sativum Right Border DNA region from Agrobacterium tumefaciens containing the right border sequence used for transfer of the T-DNA

rop Coding sequence for repressor of primer protein used for maintenance of plasmid copy number in E. coli RQ risk quotient RT room temperature SAP shrimp alkaline phosphateMonsanto Company 10-SY-210U 31 of 721 SCI-GROW Screening Concentration in Ground Water SCN soybean cyst nematode

SD standard deviation SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis

S.E. standard error SGF simulated gastric fluid

SIF simulated intestinal fluid S. maltophilia Stenotrophomonas maltophilia soybean Glycine max (L.) Merr. T-DNA transfer(ed) DNA

TBS tris buffered saline TES threatened and endangered species

TEV 5 ′ non-translated region from the Tobacco Etch RNA virus genome that is involved in regulating gene expression

TFA trifluoroacetic acid TIU trypsin inhibitor units Tm melting temperature

TMRC theoretical maximum resiude concentration TMB 3,3',5,5' tetramethyl-benzidine TRED tolerance reregistration eligibility decision TSSP tissue-specific pool TUG Technology Use Guide

U.S. United States of America USDA U.S. Department of Agriculture v/v volume per volume

VMD volume median diameter w/v weight per volume

WAT weeks after treatment

WPS Worker Protection standard WSSA Weed Science Society of AmericaMonsanto Company 10-SY-210U 32 of 721 

### I. RATIONALE FOR THE DEVELOPMENT OF MON 87708

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

The Animal and Plant Health Inspection Serv ice (APHIS) of the United States (U.S.) Department of Agriculture (USDA) has res ponsibility, under the Plant Protection Act (Title IV Pub. L. 106-224, 114 Stat. 438, 7 U.S.C. § 7701-7772), to prevent the introduction and dissemination of plant pe sts into the U.S. APHIS regulation 7 CFR § 340.6 provides that an applicant may petition APHIS to evaluate submitted data to determine that a particular regulated arti cle does not present a pl ant pest risk and no longer should be regulated. If APHIS dete rmines that the regulated article does not present a plant pest risk, the petition is gr anted, thereby allowing unr estricted introduction of the article. A listing of all regulated MON 87708 fiel d trials conducted under USDA notification can be found in Appendix A.

Monsanto Company is submitting this requ est to APHIS for a determination of nonregulated status for new biotechnology- derived soybean product, MON 87708, any progeny derived from crosses between MON 87708 and conventional soybean, and any progeny derived from crosses of MON 87708 with biotechnology-derived soybean that have previously been granted nonre gulated status under 7 CFR Part 340.

I.B. Rationale for the Development of Dicamba-Tolerant Soybean MON 87708

The Roundup Ready ® 

### soybean system permits over- the-top applicat ion of Roundup ®


### agricultural herbicides containing the active ingredient glyphosate for effective weed

control. The value of th e Roundup Ready soybean system ha s been demonstrated by the significant growth in the number of plante d acres since its intr oduction in 1996. Today more than 90% of all soybean acres grow n in the U.S. are Roundup Ready (USDA-NASS, 2009c). The Roundup Ready soybean system delivers effective broad spectrum weed control, provides flexibility of applicati on timing, has facilitated increased adoption of reduced tillage practices, and has resulted in increased grower income (Carpenter and Gianessi, 2001; Bonny, 2008; Hu rley et al., 2009). A dditionally, the Roundup Ready soybean system provides incremental envi ronmental benefits (Bonny, 2008; Brookes and Barfoot 2009), and glyphosate, as concluded by the U.S. EPA (1993), has a favorable safety profile. Continued use of the Roundup Ready soybean system will maintain effective and familiar weed control management practices that are fully compatible with all current tillage and land mana gement systems including cons ervation tillage practices. Growth of conservation tillage in the U.S. was greatly accelerated with the introduction of glyphosate-tolerant crops in large part because of the broad spectrum postemergence control offered by glyphosat e (Price et al. 2011).

As with all herbicides used in agriculture, there is potential for weeds to develop

resistance to the herbicide over time. 1 

### If unmanaged, herbicide resistance can become a limiting factor in crop production. Glyphosate has had few cases of weed resistance,

1 http://www.weedscience.org/In.aspMonsanto Company 10-SY-210U 33 of 721 

### particularly in relation to othe r herbicides. In the U.S., while there have been thirteen confirmed glyphosate-resistant weeds (Heap, 2011), glyphosate st ill controls more than 160 weed species (Roundup WeatherMax he rbicide label, EPA Reg. No.524-537) and remains an extremely valuable tool for U.S, soybean crop production. Herbicide resistant

weeds are those listed on the Internationa l Survey of Resistant Weeds website ( www.weedscience.org ). Additionally, studies have shown that resistance can be postponed, contained and managed through good management practices. One of the management practices most often recomm ended by University/Cooperative Extension Service and industry is the use of multiple herbicide modes-of-action. Simultaneously using two herbicides with different modes-of -action significantly re duces the probability of weeds developing resistance to either or both herbicides (Powles et al., 1996; Beckie and Reboud, 2009). Other recommended manage ment practices to manage herbicide resistance includes the use of multiple herbicide modes-of-action in sequence, and/or the inclusion of mechanical or cultural practices in addition to the use of an herbicide.

Monsanto Company has developed biotec hnology-derived soybean MON 87708 that is tolerant to dicamba (3,6-di chloro-2-methoxybenzoic acid) he rbicide. Dicamba is a synthetic auxin herbicide that kills plants by mimicking naturally-occurring plant growth hormones called auxins, thereby destroying ti ssue through uncontrolle d cell division and growth (Ahrens, 1994). Dicamba’s mode-of- action is different from glyphosate, and it provides efficacious control of broadleaf weeds and is co mplementary to glyphosate on

hard-to-control weeds such as common lamb squarters, hemp sesbania, morning glory species, nightshade, Pennsylvania smartweed, prickly sida, velvetleaf, waterhemp, and wild buckwheat (Johnson et al., 2010). Addi tionally, dicamba provides effective control of herbicide-resistant broadleaf weeds, in cluding glyphosate-resist ant weeds such as marestail, common ragweed, giant ragweed, palmer pigweed, and waterhemp (Johnson et al., 2010). Hard-to-control weed s generally require a higher ra te and/or application at a smaller growth stage in order to consistently achieve commercially acceptable control. Refer to the Roundup WeatherMax label (U.S . EPA Reg. No. 524-537) for a listing of

these weeds. Herbicide resistant weeds are th ose listed on the International Survey of Resistant Weeds website ( www.weedscience.org ). Since its intr oduction in 1967, only four species with known dicamba-resistant biotypes have been identified in North America (Heap, 2011).

MON 87708 will be combined with MON 89788 (Roundup Ready 2 Yield soybean) utilizing traditional breeding techniques. Dicam ba is an effective broadleaf herbicide and the potential use of dicamba and glyphosate herb icides at the same time in mixtures for weed control will provide growers greater app lication flexibility prior to planting as well as in-crop for greater consistency of contro l in both conventional and conservation tillage situations (Johnson et al., 2010). Use of dicam ba, in addition to glyphosate and the other herbicide options currently labeled for us e on soybean, provides more options to implement diversified weed management progr ams to control a broad spectrum of grass and broadleaf weed species (Johnson et al, 2010). Successful adoption of the dicamba tolerance trait, into the R oundup Ready soybean system, will pr ovide: 1) growers with an opportunity for an efficient, e ffective weed management system ; 2) an effective tool for the management of glyphosate resistant weeds that will help to conserve reduced tillage practices; 3) an option to delay or preven t further resistance to glyphosate and otherMonsanto Company 10-SY-210U 34 of 721 

### critically important soybean herbicides, in particular herbicides in the ALS and PPO class of chemistry; 4) excellent crop safety; a nd 5) soybean growers with effective weed control systems necessary for production yiel ds to meet the growing needs of the food, feed, and industrial markets.

MON 87708 expresses a mono-oxy genase enzyme that rapi dly demethylates dicamba rendering it inactive, thereby c onferring tolerance to dicamba. The demethylation of dicamba produces 3,6-dichlorosalicylic ac id (DCSA), a known soybean, soil, and livestock metabolite whose safety has been evaluated by the Environmental Protection Agency (U.S. EPA). DCSA, in addition to dicamba, is included in the current 10 ppm pesticide residue tolerance for soybean seed th at supports the existing uses of dicamba on conventional soybean (40 CFR § 180.227). Even with the expanded use of dicamba on MON 87708, compared to conventional soybean uses, the rapid metabolism of dicamba

results in residues in dicamba-treated M ON 87708 seed, including the DCSA metabolite, that are well below the established 10 ppm to lerance, and therefore no modification to the existing soybean seed tolerance is needed. Consequently, only a pproval for the expanded use pattern of dicamba on MON 87708 has been requested of EPA. Furthermore, the U.S., Canada and the EU have recently comp leted reviews of dicamba where the safety

of dicamba has been confir med (U.S. EPA, 2009; PMRA, 2008; European Commission, 2008). The proposed use pattern of dicamba in MON 87708 falls within the use pattern criteria (rates and methods) evaluated and approved by EPA in association with existing dicamba agricultural uses.

I.C. Submissions to Other Regulatory Agencies

Under the Coordinated Framework for Regul ation of Biotechnology, the responsibility for regulatory oversight of biotechnology -derived crops th at do not include plant-incorporated protectan ts falls on two federal agen cies: U.S. Food and Drug Administration (FDA) and United States Department of Agriculture (USDA). Deregulation of MON 87708 by USDA constitute s only one component of the overall regulatory oversight and review of this product. As a practical matter, MON 87708

cannot be released and marketed until FDA a nd USDA have completed their reviews and assessments under their respective jurisdicti ons. Additionally, EPA must complete its review and assessments prior to a pproving the use of dicamba on MON 87708.

I.C.1. Submission to FDA

MON 87708 falls within the scope of th e 1992 FDA policy statement concerning regulation of products derive d from new plant varieties, including those developed through biotechnology (U.S. FDA, 1992). In co mpliance with this policy, Monsanto has

initiated a consultation with the FDA (BNF No. 125) on th e food and feed safety and compositional assessment of MON 87708. A safe ty and nutritional assessment summary document for MON 87708 (BNF No. 125) was submitted to F DA on November 9, 2010. FDA completed the consultation pr ocess for MON 87708 on October 11, 2011. 2

2 http://www.accessdata.fda.gov /scripts/fcn/fcnDeta ilNavigation.cfm?rpt=bioListing&id=86Monsanto Company 10-SY-210U 35 of 721 

### I.C.2. Submission to EPA

The EPA has authority over the use of pesticidal substances under the Federal Insecticide, Fungicide and Rodenticide Act (FIFRA), as amended (7 U.S.C. § 136 et seq. ). Monsanto has submitted to the EPA an application to amend Registration Number 524-582 to register a new use pattern for dicamba on MON 87708. The new use pattern facilitates a wider window of application of dicamba on MON 87708, allowing dicamba to be applied preemergence through crop emergence (crackin g) and in-crop postemergence through the early R1/R2 reproductive phase. EPA has review ed the safety of dicamba and DCSA, the primary metabolite in MON 87708, during the reregistration of dicamba in 2006. EPA concluded in the 2006 dicamba Reregistration Eligibility Decision (RED) document that

risks to human health and the environment a ssociated with exposur e to dicamba and its metabolites, including DCSA, were below the Agency’s level of concern for all registered uses of dicamba including conve ntional soybean (U.S. EPA, 2009). Dicamba residues on soybean seed (less than 0.07 ppm average residue and less than 0.5 ppm maximum residue) resulting from its applic ation on MON 87708 at the maximum labeled use rate are well below the established 10 ppm soybean seed pesticide residue tolerance. Therefore, a change to the current soybean s eed tolerance is not needed to support the use

of dicamba on MON 87708. However Monsanto has requested the establishment of new tolerances for soybean forage and hay, which will allow for the feeding of forage and hay

to livestock. No other revisions to dicamba pesticide residue tolerances are needed including animal products such as meat or milk. Furthermore, the use of dicamba on MON 87708 does not present any new environm ental exposure scenarios not previously evaluated and deemed acceptable by EPA. Additional details regarding dicamba and its use on MON 87708 are available in Appendix M.

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

To support commercial introduction of MON 87708 in the U.S., re gulatory submissions

will be made to countries that will eventu ally commercialize or import significant quantities of soybean or its processed fract ions from the U.S. These will include submissions to a number of foreign governme nt regulatory authorities, including: Ministry of Agriculture, People’s Republic of China; Japan’s Minist ry of Agriculture, Forestry, and Fisheries, Minist ry of Environment, and the Mi nistry of Health, Labor, and Welfare; the Canadian Food Inspection Agen cy and Health Canada; the Intersectoral Commission for Biosafety of Genetically Mo dified Organisms, Mexico; the European Food Safety Authority, as well as to regulat ory authorities in other soybean importing countries with functioning re gulatory systems. As appr opriate, notifications of importation will be made to importing countries that do not have a formal approval

process.Monsanto Company 10-SY-210U 36 of 721 

### II. THE BIOLOGY OF SOYBEAN

The Organization for Economic Co-Operatio n and Development (OECD) Consensus Document (OECD, 2000) on the biology of soybean provides key information on:

 a general description of soybean bi ology, including taxonomy and morphology as well as soybean use as a crop plant

 agronomic practices in soybean cultivation

 geographic centers of origin

 reproductive biology

 cultivated soybean as a volunteer weed

 inter-species/genus introgression into relatives and interactions with other organisms, and

 a summary of the ecology of soybean

The taxonomic information for soybean is available in the USDA’s PLANTS Profile (USDA-NRCS, 2010).

To support the evaluation of the plant pe st potential of MON 87708 relative to conventional soybean, additional informati on regarding several aspects of soybean biology can be found elsewhere in this petition. This includes: agronomic practices for soybean in Section VIII; volunteer management of soybean in Section VIII.J; and interspecies/genus introgression pot ential in Section VII.C.3.

II.A. Soybean as a Crop

Soybean is the most widely grown oilseed in the world, with approximately 211 million metric tons of harvested seed produced in 2008. This represents 56% of world oilseed seed production that year. Soybean is grown as a commercial crop in over 35 countries. The major producers are the U.S., Brazil, Ar gentina, China, India, and Paraguay, accounting for approximately 94% of th e global soybean production in 2008. Approximately one-third of the 2008 wo rld soybean production was in the U.S. (Soyatech, 2010). The U.S. was also the la rgest soybean seed exporting country in 2008 (ASA, 2009).

Soybean has a long history of planting and pr oduction in North America. Soybean was originally introduced into North Ameri ca from China in 1765 and has since been reintroduced several times by scientists, seed dealers, merchants, military expeditions, and various individuals (Si ngh and Hymowitz, 1999). Conve ntional plant breeding is based on the interplay and combination of ge nes present in the particular crop genome, and soybean is limited with regard to ge netic diversity (Chung and Singh, 2008).

II.B. Characteristics of the Recipient Plant

The conventional soybean variety A 3525, used as the recipient for the dmo expression cassette insertion that produced MON 87708, wa s developed by Asgrow Seed Company.

A3525 is a mid

− maturity group III soybean variety with very high yield potential.Monsanto Company 10-SY-210U 37 of 721 

### A3525 has superior yields rela tive to varieties of similar maturity and has excellent agronomic characteristics (Steffen, 2004).

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

Soybean variety A3525 is the near isogeni c line to MON 87708 and was used as the conventional soybean comparator (hereafter referred to as the conventiona l control) in the safety assessment of MON 87708. MON 87708 and the conventional control have similar genetic backgrounds with the exception of the dmo expression cassette, thus, the

effect of the dmo expression cassette and th e expressed MON 87708 DMO could

therefore be assessed in an unbiased manner. In addition, commercial soybean varieties that were derived through conventional methods and Roundup Ready soybean varieties (hereafter referred to as commercial reference varieties) were used as reference materials to establish ranges of natural variability or responses represen tative of commercial soybean varieties. The commercial referen ce varieties used at each location were selected based on their availability and agr onomic fit for the respect ive geographic region.Monsanto Company 10-SY-210U 38 of 721 

### III. DESCRIPTION OF TH E GENETIC MODIFICATION

MON 87708 was developed through Agrobacterium tumefaciens -mediated transformation of conventional soybean A3525 meristem tissue utilizing transformation plasmid vector PV-GMHT4355. This section describes the plasmid vector, the donor genes, and the regulatory elements used in the development of MON 87708 and the deduced amino acid sequence of the MON 87708 DMO. In this section, transfer DNA (T-DNA) refers to DNA that is transferred to the plan t during transformation. An expression cassette is comprised of sequen ces to be transcribed and the regulatory elements necessary for the expression of those sequences.

III.A. The Plasmid Vector PV-GMHT4355

PV-GMHT4355 was used for the transformati on of conventional soybean to produce MON 87708 and is shown in Figure III-1; PV -GMHT4355 is approximately 11.4 kb and contains two T-DNAs, each delineated by Left a nd Right Border sequences to facilitate transformation. The first T-DNA, desi gnated as T-DNA I, contains the dmo coding sequence under regulation of the pe anut chlorotic streak virus ( PC1SV ) promoter and the

pea E9 3 ′ non-translated region. The second T- DNA, designated as T-DNA II, contains

the cp4 epsps coding sequence under the regulati on of the figwort mosaic virus ( FMV ) promoter and the pea E9 3 ′ non-translated region. Duri ng transformation, both T-DNAs were inserted into the soybean genome (S ection III.B) where T-DNA II, containing the cp4 epsps expression cassette, functioned as a marker gene for the selection of transformed plantlets. Subsequently, conven tional self-pollinated breeding methods and segregation, along with a combin ation of analytical techniques , were used to isolate those plants that contain the dmo expression cassette (T-DNA I) and did not contain the cp4

epsps expression cassette (T-DNA II).

The backbone region of PV-GMHT4355 that is outside both of the T-DNAs contains two origins of replication for maintenance of the plasmid vector in bacteria ( ori V

, ori

pBR322 ), a bacterial selectable marker gene ( aadA ), and a coding sequence for repressor of primer ( rop ) protein which is necessary for the ma intenance of the plasmid vector copy

number in E. coli . A description of the genetic elements and their prefixes ( e.g ., P-, L-, I-, TS-, OR-, B-, CS-, and T-) in PV-G MHT4355 is provided in Table III-1.Monsanto Company 10-SY-210U 39 of 721 Probe

Number Probe Type Probe

Name Start Position (bp) End Position (bp) Total Length (bp)

1 Backbone Probe B1 443 1328 886
2 Backbone probe B2 1250 2754 1505

3 Backbone Probe B3 2625 4384 1760

4 T-DNA II Probe TII-1 4796 5637 842

5 T-DNA II Probe TII-2 5575 7021 1447

6 T-DNA II Probe TII-3 6937 7761 825

7 Backbone Probe B4 8119 8289 171

8 T-DNA I Probe TI-1 8290 9523 1234

9 T-DNA I Probe TI-2 9448 10668 1221

10 T-DNA I Probe TI-3 10610 442 1185



### Figure III-1. Circular Map of Plasmid V ector PV-GMHT4355 Showing Probes 1-10
The plasmid vector PV-GMHT4355 containing the T-DNAs used in Agrobacterium -mediated transformation to produce MON 87708. Genetic elemen ts and restriction sites for enzymes used in the Southern blot analyses (with positions relative to the size of the plasmid vector) are shown on the exterior of the map. The probes used in the Southern blot analyses (labeled 1-10 on the interior of the map) are detailed in the accompanying table above.

*The Left and Right Border sequences of T-DNA II share 100% identity to those of T-DNA I, which were covered by probes 8 and 10 and thus not included in the T-DNA II probes. PV-GMHT4355

11352 bp

Aat II 6042 Bsp1286 I 386

Bsp1286 I 554

Bsp1286 I 830

Bsp1286 I 1217

Bsp1286I 2041

Bsp1286 I 2539

Bsp1286 I 3370

Bsp1286 I 3720

Bsp1286 I 4444

Bsp1286 I 5867 Bsp1286 I 7733

Kpn I 6322 Kpn I 10278

Nde I 2044 Pvu II 1814

Pvu II 5637 Pvu II 5823 Pvu II 7397 Pvu II 10380 B-Left Border

OR- ori V

CS- rop

OR- ori-pBR322

aadA

B-Left Border

T-

E9 CS- cp4 epsps TS- CTP2 L-

DnaK P-

FMV B-Right Border B-Right Border P-

PC1SV

L- TEV TS-

RbcS

CS- dmo T- E9

1

2

3

4 5 6 7 8 9 10

*

*Monsanto Company 10-SY-210U 40 of 721 

### Table III-1. Summary of Genetic Elemen ts in the Plasmid Vector PV-GMHT4355

Genetic Element Location in Plasmid (bp) Function (Reference)

T-DNA I (Present in MON 87708)

B 1 

### -Right Border 8290-8646 DNA region from Agrobacterium tumefaciens containing the Right Border sequence used for transfer of the T-DNA (D epicker et al., 1982; Zambryski et al., 1982) Intervening sequence 8647-8691 Sequence used in DNA cloning

P 2



### -PC1SV 8692-9124 Promoter for the Full-Length Transcript (FLt) of peanut chlorotic streak caulimovirus (Maiti and Shepherd, 1998) that directs transcription in plant cells Intervening sequence 9125-9144 Sequence used in DNA cloning

L 3



### - TEV 9145-9276 5 ′ non-translated region from the Tobacco Etch virus genome (Niepel and Gallie, 1999) that is involved in regulati ng gene expression Intervening sequence 9277 Sequence used in DNA cloning

TS 4



### - RbcS 9278-9520 Sequences encoding the transit peptide and the first 24 amino acids of the mature protein of the

RbcS gene from Pisum sativum (pea) (Fluhr et al., 1986) that directs transport to the DMO precursor protein of the chloroplast Intervening Sequence 9521-9529 Sequence used in DNA cloning

CS 5



### - dmo 9530-10552 Coding sequence for the dicamba mono-oxygenase from Stenotrophomonas maltophilia (Herman et al., 2005; Wang et al., 1997) Intervening Sequence 10553-10620 Sequence used in DNA cloning

T 6



### - E9 10621-11263 3 ′ non-translated region from the RbcS2 gene of

Pisum sativum (pea) encoding the Rubisco small subunit, which functions to direct polyadenylation of the mR NA (Coruzzi et al., 1984) Intervening Sequence 11264-11352 Sequence used in DNA cloning

B-Left Border 1-442 DNA region from Agrobacterium tumefaciens containing the Left Border sequence used for transfer of the T-DNA (Barker et al., 1983)Monsanto Company 10-SY-210U 41 of 721 

### Table III-1 (continued). Summary of Gene tic Elements in the Plasmid Vector

PV-GMHT4355

Genetic Element Location in Plasmid (bp) Function (Reference) Plasmid Vector Backbone (Not present in MON 87708) Intervening Sequence 443-528 Sequence used in DNA cloning

OR 7



### - ori V 529-925 Origin of replication from the broad host range plasmid RK2 for maintenance of plasmid in Agrobacterium (Stalker et al., 1981) Intervening Sequence 926-1662 Sequence used in DNA cloning

CS- rop 1663-1854 Coding sequence for repressor of primer protein derived from the ColE1 plasmid for maintenance of plasmid copy number in E. coli (Giza and Huang, 1989) Intervening Sequence 1855-2281 Sequence used in DNA cloning

OR- ori-pBR322 2282-2870 Origin of replicat ion from pBR322 for maintenance of plasmid in E. coli (Sutcliffe, 1979) Intervening Sequence 2871-3400 Sequence used in DNA cloning

aadA 3401-4289 Bacterial promoter, coding and 3 ′ UTR sequences for an aminoglycoside-modifying enzyme, 3 ′′ (9)-O-nucleotidyltransferase from transposon Tn7 (Fling et al., 1985) that confers spectinomycin and streptomycin resistance Intervening Sequence 4290-4384 Sequence used in DNA cloning T-DNA II (Not present in MON 87708)

B-Left Border 4385-4795 DNA region from Agrobacterium tumefaciens containing the Left Border sequence used for transfer of the T-DNA (Barker et al., 1983) Intervening Sequence 4796-4809 Sequence used in DNA cloning

T-E9 4810-5452 3 ′ non-translated sequence from RbcS2 gene of

Pisum sativum (pea) encoding the Rubisco small subunit, which functions to direct polyadenylation of the mR NA (Coruzzi et al., 1984) Intervening Sequence 5453-5458 Sequence used in DNA cloning

CS- cp4 epsps 5459-6826 Codon optimized coding sequence of the aroA gene from Agrobacterium spp. strain CP4 encoding CP4 EPSPS (Barry et al., 1997; Padgette et al., 1996a)Monsanto Company 10-SY-210U 42 of 721 

### Table III-1 (continued). Summary of Gene tic Elements in the Plasmid Vector

PV-GMHT4355

Genetic Element Location in

Plasmid

(bp) Function (Reference)

TS- CTP2 6827-7054 Sequences encoding th e chloroplast transit peptide region from the shkG gene of Arabidopsis

thaliana encoding EPSPS (Herrmann, 1995; Klee et al., 1987) that directs transport of the CP4 EPSPS precursor protein to the chloroplast Intervening Sequence 7055-7063 Sequence used in DNA cloning

L- DnaK 7064-7159 5 ′ non-translated leader sequence from the Petunia hybrida Hsp70 gene (Rensing and Maier, 1994) that is involved in regulating gene expression Intervening Sequence 7160-7162 Sequence used in DNA cloning

P- FMV 7163-7714 Promoter for the 35S R NA from figwort mosaic virus (Rogers, 2000) that di rects transcription in plant cells Intervening Sequence 7715-7761 Sequence used in DNA cloning

B-Right Border 7762-8118 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 8119-8289 Sequence used in DNA cloning

1 B -border.

2 P-promoter.

3 L- leader.

4 TS- targeting sequence.

5 CS-coding sequence.

6 T- 3' non-translated transcriptional termination sequence and polyadenylation signal sequences.

7 OR-origin of replication.Monsanto Company 10-SY-210U 43 of 721 

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

The Agrobacterium -mediated soybean transformati on used to produce MON 87708 was based on the method described by Martinell et al. (2002), which allows for the generation of transformed plants without ut ilization of callus. Briefly, meristem tissues were excised from the embryos of germinated conventio nal seed. After co -culturing with the Agrobacterium carrying the vector, the meristems were placed on selection medium containing glyphosate, carbenicillin, cefotaxime, and ticarcillin/clavulanate acid mixture, to inhibit the growth of untransformed plant cells and excess Agrobacterium . The meristems were then placed in media conducive to shoot and root development. Rooted plants with normal phenotypic characteristics were selected and transferred to soil for growth and further assessment.

The R 0 

### plants generated through this transfor mation were self-pollinated to produce R 1 

### plants, and the unlinked inse rtions of T-DNA I and T-DNA II were segregated. A non-lethal dose of glyphosate was applied to R 1 

### plants and those plants with minor herbicide injury were selected for further analyses, whereas plants showing no injury, indicating that they contained the cp4 epsps coding sequence from T-DNA II, were eliminated from further development. Subs equently, plants that were homozygous for T-DNA I were identified by quantitative polym erase chain reaction (PCR) analysis.

MON 87708 was selected as the lead event ba sed on superior phenotypi c characteristics, dicamba tolerance, and its molecular profile. The major development steps of MON 87708 are depicted in Figur e III-2. The result of this process was the production of marker-free, dicamba-tolerant soybean MON 87708.Monsanto Company 10-SY-210U 44 of 721 

### 

Figure III-2. Schematic of the Development of MON 87708 Transformed meristem tissue fr om A3525 with PV-GMHT4355 in Agrobacterium tumefaciens

Selected transformants and generated rooted shoots from the

transformed meristem tissues

Screening of transformed plants for the presence of T-DNA I ( dmo expression cassette) and absence of the T-DNA II ( cp4 epsps expression cassette)

Identified MON 87708 as lead candidate based on analysis of the genomic insert and evaluation of pr ogeny generations in laboratory and

field assessments Assembled Agrobacterium binary plasmid vector PV-GMHT4355 and

transferred to Agrobacterium tumefaciens strain ABIMonsanto Company 10-SY-210U 45 of 721 

### III.C. The dmo Coding Sequence and MON 87708 DMO (T-DNA I)

The dmo expression cassette (T-DNA I) pr esent in MON 87708 encodes MON 87708 DMO (Figure III-3). The dmo expression cassette contains the coding region for the

DMO from Stenotrophomonas maltophilia (Herman et al., 2005; Wang et al., 1997). The presence of MON 87708 DMO confers toleranc e to dicamba (refer to Section V.A for more details).

1 MASMISSSAV TTVSRASRGQ SAAMAPFGGL KSMTGFPVRK VNTDITSITS NGGRVKCMQV

61 WPPIGKKKFE TLSYLPPLTR D SRAMATFVR NAWYVAALPE ELSEKPLGRT ILDTPLALYR
121 QPDGVVAALL DICPHRFAPL SDGILVNGHL QCPYHGLEFD GGGQCVHNPH GNGARPASLN

181 VRSFPVVERD ALIWICPGDP ALADPGAIPD FGCRVDPAYR TVGGYGHVDC NYKLLVDNLM
241 DLGHAQYVHR ANAQTDAFDR LEREVIVGDG EIQALMKIPG GTPSVLMAKF LRGANTPVDA
301 WNDIRWNKVS AMLNFIAVAP EGTPKEQSIH SRGTHILTPE TEASCHYFFG SSRNFGIDDP
361 EMDGVLRSWQ AQALVKEDKV VVEAIERRRA YVEANGIRPA MLSCDEAAVR VSREIEKLEQ

421 LEAA



### Figure III-3. Deduced Amino Acid Sequence of the RbcS Targeting Sequence and

MON 87708 DMO The transit peptide and the first 24 amino acids of the mature protein of the RbcS gene are underlined. Accumulation of MON 87708 DMO is targeted to the chloroplasts using the RbcS transit peptide. (see Section V.A for more detail).



### III.D. The cp4 epsps Coding Sequence and the CP4 EPSPS Protein (T-DNA II)

The cp4 epsps expression cassette (T-DNA II), that is not present in MON 87708, encoded a 47.6 kDa CP4 EPSPS protein, consis ting of a single polypeptide of 455 amino acids (Padgette et al., 1996b). The cp4 epsps coding sequence is the codon optimized coding sequence of the aroA gene from Agrobacterium spp. strain CP4 encoding CP4 EPSPS (Barry et al., 1997; Padgette et al ., 1996a). CP4 EPSPS confers tolerance to glyphosate and was used as a selectable ma rker during the transformation selection process. Through conventional self-pollinat ed breeding methods and segregation, along with a combination of analytical techni ques, plants that did not contain the cp4 epsps expression cassette were isolated.

III.E. Regulatory Sequences

The dmo coding sequence in T-DNA I is under the regulation of the PC1SV promoter,

TEV leader, the RbcS targeting sequence, and the E9 3 ′ non-translated region. The

PC1SV promoter is the promoter for the Full-Le ngth Transcript (FLt) of peanut chlorotic streak caulimovirus (Maiti and Shepherd, 1998) that directs tran scription in plant cells.

The TEV leader is the 5 ′ non-translated region from the To bacco Etch virus (Niepel and Gallie, 1999) and is involved in re gulating gene expression. The RbcS targeting sequence is the sequence encoding the chloroplast transi t peptide and the first 24 amino acids of the mature protein of the ribulose-1,5-bisphos phate carboxylase oxygenase gene from pea ( Pisum sativum ) (Fluhr et al., 1986) that directs tr ansport of the DMO precursor protein to the chloroplast. The E9 3 ′ non-translated region is the 3 ′ non-translated region fromMonsanto Company 10-SY-210U 46 of 721 

### the RbcS2 gene of pea encoding th e Rubisco small subunit, wh ich functions to direct polyadenylation of the mRNA (Coruzzi et al., 1984).

T-DNA II contains the cp4 epsps coding sequence under th e regulation of the FMV
promoter, DnaK leader, the CTP2 targeting sequence, and the E9 3 ′ non-translated region.

The FMV promoter is the promoter for the 35S RNA from figwort mosaic virus (Rogers, 2000) that directs transcription in plant cells. The DnaK leader is the 5 ′ non-translated leader sequence from the Petunia hybrida Hsp70 gene (Rensing and Ma ier, 1994) that is involved in regulating gene expression. The CTP2 targeting sequenc e is the sequence encoding the chloroplast transit peptide region from the shkG gene of Arabidopsis

thaliana encoding EPSPS (Herrmann, 1995; Klee et al ., 1987) that directs transport of the CP4 EPSPS precursor protein to the chloroplast. The E9 3 ′ non-translated region is the 3 ′ non-translated region from the RbcS2 gene of pea encoding the Rubisco small subunit, which functions to direct polyadenylation of the mRNA (Coruzzi et al., 1984).

III.F. T-DNA Borders

PV-GMHT4355 contains Right an d Left Border regions (Fi gure III-1 and Table III-1)

that were derived from Agrobacterium tumefaciens (Barker et al., 1983; Depicker et al., 1982; Zambryski et al., 1982). Th e border regions each contain a 24-25 bp nick site that is the site of DNA exchange during transf ormation. The border re gions separate the T-DNA from the backbone region and are involve d in their efficient transfer into the soybean genome. Because PV-GMHT4355 is a 2T-DNA vector, it contains two Right Border regions and two Left Border regions, where one set flanks T-DNA I and the other

set flanks T-DNA II.

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

Genetic elements that exist outside of the T- DNA borders are those that are essential for

the maintenance or selection of PV-GMHT4355 in bacteria and are referred to as the plasmid backbone. The ori V , derived from the broad host pl asmid RK2, is required for the maintenance of the plasmid vector in Agrobacterium (Stalker et al., 1981), whereas

the ori-pBR322 , derived from the plasmid vect or pBR322, is required for the maintenance of the plasmid vector in E. coli (Sutcliffe, 1979). The rop is necessary for the maintenance of plasmid vector copy number in E. coli (Giza and Huang, 1989). The

aadA is a bacterial promoter and coding seque nce for an enzyme from transposon Tn7 that confers spectinomycin and streptomyc in resistance (Fling et al., 1985) in E. coli and Agrobacterium during molecular cloning. Because these elements are outside the border regions, they were not expected to be transf erred into the soybean genome. The absence of the backbone sequence in MON 87708 was conf irmed by Southern bl ot analyses (see Section IV.C).Monsanto Company 10-SY-210U 47 of 721 

### IV. CHARACTERIZATION OF THE GENETIC MODIFICATION

A multi-faceted approach was taken to characterize the genetic modification that produced MON 87708. The results confirmed that MON 87708 contains a single copy of

the dmo expression cassette (T-DNA I) that is stably integrated at a single locus and is inherited according to Mendelian principles over multiple generations (Section IV.G).

The results confirmed that no T-DNA II or plasmid vector backbone sequences are

detected in MON 87708. These conclusions ar e based on several li nes of evidence: 1) Southern blot analyses to assay the en tire soybean genome for the presence of DNA derived from PV-GMHT4355, and to confirm th at a single copy of T- DNA I was inserted at a single site and that th e insert is stably inherited; 2) DNA seque ncing analyses to determine the exact sequence of the inse rted DNA and allowed a comparison to the T-DNA I sequence in PV-GMHT4355 to confirm that only the expected sequences were integrated; and 3) a compar ison of the DNA flanking T-DNA I to the sequence of the insertion site in conventional soybean to iden tify any rearrangements that occurred at the insertion site during transforma tion. Taken together, the characterization of the genetic modification demonstrates that a single c opy of the T-DNA I was inserted at a single locus of the genome.

Southern blot analyses were used to dete rmine the number of copies and the insertion sites of T-DNA I as well as the presence or absence of T-DNA II and plasmid vector backbone sequences. The Southern blot strategy was designed to ensure that all potential inserted segments would be identified. The entire soybean genome was assayed with probes that spanned the complete plasmid v ector PV-GMHT4355 to de tect the presence

of T-DNA I as well as confirm the lack of any detectable T-DNA II and plasmid vector backbone sequences. This was accomplished by using probes that were less than 2 kb in length, ensuring 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 copies per genome equivalent. Two restri ction enzyme sets were specifically chosen to fully characterize T-DNA I and look for any pote ntial fragments of T-DNA I. This two enzyme set design also maximizes the possibili ty of detecting an insertion elsewhere in the genome that could be overlooked if that band comigrated with an expected band. Additionally, the restriction enzyme sets were chosen such that at least one enzyme from

each set resides in the known 5 ′ or 3 ′ flanking sequence and that together the enzyme sets result in overlapping segments covering the enti re insert. Therefore, at least one segment for each flank is of a predictable size and overl aps with another predic table size segment. This overlapping strategy confirms that the entire insert sequence is identified in a predictable hybridi zation pattern.

To determine the number of copies and the insertion sites of T-DNA I, and the presence or absence of T-DNA II and the plasmid vect or backbone sequences, duplicated samples that consisted of equal amounts of digested DNA were run on the agarose gel. One set of samples was run for a longer pe riod of time (long run) than a second set (short run). The long run allows for greater resolution of la rge molecular weight DNA, whereas the short

run allows the detection of small molecular weight DNA. The molecular weight markers on the left of the figures were used to estim ate the sizes of the ba nds present in the long run lanes of the Southern blots, and the mo lecular weight markers on the right of theMonsanto Company 10-SY-210U 48 of 721 

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

Southern blots.

The DNA sequencing analyses complement the Southern blot analyses. Southern blot

results demonstrated that MON 87708 contai ns a single copy of T-DNA I at a single insertion site. Sequencing of the insert and the flanking DNA confirmed the organization of the elements within the insert, determined the 5 ′ and 3 ′ insert-to-plant junctions, as well as the complete DNA sequence of the insert and adjacent DNA. In addition, DNA sequencing analyses confirmed that each geneti c element in the insert is intact and the sequence of the insert matches the corresponding sequence in PV-GMHT4355. Furthermore, genomic organization at the in sertion site was assessed by comparing the insert and flanking sequence to the inse rtion site in conventional soybean.

The stability of the T-DNA I present in MON 87708 across multiple generations (R 2 

### -R 6 

### ) was demonstrated by Southern blot analysis . Genomic DNA from five generations of MON 87708 was digested with one of the enzy me sets used for the insert and copy number analysis and was hybridized with a pr obe that detects restriction segments that encompass the entire T-DNA I. This fingerpri nt strategy consists of two border segments that assess not only the stab ility of T-DNA I, but also th e stability of genomic DNA directly adjacent to T-DNA I.

The results of these analyses for MON 87708 demonstrated that a single copy of the T-DNA I was inserted at a singl e locus of the genome. Gene rational stability analysis demonstrated that an expected Southern bl ot fingerprint of MON 87708 was maintained through five generations of the breeding hi story, thereby confirming the stability of T-DNA I in MON 87708. Results from segreg ation analyses showed heritability and stability of the insert occurred as e xpected across multiple generations, which corroborates the molecular insert stability anal ysis and establishes th e genetic behavior of the T-DNA I at a single chromosomal locus.

The Southern blot analysis confirmed that T-DNA I reported in Figure IV-1 represents the only detectable insert in MON 87708. Figure IV-1 is a linear map depicting

restriction sites within the insert as we ll as within the known soybean genomic DNA immediately flanking the insert in MON 87708. The circular map of PV-GMHT4355 annotated with the probes used in the Southern blot analysis is presented in Figure III-1. Based on the linear map of th e insert and the plasmid ma p, a table summarizing the expected DNA segments for Southern analyses is presented in Table IV-1. The genetic elements integrated in MON 87708 are summari zed in Table IV-2. The generations used are depicted in the breeding history shown in Figure IV-9. Materials and methods used for characterization of T-DNA I in MON 87708 are found in Appendix B.Monsanto CompanyFigure IV-1. Schematic Representation of the Insert andA linear map of the insert and genomic DNA flankingthe insert in MONinsert, as well as restriction sites with positionsrelative to the size of the linear map for enzymesand locations of the T-DNAI probes, which are described in Figure IIImap are the expected sizes of the DNA segments after digestioninsert and the beginning of the genomic DNA sequence flanking the 5the elements in MON 87708.10-SY-210U1. Schematic Representation of the Insert andDNA Flanking Sequences in MON 87708A linear map of the insert and genomic DNA flankingthe insert in MON87708 is shown. Identified on the map are geneticelements within theinsert, as well as restriction sites with positionsrelative to the size of the linear map for enzymesused in the Southern analyses.I probes, which are described in Figure III-1, are shown on the middle portion.Shown on the lower portion of themap are the expected sizes of the DNA segments after digestionwith respective restriction enzymes. Arrowheads ( ) indicate the end of theinsert and the beginning of the genomic DNA sequence flanking the 5′and 3′end of the insert. The arrows (→) indicate the s49 of 72187708 is shown. Identified on the map are geneticelements within theused in the Southern analyses.The relative sizesShown on the lower portion of theive restriction enzymes. Arrowheads ( ) indicate the end of the) indicate the sequence direction ofMonsanto Company 10-SY-210U 50 of 721Table IV-1. Summary Chart of the Expected DNA Segments Based on Hybridizing Probes and Restriction Enzymes Used inMON 87708 AnalysisSouthern Blot Figure IV-2IV-3IV-4IV-5IV-6IV-7IV-8IV-10Probe Used 89104561, 2, 3,and 79Probing Target Digestion Enzyme ExpectedBand Sizes (kb) on Each Southern BlotPlasmid VectorPV-GMHT4355AatII/NdeI ~7.4~7.4~4.0~7.4~4.0~7.4~4.0~7.4~7.4~4.0~7.4~7.4Probe Templates1N/A ~~2~~2~~2~~2~~2~~2~0.2~0.9~1.5~1.8~~2ConventionalControl A3525Bsp1286I/PvuIINoneNoneNoneNoneNoneNoneNoneNoneHpaI/KpnINoneNoneNoneNoneNoneNoneNoneNoneMON 87708Bsp1286I/PvuII ~2.6~2.6~1.5~1.5~1.5NoneNone None~2.6~1.5HpaI/KpnI>2.7*>2.7*~1.7~1.7~1.7NoneNone None --31Probe templates were spiked when multiple probes are used in Southern blot analysis.2‘~~’ indicates that only plasmid template was used since the Southern blot was hybridized with one probe.3‘--’ indicates that the particular restriction enzyme or the combination of the enzymes was not used in the analysis.*Southern analysis indicatesthis segment to be ~5.6 kb.Monsanto Company 10-SY-210U 51 of 721 

### Table IV-2. Summary of Ge netic Elements in MON 87708

Genetic Element 

### Location (bp) Function (Reference)

5 ′ Flanking Sequences 1-1048 DNA sequence adjacent to the 5 ′ end of the

insertion site

B 1 -Right Border * 1049-1091 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 1092-1136 Sequence used in DNA cloning

P 2

- PC1SV 1137-1569 Promoter for the Full-Length Transcript (FLt) of peanut chlorotic streak caulimovirus (Maiti and Shepherd, 1998) that directs transcription in plant

cells Intervening sequence 1570-1589 Sequence used in DNA cloning

L 3

- TEV 1590-1721 5' non-translated region from the Tobacco Etch virus genome (Niepel and Gallie, 1999) that is involved in regulating gene expression Intervening sequence 1722-1722 Sequence used in DNA cloning

TS 4

- RbcS 1723-1965 Sequences encoding the transit peptide and the first 24 amino acids of the mature protein of the RbcS gene from Pisum sativum (pea) (Fluhr et al., 1986) that directs transport of the DMO precursor protein to the chloroplast
Intervening Sequence 1966-1974 Sequence used in DNA cloning

CS 5

- dmo 1975-2997 Coding sequence for the dicamba mono-oxygenase

from Stenotrophomonas maltophilia (Herman et al., 2005; Wang et al., 1997)
Intervening Sequence 2998-3065 Sequence used in DNA cloning

T 6

- E9 3066-3708 3 ′ non-translated region from the RbcS2 gene of

Pisum sativum (pea) encoding the Rubisco small subunit, which functions to direct polyadenylation of the mRNA (Coruzzi et al., 1984) Intervening Sequence 3709-3797 Sequence used in DNA cloning

B-Left Border * 3798-4051 DNA region from Agrobacterium tumefaciens containing the Left Border sequence used for transfer of the T-DNA (Barker et al., 1983)

3 ′ Flanking Sequences 4052-5322 DNA sequence adjacent to the 3 ′ end of the

insertion site

1 B-border.

2 P-promoter.

3 L-leader.

4 TS- targeting sequence.

5 S- coding sequence.

6 T-3' non-translated transcriptional termination sequence and polyadenylation signal sequences.

*These borders are truncated.Monsanto Company 10-SY-210U 52 of 721 

### IV.A. Insert and Copy Number of T-DNA I in MON 87708

The copy number and insertion site of T-DNA I was assessed by digesting MON 87708 genomic DNA with the restriction enzyme combination Bsp1286 I/ Pvu II or Hpa I/ Kpn I and hybridizing Southern blot s with probes that span T- DNA I (Figure III-1). Each restriction digest is expected to produce a specific banding pattern on the Southern blots (Table IV-1). Since each detected se gment contains flanking genomic DNA, any additional integrated sites would produce a different banding patt ern with additional

bands.

The restriction enzyme combination Bsp1286 I/ Pvu II cuts once w ithin T-DNA I and once within each of the known geno mic DNA sequences flanking the 5 ′ and 3 ′ ends of T-DNA I (Figure IV-1). Therefore, if T-DNA I sequences are present at a single integration site in M ON 87708, the digestion with Bsp1286 I/ Pvu II was expected to generate two border segments with expected sizes of ~2.6 kb and ~1.5 kb (Figure IV-1, and Table IV-1). The ~2.6 kb restriction segment contained ge nomic DNA flanking the

5 ′ end of T-DNA I, the Right Border, the PC1SV promoter, the TEV leader, the RbcS targeting sequence, and a portion of the dmo coding sequence. The ~1.5 kb restriction segment contained a portion of the dmo coding sequence, the E9 3 ′ non-translated sequence, the Left Border, a nd genomic DNA flanking the 3 ′ end of T-DNA I.

The restriction enzyme combination Hpa I/ Kpn I cuts once within T-DNA I and once within the known genomic DNA flanking the 3 ′ end of T-DNA I (Figure IV-1). Therefore, if T-DNA I sequences are presen t at a single integrat ion site in MON 87708, the digestion with Hpa I/ Kpn I was expected to generate two border segments with expected sizes of ~1.7 kb and greater than 2.7 kb (Figure IV-1, and Table IV-1). Since

the Hpa I/ Kpn I restriction site in th e genomic DNA flanking the 5 ′ end of the insert lies outside of the known sequence, it was not possible to predic t a precise segment size. However, the segment size was determined by Southern blot analyses to be ~5.6 kb (Figures IV-2 and IV-3). The ~5.6 kb restriction segment contained genomic DNA flanking the 5 ′ end of T-DNA I, the Right Border, the PC1SV promoter, the TEV leader,

the RbcS targeting sequence, and a portion of dmo coding sequence. The ~1.7 kb restriction segment contained a portion of the dmo coding sequence, the E9 3 ′ non-translated sequence, the Left Bo rder, and genomic DNA flanking the 3 ′ end of

T-DNA I.

In the Southern blot analyses performed, each Southern blot contained a negative and a positive control. Conventional control genomic DNA digested with either the restriction enzyme combination Bsp1286 I/ Pvu II or Hpa I/ Kpn I was used as a negative control to determine if the probes hybridized to any endogenous soybean sequences. As a positive

control on the Southern blot s, PV-GMHT4355 digested with the restriction enzyme

combination Aat II/ Nde I was mixed with predigested conventional control DNA. The positive hybridization control was spiked at 0.1 and 1 genome equivalent to demonstrate sufficient sensitivity of the Southern blot. Individual Southern blots were hybridized with the following probes: probes 8, 9, and 10 (refer to Figure III-1 and Table IV-1). The results of this analysis are s hown in Figures IV-2 through IV-4.Monsanto Company 10-SY-210U 53 of 721 

### IV.A.1. Probe 8

Conventional control DNA dige sted with the restric tion enzyme combination Bsp1286 I/ Pvu II (Figure IV-2, lanes 1 and 5) or Hpa I/ Kpn I (Figure IV-2, lanes 3 and 7) and hybridized with probe 8 (Figure III-1) pr oduced no detectable hybridization bands as expected for the negative control. PV-GMHT4 355, digested with th e restriction enzyme

combination Aat II/ Nde I and mixed with conventional control DNA predigested with the restriction enzyme combination Hpa I/ Kpn I (Figure IV-2, lanes 10 and 11), produced the expected size band at ~7.4 kb (refer to Figure III-1 and Table IV-1 ). These results indicate that the pr obe is hybridizing to its target sequence.

MON 87708 DNA digested with the re striction enzyme combination Bsp1286 I/ Pvu II and hybridized with probe 8 (Figure II I-1) produced one unique band at ~2.6 kb (Figure IV-2, lanes 2 and 6). The ~2.6 kb band is the expected size for the border segment containing the 5 ′ end of T-DNA I along with the adjacent genomic DNA flanking the 5 ′ end of T-DNA I (Figure IV-1).

MON 87708 DNA digested with the restriction enzyme combination Hpa I/ Kpn I and hybridized with probe 8 (Figure III-1) produc ed one unique band at ~5.6 kb (Figure IV-2, lanes 4 and 8). The ~5.6 kb band is consistent with the exp ected band being greater than 2.7 kb for the border segment containing the 5 ′ end of T-DNA I along with the adjacent genomic DNA flanking the 5 ′ end of T-DNA I (Figure IV-1).

No additional bands were detected using probe 8. Based on the results presented in Figure IV-2, it was concluded that T-DNA I sequences covere d by probe 8 reside at a single integration locus as one copy in MON 87708.

IV.A.2. Probe 9

Conventional control DNA dige sted with the restric tion enzyme combination Bsp1286 I/ Pvu II (Figure IV-3, lanes 1 and 5) or Hpa I/ Kpn I (Figure IV-3, lanes 3 and 7) and hybridized with probe 9 (Figure III-1) pr oduced no detectable hybridization bands as expected for the negative control. PV-GMHT4 355, digested with th e restriction enzyme

combination Aat II/ Nde I and mixed with conventional control DNA predigested with the restriction enzyme combination Hpa I/ Kpn I (Figure IV-3, lanes 10 and 11), produced the expected size band at ~7.4 kb (refer to Figure III-1 and Table IV-1 ). These results indicate that the pr obe is hybridizing to its target sequence.

MON 87708 DNA digested with the re striction enzyme combination Bsp1286 I/ Pvu II and hybridized with probe 9 (Figure III-1) produced two unique bands at ~1.5 kb and ~2.6 kb (Figure IV-3, lanes 2 and 6). The ~1.5 kb band is the expected size for the border segment containing the 3 ′ end of T-DNA I along with the adjacent genomic DNA flanking the 3 ′ end of T-DNA I (Figure IV-1). The ~2.6 kb band is the expected size for the border segment containing the 5 ′ end of T-DNA I along with the adjacent genomic DNA flanking the 5 ′ end of T-DNA I (Figure IV-1).

MON 87708 DNA digested with the restriction enzyme combination Hpa I/ Kpn I and hybridized with probe 9 (Figure III-1) produ ced two unique bands at ~1.7 kb and ~5.6 kbMonsanto Company 10-SY-210U 54 of 721 

### (Figure IV-3, lanes 4 and 8). The ~1.7 kb band is the expected size for the border segment containing the 3 ′ end of T-DNA I along with the adjacent genomic DNA flanking the 3 ′ end of T-DNA I (Figure IV-1). The ~5.6 kb band is consistent with the expected band being greater than 2.7 kb for the border segment containing the 5 ′ end of T-DNA I along with the adjacent genomic DNA flanking the 5 ′ end of T-DNA I (Figure IV-1).

No additional bands were detected using probe 9. Based on the results presented in Figure IV-3, it was concluded that T-DNA I sequences covere d by probe 9 reside at a single integration locus as one copy in MON 87708.

IV.A.3. Probe 10

Conventional control DNA dige sted with the restric tion enzyme combination Bsp1286 I/ Pvu II (Figure IV-4, lanes 1 and 5) or Hpa I/ Kpn I (Figure IV-4, lanes 3 and 7) and hybridized with probe 10 (Figure III-1) produced no dete ctable hybridization bands as expected for the negative control. PV-GMHT4355, digested with the restriction enzyme combination Aat II/ Nde I and mixed with conventio nal control DNA predigested

with the restriction enzyme combination Hpa I/ Kpn I (Figure IV-4, lanes 10 and 11), produced two bands at ~4.0 kb and ~7.4 kb. Both bands were expected because probe 10

contains E9 and left border se quences that hybridized to bo th the ~4.0 kb and the ~7.4 kb fragments from the digested plasmid (refer to Figure III-1 and Table IV-1). These results indicate that the pr obe is hybridizing to its target sequence.

MON 87708 DNA digested with the re striction enzyme combination Bsp1286 I/ Pvu II and hybridized with probe 10 (Figure II I-1) produced a unique band at ~1.5 kb (Figure IV-4, lanes 2 and 6). The ~1.5 kb band is the expected size for the border segment containing the 3 ′ end of T-DNA I along with the adjacent genomic DNA flanking the 3 ′ end of T-DNA I (Figure IV-1).

MON 87708 DNA digested with the restriction enzyme combination Hpa I/ Kpn I and hybridized with probe 10 (Figure III-1) produ ced a unique band at ~1.7 kb (Figure IV-4, lanes 4 and 8). The ~1.7 kb band is the expe cted size for the border segment containing

the 3 ′ end of T-DNA I along with the ad jacent genomic DNA flanking the 3 ′ end of T-DNA I (Figure IV-1).

No additional bands were detected using pr obe 10. Based on the results presented in Figure IV-4, it was concluded that T-DNA se quences covered by probe 10 reside at a single integration locus as one copy in MON 87708.Monsanto Company 10-SY-210U 55 of 721 

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

T-DNA I in MON 87708: Probe 8 The blot was hybridized with a 32 P labeled T-DNA I probe that spans a portion of the T-DNA I sequence (Probe 8, Figure III-1). Each lane cont ains approximately 10 μg of digested genomic DNA isolated from leaf tissue. Lane designations are as follows:

Lane Description 1. Conventional control ( Bsp1286 I/

Pvu II) 2. MON 87708 ( Bsp1286 I/

Pvu II) 3. Conventional control ( Hpa I/ Kpn I) 4. MON 87708 ( Hpa I/ Kpn I) 5. Conventional control ( Bsp1286 I/

Pvu II) 6. MON 87708 ( Bsp1286 I/

Pvu II) 7. Conventional control ( Hpa I/ Kpn I) 8. MON 87708 ( Hpa I/ Kpn I)

9. Blank 10. Conventional control ( Hpa I/ Kpn I) spiked with PV-GMHT4355 ( Aat II/ Nde I) (~1 genome equivalent) 11. Conventional control ( Hpa I/ Kpn I) spiked with PV-GMHT4355 ( Aat II/ Nde I) (~0.1 genome equivalent)

Arrows denote sizes of DNA, in kilobase pairs, obtained from molecular weight markers on ethidium bromide stained gel. 1 2 3 4 5 6 7 8 9 10 11

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 LONG RUN SHORT RUNMonsanto Company 10-SY-210U 56 of 721 

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

T-DNA I in MON 87708: Probe 9 The blot was hybridized with a 32 P labeled T-DNA I probe that spans a portion of the T-DNA I sequence (Probe 9, Figure III-1). Each lane cont ains approximately 10 μg of digested genomic DNA isolated from leaf tissue. Lane designations are as follows:

Lane Description 1. Conventional control ( Bsp1286 I/

Pvu II) 2. MON 87708 ( Bsp1286 I/

Pvu II) 3. Conventional control ( Hpa I/ Kpn I) 4. MON 87708 ( Hpa I/ Kpn I) 5. Conventional control ( Bsp1286 I/

Pvu II) 6. MON 87708 ( Bsp1286 I/

Pvu II) 7. Conventional control ( Hpa I/ Kpn I) 8. MON 87708 ( Hpa I/ Kpn I)

9. Blank 10. Conventional control ( Hpa I/ Kpn I) spiked with PV-GMHT4355 ( Aat II/ Nde I) (~1 genome equivalent) 11. Conventional control ( Hpa I/ Kpn I) spiked with PV-GMHT4355 ( Aat II/ Nde I) (~0.1 genome equivalent)

Arrows denote sizes of DNA, in kilobase pairs, obtained from molecular weight markers on ethidium bromide stained gel. 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 LONG RUN SHORT RUNMonsanto Company 10-SY-210U 57 of 721 

### Figure IV-4. Southern Blot Analysis to Determine Insert and Copy Number of

T-DNA I in MON 87708: Probe 10 The blot was hybridized with a 32 P labeled T-DNA I probe that spans a portion of the T-DNA I sequence (Probe 10, Figure III-1). Each lane cont ains approximately 10 μg of digested genomic DNA isolated from leaf tissue. Lane designations are as follows:

Lane Description 1. Conventional control ( Bsp1286 I/

Pvu II) 2. MON 87708 ( Bsp1286 I/

Pvu II) 3. Conventional control ( Hpa I/ Kpn I) 4. MON 87708 ( Hpa I/ Kpn I) 5. Conventional control ( Bsp1286 I/

Pvu II) 6. MON 87708 ( Bsp1286 I/

Pvu II) 7. Conventional control ( Hpa I/ Kpn I) 8. MON 87708 ( Hpa I/ Kpn I)

9. Blank 10. Conventional control ( Hpa I/ Kpn I) spiked with PV-GMHT4355 ( Aat II/ Nde I) (~1 genome equivalent) 11. Conventional control ( Hpa I/ Kpn I) spiked with PV-GMHT4355 ( Aat II/ Nde I) (~0.1 genome equivalent)

Arrows denote sizes of DNA, in kilobase pairs, obtained from molecular weight markers on ethidium bromide stained gel. 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 LONG RUN SHORT RUNMonsanto Company 10-SY-210U 58 of 721 

### IV.B. Southern Blot Analysis to Det ermine the Presence or Absence of T-DNA II Sequences in MON 87708

To determine the presence or absen ce of T-DNA II sequences, MON 87708 and conventional control genomic DNA were di gested with the re striction enzyme

combination Bsp1286 I/ Pvu II or Hpa I/ Kpn I and Southern blots were hybridized with probes that span the T-DNA II sequence (Figur e III-1). As a pos itive control on the Southern blots, PV-GMHT4355 digested wi th the restriction enzyme combination

Aat II/ Nde I was mixed with predigested co nventional control DNA. The positive hybridization control was sp iked at 0.1 and 1 genome equivalent to demonstrate sufficient sensitivity of the Southern blot. Each blot was hybridized with one of three overlapping probes spanning the T-DNA II seque nce other than the two border regions that share the same sequences as present in T-DNA I (Probes 4, 5 and 6, Figure III-1). If T-DNA II sequences were present in MON 87708, then probing with the T-DNA II sequences should result in unique hybridizing ba nds. The results of this analysis are shown in Figures IV-5 through IV-7.

IV.B.1. Probe 4

Conventional control DNA digested with Bsp1286 I/ Pvu II (Figure IV-5, lanes 1 and 5)

or Hpa I/ Kpn I (Figure IV-5, lanes 3 and 7) and hybridized with probe 4 showed no detectable hybridization bands, as expected for the negative cont rol. PV-GMHT4355, previously digested with Aat II/ Nde I and mixed with conventional control DNA predigested with Hpa I/ Kpn I (Figure IV-5, lanes 10 and 11), produced two bands at ~4.0 kb and ~7.4 kb. Both bands were expected because probe 4 contains E9 sequence that hybridized to both the ~4.0 kb and the ~ 7.4 kb fragments from the digested plasmid (refer to Figure III-1 and Table IV-1). These results indicate that the probe is hybridizing to its target sequence.

MON 87708 DNA digested with the re striction enzyme combination Bsp1286 I/ Pvu II and hybridized with probe 4 (Figure II I-1) produced one unique band at ~1.5 kb (Figure IV-5, lanes 2 and 6) . MON 87708 DNA digested with Hpa I/ Kpn I and hybridized with probe 4 (Figure III-1) produc ed one unique band at ~1.7 kb (Figure IV-5, lanes 4 and 8). Probe 4 contains the E9 3 ′ non-translated region sequence that is also contained in T-DNA I (Figure III- 1). Therefore, probe 4 was expected to hybridize to the ~1.5 kb and ~1.7 kb fragments (Figure IV-1) derived from the T-DNA I insert. These bands were also detected by probe 10 (Figur e IV-4, lanes 2 and 6, and lanes 4 and 8). Any T-DNA II sequences other than those asso ciated with T-DNA I would be detected as novel bands. No unexpected bands were det ected indicating that MON 87708 contains no detectable T-DNA II elements covered by probe 4.

IV.B.2. Probe 5

Conventional control DNA dige sted with the restric tion enzyme combination Bsp1286 I/ Pvu II (Figure IV-6, lanes 1 and 5) or Hpa I/ Kpn I (Figure IV-6, lanes 3 and 7) and hybridized with probe 5 (Figure III-1) s howed no detectable hybridization bands, as expected for the negative control. PV -GMHT4355, previously digested with Aat II/ Nde IMonsanto Company 10-SY-210U 59 of 721 

### and mixed with conventional control DNA predigested with Hpa I/ Kpn I (Figure IV-6, lanes 10 and 11), produced tw o expected size bands at ~4.0 kb and ~7.4 kb (refer to Figure III-1 and Table IV-1). These results indicate that the probe is hybridizing to its

target sequence.

MON 87708 DNA digested with the re striction enzyme combination Bsp1286 I/ Pvu II (Figure IV-6, lanes 2 and 6) or Hpa I/ Kpn I (Figure IV-6, lanes 4 and 8) and hybridized with probe 5, produced no dete ctable hybridization bands. These results indicate that

MON 87708 contains no detectable T- DNA II elements covered by probe 5.

IV.B.3. Probe 6

Conventional control DNA digested with Bsp1286 I/ Pvu II (Figure IV-7, lanes 1 and 5)

or Hpa I/ Kpn I (Figure IV-7, lanes 3 and 7) and hybr idized with probe 6 (Figure III-1) showed no detectable hybridiz ation bands, as expected fo r the negative control. PV-GMHT4355 previously digested with Aat II/ Nde I and mixed with conventional control DNA predigested with Hpa I/ Kpn I (Figure IV-6, lanes 10 and 11) produced one expected size band at ~7.4 kb (refer to Figur e III-1, and Table IV-1). These results indicate that the probe is hybridiz ing to its target sequence.

MON 87708 DNA digested with the re striction enzyme combination Bsp1286 I/ Pvu II (Figure IV-7, lanes 2 and 6) or Hpa I/ Kpn I (Figure IV-7, lanes 4 and 8) and hybridized with probe 6 produced no detectable hybridizat ion bands. These results indicated that

MON 87708 contains no detectable T- DNA II elements covered by probe 6.Monsanto Company 10-SY-210U 60 of 721 

### Figure IV-5. Southern Blot Analysis to Detect the Presence or Absence of T-DNA II Sequences in MON 87708: Probe 4 The blot was hybridized with a 32 P labeled T-DNA II probe that spans a portion of the T-DNA II sequence (Probe 4, Figure III-1). Each lane cont ains approximately 10 μg of digested genomic DNA isolated from leaf tissue. Lane designations are as follows:

Lane Description 1. Conventional control ( Bsp1286 I/

Pvu II) 2. MON 87708 ( Bsp1286 I/

Pvu II) 3. Conventional control ( Hpa I/ Kpn I) 4. MON 87708 ( Hpa I/ Kpn I) 5. Conventional control ( Bsp1286 I/

Pvu II) 6. MON 87708 ( Bsp1286 I/

Pvu II) 7. Conventional control ( Hpa I/ Kpn I) 8. MON 87708 ( Hpa I/ Kpn I)

9. Blank 10. Conventional control ( Hpa I/ Kpn I) spiked with PV-GMHT4355 ( Aat II/ Nde I) (~1 genome equivalent) 11. Conventional control ( Hpa I/ Kpn I) spiked with PV-GMHT4355 ( Aat II/ Nde I) (~0.1 genome equivalent)

Arrows denote sizes of DNA, in kilobase pairs, obtained from molecular weight markers on ethidium bromide stained gel. 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 LONG RUN SHORT RUNMonsanto Company 10-SY-210U 61 of 721 

### Figure IV-6. Southern Blot Analysis to De tect the Presence or Absence of T-DNA II Sequences in MON 87708: Probe 5 The blot was hybridized with a 32 P labeled T-DNA II probe that spans the coding region of the T-DNA II sequence (Probe 5, Figure III-1). Each la ne contains approximately 10 μg of digested genomic DNA isolated from leaf tissue. Lane designations are as follows:

Lane Description 1. Conventional control ( Bsp1286 I/

Pvu II) 2. MON 87708 ( Bsp1286 I/

Pvu II) 3. Conventional control ( Hpa I/ Kpn I) 4. MON 87708 ( Hpa I/ Kpn I) 5. Conventional control ( Bsp1286 I/

Pvu II) 6. MON 87708 ( Bsp1286 I/

Pvu II) 7. Conventional control ( Hpa I/ Kpn I) 8. MON 87708 ( Hpa I/ Kpn I)

9. Blank 10. Conventional control ( Hpa I/ Kpn I) spiked with PV-GMHT4355 ( Aat II/ Nde I) (~1 genome equivalent) 11. Conventional control ( Hpa I/ Kpn I) spiked with PV-GMHT4355 ( Aat II/ Nde I) (~0.1 genome equivalent)

Arrows denote sizes of DNA, in kilobase pairs, obtained from molecular weight markers on ethidium bromide stained gel. 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 LONG RUN SHORT RUNMonsanto Company 10-SY-210U 62 of 721 

### Figure IV-7. Southern Blot Analysis to De tect the Presence or Absence of T-DNA II Sequences in MON 87708: Probe 6 The blots were hybridized with a 32 P labeled T-DNA II probe that spans a portion of the T-DNA II sequence (Probe 6, Figure III-1). Each la ne contains approximately 10 μg of digested genomic DNA isolated from leaf tissue. Lane designations are as follows:

Lane Description 1. Conventional control ( Bsp1286 I/

Pvu II) 2. MON 87708 ( Bsp1286 I/

Pvu II) 3. Conventional control ( Hpa I/ Kpn I) 4. MON 87708 ( Hpa I/ Kpn I) 5. Conventional control ( Bsp1286 I/ Pvu II) 6. MON 87708 ( Bsp1286 I/

Pvu II) 7. Conventional control ( Hpa I/ Kpn I) 8. MON 87708 ( Hpa I/ Kpn I)

9. Blank 10. Conventional control ( Hpa I/ Kpn I) spiked with PV-GMHT4355 ( Aat II/ Nde I) (~1 genome equivalent) 11. Conventional control ( Hpa I/ Kpn I) spiked with PV-GMHT4355 ( Aat II/ Nde I) (~0.1 genome equivalent)

Arrows denote sizes of DNA, in kilobase pairs, obtained from molecular weight markers on ethidium bromide stained gel. 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 LONG RUN SHORT RUNMonsanto Company 10-SY-210U 63 of 721 

### IV.C. Southern Blot Analysis to De termine the Presence or Absence of PV-GMHT4355 Backbone Sequences in MON 87708

To determine the presence or absen ce of PV-GMHT4355 backbone sequences, MON 87708 and conventional control genomic DNA were digested with the restriction enzyme combination Bsp1286 I/ Pvu II or Hpa I/ Kpn I and Southern blots were hybridized with probes that span the plasmi d vector backbone sequence (Figure III-1). As a positive control on the Southern blots, digested PV -GMHT4355 and probe templates generated from PV-GMHT4355 were used. Approximately 1 genome equivalent of PV-GMHT4355 digested with the restriction enzyme combination

Aat II/ Nde I was mixed with predigested conven tional control DNA. As an additional positive control, approximately 0.1 and 1 genome equivalent of probe templates (Figure III-1, probes 1, 2, 3, and 7) genera ted from PV-GMHT4355 were mixed with predigested conventional control DNA. The bl ot was hybridized with probes 1, 2, 3, and 7 (Figure III-1). If backbone sequences are present in MON 87708, then probing with backbone probes should result in hybridizing bands. The results of this analysis are shown in Figure IV-8.

IV.C.1. Plasmid Vector Backbone Probes 1, 2, 3, and 7

Conventional control DNA dige sted with the restric tion enzyme combination Bsp1286 I/ Pvu II (Figure IV-8, lanes 1 and 5) or Hpa I/ Kpn I (Figure IV-8, lanes 3 and 7) and hybridized simultaneously with the probes 1, 2, 3, and 7 (Figure III-1) spanning the entire backbone sequence of PV-GMHT4355 showed no detect able hybridization bands, as expected for the negative control. PV-GMHT4355, previously digested with

Aat II/ Nde I and mixed with conventiona l control DNA predigested with Hpa I/ Kpn I (Figure IV-8, lane 10), produced two expected size bands at ~4.0 kb and ~7.4 kb (refer to Figure III-1 and Table IV-1). In addition, there are two faint hybridization bands at ~4.5 kb and ~11 kb (Figure IV-8, lane 10). Th e ~4.5 kb band was likely due to an artifact that occurred during the electrophoresis , and the ~11 kb band was likely due to undigested plasmid DNA or an artifact that occurred during the electrophoresis. Since these faint bands appeared only in the plasmid spike and the expected bands were observed, they have no negative impact on the conclusions made from this blot. Probe template spikes of probes 1, 2, 3, and 7 (Figure III-1) generated from PV-GMHT4355 mixed with conventional control DNA predigested with Hpa I/ Kpn I (Figure IV-8, lanes 11 and 12) produced th e expected size bands at ~0.2 kb, ~0.9 kb, ~1.5 kb, and ~1.8 kb, respectively. The 0.1 genome equivalent copy of the expected ~0.2 kb band was not observed on the exposure of the Southern blot that is reported in Figure IV-8, lane 12; however, the band was observed on the same bl ot with a longer exposure. These results indicate that the probe s are hybridizing to thei r target sequences.

MON 87708 DNA digested with the re striction enzyme combination Bsp1286 I/ Pvu II (Figure IV-8, lanes 2 and 6) or Hpa I/ Kpn I (Figure IV-8, lanes 4 and 8) and hybridized simultaneously with probes 1, 2, 3, and 7 pr oduced no detectable bands. The data

indicate MON 87708 contains no detectable backbone sequences from PV-GMHT4355.Monsanto Company 10-SY-210U 64 of 721 

### Figure IV-8. Southern Blot Analysis to Determine the Presence or Absence of PV-GMHT4355 Backbone Sequences in MON 87708: Probes 1, 2, 3, and 7 The blot was hybridized simultaneously with four 32 P labeled backbone probes (Probes 1, 2, 3, and 7, Figure III-1). Each lane contains approxi mately 10 μg of digested genomic DNA isolated from leaf tissue. Lane designations are as follows:

Lane Description 1. Conventional control ( Bsp1286 I/

Pvu II) 2. MON 87708 ( Bsp1286 I/

Pvu II) 3. Conventional control ( Hpa I/ Kpn I) 4. MON 87708 ( Hpa I/ Kpn I) 5. Conventional control ( Bsp1286 I/

Pvu II) 6. MON 87708 ( Bsp1286 I/

Pvu II) 7. Conventional control ( Hpa I/ Kpn I) 8. MON 87708 ( Hpa I/ Kpn I)

9. Blank 10. Conventional control ( Hpa I/ Kpn I) spiked with PV-GMHT4355 ( Aat II/ Nde I) (~1 genome equivalent) 11. Conventional control ( Hpa I/ Kpn I) spiked with probe templates (~1 genome equivalent) 12. Conventional control ( Hpa I/ Kpn I) spiked with probe templates (~0.1 genome equivalent)

Arrows denote sizes of DNA, in kilobase pairs, obtained from molecular weight markers on ethidium bromide stained gel. 23.1

9.4

6.6

4.4

2.3

2.0 1 2 3 4 5 6 7 8 9 10 11 12

23.1

9.4

6.6

4.4

2.3

0.6 2.0 LONG RUN SHORT RUNMonsanto Company 10-SY-210U 65 of 721 

### IV.D. Organization and Sequence of the Insert and Adjacent DNA in MON 87708

The organization of the elements within the T-DNA I was confirmed by DNA sequence analyses. PCR primers were designed with the intent to amplify two overlapping regions of the DNA that span the entire length of T-DNA I (Figure B-1, Appendix B). The amplified DNA segments were subjected to DNA sequencing analyses . The T-DNA I in MON 87708 is 3003 bp and matches the sequenc e of plasmid vector PV-GMHT4355, as

described in Tables III-1 and IV-2.

IV.E. PCR and DNA Sequence Analyses to Examine the MON 87708 Insertion Site

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

MON 87708 and conventional control to exam ine the insertion si tes. The PCR was performed with one primer specific to the genomic DNA sequence flanking the 5 ′ end of T-DNA I paired with a second primer specif ic to the genomic DNA sequence flanking

the 3 ′ end of T-DNA I (Figure B-2, Appendix B). A sequence comparison between the PCR product generated from the conventional control and the sequence generated from

the 5 ′ and 3 ′ flanking sequences of T-DNA I in MON 87708 indicates there was an 899 bp deletion and a 128 bp insertion just 5 ′ of T-DNA I, and a 35 bp insertion just 3 ′ of T-DNA I. These molecular rearrangements presumably resulted from double-stranded break repair mechanisms in the plant during the Agrobacterium -mediated transformation process (Salomon and Puchta, 1998).

IV.F. Southern Blot Analysis to Examine In sert Stability in Multiple Generations of

MON 87708

In order to demonstrate th e stability of the T-DNA I in sert present in MON 87708 through multiple generations, Southern blot analysis was performed using DNA obtained from five breeding generations of MON 87708. For referenc e, the breeding history of MON 87708 is presented in Figure IV-9. The sp ecific generations test ed are indicated in the legend of Figure IV-10. The R 3 

### generation was used for the molecular characterization analyses shown in Figures IV -2 through IV-8. To analyze stability, four additional generations were evaluated by Sout hern blot analysis and compared to the fully characterized R 3 

### generation. Genomic DNA, isolated from each of the selected generations of MON 87708 and the conventional control, was digested with the restriction enzyme combination Bsp1286 I/ Pvu II (Figure IV-1) and hybridized with probe 9 (Figure III-1). Probe 9 will dete ct both border fragments generated by the Bsp1286 I/ Pvu II digestion. Any instability associat ed with the T-DNA I insert would be

detected as novel bands within the fingerprint on the Southern blot. The Southern blot has the same positive hybridizat ion controls as described in Section IV.A. The results are shown in Figure IV-10.Monsanto Company 10-SY-210U 66 of 721 

### IV.F.1. Probe 9

Conventional control DNA dige sted with the restric tion enzyme combination Bsp1286 I/ Pvu II produced no hybridization signals (F igure IV-10, lane 1) as expected for the negative control. PV-GMHT4355, di gested with the restriction enzyme

combination Aat II/ Nde I and mixed with conventional control DNA predigested with the restriction enzyme combination Bsp1286 I/ Pvu II (Figure IV-10, lanes 8 and 9), produced the expected size band at ~7.4 kb (refer to Fi gure III-1 and Table IV-1). Additionally, there were two very faint hybridization bands in the ~1 genome equivalent plasmid vector PV-GMHT4355 spike at ~4.3 kb and ~6.5 kb observed in a longer exposure of the Southern blot (data not shown). These bands were likely due to an artifact that occurred during the electrophoresis. Since these faint bands appeared only in the plasmid vector spike and the expected ~7.4 kb band was observe d, they do not have any negative impact

on the conclusions from this Southern blot an alysis. These results indicate that the probe is hybridizing to its target sequence.

Digestion of MON 87708 genomic DNA from multiple generati ons with the restriction enzyme combination Bsp1286 I/ Pvu II and hybridized with probe 9 (Figure III-1) produced two bands at ~1.5 kb and ~2.6 kb (Figure IV-10, lanes 2-6). The ~1.5 kb band is the expected size for the border segment containing the 3 ′ end of T-DNA I along with the adjacent genomic DNA flanking the 3 ′ end of T-DNA I (Figure IV-1). The ~2.6 kb band is the expected size for the border segment containing the 5 ′ end of T-DNA I along with the adjacent genomic DNA flanking the 5 ′ end of T-DNA I (Figure IV-1). The fingerprint of the Southern blot signals from multiple generations, R 2 

### , R 4 

### , R 5 

### , and R 6 

### (Figure IV-10, lanes 2, 4, 5, and 6), of MON 87708 is consistent with the fully characterized generation R 3 

### (Figure IV-3, lanes 2 and 6; Figure IV-10, lane 3). No unexpected bands were detected, indicat ing that MON 87708 contains one copy of T-DNA I that is stably maintain ed across multiple generations.Monsanto Company 10-SY-210U 67 of 721 

### Figure IV-9. Breeding History of MON 87708

The R 3 generation was used for the molecular analy ses reported in Figures IV-2 through IV-8 and is referred to as MON 87708 in all Southern blot figures. The R 5 generation was used for development of all commercial produc ts. MON 87708 from generations R 2 , R 3 , R 4 , R 5 , and R 6 (bolded in the breeding tree) were used for analyzing the stability of T-DNA I in MON 87708 across generations (Figure IV-10).Monsanto Company 10-SY-210U 68 of 721 

### Figure IV-10. Southern Blot Analysis to Examine Insert Stab ility in Multiple

Generations of MON 87708: Probe 9 The blot was hybridized with a 32 P labeled T-DNA I probe that spans the coding region of the T-DNA I (Probe 9, Figure III-1). Each lane cont ains approximately 10 μg of digested genomic DNA isolated from leaf tissue. Lane designations are as follows:

Lane Description 1. Conventional control ( Bsp1286 I/

Pvu II)

2. R 2 generation of MON 87708 ( Bsp1286 I/

Pvu II)

3. R 3 generation of MON 87708 ( Bsp1286 I/

Pvu II)

4. R 4 generation of MON 87708 ( Bsp1286 I/

Pvu II)

5. R 5 generation of MON 87708 ( Bsp1286 I/

Pvu II)

6. R 6 generation of MON 87708 ( Bsp1286 I/

Pvu II)

7. Blank 8. Conventional control ( Bsp1286 I and Pvu II) spiked with PV-GMHT4355 ( Aat II/ Nde I) (~1 genome equivalent) 9. Conventional control ( Bsp1286 I and Pvu II) spiked with PV-GMHT4355 ( Aat II/ Nde I) (~0.1 genome equivalent)

Arrows denote sizes of DNA, in kilobase pairs, obtained from molecular weight markers on ethidium bromide stained gel. 1 2 3 4 5 6 7 8 9

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

0.5Monsanto Company 10-SY-210U 69 of 721 

### IV.G. Inheritance of the Genetic Insert in MON 87708

During development of MON 87708, segregation data were generated to assess the heritability and st ability of the T-DNA I pr esent in MON 87708. Chi-square analysis was performed over several generations to conf irm the segregation and stability of T-DNA I in MON 87708. The Chi-square analysis is based on testing the observed segregation ratio to the expected segregation ra tio according to Mendelian principles.

The MON 87708 breeding path, from which se gregation data were generated, is described in Figure IV-1 1. The transformed R 0 

### plant was self-pol linated to produce R 1 

### seed. An individual plant (#2, designate d as MON 87708), that was homozygous for a single copy of the dmo expression cassette, was identified from the R 1 

### segregating population via Invader  

### and Southern blot analysis. Invader is a non-PCR based assay that can be used to accurately quantify tran sgene copy number in plant genomes (Gupta et al., 2008).

The selected R 1 

### MON 87708 plant was self-pollinated to give rise to a population of R 2 

### plants that were repeatedly self-pollinated through the R 4 

### generation. At each generation, the fixed homozygous plants were tested fo r the expected segreg ation pattern of 1:0 (positive:negative) for the dmo expression cassette using th e Invader analysis, Southern blot analysis, and/or PCR.

At the R 4 

### generation, homozygous MON 87708 plants were bred via traditional breeding with a soybean variety th at did not contain the dmo expression cassette to produce F 1 

### hemizygous seed. The resulting F 1 

### plants were then self-pollinated to produce F 2 

### seed.

The F 2 

### plants were tested for the presence of the dmo expression cassette by Invader analysis, and hemizygous F 2 

### plants were selected and self-pollinated to produce F 3 

### seed. This process was repeated through the F 4 

### generation. The heritabili ty and stability of the

dmo expression cassette in M ON 87708 was assessed in the F 2 

### , F 3 

### , and F 4 

### generations. A total of 2413 out of 3223 plants were positive for the presence of the dmo expression

cassette in the F 2 

### generation; however, the zygosity of 200 of thos e 2413 plants could not be determined from the assay. Exclusion of these dmo -positive plants from the analysis likely would have skewed the distribution of homozygous positive: hemizygous positive:homozygous negative plants. Therefore, the segregation assessment

in the F 2 

### generation was based on the presence or absence of the dmo expression cassette which was expected to segregate at a 3:1 ( positive:negative) ratio according to Mendelian inheritance principles. Subsequently, assessment of segregation in the F 3 

### and F 4 

### generations was based on zygosity, and the dmo expression cassette was predicted to segregate at a 1:2:1 (homozygous positive:hemizygous positive :homozygous negative) ratio according to Mendelian inheritance principles.

A Chi-square ( χ 2 

### ) analysis was used to compare the observed segregation ratios to the expected ratios according to Mendeli an inheritance principles. The χ 2 

### was calculated as:

χ

2 

### = ∑ [( | o - e | )2 / e]Monsanto Company 10-SY-210U 70 of 721 

### where o = observed frequency of the phenot ype and e = expected frequency of the phenotype. The level of statistical si gnificance was predetermined to be 5%.

The results of the χ 2 

### analysis of the segregating pr ogeny of MON 87708 are presented in

Table IV-3. The χ 2 

### value for the F 2 

### , F 3 

### , and F 4 

### generations indicated no significant difference between the observed and expected segregation ratios. These results support

the conclusion that the dmo expression cassette in MON 87 708 resides at a single locus within the soybean genome and is inherited according to ex pected Mendelian inheritance principles. These results are also consistent with the molecular characterization data that

indicate MON 87708 contains a single, intact copy of the dmo expression cassette that was inserted into the soybean genome at a single locus.Monsanto Company 10-SY-210U 71 of 721Transformed and regenerated R0plantR1R2(homozygous positive)R3(homozygous positive)R4(homozygous positive) F1(hemizygous positive)Breeding path continued F2(expected segregation of 3:1)positive : negativeF3(expected segregation of 1:2:1)homozygous positive : hemizygous positive : homozygous negativeF4(expected segregation of 1:2:1)homozygous positive : hemizygous positive : homozygous negativeSelf pollinatedFigure IV-11. Breeding Path for Generating Segregation Data for MON 87708Chi-square analysis conducted on segregation data from the F2, F3, and F4generations.Note: Hemizygous positive plants in the F1, F2, F3, and F4generations were selected and self-pollinated to produce seed of the subsequent generation.Plant #2 (MON 87708) selected and self pollinatedcross with variety that did notcontain thedmoexpressioncassetteMonsanto Company 10-SY-210U 72 of 721TableIV-3. Segregation of thedmoExpression Cassette Duringthe Development of MON 87708Generation1TotalPlantsTested2Observed# PlantsPositiveObserved# PlantsNegative3:1 Segregation3Expected# PlantsPositiveExpected# PlantsNegativeχ2ProbabilityF23223 2413 810 2417.25 805.75 0.03 0.863Generation1TotalPlantsTested2Observed# PlantsHomozygousPositiveObserved# PlantsHemizygousPositiveObserved# PlantsHomozygousNegative1:2:1 SegregationExpected# PlantsHomozygousPositiveExpected# PlantsHemizygousPositiveExpected# PlantsHomozygousNegativeχ2ProbabilityF3118 29 52 37 29.5 59 29.5 2.7 0.2534F4343 83 171 89 85.75 171.5 85.75 0.2 0.89911F2, F3, and F4progeny were from self-pollinated F1,F2, and F3plants hemizygous positive for thedmoexpression cassette, respectively.2Plants were tested for the presence of thedmoexpression cassetteby Invader analysis.3Assessment of segregation in the F2generation was based on the presence or absence of thedmoexpression cassette due to an unacceptablenumber ofdmo-positive plants for which zygosity could not be determined from the assay.Monsanto Company 10-SY-210U 73 of 721 

### IV.H. Genetic Modification Characterization Conclusion

Molecular characterization of MON 87708 by Southe rn blot analyses demonstrated that a single copy of the T-DNA I sequences from the plasmid vector PV-GMHT4355 was integrated into the soybean genome at a single locus. There were no additional genetic elements from the T-DNA II or backbone sequences of the plasmid vector PV-GMHT4355 detected, linked or unlinke d to the intact T-DNA I present in

MON 87708.

The PCR and DNA sequence analyses performed on MON 87708, which confirmed the organization of the elements with in T-DNA I, demonstrated the 5 ′ and 3 ′ insert-to-plant junctions and determined the complete DNA sequence of T-DNA I and adjacent DNA sequence flanking the insert in MON 87708. Analysis of the T-DNA I insertion site

indicates that there was an 899 bp deletion of genomic DNA at the insert-to-plant DNA junction. Additionally, a 128 bp insertion was identified in the 5 ′ adjacent flanking sequence of MON 87708 and a 35 bp in sertion was identified in the 3 ′ adjacent flanking sequence of MON 87708.

Generational stability analysis by Southern blot dem onstrated that MON 87708 has been maintained through five breeding generation s, thereby confirmi ng the stability of T-DNA I in MON 87708. Results from segreg ation analyses show heritability and stability of the insert occurred as e xpected across multiple generations, which corroborates the molecular insert stability anal ysis and establishes th e genetic behavior of the T-DNA I in MON 87708 at a single chromosomal locus.Monsanto Company 10-SY-210U 74 of 721 

### V. CHARACTERIZATI ON AND SAFETY ASSESSMENT OF THE

MON 87708 DMO

Characterization of the intr oduced protein in a biotechno logy-derived crop product is important to establishing its food, feed, and environmental safety. As described in Section IV, MON 87708 contains a dmo expression cassette that upon translation results in two forms of the DMO protein; referred to as DMO and DMO+27 (Section V.A). The active form of these proteins , necessary to conf er dicamba tolerance, is a trimer comprised of three DMO monomers (Chakr aborty et al., 2005) . In MON 87708, the trimer can be comprised of DMO, DMO+27, or a combination of both. Therefore, this document will refer to both forms of the pr otein and all forms of the trimer as

MON 87708 DMO.

This section summarizes: 1) the functional ity of DMO; 2) the characterization of MON 87708 DMO; 3) the levels of MON 87708 DMO in plant tissues; 4) assessment of the potential allergenicity of MON 87708 DM O and 5) the food, feed, and environmental safety assessment of MON 87708 DMO. The data support a conclusi on that MON 87708 is safe for the environment and human or animal consumption based on several lines of evidence, all of which are summarized below.

V.A. Function of DMO and MON 87708 DMO

DMO was initially purified from Stenotrophomonas maltophilia (S. maltophilia) strain DI-6, isolated from soil at a dicamba manufacturing plant (Kru eger et al., 1989). DMO is an enzyme that catalyzes the deme thylation of dicamba to the non-herbicidal compound DCSA and formaldehyde (Chakrabor ty et al., 2005). DMO is a Rieske-type non-heme iron oxygenase, that is part of a three component system comprised of a reductase, a ferredoxin, and a terminal oxygenase, in this case the DMO. These three enzymes work together in a redox system si milar to many other oxyge nases to transport
electrons from nicotinamide adenine dinuc leotide (NADH) to oxygen and catalyze the demethylation (Behrens et al., 2007) as presented in Figure V-1.

Figure V-1. Three Components of the DMO Oxygenase System Reductase (oxidized)

Flavin

Reductase (reduced) Ferredoxin (reduced) [2Fe-2S]

Ferredoxin (oxidized) NADH

+
H +

NAD+ Oxygenase (oxidized) [2Fe-2S]

Fe+2

Oxygenase (reduced) O 2 O OH

OCH 3

Cl Cl O OH

OH

Cl Cl 3,6 DCSA

Dicamba + O

H H Dicamba Mono-Oxygenase

Dicamba Mono-Oxygenase FormaldehydeMonsanto Company 10-SY-210U 75 of 721 

### The crystal structure of a DMO has been solv ed (D'Ordine et al., 2009; Dumitru et al., 2009) and shows that the DMO monomers cont ain a Rieske [2Fe-2S] cluster domain and a non-heme iron center domain typical of a ll Rieske-type mono-oxygenases (Ferraro et al., 2005). To catalyze the demethylation of dicamba, electrons transferred from NADH are shuttled through an endogenous reductase and ferredoxin to the terminal DMO. The electrons are received by the Rieske [2Fe -2S] cluster on one DMO monomer and

transferred to the non-heme iron center at the catalytic site of an adjacent monomer (D'Ordine et al., 2009; Dumitru et al., 2009) , where it reductively activates oxygen to catalyze the final demethylation of dica mba. As a result of the reaction, 3.6 dichlorosalicylic acid (DCSA) and formalde hyde are formed. DCSA is a known soybean, soil, and livestock metabolite whose safe ty has been evaluated by the EPA. Formaldehyde is found naturally in many plan ts at levels up to several hundred ppm (Adrian-Romero et al, 1999). An assessment of the safety and potential effects of the DMO reaction products is pr ovided in Section IX.B.3.6.

V.A.1. Formation of MON 87708 DMO

DMO is targeted to chloroplasts for co-lo calization with the e ndogenous reductase and ferredoxin enzymes that supply electrons for the DMO demethylation reaction as described by Behrens et al. (2007). The MON 87708 DMO precursor pr otein contains 84 additional amino acids corresponding to a 57 amino acid Chloroplast Transit Peptide (CTP) from pea and 24 amino acids from the N-terminal coding region of the pea Rubisco small (RbcS) s ubunit to target the protein to the chloroplast (Comai et al., 1988),and three amino acids from an intervening sequence used for cloning purposes (Table IIII). It was anticipated that during translocation of the DMO precursor into chloroplasts

the introduced 84 amino acids would be fu lly cleaved resulting in the predicted Nterminus of the DMO protein. However, analys is of mature seed extracts by western blot demonstrated the presence of two immunor eactive bands (Figure C-2, Appendix C). Analysis of these two bands determined that the lower molecular weight band corresponded to the full-length DMO protei n (referred to as DMO). DMO has an apparent molecular weight of 39.8 kDa and is a single polypeptide chain of 339 amino acids. The higher molecular weight band of approximately 42 kDa corresponded to the full-length DMO protein plus 27 amino acids originating from the pea Rubisco small subunit and intervening sequence on its N-te rminus (referred to as DMO+27; 367 amino acid polypeptide). Both forms of the DMO pr otein were characterized (Appendix C).

As described previously the active form of DM O is a trimer (Chakrab orty et al., 2005; Dumitru et al., 2009). For MON 87708 DMO to be functionally active and confer dicamba tolerance to MON 87708, a trimeric stru cture is required. This trimer contains

either form of DMO or a mixture of both, and its activity was confirmed during characterization (Section V.B. and Appendix C).

V.A.2. Specificity of MON 87708 DMO

DMO has high specificity for its substrate dicam ba (D'Ordine et al., 2009; Dumitru et al., 2009). The specificity of DMO fo r dicamba is likely due to th e specific interactions that occur in the catalytic site between the dicam ba substrate and DMO. Dicamba interacts

with amino acids in the ac tive site of DMO through both the carboxylate moiety and theMonsanto Company 10-SY-210U 76 of 721 

### chlorine atoms of dicamba, which are primarily involved in orienting the substrate in the catalytic pocket. These chlorine atoms are required for catalysis (D'Ordine et al., 2009; Dumitru et al., 2009).

The possibility that MON 87708 DMO can meta bolize plant endogenous substrates was

tested in in vitro experiments using an Escherichia coli ( E. coli )-produced DMO. The E.

coli -produced DMO is similar in sequence a nd function to MON 87708 DMO, therefore it is appropriate to extend specificity data generated with the E. coli -produced DMO to MON 87708 DMO (Appendix C.2.). A set of pot ential substrates was selected based on structural similarity to dicamba and abundan ce in soybean (Janas et al., 2000), including o-anisic acid (2-methoxybenzoic acid), vani llic acid (4-hydroxy-3-methoxybenzoic acid), syringic acid (3,5-dimethoxy-4-hydroxybenzoi c acid), ferulic acid [3-(4-hydroxy-3- methoxy-phenyl)prop-2-enoic acid] a nd sinapic acid [3-(4-hydroxy-3,5- dimethoxyphenyl)prop-2-enoic acid] (Figure V-2). The disappearance of potential

substrates and the formati on of potential oxidation products were monitored using LCUV and LC-MS (Appendix C). None of the te sted substrates was metabolized by the

E.coli -produced DMO in vitro. Therefore, DMO, though structurally similar to other Rieske mono-oxygenases, is specific for dicamba (see Section V.E.3 for additional details).

Figure V-2. Dicamba and Set of Potent ial Endogenous Substrates Tested in in vitro Experiments with DMO The arrow indicates methyl group removed by DMO.



### The possibility that MON 87708 DMO can meta bolize exogenous substrates was tested

in in vivo experiments. In addition to dicamba, a total of 19 herbicides representing eight families with distinct modes-of-action, some of which are approved for use in soybean, were tested with MON 87708 and the near isogenic conventional soybean control A3525 (Table V-1). Soybean naturally has varying levels of tolerance to different herbicides. For example, soybean is tolerant to in-cr op postemergence applications of alachlor, but not atrazine. Each herbicide was applied at two spray rates, representative of potential

commercial rates needed to control broadleaf weeds, at the V2-V3 soybean growth stage and then scored a visual rating based on the amount of injury observed on the plants. Across nearly all of the herb icides tested, MON 87708 and th e conventional control were similar in their level of tolerance, indicating that these herbicides do not serve as a substrate for MON 87708 DMO. However, MON 87708 did show slightly more tolerance compared to the conventional control when treated with the three phenoxycarboxylic acid (phenoxy) synt hetic auxin herbicides: 2,4-D (2,4-dichlorophenoxy acetic acid), MCPA (2 -methyl-4-chlorophenoxy acetic acid) andMonsanto Company 10-SY-210U 77 of 721 

### 2,4-DB (2,4-dichlorophenoxy butan oic acid). Chloramben and TBA are no longer

available in the U.S and were excluded from th is testing. See Appendix C, Section C.2.

As 2,4-D is the most structurally sim ilar to dicamba of the three phenoxy auxin herbicides (2,4-D, 2,4-DB, and MCPA), it was selected as a representative for further in

vitro experimentation. Subsequent experiment s were performed to evaluate whether 2,4-D can be metabolized by E. coli -produced DMO. The presumptive product of the oxidative reaction between 2,4-D and DMO is 2,4-dichlorophenol (2,4-DCP), formed from the dealkylation of 2,4-D. The potenti al disappearance of 2,4-D and formation of 2,4-DCP were monitored using LC-UV and LC -MS (Appendix C, Section C.2). Neither the formation of 2,4-DCP nor any measurable decrease in 2,4-D were detected in the in

vitro experiment. These results indicat e that 2,4-D cannot be metabolized by E. coli

produced DMO, and demonstrate that DMO is specific to dicamba. The tolerance of

MON 87708 to other herbicides at anticipated commercial application rates is no different than the conventional soybean, ex cept for the phenoxy auxin herbicides where MON 87708 showed limited, but not commercially acceptable, tolerance when treated.

Table V-1. Herbicides Applied to MON 87708 and Conventional Control

Herbicide Active Ingredient Herbicide Chemical Family (MOA) 1



### Dicamba Benzoic acid (Synthetic Auxin) 2,4-D Phenoxycarboxylic acid (Synthetic Auxin) 2,4-DB Phenoxycarboxylic acid (Synthetic Auxin) MCPA Phenoxycarboxylic acid (Synthetic Auxin) Triclopyr Pyridinecarboxylic acid (Synthetic Auxin) Clopyralid Pyridinecarboxylic acid (Synthetic Auxin) Picloram Pyridinecarboxylic acid (Synthetic Auxin) Alachlor Chloroacetamide (Inhibition of VLCFAs)

Acetochlor Chloroacetamide (Inhibition of VLCFAs) Atrazine Triazine (Inhibition of Photosynthesis at Photosytem II) Linuron Ureas (Inhibition of Phot osynthesis at Photosytem II) Oxyfluorofen Diphenyl ether(Inhibition of PPO) Lactofen Diphenylether (Inhibition of PPO) Chlorimuron Sulfonylurea (Inhibition of ALS) Chlorsulfuron Sulfonylurea (Inhibition of ALS) Halosulfuron Sulfonylurea (Inhibition of ALS) Imazapyr Imidazolinone (Inhibition of ALS) Trifluralin Dinitroaniline (Microtubule Assembly Inhibition) Paraquat Bipyridilium (Photosys tem I electron diversion) Glyphosate Glycine (Inhibition of EPSP synthase)

1 HRAC (2009)



### V.B. Characterization of MON 87708 DMO

The safety assessment of crops derived th rough biotechnology incl udes characterization of the functional and physicoc hemical properties, and confirmation of the safety of the introduced protein. As stated previously, bot h forms of the protein and all forms of theMonsanto Company 10-SY-210U 78 of 721 

### trimer are referred to as the MON 87708 DMO. MON 87708 DMO was purified in sufficient quantities directly from the seed of MON 87708 and used in subsequent safety assessment studies. Typically protein safety studies are conducted on proteins produced in heterologous expression systems, such as E. coli . Since the MON 87708 DMO used in the subsequent safety studies was purified directly from MON 87708 DMO, equivalence evaluations between plant- produced and bacterial-produced MON 87708 DMO was not necessary. The physicochemical characte ristics and functiona l activity of the MON 87708 DMO were determined by a panel of analytical techniques, including: 1) western blot analysis to establish identity and immunoreactivity of MON 87708 DMO using an anti-DMO antibody, 2) N-terminal se quence analysis, 3) matrix-assisted laser desorption/ionization time-of-flight mass sp ectrometry (MALDI-TOF MS) to generate a tryptic peptide map of the MON 87708 DMO, 4) sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to establis h the apparent molecular weight of MON 87708 DMO, 5) glycosyl ation status of MON 87708 DMO, and 6) MON 87708 DMO activity analysis to demonstrate functiona l activity. The details of the materials, methods, and results are described in A ppendix C, while the conclusions of the

MON 87708 DMO characterization are summarized below.

The identities of both forms of the DMO pr otein produced in MON 87708 that constitute

MON 87708 DMO were confirme d by western blot analysis by probing with an antiDMO antibody, N-terminal sequencing, and MA LDI-TOF MS analysis of peptides produced after trypsin digestion. The anti body specifically detected DMO and DMO+27 on a western blot. The N-terminal sequence of the first 15 amino acid residues of both DMO and DMO+27 was identical to the predicted amino acid sequence, with the exception of the N-terminal methionine resi due. MALDI-TOF MS analyses of DMO and DMO+27 yielded peptide masses consistent with their expected sequence. The apparent molecular weights of DMO and DMO+27 were 39.8 and 42.0 kDa, respectively and neither were glycosylated. The MON 87708 DMO activity was determined by measuring the production of DCSA using dicamba as the s ubstrate, resulting in a specific activity of 62.21 nmoles DCSA/min/mg of MON 87708 DMO. Taken together, these data provide a

detailed characterization of the MON 87708 DM O isolated from the seed of MON 87708.

V.C. Expression Levels of MON 87708 DMO

The levels of MON 87708 DMO in various tissu es of MON 87708 that are relevant to the risk assessment were determined by a valid ated ELISA. Tissues of MON 87708 and the near isogenic conventional soybean cont rol A3525 were collected during the 2008 growing season from five field sites in th e U.S.: Jefferson County, Iowa; Stark County, Illinois; Clinton County, Illinoi s; Parke County, Indiana; and Berks County, Pennsylvania. These field sites were representative of soybean producing regions suitable for commercial production. At each site, three replicated plots containing MON 87708, as

well as the conventional contro l, were planted using a randomized complete block field design. Over-season leaf (OSL 1-4), root, fo rage, and seed tissues were collected from each replicated plot at all field sites (ex cept for the conventional control from Berks County, Pennsylvania where only tw o replicates were collected). A description of tissues collected is provided in Table V-2.Monsanto Company 10-SY-210U 79 of 721 

### Table V-2. Tissues Collected and Analyzed for MON 87708 DMO

Tissue Soybean Development Stage 1 

### Days After Planting

OSL-1 V3-V4 21-30

OSL-2 V5-V8 31-42

OSL-3 R2-V12 43-58

OSL-4 R5-V16 55-78

Root R6 70-91 Forage R6 70-91

Seed R8 109-147

1 

### Soybean plant growth stages described in Soybean Growth and Development (ISU, 2004).

The levels of MON 87708 DMO we re determined in all seven tissue types as described in Table V-3. The ELISA assay detected all forms of MON 87708 DMO and therefore the levels represent the total of MON 87708 DM O. The results obtained from the ELISA analysis are summarized in Table V-3 and th e details of the mate rials and methods are described in Appendix D. In summary, expr ession analysis of the samples from the 2008

U.S. field trial showed that MON 87708 DMO wa s detected in all ti ssue types across all five sites ranging from 3.9 – 180 μg/g dry we ight (dwt). The mean levels of the

MON 87708 DMO across the five sites were hi ghest in leaf (ranging from OSL-1 at 17 μg/g dwt, to OSL-4 at 69 μg/g dwt), fo llowed by forage (53 μg/g dwt), seed (47 μg/g dwt), and root (6.1 μg/g dwt). As expected for the conventional control, the

ELISA values for MON 87708 DMO were less than the limit of quantitation (LOQ) of the assay in all tissue types.Monsanto Company 10-SY-210U 80 of 721 

### Table V-3. Summary of the Levels of MON 87708 DMO in Leaf, Root, Forage, and

Seed from MON 87708 Grown in 2008 U.S. Field Trials

Tissue Type 

### MON 87708

DMO 1
Mean (SD) 2
( μ g/g fwt) 3 Range 4
( μ g/g fwt) 

### MON 87708

DMO Mean (SD) ( μ g/g dwt) 5 Range ( μ g/g dwt) LOQ/LOD ( μ g/g fwt) 6,7

OSL-1 

### 3.1 (1.9) 0.87 – 6.8 17 (7.7) 6.2 – 29 0.63/0.20

OSL-2 

### 5.2 (2.6) 1.4 – 9.8 31 (13) 12 – 54 0.63/0.20

OSL-3 

### 6.0 (2.2) 3.5 – 11 44 (14) 25 – 71 0.63/0.20

OSL-4 

### 16 (12) 4.6 – 43 69 (46) 23 – 180 0.63/0.20

Root 

### 1.9 (0.73) 1.2 – 3.6 6.1 (2.1) 3.9 – 11 0.031/0.015

Forage 

### 12 (2.5) 7.0 – 17 53 (18) 25 – 84 0.63/0.10

Seed 

### 43 (7.7) 31 – 55 47 (8.7) 34 – 59 1.3/0.21

1 Represents total for 

### MON 87708 DMO

. 2 The mean and standard deviation (SD) were calc ulated (n=15). The “n” values for the calculated mean and standard deviations represent the num ber of samples figured into the calculation.

3 Protein levels are expressed as microgram ( μ g) of protein per gram (g) of tissue on a fresh weight (fwt) basis.

4 Minimum and maximum values were determined for each tissue type.

5 Protein levels are expressed as μ g/g dwt. The dry weight values were calculated by dividing the

μ g/g fwt by the dry weight conversion fact ors obtained from moisture analysis data.

6 The limit of quantitation (LOQ) was calculate d based on the lowest DMO standard concentration. The “ng/ml” value was converted to “ μ g/g fwt” using the respective dilution

factor and tissue-to-buffer ratio.

7 The limit of detection (LOD) was calculated as the mean value plus three SD using the data generated with conventional control sample extr acts for each tissue type. The LOD value in “ng/ml” was converted to “ μ g/g fwt” using the respective dilution factor and tissue-to-buffer

ratio.Monsanto Company 10-SY-210U 81 of 721 

### V.D. Assessment of Potential Allergenicity of MON 87708 DMO

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

biochemical characteristics of the introduced protein to biochemical characteristics of known allergens (Codex Alimentarius, 2003). A pr otein is not likely to be associated with allergenicity if: 1) the protein is from a non-allergenic s ource, 2) the protein represents a very small portion of the total plant protein, 3) the protein does not share structural similarities to known allergens ba sed on the amino acid sequence, and 4) the protein is rapidly digested in mammalian ga strointestinal system s. MON 87708 DMO, as defined above, refers to all forms of the protein and the resulting trimer, has been assessed for its potential allergenicity accord ing to these safety assessment guidelines.

1) MON 87708 DMO originates from S. maltophilia , an organism that has not been reported to be a source of known allergens.

2) MON 87708 DMO represents no more th an 0.01% of the total protein in the

seed of MON 87708.

3) Bioinformatics analyses demonstrat ed that the DMO+27 form of MON 87708 DMO, that also contains the DMO seque nce, does not share amino acid sequence

similarities with known alle rgens and, therefore, is highly unlikely for DMO or DMO+27 to contain immunologically cros s-reactive a llergenic epitopes.

4) In vitro digestive fate experiments conducted with the MON 87708 DMO demonstrate that the proteins are rapidly digested in si mulated gastric fluid (SGF) and in simulated intestinal fluid (SIF).

Taken together, these data support the c onclusion that MON 87708 DMO does not pose a significant allergenic risk to humans or animals.

V.E. Safety Assessment Summary of MON 87708 DMO

Numerous factors have been considered in the safety assessment of MON 87708 DMO. A comprehensive food, feed, and environm ental safety assessment of the MON 87708

DMO was conducted. The results are summar ized below along with the conclusions

reached from the assessment.

V.E.1. The Donor Organism is Safe

The dmo gene is derived from the bacterium Stenotrophomonas maltophilia (Palleroni and Bradbury, 1993). S. maltophilia is an aerobic, ubiquit ous environmental gram negative bacterium commonly present in a quatic environments, soil, and plants. S. maltophilia is ubiquitously associated with plan ts and has been isolated from the rhizosphere of wheat, maize, grasses, be et, cucumber, chicory, potato, strawberry, sugarcane, and rapeseed (Berg et al., 1996; Berg et al., 1999; Berg et al., 2002; Denton et al., 1998; Echemendia, 2007; Juhnke and des Jardin, 1989; Juhnke et al., 1987; Lambert et al., 1987). S. maltophilia was isolated from cotton seed, bean pods, and coffee (Nunes and de Melo, 2006; Swings et al., 1983), thus, S. maltophilia can be found in a variety ofMonsanto Company 10-SY-210U 82 of 721 

### foods and feeds. It is also widespread in the home envi ronment and can be found around dishwashers, sponges, toothbrushes, flowers, pl ants, fruits, vegetables , frozen fish, milk, and poultry (Ryan et al., 2009). Strains of S. maltophilia have been found in the transient flora of hospitalized patients as a commensal organism (E chemendia, 2007). Infections caused by S. maltophilia are extremely uncommon (Cunha, 2006) and S. maltophilia can be found in healthy individuals without causing any harm to human health (Denton et al., 1998). Similar to the indigenous bacter ia of the gastrointestinal tract, S. maltophilia can be an opportunistic pathogen (Berg, 1996). As such, S. maltophilia is of low virulence in immuno-compromised patients wh ere a series of factors mu st occur for colonization by S. maltophilia on humans (Ryan et al., 2009). The ubiquitous presence of S. maltophilia in the environment, the presence in healthy indivi duals, and the incidental presence on foods without any adverse safety reports establ ishes the safety of the donor organism.

V.E.2. MON 87708 DMO Belongs to a Common Class of Mono-Oxygenases

MON 87708 DMO is classified as an oxyge nase. Oxygenases are enzymes that incorporate one or two oxygen atoms into substr ates and are widely distributed in many universal metabolic pathways (Harayama et al ., 1992). Within this large enzymatic class are mono-oxygenases that incorporate a singl e oxygen atom as a hydroxyl group with the concomitant production of water and oxidatio n of NAD(P)H (Harayama et al., 1992). Non-heme iron oxygenases, where iron is involve d in the catalytic si te, are an important class of oxygenases. Within this class are Rieske oxygenases, which contain a Rieske iron-sulfur [2Fe-2S] cluster. All Rieske non-heme iron oxygenases contain two catalytic domains, a non-heme iron domain (nh-Fe) th at is a site of oxygen activation, and a Rieske [2Fe-2S] domain (Ferraro et al., 2005). MON 87708 DMO bel ongs to this class of oxygenases which are found in diverse phyla ra nging from bacteria to plants (Ferraro et al., 2005; Schmidt and Shaw, 2001).

As discussed previously, the crystal structure of a DMO has been solved (D'Ordine et al., 2009; Dumitru et al., 2009). The crystallography results demonstrated that, similar to all Rieske non-heme iron oxygenases, DMO contai ns two catalytically important and highly conserved domains; a mononuclear non-heme iron domain (nh-Fe) that is a site of oxygen activation, and a Rieske [2 Fe-2S] domain (D'Ordine et al., 2009; Dumitru et al., 2009; Ferraro et al., 2005). The amino acids binding the non-heme iron and those that constitute the Rieske [2Fe-2S] domain in th e DMO protein are also highly conserved in these plant proteins, as is th eir spatial orientation (D'Ordin e et al., 2009; Ferraro et al., 2005). Rieske domains are ubiquitous in nume rous bacterial and plant proteins like the iron-sulfur protein of the cytochrome bc1 complex, chloroplast cytochrome b6/f complex, and choline mono-oxygenases (Breyton, 2000; Darr ouzet et al., 2004; Gr ay et al., 2004; Hibino et al., 2002; Rathinasabapathi et al ., 1997; Russell et al., 1998). The presence of two conserved domains, a Rieske [2Fe-2 S] domain and a mononuclear iron domain, suggests that all Rieske type non-heme iron oxygenases share the same reaction mechanism, by which the Rieske domain transfers electrons from the ferredoxin to the mononuclear iron to allow catalys is (Chakraborty et al., 2005; Dumitru et al., 2009; Ferraro et al., 2005). The structure and mech anistic homologies are further evidence of the evolutionary relatedness of all Rieske non-heme iron oxygenases to each other (Nam et al., 2001; Rosche et al., 1997; Werlen et al., 1996). Additionally, a FASTA alignmentMonsanto Company 10-SY-210U 83 of 721 

### search of publicly available databases us ing the DMO+27 sequence as a query yielded homologous sequences from many different spec ies, predominantly bacteria, with amino acid sequence identity ranging up to appr oximately 42%. Homo logous oxygenases are also present in plants, including such crops as rice ( Orysa sativa ), canola ( Brassica

napus ), and corn ( Zea mays ), with sequence identity up to 24%. The highest homology was observed to pheophorbide A oxyge nases from corn, canola and pea ( Pisum sativum ). Pheophorbide A oxygenase is also a Rieske-type oxygenase that plays a key role in the overall regulation of chlorophyll degradation in plants (Rodoni et al., 1997). The protein is constitutively present in all green tissues a nd, at slightly lower levels, in etiolated and non-photosynthetic tissues including seeds (Yang et al., 2004).

Therefore, MON 87708 DMO shares sequence identity and many catalytic and domain structural similarities with a wide variety of oxygenases present in bacteria and plants currently widely prevalent in the environm ent and consumed, establishing that animals and humans are extensively exposed to these types of enzymes.

V.E.3. DMO is a Dicamba-Specific Mono-Oxygenase

DMO converts dicamba to DCSA. This demethyl ation is very specif ic to dicamba, where both the carboxylate moiety and the chlorine atoms help position the substrate at the active site of the enzyme (D'Ordine et al ., 2009; Dumitru et al., 2009). Crystallography

studies of the substrate in the active site de monstrated that these chlorines function as steric “handles” that position the substrate in the proper orientation in the binding pocket (Dumitru et al., 2009). Potentia l substrates abundant in soybean (o-ani sic acid, vanillic acid, syringic acid, ferulic acid and sinapic acid) that are structurally similar to dicamba, were not metabolized by an E. coli -produced DMO in laboratory tests indicating that the DMO enzyme is specific for di camba (Section V.A.2). The E. coli -produced DMO is similar in sequence and function to MON 877 08 DMO, therefore it is appropriate to extend specificity data generated with the E. coli -produced DMO to MON 87708 DMO.

Given the limited amount of chlorinated metabo lites with structures similar to dicamba in plants and other eukaryotes (Wishart, 2010; Wishart et al., 2009) it is unlikely that MON 87708 DMO will catalyze the conversi on of other endogenous substrates. Therefore, the activity of the enzyme is specific for dicamba while it maintains many structural properties common to oxygenases th at are ubiquitous to all organisms.

V.E.4. MON 87708 DMO is Not a Known Allergen or Toxin

Bioinformatics analyses were performed to assess the allergenic potential, toxicity, or biological activity of MON 87708 DMO. The bioinformatics assessment was performed on DMO+27, which includes the amino acid sequence of DMO. The analysis

demonstrated that MON 87708 DMO does not share amino acid sequence similarities with known allergens, gliadins, glutenins, or protein toxins which could have adverse effects to human or animal health (Section V.D).Monsanto Company 10-SY-210U 84 of 721 

### V.E.5. MON 87708 DMO is Labile in in vitro Digestion Assays

MON 87708 DMO was readily dige stible in SGF and SIF. Rapid degradation of the

MON 87708 DMO in SGF and SIF makes it highly unlikely that the MON 87708 DMO

would be absorbed in the small intestine and have any adverse effects on human or

animal health.

V.E.6. MON 87708 DMO is Not a Toxin

An acute oral toxicology st udy was conducted with MON 87708 DMO

. 

### Results indicate that MON 87708 DMO did not cause any adverse effects in mice, with a No Observable Adverse Effect Level (NOAEL) of 140 mg/ kg body weight (BW), the highest dose level

tested.

Potential human health risks from consum ption of foods derived from MON 87708 were evaluated using a Margin of Exposure (MOE ) approach. A MOE was calculated between the acute mouse NOAEL (140 mg/kg BW) for the MON 87708 DMO and 95th percentile “eater-only” estimates of acute dietary expos ure determined using the Dietary Exposure Evaluation Model (DEEM-FCID version 2.03, E xponent Inc.). DEEM food consumption

data are obtained from the 1994-1996 and 1998 USDA Continuing Survey of Food Intakes by Individuals (CSFII). The MOEs for acute dietary intake of MON 87708 DMO were estimated to be 24,800 and 600 for th e general population and non-nursing infants, the sub-population with the hi ghest estimated exposure, resp ectively. These very large MOEs, in addition to the above mentione d protein safety data for MON 87708 DMO, support the conclusion that there is no meani ngful risk to human health from dietary exposure to MON 87708 DMO.

Potential health risks to animals from the presence of MON 87708 DMO in feed were evaluated by calculating an estimate of daily dietary intake (DDI). In the worst case scenario, the percentage of MON 87708 DMO consumed from MON 87708 as a percentage of the daily protein intake for a dairy cow is 0.0396% and for both the broiler and pig is less than 0.0121%. These very small levels of exposure of animals to MON 87708 DMO in their feed, in addition to the above mentioned safety data for MON 87708 DMO, support the conclusion that there is no meaningful risk to animal health when MON 87708 is pr esent in their diets.

Using the guidance provided by the FDA in its 1992 Policy Statement regarding the evaluation of New Plant Varieties, a conclusi on of “no concern” is reached for the donor organism and MON 87708 DMO. The food and feed products contai ning MON 87708 or derived from MON 87708 are as safe as soybe an currently on the market for human and animal consumption.

V.F. MON 87708 DMO Characterization and Safety Conclusion

MON 87708 DMO is an oxygenase that catalyze s the O-demethylation of the herbicide

dicamba. MON 87708 DMO was derived from S. maltophilia , which is an environmentally ubiquitous bacterium that does not pose a health risk to healthy

individuals. MON 87708 DMO is a Rieske-type mono-oxygen ase that has homologs inMonsanto Company 10-SY-210U 85 of 721 

### bacteria and plants that share many of the ty pical structural and f unctional characteristics of these types of oxygenases, while maintainin g specificity for its substrate. MON 87708 DMO was fully characterized confirming both the N-terminal and internal amino acid sequence and the lack of glycosylati on. MON 87708 DMO was isolated from

MON 87708 and was used for th e described safety studies; therefore an equivalence evaluation to the protein produced in a hetero logous expression system was not required. Expression studies using ELISA demonstrat ed that MON 87708 DMO was expressed in all tissues assayed at leve ls ranging from 3.9 – 180 μg/g dwt, representing a low percentage of the total protei n in soybean. Bioinformatics analysis determined that

MON 87708 DMO does not share amino acid sequence similarities with known allergens, gliadins, glutenins, or protein toxins. MON 87708 DMO was ra pidly digested in in vitro assays using simulated gastric and intestinal fluids and did not show any adverse effects when administered to mice via oral gavage at levels that resulted in large MOEs. Together with the safety data, these data s upport a conclusion that there is no meaningful

risk to human health from di etary exposure to MON 87708 DM O. Therefore, the food and feed products containing MON 87708 or de rived from MON 87708 are as safe as soybean currently on the market fo r human and animal consumption.Monsanto Company 10-SY-210U 86 of 721 

### VI. COMPOSITIONAL AS SESSMENT OF MON 87708

Safety assessments of biotech crops typically include comparisons of the composition of forage and whole grain of th e GM crop to that of conve ntional counterparts (Codex Alimentarius, 2003). Compositional assessments are performed using the principles and analytes outlined in the OECD consensus documents for soybean composition (OECD, 2001).

A recent review of compositional assessments conducted according to OECD guidelines that encompassed a total of seven GM crop va rieties, nine countries and eleven growing seasons concluded that incorporation of bi otechnology-derived agronomic traits has had little impact on natural variation in crop co mposition; most compositional variation is attributable to growing regi on, agronomic practices and gene tic background (Harrigan et al., 2010). Numerous scientif ic publications have furthe r documented the extensive variability in the concentrations of crop nutrients and anti-nutrients that reflect the influence of environmental and genetic factor s as well as extensiv e conventional breeding efforts to improve nutrition, agronomics and yield. (Reynolds et al., 2005). Compositional equivalence between biotech nology-derived and conventional crops provides an “equal or increased assurance of the safety of f oods derived from genetically modified plants” (OECD, 2001). The OECD consensus documents emphasize quantitative measurements of essential nutrien ts 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 safety and nutritional concerns. Levels of the components in seed and fo rage of the biotechnology-derived crop are compared to: 1) corresponding levels in a conventional comparator, the nonbiotechnology near isogenic line, grown conc urrently, under identical field conditions, and 2) natural ranges generated from an ev aluation of commercial reference varieties grown concurrently and from data pub lished in the scientific literature.

The latter comparison places any potential differences between the assessed crop and its comparator in the context of the well-document ed variation in the co ncentrations of crop

nutrients and anti-nutrients.

VI.A. Compositional Equivalence of MON 87708 Seed and Forage to Conventional Soybean

Seed and forage samples were collected from MON 87708 and the near isogenic conventional soybean control A3525 grown in a 2008 U.S. field production. Four

different commercial reference varieties were in cluded at each site of the field production to provide data on natural variability of each compositional component analyzed. The field production was conducted at five site s: Jefferson County, Io wa; Stark County, Illinois; Clinton County, Illinoi s; Parke County, Indiana; and Berks County, Pennsylvania. All soybean plants including MON 87708, the c onventional control, and the commercial

reference varieties were trea ted with maintenanc e pesticides as ne cessary throughout the growing season. In addition, MON 87708 plots were either tr eated at the V2-V3 growth stage with dicamba herbicide at the maximum in-crop label rate (0.5 lb acid equivalence (a.e.)/acre) or not treated with dicamba herbicide.Monsanto Company 10-SY-210U 87 of 721 

### Compositional analyses were conducted to assess whether levels of key nutrients and anti-nutrients in MON 87708 were equivalent to levels in the conventional control and to the composition of the commercial reference va rieties. A description of nutrients and anti-nutrients present in soybean is pr ovided in the OECD consensus document on compositional considerations for soybean (OECD, 2001). Nutrients assessed included proximates (ash, carbohydrates by calculation, moisture, protein, and fat), fiber, amino acids (18 components), fatty acids (FA, C8-C22), and vitamin E ( α -tochopherol) in seed, and proximates (ash, carbohydrates by calculation, moisture, prot ein, and fat) and fiber in forage. Anti-nutrients assessed in seed incl uded raffinose, stachyose, lectin, phytic acid, trypsin inhibitors, and is oflavones (daidzein, genist ein, and glycitein).

In all, 64 different components were measur ed (seven in forage and 57 in seed). Components that had more th an 50% of the observations below the assay limit of quantitation (LOQ) were excluded from statis tical analysis. Therefore, 50 components

for both dicamba-treated and untreated M ON 87708 were statistically assessed using a mixed-model analysis of variance method. Values for all assessed components were reported on a dry weight basis with the exception of moisture, which was reported as % fresh weight (fwt) and fatty acids, wh ich were reported as % of total FA.

For MON 87708, six statistical co mparisons to the conventional control were conducted. 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 a 5% level of significance. Data from the co mmercial 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 soybean varieties that have a history of safe consumption and that were grown concurrently with MON 87708

and the conventional control in the same trial.

For the combined-site analysis, statistically significant differences in nutrient and antinutrient components were further evaluated usi ng considerations rele vant to the safety and nutritional quality of MON 87708 when co mpared to the conven tional control A3525, the conventional counterpart w ith a history of safe cons umption: 1) the relative magnitude of the difference in the mean valu es of nutrient and an ti-nutrient components

of MON 87708 and the conventional contro l, 2) whether the MON 87708 component mean value is within the range of natural va riability of that component as represented by the 99% tolerance interval of the commercial varieties grown concurrently in the same trial, 3) analyses of the re producibility of the statistically significant combined-site component differences at indivi dual sites, and 4) assessing the differences within the

context of natural variabil ity of commercial soybean composition published in the

scientific literature and in the Internati onal Life Sciences In stitute (ILSI) Crop Composition Database (ILSI, 2006; Ridley et al., 2004).

This analysis provides a comprehensive comparative assessment of the levels of key

nutrients and anti-nutrie nts in seed, and of key nutrients in forage of MON 87708 and the conventional control, discussed in the cont ext of natural variab ility in commercialMonsanto Company 10-SY-210U 88 of 721 

### soybean. Results of the comparison indicate that the composition of the seed and forage of MON 87708 is equivalent to that of the near isogenic conventional control A3525 and within the range of natural variability of the commercial reference varieties.

VI.A.1. Composition of Soybean Seed and Forage (Treated)

VI.A.1.1. Nutrient Levels in Soybean Seed (Treated)

In the combined-site analysis of nutrient levels in seed, the following components showed no statistically signi ficant differences in mean values between MON 87708 and the conventional control: moisture, total fat, six amino acids (alani ne, lysine, methionine, serine, threonine, and tryptophan), and three fatty acids (18:0 stearic acid, 20:0 arachidic acid, and 20:1 eicosenoic aci d) (Table VI-2).

The components that showed statistically sign ificant differences in mean values between

MON 87708 and the conventional control in the combined-site analysis were: three proximates (ash, carbohydrates by calculation, and protein), 12 amino acids (arginine, aspartic acid, cystine, glutamic acid, glycine, histidine, isol eucine, leucine, phenylalanine, proline, tyrosine, and valine), three types of fiber (acid detergent fiber -ADF, neutral detergent fiber -NDF, and crude fiber), five fa tty acids (16:0 palmitic acid, 18:1 oleic acid, 18:2 linoleic acid, 18:3 linolenic acid, and 22: 0 behenic acid), and vi tamin E (Tables VI-1 and VI-2).

These statistically significant differences in nut rients were evaluated using considerations relevant to the safety and nutritional qua lity of MON 87708 when compared to the

conventional control:

1) All nutrient component differences obser ved in the combined-site analysis, whether reflecting increased or decreas ed MON 87708 mean values with respect

to the conventional control were small. Relative magnitude of differences ranged from 2.65 to 7.91% for amino acids, 1.51 to 8.19% for fatty acids, 15.13% for vitamin E, and 2.41 to 12.37% for proximates and fibers.

2) Mean values for all of these statistica lly different nutrient components from the combined-site analysis of MON 87708 were within the 99% to lerance interval

established from the commercial referenc e varieties grown concurrently and were, therefore, within the range of natural va riability of that co mponent in commercial soybean varieties with a history of safe consumption (Tables VI-1 and VI-2).

3) Assessment of the reproducibi lity of the combined-site differences at the five

individual sites showed: statistically significant di fferences for carbohydrates by calculation, crude fiber, cystine, and glycine at one site; aspartic acid, phenylalanine, proline, tyrosine, valine, 16:0 palmitic acid, and 18:2 linoleic acid at two sites; protein, arginine, glutamic acid, histidine, isoleucine, leucine, and 22:0 behenic acid at three sites; vitamin E at four sites; an d 18:1 oleic acid and

18:3 linolenic acid differed across all five sites. Although they were different in the combined-site analysis, no differences were observed for ash, ADF or NDF atMonsanto Company 10-SY-210U 89 of 721 

### any of the individual sites. Individual site mean values of MON 87708 for all nutrient components with sta tistically significant differe nces fell within the 99%

tolerance interval established from th e commercial reference varieties grown concurrently and were, therefore, within the range of natural variability of that component in commercial soybean varieties with a history of safe consumption.

4) All mean values of MON 87708 for all nu trient components were within the context of the natural variability of co mmercial soybean composition as published

in the scientific literature and availa ble in the ILSI Crop Composition Database (ILSI, 2006; Ridley et al., 2004).

Thirteen of the 24 differences between MON 87708 and the conventional control observed in the combined-site data analysis were attributable to small differences in protein and 12 individual amino acids (all expressed as % dw t). The relative magnitude of the difference between the mean protei n values for MON 87708 and the conventional control was small (a decrease of 3.65% in the combined-site analysis for MON 87708) and reached statistical significance at only three of the five individual sites. Correspondingly, differences in all amino acids were small and not observed consistently as statistically significant differences at all individual sites. Eleven of the 12 amino acids

observed to be different in the combined -site analysis were decreased (2.65-7.91%)

relative to the conventional control and, as with protei n, statistically significant differences were not consistently observed at all individual sites. Cystine showed a relative increase of 3.01% but was statistically significantly different at only one site.

Four of the six amino acids (alanine, lysine, serine, and threonine) not observed to be statistically different in the combined-site an alysis also showed modest decreases ranging from ~ 1.5-2.3% (Table VI-2) consistent with the directionality of the changes observed in protein content. Overall, observed differe nces in protein and am ino acid levels are not considered to be meaningful from a food and feed safety and nutritional perspective because they were small, and the mean MON 87708 values were within the 99% tolerance interval established by the commerc ial reference varieties grown concurrently

in the same trial.

Five of the combined-site differences be tween MON 87708 and the conventional control were attributable to fatty acid levels (all ex pressed as % total FA) in seed, whereas total fat content was not statistically significantl y different. For 18:1 oleic acid and 18:3 linolenic acid, the relative magnitude of differences between the mean values for

MON 87708 and conventional control were sm all in the combined-site analysis (a

decrease of 8.19% and an increase of 6.65% compared to the c onventional control, respectively) and at the five individual sites (levels were <11% decreased for 18:1 oleic

acid and <10% increased for 18:3 linolenic aci d at all sites compared to conventional control) (Tables VI-2, E-4, E- 7, E-10, E-13, and E-16).

By comparison, the observed differences between MON 87708 and conventional control

for 18:1 oleic and 18:3 linolenic acids are markedly less than differences in soybean varieties developed through conventional breedi ng (Fehr, 2007; Clemente and Cahoon, 2009). The average relative levels of 18:3 linolenic acid in commercial soybean are approximately 10% total FA, while the aver age relative level of 18:1 oleic acid inMonsanto Company 10-SY-210U 90 of 721 

### commercial soybean is approximately 18-25% total FA. In the compositional analysis presented here, the values of FA components in the conventional control, when assessed as individual replicates across all five individual sites, ra nged from 19.6 to 22.4% total

FA for 18:1 oleic acid and from 8.4 to 10.1% total FA for 18: 3 linolenic acid (Table VI-2). The values from the commer cial reference varieties ranged from 17.9 to

25.3% total FA for 18:1 oleic acid and 7.4 to 11.4% total FA for 18:3 linolenic acid (Table VI-2). Additionally, literature data from Lundry et al. (2008) and Berman et al. (2009) and the ILSI Crop Composition Data base (ILSI, 2006; Ridley et al., 2004) highlight the extensive natural variability in fatty acid levels in s oybean, as presented in Table VI-9. The small relative magnitudes of the differences in 18:3 linolenic acid and 18:1 oleic acid compared to the conventiona l control as well the broad range of these fatty acids present in commercial soybean vari eties, suggest that the differences are not meaningful to food and feed safety and nutritional quality in MON 87708.

The relative magnitudes of differences betw een the mean values for MON 87708 and the

conventional control for the othe r three ftty acids observed in the combined-site analysis were small (2.29% increase for 16:0 palmitic acid, 1.51% increase for 18:2 linoleic acid and a 4.70% decrease for 22:0 behenic acid). Th e small magnitude of differences as well

as the lack of statistical differences acro ss all individual sites (Tables VI-2, E-4, E-7, E-10, E-13, and E-16) further confirmed that the differences observed in fatty acid composition are not meaningful to food and feed safety and nutritional quality.

One of the combined-site differences observed between MON 87708 and the

conventional control was attr ibutable to vitamin E (expr essed as mg/100g dwt). The relative magnitude of difference between the mean values of MON 87708 and

conventional control for vitamin E in the co mbined-site analysis was an increase of 15.1% with respect to the conventional contro l (Tables VI-1).

Levels of vitamin E are known to be affect ed by environmental growing conditions (E) and germplasm (G) as demonstrated in results from recent assessments on soybean varieties grown at three locations in the U.S. over a period of four years (Britz et al., 2008) and across six environments in Eastern Canada in a single year (Seguin et al., 2009). Britz et al. (2008) showed more than a two-fold variation in levels across their study (units expresse d as the ratio of α -tocopherol (vitamin E) to total tocopherol content). Vitamin E values in Seguin et al. (2009) ranged from 0.87 to 3.32 mg/100g dwt. Both

assessments showed that G and E effects as well as G × E interaction effects influenced vitamin E content. In the compositional analysis presented here, values of vitamin E in the conventional control, when assessed as indi vidual replicates acro ss all sites, ranged by as much as 0.89 to 2.11 mg/100g dwt (Table VI-2). Ranges of vitamin E values from the concurrently grown commercial reference va rieties were even greater and ranged from 0.69 to 2.91 mg/100g dwt (Table VI-2). Literature data from other compositional assessments (Berman et al., 2009; Lundry et al., 2008; ILSI, 2006; Ridley et al., 2004) that further highlight the extensive natural va riability in vitamin E levels in soybean are presented in Table VI-9. Theref ore, given this established va riability of vitamin E levels in conventional soybean and the fact that s oybean is not an importa nt nutritional source of vitamin E in human or animal diets, th is increase in vitamin E levels in MON 87708Monsanto Company 10-SY-210U 91 of 721 

### compared to the conventional control s upports the conclusion that this observed difference is not meaningful to food a nd feed safety and nutritional quality.

The remaining combined-site differences between MON 87708 and the conventional control were attributable to two proximate s (ash and carbohydrates by calculation) and three fibers (ADF, NDF, and cr ude fiber). The relative ma gnitude of these increases were small (2.41% to 12.37%) and there was no consistency of these combined-site differences at the individual sites (carbohydr ates by calculation and crude fiber were different at only one site, wh ereas ash, ADF and NDF were not different at any of the individual sites). The combined-site mean values for these nut rient components also

were within the 99% tolerance interval established from the commercial reference varieties grown concurrently establishing that these differences are not meaningful to food and feed safety and nutrition.

In summary, statistical analyses found no cons istent differences across sites in the levels of nutrient components in seed from MON 87708 and the conventional control, except for differences in 18:1 oleic acid, 18:3 linol eic acid, and vitamin E levels that were of small magnitude and were within the natura l variability of the concurrently grown commercial soybean varieties. These data support the conclusi on that MON 87708 is compositionally equivalent to conventional soybean.

VI.A.1.2. Anti-Nutrient Levels in Soybean Seed (Treated)

In the combined-site analysis, no statistically significant differenc es were observed in four of the eight anti-nutrient component comp arisons (lectin, trypsin inhibitors, genistein, and glycitein) between MON 87708 and the conventional control. Statistically significant differences were observed betw een MON 87708 and the conventional control in the other four anti-nutrient components that were measured (Table s VI-1 and VI-3).

The differences included decreased mean va lues for phytic acid, raffinose, stachyose, and

an increased mean level of daidzein, compared to the conventional control.

The statistically significant differences in anti-nutrients were evaluated using considerations relevant to the safety and nutritional qual ity of MON 87708 when compared to the conventional control:

1) All anti-nutrient component differences observed in the combined-site analysis, whether reflecting increased or decreas ed MON 87708 mean values with respect

to the conventional control we re small. Relative magnit ude of differences in the combined-site analysis for the anti-nutri ents that were decreased in MON 87708 ranged from 6.1% (phytic acid) to 7.73% (raffinose). The relative magnitude of difference (increase) in daidzein was 11.5%.

2) MON 87708 mean values for these anti-nu trient components from the combinedsite analysis were within the 99% tolerance interval established from the

commercial reference varietie s concurrently grown in th e same trial and, therefore were within the range of natural variability of these components in commercial soybean varieties with a history of safe consumption (Tables V1-1 and V1-3).Monsanto Company 10-SY-210U 92 of 721 

### 3) Assessment of the reproducibi lity of the combined-site differences at the five

individual sites showed no consistent pattern across sites. A statistically significant decrease was observed for stac hyose at one site and phytic acid at two sites, whereas a significant increase was seen for daidzein at two sites. No

differences for raffinose were observed at any of the individual sites. Mean

values for all of the above anti- nutrient components in MON 87708 at the

individual sites were within the 99% tolerance interval established from the concurrently grown commercial reference varieties.

4) All mean values of MON 87708 for all anti-n utrients were with in the context of the natural variability of commercial soybean composition as published in the

scientific literature and available in the ILSI Crop Composition Database (ILSI, 2006; Ridley et al., 2004).

In summary, statistical analyses found no cons istent differences across sites in the levels of anti-nutrient components in seed from MON 87708 and the conventional control. Thus, a comprehensive evaluation of anti-nutrient components in seed support the conclusion that MON 87708 is compositionally equivalent to conventional soybean.

VI.A.1.3. Nutrient Levels in Soybean Forage (Treated)

In the combined-site analysis of forage, six of the seven nutrient component comparisons

did not have a statistica lly significant difference between MON 87708 and the conventional control (Tables V I-1 and VI-4). The only stat istical difference was for the ADF mean value and it was evaluated using c onsiderations relevant to the safety and nutritional quality of MON 87708 when compar ed to the conventi onal control.

1) The relative magnitude of difference in ADF, with respect to the conventional control, was small with an increase of 10.45%.

2) The mean value for ADF from the comb ined-site analysis of MON 87708 was

within the 99% tolerance interval esta blished from the commercial reference varieties grown concurrently in the same trial and, therefore within the range of natural variability of that component in commercial soybean varieties with a history of safe consumption (Tables V1-1 and V1-4).

3) Assessment of the reproducibility of the combined-site difference of ADF across the individual sites showed no statistically significant differences at any of the

five individual sites.

4) The level of ADF was within the natural variability observed for commercial soybean varieties as published in the scientif ic literature and available in the ILSI Crop Composition Database (ILSI , 2006; Ridley et al., 2004).

In summary, statistical analyses found no cons istent differences across sites in the levels of nutrient components in forage from MON 87708 and the conventiona l control. Thus, a comprehensive evaluation of nutrient compone nts in forage supports the conclusion that MON 87708 is compositionally equiva lent to conventional soybean.Monsanto Company 10-SY-210U 93 of 721 

### VI.A.2. Composition of Soybean Seed and Forage (Untreated)

VI.A.2.1. Nutrient Levels in Soybean Seed (Untreated)

In the combined-site analysis of nutrient levels in seed, the following components showed no statistically significant differe nces in mean values between MON 87708 (untreated) and the conventi onal control: ash, carbohydrates by calculation, moisture, total fat, crude fiber, ten amino acids (alani ne, aspartic acid, glycin e, histidine, lysine, methionine, serine, threonine, tryptophan, tyrosi ne), and three fatty acids (18:0 stearic acid, 20:0 arachidic acid, and 20:1 ei cosenoic acid) (Table VI-6).

The components that showed statistically sign ificant differences in mean values between MON 87708 (untreated) and the conventional cont rol in the combined-site analysis were: protein, eight amino acids (arginine,cystin e, glutamic acid, isoleucine, leucine, phenylalanine, proline, and valine), two types of fiber (acid detergent fiber (ADF), and neutral detergent fiber (NDF)), five fatty acids (16:0 palmitic acid, 18:1 oleic acid, 18:2 linoleic acid, 18:3 linolenic ac id, and 22:0 behenic acid), an d vitamin E (Tables VI-5 and VI-6).

These statistically significant differences in nut rients were evaluated using considerations relevant to the safety and nutritional quality of MON 87708 (untreat ed) when compared

to the conventional control:

1) All nutrient component differences obs erved in the combined-site analysis, whether reflecting increased or decreased MON 87708 (untreated) mean values with respect to the conventional cont rol were small. Relative magnitude of differences ranged from 2.27 to 5.88% or protein and amino acids, 1.45 to 7.60% for fatty acids, 18.16% for vitamin E, and 3.99 to 6.33% for fibers.

2) Mean values for all of these statisti cally different nutrien t components from the combined-site analysis of MON 87708 (untreated ) were within the 99% tolerance interval

established from the commercial referen ce varieties grown concurrently and were, therefore, within the range of natural vari ability of that component in commercial soybean varieties with a history of safe consumption (Tables VI-5 and VI-6).

3) Assessment of the reproduc ibility of the combined-site differences at the five individual sites showed: statistically signi ficant differences for cystine, isoleucine, valine, and 16:0 palmitic acid at one site; prot ein, arginine, glutamic acid, and leucine at two sites; phenylalanine, proline, 18:2 linolei c acid, and 22:0 behenic acid at three sites; 18:1 oleic acid, 18:3 linolenic acid, and vitamin E at four sites. No components were statistically significantly different at all five sites. Although they were different in the combined site analysis, no differences were observed for ADF or NDF at any of the

individual sites. Individual site mean values of MON 87708 (untreated) for all nutrient components with statistic ally significant differences fell w ithin the 99% tolerance interval

established from the commercial referen ce varieties grown concurrently and were, therefore, within the range of natural vari ability of that component in commercial soybean varieties with a hi story of safe consumption.Monsanto Company 10-SY-210U 94 of 721 

### 4) All mean values of MON 87708 (untreated) for all nutrient components were

within the context of the na tural variability of commer cial soybean composition as published in the scientific literature and av ailable in the ILSI Crop Composition Database (ILSI, 2006; Ridley et al., 2004).

Nine of the 17 differences between MON 87708 (untreated) and the conventional control observed in the combined-site data analysis were attributable to small differences in protein and eight individual amino acids (all expressed as % dwt). The relative magnitude of the difference between the mean protein values for MON 87708 (untreated)

and the conventional control was small (a decrease of 2.94% in the combined-site analysis for MON 87708 (untreated) and reached statistical significance at only two of the five individual sites. Correspondingly, di fferences in all amino acids were small and not observed consistently as st atistically significant differences at all indivi dual sites.

Seven of the 8 amino acids observed to be di fferent in the combined-site analysis were decreased (2.27 to 5.88%) relative to the conventional contro l. Cystine showed a relative

increase of 3.27% but was statis tically signific antly different at only one site. Overall, observed differences in protein and amino acid levels are not c onsidered to be meaningful from a food and feed safety a nd nutritional perspective because they were small, and the mean MON 87708 (untreated) values were within the 99% tolerance interval established by the commercial reference varieties grow n concurrently in the same trial.

Five of the combined-site differences between MON 87708 (untreated) and the

conventional control were attribut able to fatty acid levels (all expressed as % total FA) in seed, whereas total fat content was not statistically significantly different. For 18:1 oleic acid and 18:3 linolenic acid, the relative ma gnitude of differences between the mean values for MON 87708 (untreated) and conventi onal control were small in the combinedsite analysis (a decrease of 7.60% and an increase of 5.78% compared to the conventional control, respectively) and at the individual sites (levels were <12% decreased for 18:1

oleic acid and <8% increased for 18:3 linolenic acid at all sites compared to conventional control) (Tables VI-6, E-19, E-22, E-25, E-28, and E-31).

By comparison, the observed differen ces between MON 87708 (untreated) and

conventional control for 18:1 oleic and 18:3 linolenic acids are markedly less than differences in soybean varieties developed through conventional breeding (Clemente and Cahoon, 2009; Fehr, 2007). The average relati ve levels of 18:3 linolenic acid in commercial soybean are approximately 10% tota l FA, while the average relative level of

18:1 oleic acid in commercial soybean is approximately 18-25% total FA. In the compositional analysis presented here, the va lues of FA components in the conventional control, when assessed as individual replic ates across all five i ndividual sites, ranged

from 19.6 to 22.4% total FA for 18:1 oleic acid and from 8.4 to 10.1% total FA for 18:3 linolenic acid (Table VI-6). The values fr om the commercial reference varieties ranged

from 17.9 to 25.3% total FA for 18:1 oleic acid and 7.4 to 11.4% total FA for 18:3 linolenic acid (Table VI-6). Additionally, li terature data from L undry et al. (2008) and Berman et al. (2009) and the ILSI Crop Co mposition Database highlight the extensive natural variability in fatty acid levels in s oybean, as presented in Table VI-9. The small relative magnitudes of the differences in 18:3 linolenic acid and 18:1 oleic acid compared to the conventional control as well the broad range of these fatty acids present inMonsanto Company 10-SY-210U 95 of 721 

### commercial soybean varieties, s uggest that the differences ar e not meaningful to food and feed safety and nutritional qual ity in MON 87708 (untreated).

The relative magnitudes of differences between the mean values for MON 87708 (untreated) and the conventional control for the other three fatty acids observed in the combined-site analysis were small (2.37% increase for 16:0 palmitic acid, 1.45% increase

for 18:2 linoleic acid and a 3.71% decrease for 22:0 behenic acid). The small magnitude

of differences as well as the lack of statis tical differences across all individual sites (Tables VI-6, E-19, E-22, E28 and E-28) furthe r confirmed that the differences observed in fatty acid composition are not meaningful to food and feed safety and nutritional quality.

One of the combined-site differences obs erved between MON 87708 (untreated) and the

conventional control was attr ibutable to vitamin E (expr essed as mg/100g dwt). The relative magnitude of difference between the mean values of MON 87708 (untreated) and

conventional control for vitamin E in the co mbined-site analysis was an increase of 18.16% with respect to the conventi onal control (Table VI-6).

Levels of vitamin E are known to be affect ed by environmental growing conditions and germplasm as demonstrated in results fr om recent assessments on soybean varieties grown at three locations in the U.S. over a period of four years (Britz et al., 2008) and

across six environments in Eastern Canada in a single year (Seguin et al., 2009). Britz et al. (2008) showed more than a two-fold va riation in levels acr oss their study (units expressed as the ratio of α tocopherol (vitamin E) to tota l tocopherol content). Vitamin E values in Seguin et al. (2009) ranged from 0.87 to 3.32 mg/100g dwt. In the compositional analysis presented here, values of vitamin E in the conventional control,

when assessed as individual re plicates across all sites, ra nged by as much as 0.89 to 2.11 mg/100g dwt (Table VI-6). Ranges of vita min E values from the concurrently grown

commercial reference varieties were even greater and ranged from 0.69 to 2.91 mg/100g dwt (Table VI-6). Literature data from ot her compositional assessments (Berman et al., 2009; ILSI, 2006; Lundry et al., 2008; Ridley et al ., 2004) that furt her highlight the extensive natural variability in vitamin E levels in soybean are presented in Table VI-9. Therefore, given this established variability of vitamin E levels in conventional soybean and the fact that soybean is not an important nutritional source of vitamin E in human or animal diets, this increase in vitamin E leve ls in MON 87708 (untreated) compared to the conventional control supports the conclusion that this observed difference is not meaningful to food and feed safety and nutritional quality.

The remaining combined-site differences between MON 87708 (untreated) and the

conventional control were attributable to two fibers (ADF and NDF). The relative magnitude of these increases were small (3.99% to 6.33%) and ADF and NDF were not different at any of the individual sites. The combined-site mean values for these nutrient components also were within the 99% toleran ce interval established from the commercial reference varieties grown concurrently es tablishing that these differences are not meaningful to food and feed safety and nutrition.Monsanto Company 10-SY-210U 96 of 721 

### In summary, statistical analyses found no cons istent differences across sites in the levels of nutrient components in seed from MON 87708 (untre ated) and the conventional control, except for differences in 18:1 olei c acid, 18:3 linoleic acid, and vitamin E levels that were of small magnitude and were within the natural variability of the concurrently grown commercial soybean varieties. These data support the conclusion that MON 87708 (untreated) is compositionally equivalent to conventional soybean.

VI.A.2.2. Anti-Nutrient Levels in Soybean Seed (Untreated)

In the combined-site analysis, no statistically significant differenc es were observed in five of the eight anti-nutri ent component comparisons (l ectin, phytic acid, raffinose, stachyose, and glycitein) betw een MON 87708 (untreated) and the conventional control. Statistically significant differences were observed between MON 87708 (untreated) and

the conventional control in the other three an ti-nutrient components that were measured (Tables VI-5 and VI-7). The differences included increased mean values trypsin inhibitors, daidzein, and genistein comp ared to the convent ional control.

The statistically significant differences in anti-nutrients were evaluated using

considerations relevant to the safety and nutritional qua lity of MON 87708 (untreated) when compared to the conventional control:

1) All anti-nutrient component differences observed in the combined-site analysis reflected increased MON 87708 (untreated) mean values with respect to the conventional control, but were all less than 20%. Relativ e magnitude of differences in the combinedsite analysis for the anti-nut rients were 15.37% (trypsin in hibitor), 17.24% (daidzein) and 11.59% (genistein).

2) MON 87708 (untreated) mean values for these anti-nutrient components from the combined-site analysis were within the 99 % tolerance interval established from the commercial reference varieties concurrently grown in the same trial and, therefore were within the range of natural variability of these compone nts in commercial soybean varieties with a history of safe c onsumption (Tables VI-5 and VI-7).

3) Assessment of the reproduc ibility of the combined-site differences at the five

individual sites showed no c onsistent pattern across sites. None of the anti-nutrient components found to be different in the comb ined site analysis were observed to be statistically different at more th an one of the five individual s ites. Mean values for all of the above anti-nutrient components in M ON 87708 (untreated) at the individual sites

were within the 99% tolerance interval established from the concurrently grown

commercial reference varieties.

4) All mean values of MON 87708 (untreated) for all anti-nutrients were within the context of the natural variability of comme rcial soybean composition as published in the

scientific literature and available in th e ILSI Crop Composition Database (ILSI, 2006; Ridley et al., 2004).

In summary, statistical analyses found no cons istent differences across sites in the levels of anti-nutrient components in seed from MON 87708 (untr eated) and the conventionalMonsanto Company 10-SY-210U 97 of 721 

### control. Thus, a comprehens ive evaluation of anti-nutrien t components in seed support the conclusion that MON 87708 is compositionally equivalent to conventional soybean.

VI.A.2.3. Nutrient Levels in Soybean Forage (Untreated)

In the combined-site analysis of fora ge, none of the seven nutrient component comparisons had a statistica lly significant difference between MON 87708 (untreated) and the conventional control (T ables VI-5 and VI-8). In summary, statistical analyses

found no consistent differences across sites in the levels of nutrient components in forage from MON 87708 (untreated) and the conventi onal control. Thus, a comprehensive evaluation of nutrient components in fora ge supports the conclusion that MON 87708 (untreated) compositionally equiva lent to conventional soybean.Monsanto Company 10-SY-210U 98 of 721Table VI-1. Summary of Differences (α=0.05) for the Comparison of SoybeanComponent Levels for MON 87708 (Treated)vs. Conventional ControlMean Difference(MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean3Control4MeanMean Difference(% of Control)Significance(p-Value)MON 87708RangeCommercialTolerance Interval5Statistically Significant Differences Observed in Combined-Site AnalysisSeed Proximate (% dwt)Ash5.245.122.410.0314.94 - 5.694.74, 6.01Carbohydrates37.9336.643.500.01235.65 - 39.2132.07, 40.08Protein40.8642.41-3.650.01639.00 - 42.5335.50, 45.19Seed Fiber (% dwt)Acid Detergent Fiber13.5512.865.300.00912.45 - 15.5710.06, 18.04Crude Fiber8.297.3712.37<0.0016.23 - 9.655.76, 10.76Neutral Detergent Fiber15.2914.346.630.02813.11 - 17.8311.36, 19.38Seed Amino Acid (% dwt)Arginine3.303.58-7.910.0063.09 - 3.502.55, 3.83Monsanto Company 10-SY-210U 99 of 721Table VI-1 (continued). Summary of Differences (α=0.05) for the Comparison of Soybean Component Levels for MON 87708(Treated) vs. Conventional ControlMean Difference(MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean3Control4MeanMean Difference(% of Control)Significance(p-Value)MON 87708RangeCommercialTolerance Interval5Statistically Significant Differences Observed in Combined-Site AnalysisSeed Amino Acid (% dwt)Aspartic Acid4.634.78-3.180.0164.44 - 4.804.04, 5.13Cystine0.610.593.01<0.0010.58 - 0.630.50, 0.68Glutamic Acid7.387.69-4.030.0107.05 - 7.736.28, 8.30Glycine1.761.81-2.650.0201.67 - 1.831.53, 1.92Histidine1.061.09-3.070.0171.02 - 1.100.93, 1.16Isoleucine1.881.95-3.580.0061.75 - 1.971.65, 2.06Leucine3.063.17-3.370.0082.93 - 3.192.72, 3.39Phenylalanine2.062.13-3.330.0341.92 - 2.181.80, 2.30Monsanto Company 10-SY-210U 100 of 721Table VI-1 (continued). Summary of Differences (α=0.05) for the Comparison of Soybean Component Levels for MON 87708(Treated) vs. Conventional ControlMean Difference(MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean3Control4MeanMean Difference(% of Control)Significance(p-Value)MON 87708RangeCommercialTolerance Interval5Statistically Significant Differences Observed in Combined-Site AnalysisSeed Amino Acid (% dwt)Proline1.992.05-3.240.0171.90 - 2.091.65, 2.26Tyrosine1.371.42-3.470.0481.28 - 1.461.24, 1.50Valine1.982.06-3.890.0061.82 - 2.091.72, 2.20Seed Fatty Acid (% Total FA)16:0 Palmitic11.5911.332.290.00211.25 - 12.168.44, 12.5618:1 Oleic19.2020.91-8.19<0.00117.85 - 19.9415.73, 27.1918:2 Linoleic54.4053.591.510.01053.42 - 55.6748.61, 59.3718:3 Linolenic10.129.496.65<0.0018.99 - 10.886.01, 12.5822:0 Behenic0.270.28-4.700.0010.25 - 0.290.24, 0.40Monsanto Company 10-SY-210U 101 of 721Table VI-1 (continued). Summary of Differences (α=0.05) for the Comparison of Soybean Component Levels for MON 87708(Treated) vs. Conventional ControlMean Difference(MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean3Control4MeanMean Difference(% of Control)Significance(p-Value)MON 87708RangeCommercialTolerance Interval5Statistically Significant Differences Observed in Combined-Site AnalysisSeed Vitamin (mg/100g dwt)Vitamin E1.411.2315.130.0011.08 - 2.170, 3.49Seed Anti-nutrient (% dwt)Phytic Acid1.301.39-6.140.0431.08 - 1.510.77, 1.91Raffinose0.430.47-7.730.0450.32 - 0.590.13, 0.70Stachyose3.363.62-7.240.0113.07 - 4.022.30, 4.07Seed Isoflavone (μg/g dwt)Daidzein1494.971340.7111.510.046899.83 - 2305.260, 2271.38Forage Fiber (% dwt)Acid Detergent Fiber30.5827.6910.450.02123.30 - 45.1116.54, 41.80Monsanto Company 10-SY-210U 102 of 721Table VI-1 (continued). Summary of Differences (α=0.05) for the Comparison of Soybean Component Levels for MON 87708(Treated) vs. Conventional ControlMean Difference(MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean3Control4MeanMean Difference(% of Control)Significance(p-Value)MON 87708RangeCommercialTolerance Interval5Statistically Significant Differences Observed in Five Individual SitesSeed Fatty Acid (% Total FA)18:1 Oleic Site IARL19.3821.67-10.580.00119.07 - 19.7315.73, 27.1918:1 Oleic Site ILCY19.7421.57-8.460.01119.44 - 19.9415.73, 27.1918:1 Oleic Site ILWY19.5221.14-7.660.01019.34 - 19.6415.73, 27.1918:1 Oleic Site INRC18.7820.19-6.96<0.00118.58 - 18.9515.73, 27.1918:1 Oleic Site PAHM18.5820.01-7.130.01517.85 - 19.4215.73, 27.1918:3 Linolenic Site IARL10.6410.045.940.03310.58 - 10.746.01, 12.5818:3 Linolenic Site ILCY9.078.585.780.0078.99 - 9.166.01, 12.5818:3 Linolenic Site ILWY10.5410.054.920.02610.51 - 10.596.01, 12.58Monsanto Company 10-SY-210U 103 of 721Table VI-1 (continued). Summary of Differences (α=0.05) for the Comparison of Soybean Component Levels for MON 87708(Treated) vs. Conventional ControlMean Difference(MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean3Control4MeanMean Difference(% of Control)Significance(p-Value)MON 87708RangeCommercialTolerance Interval5Statistically Significant Differences Observed in Five Individual SitesSeed Fatty Acid (% Total FA)18:3 Linolenic Site INRC10.039.317.65<0.0019.89 - 10.106.01, 12.5818:3 Linolenic Site PAHM10.339.479.020.0069.91 - 10.886.01, 12.58Statistically Significant Differences Observed in Four Individual SitesSeed Vitamin (mg/100g dwt)Vitamin E Site IARL1.150.9422.250.0331.10 - 1.220, 3.49Vitamin E Site ILCY2.131.8614.430.0382.10 - 2.170, 3.49Vitamin E Site ILWY1.180.9424.640.0111.08 - 1.260, 3.49Vitamin E Site PAHM1.321.237.900.0101.21 - 1.540, 3.49Statistically Significant Differences Observed in Three Individual SitesSeed Proximate (% dwt)Protein Site ILCY40.1741.72-3.720.04739.44 - 40.9635.50, 45.19Monsanto Company 10-SY-210U 104 of 721Table VI-1 (continued). Summary of Differences (α=0.05) for the Comparison of Soybean Component Levels for MON 87708(Treated) vs. Conventional ControlMean Difference(MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean3Control4MeanMean Difference(% of Control)Significance(p-Value)MON 87708RangeCommercialTolerance Interval5Statistically Significant Differences Observed in Three Individual SitesSeed Proximate (% dwt)Protein Site ILWY40.8841.99-2.640.04240.56 - 41.3735.50, 45.19Protein Site PAHM40.2543.69-7.860.00239.00 - 41.0535.50, 45.19Seed Amino Acid (% dwt)Arginine Site ILWY3.303.57-7.580.0023.24 - 3.332.55, 3.83Arginine Site INRC3.443.72-7.370.0113.39 - 3.502.55, 3.83Arginine Site PAHM3.253.88-16.130.0013.09 - 3.362.55, 3.83Glutamic Acid Site ILCY7.437.61-2.380.0327.27 - 7.546.28, 8.30Glutamic Acid Site ILWY7.297.51-2.860.0027.20 - 7.356.28, 8.30Glutamic Acid Site PAHM7.288.00-9.080.0037.06 - 7.406.28, 8.30Monsanto Company 10-SY-210U 105 of 721Table VI-1 (continued). Summary of Differences (α=0.05) for the Comparison of Soybean Component Levels for MON 87708(Treated) vs. Conventional ControlMean Difference(MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean3Control4MeanMean Difference(% of Control)Significance(p-Value)MON 87708RangeCommercialTolerance Interval5Statistically Significant Differences Observed in Three Individual SitesSeed Amino Acid (% dwt)Histidine Site ILCY1.061.08-1.840.0221.04 - 1.070.93, 1.16Histidine Site ILWY1.051.07-1.620.0191.05 - 1.050.93, 1.16Histidine Site PAHM1.051.13-7.520.0021.02 - 1.060.93, 1.16Isoleucine Site ILCY1.891.97-3.980.0101.87 - 1.931.65, 2.06Isoleucine Site ILWY1.871.90-1.220.0041.85 - 1.891.65, 2.06Isoleucine Site PAHM1.852.00-7.590.0141.79 - 1.901.65, 2.06Leucine Site ILCY3.093.17-2.420.0023.04 - 3.142.72, 3.39Leucine Site ILWY3.023.10-2.49<0.0013.00 - 3.042.72, 3.39Monsanto Company 10-SY-210U 106 of 721Table VI-1 (continued). Summary of Differences (α=0.05) for the Comparison of Soybean Component Levels for MON 87708(Treated) vs. Conventional ControlMean Difference(MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean3Control4MeanMean Difference(% of Control)Significance(p-Value)MON 87708RangeCommercialTolerance Interval5Statistically Significant Differences Observed in Three Individual SitesSeed Amino Acid (% dwt)Leucine Site PAHM3.033.28-7.420.0022.96 - 3.092.72, 3.39Seed Fatty Acid (% Total FA)22:0 Behenic Site IARL0.260.28-5.490.0220.25 - 0.270.24, 0.4022:0 Behenic Site ILWY0.260.28-6.670.0080.26 - 0.270.24, 0.4022:0 Behenic Site INRC0.280.29-4.850.0380.27 - 0.290.24, 0.40Statistically Significant Differences Observed in Two Individual SitesSeed ProximateMoisture (% fwt) Site ILWY6.966.1612.990.0226.80 - 7.174.27, 9.58Moisture (% fwt) Site PAHM7.8410.50-25.30<0.0017.38 - 8.474.27, 9.58Seed Amino Acid (% dwt)Aspartic Acid Site ILWY4.594.67-1.900.0114.55 - 4.614.04, 5.13Monsanto Company 10-SY-210U 107 of 721Table VI-1 (continued). Summary of Differences (α=0.05) for the Comparison of Soybean Component Levels for MON 87708(Treated) vs. Conventional ControlMean Difference(MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean3Control4MeanMean Difference(% of Control)Significance(p-Value)MON 87708RangeCommercialTolerance Interval5Statistically Significant Differences Observed in Two Individual SitesSeed Amino Acid (% dwt)Aspartic Acid Site PAHM4.564.94-7.650.0024.45 - 4.634.04, 5.13Phenylalanine Site ILWY2.012.07-2.950.0461.96 - 2.061.80, 2.30Phenylalanine Site PAHM2.042.21-7.960.0102.00 - 2.071.80, 2.30Proline Site ILWY1.942.05-5.090.0201.93 - 1.961.65, 2.26Proline Site PAHM1.982.10-5.980.0161.94 - 2.001.65, 2.26Threonine Site ILWY1.521.55-1.690.0051.51 - 1.531.40, 1.69Threonine Site PAHM1.551.62-4.230.0291.52 - 1.571.40, 1.69Tyrosine Site INRC1.381.44-4.490.0441.35 - 1.431.24, 1.50Monsanto Company 10-SY-210U 108 of 721Table VI-1 (continued). Summary of Differences (α=0.05) for the Comparison of Soybean Component Levels for MON 87708(Treated) vs. Conventional ControlMean Difference(MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean3Control4MeanMean Difference(% of Control)Significance(p-Value)MON 87708RangeCommercialTolerance Interval5Statistically Significant Differences Observed in Two Individual SitesSeed Amino Acid (% dwt)Tyrosine Site PAHM1.351.49-9.430.0111.28 - 1.431.24, 1.50Valine Site ILCY1.962.05-4.370.0131.94 - 2.011.72, 2.20Valine Site PAHM1.952.13-8.170.0121.89 - 2.001.72, 2.20Seed Fatty Acid (% Total FA)16:0 Palmitic Site IARL11.4911.004.470.00111.44 - 11.548.44, 12.5616:0 Palmitic Site ILWY11.2611.042.020.01711.25 - 11.278.44, 12.5618:2 Linoleic Site ILCY54.5453.262.400.02154.45 - 54.7048.61, 59.3718:2 Linoleic Site INRC54.9854.431.000.01954.80 - 55.1448.61, 59.37Monsanto Company 10-SY-210U 109 of 721Table VI-1 (continued). Summary of Differences (α=0.05) for the Comparison of Soybean Component Levels for MON 87708(Treated) vs. Conventional ControlMean Difference(MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean3Control4MeanMean Difference(% of Control)Significance(p-Value)MON 87708RangeCommercialTolerance Interval5Statistically Significant Differences Observed in Two Individual SitesSeed Anti-nutrient (% dwt)Phytic Acid Site IARL1.361.53-11.280.0181.33 - 1.380.77, 1.91Phytic Acid Site ILWY1.401.55-9.340.0301.33 - 1.460.77, 1.91Seed Isoflavone (μg/g dwt)Daidzein Site ILWY1458.081271.6014.670.0041416.31 -1535.980, 2271.38Daidzein Site INRC1683.501419.4018.610.0491593.24 -1777.490, 2271.38Glycitein Site ILWY111.7779.7040.23<0.001109.88 - 113.8631.24, 233.60Glycitein Site INRC111.5198.4213.310.016110.91 - 112.2831.24, 233.60Forage Proximate (% dwt)Protein Site IARL25.2123.009.630.04324.71 - 25.5215.69, 26.63Monsanto Company 10-SY-210U 110 of 721Table VI-1 (continued). Summary of Differences (α=0.05) for the Comparison of Soybean Component Levels for MON 87708(Treated) vs. Conventional ControlMean Difference(MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean3Control4MeanMean Difference(% of Control)Significance(p-Value)MON 87708RangeCommercialTolerance Interval5Statistically Significant Differences Observed in Two Individual SitesForage Proximate (% dwt)Protein Site INRC21.7823.33-6.630.01920.99 - 22.5115.69, 26.63Statistically Significant Differences Observed in One Individual SiteSeed Proximate (% dwt)Carbohydrates Site PAHM38.3035.238.710.00837.69 - 38.6532.07, 40.08Seed Fiber (% dwt)Crude Fiber Site INRC8.066.8917.030.0097.76 - 8.475.76, 10.76Seed Amino Acid (% dwt)Alanine Site PAHM1.751.86-5.810.0101.74 - 1.771.56, 1.91Cystine Site PAHM0.620.594.790.0240.60 - 0.630.50, 0.68Glycine Site PAHM1.731.86-6.780.0041.69 - 1.751.53, 1.92Monsanto Company 10-SY-210U 111 of 721Table VI-1 (continued). Summary of Differences (α=0.05) for the Comparison of Soybean Component Levels for MON 87708(Treated) vs. Conventional ControlMean Difference(MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean3Control4MeanMean Difference(% of Control)Significance(p-Value)MON 87708RangeCommercialTolerance Interval5Statistically Significant Differences Observed in One Individual SiteSeed Amino Acid (% dwt)Lysine Site PAHM2.602.75-5.390.0092.53 - 2.652.33, 2.84Serine Site ILWY1.982.06-3.830.0031.97 - 2.001.78, 2.27Tryptophan Site ILCY0.510.486.210.0240.49 - 0.530.38, 0.52Seed Anti-nutrient (% dwt)Lectin (H.U./mg dwt) Site ILWY1.102.33-52.880.0450.59 - 1.510, 7.73Stachyose Site INRC3.143.46-9.180.0433.12 - 3.172.30, 4.07Forage Proximate (% dwt)Carbohydrates Site PAHM70.9565.817.810.01569.23 - 73.3160.69, 73.46Moisture (% fwt) Site PAHM74.2774.91-0.860.02173.40 - 75.4062.08, 89.801dwt = dry weight; fwt = fresh weight; FA= fatty acid; H.U. = Hemagglutinating Units.2MON 87708 was treated with dicamba.3Mean = least-square mean.4Control refers to the near isogenic conventional soybean control A3525.5With 95% confidence, interval contains 99% of the values expressed in the population of commercial referencevarieties. Negative limits set to zero.Monsanto Company 10-SY-210U 112 of 721Table VI-2.Statistical Summary of Combined-Site Soybean Seed Nutrients for MON 87708 (Treated)vs. Conventional ControlDifference (MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Proximate (% dwt)Ash5.24 (0.067)5.12 (0.067)0.12 (0.055)0.011, 0.240.0314.74, 6.01(4.94 - 5.69)(4.73 - 5.47)(-0.28 - 0.45)(4.93 - 5.88)Carbohydrates37.93 (0.50)36.64 (0.50)1.28 (0.40)0.36, 2.200.01232.07, 40.08(35.65 - 39.21)(34.11 - 38.45)(-0.38 - 4.07)(33.82 - 39.26)Moisture (% fwt)6.88 (0.65)7.14 (0.65)-0.26 (0.52)-1.46, 0.940.6294.27, 9.58(5.17 - 8.47)(5.79 - 10.60)(-3.12 - 1.43)(5.50 - 9.23)Protein40.86 (0.39)42.41 (0.39)-1.55 (0.51)-2.73, -0.370.01635.50, 45.19(39.00 - 42.53)(40.69 - 43.85)(-4.84 - 0.088)(37.06 - 43.42)Total Fat15.97 (0.59)15.84 (0.59)0.13 (0.31)-0.58, 0.840.69112.33, 24.10(14.00 - 18.56)(14.40 - 18.39)(-1.90 - 2.37)(15.47 - 21.34)Fiber (% dwt)Acid Detergent Fiber13.55 (0.40)12.86 (0.40)0.68 (0.25)0.18, 1.190.00910.06, 18.04(12.45 - 15.57)(11.62 - 14.57)(-0.71 - 2.13)(12.07 - 17.46)Monsanto Company 10-SY-210U 113 of 721Table VI-2 (continued). Statistical Summary of Combined-Site Soybean Seed Nutrients for MON 87708 (Treated) vs.Conventional ControlDifference (MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Fiber (% dwt)Crude Fiber8.29 (0.26)7.37 (0.26)0.91 (0.26)0.40, 1.43<0.0015.76, 10.76(6.23 - 9.65)(6.05 - 8.64)(-0.34 - 2.67)(6.35 - 11.31)Neutral Detergent Fiber15.29 (0.59)14.34 (0.59)0.95 (0.41)0.11, 1.790.02811.36, 19.38(13.11 - 17.83)(11.81 - 17.99)(-1.31 - 4.57)(11.66 - 19.45)Amino Acid (% dwt)Alanine1.76 (0.018)1.80 (0.018)-0.037 (0.017)-0.075, 0.00180.0591.56, 1.91(1.66 - 1.83)(1.69 - 1.90)(-0.16 - 0.042)(1.59 - 1.86)Arginine3.30 (0.069)3.58 (0.069)-0.28 (0.078)-0.46, -0.100.0062.55, 3.83(3.09 - 3.50)(3.19 - 3.93)(-0.83 - 0.0059)(2.88 - 3.74)Aspartic Acid4.63 (0.044)4.78 (0.044)-0.15 (0.050)-0.27, -0.0370.0164.04, 5.13(4.44 - 4.80)(4.46 - 5.01)(-0.56 - 0.12)(4.22 - 4.94)Cystine0.61 (0.0049)0.59 (0.0049)0.018 (0.0046)0.0085, 0.027<0.0010.50, 0.68(0.58 - 0.63)(0.56 - 0.62)(-0.0071 - 0.053)(0.53 - 0.66)Monsanto Company 10-SY-210U 114 of 721Table VI-2 (continued). Statistical Summary of Combined-Site Soybean Seed Nutrients for MON 87708 (Treated) vs.Conventional ControlDifference (MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Amino Acid (% dwt)Glutamic Acid7.38 (0.085)7.69 (0.085)-0.31 (0.093)-0.53, -0.0950.0106.28, 8.30(7.05 - 7.73)(7.12 - 8.14)(-1.09 - 0.17)(6.69 - 7.92)Glycine1.76 (0.016)1.81 (0.016)-0.048 (0.017)-0.086, -0.00960.0201.53, 1.92(1.67 - 1.83)(1.70 - 1.89)(-0.20 - 0.042)(1.58 - 1.84)Histidine1.06 (0.0095)1.09 (0.0095)-0.033 (0.011)-0.059, -0.00760.0170.93, 1.16(1.02 - 1.10)(1.02 - 1.14)(-0.12 - 0.031)(0.95 - 1.13)Isoleucine1.88 (0.019)1.95 (0.019)-0.070 (0.019)-0.11, -0.0260.0061.65, 2.06(1.75 - 1.97)(1.79 - 2.04)(-0.24 - 0.11)(1.68 - 2.02)Leucine3.06 (0.029)3.17 (0.029)-0.11 (0.031)-0.18, -0.0350.0082.72, 3.39(2.93 - 3.19)(2.96 - 3.32)(-0.36 - 0.072)(2.80 - 3.27)Lysine2.64 (0.019)2.68 (0.019)-0.041 (0.023)-0.094, 0.0120.1102.33, 2.84(2.53 - 2.71)(2.54 - 2.77)(-0.23 - 0.090)(2.38 - 2.74)Monsanto Company 10-SY-210U 115 of 721Table VI-2 (continued). Statistical Summary of Combined-Site Soybean Seed Nutrients for MON 87708 (Treated) vs.Conventional ControlDifference (MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Amino Acid (% dwt)Methionine0.58 (0.0053)0.58 (0.0053)0.00012 (0.0062)-0.013, 0.0130.9850.50, 0.64(0.53 - 0.60)(0.53 - 0.60)(-0.039 - 0.071)(0.52 - 0.63)Phenylalanine2.06 (0.028)2.13 (0.028)-0.071 (0.028)-0.13, -0.00670.0341.80, 2.30(1.92 - 2.18)(1.95 - 2.27)(-0.27 - 0.048)(1.85 - 2.21)Proline1.99 (0.021)2.05 (0.021)-0.067 (0.022)-0.12, -0.0150.0171.65, 2.26(1.90 - 2.09)(1.89 - 2.13)(-0.17 - 0.065)(1.74 - 2.16)Serine2.04 (0.023)2.09 (0.023)-0.048 (0.026)-0.11, 0.0130.1051.78, 2.27(1.92 - 2.12)(1.95 - 2.21)(-0.19 - 0.054)(1.90 - 2.18)Threonine1.56 (0.015)1.58 (0.015)-0.023 (0.015)-0.058, 0.0120.1691.40, 1.69(1.48 - 1.62)(1.51 - 1.64)(-0.10 - 0.052)(1.47 - 1.64)Tryptophan0.47 (0.0085)0.46 (0.0085)0.0070 (0.0097)-0.015, 0.0290.4940.38, 0.52(0.44 - 0.53)(0.43 - 0.50)(-0.035 - 0.064)(0.39 - 0.50)Monsanto Company 10-SY-210U 116 of 721Table VI-2 (continued). Statistical Summary of Combined-Site Soybean Seed Nutrients for MON 87708 (Treated) vs.Conventional ControlDifference (MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Amino Acid (% dwt)Tyrosine1.37 (0.018)1.42 (0.018)-0.049 (0.021)-0.098, -0.000460.0481.24, 1.50(1.28 - 1.46)(1.34 - 1.52)(-0.20 - 0.078)(1.26 - 1.49)Valine1.98 (0.020)2.06 (0.020)-0.080 (0.022)-0.13, -0.0300.0061.72, 2.20(1.82 - 2.09)(1.90 - 2.17)(-0.27 - 0.13)(1.73 - 2.13)Fatty Acid (% Total FA)16:0 Palmitic11.59 (0.16)11.33 (0.16)0.26 (0.060)0.12, 0.400.0028.44, 12.56(11.25 - 12.16)(10.92 - 12.08)(-0.15 - 0.62)(9.40 - 11.54)18:0 Stearic4.06 (0.10)4.04 (0.10)0.028 (0.049)-0.085, 0.140.5842.90, 5.19(3.60 - 4.40)(3.67 - 4.31)(-0.19 - 0.42)(3.24 - 4.67)18:1 Oleic19.20 (0.30)20.91 (0.30)-1.71 (0.19)-2.15, -1.27<0.00115.73, 27.19(17.85 - 19.94)(19.60 - 22.44)(-2.71 - -0.90)(17.88 - 25.31)18:2 Linoleic54.40 (0.37)53.59 (0.37)0.81 (0.24)0.25, 1.370.01048.61, 59.37(53.42 - 55.67)(52.33 - 54.99)(-0.59 - 1.68)(50.95 - 56.68)Monsanto Company 10-SY-210U 117 of 721Table VI-2 (continued). Statistical Summary of Combined-Site Soybean Seed Nutrients for MON 87708 (Treated) vs.Conventional ControlDifference (MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Fatty Acid (% Total FA)18:3 Linolenic10.12 (0.27)9.49 (0.27)0.63 (0.072)0.46, 0.80<0.0016.01, 12.58(8.99 - 10.88)(8.42 - 10.14)(0.36 - 1.20)(7.43 - 11.37)20:0 Arachidic0.26 (0.0052)0.26 (0.0052)-0.0012 (0.0031)-0.0082, 0.00590.7070.19, 0.34(0.23 - 0.27)(0.24 - 0.27)(-0.013 - 0.020)(0.20 - 0.30)20:1 Eicosenoic0.093 (0.017)0.090 (0.017)0.0029 (0.0042)-0.0056, 0.0110.4950.022, 0.24(0.069 - 0.16)(0.068 - 0.17)(-0.010 - 0.050)(0.065 - 0.17)22:0 Behenic0.27 (0.0038)0.28 (0.0038)-0.013 (0.0029)-0.020, -0.00660.0010.24, 0.40(0.25 - 0.29)(0.27 - 0.30)(-0.023 - 0.0024)(0.28 - 0.36)Vitamin (mg/100g dwt)Vitamin E1.41 (0.18)1.23 (0.18)0.19 (0.038)0.098, 0.270.0010, 3.49(1.08 - 2.17)(0.89 - 2.11)(0.018 - 0.42)(0.69 - 2.91)1dwt = dry weight; fwt = fresh weight; FA = fatty acid.2MON 87708 was treated with dicamba.3Mean (S.E.) = least-square mean (standard error).4Control refers to the near isogenic conventional soybean control A3525.5With 95% confidence, interval contains 99% of the values expressed in the population of commercial referencevarieties. Negative limits set to zero.Monsanto Company 10-SY-210U 118 of 721Table VI-3. Statistical Summary of Combined-Site SoybeanSeed Anti-Nutrients for MON87708 (Treated) vs. ConventionalControlDifference (MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Anti-nutrientLectin (H.U./mg dwt)3.17 (0.76)3.16 (0.76)0.013 (0.67)-1.54, 1.570.9840, 7.73(0.59 - 10.27)(0.46 - 10.38)(-4.27 - 8.13)(0.68 - 8.34)Phytic Acid (% dwt)1.30 (0.071)1.39 (0.071)-0.085 (0.035)-0.17, -0.00340.0430.77, 1.91(1.08 - 1.51)(1.09 - 1.62)(-0.29 - 0.15)(1.00 - 1.64)Raffinose (% dwt)0.43 (0.038)0.47 (0.038)-0.036 (0.018)-0.072, -0.000770.0450.13, 0.70(0.32 - 0.59)(0.36 - 0.60)(-0.24 - 0.069)(0.26 - 0.59)Stachyose (% dwt)3.36 (0.078)3.62 (0.078)-0.26 (0.099)-0.46, -0.0620.0112.30, 4.07(3.07 - 4.02)(3.07 - 4.15)(-1.00 - 0.40)(2.50 - 3.94)Trypsin Inhibitor (TIU/mg dwt)32.27 (1.40)30.37 (1.40)1.90 (1.79)-2.23, 6.040.31922.05, 41.12(26.09 - 39.27)(25.22 - 34.22)(-4.76 - 8.72)(22.81 - 44.56)Isoflavone (μg/g dwt)Daidzein1494.97 (155.94)1340.71 (155.94)154.26 (65.62)2.95, 305.570.0460, 2271.38(899.83 - 2305.26)(762.49 - 1729.91)(-258.27 - 795.19)(451.33 - 2033.05)Monsanto Company 10-SY-210U 119 of 721Table VI-3 (continued). Statistical Summary of Combined-Site Soybean Seed Anti-Nutrients for MON 87708 (Treated) vs.Conventional ControlDifference (MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Isoflavone (μg/g dwt)Genistein967.01 (90.36)886.57 (90.36)80.44 (41.86)-4.30, 165.190.06278.36, 1869.48(594.13 - 1496.78)(588.17 - 1162.01)(-185.98 - 513.56)(533.88 - 1726.03)Glycitein108.01 (5.24)95.85 (5.24)12.16 (6.91)-3.77, 28.090.11631.24, 233.60(77.67 - 119.09)(68.68 - 122.09)(-43.86 - 50.41)(73.61 - 231.75)1dwt = dry weight; H.U. = Hemagglutinating Units; TIU = Trypsin Inhibitor Units.2MON 87708 was treated with dicamba.3Mean (S.E.) = least-square mean (standard error).4Control refers to the near isogenic conventional soybean control A3525.5With 95% confidence, interval contains 99% of the values expressed in the population of commercial referencevarieties. Negative limits set to zero.Monsanto Company 10-SY-210U 120 of 721Table VI-4. Statistical Summary of Combined-Site Soybean Forage Nutrientsfor MON 87708 (Treated) vs. ConventionalControlDifference (MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Proximate (% dwt)Ash7.29 (0.54)7.39 (0.54)-0.10 (0.27)-0.71, 0.510.7123.36, 10.84(5.94 - 9.65)(6.10 - 10.46)(-0.89 - 1.56)(5.20 - 9.81)Carbohydrates66.48 (1.03)65.66 (1.04)0.83 (0.96)-1.40, 3.050.41460.69, 73.46(62.21 - 73.31)(62.91 - 67.94)(-3.95 - 6.90)(62.73 - 71.72)Moisture (% fwt)75.63 (1.82)75.55 (1.82)0.081 (0.27)-0.55, 0.710.77562.08, 89.80(72.40 - 82.80)(71.60 - 82.70)(-1.40 - 1.30)(70.40 - 84.10)Protein21.52 (0.95)22.32 (0.95)-0.80 (0.80)-2.67, 1.070.35015.69, 26.63(15.23 - 25.52)(20.88 - 24.11)(-6.26 - 2.75)(18.50 - 25.86)Total Fat4.67 (0.66)4.64 (0.66)0.032 (0.26)-0.57, 0.630.9040, 10.04(2.00 - 7.34)(2.01 - 6.72)(-0.68 - 1.96)(1.57 - 7.99)Fiber (% dwt)Acid Detergent Fiber30.58 (1.79)27.69 (1.80)2.89 (1.19)0.45, 5.340.02116.54, 41.80(23.30 - 45.11)(21.79 - 38.15)(-4.78 - 16.24)(20.98 - 39.23)Monsanto Company 10-SY-210U 121 of 721Table VI-4 (continued). Statistical Summary of Combined-Site Soybean Forage Nutrients for MON 87708 (Treated) vs.Conventional ControlDifference (MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Fiber (% dwt)Neutral Detergent Fiber29.63 (1.68)30.49 (1.70)-0.86 (1.22)-3.65, 1.940.50320.28, 44.03(24.21 - 38.51)(23.66 - 39.42)(-8.13 - 11.03)(24.81 - 42.80)1dwt = dry weight; fwt = fresh weight.2MON 87708 was treated with dicamba.3Mean (S.E.) = least-square mean (standard error).4Control refers to the near isogenic conventional soybean control A3525.5With 95% confidence, interval contains 99% of the values expressed in the population of commercial referencevarieties. Negative limits set to zero.Monsanto Company 10-SY-210U 122 of 721Table VI-5. Summary of Differences (α=0.05) for the Comparison of Soybean Component Levels for MON 87708 (Untreated)vs. Conventional ControlMean Difference(MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean3Control4MeanMean Difference(% of Control)Significance(p-Value)MON 87708RangeConventionalTolerance Interval5Statistical Differences Observed in Combined-Site AnalysisSeed Proximate (% dw)Protein41.1742.41-2.940.04039.96 - 43.0635.50, 45.19Seed Fiber (% dw)Acid Detergent Fiber13.3812.863.990.04611.01 - 15.7210.06, 18.04Neutral Detergent Fiber15.2414.346.330.03512.91 - 18.3811.36, 19.38Seed Amino Acid (% dw)Arginine3.373.58-5.880.0263.12 - 3.602.55, 3.83Cystine0.610.593.27<0.0010.59 - 0.640.50, 0.68Glutamic Acid7.467.69-3.030.0377.25 - 7.886.28, 8.30Isoleucine1.901.95-2.420.0391.80 - 2.021.65, 2.06Monsanto Company 10-SY-210U 123 of 721Table VI-5 (continued). Summary of Differences (α=0.05) for the Comparison of Soybean Component Levels for MON 87708(Untreated) vs. Conventional ControlMean Difference(MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean3Control4MeanMean Difference(% of Control)Significance(p-Value)MON 87708RangeConventionalTolerance Interval5Statistical Differences Observed in Combined-Site AnalysisSeed Amino Acid (% dw)Leucine3.103.17-2.270.0493.01 - 3.242.72, 3.39Phenylalanine2.062.13-3.040.0481.98 - 2.141.80, 2.30Proline1.982.05-3.390.0141.89 - 2.051.65, 2.26Valine2.002.06-2.760.0301.90 - 2.131.72, 2.20Seed Fatty Acid (% Total FA)16:0 Palmitic11.6011.332.370.00211.02 - 12.158.44, 12.5618:1 Oleic19.3220.91-7.60<0.00118.26 - 20.7315.73, 27.1918:2 Linoleic54.3753.591.450.01252.18 - 55.6248.61, 59.3718:3 Linolenic10.049.495.78<0.0018.94 - 10.906.01, 12.58Monsanto Company 10-SY-210U 124 of 721Table VI-5 (continued). Summary of Differences (α=0.05) for the Comparison of Soybean Component Levels for MON 87708(Untreated) vs. Conventional Control (continued)Mean Difference(MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean3Control4MeanMean Difference(% of Control)Significance(p-Value)MON 87708RangeConventionalTolerance Interval5Statistical Differences Observed in Combined-Site AnalysisSeed Fatty Acid (% Total FA)22:0 Behenic0.270.28-3.710.0060.25 - 0.290.24, 0.40Seed Vitamin (mg/100g dw)Vitamin E1.451.2318.16<0.0011.11 - 2.270, 3.49Seed Anti-nutrientTrypsin Inhibitor (TIU/mg dw)35.0330.3715.370.03123.32 - 51.5022.05, 41.12Seed Isoflavone (μg/g dw)Daidzein1571.791340.7117.240.007910.73 - 2297.580, 2271.38Genistein989.28886.5711.590.018654.16 - 1469.1378.36, 1869.48Statistical Differences Observed in More than One Individual SiteSeed Fatty Acid (% Total FA)18:1 Oleic Site IARL19.2821.67-11.020.00118.77 - 19.6815.73, 27.19Monsanto Company 10-SY-210U 125 of 721Table VI-5 (continued). Summary of Differences (α=0.05) for the Comparison of Soybean Component Levels for MON 87708(Untreated) vs. Conventional ControlMean Difference(MON 87708minus Control)Analytical Component (Units)1MON 877082Mean3Control4MeanMean Difference(% of Control)Significance(p-Value)MON 87708RangeConventionalTolerance Interval5Statistical Differences Observed in More than One Individual SiteSeed Fatty Acid (% Total FA)18:1 Oleic Site ILCY19.8521.57-7.980.01319.62 - 20.2215.73, 27.1918:1 Oleic Site INRC18.8020.19-6.90<0.00118.63 - 18.9615.73, 27.1918:1 Oleic Site PAHM18.4520.01-7.780.01118.26 - 18.8015.73, 27.1918:3 Linolenic Site ILCY9.098.585.960.0068.94 - 9.236.01, 12.5818:3 Linolenic Site ILWY10.6510.055.950.01410.37 - 10.906.01, 12.5818:3 Linolenic Site INRC10.059.317.94<0.00110.00 - 10.106.01, 12.5818:3 Linolenic Site PAHM10.039.475.870.0269.74 - 10.186.01, 12.58Seed Vitamin (mg/100g dw)Vitamin E Site IARL1.260.9433.640.0081.11 - 1.420, 3.49Monsanto Company 10-SY-210U 126 of 721Table VI-5 (continued). Summary of Differences (α=0.05) for the Comparison of Soybean Component Levels for MON 87708(Untreated) vs. Conventional ControlMean Difference(MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean3Control4MeanMean Difference(% of Control)Significance(p-Value)MON 87708RangeConventionalTolerance Interval5Statistical Differences Observed in More than One Individual SiteSeed Vitamin (mg/100g dw)Vitamin E Site ILCY2.211.8618.850.0162.11 - 2.270, 3.49Vitamin E Site ILWY1.150.9421.980.0171.11 - 1.200, 3.49Vitamin E Site PAHM1.371.2312.000.0021.26 - 1.590, 3.49Seed Amino Acid (% dw)Phenylalanine Site ILCY2.072.13-3.030.0242.05 - 2.081.80, 2.30Phenylalanine Site INRC2.082.20-5.540.0372.03 - 2.141.80, 2.30Phenylalanine Site PAHM2.082.21-6.070.0252.03 - 2.111.80, 2.30Proline Site ILCY1.952.06-5.340.0041.90 - 1.981.65, 2.26Proline Site INRC2.012.06-2.630.0201.99 - 2.041.65, 2.26Monsanto Company 10-SY-210U 127 of 721Table VI-5 (continued). Summary of Differences (α=0.05) for the Comparison of Soybean Component Levels for MON 87708(Untreated) vs. Conventional ControlMean Difference(MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean3Control4MeanMean Difference(% of Control)Significance(p-Value)MON 87708RangeConventionalTolerance Interval5Statistical Differences Observed in More than One Individual SiteSeed Amino Acid (% dw)Proline Site PAHM2.002.10-5.090.0281.93 - 2.041.65, 2.26Seed Fatty Acid (% Total FA)18:2 Linoleic Site IARL54.3052.703.040.02553.70 - 55.3448.61, 59.3718:2 Linoleic Site ILCY54.3153.261.970.04053.67 - 54.6348.61, 59.3718:2 Linoleic Site INRC54.9454.430.930.02454.79 - 55.1348.61, 59.3722:0 Behenic Site IARL0.260.28-4.960.0300.26 - 0.270.24, 0.4022:0 Behenic Site INRC0.280.29-5.540.0250.27 - 0.280.24, 0.4022:0 Behenic Site PAHM0.260.27-4.780.0180.25 - 0.270.24, 0.40Monsanto Company 10-SY-210U 128 of 721Table VI-5 (continued). Summary of Differences (α=0.05) for the Comparison of Soybean Component Levels for MON 87708(Untreated) vs. Conventional ControlMean Difference(MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean3Control4MeanMean Difference(% of Control)Significance(p-Value)MON 87708RangeConventionalTolerance Interval5Statistical Differences Observed in More than One Individual SiteSeed Proximate (% dw)Moisture (% fw) Site IARL7.846.0729.210.0107.39 - 8.734.27, 9.58Moisture (% fw) Site PAHM9.5310.50-9.240.0119.21 - 9.914.27, 9.58Protein Site INRC41.4743.58-4.850.04940.22 - 43.0635.50, 45.19Protein Site PAHM40.3843.69-7.570.00239.96 - 40.9735.50, 45.19Seed Amino Acid (% dw)Arginine Site INRC3.403.72-8.500.0073.35 - 3.502.55, 3.83Arginine Site PAHM3.393.88-12.750.0023.35 - 3.442.55, 3.83Glutamic Acid Site ILCY7.447.61-2.240.0397.33 - 7.516.28, 8.30Glutamic Acid Site PAHM7.428.00-7.240.0077.32 - 7.506.28, 8.30Monsanto Company 10-SY-210U 129 of 721Table VI-5 (continued). Summary of Differences (α=0.05) for the Comparison of Soybean Component Levels for MON 87708(Untreated) vs. Conventional ControlMean Difference(MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean3Control4MeanMean Difference(% of Control)Significance(p-Value)MON 87708RangeConventionalTolerance Interval5Statistical Differences Observed in More than One Individual SiteSeed Amino Acid (% dw)Leucine Site ILCY3.113.17-1.820.0083.08 - 3.132.72, 3.39Leucine Site PAHM3.103.28-5.460.0093.08 - 3.112.72, 3.39Lysine Site ILWY2.682.631.760.0062.66 - 2.692.33, 2.84Lysine Site PAHM2.642.75-4.020.0252.62 - 2.652.33, 2.84Seed Isoflavone (μg/g dw)Glycitein Site ILWY96.9679.7021.650.00192.19 - 103.2531.24, 233.60Glycitein Site INRC119.2798.4221.190.003113.04 - 124.2431.24, 233.60Statistical Differences Observed in One Individual SiteSeed Amino Acid (% dw)Alanine Site PAHM1.791.86-3.650.0461.76 - 1.811.56, 1.91Monsanto Company 10-SY-210U 130 of 721Table VI-5 (continued). Summary of Differences (α=0.05) for the Comparison of Soybean Component Levels for MON 87708(Untreated) vs. Conventional ControlMean Difference(MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean3Control4MeanMean Difference(% of Control)Significance(p-Value)MON 87708RangeConventionalTolerance Interval5Statistical Differences Observed in One Individual SiteSeed Amino Acid (% dw)Aspartic Acid Site PAHM4.664.94-5.620.0084.64 - 4.694.04, 5.13Cystine Site INRC0.620.595.300.0290.60 - 0.630.50, 0.68Glycine Site PAHM1.771.86-4.840.0141.76 - 1.781.53, 1.92Histidine Site PAHM1.071.13-5.370.0081.07 - 1.080.93, 1.16Isoleucine Site PAHM1.892.00-5.670.0351.88 - 1.911.65, 2.06Serine Site ILWY2.102.062.110.0262.09 - 2.111.78, 2.27Threonine Site ILWY1.591.552.85<0.0011.58 - 1.601.40, 1.69Valine Site PAHM2.002.13-5.930.0352.00 - 2.001.72, 2.20Monsanto Company 10-SY-210U 131 of 721Table VI-5 (continued). Summary of Differences (α=0.05) for the Comparison of Soybean Component Levels for MON 87708(Untreated) vs. Conventional ControlMean Difference(MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean3Control4MeanMean Difference(% of Control)Significance(p-Value)MON 87708RangeConventionalTolerance Interval5Statistical Differences Observed in One Individual SiteSeed Fatty Acid (% Total FA)16:0 Palmitic Site IARL11.5111.004.670.00111.39 - 11.638.44, 12.5620:1 Eicosenoic Site ILCY0.150.16-4.820.0280.15 - 0.160.022, 0.24Seed Anti-nutrientTrypsin Inhibitor (TIU/mg dw) Site ILWY38.9329.7330.950.03337.89 - 39.4922.05, 41.12Forage Proximate (% dw)Ash Site INRC6.206.95-10.700.0475.87 - 6.723.36, 10.84Moisture (% fw) Site IARL83.4781.971.830.02782.70 - 84.1062.08, 89.80Protein Site IARL25.7623.0012.020.02224.63 - 27.0415.69, 26.631dw = dry weight; fw = fresh weight; FA = fatty acid; TIU = Trypsin Inhibitor Units.2 MON 87708 plants were not sprayed with dicamba, but received another conventional treatmentas was done for the conventionalcontrol.3Mean = least-square mean.4Control refers to the non-biotechnology derived, conventional control (A3525).5With 95% confidence, interval contains 99% of the values expressed in the population of conventional substances. Negative limits set to zero.Monsanto Company 10-SY-210U 132 of 721Table VI-6. Statistical Summary ofCombined-Site Soybean Seed Nutrients for MON 87708 (Untreated) vs. ConventionalControlDifference (MON 87708 minus Control)Analytical Component (Units)1MON 87708 2Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)ConventionalTolerance Interval5(Range)Proximate (% dw)Ash5.22 (0.067)5.12 (0.067)0.10 (0.055)-0.010, 0.210.0734.74, 6.01(4.89 - 5.51)(4.73 - 5.47)(-0.28 - 0.55)(4.93 - 5.88)Carbohydrates37.30 (0.50)36.64 (0.50)0.66 (0.40)-0.26, 1.580.13832.07, 40.08(35.27 - 39.79)(34.11 - 38.45)(-1.89 - 4.52)(33.82 - 39.26)Moisture (% fw)7.30 (0.65)7.14 (0.65)0.16 (0.52)-1.04, 1.360.7714.27, 9.58(5.68 - 9.91)(5.79 - 10.60)(-1.19 - 2.89)(5.50 - 9.23)Protein41.17 (0.39)42.41 (0.39)-1.25 (0.51)-2.42, -0.0690.04035.50, 45.19(39.96 - 43.06)(40.69 - 43.85)(-3.89 - 0.47)(37.06 - 43.42)Total Fat16.32 (0.59)15.84 (0.59)0.48 (0.31)-0.23, 1.190.15512.33, 24.10(13.95 - 18.66)(14.40 - 18.39)(-1.73 - 1.70)(15.47 - 21.34)Fiber (% dw)Acid Detergent Fiber13.38 (0.40)12.86 (0.40)0.51 (0.25)0.0084, 1.020.04610.06, 18.04(11.01 - 15.72)(11.62 - 14.57)(-0.72 - 2.45)(12.07 - 17.46)Monsanto Company 10-SY-210U 133 of 721Table VI-6 (continued). Statistical Summary of Combined-Site Soybean Seed Nutrients for MON 87708 (Untreated) vs.Conventional ControlDifference (MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)ConventionalTolerance Interval5(Range)Fiber (% dw)Crude Fiber7.50 (0.26)7.37 (0.26)0.13 (0.26)-0.39, 0.640.6205.76, 10.76(6.14 - 8.89)(6.05 - 8.64)(-1.33 - 1.19)(6.35 - 11.31)Neutral Detergent Fiber15.24 (0.59)14.34 (0.59)0.91 (0.41)0.064, 1.750.03511.36, 19.38(12.91 - 18.38)(11.81 - 17.99)(-2.27 - 4.17)(11.66 - 19.45)Amino Acid (% dw)Alanine1.78 (0.018)1.80 (0.018)-0.025 (0.017)-0.063, 0.0140.1741.56, 1.91(1.72 - 1.84)(1.69 - 1.90)(-0.10 - 0.043)(1.59 - 1.86)Arginine3.37 (0.069)3.58 (0.069)-0.21 (0.078)-0.39, -0.0310.0262.55, 3.83(3.12 - 3.60)(3.19 - 3.93)(-0.55 - 0.051)(2.88 - 3.74)Aspartic Acid4.68 (0.044)4.78 (0.044)-0.10 (0.050)-0.22, 0.0140.0784.04, 5.13(4.57 - 4.90)(4.46 - 5.01)(-0.35 - 0.10)(4.22 - 4.94)Cystine0.61 (0.0049)0.59 (0.0049)0.019 (0.0046)0.010, 0.029<0.0010.50, 0.68(0.59 - 0.64)(0.56 - 0.62)(-0.0068 - 0.047)(0.53 - 0.66)Monsanto Company 10-SY-210U 134 of 721Table VI-6 (continued). Statistical Summary of Combined-Site Soybean Seed Nutrients for MON 87708 (Untreated) vs.Conventional ControlDifference (MON 87708 minus Control)Analytical Component (Units)1MON 87708 2Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)ConventionalTolerance Interval5(Range)Amino Acid (% dw)Glutamic Acid7.46 (0.085)7.69 (0.085)-0.23 (0.093)-0.45, -0.0180.0376.28, 8.30(7.25 - 7.88)(7.12 - 8.14)(-0.70 - 0.12)(6.69 - 7.92)Glycine1.77 (0.016)1.81 (0.016)-0.034 (0.017)-0.073, 0.00420.0741.53, 1.92(1.74 - 1.85)(1.70 - 1.89)(-0.13 - 0.041)(1.58 - 1.84)Histidine1.07 (0.0095)1.09 (0.0095)-0.021 (0.011)-0.047, 0.00440.0920.93, 1.16(1.05 - 1.11)(1.02 - 1.14)(-0.073 - 0.029)(0.95 - 1.13)Isoleucine1.90 (0.019)1.95 (0.019)-0.047 (0.019)-0.091, -0.00280.0391.65, 2.06(1.80 - 2.02)(1.79 - 2.04)(-0.22 - 0.085)(1.68 - 2.02)Leucine3.10 (0.029)3.17 (0.029)-0.072 (0.031)-0.14, -0.000240.0492.72, 3.39(3.01 - 3.24)(2.96 - 3.32)(-0.23 - 0.055)(2.80 - 3.27)Lysine2.65 (0.019)2.68 (0.019)-0.026 (0.023)-0.079, 0.0270.2952.33, 2.84(2.60 - 2.76)(2.54 - 2.77)(-0.13 - 0.083)(2.38 - 2.74)Monsanto Company 10-SY-210U 135 of 721Table VI-6 (continued). Statistical Summary of Combined-Site Soybean Seed Nutrients for MON 87708 (Untreated) vs.Conventional ControlDifference (MON 87708 minus Control)Analytical Component (Units)1MON 87708 2Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)ConventionalTolerance Interval5(Range)Amino Acid (% dw)Methionine0.58 (0.0053)0.58 (0.0053)0.0013 (0.0062)-0.011, 0.0140.8340.50, 0.64(0.55 - 0.61)(0.53 - 0.60)(-0.036 - 0.056)(0.52 - 0.63)Phenylalanine2.06 (0.028)2.13 (0.028)-0.065 (0.028)-0.13, -0.000670.0481.80, 2.30(1.98 - 2.14)(1.95 - 2.27)(-0.18 - 0.023)(1.85 - 2.21)Proline1.98 (0.021)2.05 (0.021)-0.070 (0.022)-0.12, -0.0180.0141.65, 2.26(1.89 - 2.05)(1.89 - 2.13)(-0.17 - 0.043)(1.74 - 2.16)Serine2.07 (0.023)2.09 (0.023)-0.021 (0.026)-0.081, 0.0390.4491.78, 2.27(1.91 - 2.11)(1.95 - 2.21)(-0.19 - 0.073)(1.90 - 2.18)Threonine1.57 (0.015)1.58 (0.015)-0.0093 (0.015)-0.044, 0.0260.5561.40, 1.69(1.49 - 1.61)(1.51 - 1.64)(-0.088 - 0.052)(1.47 - 1.64)Tryptophan0.47 (0.0085)0.46 (0.0085)0.0095 (0.0097)-0.013, 0.0320.3590.38, 0.52(0.41 - 0.50)(0.43 - 0.50)(-0.053 - 0.049)(0.39 - 0.50)Monsanto Company 10-SY-210U 136 of 721Table VI-6 (continued). Statistical Summary of Combined-Site Soybean Seed Nutrients for MON 87708 (Untreated) vs.Conventional ControlDifference (MON 87708 minus Control)Analytical Component (Units)1MON 87708 2Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)ConventionalTolerance Interval5(Range)Amino Acid (% dw)Tyrosine1.41 (0.018)1.42 (0.018)-0.015 (0.021)-0.064, 0.0340.5061.24, 1.50(1.35 - 1.48)(1.34 - 1.52)(-0.086 - 0.098)(1.26 - 1.49)Valine2.00 (0.020)2.06 (0.020)-0.057 (0.022)-0.11, -0.00690.0301.72, 2.20(1.90 - 2.13)(1.90 - 2.17)(-0.25 - 0.097)(1.73 - 2.13)Fatty Acid (% Total FA)16:0 Palmitic11.60 (0.16)11.33 (0.16)0.27 (0.060)0.13, 0.410.0028.44, 12.56(11.02 - 12.15)(10.92 - 12.08)(-0.094 - 0.59)(9.40 - 11.54)18:0 Stearic4.04 (0.10)4.04 (0.10)0.0057 (0.049)-0.11, 0.120.9092.90, 5.19(3.55 - 4.57)(3.67 - 4.31)(-0.23 - 0.38)(3.24 - 4.67)18:1 Oleic19.32 (0.30)20.91 (0.30)-1.59 (0.19)-2.03, -1.15<0.00115.73, 27.19(18.26 - 20.73)(19.60 - 22.44)(-2.82 - -0.045)(17.88 - 25.31)18:2 Linoleic54.37 (0.37)53.59 (0.37)0.78 (0.24)0.22, 1.340.01248.61, 59.37(52.18 - 55.62)(52.33 - 54.99)(-0.98 - 2.61)(50.95 - 56.68)Monsanto Company 10-SY-210U 137 of 721Table VI-6 (continued). Statistical Summary of Combined-Site Soybean Seed Nutrients for MON 87708 (Untreated) vs.Conventional ControlDifference (MON 87708 minus Control)Analytical Compoent (Units)1MON 87708 2Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)ConventionalTolerance Interval5(Range)Fatty Acid (% Total FA)18:3 Linolenic10.04 (0.27)9.49 (0.27)0.55 (0.072)0.38, 0.72<0.0016.01, 12.58(8.94 - 10.90)(8.42 - 10.14)(-0.18 - 1.01)(7.43 - 11.37)20:0 Arachidic0.26 (0.0052)0.26 (0.0052)-0.0013 (0.0031)-0.0083, 0.00580.6840.19, 0.34(0.23 - 0.29)(0.24 - 0.27)(-0.014 - 0.022)(0.20 - 0.30)20:1 Eicosenoic0.092 (0.017)0.090 (0.017)0.0011 (0.0042)-0.0074, 0.00960.7950.022, 0.24(0.065 - 0.16)(0.068 - 0.17)(-0.013 - 0.058)(0.065 - 0.17)22:0 Behenic0.27 (0.0038)0.28 (0.0038)-0.010 (0.0029)-0.017, -0.00380.0060.24, 0.40(0.25 - 0.29)(0.27 - 0.30)(-0.023 - 0.0054)(0.28 - 0.36)Vitamin (mg/100g dw)Vitamin E1.45 (0.18)1.23 (0.18)0.22 (0.038)0.14, 0.31<0.0010, 3.49(1.11 - 2.27)(0.89 - 2.11)(0.0086 - 0.49)(0.69 - 2.91)1dw = dry weight; fw = fresh weight; FA = fatty acid.2MON 87708 plants were not sprayed with dicamba, but received another conventional treatment as was done for the conventional control.3Mean (S.E.) = least-square mean (standard error).4Control refers to the non-biotechnology derived, conventional control (A3525).5With 95% confidence, interval contains 99% of the values expressed in the population of conventional substances. Negative limits set to zero.Monsanto Company 10-SY-210U 138 of 721Table VI-7. Statistical Summary of Combined-Site Soybean Seed Anti-Nutrientsfor MON 87708 (Untreated) vs. ConventionalControlDifference (MON 87708 minus Control)Analytical Component (Units)1MON 87708 2Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)ConventionalTolerance Interval5(Range)Anti-nutrientLectin (H.U./mg dw)3.05 (0.76)3.16 (0.76)-0.12 (0.67)-1.67, 1.440.8670, 7.73(1.18 - 6.35)(0.46 - 10.38)(-7.83 - 3.07)(0.68 - 8.34)Phytic Acid (% dw)1.33 (0.071)1.39 (0.071)-0.060 (0.035)-0.14, 0.0220.1290.77, 1.91(1.05 - 1.48)(1.09 - 1.62)(-0.21 - 0.26)(1.00 - 1.64)Raffinose (% dw)0.46 (0.038)0.47 (0.038)-0.0069 (0.018)-0.042, 0.0290.6970.13, 0.70(0.34 - 0.58)(0.36 - 0.60)(-0.065 - 0.056)(0.26 - 0.59)Stachyose (% dw)3.48 (0.078)3.62 (0.078)-0.15 (0.099)-0.35, 0.0540.1472.30, 4.07(2.94 - 3.85)(3.07 - 4.15)(-0.82 - 0.53)(2.50 - 3.94)Trypsin Inhibitor (TIU/mg dw)35.03 (1.40)30.37 (1.40)4.67 (1.79)0.53, 8.800.03122.05, 41.12(23.32 - 51.50)(25.22 - 34.22)(-2.94 - 18.17)(22.81 - 44.56)Isoflavone (μg/g dw)Daidzein1571.79 (155.94)1340.71 (155.94)231.08 (65.62)79.76, 382.390.0070, 2271.38(910.73 - 2297.58)(762.49 - 1729.91)(-187.35 - 691.83)(451.33 - 2033.05)Monsanto Company 10-SY-210U 139 of 721Table VI-7 (continued). Statistical Summary of Combined-Site Soybean Seed Anti-Nutrientsfor MON 87708 (Untreated) vs.Conventional ControlDifference (MON 87708 minus Control)Analytical Component (Units)1MON 87708 2Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)ConventionalTolerance Interval5(Range)Isoflavone (μg/g dw)Genistein989.28 (90.36)886.57 (90.36)102.71 (41.86)17.96, 187.460.01878.36, 1869.48(654.16 - 1469.13)(588.17 - 1162.01)(-116.63 - 400.27)(533.88 - 1726.03)Glycitein110.40 (5.24)95.85 (5.24)14.55 (6.91)-1.38, 30.480.06831.24, 233.60(83.25 - 133.73)(68.68 - 122.09)(-2.04 - 37.48)(73.61 - 231.75)1dw = dry weight; H.U. = Hemagglutinating Units; TIU = Trypsin Inhibitor Units.2MON 87708 plants were not sprayed with dicamba, but received another conventional treatment as was done for the conventional control.3Mean (S.E.) = least-square mean (standard error).4Control refers to the non-biotechnology derived, conventional control (A3525).5With 95% confidence, interval contains 99% of the values expressed in the population of conventional substances. Negative limits set to zero.Monsanto Company 10-SY-210U 140 of 721Table VI-8. Statistical Summary ofCombined-Site Soybean Forage Nutrients for MON 87708 (Untreated) ConventionalControlDifference (MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)ConventionalTolerance Interval5(Range)Proximate (% dw)Ash7.01 (0.54)7.39 (0.54)-0.39 (0.27)-0.99, 0.220.1833.36, 10.84(4.92 - 9.45)(6.10 - 10.46)(-1.57 - 0.97)(5.20 - 9.81)Carbohydrates65.55 (1.03)65.66 (1.04)-0.11 (0.96)-2.33, 2.120.91360.69, 73.46(61.64 - 71.05)(62.91 - 67.94)(-6.11 - 4.64)(62.73 - 71.72)Moisture (% fw)75.81 (1.82)75.55 (1.82)0.25 (0.27)-0.37, 0.880.37962.08, 89.80(72.30 - 84.10)(71.60 - 82.70)(-1.00 - 2.70)(70.40 - 84.10)Protein22.70 (0.95)22.32 (0.95)0.38 (0.80)-1.49, 2.250.64815.69, 26.63(16.28 - 27.04)(20.88 - 24.11)(-5.21 - 4.89)(18.50 - 25.86)Total Fat4.70 (0.66)4.64 (0.66)0.055 (0.26)-0.55, 0.660.8380, 10.04(2.61 - 6.52)(2.01 - 6.72)(-1.38 - 1.63)(1.57 - 7.99)Fiber (% dw)Acid Detergent Fiber27.72 (1.79)27.69 (1.80)0.036 (1.19)-2.41, 2.480.97616.54, 41.80(23.32 - 34.63)(21.79 - 38.15)(-7.68 - 4.38)(20.98 - 39.23)Monsanto Company 10-SY-210U 141 of 721Table VI-8 (continued). Statistical Summary of Combined-Site Soybean Forage Nutrients for MON 87708 (Untreated) vs.Conventional ControlDifference (MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)ConventionalTolerance Interval5(Range)Fiber (% dw)Neutral Detergent Fiber30.98 (1.68)30.49 (1.70)0.49 (1.22)-2.31, 3.290.69820.28, 44.03(25.38 - 37.80)(23.66 - 39.42)(-5.93 - 10.81)(24.81 - 42.80)1dw = dry weight; fw = fresh weight.2MON 87708 plants were not sprayed with dicamba, but received another conventional treatment as was done for the conventional control.3Mean (S.E.) = least-square mean (standard error).4Control refers to the non-biotechnology derived, conventional control (A3525).5With 95% confidence, interval contains 99% of the values expressed in the population of conventional substances. Negative limits set to zero.Monsanto Company 10-SY-210U 142 of 721 

### Table VI-9. Literature and ILSI Database Ranges for Components in Soybean Seed and Forage

Seed Tissue Components 1 Literature Range 2 ILSI Range 3

Seed Nutrients Proximates (% dwt)

Ash 4.61 – 6.32 a ; 4.32 – 5.88 b 3.89 – 6.99 Carbohydrates by calculation 32.75 – 40.98 a ; 29.88 – 43.48 b 29.6 – 50.2 Moisture (% fwt) 6.24 – 12.10 a ; 5.44 – 11.70 b 4.7 – 34.4

Protein 34.78 – 43.35 a ; 32.29 – 42.66 b 33.19 – 45.48

Total Fat 14.40 – 20.91 a ; 15.10 – 23.56 b ; 15.5 c – 24.7 c 8.10 – 23.56

Fiber (% dwt) Acid Detergent Fiber 9.22 – 26.26 a ; 11.81 – 19.45 b 7.81 – 18.61
Neutral Detergent Fiber 10.79 – 23.90 a ; 13.32 – 23.57 b 8.53 – 21.25

Amino Acids (% dwt)

Alanine 1.62 – 1.89 a ; 1.43 – 1.93 b 1.51 – 2.10 Arginine 2.57 – 3.34 a ; 2.15 – 3.05 b 2.29 – 3.40 Aspartic acid 4.16 – 5.02 a ; 4.01 – 5.72 b 3.81 – 5.12 Cystine/Cysteine 0.52 – 0.69 a ; 0.41 – 0.71 b 0.37 – 0.81

Glutamic acid 6.52 – 8.19 a ; 5.49 – 8.72 b 5.84 – 8.20 Glycine 1.59 – 1.90 a ; 1.41 – 1.99 b 1.46 – 2.00

Histidine 0.96 – 1.13 a ; 0.86 – 1.24 b 0.88 – 1.18

Isoleucine 1.59 – 2.00 a ; 1.41 – 2.02 b 1.54 – 2.08

Leucine 2.79 – 3.42 a ; 2.39 – 3.32 b 2.59 – 3.62 Lysine 2.36 – 2.77 a ; 2.19 – 3.15 b 2.29 – 2.84

Methionine 0.45 – 0.63 a ; 0.39 – 0.65 b 0.43 – 0.68 Phenylalanine 1.82 – 2.29 a ; 1.62 – 2.44 b 1.63 – 2.35

Proline 1.83 – 2.23 a ; 1.63 – 2.25 b 1.69 – 2.28

Serine 1.95 – 2.42 a ; 1.51 – 2.30 b 1.11 – 2.48

Threonine 1.44 – 1.71 a ; 1.23 – 1.74 b 1.14 – 1.86 Tryptophan 0.30 – 0.48 a ; 0.41 – 0.56 b 0.36 – 0.50 Tyrosine 1.27 – 1.53 a ; 0.74 – 1.31 b 1.02 – 1.61

Valine 1.68 – 2.11 a ; 1.50 – 2.13 b 1.60 – 2.20

Fatty Acids (% total FA) 8:0 Caprylic not available 0.148 – 0.148 10:0 Capric 0.15 – 0.27 b not available

12:0 Lauric not available 0.082 – 0.132 14:0 Myristic 0.063 – 0.11 b 0.071 – 0.238 14:1 Myristoleic not available 0.121 – 0.125

15:0 Pentadecanoic not available not available

15:1 Pentadecenoic not available not available

16:0 Palmitic 9.80 – 12.63 b 9.55 – 15.77

16:1 Palmitoleic 0.055 – 0.14 b 0.086 – 0.194 17:0 Heptadecanoic 0.076 – 0.13 b 0.085 – 0.146 17:1 Heptadecenoic 0.019 – 0.064 b 0.073 – 0.087

18:0 Stearic 3.21 – 5.63 b 2.70 – 5.88

18:1 Oleic 16.69 – 35.16 b 14.3 – 32.2

18:2 Linoleic 44.17 – 57.72 b 42.3 – 58.8

18:3 Gamma Linolenic not available not available

18:3 Linolenic 4.27 – 9.90 b 3.00 – 12.52Monsanto Company 10-SY-210U 143 of 721 

### Table VI-9 (continued). Literature and ILSI Database Ranges for Components in Soybean Seed and Forage

Seed Tissue Components 1 Literature Range 2 ILSI Range 3

Seed Nutrients Fatty Acids (% total FA)

20:0 Arachidic 0.35 – 0.57 b 0.163 – 0.482

20:1 Eicosenoic 0.13 – 0.30 b 0.140 – 0.350

20:2 Eicosadienoic 0.016 – 0.071 b 0.077 – 0.245

20:3 Eicosatrienoic not available not available

20:4 Arachidonic not available not available

22:0 Behenic 0.35 – 0.59 b 0.277 – 0.595

22:1 Erucic not available not available

Vitamins (mg/100g dwt)

Vitamin E 1.29 – 4.80 a ; 1.12 – 8.08 b 0.19 – 6.17

Seed Anti-Nutrients Lectin (H.U./mg fwt) 0.45 – 10.87 a ; 0.090 – 11.18 b 0.09 – 8.46 Trypsin Inhibitor (TIU/mg dwt) 20.79 – 59.03 a ; 18.14 – 42.51 b 19.59 – 118.68

Phytic Acid (% dwt) 0.41 – 1.92 a ; 0.81 – 2.66 b 0.63 – 1.96 Raffinose (% dwt) 0.26 – 0.84 a ; 0.43 – 1.85 b 0.21 – 0.66 Stachyose (% dwt) 1.53 – 3.04 a ; 1.97 – 6.65 b 1.21 – 3.50

Isoflavones ( μ g/g dwt) (mg/kg dwt)

Daidzein 224.03 – 1571.91 a ; 198.95 – 1458.24 b 60.0 – 2453.5

Genistein 338.24 – 1488.89 a ; 148.06 – 1095.57 b 144.3 – 2837.2 Glycitein 52.72 – 298.57 a ; 32.42 – 255.94 b 15.3 – 310.4

Forage Tissue Components 1 Literature Range 2 ILSI Range 3

Forage Nutrients

Proximate (% dwt)

Ash 5.28 – 9.24 a ; 4.77 – 8.54 b 6.72 – 10.78 Carbohydrates by calculation 62.25 – 72.30 a ; 60.61 – 77.26 b 59.8 – 74.7 Moisture (% fwt) 68.50 – 78.40 a ; 62.76 – 80.20 b 73.5 – 81.6

Protein 16.48 – 24.29 a ; 12.68 – 23.29 b 14.38 – 24.71

Total Fat 2.65 4 – 9.87 a ; 2.96 – 7.88 b 1.302 – 5.132

Fiber (% dwt) Acid Detergent Fiber 23.86 – 50.89 a ; 25.49 – 47.33 b not available Neutral Detergent Fiber 19.61 – 43.70 a ; 30.96 – 54.55 b not available

1 fwt = fresh weight; dwt = dry weight; H.U. = hemagglutinating unit; TIU = trypsin inhibitor unit.

2 Literature range references: a( Lundry et al., 2008); b( Berman et al., 2009); c( OECD, 2001).

4 ILSI Crop Composition Database (2006).Monsanto Company 10-SY-210U 144 of 721 

### VI.B. Compositional Assessment Conclusion

Analyses of nutrient and anti- nutrient levels in both di camba-treated and untreated MON 87708 and the near isogenic conventiona l control A3525 were conducted to assess compositional equivalence. The tissues anal yzed included seed and forage harvested from plants grown at five field sites in the U.S. during the 2008 field season. The composition analysis, conducted in accordance with OECD guidelines, also included

measurement of nutrients and anti-nutrients in the commercial reference varieties concurrently grown with MON 87708 to provide data on natural va riability of each compositional component. All soybean pl ants including MON 87708, the conventional control, and the commercial reference varietie s were treated with maintenance pesticides as necessary throughout the gr owing season. In addition, MON 87708 plots were either treated at the V2-V3 growth stage with dicam ba herbicide at the maximum in-crop label rate (0.5 lb a.e./acre) or not tr eated with dicamba herbicide.

For MON 87708 treated, the combined-site anal ysis of both seed and forage showed no statistically significant diffe rences between MON 87708 and conventional control for 21 (42.0%) of the 50 mean value comparisons. Of the statistically significant differences observed, one was from the forage analysis, and 28 were from the seed analysis. Nutrient component differences in seed include d mean values fo r ash, carbohydrates by calculation, protein and 12 amino acids, five fatty acids, ADF, NDF, crude fiber, and vitamin E. In the combined-site analysis, all nutrient component differences in seed

between MON 87708 and the conventional control were of small relative magnitude with respect to the conventional control and, wh ether increased or decreased, ranged from 1.51% to 12.37% for the three proximates, amino acids, fatty acids, and fibers, and

15.13% for vitamin E. Two of the nutrient components in the comb ined-site analysis (decreased levels of 18:1 oleic ac id and increased levels of 18:3 linolenic acid) were also observed to be statistically different at all five indivi dual sites, and one nutrient component (vitamin E) was observed to be increa sed at four of the five individual sites as in the combined-site analysis. The other co mbined-site differences occurred at fewer or

none of the individual sites. Anti-nutrient component differences in seed were observed in mean values for phytic acid, raffinose, stachyose, and daidzein. In the combined-site analysis, all anti-nutrient component diffe rences in seed between MON 87708 and the

conventional control were of small relative magnitude, with respect to the conventional control, and ranged from a 6.14% decrease (phytic acid) to an 11.51% increase (daidzein). None of the anti-nutrient components were obser ved to be statistically different at more

than two of the five individua l sites. The only nutrient co mponent difference in forage for the combined-site analysis was observe d in ADF and its relative magnitude of difference, with respect to the conventi onal control, was 10.45%. No differences

between MON 87708 and the conventional contro l ADF mean values were observed at any of the five individual sites. M ean values of MON 87708 components with statistically significant differe nces to the conventional cont rol were all within the 99%

tolerance interval established from th e commercial reference varieties grown concurrently and at the same field sites, as well as ranges in the scientific literature and the ILSI Crop Composition Database.Monsanto Company 10-SY-210U 145 of 721 

### For MON 87708 (untreated) the combined-site an alysis of both seed and forage showed no statistically significant differences be tween MON 87708 (untreated) and conventional control for 30 (60.0%) of the 50 mean value co mparisons. Of the statistically significant differences observed, none were from the fo rage analysis, and 20 were from the seed analysis. Nutrient component differences in seed included mean values for protein and eight amino acids, five fatty acids, ADF, NDF, and vitamin E. In the combined-site analysis, all nutrient component differences in seed betw een MON 87708 (untreated) and the conventional control were of small relative magnitude with respect to the conventional control and, whet her increased or decreased, ranged from 1.45% to 7.60% for protein and amino acids, fatty acids, and fibers, and 18.16% for vitamin E. None of the nutrient components in the combined-site analysis were observed to be statistically

different at all five individual sites. Anti-nutrient co mponent differences in seed were observed in mean values for trypsin inhibito r, daidzein, and genistein. In the combinedsite analysis, all anti-nutri ent component differences in seed between MON 87708 (untreated) and the conv entional control were of small relative magnitude, with respect to the conventional control, and ranged fr om an 11.59% increase (genistein), 15.37% increase (trypsin inhibitor), and a 17.24% increase (daidzein) . None of the anti-nutrient components from the combined-site analysis were observed to be statistically different at

more than one of the five individual sites. No nutrient component differences in forage for the combined-site analysis were observe d. Mean values of MON 87708 components with statistically signi ficant differences to the conventio nal control were all within the

99% tolerance interval established from the commercial reference varieties grown concurrently and at the same field sites, as well as ranges in the scientific literature and the ILSI Crop Composition Database.

In summary, a comprehensive evaluation of ke y nutrients and anti-nu trients in seed and key nutrients in forage for both dicamba- treated and untreated MON 87708 supports the conclusion that soybean seed and forage produced from MON 87708 are compositionally equivalent to that of conventional soybean a nd that neither the dicamba tolerance trait inMON 87708, nor the dicamba herbicide treatment, applied according to maximum incrop label rates (including the associated di camba residue levels) have a meaningful impact on the composition and therefore on th e food and feed safety or the nutritional quality of MON 87708 compared to conventional soybean.Monsanto Company 10-SY-210U 146 of 721 

### VII. PHENOTYPIC, AGRONOMIC, AND ENVIRONMENTAL

INTERACTIONS ASSESSMENT

This section provides an assessment of th e phenotypic, agronomic, and environmental interaction characteristics, including plant-sy mbiont associations of both dicamba treated and untreated MON 87708 compared to the near isogenic conventi onal soybean control A3525. Analysis of the untreat ed MON 87708 allows an assessm ent of the effect of the inserted gene only. However, for this product, since DCSA and formaldehyde are reaction products formed in the presence of dicamba herbicide, and since salicylic acid and formaldehyde are known to be involved in plant defense responses, dicamba treated MON 87708 data are supplied as well. Th e data support a determination that MON 87708 is similar to conventional soyb ean with the exception of the dicamba tolerance trait and, therefore, is no more likely to pose a plan t pest risk than conventional soybean. These conclusions are based on the results of multiple evaluations.

Phenotypic, agronomic, and environmenta l interaction charac teristics of both

dicamba-treated and untreated MON 87708 were evaluated in a comparative manner to assess plant pest potential. These assessmen ts included evaluation of seed germination characteristics, plant growth and developm ent characteristics, observations for plant responses to abiotic stress, plant-diseas e and plant-arthropod interactions, pollen characteristics, and plant-symbiont inter action characteristics. Results from the phenotypic, agronomic, and environmental in teractions assessment demonstrate that MON 87708 does not possess weedy characteristic s, increased susceptib ility or tolerance to specific abiotic stress, di seases, or arthropods, or charac teristics that would confer a plant pest risk compared to the conventional control.

VII.A. Characteristic s Measured for Assessment

A detailed phenotypic description of the regulat ed article is requested as part of the petition for determination of nonregulated status in 7 CF R § 340.6 including differences from the unmodified recipient organism that w ould “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 characteriz ation of MON 87708, data were collected to provide a detailed phenotypic, agronomic, a nd environmental intera ction description of

both dicamba-treated and untreated MON 87708 and included an evaluation of specific

characteristics related to altered weed iness or plant pest potential.

The plant characterization and assessment of MON 87708 encompassed six general data categories: 1) seed germination, dormancy, a nd emergence; 2) vegetative growth; 3) reproductive development (including pollen char acteristics); 4) seed retention on the plant and lodging; 5) plant response to abioti c stress and interactions with diseases and arthropods; and 6) plant-symb iont interactions. An overv iew of the characteristics assessed is presente d in Table VII-1.Monsanto Company 10-SY-210U 147 of 721 

### The data were evaluated from a basis of familiarity (OECD, 1993) and were comprised of a combination of field, greenhouse, and labor atory assessments conduc ted by scientists who are familiar with the production and ev aluation of soybean. In each of these assessments, the dicamba-treated and/or un treated MON 87708 was compared to the near isogenic conventional soybean control A3525 that has a genetic background similar to MON 87708 but does not possess the dicamba to lerance trait. In addition, multiple commercial reference varieties (see Appendices F-I and Tables F-1, G-1, and I-1) were included to provide a range of comparative values that are representative of existing commercial soybean varieties for each measured phenotypic, agronomic, and

environmental interaction characteristic. Co mmercial reference s oybean varieties are developed through a process of selecting a nd breeding for various desirable soybean characteristics and can provide a range of natural variability for characteristics and context for interpreting experimental results.Monsanto Company 10-SY-210U 148 of 721 

### Table VII-1. Phenotypic, Agronomic, and Environmental Interaction

Characteristics Evaluated in U.S. Field Trials, Laboratory, or Greenhouse Tests

Data
category Characteristics

measured (associated section where discussed) Evaluation timing (setting of evaluation) 1 Evaluation description (measurement endpoints)

Seed germination, dormancy,

and

emergence Normal germinated (VII.C.1) Day 5 and 8 (20/30°C) (laboratory) Percentage of seed producing seedlings exhibiting normal developmental

characteristics

Abnormal germinated (VII.C.1) Day 8 (20/30°C) (laboratory) Percen tage of seed producing seedlings

that could not be cl assified as normal germinated

Germinated (VII.C.1) Day 5, 8, and 13 (10, 20, 30, 10/20 and 10/30°C) (laboratory) Percentage of seed that had germinated normally and abnormally

Dead (VII.C.1) Day 5 and 8 (10, 20, 30, 10/20, 10/30, and 20/30°C); Day 13 (10, 20, 30, 10/20 and 10/30°C) (laboratory) Percentage of seed that had visibly

deteriorated and become soft to the touch(also included non-viable hard and nonviable firm-swollen seed)

Viable hard (VII.C.1) Day 8 (20/30°C); Day 13 (10, 20, 30, 10/20 and 10/30°C) (laboratory) Percentage of seed that did not imbibe

water and remained hard to the touch (viability determined by a tetrazolium

test 2 )

Viable firm-swollen (VII.C.1) Day 8 (20/30°C); Day 13 (10, 20, 30, 10/20 and 10/30°C) (laboratory) Percentage of seed th at imbibed water and

were firm to the touch but did not germinate (viability determined by a

tetrazolium test 2 ) Early stand count (VII.C.2.3) V2 - V4 (Field) Number of emerged plants in two rows,

standardized to 20 ft rows

Final stand count (VII.C.2.3) Maturity, R8 (Field) Number of plants in two rows,

standardized to 20 ft rows

Vegetative growth Seedling vigor (VII.C.2.3) V2 - V4 (Field) Rated on a 1-9 scale, where 1 = excellent, 5 = average, and 9 = poor vigor Growth stage

assessment (VII.C.2.3) Every 2-3 weeks, V2-R8 (Field) Average soybean plant growth stage per plot

Flower color (VII.C.2.3) Flowering, R2 (Field) Color of flowers: purple, white, or mixed

Plant pubescence 3
(VII.C.2.3) Maturity, R8 (Field) Pubescen ce on plants in each plot categorized as hairy or hairless Plant height (VII.C.2.3) Maturity, R8 (Field) Distance (in) from the soil surface to the uppermost node on the main stem of five representative plants per plot

Reproductive development Days to 50% flowering (VII.C.2.3) Flowering, R1-R2 (Field) Calendar day number (days from January 1) when approximately 50% of the plants in each plot were flowering Pollen viability (VII.C.3) Flowering, R1-R2 (laboratory) Percen tage of viable pollen based on pollen grain staining characteristics Pollen morphology (VII.C.3) Flowering, R1-R2 (laboratory) Diamet er (μm) of viable pollen grains

Seed moisture (VII.C.2.3 Harvest (Field) Percent moisture content of harvested seed

100 seed weight (VII.C.2.3) Harvest (Field) Mass (g) of 100 harvested seed

Test weight 3
(VII.C.2.3) Harvest (Field) Mass (lb) of a bushel of harvested seed

Yield (VII.C.2.3) Harvest (Field) Bushels of harvested seed per acre, adjusted to 13% moistureMonsanto Company 10-SY-210U 149 of 721 

### Table VII-1 (continued). Phenotypic, Ag ronomic and Environmental Interaction

Characteristics Evaluated in U.S. Field Trials, Laboratory or Greenhouse Tests

Data
category Characteristics

measured (associated section where discussed) Evaluation timing (setting of evaluation) 1 Evaluation description (measurement endpoints)

Seed

retention and lodging Lodging (VII.C.2.3) Maturity, R8 (Field) Rated on 0-9 scale, where 0 = completely erect and 9 = completely flat or lodged Pod shattering (VII.C.2.3) Maturity, R8 (Field) Rated on 0-9 scale, where 0 = no shattering and 9 = completely shattered

Plantenvironment

interactions Plant response to

abiotic stress (VII.C.2.4) Four times per growing season (Field) Qualitative assessment of each plot, with rating on a 0-9 scale, where 0 = no symptoms and 9 = severe symptoms Disease damage (VII.C.2.4) Four times per growing season (Field) 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.2.4) Four times during growing season (Field) Damage assessed on upper four nodes of 10 representative plants per plot using arthropod-specific 0-5 rating scales of increasing severity Arthropod

abundance (VII.C.2.4) 3 Four times during growing season (Field) Quantitative assessment of pest and beneficial arthropods

Plantsymbiont

interactions 3 Biomass (VII.C.4) 6 weeks after emergence (Greenhouse) Nodule, root, and shoot dry weight (g/plant)

Nodule number (VII.C.4) 6 weeks after emergence (Greenhouse) Nodule number

Total nitrogen (VII.C.4) 6 weeks after emergence (Greenhouse) Shoot total nitrogen (% and g/plant)

1 Soybean plant growth stages were determined usin g descriptions and guidelines outlined in Soybean Growth and Development (ISU, 2004).

2 Viability of hard and firm-swollen seed were de termined by a tetrazolium test (AOSA, 2000).

3 Plant pubescence, test weight, ar thropod abundance, and plant symbiont interactions were recorded only
in the 2008 (untr eated) field trials.Monsanto Company 10-SY-210U 150 of 721 

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

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

Expert knowledge and experience with conve ntionally bred soybean was the basis for selecting appropriate endpoints and estimatin g the range of responses that would be considered typical for soybean. As su ch, both dicamba-trea ted and untreated MON 87708 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 wellunderstood weedy characteristics ( e.g. , seed dormancy, pre-harvest seed loss characteristics, and lodging) was used to asse ss whether there is an increase in weediness of MON 87708, an element of APHIS’s plan t 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 assessment. Based on all of the data collected, an assessment was made to determine if MON 87708 is likely to pose an increased plant pest risk compared to conventional soybean. Prior to analysis,

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

were identified.

VII.B.1. Interpretation of Detected Differences Criteria

Comparative plant characterization data from a biotechnology-derived crop and the conventional control are interpre ted in the context of contribu tions to increased plant pest potential as assessed by APHIS. Under the fr amework of familiarity, characteristics for which no differences are detected support a conclusion of no increased plant pest potential of the biotechnology-derived cr op compared to the conventional crop.

Characteristics for which differences are dete cted are considered in a step-wise method (Figure VII-1). All detected differences for a characteristic are considered in the context

of whether or not the difference would in crease the plant pest potential of the biotechnology-derived crop. Ultimately, a wei ght of evidence approach considering all

characteristics and data is used for the overa ll risk assessment of differences and their significance. Figure VII-1 illustrates the step wise assessment process employed in detail:Monsanto Company 10-SY-210U 151 of 721 Note: A “no” answer at any step indicates that the characteristic does not contribute to a biological or ecological change for the crop in terms of plant pest potential and subsequent steps are not considered. If the answer is “yes” or “uncertain”, the subsequent step is considered.



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

Steps 1 and 2 - Evaluate Detected St atistically Significant Differences

Data on each measured characteristic are sta tistically analyzed, where appropriate, within

each individual site and in a combined-site analysis, in which the data are pooled among sites. All statistically significant differences are evaluated and considered in the context of a change in plant pest potential. Differen ces detected in individual-site analyses that are not consistently observed across multiple en vironments in the combined-site analysis are considered not biologically meaningful in terms of plant pest po tential and, therefore, are not further considered in subsequent step s. Any difference detected in the combinedsite analysis is further assessed.

Step 3 - Evaluate Differences Relative to Commercial Reference Varieties Range

If a difference for a characteristic is dete cted in the combined-site analysis across multiple environments, then the mean value of the biotechnology-derived crop for the

characteristic is assessed relative to the commercial reference varieties. 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 ecological 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 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 3 Step 1Monsanto Company 10-SY-210U 152 of 721 

### Step 4 - Evaluate Differences in the Context of the Crop

If the mean value of the biotechnology-deri ved crop is outside the variation of the commercial reference varieties ( e.g. , reference range), the mean value of the biotechnology-derived crop for th e characteristic is assesse d relative to known values common for the crop ( e.g. , published values).

Step 5 - Plant Pest Potential

If the mean value of the biotechnology-deri ved crop is outside the range of values common for the crop, the detected difference fo r the characteristic 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, 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 popula tions of the crop or to a sexually-compatible species.Monsanto Company 10-SY-210U 153 of 721 

### VII.C. Comparative Assessments of the Ph enotypic, Agronomic, and Environmental

Interaction Characteristics of MON 87708

This section provides the results of compar ative assessments condu cted in re plicated laboratory, greenhouse, and/or multi-site field experime nts to provide a detailed phenotypic, agronomic, and environmental in teraction description of MON 87708. The

MON 87708 characteristics evaluated in these assessments included: seed dormancy and germination characteristics (Section VII. C.1), plant phenotypic and environmental

interaction observations under field conditions (Section VII.C.2), po llen characteristics (Section VII.C.3), and symbiont interactions (Section VII.C.4). Additional details for each assessment are provided in Appendices F through I.

VII.C.1. Seed Dormancy and Germination Characteristics

USDA-APHIS considers the potential for weed iness to constitute a plant pest factor (7 CFR § 340.6). Seed germination and dor mancy mechanisms vary among species and their genetic basis tends to be complex. Seed dormancy ( e.g. , hard seed) is an important

characteristic that is often associated with plants that are considered weeds (Anderson, 1996; Lingenfelter and Hartwig, 2003); however , it is not uncommon to observe low

levels of hard seed in conventional s oybean (Mullin and Xu, 2001). Standardized germination assays are available and routinely used to measure the germination characteristics of soybean seed. The Associat ion of Official Seed Analysts (AOSA), an internationally recognized s eed testing organization, recomme nds a temperature range of 20/30°C as optimal for testing the germinati on characteristics of soybean seed (AOSA, 2007).

Comparative assessments of seed dormanc y and germination characteristics were

conducted on MON 87708 and the conventional control. In addition, eight commercial reference varieties were included to provide a range of comparative values that are representative of existing comm ercial soybean varieties. The seed lots for MON 87708, conventional control, and the commercial refere nce varieties were produced in replicated field trials during 2008 in Iowa, Illinois, a nd Missouri, geographic areas which represent environmentally relevant conditions for soybean production for this product. In addition to the AOSA recommended temperature range of 20/30°C, seed was tested at five additional temperature regimes of 10, 20, 30, 10/20, and 10/30°C to assess seed germination properties. The details of the materials, experimental methods, and germination data from all individual pr oduction sites are presented in Appendix F.

In a combined-site analysis, in which the data were pooled am ong the three seed production sites, no statistically significant differences (5% level of significance) were

detected between MON 87708 and the conventiona l control for percent viable hard seed or percent viable firm-swollen seed in any temperature regime (Table VII-2). Within some temperature regimes, it was not possibl e to conduct an analysis of variance for percent viable firm-swollen seed due to no vari ance in the data. For these data, the values

for MON 87708 and the conventional control were all zero, indicating no biological differences. No statistically significant differences we re detected between MON 87708Monsanto Company 10-SY-210U 154 of 721 

### and the conventional control for percent germin ated or percent dead seed in the 20, 30, 10/20, and 20/30°C temperature regimes.

Four statistically si gnificant differences were de tected between MON 87708 and the

conventional control in the combined-site an alysis (Table VII-2). MON 87708 had lower percent germinated seed than the conven tional control at 10°C (98.9% vs. 99.7%) and 10/30°C (98.6% vs. 99.7%). Concurrentl y, MON 87708 had higher pe rcent dead seed than the conventional control at 10°C (0.8% vs. 0.2%) and 10/30°C (1.4% vs. 0.3%). The differences in percent germinated a nd dead seed of MON 87708 were small in magnitude, and the mean values of MON 87708 were all within the range of the

commercial reference varietie s produced in the field trials along with MON 87708 and the conventional control. Furthermore, lowe r percent germinated seed and higher percent

dead seed would not contribute to increased weediness.

The biological characterist ics evaluated were used to characterize MON 87708 in the context of plant pest risk assessment. Based on the seed dormancy and germination characteristics assessed, the results, part icularly MON 87708’s lack of hard seed, demonstrate there were no changes indicativ e of increased weediness or plant pest potential of MON 87708 compared to conventional soybean.Monsanto Company 10-SY-210U 155 of 721 

### Table VII-2. Combined-Site Comparison of MON 87708 to Conventional Control for Seed Dormancy and Germination Characteristics

Temperature Regime Germination

Characteristic 1 Mean % (S.E.) 2 Reference Range 3

MON 87708 Control Min. Max.

10 °C Germinated 98.9 (0 .4)* 99.7 (0.2) 94.4 99.8

Viable Hard 0.0 (0.0) 0.0 (0.0) 0.0 0.3

Dead 0.8 (0.4)* 0.2 (0.1) 0.2 5.3

Viable Firm-swollen 0.3 (0.1) 0.2 (0.1) 0.0 0.4

20 °C Germinated 99.3 (0.4) 99.3 (0.3) 95.3 100.0

Viable Hard 0.0 (0.0) 0.1 (0.1) 0.0 0.4

Dead 0.8 (0.4) 0.6 (0.3) 0.0 4.8

Viable Firm-swollen 0.0 (0.0) 0.0 (0.0) 0.0 0.3

30 °C Germinated 98.7 (0.6) 99.3 (0.3) 93.3 100.0

Viable Hard 0.0 (0.0) 0.0 (0.0) 0.0 0.1

Dead 1.3 (0.6) 0.7 (0.3) 0.0 6.8

Viable Firm-swollen 0.0 (0.0)† 0.0 (0.0) 0.0 0.0

10/20 °C Germinated 99.3 (0.3) 99.3 (0.3) 95.9 100.0

Viable Hard 0.1 (0.1) 0.1 (0.1) 0.0 0.4

Dead 0.6 (0.3) 0.6 (0.3) 0.0 3.9

Viable Firm-swollen 0.0 (0.0) 0.1 (0.1) 0.0 0.3

10/30 °C Germinated 98.6 (0.5)* 99.7 (0.2) 95.9 99.8

Viable Hard 0.0 (0.0) 0.1 (0.1) 0.0 0.0

Dead 1.4 (0.5)* 0.3 (0.2) 0.3 4.1

Viable Firm-swollen 0.0 (0.0) 0.0 (0.0) 0.0 0.1

20/30 °C Normal Germinated 95.9 (1.2) 96.6 (1.0) 86.0 99.0

Abnormal Germinated 2.9 (0.7) 2.8 (0.8) 0.8 9.8

Viable Hard 0.0 (0.0) 0.0 (0.0) 0.0 0.3

Dead 1.2 (0.5) 0.7 (0.3) 0.3 4.1

Viable Firm-swollen 0.0 (0.0)† 0.0 (0.0) 0.0 0.0

Note: The data in this table are the combined-site re sults for the seeds from three 2008 field sites. The experimental design was a split-plot where the whole- plot treatment was seed production site and the subplot treatment was seed material ( i.e ., MON 87708, conventional control, or commercial reference varieties). *Indicates a statistically signifi cant difference between MON 87708 and the conventional control ( α =0.05). † No statistical comparisons were made due to lack of variability in the data.

1 Germinated seed in the AOSA temperature regime (20/30°C) were categorized as either normal germinated or abnorma l germinated seed.

2 Means based on twelve replicates (n = 12) of 100 seeds. The total percentage of all germination

characteristics of MON 87708 or conventional control in some temperature regimes is greater than 100.0% due to numerical rounding of the means. S.E. = Standard Error.

3 Minimum and maximum mean values from among eight commercial reference varieties.Monsanto Company 10-SY-210U 156 of 721 

### VII.C.2. Phenotypic, Agronomic, and En vironmental Interaction Characteristics

Evaluated under Field Conditions

Plant growth, development, and yield ch aracteristics were evaluated under field conditions as part of the plant characteri zation assessment of MO N 87708. These data were developed to provide USDA-APHIS w ith a detailed description of MON 87708

relative to the conventional --control and comm ercial reference varieties. According to 7 CFR § 340.6, as part of the petition to s eek deregulation, a petitioner must submit “a detailed description of the phe notype of the regulated article.” This information is being provided to assess whether there are phenot ypic differences between MON 87708 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 w eediness of MON 87708, an element of APHIS’s plant pest determination. Environmental interactions were also assessed as an indirect indicator of phenotypic changes to MON 8770 8 compared to the same comparators

described above and are also considered in the plant pest assessment.

VII.C.2.1. Phenotypic, Agronomic, and En vironmental Interaction Characteristics

for Untreated MON 87708 Evaluate d under 2008 Field Conditions

Data were collected from field trials located at 16 field sites in the U.S. and two field sites in Canada during 2008 to evaluate phenotypic, agronomic, and environmental

interaction characteristics. These 18 field sites provided a di verse range of environmental and agronomic conditions representative of commercial soybean production areas in North America (Table VII-3). The experiments were arranged as randomized complete block designs with three replications at each field site. All plots of MON 87708, the conventional control, and the commercial refe rence varieties at each site were uniformly managed in order to assess whether the introdu ction of the dicamba tolerance trait altered the phenotypic and agronomic characteristics or the envi ronmental interactions of MON 87708 compared to the conventional cont rol. Therefore, dicamba herbicide was not applied to MON 87708 during the study. A description of the evaluated phenotypic

and environmental interaction characteristic s 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 pr esented and discussed in Appendix G, while the combined-site analyses are summarized be low. The results of this assessment

demonstrated that the introduction of th e dicamba tolerance trait did not alter MON 87708 compared to the conventional contro l in terms of weediness. The lack of differences in plant response to abiotic stre ss, disease damage, arthropod-related damage, and pest and beneficial arthropod abundance further support the conclusion that the

introduction of the dicamba tolerance trait is no t likely to result in increased plant pest potential or an altered environmental im pact from untreated MON 87708 compared to conventional soybean.Monsanto Company 10-SY-210U 157 of 721 

### Table VII-3. 2008 Field Phenotypic Eval uation Sites for Untreated MON 87708

Location Location

Code USDA-APHIS

Notification Number Jackson County, Arkansas AR 08-072-110n Norfolk County, Ontario, Canada Can1 N/A Kent County, Ontario, Canada Can2 N/A Jefferson County, Iowa IA1 08-058-101n Benton County, Iowa IA2 08-058-101n Howard County, Iowa IA3 08-072-110n Clinton County, Illinois IL1 08-058-101n Stark County, Illinois IL2 08-058-101n Boone County, Indiana IN1 08-072-110n Clinton County, Indiana IN2 08-072-110n Parke County, Indiana IN3 08-058-101n Pawnee County, Kansas KS 08-072-110n Ottawa County, Michigan MI 08-072-110n Shelby County, Missouri MO1 08-072-110n Macon County, Missouri MO2 08-072-110n York County, Nebraska NE 08-072-110n Berks County, Pennsylvania PA 08-058-101n Walworth County, Wisconsin WI 08-058-101n N/A = Not applicable, trial was conducted un der a Canadian confined research field testing permit.

VII.C.2.2. Phenotypic, Agronomic, and En vironmental Interaction Characteristics

for Dicamba-Treated Evaluate d under 2009 Field Conditions

In addition to the data from 2008 field tria ls, data were collected from field trials conducted in 2009 at eight sites within U.S. soybean production regions (Table VII-4). MON 87708, the conventional control variet y A3525, and three commercially-released reference soybean varieties were evaluated at each site. A total of 14 reference varieties were evaluated among the sites. The experimental design at each site was a randomized complete block with four replications. A ll plots of MON 87708, the conventional control,

and the commercial reference varieties at each site were uniformly managed in order to

assess whether the introduction of the dicamba tolerance trait in the presence of dicamba altered the phenotypic and agronom ic characteristics or the environmental interactions of MON 87708 compared to the conventional control. Therefore, MON 87708, in each replication at all sites, recei ved an application of a commer cial formulation of dicamba (Clarity) at 0.5 pound a.e dicamba per acre. Treated MON 87708 was compared to the control within each site (i.e., individual-site analysis) and in a combined-site analysis, in which the data were pooled across the eight sites, for 12 plant characteristics. The minimum and maximum mean values (refere nce range) were determined from the references to provide phenot ypic characteristic and enviro nmental interaction values representative of commerc ial soybean varieties.Monsanto Company 10-SY-210U 158 of 721 

### Table VII-4. 2009 Field Phenotypic E valuation Sites for Dicamba Treated

MON 87708

County, State Location

Code USDA-APHIS

Notification Number Jackson County, Arkansas ARNE 09-061-108n Jefferson County, Iowa IARL 09-061-108n Clinton County, Illinois ILCY 09-061-108n Stark County, Illinois ILWY 09-061-108n Parke County, Indiana INRC 09-061-108n Boone County, Indiana INSH 09-061-108n Pawnee County, Kansas KSLA 09-061-108n York County, Nebraska NEYO 09-061-108n

VII.C.2.3. Field Phenotypic and Agronomic Characteristics

VII.C.2.3.1. 2008 Field Phenotypic and Agronomic Characteristics for Untreated

MON 87708

A total of 14 phenotypic and agronomic characte ristics were evaluated (Table VII-5 and Table G-5 of Appendix G). In a combined-sit e analysis in which the data were pooled among the sites, no statistica lly significant differences we re detected (5% level of significance) between untreated MON 87708 and the conventional control for early stand count, seedling vigor, days to 50% flower ing, lodging, pod shatteri ng, final stand count, seed moisture, test weight, or yield (Table VII-5). A statistically significant difference

was detected between untre ated MON 87708 and the conve ntional control for plant height and 100 seed weight in the combin ed-site analysis. Untreated MON 87708 was 6% taller (33.5 vs. 31.6 inches) and had 3.3% lower 100 seed weight than the conventional control (15.0 vs. 15.5 grams). Howe ver, the differences in plant height and 100 seed weight were small in magnitude, a nd the mean values of untreated MON 87708 for both plant height and 100 seed weight were within the range of the commercial reference varieties grown in the trials along with untreated MON 87708 and the

conventional control. The difference in 100 seed weight did not result in a statistically significant difference in final yi eld, and it is unlikely that a di fference in seed weight or plant height would contribute to increased weediness of MON 87708 compared to the

conventional control.

Flower color, plant pubescence, and plant grow th stage data were categorical and were not statistically analyzed; however, at each site, all plants of untreated MON 87708 and the conventional control had purple flow ers and hairy pubescen ce as expected. Additionally, untreated MON 87708 and the conventional contro l were within the same range of plant growth stages for 131 out of the 132 growth stage gobservations among all sites (Appendix G; Table G-4). During the second observation at th e WI site, untreated MON 87708 plants were at the V5 growth stag e while the conventional control was at V4.Monsanto Company 10-SY-210U 159 of 721 

### The growth stage of untreated MON 87708, however, was within the range of growth stages observed for the commercial refere nce varieties grown concurrently (V4

− V5). Thus, there were no biologically meaningful differences in plant development observed

between untreated MON 87708 and the conventional control.

Table VII-5. Combined-Site Comparison of Untreated MON 87708 to Conventional Control during 2008 for Phenotypic and Agronomic Characteristics

Phenotypic Characteristic (units) Mean (S.E.) Reference Range 1



### MON 87708 Control 2 

### Minimum Maximum Early stand count (#/plot) 271.1 (9.0) 265.2 (9.8) 195.7 452.5 Seedling vigor (1-9 scale) 3.5 (0.2) 3.4 (0.2) 1.8 4.5 Days to 50% flowering 3 

### 206.4 (1.2) 206.2 (1.3) 199.6 216.7

Flower color 4 

### Purple Purple Purple Purple Plant pubescence 4 

### Hairy Hairy Hairy Hairy Plant height (in.) 33.5 (0.9)* 31.6 (0.8) 25.4 42.4 Lodging (0-9 scale) 1.2 (0.2) 0.9 (0.1) 0.1 2.9 Pod shattering (0-9 scale) 0.1 (0.0) 0.1 (0.0) 0.0 0.3 Final stand count (#/plot) 251.1 (8.4) 243.4 (8.7) 178.3 338.0 Seed moisture (%) 11.8 (0.3) 11.7 (0.3) 10.4 13.9 100 seed weight (g) 5 

### 15.0 (0.2)* 15.5 (0.2) 14.2 18.7 Test weight (lb/bu) 56.2 (0.3) 55.9 (0.3) 53.6 57.6 Yield (bu/A) 55.4 (2.0) 55.1 (2.3) 37.7 72.7
Note: The experimental design was a randomized comp lete block. S.E. = Standard Error. Means

based on n = 54 for MON 87708 and n = 53 for the conventional control with exception of 100 seed weight for the control where n = 52. * Indicates a statistically significant difference between MON 87708 and the conventional control ( α =0.05).

1 Reference ranges were determ ined from the minimum and ma ximum mean values from among

18 commercial reference varieties.

2 Excessive water damage from heavy precipitation ear ly in the season at the WI site resulted in a poor stand in one replicate of the conventional cont rol. Therefore, the data for all characteristics from the single replicate at the WI site were excluded from the statistical analysis.

3 Calendar day number (from 1 Jan 2008) when approxim ately 50% of the plants in each plot were

flowering.

4 Flower color and plant pubescence data were categorical and not statistically analyzed (see Appendix G, Table G-3).

5 Data on 100 seed weight were inadvertently not co llected from one replicate of the conventional

control at the MO1 site.Monsanto Company 10-SY-210U 160 of 721 

### VII.C.2.3.2. 2009 Field Phenotypic and Agronomic Characteristics for

Dicamba-Treated MON 87708

A total of 12 phenotypic and agronomic characte ristics were evaluated (Table VII-6 and Table G-6 of Appendix G). In the combined -site analysis, no statistically significant

differences were detected between treated MON 87708 and the conventional control for early stand count, seedling vigor, days to 50% flowering, plant height, lodging, pod shattering, final stand count, seed moisture, or yield (Table VII-6). One statistically significant difference was detected between treated MON 87708 and the control, where MON 87708 had a lower 100 seed weight than the control (14.6 vs. 15.6 g). The difference in 100 seed weight is relatively small in magnitude and the mean 100 seed weight of treated MON 87708 was slightly below the reference range. It is unlikely that a difference in 100 seed weight would contri bute to increased weed potential of MON 87708 when treated with dicamba compared to conventional soybean. Flower color and plant growth stage data were categorical and were not statistically analyzed; however, at each site, all plants of treated MON 87708 and the control had purple flowers as expected. Additionally, treated MON 87708 and the control were within the same range of plant growth stages for all growth stage observations among the sites. Thus, there were no biologically-meaningful differences in plant development observed between treated MON 87708 and conventional soybean.

The results of this assessment demonstrated that the introduction of the dicamba tolerance trait and the associated dicamba applicatio n does not alter MON 87708 compared to the

conventional control in terms of weediness. The lack of differences in plant response to abiotic stress, disease damage, and arthro pod-related damage further support the conclusion that the introduction of the dicamba tolerance trait is not likely to result in increased plant pest potential or an altere d environmental impact for dicamba-treated MON 87708 compared to conventional soybean.Monsanto Company 10-SY-210U 161 of 721 

### Table VII-6. Combined-Site Comparison of Dicamba Treated MON 87708 to Conventional Control during 2009 for Phen otypic and Agronomic Characteristics

Phenotypic Characteristic (units) MON 87708 (S.E.) Reference Range 1



### MON 87708 Control Minimum Maximum
Early stand count (#/plot) 298.9 (5.20) 301.1 (3.58) 263.4 340.8 Seedling vigor (1-9 scale) 3.0 (0.33) 3.0 (0.31) 1.0 4.5 Days to 50% flowering 2 

### 214.7 (1.12) 214.6 (1.19) 205.0 226.0 Plant height (in) 31.6 (0.78) 31.2 (0.92) 25.3 38.3 Lodging (1-9 scale) 2.2 (0.27) 2.4 (0.31) 1.0 4.5 Pod shattering (1-9 scale) 1.1 (0.05) 1.1 (0.07) 1.0 1.5 Final stand count (#/plot) 264.8 (6.81) 266.8 (5.20) 219.5 305.5 Seed moisture (%) 13.1 (0.38) 13.4 (0.37) 11.1 17.0 100 seed weight (g) 14.6* (0.23) 15.6 (0.23) 15.0 17.7 Yield (bu/ac) 46.7 (2.37) 46.8 (2.23) 29.4 59.9

Note: The experimental design was a randomized co mplete block with four replications. Site codes are as follows: ARNE = Jackson County, AR; IARL = Jefferson County, IA; ILCY = Clinton County, IL; ILWY = Stark County, IL; INRC = Parke County, IN; INSH = Boone County, IN; KSLA = Pawnee County, KS; NEYO = York County, NE * Statistically significant differences ( α =0.05) between MON 87708(T) and the conventional soybean control.

1 Reference range = Minimum and maximum me an values among the 14 commercially-released reference soybean varieties.

2 Calendar day number (days after 1 Jan 2009) when approximately 50% of the plants in each plot were flowering.Monsanto Company 10-SY-210U 162 of 721 

### VII.C.2.3.3. Field Phenotypic and Ag ronomic Characteri stics for Both

Dicamba-Treated and Untreated MON 87708 - Conclusion

The phenotypic and agronomic characteristics we re used to provide a detailed description

of both dicamba-treated and unt reated MON 87708 compared to the conventional control. A subset of these characteristics was useful to assess the weediness potential of MON 87708. Based on the assessed phenotypic and agronomic characteristics, the results support a determination that both dicamba-treated and untreated MON 87708 are similar to conventional soybean and are no more weedy or likel y to pose a plant pest risk than conventional soybean.

VII.C.2.4. Environmental Interaction Characteristics

USDA-APHIS considers the environmental in teraction of the biotechnology-derived crop compared to its conventional counterpart to determine the potential for increased plant pest characteristics. Evaluations of environm ental interactions were conducted as part of the plant characteri zation for untreated MON 87708. In the 2008 North American field trials conducted for evaluation of phenot ypic and agronomic characteristics of MON 87708, data were also collected on plan t response to abiotic stress (drought, wind, nutrient deficiency, etc.), disease damage, arthropod-rela ted damage, and arthropod abundance (Appendix G; Tables G-9, G-10, G-11, G-12, and G-13) . Similarly these data, except for arthropod abundance, were also collected for the 2009 U.S. field trials (Appendix G; Tables G-14, G-15, and G-16). These data were used as part of the environmental consequences (Section IX) to assess plant pest poten tial and provide an indication of potential effect s of untreated MON 87708 on non- target organisms (NTOs) and threatened and endangered species compared to the conventional control. In addition, multiple commercial reference varieties were in cluded in the analysis to establish a range of natural variability for each assessed characte ristic. The results of the field evaluations

showed that the dicamba to lerance trait did not unexpectedly alter the assessed

environmental interactions of untreated MON 87708 compared to the conventional control. Additionally, results of field evaluations showed that the dicamba tolerance trait in the presence of dicamba herbicide did not unexpectedly alter the assessed

environmental interactions of treated MON 87708 compared to the conventional control. The lack of significant biologi cally meaningful differences in plant response to abiotic stress, disease damage, arthropod-related damage, and pest and beneficial arthropod abundance support the conclusion that the introducti on of the dicamba tolerance trait is unlikely to result in increased plant pest potential or an altered environmental impact from MON 87708 compared to conventional soybean.

In the 2008 field trials, the observations of plant response to abiotic stress, disease damage, and arthropod-related damage were performed four times during the growing season at all 18 sites, a nd arthropod abundance was as sessed quantitatively from collections performed four times during the growing season at f our of the 18 sites ( i.e.

, IL2, IN1, MI, and MO1 sites). In the 2009 field trials, the observations of plant responseMonsanto Company 10-SY-210U 163 of 721 

### to abiotic stress, disease damage, and arth ropod-related damage were performed four times during the growing seas on at all eight sites.

The assessed stressors (abiotic, diseases, and arthropods) were at natural levels as no artificial infestation or impos ed abiotic stress was used and, therefore, typically varied

between observations at a site and among sites. Abiotic st ress and disease damage data were collected from each plot using a non-speci fic 0 – 9 scale of increasing severity of observed damage. The 0 – 9 scale was not de signed to rate specific damage symptoms. However, the non-specific scale was utilized to allow for the evaluation of the wide variety of potential abiotic stressor and disease damage symptoms potentially occurring

across the season and across sites. Due to the non-specific nature of the scale used, the data were not statistically analyzed but rath er assessed qualitatively and placed into one of the following categories: none, slight, moderate, or severe. The response of

MON 87708 and the conventional control to an abiotic stress or dise ase were considered different on a particular observation date at a site if the range of injury severity to MON 87708 did not overlap with the range of in jury severity to the control across all three replications ( e.g. , “none” vs. “slight-moderate” rating). For each observation at a site, the range of injury severity across the commercial reference varieties provided assessment data that are repr esentative of commercial soybean varieties. Arthropodrelated damage was assessed from each plot on the upper four nodes of 10 representative plants using a 0 – 5 pest-specific rating scale of increasing severity of observed damage. These numerical data along with the quan titative arthropod abun dance data were subjected to statistical analysis.

VII.C.2.4.1. 2008 Environmental Interact ion Characteristics for Untreated

MON 87708

In an assessment of abiotic stress respons e and disease damage, no differences were

observed between untreated MON 87708 and the conventional control for 193 out of 194 comparisons for the assessed abiotic stressor s or for any of the 215 comparisons for the assessed diseases among all observations at the sites (Appendix G; Tables G-9 and G-10).

One difference was observed in abiotic stress response duri ng the fourth observation at the WI site where minor wind damage was observed in MON 87708 (“slight” rating) and no wind damage was observed in the conven tional control or commercial reference varieties (“none” rating). The difference, however, was not observed during any of the other 29 wind damage observations among the s ites. Thus, the small difference in wind 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 increased weediness or plant pest potential or an altered environmental impact from untreated MON 87708 compared to conventional soybean.

In an assessment of arthropod-related dama ge, no statistically si gnificant differences were detected (5% level of significance) between untreated MON 87708 and the

conventional control for 89 out of 95 co mparisons for the assessed arthropods (Appendix G; Table G-11). Statistical co mparisons could not be made between

MON 87708 and the conventional control for 121 additional arthropod-related damage comparisons due to no variance in the data; however, the means for MON 87708 and theMonsanto Company 10-SY-210U 164 of 721 

### conventional control were the same value for these comparisons, indicating no biological

differences. A total of six st atistically significant differences involving four taxa were

detected between MON 87708 and the conventional control. MON 87708 had less damage than the conventional control from aphi ds in the third observation at the IA2 site (0.3 vs. 0.5 rating) and second observation at the IA3 site (0.8 vs. 0.9 rating). MON 87708 had less damage than the conventi onal control from blister beetles in the second observation at the MO1 site (0.1 vs . 0.4 rating) and more damage than the conventional control from potato leafhopper in the first observati on at the PA site (1.1 vs. 0.6 rating). MON 87708 had less damage than the conventional control from Japanese

beetle in the second observation at the IN1 si te (0.6 vs. 0.9 rating) and more damage in the fourth observation at the PA site (0.6 vs. 0.4 rating). The mean damage ratings for MON 87708 were within the range of the commercial referenc e varieties grown concurrently with MON 87708 and the conventi onal control for all differences detected in arthropod-related damage with the exceptio n of the differences in Japanese beetle damage at the IN1 and PA sites. The mean Japanese beetle damage rating for MON 87708 at the IN1 site was slightly lowe r than the range of commercial reference varieties for the difference detected (MON 87708 mean = 0.6 rating; range of commercial reference varieties = 0.7 – 0.9 ratings) and sl ightly higher than th e range of commercial

reference varieties for the difference dete cted at the PA site (MON 87708 mean = 0.6 rating; range of commercial reference varieties = 0.3 – 0.5 ratings). Furthermore, the

differences detected in arthr opod-related damage were all small in magnitude and were

not consistent across ob servations or sites. These re sults support a conclusion that the detected differences in arthropod-related da mage were not indicative of a consistent response associated with the trait and were not considered biologi cally meaningful in terms of increased weediness or plant pest potential or an altered environmental impact from untreated MON 87708 compared to conventional soybean.

In an assessment of pest and beneficial ar thropod abundance, no stat istically significant differences were detected (5% level of significance) between unt reated MON 87708 and

the conventional control for 142 out of 151 comparisons, including 74 arthropod pest abundance comparisons and 77 beneficial arthropod abundance comparisons, among the collection intervals at the four sites (Appe ndix G; Tables G-12 a nd G-13). Statistical comparisons could not be made between MON 87708 and the conventional control for eight additional comparisons, including thre e arthropod pest abundance comparisons and five beneficial arthropod abunda nce comparisons, due to no va riance in the data; however,

the means for MON 87708 and the conventional control were the same value for these comparisons, indicating no biologic al differences. A total of ni ne statistically significant

differences were detected between M ON 87708 and the conventional control for arthropod abundance, including seven for pest arthropods and two for beneficial arthropods.

The seven differences detected for pest arthropod abundance included observations for green cloverworm, Japanese beetles, a nd stink bugs (Table G-12). MON 87708 had lower green cloverworm abundance than the c onventional control in the first collection from the IL2 site (0.0 vs. 2.0 per plot), thir d collection from the IN 1 site (7.0 vs. 11.3 per plot), and fourth collection from the MI site (0.0 vs. 0.7 per pl ot); and higher green

cloverworm abundance than the conventional cont rol in the first colle ction from the MIMonsanto Company 10-SY-210U 165 of 721 

### site (1.7 vs. 0.0 per plot). MON 87708 had lo wer Japanese beetle abundance than the

conventional control in the firs t collection from the MI site (2.3 vs. 8.0 per plot). In addition, MON 87708 had lower stink bug abundance than the conventional control in the third collection from the IL2 site (0.0 vs. 1.3 per plot) and higher stink bug abundance in the third collection from the IN1 site ( 2.0 vs. 0.0 per plot). The mean arthropod

abundance values for MON 87708 were within the range of the commercial reference varieties grown concurrently with MON 87708 and the conventional control for all differences detected with the exception of the difference detected for green cloverworm abundance at the IL2 site (MON 87708 mean = 0.0 per plot; range of commercial reference varieties = 0.3 – 2.7 per plot) a nd the IN1 site (MON 87708 = 7.0 per plot; range of commercial reference varieties = 8.0 – 13.0 per plot) and stink bug abundance at the IN1 site (MON 87708 = 2.0 per plot; range of commercial reference varieties = 0.0 – 0.7 per plot). Furthermore, the differences de tected in green cloverw orm, Japanese beetle, and stink bug abundance were all small in ma gnitude and were not detected in other collections or sites where these pests were present. These results support a conclusion that the detected differences in pest ar thropod abundance were not indicative of a consistent response associated with the tr ait and were not considered biologically meaningful in terms of increased weedine ss or plant pest potential or an altered environmental impact from MON 87708 compared to conventional soybean.

The two differences detected for beneficial arthropod abundance in cluded observations

for Araneae (spiders) and Nabis spp. (Table G-13). MON 87708 had lower Araneae abundance (0.0 vs. 3.0 per plot) and higher Nabis spp. abundance (4.7 vs. 1.7 per plot)

than the conventional control in the fourth collection from the MI site. The mean

Araneae abundance value for MON 87708 was slig htly lower than the range of commercial reference varieties (0.7 – 1.0 per plot), while the mean Nabis spp. abundance value for MON 87708 was within the range of commercial reference varieties (2.0 – 6.3 per plot). Furthermore, the differences detected for both Araneae and Nabis spp. abundance were small in magnitude and were no t consistent across collections or sites. The results support a conclusion that the detected differences in beneficial arthropod

abundance were not indicative of a consistent response a ssociated with the dicamba

tolerance trait and were not considered biol ogically meaningful in terms of increased weediness or plant pest potential or an al tered environmental impact from untreated MON 87708 compared to conventional soybean.

VII.C.2.4.2. 2009 Environmental Interact ion Characteristics for Treated

MON 87708

In an assessment of plant re sponse to abiotic stressors and disease damage for the 2009 field trials (Appendix G; Tabl e G-14 and G-15), no differences were observed between

treated MON 87708 and the conventional contro l for 181 of 182 comparisons (including 89 abiotic stress response and 92 disease dama ge comparisons) among all observations at

the sites. One difference was observed be tween treated MON 87708 and the control for white mold during a single observation (slight vs. none). The damage rating for treated

MON 87708 was outside of the reference range (no damage was observed in the references), and this difference was not obs erved in any of the other two white mold

evaluations across the sites.Monsanto Company 10-SY-210U 166 of 721 

### In an assessment of arthropod-related dama ge (Appendix G; Table G-16) there were a total of 93 comparisons. No statistically si gnificant differences were detected between

treated MON 87708 and the control for 56 ou t of 59 comparisons. In addition, no numerical differences were observed for the 34 comparisons for which p -values could not be generated due to lack of variability in the data. Treated MON 87708 had lower bean leaf beetle damage than the control for Ob servation 3 at the KSLA site (0.00 vs. 0.08), and greater grasshopper damage for Observatio n 3 at the INRC site (0.45 vs. 0.10) and at the KSLA site (0.20 vs. 0.03). Th e mean damage ratings for bean leaf beetle damage and grasshopper damage at the KSLA site were wi thin the respective re ference ranges. For the remaining difference, the mean damage rating for grasshopper damage at the INRC

site from treated MON 87708 was outside the reference range; however, this difference

was not consistent across observations or site s. Thus, there was not a consistent response

associated with the dicamba tolerance trait or the herbicide treatment, and the results are not considered biologically meaningful in terms of adverse environmental impacts of treated MON 87708 compared to the conventional soybean.

VII.C.2.4.3. Environmental Interaction Char acteristics for Both Dicamba Treated

and Untreated MON 87708 - Conclusion

The results of the 2008 field evaluations showed that the dicamba tolerance trait did not unexpectedly alter the assessed environmenta l interactions of untreated MON 87708 compared to the conventional control. Additionally, the results of the 2009 field

evaluations showed that the dicamba tolerance trait in the presence of dicamba herbicide did not unexpectedly alter the assessed e nvironmental interactions of MON 87708 compared to the conventional control. The la ck of significant biol ogical differences in plant responses to abiotic stress, disease damage, arthropod-related damage for both dicamba treated and untreated MON 87708, and pest and beneficial arthropod abundance for untreated MON 87708 support the conclusi on that the introduction of the dicamba tolerance trait is unlikely to result in increased plant pest potential compared to conventional soybean.

VII.C.3. Pollen Characteristics

USDA-APHIS considers the potential for gene flow and introgression of the biotechnology-derived trai t into other soybean varieties and wild re latives to determine the potential for increased wee dy or invasive characteristics of the receiving species. Pollen morphology and viability information are pertinent to this assessment and, therefore, were assessed for MON 87708. In addition, charac terization of pollen produced by MON 87708 and the conventional contro l is relevant to th e plant pest risk

assessment because it adds to the detailed description of the phenotype of MON 87708 compared to the conventional control.

The purpose of this evaluation was to as sess the morphology and viability of pollen collected from MON 87708 compared to that of the conventional control. Pollen was collected from MON 87708, the conventional c ontrol, and four commercial reference varieties grown under similar agronomic conditions in a field trial in Illinois. The trial was arranged in a randomized complete block design with three replications. TwentyMonsanto Company 10-SY-210U 167 of 721 

### flowers were collected from each plot. Po llen was extracted, combined among flowers collected from the same plot, and stained with Alexander’s stai n (Alexander, 1980). Pollen viability was evaluated for each sample, and pollen grain diameter was measured for ten representative viable pollen grains per replication. General morphology of the pollen was observed for each of the three replications of MON 87708, the conventional control, and the commercial refere nce varieties (see Appendix H).

No statistically significant differences were detected (5% level of significance) between

MON 87708 and the conventional control for percent viable pollen or pollen grain diameter (Table VII-7). Furthermore, no vi sual differences in general pollen morphology

were observed between MON 87708 and the conventional control. These results

demonstrate that the introducti on of the dicamba tolerance tr ait did not alte r the overall morphology or viability of MON 87708 pollen co mpared to the conventional control. The pollen characterization data contribute to the detail ed phenotypic description of MON 87708 compared to the conventional cont rol. The result supports an overall

conclusion that MON 87708 is similar to conven tional soybean and is no more likely to pose a plant pest risk th an conventional soybean.

Table VII-7. Pollen Characteristics of MON 87708 Compared to Conventional

Control

Pollen

Characteristic Mean (S.E.) 1 

### Reference Range 2



### MON 87708 Control Minimum Maximum

Viability (%) 99.3 (0.3) 98.4 (0.9) 98.1 98.4

Diameter (μm) 24.4 (0.5) 24.3 (0.7) 23.4 24.3

Note: No statistically significant differenc es were detected between MON 87708 and the conventional control ( α =0.05).

1 Means based on n = 3. S.E. = Standard Error

2 Reference ranges were determ ined from the minimum and ma ximum mean value from among

the four commercial reference varieties.



### VII.C.4. Symbiont Interactions

As part of the plant pest risk assessment, USDA-APHIS considers the impact of the biotechnology-derived crop on pl ant pest potential and the e nvironment as well as on agricultural or cultivat ion practices compared to its conve ntional counterpart. Changes in the symbiotic relationship with rhizosphere-inhabiting bacteria Rhizobiaceae and Bradyrhizobiaceae could directly impact pest potenti al, the environment, or cultivation practices ( i.e. , the need to add additional nitrogen to sustain soybean production). Thus, the purpose of this evaluation was to assess whether the introduction of the dicamba

tolerance trait a ltered the symbiotic inte raction of MON 87708 with Bradyrhizobium
japonicum ( B. japonicum ) compared to that of the conventional control.Monsanto Company 10-SY-210U 168 of 721 

### Members of the bacterial family Rhizobiaceae and Bradyrhizobiaceae form a highly complex and specific symbiotic relationship with leguminous plants, including soybean (Gage, 2004). The nitrogen-fixing plant-micr obe symbiosis results in the formation of root nodules, providing an environment in whic h differentiated bacteria called bacteroids are capable of reducing or fi xing atmospheric nitrogen. Th e product of nitrogen fixation, ammonia, then can be utilized by the plant. In soybean, atmospheric nitrogen is fixed into ammonia through a symbiotic association with the bacterium B. japonicum . As a result of this relationship, nitrogen inputs are typically no t necessary for agricultural production of soybean.

The relative effectiveness of the symbiotic association be tween a leguminous plant and its rhizobial symbiont can be assessed by various methods. Assessment of nodule number and mass along with plant growth an d nitrogen status ar e commonly used to assess differences in the symbiotic associ ation between a legume and its associated rhizobia (Israel et al., 1986). It should be noted, however, that nodule number relative to nodule dry weight may be variable in soyb ean experiments because nodules may be larger in diameter and less numerous, while others are not as developed (smaller) but more abundant (Appunu and Dhar, 2006; Israel et al., 1986).

MON 87708, the conventional control, and si x commercial reference varieties were produced from seeds planted in pots containi ng nitrogen-deficient potting medium grown in a greenhouse. Seeds were inoculated with a solution of B. japonicum . The pots were arranged in a randomized complete block design with eight replicates . At six weeks after emergence, plants were excised at the surf ace of the potting medium , and shoot and root plus nodule material were removed from the pots. Nodules were separated from roots prior to enumeration and determination of dry weight. MON 87708 was compared to the conventional control for key ch aracteristics related to their associati on with the soybeanB. japonicum symbiosis. Detailed information on materials and methods used for the symbiont evaluation is pres ented in Appendix I.

No statistically significant differences were detected (5% level of significance) between

MON 87708 and the conventional control for any of the measured characteristics, including nodule number, nodule dry weights, root dry weights, s hoot dry weights, and shoot total nitrogen (percent and mass) (Table VII-8).

Based on the assessed characteristics, th e results support the conclusion that the introduction of the dicamba tolerance trait does not alter the symbiotic relationship

between B. japonicum and MON 87708 compared to the conventional control. Thus, these data further support a conclusion of no change in plant pest potential and no expected impact to cultivation practices relative to nitrogen inputs for MON 87708 compared to conventional soybean.Monsanto Company 10-SY-210U 169 of 721 

### Table VII-8. Symbiont Interaction A ssessment of MON 87708 and Conventional

Control

Characteristic Mean (S.E.) 1 

### Reference Range 2



### MON 87708 Control Minimum Maximum

Nodule Number (per plant) 

### 264 (25) 238 (13) 148 346

Nodule Dry Weight (g/plant) 

### 0.58 (0.03) 0.56 (0.02) 0.43 0.66

Root Dry Weight (g/plant) 

### 1.33 (0.07) 1.15 (0.04) 1.09 1.89

Shoot Dry Weight (g/plant) 

### 6.95 (0.46) 6.03 (0.23) 5.93 8.68

Shoot Total Nitrogen (% dwt) 

### 4.46 (0.05) 4.48 (0.06) 3.51 4.26

Shoot Total Nitrogen (g) 

### 0.31 (0.02) 0.27 (0.01) 0.25 0.32

Note: Pots were arranged in a greenhouse in a randomized complete block design. No statistically significant differences were de tected between MON 87708 and the conventional control ( α =0.05).

1 Means based on n = 8. S.E. = Standard Error

2 Reference ranges were determ ined from the minimum and ma ximum mean value from among

the six commercial reference varieties.



### VII.D. Phenotypic, Agronomic, and Environmental Interactions Assessment

Conclusion

An extensive and robust set of information and data were used to assess whether the

introduction of the dicamba to lerance trait or the introducti on of the dicamba tolerance trait in the presence of dicamba herbicide a ltered the plant pest potential of MON 87708 compared to the conventional control. Phenotypic, agronomic, and environmental

interaction characteristics of both dicam ba-treated and untre ated MON 87708 were evaluated and compared to those of the conve ntional control and c onsidered within the variation among commercial re ference varieties. These assessments included plant growth and development charact eristics; seed dormancy and germination characteristics; pollen characteristics; observations of abio tic stress response, disease damage, arthropodrelated damage and arthropod abundance; and pl ant-symbiont interaction characteristics. Results from the phenotypic, agronomic, an d environmental interactions assessment demonstrate that MON 87708 does not posse ss weedy characteristics, increased susceptibility or tolerance to specific ab iotic stress, diseases, or arthropods, or characteristics that would confer a plant pest risk or significant environmental impact compared to conventional soybean.Monsanto Company 10-SY-210U 170 of 721 

### VIII. U.S. AGRO NOMIC PRACTICES

VIII.A. Introduction

As part of the plant pest assessment re quired by 7 CFR § 340.6(c)(4), impacts to agricultural and cultivation practices must be considered. This section provides a summary of current agronomic practices in the U.S. fo r producing soybean and is included in this petition as a baseline to a ssess possible impacts to agricultural practices

due to the cultivation of MON 87708. Di scussions include soybean production, seed production, plant growth and development, general management practices during the season, management of weeds, insects and diseases, soybean rotational crops, and volunteer soybean management. Information pr esented in Section VII.C.2 demonstrated that MON 87708 is no more susceptible to dise ases or pests than conventional soybean. Additionally data presented in Section VII.C sh ow that, with the exception of tolerance to the herbicide dicamba, MON 87708 is phenot ypically equivalent to conventional soybean. Thus, there are no changes to the inputs needed for MON 87708, and no specific impacts to most of the agronomic practices employed for production of soybean. In the areas where there is potential fo r impact on agronomic practices from the deregulation of MON 87708, the scope and magnitude of those impacts will be discussed.

Soybean is planted in over 30 states, demonstrat ing its wide adaptation to varied soils and climate. The soil, moisture, and temper ature requirements for producing soybean are generally similar to those for corn, and thus th e two crops share a similar cultivation area. Proper seedbed preparation, appropriate variet y selection, appropriate planting dates and plant population, and good integr ated pest management practices are important for optimizing the yield potential and economic return for soybean.

Annual and perennial weeds are perceived to be the greatest pest problem in soybean production (Aref and Pike, 1998). Weeds compete with soybean for water, nutrients, and light resulting in substantial yield losses wh en left uncontrolled. Weed species in soybean vary from region to region and st ate to state. Economic thresholds for controlling weeds in soybean require some fo rm of weed management practice on all soybean acreage. Weed manage ment practices include mechanical tillage, crop rotations, cultural practices, and herbicide application. Numerous selective herbicides are available for preplant, preemergence, and postemergence control of annual and perennial weeds in soybean. Approximately 98% of the soybean acreage in the U.S. receives an herbicide application (USDA-NASS, 2006). Soybean insect s and diseases generally are considered less problematic, although infestations can reach economic thresholds requiring

treatment.

Volunteer soybean, i.e. , soybean plants that have germin ated and emerged unintentionally in a subsequent crop, are not considered a significant concern in rotational crops primarily because of climatic conditions and adequate control of volunteer soybean from tillage practices. Additionally, mechanical a nd chemical control methods are available to manage the occasional volunteer soybean plant. Due to its lack of weediness potential, introduction of MON 87708 in the soybean pr oduction system would have a negligible impact on managing soybean volunteer plants in rotational crops su ch as corn, cotton,Monsanto Company 10-SY-210U 171 of 721 

### and wheat. The numerous control measures that are effective on conventional and Roundup Ready soybean volunteer plants will continue to be effective on volunteer MON 87708 plants when they arise.

As shown in Sections VI and VII, with th e exception of the dicamba tolerance trait, no phenotypic, compositional, or environm ental differences between MON 87708 and conventional soybean have b een observed. Moreover herb icide-tolerant soybean is currently grown on 91% of U.S. soybean acres (USDA-NASS, 2009c ). MON 87708 will

facilitate a wider window of application of dicamba (at planting and in-crop) and will replace or supplement the use of other soybean he rbicides. Therefore, it is not anticipated

that commercialization of MON 87708 in the U.S. would have a notable impact on current soybean cultivation practices, includi ng the management of weeds, diseases, and insects other than the in-crop use of dicamba in soybean production.

VIII.B. Overview of U.S. Soybean Production

VIII.B.1. Soybean Production

Soybean first entered North America in the 18 th 

### century (Hoeft et al., 2000). During the 1930s, soybean started to be processed industria lly in the U.S. for edible oil and protein meal. In 2008, soybean represented 56 percen t of world oilseed production, and about a third of those soybeans were produced in the U.S. (ASA, 2009). In 2008, the U.S. exported 1.16 billion bushels (31.6 million me tric tons) of soybean, which accounted for 40 percent of the world's soybean exports (A SA, 2009). In total, the U.S. exported $20 billion worth of soybean and soybean produc ts globally in 2008 (ASA, 2009). China was the largest export market for U.S. soybean w ith purchases totalli ng $7.2 billion. Mexico was the second largest export ma rket with purchases of $1.7 billion. Other significant markets include the European Union ($1.6 billion) and Japan ($1.3 billion).

Approximately 94% of the world’s soybean seed supply was crushed to produce soybean meal and oil in 2008 (Soyatec h, 2010), and the majority wa s used to supply the feed industry for livestock use or the food indus try for edible vegetable oil and soybean protein isolates.

The productivity of soybean is highly dependent upon soil and climatic conditions. In the U.S., the soil and climatic requirements for gr owing soybean are very similar to corn. The soils and climate in the Midwestern, Easter n, and portions of the Great Plains regions of the U.S. provide sufficient water under normal climatic conditions to produce a soybean crop. The general water requireme nt for a high-yielding soybean crop is approximately 20 inches of water during the growing season (Hoeft et al., 2000). Soil texture and structure are key components dete rmining water availability in soils, where medium-textured soils hold more water, allo wing soybean roots to penetrate deeper in medium-textured soils than in clay soils. Ir rigation is used on appr oximately 9% of the soybean acreage to supplement the water s upply during dry periods in the Western and Southern soybean growing re gions (USDA-ERS, 2008).Monsanto Company 10-SY-210U 172 of 721 

### Most of the soybean acreage is grown as a full-season crop. Approximately 8% of the soybean acres are planted in a double-crop sy stem following winter wheat south of 35o North latitude (Boerma and Specht, 2004). However, this percentage can vary significantly from year to year. The decisi on to plant double-crop soybean is influenced by both agronomic and economic factors. Agro nomic factors include harvest date of the wheat crop, which determines the double-crop soybean planting date , and available soil

moisture. Economic factors include expected soybean price and anticipated economic return (Boerma and Specht, 2004).

The U.S. soybean acreage in the past 10 year s has varied from a pproximately 64.7 to 75.7 million acres, with the lowest acreage recorded in 2007 and the highest in 2008 (Table VIII-1). Average soybean yields have vari ed from 33.9 to 43.3 bushels per acre over this same time period. Annual soybean production ranged from 2.45 to 3.19 billion bushels over the past ten years. According to data from USDA-NASS (2009a), soybean was planted on approximately 75.7 million acres in the U.S. in 2008, producing 2.96 billion bushels of soybean (Table V III-1). Soybean acreage and production in 2008 was up from 2007, mainly due to a decrease in corn ac reage. The value of soybean reached $27.4 billion in the U.S. in 2008 (USDA-NASS, 2009b) . In comparison, corn and wheat values in 2008 were $47.37 and $16.57 billion, resp ectively (USDA-NASS, 2009a, b).

For purposes of this agronomic practices di scussion, soybean production is divided into three major soybean growing regions accounting for 99.1% of the 2008 U.S. soybean acreage: Midwest/Great Plains region (IL , IN, IA, KS, KY, MI, MN, MO, NE, ND, OH, SD, and WI), Southeast regi on (AL, AR, GA, LA, MS, NC, SC, and TN) and the Eastern Coastal region (DE, MD, NJ, NY, PA, and VA) (Table VIII-2). The vast majority of soybean was grown in the Midwes t region, representing 82.1% of the total U.S. acreage. The Southeast and Eastern Coastal regions represented 14.3% and 2.7% of the acreage, respectively. Among the three regions, the Midwest region produced the highest average yield at 38.6 bushels per acre in 2008, and aver age state yields in this region ranged from 28.0 to 47.0 bushels per acre. The average yield in th e Southeast region was 34.4 bushels per acre, with states within this region av eraging from 30.0 to 40.0 bushels per acre. The average yield in the Eastern Coastal region was 34.1 bushels per acre, with individual state averages ranging from 27.5 to 46.0 bushels per acre.

Managing input costs is a major component to the economics of producing a soybean crop (Helsel and Minor, 1993). Key decisions on input costs include choosing what soybean varieties to plant, amounts of fertiliz er to apply, and what herbicide program to use. The average operating cost for pr oducing soybean in the U.S. in 2008 was $127.79 per acre, according to statistics comp iled by the USDA-Economic Research Service (USDA-ERS, 2008). The value of the producti on less operating cost was reported to be $318.66 per acre. A summary of potential pro duction costs and return s are presented in

Table VIII-3.Monsanto Company 10-SY-210U 173 of 721 

### Table VIII-1. Soybean Production in the U.S., 1999 – 2008 1



### Year Acres

Planted (×1000) Acres

Harvested

(×1000) Average

Yield (bushels/acre) Total

Production (×1000 bushels) Value (billions $)
2008 75,718 74,641 39.6 2,959,174 27.40 2007 64,741 64,146 41.7 2,677,117 26.97 2006 75,522 74,602 42.7 3,188,247 20.42 2005 72,142 71,361 43.3 3,086,432 16.93 2004 75,208 73,958 42.2 3,123,686 17.89 2003 73,404 72,476 33.9 2,453,665 18.01 2002 73,963 72,497 38.0 2,756,147 15.25 2001 74,075 72,975 39.6 2,890,682 12.61 2000 74,266 72,408 38.1 2,757,810 12.47 1999 73,730 72,446 36.6 2,653,758 12.21

Ave. 73,277 72,151 39.6 2,854,672 18.02

1 Source is USDA-NASS (2009a,b).Monsanto Company 10-SY-210U 174 of 721 

### Table VIII-2. U.S. Soybean Production by Region and State in 2008 1

Region/State Acres Planted (thousands) Acres
Harvested (thousands) Average Yield (bushels/acre) Total Production (×1000 bushels) Value (billions $) Midwest Region

Illinois 9,200 9,100 47.0 427,700 4.00

Indiana 5,450 5,430 45.0 244,350 2.27

Iowa 9,750 9,670 46.0 444,820 4.29

Kansas 3,300 3,250 37.0 120,250 1.03
Kentucky 1,390 1,380 34.0 46,920 0.42
Michigan 1,900 1,890 37.0 69,930 0.64

Minnesota 7,050 6,950 38.0 264,100 2.54

Missouri 5,200 5,030 38.0 191,140 1.72

Nebraska 4,900 4,860 46.5 225,990 2.12

North Dakota 3,800 3,760 28.0 105,280 0.96

Ohio 4,500 4,480 36.0 161,280 1.55

South Dakota 4,100 4,060 34.0 138,040 1.25

Wisconsin 1,610 1,590 35.0 55,650 0.51

Region Totals 62,150 61,450 38.6 2,495,450 23.30
Southeast Region

Alabama 360 350 35.0 12,250 0.12

Arkansas 3,300 3,250 38.0 123,500 1.09

Georgia 430 415 30.0 12,450 0.11

Louisiana 1,050 950 33.0 31,350 0.29

Mississippi 2,000 1,960 40.0 78,400 0.69

North Carolina 1,690 1,670 33.0 55,110 0.47

South Carolina 540 530 32.0 16,960 0.15

Tennessee 1,490 1,460 34.0 49,640 0.43

Region Totals 10,860 10,585 34.4 379,660 3.35
Eastern Coastal Region

Delaware 195 193 27.5 5,308 0.05

Maryland 495 485 30.0 14,550 0.13

New Jersey 92 90 29.0 2,610 0.02

New York 230 226 46.0 10,396 0.09
Pennsylvania 435 430 40.0 17,200 0.15
Virginia 580 570 32.0 18,240 0.16

Region Totals 2027 1994 34.1 68,304 0.60Monsanto Company 10-SY-210U 175 of 721 

### Table VIII-3. U.S. Soybean Production Costs and Returns in 2008 1

Production Cost or Return Cate g or y Itemized Costs Return per
Planted Acre ( $ USD )

Total Gross Value of Production 446.45

O p eratin g Costs: Seed 44.35

Fertilize r 25.12

Chemicals 15.73

Custom o p erations 6.56
Fuel, lube and electricit y 20.20

Re p airs 12.91

Purchased irri g ation wate r 0.12

Interest on o p eratin g ca p ital 2.80
Total, o p eratin g costs 127.79

Allocated overhead: Hired labo r 2.07 Opportunity cost of unpaid

g rower’s labo r 16.77

Capital recovery of machinery
and e q ui p men t 70.98

Opportunity cost of land (rental rate) 94.58

Taxes and insurance 9.64

General farm overhead 14.29
Total, allocated overhead 208.35

Total cost listed 336.13

Value of production less total cost
listed 110.32

Value of production less operating

costs 318.66

Supporting Information: Yield = 43 bushels/acre, Price = $10.48/bushel, Enterprise size = 303 planted acres, Irrigated = 9%, Dry land = 91%.

1 Source is USDA-ERS (2008).Monsanto Company 10-SY-210U 176 of 721 

### VIII.B.2. Soybean Seed Production

Standardized seed production practices are responsible for maintaining high-quality seed stocks, an essential basis for U.S. agricultu re. By the early 20th century, agronomists learned how to develop specific plant varieties with desirable traits. In the U.S., state agricultural experiment stations developed many seed varietie s that were distributed to growers for use. Seed was saved by grower s and later sold to ne ighbors; however, the

desirable traits of the varieties often we re lost through random genetic changes and contamination with other crop a nd weed seed (Sundstrom et al., 2002). The value of seed quality (including genetic purity, vigor, and pr esence of weed seed, seed-borne diseases, and inert materials, such as dirt) was quickly identified as a major factor impacting crop yields. States developed seed laws and certif ication agencies to ensure that purchasers

who received certified seed could be assured that the seed met established seed quality standards (Bradford, 2006). The federal governme nt passed the U.S. Federal Seed Act of 1939 to recognize seed certification and the esta blishment of official certifying agencies. Regulations first adopted in 1969 under the Fede ral Seed Act recognize land history, field isolation, and varietal purity standards for foundation, registered, an d certified seed. Under international agreements such as the Organization for Economic Co-Operation and Development (OECD) scheme, the U.S. an d other countries mutually recognize minimum seed quality standards (Bradford, 2006). The Association of Official Seed Certifying Agencies (AOSCA) represents state and private seed certification organizations in the U.S., and includes inte rnational member coun tries in North and South America, Australia, and New Zealand.

Soybean seed is separated into four seed cl asses: 1) breeder, 2) foundation, 3) registered, and 4) certified (AOSCA, 2009). Breeder seed is seed di rectly controlled by the originating or sponsoring plan t breeding organization or firm . Foundation seed is firstgeneration seed increased from breeder seed and is handled in a manner to maintain specific levels of varietal pu rity and identity. Register ed seed is the progeny of

foundation seed that is handled to maintain satisfactory varietal purity and identity. Certified seed is the progeny of breeder, foundation or registered seed, and is typically two generations removed from foundation see d. While not all soybean seed sold to growers is officially certified, commercial soybean seed sold and planted for typical soybean production is produced pr edominately to meet or exceed certified seed standards. This section of the petition will provide a broad overview of the practices used in producing certified seed.

Soybean seed breeders and producers have put in place practical measures to assure the quality and genetic purity of soybean varietal seed for commercial planting. The need for such systems arose from the recognition that the quality of improved soybean varieties quickly deteriorated in the absence of mon itoring for quality and genetic purity (CAST, 2007). Seed certification programs were ini tiated in the early 1900s in the U.S. to preserve the genetic identity and variety purity of seed. There are special land requirements, seed stock eligibility requirements, field inspections and seed labeling

standards for seed certification. Seed certific ation services are available through various state agencies affiliated with AOSCA. Large seed producers implement their own seed quality assurance programs. However, large seed producers often will utilize the servicesMonsanto Company 10-SY-210U 177 of 721 

### of state certifying agencies as a third party source to perfor m certain field inspections and

audits.

U.S soybean production for all purposes ha s varied from appr oximately 64.7 to 75.7 million acres in the past ten years (USDA- NASS, 2009a; Table VIII-1). To plant this area of soybean acreage require s 105 to 125 million units (50 lbs/unit) of soybean seed.

This seed volume includes allowances for seed losses due to weat her, poor yields, and quality issues. Additional allowances are included for distribution excess, seed returns, replants, and potential increases in soybean acreage. Assuming an average soybean yield of 45 bushels, or 54 units (50 lbs/unit) per ac re, 1.9 to 2.3 million acres would be required to produce this volume of commercial certified soybean seed each year.

Certified soybean seed is produced througho ut most of the U.S. soybean-growing regions. Soybean varieties are developed and adapted to certain ge ographical zones and are separated into ten maturity groups – Group 00 to Group VIII (see Section VIII.C). Seed production for these matur ity groups is grown in the respective geographical zone for each maturity group. However, the pr oduction areas generally are on the northern edge of the respective zone to mi nimize incidences of disease.

Soybean seed is produced by a number of comp anies that produce and sell seed, such as Monsanto Company, Pioneer Hi-Bred Interna tional, Syngenta Seeds, Kruger Seed Co., and Becks Hybrids. In addi tion, certified seed is produced by toll seed producers, or tollers, which are companies that produce but do not directly sell cert ified seed, such as Remington Seeds LLC and Precision Soya. Seed companies and tollers in turn contract acreage with growers to produce the needed amount of soybean seed. Seed production or processing plants at these seed companies identify local soybean growers to produce the seed and also monitor and inspect seed fi elds throughout the grow ing season. The seed production plants also clean, condition, and ba g the harvested soybean seed as well as monitor and inspect all the pr ocesses at the plant. Produc tion plants typically produce between 100,000 units to 2,000,000 units of soybean seed. Production plants will produce the various soybean varieties in differe nt climates or environments to spread production risks.

The entire seed production process at the majority of the seed companies and tollers operate using International Or ganization for Standardization (ISO) certification standards and, therefore, include internal and external audits (ISO, 2009). ISO standards ensure

desirable characteristics of seeds and servic es, 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 or ganization can consistently deliver excellent product or services. The standards not only must meet the customer’s requirements and applicable seed regulatory requirements, bu t must aim to enhance customer satisfaction and achieve continual improvement of its performance in pursuit of these objectives (ISO, 2009).

The field operations and management practices for producing soybean seed are similar to normal soybean production. However, special a ttention is needed in certain areas to produce seed with high quality, high germina tion rates, and high genetic purity (HelselMonsanto Company 10-SY-210U 178 of 721 

### and Minor, 1993). General guide lines specific for seed prod uction are discussed below. Importantly, the seed production field should no t have been planted with soybean in the previous crop season in order to avoid pot ential volunteer soybean plants (even though the risk of soybean volunteer plants is negligible) and to en sure genetic purity.

Very early planting is typically avoided b ecause the seed produced from early planting often results in poorer quality seed (Helsel a nd Minor, 1993). Every effort must be made to eliminate weeds in a seed field through the use of herbicides and cultivation practices to prevent weed seed in the harvested soybean seed. Fields are scouted frequently for insect pests and insecticides are applied when insect pest infestations reach economical

threshold levels. Foliar-a pplied fungicides should be considered when disease infestations are predicted in the area. Harvest should oc cur as soon as the mature soybean seed reaches 13% moisture content. Harvesting soybean seed with less than 13% moisture can cause damage to the seed co at and result in split soybean seed that can affect germination and viability. Harvesting equipment must be adjusted to minimize or avoid seed damage. Harvesting equipment mu st be cleaned before entering the seed fields to assure genetic purity. Certain seed handling equipment, such as auger elevators, should be avoided because they can increase seed damage.

Field inspections are vital to ensure the soybean 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 soybean growing season to vi sually evaluate variety purity, ensure soybean plants are developing properly, and fiel ds are maintained free of weeds, insects, and diseases. The fields are also mapped to ensure the seed field has the minimum federal isolation requirement of five feet as a physical barrier (AOSCA, 2009). Some states and seed producers have a stricter isolation requirement of 10 feet.

Production plant personnel make every effort to avoid mechanical damage to the harvested seed during the scr eening, cleaning, and bagging process. Specific methods are used to assure the genetic purity and the iden tity of the seed is maintained throughout the handling and storage operation. Bin inspections and sample collections are conducted at storage locations at the seed production plant to examine th e physical characteristics of the soybean seed and to ensure proper bin cleanout. Seed is inspected for appearance, disease, discoloration, seed coat, mechanical damage, inert matter, and weed seed. Warm and cold germination tests are conducted on all seed lots to verify acceptable germination rates. Many seed companies will also conduc t tetrazolium staining tests to assess seed viability.

Commercially certified soybean seed must m eet state and federal seed standards and labeling requirements. AOSCA standards for ce rtified soybean seed are as follows: 98% pure seed (minimum), 2% inert matter (max imum), 0.05% weed seed (maximum, not to exceed 10 per lb.), 0.60% total of other crop seeds (maximum), 0.5% other varieties (maximum, includes off-colored beans and off-type seeds), 0.10% other crop seeds (maximum, not to exceed three per lb.), and 80% germination and hard seed (minimum) (AOSCA, 2009). State seed certification standa rds vary slightly from state to state and

can be more restrictive than th e seed standard s of AOSCA.Monsanto Company 10-SY-210U 179 of 721 

### When deregulated, MON 87708 seed will be produced in the same manner as commercially certified soybean seed, such that it will meet all state and federal seed standards and labeling requirements.

VIII.C. Production Management Considerations

VIII.C.1. Pre-Season

Well in advance of planting a soybean crop, decisions are made regarding the planned crop rotation, the tillage system and row sp acing that will be im plemented, the planting equipment that will be used, the seed or vari ety that will be planted, and soil fertility management requirements. Many of the decisi ons in this area are made prior to or immediately after harvest of th e previous crop. There are ma ny benefits to crop rotation, with the majority of the soybean acreage plan ted in a two-year corn-soybean rotation (see Section VIII.I). Crop rotation is generally a long-term decision, but the rotation sequence can be modified to take advantage of a pa rticular economic or market opportunity. The decision to plant soybean in a conservation tillage or no-till system may require special equipment and will be made long before plan ting. In addition, this decision on tillage system usually will be a long-term commitment, provided the system is successful. A decision to change row spacing is a similar l ong-term commitment that generally requires new equipment.

The benefits of conservation tillage or no-till systems are well documented and include reduced soil erosion, reduced fuel and labor costs, and co nservation of soil moisture (CTIC, 2011). In 2004, approximately 27.5 million acres (39.6%) of soybean were planted in a no-till system (CTI C, 2007). Slow soybean emer gence and growth leading to lower yields have been some of the concer ns associated with adoption of conservation tillage systems in soybean, especially no-ti ll. Research in Wi sconsin and Minnesota shows that soil temperatures can be four to five degr ees colder in no-till than conventional tillage systems, wh ich can slow seedling emergence, but have little effect on soybean yield (Pedersen, 2008a). Improved planters for establishment of good soybean populations and planting Roundup Ready soyb ean allowing the use of glyphosate to effectively control weeds in no -till fields have made no-till a viable production system for soybean (Pedersen, 2008a). Extension special ists still recommend some spring tillage on fine-textured and poorly draine d soils for proper seedbed prep aration (Pedersen, 2008a).

Most field crops, including soybean, respond well to fertilizer when pl anted in soils with low fertility levels. Soybean requires 16 essential elements for growth and development. Deficiencies in any of these elements can re duce yields (Hoeft et al., 2000). The primary or major essential nutrients are nitrogen, phosphorus and potassium. The soybean plant is a member of the legume family, like alfa lfa and clover, and fixes a significant portion of its own nitrogen through the symbiotic relationship with the nitrogen-fixing Bradyrhizobia bacteria ( Bradyrhizobium japonicum ) that live in the nodules on its roots. Bradyrhizobia are unicellar, microscopic bact eria that invade th e soybean plant through its root hairs (Hoeft et al., 2000). The plant responds to this invasion by forming nodules

which contain colonies of bact eria. Once established on the s oybean root, bacteria in the nodule take gaseous nitrogen from the atmosphere and fix it in forms easily used by theMonsanto Company 10-SY-210U 180 of 721 

### soybean plant. Since these bacteria are not native to U.S. soils and would not normally be found in these soils, inoculation of th e soybean seed with these bacteria is recommended when soybean has not been grow n in a field for three to five years. Nitrogen fertilizer applications at planting generally do not improve yield and decrease nodulation while increasing the plant’s depend ency on the soil for nitrogen (Pedersen, 2008a). Therefore, nitrogen fertilizer is sel dom applied prior to planting a soybean crop.

Soil tests are the only reliable way to determine the pH , phosphorus, and potassium levels in the soil. Liming and fertilizer requiremen ts subsequently are determined based on soil

test results. Ideal soil test results for corn are also ideal for soybean (Scott and Aldrich, 1970). In corn-soybean rotations in the Mi dwest, phosphorus and potassium fertilizers are applied prior to a corn crop in accordan ce with soil test recommendations, but are seldom applied prior to a soybean crop. Howeve r, in some of the southern growing areas, differences in crop rotations and soil types may require a fertilizer application prior to planting soybean.

Although not common, deficiencies in soil ca n occur in secondary nutrients (calcium, magnesium, and sulfur) or micronutrients (boron, chloride, copper, iron, manganese, molybdenum, and zinc). The availa bility of soil nutri ents is dependent on soil acidity or pH level. Because soybean is adversely aff ected when the pH is below approximately 5.8 (Hoeft et al., 2000), soil pH should be main tained at about 6.0 to 6.5 through the addition

of limestone.

Soybean varieties are developed and adapte d to certain geographical zones and are separated into ten maturity groups – Group 00 to Group VIII (Pimentel, 1991; Zhang et al., 2004). Groups 00 and 0 are the earliest maturity groups a nd are adapted best to the

area north of latitude 46o North. Succeedin g groups are adapted further south with Groups I and II within latitudes 41o and 46o North, and Group III within latitudes 38o and 41o North. Group 00 through Group IV soybean va rieties are planted in the Midwest and Eastern Coastal regions. Groups II, III a nd IV account for approximately 76% (24%, 36%, and 16%, respectively) of the soybean pl anted in the U.S. (T. Schlueter, personal communication, August 2008). Groups IV through V III are planted in the southern states with Groups V, VI and VII representing 7% , 2%, and 2% of the planted soybean, respectively (T. Schlueter, pers onal communication, August 2008).

Soybean variety selection is cr ucial for high yield and quality, and is the foundation of an effective management plan (Pedersen, 2008a). Characteristics to consider in selecting a variety include maturity, yield potential, dise ase and pest resistance, iron deficiency tolerance (chlorosis), lodging sc ore, height, and specific soyb ean quality traits, such as protein and oil content. If a field has a hist ory of a particular di sease or pest, planting soybean varieties that have resistance or tole rance to these pests and diseases can be an

effective and economical method of control.

VIII.C.2. Planting and Early Season

An understanding of the growth stages of soybe an is also important for the proper timing of certain management practices, such as he rbicide and insecticide applications. InMonsanto Company 10-SY-210U 181 of 721 

### addition, the impact of certain weather conditions, insect pe sts, and diseases on soybean yield is dependent on growth stage. The syst em of soybean growth stages divides plant development into vegetative (V) and repr oductive (R) stages (Pedersen, 2008a). The vegetative stages begin with VE, which designa tes emergence. V stages continue and are numbered according to how many fully deve loped trifoliate leaves are present ( i.e. , V1, V2, etc.). The reproductive (R) stages begin at flowering (R1) and include pod development and plant maturation. Full maturity is designated as R8.

Adequate soil moisture and warm temperatures facilitate rapid seed germination and emergence. The ideal soil temperature for soybean germination and emergence is 77oF (Pedersen, 2008a). However, waiting for soils to reach this soil temperature will delay planting beyond the optimum planting date th at will maximize yield. Soybean can germinate at a soil temperature of 50oF when planted at a depth of two inches. However, emergence is slow and can take up to three weeks in northern climates. Because of fluctuations in soil temperature in early spring, soil temperature should not be the only criteria for optimum planting time. Planti ng into a good seedbed is the most important consideration. Planting into soil that is too wet will reduce emergence and plant population, and can lead to reduced yield.

Planting date has the greatest impact on yiel d, according to research conducted in the Northern states (Hoeft et al ., 2000). Highest yields are gene rally obtained when planting in early to mid May. Yields begin to declin e quite rapidly when planting is delayed until late May. For example, the optimum planting dates for soybean in Io wa are the last week of April in the southern two-thirds of the st ate and the first week of May in the northern one-third of the state (Pederse n, 2008a). In the Southern U. S., planting adapted varieties before late April results in shorter plants and, in many cases, lower yields than when the same varieties are planted in May or early June. Planting after early June generally decreases plant height and yield due to water shortages in Ju ly and August.

Variations in plant spacing through row spaci ng and plant populati on have a significant effect on canopy development and soybean yield. Row spacing is important to maximize soybean yield. Research in the Midwest over the past 20 years cons istently shows that row spacing of less than 20 in ches is preferred for soybean regardless of tillage system, rotation sequence or planting date (Pederse n, 2008a). In the Southern states, the advantage from narrow rows is less c onsistent and less beneficial. In 2000, approximately 40% of soybean was planted in row spacing of 10 inches or less, 27% in 10.1 to 28.5 inches, and 33% in rows wider than 28.5 inches (Hoeft et al., 2000).

Soybean has the ability to produce good yield over a wide range of plant populations. Most soybean varieties have the ability to branch and adjust the number of pods on branches to compensate for large differences in seeding rate. Maximum yields generally require planting rates that resu lt in about 2.5 to 5 plants per square foot (Hoeft et al., 2000). Therefore, a full stand of soybean is approximately eight to ten plants per foot of row at harvest for 40-inch rows, six to eight pl ants per foot of row in 30-inch rows, four to six plants in 20-inch rows, and two to three plants in 10-inch rows. This translates to 109,000 to 218,000 plants per acre at harvest. Higher populations are recommended in narrow rows for maximum yields because plan ts are more uniformly spaced in narrowMonsanto Company 10-SY-210U 182 of 721 

### rows. Seeding rates are gene rally 10 to 25% higher than the desired harvest population, especially in no-till fields, to account fo r the losses in germination, emergence, and seedling diseases. The accuracy of the planti ng equipment also can impact the decision on seeding rate. Soybean seed traditionally has been sold by weight. Therefore, the grower must know the number of seeds per pound for the particular soybean varieties being planted for accurate seeding rates.

Treating soybean seed with a fungicide ( e.g. , metalaxyl or mefenoxam) to prevent damping-off diseases may be beneficial when planting in cold, we t soils, using reduced till and no-till planting systems, and when pl anting seed with a low germination rate (<80%) or low seed vigor (Pedersen, 2008a).

Annual and perennial weeds are considered to be the greates t pest problem in soybean production (Aref and Pike, 1998). In order to maximize yields , weeds must be controlled during the early growth stages of soybean because weeds compete with soybean for water, nutrients, and light. There have been many studies examining the loss in soybean yield due to weed competition. The amount of loss is dependent upon the species, time when the weed is growing with the crop, and crop cultural practices, particularly row spacing. A sampling of these studies show that soybean yield reductions are generally in the range from 10% to 50% (N orsworthy et al., 2002; Shurtl eff and Coble, 1985; Vail et al., 1993, Hock et al., 2006). In one study, yiel d loss due to several annual grasses ranged from 13% to 16% while yield losses due to various annual broadleaf weeds ranged from 23% to 52% (Hock et al. 2006). A combination of tillage and herbicides are used to control weeds throughout the gr owing season (Section VIII.F).

VIII.C.3. Mid to Late Season

Ideal daytime temperatures for soybean grow th are between 75oF and 85oF (Hoeft et al., 2000). Warmer temperatures result in larger plants and earlier flowering. Sustained temperatures below 75oF will delay the beginning of flowering significantly. Seed set also is affected by temperature. Seed set is generally good when pollination follows night temperatures around 70oF. Soybean vari eties differ in their response and tolerance

to temperatures.

Soybean is photoperiod sensitive, which mean s that it transitions from vegetative to flowering stage in direct re sponse to length of daylight (Scott and Aldrich, 1970). Most soybean varieties begin flowering soon after th e day length begins to shorten. Flowering of southern varieties is initiated by a shorte r day than that of varieties adapted to the north. The extent of vegetative growth occu rring after the initiation of flowering depends not only on environmental factors but also th e growth habit. Soybean varieties are described as either indeterminate or determinate in their growth habit (Scott and Aldrich, 1970). Indeterminate varieties in crease their height by two to four times after flowering begins. Indeterminate varieties are typically grown in the northern and central U.S. Determinate varieties increase their height ve ry little after flower ing and generally are grown in the southern U.S. Indeterminate and determinate varieties also differ in flowering characteristics. Indeterminate plants generally bloom first at the fourth or fifthMonsanto Company 10-SY-210U 183 of 721 

### node and progress upward. Flowering on determin ate plants begins at the eight or tenth node and progresses both downward and upward.

The first appearance of flowers signals th e beginning of the reproductive stage, namely the R1 stage (Hoeft et al., 2000). The reprodu ctive period consists of flowering, pod set,

and seed formation. Climatic conditions such as temperature and moisture supply during the flowering period will affect the number of flowers. Th e soybean plant does not form a pod from each flower. It is common for the soybean plant to have 75% of the flowers fail to develop a pod (Scott and Aldrich, 1970). This characteristic makes soybean less susceptible than corn to s hort periods of adverse weat her during flowering. Under normal conditions, pod set occurs over about a three week period. G ood soil moisture is most critical during the pod-f illing stages to prevent pod a bortion and to ensure high yields (Hoeft et al., 2000). A nother critical requirement duri ng the seed-filling stages is a high rate of photosynthesis to maximize yiel d. High humidity and temperatures during seed development and maturity can result in poor seed qua lity because these conditions promote the development of reproductive-stage diseases.

VIII.C.4. Harvest Season

When dry matter accumulation ends, the plant is considered to be physiologically mature. The seed moisture content is approximately 55 to 60% at this stage (Hoeft et al., 2000). At this stage, namely R7, at least one normal pod on the plant reaches the mature pod color. Under warm and dry w eather conditions, seed moisture content will drop to 13 to 14% in 10 to 14 days from physiological maturity (Hoeft et al., 2000). Soybean can be harvested when the moisture content drops be low 15%. However, s oybean should be at

13% moisture to be stored without artific ial drying (Scott and Aldrich, 1970). Moisture content below 12% may increase seed cracking and seed coat damage.

Pre-harvest losses are influenced by soybean variety, weather, and timeliness of harvest (Scott and Aldrich, 1970). Time ly harvest when the moisture content is 13 to 14% also will minimize losses. Proper operation and adjustment of the combine is essential to minimizing harvest losses in the field.

VIII.D. Management of Insects

Although insects are rated as less problematic than weeds in U.S. soybean production, management of insect pests during the growth and development of soybean is important for protecting the yield of soybean (Aref a nd Pike, 1998). Understanding the impact of insects on soybean growth is essential fo r proper management (Higley and Boethel, 1994). It is important to unders tand the way that insects inju re soybean as well as how the soybean plant responds to insect injury. Insect inju ry can impact yield, plant maturity, and seed quality. Insect injury in soybean seldom reaches levels to cause an economic loss, as indicated by the low percenta ge (16%) of soybean acr eage that receives an insecticide treatment (USDA-NASS, 2007b).

Characterizing soybean responses to insect in jury is essential in establishing economic injury levels (Higley and Boet hel, 1994). Most often, soybean insects are categorized orMonsanto Company 10-SY-210U 184 of 721 

### defined by the plant parts they injure, namely root-feeding, stem-feeding, leaf-feeding, or pod-feeding insects. The root- and stem-feedi ng insect groups are often the hardest to scout and typically are not det ected until after they have cau sed their damage. The leaffeeding insects comprise the bi ggest group of soybean insect pe sts, but not necessarily the most economically damaging insects. Resear ch on defoliation has determined that a major effect of leaf injury is to reduce light intercepti on by the soybean canopy which in turn can have a significant effect on yield (Higley and Boethel, 1994). Soybean has an extraordinary capacity to withstand consider able defoliation early in the season without significant yield loss. By contrast, defolia tion during the flowering and pod filling stages poses a greater threat to yiel d because the soybean plant has less time to compensate for injury compared to other growth stages. Re search indicates that the soybean plant can sustain a 35% leaf loss prior to the pre-bl oom period without lowe ring yield (NDSU, 2002). However, from pod-set to maturity, th e plant can tolerate only a 20% defoliation level before yield is impacted.

VIII.E. Management of Diseases and Other Pests

More than 100 pathogens are known to affect soybean, of which 35 are considered to be of economic importance (Heatherly and Hodge s, 1999). The estimated yield losses to soybean diseases in the U.S. were 12.5, 13.2, and 13.0 million metric tons in 2008, 2009, and 2010, respectively ( Wrather and Koe nning, 2011), which equated to 15.5%, 14.4%, and 14.4% of total soybean production, respect ively (ASA, 2011). Pathogens can affect all parts of the soybean plant, resulting in reduced quality and yield. The extent of losses depends upon the pathogen, the state of plan t development and health when infection occurs, the severity of the di sease on individual pl ants, and the number of plants affected (Heatherly and Hodges, 1999).

One or more diseases can generally be f ound in fields wherever soybean is grown (Heatherly and Hodges, 1999). However, a path ogen may be very destructive one season and difficult or impossible to find the next season. The extent and severity of soybean diseases depend on the degree of compatibility between the host and the pathogen and the

influence of the environment.

According to field surveys conducted in soybean-producing states during 1996 to 2010, soybean cyst nematode (SCN), Heterodera glycines , caused the greatest soybean yield losses (Wrather and Koenning, 2011). Phytophthora root and stem rot ( Phytophthora
sojae ), brown spot ( Septoria glycines ), charcoal rot ( Macrophomina phaseolina ),

Sclerotinia stem rot ( Sclerotinia sclerotiorum ), seedling diseases, and sudden death syndrome ( Fusarium solani f.sp. glycines ) followed in economical importance. As expected, yield losses vary by region. Sclerotinia stem rot caused yield losses in several Northern states, but not in other states. Rhizoctonia foliar blight losses were greatest in Arkansas, Louisiana, and Texas where humidit y and temperature conditions are suitable for disease development (Wrather et al., 2001).

Selecting resistant varieties is the primar y tool growers have for disease control (Heatherly and Hodges, 1999). Resistant varieties may have morphological or physiological characteristics that provide immunity, resistance, toleran ce or avoidance toMonsanto Company 10-SY-210U 185 of 721 

### certain pathogens. Cultural practices can al so play an important role in disease management by reducing initial inoculums or reducing the rate of disease development (Heatherly and Hodges, 1999). Preplant tillag e can bury crop residue, which encourages the decomposition of fungal-resting structures . Crop rotation is routinely recommended as a disease-management strategy. Rotating cr ops interrupts the disease cycle and allows time for the decomposition of inoculums. One exception is Rhizontonia spp., a soilinhabitant pathogen that grows on a wide vari ety of crops and can survive sufficiently in the soil to make crop rotation as a means of controlling this pest impractical. Row spacing, plant population, and planting date al so can be changed to manage soybean

diseases.

Soybean cyst nematode is one of the most damaging pathogens of soybean throughout the soybean growing regions of the U.S. (P edersen, 2008b). Losses have been estimated to be at about $1.5 billion in the U.S. (Peder sen, 2008a). SCN can cause yield losses up to 50%, where this pest in 2004 alone caused an estimated loss of 50 million bushels of soybean in Iowa (Pedersen, 2008c). Soybean cyst nematodes feed on the roots, causing severely stunted and yellow plants. The simplest, least expensive method to reduce populations of this pest is to rotate soybean with a non-host crop such as corn, small grains, or sorghum. Planting resistant varieties is regarded as the best and most effective management practice to prevent losses from th is pest. Several public and private soybean

varieties offer sources of resi stance to certain races of nematode. Alternating varieties

with different sources of resist ance also is beneficial.

High-quality seed is essential for control ling seedling diseases. The most important seedling diseases in soybean are Phytophthora spp., Pythium spp., Rhizoctonia spp., and

Fusarium spp. (Pedersen, 2008a). Many soybean va rieties demonstrate resistance to specific taxonomic races of Phytophthora . Treating soybean seed with a fungicide ( e.g. , metalaxyl or mefenoxam) is effective agai nst damping-off dis ease (seedling blight) caused by common soil fungi, such as Phytophthora spp

. and Pythium spp. Fungicide

seed treatments are recommended where there is a history of these seedling diseases.

Asian soybean rust is a foliar fungal dise ase that typically infests soybean during reproductive stages of development and can cause defoliation and reduce yields significantly in geographies su ch as Brazil (Dorrance et al., 2007). Soybean rust is caused by the fungus Phakopsora pachyrhizi . This disease in the U.S. was first detected in Louisiana in 2004 (LSU, 2009). At this ti me, foliar application of fungicides is the standard disease-management practice to li mit yield losses due to soybean rust.

Foliar fungicide applications can effectively reduce the incidence of many fungal diseases (Heatherly and Hodges, 1999). Howe ver, the economic return from a fungicide application may be limited to select soybean production systems; for example, high-yield environments or when producing soybean seed. According to USDA-NASS (2007b) statistics, fungicides were a pplied on approximately 4% of the soybean acreage in 2006.Monsanto Company 10-SY-210U 186 of 721 

### VIII.F. Weed Management

Annual weeds are perceived to be the grea test pest problem in soybean production, followed by perennial weeds (Aref and Pike, 1998). Soybean insects and diseases are rated less problematic but may reach economic thresholds requirin g treatment. Weed control in soybean is essential to optimizing yields. Weeds compete with soybean for light, nutrients, and soil moisture. Weeds can harbor insects and di seases, and also can interfere with harvest, caus ing extra wear on harvest e quipment (Pedersen, 2008a). The primary factors affecting soybean yield loss fr om weed competition are the weed species, weed density, and the duration of the competitio n. When weeds are left to compete with soybean for the entire growing season, yield losses can excee d 75% (Dalley et al., 2001). Generally, the competition between crops and weed s increases with higher levels of weed density. The time period that weeds compete with the soybean crop influences the level of yield loss. In general, early season weed competition will have the greatest negative impact on yield (Dalley et al., 2001). S oybean plants withstand early-season weed competition longer than corn without affec ting yield, and the canopy closes earlier in soybean than corn. In addition, canopy closur e is much sooner when soybean is drilled or planted in narrow rows. The most comm on weeds in soybean for each of the three major U.S. growing regions are presente d in Tables VIII-4, VIII-5 and VIII-6.

Crop rotations and environment have a significant impact on the adaptation and

occurrence of weeds in soybean. Foxtail spp. ( Setaria spp

. ), pigweed ( Amaranthus spp.), velvetleaf ( Abutilon theophrasti ), lambsquarters ( Chenopodium album ), and cocklebur ( Xanthium strumarium ) are common weeds in Midwest corn and soybean fields. However, growers consider giant ragweed ( Ambrosia artemisiifolia ), lambsquarters, Canada thistle ( Cirsium arvense) , cocklebur, and velvetleaf to be the top five most problematic weeds in corn and soybean becau se of difficulty controlling these weeds (Nice and Johnson, 2005). In a recent survey of growers u tilizing glyphosate-tolerant crops, pigweed, morningglory ( Ipomoea spp.), Johnsongrass ( Sorghum halepense ), ragweed spp. ( Ambrosia spp.), foxtail, and velvetleaf were mentioned as the most problematic weeds, depending on the state and cropping system (Kruger et al., 2009). With the exception of morningglory and pigw eed, these problematic weed species were present before the introducti on of glyphosate-tolerant crops, and some improvement in weed control was realized after the impl ementation of glyphosate-tolerant cropping systems (Kruger et al., 2009). Common waterhemp ( Amaranthus rudis ) and ragweed were the most frequently mentioned problem atic weeds in glyphosat e-tolerant crops in Illinois, Indiana and Iowa.

The most frequently reported common weeds in the Southeast region were morningglory ( Ipomoea spp.), prickly sida ( Sida spinosa ), johnsongrass ( Sorghum halepense ), sicklepod ( Cassia obtusifolia ), and broadleaf signalgrass ( Brachiaria platyphylla ) (Webster et al., 2005, 2009). Morningglor y, sicklepod, and pigweed are the most frequently mentioned problematic weeds in glyphosate-tolerant crop s in Mississippi and North Carolina (Kruger et al., 2009).

Cultural and mechanical weed control practices can be important components of an effective weed management program (L oux et al., 2009). Crop rotation, narrow rowMonsanto Company 10-SY-210U 187 of 721 

### spacing and planting date are a few of th e crop management practices that are implemented to provide the crop with a co mpetitive edge over weeds. Although the primary purpose of tillage is for seedbed prepar ation, tillage is still used to supplement

weed control with selective herbicides in soybean production. Approximately 98% of the soybean acreage received an herbicide app lication in 2006, indicati ng the importance of

excellent weed control in maximizing soybean yiel d (USDA-NASS, 2007b).

Herbicide-tolerant soybean was introduced to provide grower s with additional options to improve crop safety and/or improve weed c ontrol. The Roundup Ready soybean system (planting Roundup Ready soybean and applying glyphosate in crop to provide primary weed control) was introduced in 1996 and has become the st andard weed control program in U.S. soybean production and is utili zed on 91% of U.S. soybean acreage (USDANASS, 2009c).

Herbicides provide effective and economical co ntrol of weeds in soybean. The risk of weeds developing resistance to herbicides and the potential impact of resistance on the usefulness of an herbicide vary greatly acr oss different mechanisms of action and are dependent on a combination of factors, such as selection pressure, herbicide soil residual activity, herbicide chemistry, prolific seed pr oduction and high genetic variation in plants (see Appendix K for a more detailed discussi on of herbicide resistance in weeds). Weedresistance management programs that integr ate the use of herbicides with different

mechanisms of action and short residual activ ity times in soil reduce selection pressure exerted on weed species (Prather et al., 2000). Crop rotation can also be beneficial in managing resistance because it may allow the grower to manipulate planting times to avoid early-season weed germination and to use mechanical as well as chemical weed control methods (Jordan et al., 1995). As desc ribed in Appendix K, when utilized in an integrated manner, these management practices can be used to impede the development

of herbicide resistance in weeds.Monsanto Company 10-SY-210U 188 of 721 

### Table VIII-4. Common Weeds in Soybean Production: Midwest Region

Foxtail spp. (12) 1 

### Ragweed, giant (3) Dandelion (1) Pigweed spp. (11) Shattercane (3) Johnson grass (1) Velvetleaf (11) Quackgrass (3) Milkweed, honeyvine (1) Lambsquarters (10) Buckwheat, w ild (2) Nightshade, hairy (1) Cocklebur (9) Crabgrass spp. (2) Oats, wild (1) Ragweed, common (7) Kochia (2) Pokeweed, common (1) Smartweed spp. (6) Mustard, wild (2) Prickly sida (1) Morningglory spp. (5) Nightshade, Eastern black (2) Proso millet, wild (1) Sunflower, spp. (5) Palmer pigweed (2) Sandbur, field (1) Waterhemp spp. (5) Canada thistle (1) Venice mallow (1) Horseweed (marestail) (3) Chickw eed (1) Volunteer cereal (1) Panicum, fall (3) Cupgrass, w oolly (1) Volunteer corn (1)

1 Number provided in parenthesis is the number of states out of the thirteen total states in the Midwest region reporting each weed as a common weed.

Sources: IL: University of Illinois (2002) and Aaron Hage r, Extension Weed Specialist, University of Illinois - Personal Communication (2006).

IN: 2003-2005 Statewide Purdue Horseweed Weed Survey, Special database query and personal communication (2006), Bill Johnson, Extension Weed Specialist, Purdue University. IA, MN, OH, WI: WSSA, 1992. KS: Dallas Petersen, Extension Weed Specialist, Kansas State - Personal communication (2006). KY, MO: Webster et al., 2005. MI: Davis et al., 2005. NE: Alex Martin, Extension Weed Specialist, Un iversity of Nebraska – Personal communication (2006). ND: Zollinger, 2000. SD: Michael Moechnig, Extension Weed Specialist, South Dakota State University – Personal communication (2006).Monsanto Company 10-SY-210U 189 of 721 

### Table VIII-5. Common Weeds in Soybean Production: Southeast Region

Morningglory spp. (8) 1 

### Goosegrass (3) Cutleaf evening-primrose (1) Crabgrass spp. (6) Johnsongrass (3) Groundcherry (1) Prickly sida (6) Ragwee d, common (3) Henbit (1) Nutsedge spp. (6) Cocklebur (2) Lambsquarters (1) Sicklepod (5) Florida beggarweed (2) Ragweed, giant (1) Signalgrass, broadleaf (5) Hemp sesbania (2) Smartweed (1) Palmer pigweed (4) Horseweed (mar estail) (2) Spurge, nodding/hyssop (1) Pigweed spp. (4) Texas millet (2) Spurge, Prostrate (1) Barnyard grass (3) Browntop m illet (1) Tropic croton (1) Florida pusely (3) Copperleaf, hophorn (1)

1 Number provided in parenthesis is the number of states out of the eight total states in the Southeast region reporting each weed as a common weed.

Sources: AL, AR, GA, LA, NC, SC: Webster et al., 2009. MS, TN: Webster et al., 2005.



### Table VIII-6. Common Weeds in Soybean Production: Eastern Coastal Region

Foxtail spp. (6) 1 

### Morningglory spp. (4) Dandelion (1) Ragweed, common (6) Panicum, fall (4) Goosegrass (1) Velvetleaf (6) Crabgrass spp. (3) Johnson grass (1) Lambsquarters (5) Nutsedge spp. (3 ) Nightshade, Eastern black (1) Pigweed spp. (5) Quackgra ss (2) Prickly sida (1) Cocklebur (4) Canada thistle (1) Shattercane (1) Jimson weed (4) Burcucumber (1) Smartweed spp. (1)

1 Number provided in parenthesis is the number of states out of the six total states in the Eastern Coastal region reporting each weed as a common weed. Data were not available for DE in soybean.

Sources: DE, MD, NJ, PA: WSSA, 1992. NY: Russell Hahn, Extension Weed Specialist, Cornell University – Personal Communication (2006). VA: Webster et al., 2009.



### VIII.F.1. Methods of Weed Control in Soybean

Mechanical methods of weed control including tillage have been used for centuries to control weeds in crop production. Spring or fall preplant tillage and in-crop shallow cultivation can effectively reduce the compe titive ability of weeds by burying the plants, disturbing or weakening their root systems, or causing sufficient phys ical injury to kill the weeds. Research in the early 1900s cen tered on determining the economic benefits of removing weeds with the use of cultivation (Klingman et al., 1975) . A consequence of in-crop cultivation for weed control can be injury to crop roots and moisture loss. Selective herbicides have proved more e fficacious and reduced the need for in-crop tillage or cultivation to control weeds in soybean production. The development of selective herbicides has progressed rapidly since the introduction of the first syntheticMonsanto Company 10-SY-210U 190 of 721 

### herbicide (2,4-D) for weed control in corn in the early 1940s. Although the primary purpose of tillage is for seedbed preparation, tillage still is used to supplement weed control with selective herbicides in soybean production.

Alanap (1949), amiben (1958), trifluralin (1959), linuron (19 60), and alachlor (1966) led the way for numerous selective herbicides in soybean (Agranova, 2010). Bentazone (1968) was one of the early selective poste mergence herbicides used in soybean production. By the early 1990s, there were ov er 70 registered herbicides or premix herbicides for weed control in soybean (Gianessi et al., 2002). Table VIII-7 provides a summary of herbicide use in soybean produc tion in the U.S. from 1995 through 2001. Weed control programs in soybean productio n during this time period consisted of preemergence herbicides used alone or in a tank mixture with other preemergence herbicides. Applications were made as prep lant incorporated or preemergence surface applications prior to or at planting. Tank mi xtures of two preemergence herbicides were used to broaden the spectrum of control to both grasses and broadl eaf weed species. Preemergence herbicides are followed by poste mergence applications to control weed escapes that emerge later in the crop. To tal postemergence programs were seldom used in soybean production prior to 1995. For soybean planted in a no-till system, an additional preplant burndown herbicide appl ication for broad-spectrum control of existing weeds at time of planting was also applied. Therefore, multiple herbicides and/or multiple applications were generally made in soybean production. The average number of herbicide applica tions per acre in soybean rose from 1.5 in 1990 to 1.7 applications in 1995 reflecting the use of at-plant and posteme rgence applications or two postemergence applications (Gianessi et al., 2002).

It is important to understand he rbicide use in 1995, as this wa s prior to the introduction of Roundup Ready soybean system. The most wi dely used herbicides in 1995 were the sulfonylurea (chlorimuron, thifensulfuron) and imidazolinone (imazethapyr, imazaquin)

herbicide classes that are a pplied preemergence and posteme rgence in a soybean crop.

These two classes of herbic ides, both acetolactate synthase (ALS) inhibitors, were applied on approximately 87% of the soybean treated acres in 1995 (T able VIII-7). The dinitroanaline herbicides (tri fluralin and pendimethalin) were the second most widely used preemergence herbicides. Selective postemergence herbicides were used on 52% of the treated acres and were generally either eff ective on grass species or broadleaf species. Sethoxydim, clethodim, quizalofop, and fluazi fop were among the postemergence grass

herbicides. Acifluorfen and bentazon were the main posteme rgence broadleaf herbicides. Glyphosate was used on 20% of the treated acres, mainly as a preplant burndown treatment, but it also was used in spot treatments or ropewick applications to control weed escapes or volunteer corn in soybean.

Herbicide programs used in conventional s oybean have not changed significantly since 1995, with many of the traditio nal herbicides still in use. Although, new active ingredients have been intr oduced, including carfentrazone , sulfentrazone, flufenacet, flumetsulam, flumiclorac, flumioxazin, cl oransulam, and imazamox. These new active ingredients improve the level or spectrum of weed control. Numerous products have been introduced that are a pre-mixture pr oduct of two active i ngredients for broad spectrum weed control. Some of the new act ive ingredients and pre-mixtures are moreMonsanto Company 10-SY-210U 191 of 721 

### effective in controlling waterhemp, ALS-resistan t weeds, and other hard-to-control weeds. Hard-to-control weeds generally require a high er rate and/or application at a smaller growth stage in order to consistently achie ve commercially acceptable control. Refer to the Roundup WeatherMax label (U.S. EPA Reg. No. 524-537) for a listing of these

weeds. Herbicide resistant weeds are those li sted on the International Survey of Resistant Weeds website (www.weedscience.org).

Table VIII-8 provides a summary of the herbicide use in soybean in the U.S. in 2006. In 2006, herbicide-tolerant soybean (glyphosate-tolerant) was pl anted on 89% of the 75.5 million acres of soybean (USDA-NASS, 2007a ). With the high percentage of glyphosate-tolerant soybean and the additional use of glyphosate for preplant burndown applications on both glyphosate-tolerant and c onventional soybean, it is not surprising that glyphosate was used on 97% of the total soybean acres in 2006. The percentage of herbicide-tolerant soybean has subsequen tly increased to 91% in 2009 (USDA-NASS, 2009c). The remaining preemergence and postemergence herbicides are utilized in conventional soybean as well as glyphosate- tolerant soybean. A grower survey

conducted in 2006 showed that 15 to 21% of growers applied non-glyphosate herbicides

as another mode-of-action in addition to glyphosate for weed control in glyphosatetolerant soybean (Givens et al., 2009). These non-glyphosat e herbicides were applied prior to planting, at planting and poste mergence in soybean. The non-glyphosate herbicides mainly included a pplications of chlorimuron, flumiclorac, pendimethalin, imazethapyr, and 2,4-D, which were commonly used herbicides in weed management programs prior to the introduction of glyphos ate-tolerant soybean. Although these nonglyphosate herbicides were applied to s upplement the weed control provided by glyphosate, researchers report that approximately 40 to 55 % of the growers utilizing glyphosate-tolerant crops indicat e that rotating herb icides or tank mi xing glyphosate with

other herbicides is an eff ective management practice to minimize glyphosate resistance development (Johnson et al., 2009). It should be noted that in 2006 approximately 16,000 lbs of dicamba was used in soybean production which would be a sufficient amount of dicamba to treat 64,000 acres assuming the average application rate of 0.25 lb dicamba acid equivalent (a.e.) per acre (see TableVIII-7). Dicamba is currently labeled only for preplant and preharvest applications in soybean, where restri ctions on days after preplant treatment are required due to insufficient ability of soybean to tolerate applications of the herbicide, referred to as “crop tolerance.” Similarly, dicamba currently cannot be used in-crop postemergenc e applications on soybean due to a lack of crop tolerance.

Tables VIII-9 and VIII-10 provide a summary of the crop tolerance to herbicides applied in soybean production and the efficacy of th ese herbicides on 25 of the common weed species identified in Section VIII.F. These tables list only the most commonly used herbicides in soybean production. Glyphosate applied postemergence (as part of the Roundup Ready soybean system) and four other herbicides applied either preemergence or postemergence have the highest crop tolerance rating of excellent. The other herbicides are rated only good to poor. Seldom would one fiel d or farm have all 25 weed species, but they generally have a mixture of grass and broadleaf weed species. These ratings can be used by growers to facilitate the selection of an he rbicide program for a soybean crop, which offers the best overall c ontrol of the weed species. Based on TablesMonsanto Company 10-SY-210U 192 of 721 

### VIII-9 and VIII-10, glyphosate is considered to have better control (>80%) on more grass and broadleaf weed species than any other herbicide. Glyphosate/imazethapyr has the next highest overall rating, but it is rated onl y good on crop tolerance. S-Metolachlor and pendimethalin are rated high on many grass sp ecies, but are rated low on most of the broadleaf weed species. Chlori muron/tribenuron, fomesafen, and flumioxazin/cloransulam are rated high on th e broadleaf species, but are rated low on grass species.

Table VIII-7. Herbicide Use in Soyb ean in the U.S. from 1995 through 2001 1

Percent-Treated Acres Active Ingredient 1995 1996 1997 1998 1999 2000 2001
2,4-D 10 13 8 7 5 5 4 2,4-DB 1 <1 1 <1 <1 NA NA

Acifluorfen 12 11 12 7 3 3 3

Alachlor 4 5 3 2 2 1 <1

Bentazon 12 11 11 7 4 2 1

Chlorimuron 16 14 13 12 12 10 5

Clethodim 5 7 4 4 5 4 4

Clomazone 4 3 5 4 1 <1 <1

Cloransulam NA NA NA 1 5 4 5

Dimethenamid 1 1 1 1 <1 <1 NA

Ethalfluralin 1 1 <1 NA <1 <1 NA Fenoxaprop 6 4 6 4 4 4 3 Fluazifop 10 7 7 5 4 5 3

Flumetsulam 2 2 4 2 2 2 <1

Flumiclorac NA 2 1 <1 <1 <1 <1

Fomesafen 4 5 6 6 4 7 7 Glyphosate 20 25 29 47 62 66 76

Imazamox NA NA NA 7 3 6 5 Imazaquin 15 15 13 8 5 4 2 Imazethapyr 44 43 38 17 16 12 9

Lactofen 5 8 4 2 2 2 1

Linuron 2 1 1 <1 <1 <1 NA

Metolachlor 7 5 7 4 4 2 NA

Metribuzin 11 9 10 6 5 4 2 Paraquat 2 1 2 1 1 <1 NA

Pendimethalin 26 27 25 18 14 11 10 Quizalofop 6 7 4 3 1 <1 <1

S-Metolachlor NA NA NA NA NA NA <1 Sethoxydim 7 9 7 5 3 2 1

Sulfentrazone NA NA NA 3 4 4 5

Thifensulfuron 12 10 9 5 5 6 2

Trifluralin 20 22 21 16 14 14 7

1 Source is Gianessi et al. (2002).Monsanto Company 10-SY-210U 193 of 721Table VIII-8. Agricultural Chemical Applications Registered for Soybean Use in AR, IA, IL, IN, KS, KY, LA, MI, MN, MS, MO,NE, NC, ND, OH, SD, TN, VA, and WI in 20061HerbicideChemical FamilyMode-of-Action(MOA)PercentTreated AcresTotal AreaApplied(Percent/MOA)QuantityApplied(1000 lbs)Total QuantityApplied(1000 lbs/MOA)GlyphosateglycineEPSPS inhibitor4972,84192,856Glyphosate, amm. Saltglycine*142Glyphosate, iso. saltglycine9288,903Sulfosateglycine1970PendimethalindinitroanalineTubulin inhibitor351,8943,348Trifluralindinitroanaline21,454BentazonbenzothiadiazinonePSII inhibitor*370577Metribuzintriazinone2437Sulfentrazonetriazolinone170Chlorimuron-ethylsulfonylureaALS inhibitor41152265Cloransulam-methyltriazolopyrimidine117Flumetsulamtriazolopyrimidine*8Imazamoximidazolinone*9Imazaquinimidazolinone166Imazethapyrimidazolinone3100Imazethapyr, ammon.Imidazolinone*5Thifensulfuronsulfonylurea13Tribenuron-methylsulfonylurea15AlachlorchloroacetamideCell divisioninhibitor*14851,402S-Metolachlorchloroacetamide1837Flufenacetoxyacetamide*80ParaquatbipyridiliumPSI disruption11335335Monsanto Company 10-SY-210U 194 of 721Table VIII-8 (continued). Agricultural Chemical Applications Registered for Soybean Use in AR, IA, IL, IN, KS, KY, LA, MI, MN,MS, MO, NE, NC, ND, OH, SD, TN, VA, and WI in 20061HerbicideChemical FamilyMode-of-Action(MOA)PercentTreated AcresTotal AreaApplied(Percent/MOA)QuantityApplied(1000 lbs)Total QuantityApplied(1000 lbs/MOA)ClethodimcyclohexenoneACCase inhibitor34190266Fenoxaproparyloxyphenoxypropionate*9Fluazifop-P-butylaryloxyphenoxypropionate143Quizalofop-P-ethylaryloxyphenoxypropionate*14Sethoxydimcyclohexenone*10Acifluorfendiphenyl etherPPO inhibitor*647565Carfentrazone-ethyltriazolinones* 10Flumiclorac-pentylN-phenylphthalimide117FlumioxazinN-phenylphthalimide3138Fomesafendiphenyl ether2330Lactofendiphenyl ether*232,4-D, 2-EHEphenoxySynthetic auxin7102,5053,5422,4-D, dimeth. saltphenoxy39532,4-D (butoxy ester)phenoxy*68Dicamba, digly saltbenzoic acid*16Total103,156* Area receiving application is less than 0.5 percent.1Data derived from USDA-NASS (2007b). Planted acreage for thenineteen primary soybean production states was 72.9 million acres, whichrepresented 96.5% of total planted acres.Monsanto Company 10-SY-210U 195 of 721Table VIII-9. Crop Tolerance and Common Grass Weed Responses to Herbicides Applied in Soybean ProductionCommon Grass Weeds1,2Herbicide/Application CT3BY BS CG FP FT GG SC JGs JGr RR NSyPreplant IncorporatedTrifluralin 1 9 9 9 9 9 9 7 7 3 9 -Preplant or PreemergenceChlorimuron/tribenuron 2 - 8 - - - 7 - - 2 8 -Cloransulam 0 - NA - - - NA - - NA NA -Flumioxazin 2 - 5 - - - 5 - - 0 8 -Flumioxazin/cloransulam 2 - 5 - - - 5 - - 0 8 -Imazaquin 1 - 7 - - - 5 - - 2 5 6Imazethapyr 1 6 NA 7 7 7 NA 6 6 NA NA -Metribuzin 2 2 6 6 5 6 7 6 - 0 4 -Pendimethalin 2 8 9 9 9 8 9 7 7 3 4 -s-Metolachlor 1 8 8 9 8+ 8+ 9 - - 0 3 8+PostemergenceBentazon 1 - 0 - - - 0 - - 0 NA 8+Chlorimuron 2 - 0 - - - 0 - - 0 0 8Clethodim 0 9 9 8+ 9 9 9 9 9 9 8 -Cloransulam 1 - 0 - - - 0 - - 0 0 6Clorimuron/thifensulfuron 0** - NA - - - NA - - NA NA 8Fluazifop/fenoxaprop 0 9 8 8+ 9 9 9 9 9 9 7 -Flumiclorac 2 - NA - - - NA - - NA NA -Fomesafen 2 - 3 - - - 3 - - 3 0 -Glyphosate 0* 8+ 9 8+ 8+ 9 8 8 9 9 8 7Glyphosate/imazethapyr 1* 9 NA 8+ 9 9 NA 9 9 NA NA 7Imazamox 2 6 NA 7 7 7-8+ NA - - NA NA -Imazethapyr 1 6 7 7 7 7-8 5 8 8 6 4 -Lactofen 3 - 4 - - - 4 - - 2 0 -Thifensulfuron 2 - NA - - - NA - - NA NA -1All weed control ratings except for BS, GG, JGr and RR are fromthe 2009 Weed Control Guide for Ohio and Indiana, Ohio State University and Purdue University (Loux et al., 2009).Ratings for BS, GG, JGr and RR are from the 2009 Weed Control Guidelines for Mississippi, Mississippi State University (MSU, 2010). Weed control rating for weeds, except BS, GG, andRR, are: 9 = 90% to 100%, 8 = 80% to 90%, 7 = 70% to 80%, 6 = 60%to 70%, - = less than 60% control, not recommended. Weed control ratings for BS, GG, and RR are: 9-10 = excellent,7-8 = good, 4-6 = fair, 0-3 = none to slight. Ratings assume the herbicides are applied in the manner suggested in the guidelines and according to the label under optimum growing conditions.2Weed species: BY = barnyardgrass, BS = broadleaf signalgrass,CG = crabgrass, FP = fall panicum, FT = giant and yellow foxtail, GG = goosegrass, SC = shattercane, JGs = seedlingJohnsongrass, JGr = rhizome Johnsongrass,RR = red rice, and NSy = yellow nutsedge.3All crop tolerance ratings are from the 2009 Weed Control Guide forOhio and Indiana, Ohio StateUniversity and Purdue University (Loux et al., 2009). Crop tolerance (CT) rating: 0 = excellent, 1 =good, 2 = fair, 3 = poor.NA denotes not available. *Rating basedon application to Roundup Ready soybean. **Ratings based on application to STS soybean.Monsanto Company 10-SY-210U 196 of 721Table VIII-10. Common Broadleaf Weed Responses toHerbicides Applied inSoybean ProductionCommon Broadleaf Weeds1,2Herbicide/ApplicationBNCBCRGRHSLQMGPAPWPSSPSWVLWHPreplant Incorporated OnlyTrifluralin----08+27904--8Preplant or PreemergenceChlorimuron/tribenuron-897998897NA98+-Cloransulam-897NA9NANA9NANA88+-Flumioxazin9-7-996-89987778Flumioxazin/cloransulam9897997-8998798+8Imazaquin9887096-8999597-Imazethapyr976-NA9NANA9NANA98-Metribuzin---7972-89898997Pendimethalin----08+27942--7s-Metolachlor8---0608842--7PostemergenceBentazon-976472-94-8098+-Chlorimuron-987+8-8-9692788-Cloransulam-9993-8-92-2789-Clorimuron/thifensulfuron-987+NA8NANA9NANA99-Flumiclorac-77-NA7NANA7NANA-97Fomesafen878+89-8-98923769Glyphosate898+8787-99978888Glyphosate/imazethapyr998+8+NA8+NANA9NANA998Imazamox9878NA8NANA9NANA89-Imazethapyr9967067-9696099-Lactofen8+8989-8-98985679Thifensulfuron-6--NA8NANA9NANA89-1All weed control ratings except for HS, MG, PA, PS, and SP are from the 2009 Weed Control Guide for Ohio and Indiana, Ohio State University and PurdueUniversity (Loux et al., 2009). Ratings for HS, MG, PA, PS, andSP are from the 2009 Weed Control Guidelines for Mississippi,Mississippi State University(MSU, 2010). Weed control ratings for weeds, except HS, MG, PA,PS, and SP, are: 9 = 90% to 100%, 8 = 80% to 90%, 7 = 70% to80%, 6 = 60% to 70%, - =less than 60% control, not recommended. Weed control ratings for HS, MG, PA, PS, and SP are: 9-10 = excellent, 7-8 = good, 4-6 = fair, 0-3 = none to slight.Ratings assume the herbicides are applied in the manner suggested in the guidelines and according to the label under optimum growing conditions.2Weed species: BN = black nightshade, CB = cocklebur, CR = common ragweed, GR = giant ragweed, LQ = lambsquarters, MG = morningglory spp., HS =hemp sesbania, PA = palmer and spiny pigweed, PW = pigweed, PS= prickly sida, SP = sicklepod, SW = smartweed, VL = velvetleaf,and WH = waterhemp.NA denotes not available.Monsanto Company 10-SY-210U 197 of 721 

### VIII.G. Dicamba Herbicide Use in the U.S.

Dicamba was approved by the U.S. EPA for agri cultural uses in 1967 (U.S. EPA, 2009).

Dicamba is formulated as a stand-alone herbicide product and marketed by several companies under various trade names such as Banvel ® 

### , Clarity ® 

### , Diablo ® 

### , Rifle ® 

### , and Sterling ® 

### that are various salt formulations of dicamba. These dicamba products can be tank mixed with one or more active ingred ients depending on the treated crop. For example, Clarity can be tank mixed with ov er 75 herbicide products in labeled crops. Additionally, dicamba is formulated as a re gistered premix product with one or more

other herbicide active ingredients such as glyphosate, 2,4-D, diflufenzopyr, atrazine, nicosulfuron, metsulfuron, primsulfuron, tria zulfuron, rimsulfuron and halosulfuron. Dicamba herbicide ( e.g. , Clarity – diglycolamine (DGA) salt of dicamba ) is currently labeled for weed control in soybean, corn, cotton, sorghum, wheat, barley, oats, millet, pasture, rangeland, asparagus, sugarcane, turf , grass grown for seed , conservation reserve programs, and fallow croplands. Table VII I-11 provides a summary of dicamba-treated acres (crop acreage that has dicamba applie d to it) and the amount of dicamba active ingredient applied for all labeled crops each year from 1990 through 2008. Dicambatreated acreage has ranged from 17.4 to 36.3 million acres during this period. Usage of dicamba peaked during the period of 1994 through 1997, where 1994 was the peak year

when 36.3 million acres were treated with 9.4 million pounds of dicamba. Since 1994, the use of dicamba has steadily declined to 20.2 million treated-acres with 2.7 million pounds in 2008 due to the competitive market introductions of sulf onylurea herbicides (chlorsulfuron, metsulfuron-methyl, and thif ensulfuron-methyl) in wheat, new broadleaf herbicide active ingredients in corn, and R oundup Ready corn. Usage in cotton is one exception, where dicamba-treate d acres (preplant applicati ons) have increased from 140,000 to 590,000 acres from 2004 to 2008 (AgroTrak, 2009).

Table VIII-12 provides a summary of the di camba-treated acres by crop in 2008. Approximately 20.2 million acres were treate d with dicamba in 2008. Over 8 million acres of corn were treated, wh ich is 40.1% of the total dicamb a-treated acres for all crops. The next highest levels of treated acres ar e in wheat (25.2%) and fallow land (14.9%). The crops with the highest percentage of dicamba-treated acres are sugarcane (21.9%), fallow land (19.2%), sorghum (15.8%), and wheat (8.4%). Although corn represents the crop with the highest dicamba-treated acres, only 9.4% of the tota l corn acreage was treated with dicamba in 2008. For comparison, the treated percentage in corn was at approximately 29% as recently as 2000 (USDA-NASS, 2001).

Approximately 2.67 million pounds of dicamba active ingredient were applied for all agricultural uses in 2008 (Table VIII-12). Th e distribution of dicamba active ingredient

across the various labeled uses is similar to the distribution of treated acres. Based on USDA-NASS (2004, 2006, 2007b, 2008) statistics, dicamba app lication rates ranged from 0.03 to 0.25 pounds per acre with the number of applications ranging from 1 to 1.2 applications per cropping season (Table VIII-13) . Dicamba rates are the lowest in barley, wheat, and oats, where typically more than on e application is made in these crops per cropping season. The average application rate in corn is 0.19 pounds of dicamba per acre with slightly over one application per season.Monsanto Company 10-SY-210U 198 of 721 

### Dicamba is currently labeled for use in conventional or Roundup Ready soybean, although dicamba use is extremely limited becaus e applications are restricted to very early preplant and/or preharve st applications due to soybe an tolerance concerns. The dicamba-treated acreage in 2008 soybean production was approximately 530,000 acres that represented 0.7% of th e total soybean acreage.

Table VIII-11. Dicamba Use in All Labeled Crops from 1990 to 2008 1

Year Treated Acres (000,000 acres) Dicamba (a.e.) (000,000 lbs)

1990 26.8 6.7

1991 24.5 6.3

1992 30.3 7.4

1993 27.7 7.0

1994 36.3 9.4

1995 34.3 8.7

1996 33.3 8.2

1997 33.1 8.6

1998 32.2 8.0

1999 29.8 6.3

2000 29.4 5.4

2001 30.6 5.4

2002 29.4 5.0

2003 27.1 4.3

2004 22.3 3.9

2005 21.3 3.4

2006 17.4 2.7

2007 18.6 2.7

2008 20.2 2.7

1 

### Source is AgroTrak (2009). Shaded bar indicates the year w ith maximum dicamba-treated acres.Monsanto Company 10-SY-210U 199 of 721Table VIII-12. Dicamba-Treated Acres and Amounts Applied to Labeled Crops and Uses in 20081CropTotal CropAcres (000)DicambaTreated Acres(000)DicambaTreated Acres(% of Total)2DicambaTreated Crop(%)Dicamba Pounds(000 a.e.)Asparagus 34 1 <0.1 1.8 0.1Barley 3,868 211 1.0 5.5 23Corn 87,245 8,115 40.1 9.4 961Cotton 9,309 590 2.9 6.3 139Fallow 15,751 3,018 14.9 19.2 420Pastureland 96,151 1,218 6.0 1.3 254Sorghum 7,035 1,114 5.5 15.8 137Soybean 74,405 530 2.6 0.7 118Sugarcane 810 177 0.9 21.9 40Wheat, all 60,835 5,094 25.2 8.4 549All other uses NA 139 NA NA 30Total 20,207 2,670NA denotes not applicable.1Source is AgroTrak (2009).2The percentage of the total dicamba-treated acres for all labeled crops and uses.Table VIII-13. Dicamba Applications – Average Number and Rates to Labeled Crops1Crop# of DicambaApplicationsRate of Dicamba perApplicationRate of Dicamba perCrop YearCorn 1.02 0.188 0.192Cotton 1.00 0.191 0.192Sorghum 1.05 0.205 0.215Soybean 1.00 0.250 0.250Barley 1.20 0.060 0.080Wheat, spring 1.13 0.032 0.085Wheat, winter 1.20 0.122 0.149Oats 1.00 0.088 0.0881USDA-NASS, 2004 (sorghum), 2006 (cornand oats), 2007b (soybean, barleyand wheat), and 2008 (cotton)Monsanto Company 10-SY-210U 200 of 721 

### VIII.G.1. Dicamba Applicatio n Timing for Labeled Crops

Label recommendations on the application timi ng of dicamba are highly dependent on the crop being treated to ensure adequate crop safe ty. Many of the field crops currently labeled for dicamba, such as soybean and cotton, includ e preplant applicatio ns but certain timing intervals are required between application a nd planting to avoid crop injury. However, dicamba can be applied in other field crops such as corn, sor ghum, barley, and wheat either as preplant or postemergence applications w ith restrictions regarding the crop stage of growth. Broadcast applications in corn can be made up to the 5-leaf stage or 8 inches tall, whichever occurs first. Sorghum can be treated with broadcas t applications from after the spike stage up to the 5-leaf stage or 8 inches tall 3 

### . These applications are considered earlypostemergence since they occur relatively early in the grow ing season, typically ending sometime in June. Post-directed applications can be made in corn (up to 36 inches tall) and sorghum (15 inches tall), which would be much later in the season than an earlypostemergence application. However, growers se ldom make these applications in corn and sorghum since post-directed applications requi re special spraying equipment and equipment setup. Applications in wheat and barley must be made prior to the jo inting stage except in spring seeded wheat where application can be ma de up to the 6-leaf stage of wheat. These applications are relatively early in the spring or late in the fall.

Preharvest applications of dicamba are pe rmitted in several crops including soybean, sorghum, barley, and wheat. Preharvest applicat ions are only allowed af ter the crops reach a certain maturity stage and then harvesting must be delayed for a given time period depending on the crop 4 

### . This type of application is infre quently used since it is considered a

rescue or harvest-aid treatment intended to remove weeds which interfere with the harvesting equipment or operation.

Table VIII-14 provides a summary of the appli cation timings of dicamba in labeled crops. Approximately 24% of the dicamba treated acres are treated either in the fall or spring as preplant applications to the crop. Over 50% of the treated acres ar e treated postemergence. Postemergence applications represent the pr imary timing in corn, sorghum, and wheat.

3 Clarity product label can be found at: http://www.cdms.net/LDat/ld797002.pdf.

4 Clarity label specifies that preharvest application requires that soybean pods must have reached mature brown color and at least 75% leaf drop has occurred. Harvest of soybean is allowed 14 or more days after preharvest application.Monsanto Company 10-SY-210U 201 of 721Table VIII-14. Dicamba-Treated Acres (000)by Application Timing and Crop in 20081Application Timing2CropFallPreplantSpringPreplantAt PlantingPre-CropEmergenceIn-CropPostemergenceOtherTimings3TotalsAsparagus - - - - - 11Barley 8 41 - - 162 -211Corn 136 851 71 285 6,771 -8,114Cotton 41 549 - - - -590Fallow 647 - - - - 2,3713,018Sorghum 439 380 12 81 202 -1,114Soybean 43 486 - - - -529Sugarcane 13 - - 15 149 -177Wheat, spring 341 193 - 26 791 -1,351Wheat, winter 608 - 13 71 3,050 -3,742Pastures 84 - - - - 1,1341,218Totals 2,360 2,500 96 478 11,125 3,506 20,065% of Applications 11.8 12.5 0.5 2.4 55.4 17.5- denotes no application at the timing for the listed crop.1Source is AgroTrak (2009).2Dicamba-treated acres are expressed as dicamba a.e.3Application timing could be throughout the season since applicationsare made between harvests or are not dependent on a specificstage of growth.Monsanto Company 10-SY-210U 202 of 721 

### VIII.G.2. Distribution of Dicamba Use in the U.S.

Table VIII-15 provides a summary of the di camba-treated acres by crop for each of the states in the U.S. soybean gr owing regions. As expected, based on the dicamba use data previously presented, over one-half of the di camba-treated acres (58%) are in the Midwest region representing approximately 11.8 milli on acres. The primary dicamba-treated crops/uses in this region ar e corn, wheat, barley, and fallo w. Although the Plains and Western States regions are not considered a soybean producing region, it represents a large portion (32%) of the dicamba-tr eated acres (6.4 million). On ly 1.4 and 0.6 million acres are

treated with dicamba in the Southeast and Ea stern Coastal regions, respectively. Soybean producing states with over one million dicamba-t reated acres include Illinois, Kansas, and North Dakota. The state of Kansas, which grows many of the primary dicamba-labeled crops, has the largest amount of dicamba-treated acres at a pproximately 4.6 million acres (22.6% of total dicamba-treated acres), which is 2.5 times more than the next largest use states of North Dakota, Colorado, Montana, and Texas. Dicamba is used on less than 1% of U.S. soybean acres, but in Arkansas dicamba is used on 12.6% of th e soybean acres, and accounts for the majority of dicamba-treated soybean acres in the U.S. (~78%). The primary reason for the relatively higher use of dicamba in Arkansas, compared to other soybean producing states, is that dicamba is being recommended by academics and extension agents in the Delta to c ontrol glyphosate resi stant marestail.

The maps presented in Figures VIII-1 and V III-2 provide a visual illustration of the

historical distribution and inte nsity of dicamba-treated acres for all labeled crops and the current U.S. soybean production acres at the co unty level, respectively. By overlaying the geographical representations from Figures VI II-1 and VIII-2, it can be concluded that the historical use and intensity of total dicamba -treated acres align w ith soybean acreage.Monsanto Company 10-SY-210U 203 of 721Table VIII-15. Dicamba-Treated Acres by State and Labeled Crop in 20081CropAcres Treated With Dicamba(000)Region/StateSoybeanAcres(000)TotalDicambaAcres(000)PercentU.S.DicambaAcres2CornSmallGrains3FallowPastureCottonSorghumSoybeanOtherCrops4Midwest RegionIllinois 9,200 1,213 6.0 1,204 - - - - 3 6 -Indiana 5,450 494 2.4 489 - - - - - 5 -Iowa 9,750 580 2.9 576 - - 4 - - - -Kansas 3,300 4,558 22.6 658 2,198 901 30 2 675 61 33Kentucky 1,390 36 0.2 36 - - - - - - -Michigan 1,900 294 1.5 285 8 - - - - - 1Minnesota 7,050 628 3.1 606 21 - <1 - - - -Missouri 5,200 341 1.7 205 <1 - 36 99 - - -Nebraska 4,900 946 4.7 558 222 134 6 - <1 21 4North Dakota 3,800 1,670 8.3 740 846 64 - - - 9 11Ohio 4,500 270 1.3 247 23 - - - - - -South Dakota 4,100 502 2.5 183 88 198 - - 16 13 4Wisconsin 1,610 261 1.3 250 - - 11 - - - -Region Totals 62,150 11,793 58.4 6,037 3,407 1,297 88 101 695 115 53Monsanto Company 10-SY-210U 204 of 721Table VIII-15 (continued). Dicamba-Treated Acres by State and Labeled Crop in 20081CropAcres Treated With Dicamba(000)Region/StateSoybeanAcres(000)TotalDicambaAcres(000)PercentU.S.DicambaAcres2CornSmallGrains3FallowPastureCottonSorghumSoybeanOtherCrops4Southeast RegionAlabama 360 119 0.6 54 - - 14 51 - - -Arkansas 3,300 791 3.9 - - - 92 262 - 415 22Georgia 430 32 0.2 17 - - 15 - - - -Louisiana 1,050 215 1.1 17 - 23 - - - - 175Mississippi 2,000 102 0.5 - - - 50 52 - - -North Carolina 1,690 53 0.3 39 - - 14 - - - -South Carolina 540 6 0.0 - - - - - - - 6Tennessee 1,490 102 0.5 5 - - <1 96 - - -Region Totals 10,860 1,420 7.0 132 - 23 186 461 - 415 203Eastern Coastal RegionDelaware 195 0 0 - - - - - - - -Maryland 495 268 1.3 268 - - - - - - -New Jersey 92 0 0 - - - - - - - -New York 230 62 0.3 62 - - - - - - -Pennsylvania 435 170 0.8 168 - - 2 - - - -Virginia 580 118 0.6 104 5 - 9 - - - -Region Totals 2,027 618 3.1 602 5 - 11 - - - -Monsanto Company 10-SY-210U 205 of 721Table VIII-15 (continued). Dicamba-Treated Acres by State and Labeled Crop in 20081CropAcres Treated With Dicamba(000)expressed as a.e.Region/StateSoybeanAcres(000)TotalDicambaAcres(000)PercentU.S.DicambaAcres2CornSmallGrains3FallowPastureCottonSorghumSoybeanOtherCrops4Plains and Western States RegionCalifornia095 0.5 32 63 -- - - - -Colorado01,606 8.0 248 253 958 3 - 122 - 22Florida327142 0.7 - - - 140 - - - 2Idaho0170 0.8 114 51 <1 <1 - - - 4Montana01,657 8.2 - 955 686 - - - - 16New Mexico046 0.2 37 - - - - 9 - -Oklahoma400622 3.1 8 84 5 409 - 113 - 3Oregon0222 1.1 - 218 - 4 - - - -Texas2301,641 8.1 850 212 14 360 27 175 - 3Utah022 0.1 3 <1 12 - - - - 6Washington096 0.5 - 55 31 5 - - - 5Wyoming057 0.3 54 - 3 - - - - -Region Totals9576,376 31.6 1,346 1,892 1,710 934 27 419 61U.S. Totals 75,994 20,207 100 8,117 5,304 3,030 1,207 589 1,114 530 317- denotes no dicamba-treated acres for listed crop/use.1Source is AgroTrak (2009).2Percent of total U.S. Dicamba Acres = Total Dicamba-Treated Acres (per state)/U.S. Total Dicamba-Treated Acres (20,207,000) ×1003Small grains include barley,winter wheat and spring wheat.4Other labeled crops include asparagus and sugarcane.Monsanto Company 10-SY-210U 206 of 721 

### Figure VIII-1. Mean Annual Dicamba Use for Ag ricultural Uses in the U.S. During

1999-2004 1

1 Based on 2002 Census of Agriculture county crop acreage (USGS, 2004).



### Figure VIII-2. Planted Soybean Acre age by County in the U.S. in 2009 1

1 Source is USDA-NASS (2010).Monsanto Company 10-SY-210U 207 of 721 

### VIII.G.3. Potential Impacts to Adjacent Crops

U.S. EPA considers possible effects from of fsite movement as part of the pesticide registration process. In orde r to approve the use of a pest icide (herbicide) under FIFRA,

U.S. EPA must conclude that no unreasonable adve rse effects on non-tar get vegetation will

result from offsite movement when the herbic ide is used according to the product label. Thus, when herbicides are applied in accordance with the pertinent labe l restrictions, offsite impacts can be avoided. EPA reassessed the pot ential risks to non-target plants in its analysis in the dicamba RED, and it concluded that no specific additional drift mitigations were needed to support the continued registra tion of all dicamba us es (U.S. EPA, 2009). Since the proposed use pattern for dicamba on MON 87708 is consistent with use patterns evaluated and deemed eligible for reregistrati on in the dicamba RED, it is reasonable to

conclude that dicamba use on MON 87708 meets the FIFRA standards related to offsite movement and does not pose any gr eater risk to non-target ve getation over existing dicamba agricultural uses approved by EPA when used according to the product label.

Growers and commercial herbicide applicators have been applying dicamba to agricultural row crops for over 40 years. This practice has provided valuable experience and knowledge on the proper application of dicamba for eff ective weed control and also for minimizing offsite movement to sensitive crops. Dicamba herbicide spray drift can be reduced during application by using industry st andard procedures for mini mizing spray drift. These procedures include making applications with coarse droplet size th rough appropriate nozzle selection, using lower spray pressure, applying at the lowest nozzle height that provides uniform coverage, and making applications wh en wind speeds are low and consistent in direction (SDTF, 1997). In addition, grower s and commercial appl icators are legally required to follow label requirements and are ed ucated by university sp ecialists and industry representatives on the proper application equipment, equi pment setup, and climatic conditions to maximize herbicide perfor mance and minimize offsite movement of herbicides. For example, w ith the introduction of Roundup Rea dy crops and the subsequent increase in glyphosate use, uni versity specialists conducted extensive education programs on proper application procedures and precauti ons (Dr. W. Johnson – Purdue University, 2010 personal communication). Equipment manuf acturers have developed spray nozzles that provide uniform coverage for effectiv e weed control while applying larger spray droplets to reduce the potential for particle drift. Similarly, offsite movement of dicamba has been managed with the knowledge of the proper spray equipment and equipment setup, climatic conditions for accurate, on-target ap plications, and based on the requirements for applying dicamba at an appropria te distance from adjacent crops and plants (Jordan et al., 2009).

Monsanto plans to further address the us e of dicamba on MON 87708 with US EPA to evaluate whether any additional measures may be appropriate to further address potential

drift and offsite movement.Monsanto Company 10-SY-210U 208 of 721 

### VIII.H. Dicamba-Tolerant Soybean MON 87708

Monsanto has developed a new herbicide-to lerant soybean, MON 87708, to offer growers an expanded use of the he rbicide dicamba in soybean production. MON 87708 will facilitate a wider window of dicamba app lication in soybean, allowing preemergence application up to the day of crop emerge nce (cracking) and in-crop postemergence applications through the R1 /R2 growth stage. MON 87708 will be combined with MON 89788 (Roundup Ready 2 Yield soybean) utiliz ing traditional breeding techniques. The combination of herbicide-tolerance traits will allow the pre- and postemergence use of both dicamba and glyphosate herbicides in an integrated weed management program to control a broad spectrum of grass and broa dleaf weed species (Johnson et al., 2010). Increasing postemergence herbicide options is important, especially in conservation tillage situations, where the performance consistenc y of postemergence herbicides has generally been greater than that of soil active residual products. Dicamba will improve the control of glyphosate’s hard-to-control broadleaf weeds ( e.g. , common lambsquarters, hemp sesbania, morningglory species, nightshade, Pennsylva nia smartweed, prickly sida ̧ and wild buckwheat) and also offer an effective cont rol option for glyphosate -resistant broadleaf weed species, namely marestail, common ra gweed, giant ragweed, palmer pigweed, and waterhemp (Johnson et al., 2010). Dicamba will al so offer an effective control option for broadleaf species resistant to ALS and PPO chem istries. In the case of PPO resistance, a primary dicamba benefit will be to provide options for delaying the further spread of PPO resistant amaranthus species (University of Tennessee, 2010)

.



### Upon integration of MON 87708 into the Roundup Ready soybean system, growers will have the ability to continue to use establis hed soybean production practices including crop rotation, tillage systems, labeled herbicides , and row spacing, thereby using the same planting and harvesting machinery currently bei ng utilized. Growers will also continue to have the flexibility and simplicity in weed control provided by gl yphosate that will allow growers to continue to reap the environmen tal benefits associated with the use of conservation-tillage that is facilitated by th e use of glyphosate for postemergence weed control in the Roundup Ready soybean system (CTIC, 2011; CTIC, 2004).

Current labeled uses of dicamba in soybean are limited to early preplant and late postemergence (preharvest) applications. Signi ficant planting restrictions exist in soybean for preplant applications of dicamba, including a maximum application rate of 0.5 lbs a.e. per acre, a 28-day interval be tween application and planting soybean, and a minimum of one inch of rainfall must occur before planting soybean to avoid soybean injury. Monsanto has submitted an application to U.S. EPA to amend Registration Number 524-582, a DGA salt formulation, to remove all preem ergence planting restrictions (intervals and rainfall) and to allow in-crop postemergence dicamba app lications to MON 87708 through the R1/R2 growth stage of soybean. Once approved, grow ers would be authorized to apply dicamba alone or in mixtures with glyphosate or other herbicides for preplant or in-crop postemergence applications on MON 87708. Dicamba would be authorized to be applied preemergence up to crop emergence as a single a pplication or split applications up to a total of 1.0 lb a.e. per acre, and up to two postemergence ap plications up to 0.5 lb a. e. per acre each through the R1/R2 growth stage of soybea n. The maximum annual application rate of dicamba on MON 87708 is 2.0 lb dicamba a.e. pe r acre. Furthermore, Monsanto’s proposedMonsanto Company 10-SY-210U 209 of 721 

### dicamba label does not allow aerial applic ations of dicamba on MON 87708, a stewardship measure intended to address potential offsite impacts. As indicated, Monsanto also plans to

further address the use of dicamba on MON 87 708 with US EPA to evaluate whether any additional measures may be ap propriate to further address potential drift and offsite

movement.

The potential increase in dicamba use in U.S. soybean production upon deregulation of MON 87708 was assessed by estimating the to tal dicamba use acro ss soybean acres. Assuming 100% adoption of MON 87708 acr oss all U.S. soybean acreage 6 

### and an application of dicamba at the maximum labele d use rate on all soybean acres, dicamba use on MON 87708 could potentially total 150 milli on pounds. In practice, however, a single early season in-crop app lication per year of dica mba at 0.38 lb a.e. pe r acre is expected on the majority of MON 87708 planted acres. Ho wever, in no-till or conservation tillage soybean systems, an additional preplant application at 0.50 lb a.e. per acre could also be common practice, and in areas where gl yphosate resistant weeds, especially Ambrosia and

Amaranthus species, are present two in-crop applica tions at 0.5 lb a.e. each may be needed in some situations. These anticipated use patterns represent a high-end estimate for prediciting dicamba use associated with MON 87708 integrated with the Roundup Ready soybean system.

Furthermore, consistent with recommendations by academics and weed scientists, Monsanto

will recommend the use of a third herbicide m ode-of-action with soil residual activity as part of a comprehensive weed resistance manage ment program to assure that at least two

effective modes-of-action are al ways used in the cultivated soybean field. A summary of the anticipated weed control recommenda tions for the combined MON 87708 and Roundup Ready soybean system is provided in Table VIII-16.

In 2010, Monsanto conducted an in formal survey of weed scie ntists across the country to estimate the number of crop acres with glyphosa te resistant weed popu lations. Based upon this survey it was estimated that approxima tely 14-16 million acres of planted row-crops (i.e. corn, soybeans, cotton) ha d populations of glyphosate resist ant weeds. Of these acres, the majority of acres (~ 10 million) are infested with glyphosate resistant marestail populations where a preplant application of dicamba and glyphosate de scribed above will be

effective for control. The remainder of resistant acres (~5 million) have resistant Ambrosia (common and giant ragweed) and Amaranthus (palmer pigweed and water hemp,) species present. A conservative estimate of 5 million resistant acres is assumed for this assessment, which overestimates current resistant acres in soybean producing areas and also accounts for potential increases in resistant acres because not all resistant crop acre s would be planted to soybean in any given year.

6 Based on approximately 75 million acres planted to soybean in 2008, see Table VIII-1.Monsanto Company 10-SY-210U 210 of 721 

### Table VIII-16. Anticipated Weed Management Recommendations for MON 87708 Combined with the Roundup Ready 2 Yield Soybean System 1

Application Timing Conventional Tillage 2 Conservation Tillage 2

(No-till or reduced till)

No GR

Weeds GR Weeds or Suspected

GR Weeds No GR

Weeds GR Weeds or Suspected

GR Weeds

Option 1 3 Option 2 4 Option 1 3 Option 2 4

Preemergence (burndown, at planting) 5 Residual Residual Residual Residual

+ Glyphosate

+
Dicamba Residual

+ Glyphosate

+
Dicamba Residual

+ Glyphosate

+
Dicamba

Postemergence 1 (V1-V3) Glyphosate

+
Dicamba Glyphosate

+
Dicamba Glyphosate

+
Dicamba Glyphosate

+
Dicamba Glyphosate

+
Dicamba Glyphosate

+
Dicamba

Postemergence 2 (V4-R2) --- Glyphosate

+
Dicamba --- --- Glyphosate

+
Dicamba ---

1 The anticipated use patterns represent a high-end es timate for prediciting dicamba use associated with

MON 87708 integrated with the Roundup Ready soybean system. Actual weed cont rol practices by growers will vary depending on the specific weed spectrum and agronomic situation of the individual soybean field, specifically dicamba use could be lower especially for the preemergence and second postemergence applications.

2 Average rate for dicamba is 0.38 pound a.e. per acre exce pt for fields with glyphos ate resistant (GR) species

where a 0.5 pound a.e. per acre postemergence applicati on rate will be recommended. In some situations, the second postemergence application may not be needed.

3 Option 1 would be used for more aggressive glyphosate resistant weed species, such as Ambrosia or

Amaranthus species.

4 Option 2 would be used for less aggressive glyphosa te resistant weed species, such as marestail.

5 Monsanto and academics recommend the use of soil residu als as part of a comprehensive weed resistance

management program to ensure that two effective herbicide modes-of-action are used in soybean and to provide protections against additional resistance development to existing soybean herbicides.



### Assuming the anticipated use rate of 0.5 lb a.e. per acre dicamba for preemergence applications and 0.38 lb a.e. per acre dicamba for postemergence applications, and using a conservative assumption that MON 87708 has 100% adoption across all U.S. geographies and conservation tillage systems are used on ap proximately 40% of the U.S. soybean acres (CTIC, 2007), dicamba use on MON 87708 woul d total approximately 44 million pounds. When considering a more realistic adoption rate for MON 87708 of 40% (refer to Section VIII.H.2), dicamba use on MON 87708 would total approximately 17 million pounds. In

areas where resistant Ambrosia and Amaranthus species are present requiring two in-crop applications, the use of an additional 5 milli on pounds of dicamba per year is estimated.

It is anticipated that dicamba applications will continue for currently labeled crops at the dicamba-treated acreage levels and amounts presented in Table VIII-12, such that the

dicamba treatment to MON 87708 will there by result in a total U.S. dicamba use of approximately 25 million pounds annually. Th is level of dicamba use would beMonsanto Company 10-SY-210U 211 of 721 

### approximately double the historical peak level (Table VIII-11) experienced since dicamba’s

introduction in 1967.

Upon integration of MON 87708 into the Roundup Ready soybean system, dicamba will provide excellent control of numerous a nnual and perennial broadleaf weed species, including populations of broadleaf weeds that are resistant to ALS, atrazine, or glyphosate

herbicides. Table VIII-17 shows weed cont rol ratings for dicamba, glyphosate and several glyphosate tank mixtures when applied as a preplant burndown application to common broadleaf weed species found in soybean fields of the Midwest and Southeast regions. An application of dicamba alone pr ovides effective control of a br oad spectrum of winter and summer annual and perennial broadleaf weed species. In comparison, glyphosate alone provides excellent control of many grass spec ies in addition to many of the annual and perennial broadleaf species liste d. However, dicamba provides a higher level of control of certain broadleaf weeds including common la mbsquarters, Pennsylvania smartweed, red clover, alfalfa, marestail, hairy vetch, and prickly lettuce. Dicamba will be very complementary in mixtures with glyphosate for weed control in a preplant application (Johnson et al, 2010) and will offer growers e qual or superior weed control to other glyphosate mixtures because it offers reduced potential herbicide antagonism, improved efficacy and broader weed spectrum.

The dicamba tolerance trait in MON 87708 will permit in-crop applications of dicamba to soybean with excellent crop safety (crop tole rance). Dicamba will also complement the weed control of in-crop applications of glyphosate when applied as a mixture or in sequence. Table VIII-18 shows common broadlea f weed responses to dicamba compared to glyphosate and several glyphosate la beled tank mixtures in soybean. Since dicamba is not currently labeled for in-crop a pplications in soybean, weed co ntrol ratings for dicamba were taken from labeled in-crop applications of dicamba in corn for comparison purposes. Glyphosate will continue to provide broad spectr um control of annual grasses and broadleaf weeds, while dicamba will provide improved control of common ragweed, giant ragweed, hemp sesbania, morningglory species, and prickly sida. As presented in Table VIII-17, dicamba is more effective in controlling marest ail then glyphosate. Likewise, in comparison to glyphosate, dicamba is expected to also im prove the control of lambsquarters, eastern black nightshade, kochia, palmer pigweed , and wild buckwheat. In addition to complementing the weed control of glyphosate, dicamba will provide another mode of action in the Roundup Ready soybean system to lower the poten tial risk of weeds developing resistance to gl yphosate (see Section L.5.3.3 of Appendix L). Furthermore, dicamba will provide an alternative mode of action for control of broadleaf weeds with populations known to be resistant to ALS and PP O classes of herbicides (see Table VIII-8 for herbicide listings).

Application of both dicamba and glyphosate to MON 87708 integrated with the RoundupReady soybean system will provide effective control of both dicamba- and glyphosateresistant broadleaf weeds (Johnson et al, 2010). In the U.S., kochia ( Kochia scoparia ) and prickly lettuce ( Lactula serriola ) are the only species with biot ypes confirmed to be resistant to dicamba after 40+ years of use (Heap, 2009) . Additionally, a populat ion of lambsquarters ( Chenopodium album ) has been confirmed as resistan t in New Zealand, and in Canada common hempnettle ( Galeopsis tetrahit ) and wild mustard ( Sinapis arvensis ) have beenMonsanto Company 10-SY-210U 212 of 721 

### confirmed as resistant, for a total of five sp ecies worldwide with confirmed resistance to dicamba. Glyphosate has been sh own to provide good to excellent control of all five of

these broadleaf weeds. In addition, there are 3 speci es (spreading dayflower ( Commelina diffusa ), field bindweed ( Convolvulus arvensis ) and wild carrot ( Daucus carota )) in the U.S. with confirmed resistance to 2,4-D. Of the dicamba and 2,4-D species with known resistance in the U.S. and Canada, cross resistance between dicamba and 2,4-D has only been documented in wild mustard. However, cr oss resistance within the other species can not be totally ruled out nor assu med to be present. Currently in the U.S., six grass species and seven broadleaf species have been confirme d to have resistance to glyphosate. Dicamba provides good to excellent control of all seven of these broadleaf species. None of these broadleaf weed biotypes have been shown to have populations that are resistant to both glyphosate and dicamba. However, there are kno wn resistant populations of kochia that are either resistant to glyphosate or to dicamba , but no population with known resistance to both glyphosate and dicamba. Since there is no cros s resistance between dicamba and glyphosate either product can be effective on kochia populations resistant to the other. A more detailed discussion regarding the potential development of dicamba resistance in weeds can be found in Appendix K.

The introduction of MON 87708 into the Roundup Ready soybean system will allow dicamba to effectively compete with and pr ovide an additional mode-of-action to the

alternative herbicides that are currently used in combination w ith glyphosate in preplant and in-crop postemergence applications in soybean (for a comparison of dicamba to alternative non-glyphosate herbicides, see Appendix L). This will provide an additional mode-of action into the soybean weed management system that was previously not available. A comparison of dicamba to alternative herbicides, in terms of human health effects ( acute toxicity, cancer risk, chronic risk, and risk to infants and children), ecologica l effects (aquatic animal risk and aquatic plant risk), weed management ef ficacy, herbicide-resist ant weed frequency, rotational crop restrictio ns, and the potential for injury to the soybean crop, is presented in Table VIII-19. In this analysis, dicamba of fers an improved risk profile over each alternative herbicide in at least one and up to five of the comparative categories; however all

alternative herbicides are safe when used according to label direc tions. Additionally, dicamba is expected to offer greater benefits than some alternative herbicides as a supplement to glyphosate for in-crop applic ations on MON 87708 since planting interval restrictions following preplant applications of dicamba in soybean will be removed and allowing dicamba to be applied through plan ting and up to crop emergence (cracking). Dicamba will have greater flexibility for preplant applications than current preplant applications of 2,4-D and potentially will repl ace some 2,4-D applications in soybean. The superior broadleaf weed control provided by dicamba and excellent crop tolerance when applied to MON 87708 will provid e an additional mode-of-acti on and an alternative to the

herbicides used for broadleaf weed control in soybean, partic ularly acifluorfen, lactofen, chlorimuron, and flumiclorac. The human health and environmental safety of dicamba relative to other non-glyphosate al ternative herbicides is disc ussed in greater detail in Appendix L. Considering the characteristics of dicamba from a weed control, compatibility with glyphosate, and human and environmental safety perspective, it is concluded that MON 87708 will complement the established safety and efficacy of glyphosate use in the Roundup Ready soybean system.Monsanto Company 10-SY-210U 213 of 721Table VIII-17. Common Broadleaf Weed Responses to Preplant Burndown HerbicidesCommon Broadleaf Weeds1,2Herbicide/Application LQ CR GR SW CCM,SP CT RC AL HV MT PLDN,HB DL CGSpring Preplant Application2,4-D (0.5 lb/1.0 lb) - - - - - 9 -/66/8 -/7 6/8 8/9 8/9 -/8 6/7 9/9Dicamba999967-98879-7-Dicamba + 2,4-D 9 9 9 9 6 9 6 9 8 9 9 9 - 8 9Glyphosate898778676668-77Glyphosate + 2,4-D 9 9 9 8 7 9 6 8 8 8 8+ 9 6 8 9Glyphosate + Canopy 8 9 9 9 7 8 6 7 6 6 8 8+ 9 8+ 9Glyphosate + Canopy + 2,4-D 9 9 9 9 7 9 6 8 8 8 9 9 9 8+ 9Glyphosate + Gangster + 2,4-D 9 9 9 9 7 9 6 8 8 8 9 9 8 8 9Glyphosate + Python + 2,4-D 9 9 9 8 7 9 6 8 8 8 9 9 6 8 9Glyphosate + Scepter + 2,4-D 9 9 9 8 7 9 6 8 8 8 8+ 9 6 8 9Gly + Sonic/Authority First +2,4-D9 9 9 9 7 9 6 8 8 8 9 9 8 8 9Glyphosate + Valor + 2,4-D 9 9 9 8 7 9 6 8 8 8 8+ 9 8 7 91All weed control ratings are from the 2009 Weed Control Guide for Ohio and Indiana – Weed Responses to Burndown Herbicides,Ohio State University and Purdue University (Loux et al., 2009). Weedcontrol ratings for weeds are:9 = 90% to 100%, 8 = 80%to90%, 7 = 70% to 80%, 6 = 60% to 70%, and -= less than 60% control, notrecommended. Ratings assume the herbicides are appliedin the manner suggested in the guidelines and according to the label under optimum growing conditions.2Weed species: LQ = lambsquarters, CR = common ragweed, GR = giant ragweed, SW = annual smartweed, CC = commonchickweed, M & SP = mustard and shephard’spurse, CT = Canada thistle, RC = red clover, AL = alfalfa, HV = hairy vetch, MT =marestail, PL = prickly sida, DN & HB = deadnettle& henbit, DL = dandelion,and CG = crested groundselMonsanto Company 10-SY-210U 214 of 721Table VIII-18. Common Broadleaf Weed Responses to Dicamba Compared to Labeled Postemergence Herbicides in SoybeanProductionCommon Broadleaf Weeds1,2Herbicide/Application BN CB CR GRHS LQ MG PA PW PS SP SW VL WHPostemergenceBentazon - 9 7 6 4 7 2-9 4 - 8 0 9 8+ -Chlorimuron - 9 8 7+ 8 - 8-9 6 9 2 7 8 8 -Cloransulam - 9 9 9 3 - 8-9 2 - 2 7 8 9 -Chlorimuron/thifensulfuron - 9 8 7+NA 8 NA NA 9 NA NA 9 9 -Dicamba38999989988887+8Flumiclorac - 7 7 - NA 7 NA NA 7 NA NA - 9 7Fomesafen 8 7 8+ 8 9 - 8-9 8 9 2 3 7 6 9Glyphosate898+8787-99978888Glyphosate/imazethapyr998+8+NA8+NANA9NANA998Imazamox 9 8 7 8 NA 8 NA NA 9 NA NA 8 9 -Imazethapyr 9 9 6 7 0 6 7-9 6 9 6 0 9 9 -Lactofen 8+ 8 9 8 9 - 8-9 8 9 8 5 6 7 9Thifensulfuron - 6 - - NA 8 NA NA 9 NA NA 8 9 -1All weed control ratings except for HS, MG, PA, PS, and SP are from the 2009 Weed Control Guide for Ohio and Indiana – OhioState University and Purdue University (Loux et al., 2009). Ratings for HS, MG, PA, PS, and SPare from the 2009 Weed ControlGuidelines for Mississippi, Mississippi State University (MSU, 2010), except for dicambaratings for PA are from the 2010 WeedControl Manual for Tennessee (University ofTennessee, 2010). Weed control ratingsfor weeds, except HS, MG, PA, PS, and SP,are: 9 = 90% to 100%, 8 = 80% to 90%, 7 =70% to 80%, 6 = 60% to 70%, and - = less than 60% control, not recommended. Weedcontrol ratings for HS, MG, PA, PS, and SPare: 9-10 = excellent, 7-8 = good, 4-6 = fair, 0-3 = none to slight. Ratings assume theherbicides are applied in the manner suggested in the guidelines and according to the label under optimum growing conditions. NAdenotes not available.2Weed species: BN = black nightshade, CB = cocklebur, CR = common ragweed, GR = giant ragweed, LQ = lambsquarters, MG =morningglory spp., HS = hemp sesbania, PA = palmer and spiny pigweed, PW = pigweed, PS= prickly sida, SP = sicklepod, SW =smartweed, VL = velvetleaf, and WH = waterhemp3Weed control ratings for dicamba are from postemergence applications in corn.Monsanto Company 10-SY-210U 215 of 721Table VIII-19. Summary of Comparative Analysis of Dicamba and Alternative HerbicidesHerbicide ActiveIngredientMode-ofAction1Human Health Risk MeasuresAquatic NonTarget SpeciesRisk Measures KnownResistantWeedSpecies2HerbicidalEfficacy(< 50% ofdicamba)LongRotationalCropRestrictionSeriousCropInjuryPotentialNumberof“Yes”Entries3Numberof “No”Entries4AcuteToxicityRiskCancerRiskChronicRiskInfants &ChildrenRiskAquaticAnimalRiskAquaticPlantRisk2,4-D acid / esters Aux (4)Yes Yes YesNeutralYes Yes28Neutral Neutral Neutral602,4-DB Aux (4) Neutral Neutral Neutral Neutral Neutral NeutralYesNeutral Neutral20imazethapyr ALS (2) No Neutral Neutral Neutral Neutral NeutralYes(107)Yes YesNeutral31cloransulam-methyl ALS (2) No Neutral Neutral Neutral Neutral NeutralYes YesNeutral41chlorimuron-ethyl ALS (2) No Neutral Neutral Neutral Neutral NAYes YesNeutral31thifensulfuron ALS (2) No Neutral Neutral Neutral NeutralYes Yes YesNeutral41imazaquin ALS (2) No No Neutral NeutralNeutral NAYes YesNeutral32imazamox-ammonium ALS (2) No Neutral No Neutral Neutral NeutralYes YesNeutral42flumioxazin PPO (14)Neutral NeutralYesNeutralYes Yes5Yes YesNeutral50fomesafen PPO (14)YesNeutralYesNeutral NeutralNeutral NeutralYes Yes 40flumiclorac-pentyl PPO (14) NoNo Neutral Neutral Neutral NAYesNeutral Neutral12sulfentrazone PPO (14)YesNeutral NeutralNeutral NeutralYes YesNeutral Neutral40lactofen PPO (14) NoYesNeutralYes Yes Yes5 Neutral NeutralYes 51fluthiacet-methyl PPO (14) NoYesNeutral Neutral Neutral Neutral 5YesNeutralYes 31acifluorfen sodium PPO (14)Yes YesNeutralYesNeutral Neutral 5YesNeutralYes 50glufosinate-ammonium Glu (10)YesNoYesNeutral NeutralNeutral No reports Neutral Neutral Neutral30paraquat dichloride BiPyr (22)YesNoYesNeutral NeutralYes24YesNeutral Neutral40mesotrione HPPD (28)No Neutral NeutralYesNeutralYesNo reportsYes YesNeutral41Refer to Appendix L for additional details on the comparison of alternative herbicides to dicamba.“Yes”, indicates that dicamba has an improved risk profile basedon presented categories. Entries not indicated with a “Yes” mean that dicamba iseither comparable or less favorable thanthe alternative herbicide. ”Neutral” entries indicate similar risks exist for dicambaand the alternativeherbicide. “No” means the alternative herbicide offers a risk benefit compared to dicamba1Mode-of-Action based upon WSSA (2010) classifications.2A listing of the worldwide numbers of known resistant weeds for each herbicide based on its mode-of-action group. Dicamba hasfive knownresistant weed biotypes worldwide (www.weedscience.org/summary/MOASummary.aspAccessed May 28, 2010). A “Yes” indicates that theMonsanto Company 10-SY-210U 216 of 721number of resistant weeds in this herbicide class is many morethan the known five dicamba resistant species biotypes. A comparison of eachindividual herbicide in the class is not provided. See Section L.5.3.3 in Appendix L.3Number of “Yes” entries in each row is a summation for all thecategories assessed, indicating a total score for improved riskprofile for dicamba.4Number of “No” entries in each row is a summation for all thecategories assessed, indicating a total score for worse risk profile for dicamba.NA – not availableMonsanto Company 10-SY-210U 217 of 721 

### VIII.H.1. Potential Impact of Dicamba Application Timing to MON 87708

The proposed label directions for dicamba a pplications in MON 87708 will permit preplant applications up to the crop emergence and in -crop postemergence app lications up to the R1/R2 soybean growth stage. Most in-crop app lications are expected to be made up to the V4 growth stage, when germinated weeds ar e more easily and economically controlled and broadcast applications can be uniformly applied to the weeds (reduc ed crop canopy compared to later vegetative growth stages); with limited applications up to R1/R2, where this application is primarily anticipated to control missed weeds, treat a flush of broadleaf weeds germinating after the V4 application, orfor control of more aggressive glyphosateresistant weed species such as Amaranthus or Ambrosia species. Currently, in-crop postemergence applications of dicamba, excep t for preharvest ap plications, are not permitted or practical on commerci ally available soybean due to the lack of crop tolerance. An application of dicamba at or following the V4 soybean growth stage is expected to be later in the growing season than the current latest in-crop application timings to corn, sorghum, and wheat.

To assess the potential impact related to an an ticipated difference in application timing of dicamba to MON 87708 compared to current labe led uses of dicamba, the latest timing of in-crop postemergence dicamba app lications in corn was assessed. The comparison to corn application timing is pertinent since corn is grown in the sa me general areas where soybean is grown and a high per centage of the total dicamba-treated acres occur in corn (see Table VIII-15). Table VIII-20 provides the typical date and days from planting for corn and soybean to reach various growth stages. According to USDA-NASS (2009e) planting progress reports, the dates at which 50% of th e corn and soybean are planted in Illinois are April 25 and May 17, respectively. The current dicamba label 7 

### specifies an allowable use pattern relative to corn growth stages as defined by the number of leaves and plant height. Dicamba can be broadcast applied to corn up to a height of 8 inches, which is roughly equivalent to the 5-leaf stage. To relate th ese application timings to soybean, it is necessary to convert the timing of the 5-leaf stage of co rn to a comparable soybean vegetative growth stage (V stage). The 5-leaf stage of corn is equivalent to a V4 growth stage (Personal communication, Dr. R. Nielsen – Purdue University, 2010). Corn will generally reach this growth stage in 37 days from planting based on central Illinois temperatures. Presuming corn planting occurs on April 25, corn would reach the comparable V4 stage on June 1. Soybean will typically reach the V4 growth st age in 35 days. Presuming soybean planting on average occurs on May 17, soybean will reach the V4 stage on approximately June 21. Therefore, based on planting and environmental da ta from central Illinois, the most likely application timing for dicamba to MON 87708 is approximately 20 days later than the current latest application timing for corn. The timing difference is expected to be applicable to much of the Midwest region where th e majority of the soybean is grown.

7 Clarity product label can be found at:http://www.cdms. net/Images/acroiconwblue.gif. Label specifies when applying to corn not to apply Clarity when soybean are growing nearby if any of the following conditions exist: 1) corn is more than 24” tall, 2) soybean is more than 10” tall, and 3) soybean has begun to bloom.Monsanto Company 10-SY-210U 218 of 721 

### Table VIII-20. Dates and Days to Reach Va rious Growth Stages in Corn and Soybean

in Central Illinois

Corn 1 Soybean 2

Growth Stage Date & Calendar Day Days From Planting Date & Calendar Day Days From Planting

Planting April 25 (115) 3 May 17 (137) 3
VE May 10 (130) 15 May 27 (147) 10 VC June 1 (152) 15 V1 May 27 (137) 22 June 6 (157) 20 V2 May 22 (142) 27 June 11(162) 25 V3 May 27 (147) 32 June 16 (167) 30

V4 June 1 (152) 37 June 21 (172) 35 V5 June 5 (156) 41 June 26 (177) 40 V6 June 9 (160) 45 June 29 (180) 43 V7 June 13 (164) 49 July 2 (183) 46 V8 June 17 (168) 53 July 5 (186) 49

1 

### Corn growth based on Nielsen (2008) and av erage growing degree days at Champaign, IL (1971-2000).

2 

### Soybean growth is based on Naeve (2005).

3 

### Average date when 50% of the crop acreage wa s planted in Il linois during the 5-year period 2004-2008 (USDA-NASS, 2009d).

Corn and soybean planting dates and climates are considerably different in the Southeast region states than the Midwest region states. Thus, a second analysis was conducted to assess the potential difference in timing betwee n current dicamba applications in corn and the likely dicamba applicati on to MON 87708 in a southern location. Table VIII-21 provides the typical date and days for plantings of corn and soybean to reach various growth stages in western Tennessee. The dates at which 50% of the corn and soybean are planted in Tennessee are April 15 and May 25, respectivel y. Corn will generally reach the 5-leaf growth stage (comparable to the V4 soybean gr owth stage) in approximately 30 days from planting based on western Tennessee’s climate. Typical corn plantings occur on April 15, such that corn will reach the V4 stage on Ma y 15. Soybean will reach the V4 growth stage in 27 days. Typical soybean pl anting occurs on May 25, such that soybean will typically reach the V4 stage on June 21. Therefore, based on planting and environmental data from western Tennessee, the expected postemerge nce dicamba application timing (V4 growth stage) to MON 87708 is approxim ately 37 days later than the current latest application timing for corn. Although the difference between application timings is greater in this southern location compared to a northern loca tion, the estimated dates for the V4 stage in soybean for the two locations are the same.

As presented in Appendix M, Monsanto has submitted an application to EPA to amend Registration Number 524-582 to add the new use on MON 87708 to the product label. Registration Number 524-582 is a DGA salt formul ation of dicamba, which is a formulation with low volatility. Monsanto also plans to further address the use of dicamba on MON 87708 with EPA to evaluate whether any a dditional measures may be appropriate. In addition, Monsanto in cooperatio n with public sector scien tists and organizations willMonsanto Company 10-SY-210U 219 of 721 

### implement robust stewardship programs to assist in the education of a pplicators on the best application practices to minimize the potential for offsite movement, including the use of low volatility formulations.Monsanto Company 10-SY-210U 220 of 721 

### Table VIII-21. Dates and Days to Reach Va rious Growth Stages in Corn and Soybean

in Western Tennessee

Corn 1 Soybean 2

Growth

Stage Date & Calendar Day Days From Planting Date & Calendar Day Days From Planting

Planting April 15 (108) 3 May 25(148) 3
VE April 25 (118) 10 June 1 (155) 7 VC June 5 (159) 11 V1 May 1 (124) 16 June 9 (163) 15 V2 May 6 (129) 21 June 13 (167) 19 V3 May 10 (133) 25 June 17 (171) 23

V4 May 15 (138) 30 June 21 (175) 27 V5 May 19 (142) 34 July 25 (179) 31 V6 May 23 (146) 38 July 28 (182) 34 V7 May 26 (149) 41 July 1 (185) 37 V8 May 30 (153) 45 July 4 (188) 40

1 

### Corn growth based on Nielsen (2008) and 30- year average growing degree days at West Memphis, TN.

2 

### Soybean growth is based on Naeve (2005).

3 

### Average date when 50% of the crop acreage was planted in Tennessee during the 5-year period 2004-2008 (USDA-NASS, 2009d).

Table VIII-22. Average Daily Temperatures in Major Soybean Producing States 1

Average Daily Temperature (°F)

April May June July

IN 51 62 71 74

IA 49 60 70 74

IL 52 62 72 75

MN 42 56 65 69

MO 55 64 73 78

OH 50 60 69 73

AR 60 69 76 81

NE 48 59 69 74

ND 42 55 64 69

SD 45 57 66 72

1 

### Earth System Research Laboratory of Nati onal Oceanic and Atmospheric Administration (NOAA-ESRL 2011).Monsanto Company 10-SY-210U 221 of 721 

### Table VIII-23. Average Relative Humidity (1971-2000) in Major Soybean Producing

States 1

Morning Afternoon April May June July April May June July

IN 78 80 81 84 56 57 57 59

IA 77 78 81 85 61 62 64 66

IL 73 75 76 79 60 60 61 63

MN 75 76 81 84 59 58 62 64

MO 76 82 84 84 61 66 66 65

OH 76 78 80 83 54 54 55 55

AR 80 86 86 85 61 61 64 63

NE 77 79 81 81 56 58 58 58

ND 77 76 81 81 59 56 60 58

SD 78 79 81 82 58 58 60 57

1 

### National Climatic Data Center of Nationa l Oceanic and Atmosphe ric Administration (NOAA-NCDC 2011).

VIII.H.2. Impact on Dicamba Use in U.S. Soybean Production Following Integration

of MON 87708 into the Roundu p Ready Soybean System

Upon integration of MON 87708 into th e Roundup Ready soybean system, dicambaherbicide use will be expanded to in-crop postemergence applications for those hard-tocontrol and herbicide-resistant broadleaf w eeds found in U.S. soybean production. The impact that MON 87708 will have on overall dicamba use will be dependent upon the level of MON 87708 adoption by growers. Thus, the extent of dicamba-treated soybean acreage following the deregulation of MON 87708 is diffi cult to forecast. Monsanto estimates dicamba-treated acres to ultimately be in the range of 30 to 50% of the total U.S. soybean

acres. This estimate is based on a number of factors: 1) the percentage of non-glyphosate herbicides currently used in Roundup Ready s oybean, 2) current and historical use of dicamba in corn, 3) the development of glyph osate-resistant weeds in soybean cultivation areas, 4) the effectiveness of other non-gl yphosate herbicides used in the Roundup Ready soybean system, 5) compliance with EPA mandatory label instructions may have the practical affect of limiting the use in some soybean growing areas; and 6) the foreseeable future introduction of new competitive bi otechnology-derived traits in soybean.

Approximately 15 to 21% of growers used a non-glyphosate herbicide in addition to glyphosate in the Roundup Ready soybean syst em in 2005 (Givens et al., 2009). The reasons growers stated for their use of a non-glyphosate herbicid e in the Roundup Ready soybean system were generally for early weed control, residual weed control, improvement in the control of specific hard-to-contro l weeds, and/or glyphos ate-resistant weed management. Frisvold et al. (2009) reported th at in 2007 approximately 30% of the growers often or always use herbicides with di fferent modes-of-action in the Roundup Ready soybean system. The future use of non-glyphosat e herbicides would be expected to increaseMonsanto Company 10-SY-210U 222 of 721 

### in order to support the management of glyphos ate-resistant weeds. Additionally, grower educational programs on weed resistance management conducted by industry and universities encourage the use of non-glyphosat e herbicides with a lternative modes-ofaction in Roundup Ready cropping systems as a pr oactive measure to minimize the potential for development of glyphosate-resistant weeds (Beckie, 2006; Powles, 2008). These programs, along with Monsanto’s support for the use of anothe r herbicide mode-of-action to control glyphosate-resistant weeds, will likel y drive a further increase in non-glyphosate herbicides applied in soybean production.

A second factor that can be used as an indica tor of potential future dicamba-treated soybean acreage is the current and historical use of di camba in corn. Dicamba has been applied for many years in conventional and Roundup Rea dy corn for supplemental broadleaf weed control very similar to the proposed uses of dicamba on MON 87708. Therefore, information on dicamba use in corn will prov ide an indication of the potential demand for dicamba applied to MON 87708 when planted as a combined trait with Roundup Ready 2 Yield soybean. The use of non-glyphosate herb icides in Roundup Ready corn was reported to be 43 to 44% in 2005, which is significa ntly higher than pres ently used in Roundup Ready soybean (Givens et al., 2009). More rece nt market research data indicate a higher adoption rate of non-glyphosate herbicides in the corn, where market estimates were around 70% in 2010 (Agro Trak, 2010). Although, the reasons for using a non-glyphosate herbicide in the Roundup Ready corn system are the same as in Roundup Ready soybean, the benefits of using residual herbicides ar e greater in Roundup Ready corn because of the need for early season weed control and greater application flexibility with postemergence application of glyphosate (Dalley et al., 2001). Arguably for these reasons, the use of nonglyphosate herbicides applie d to MON 87708 as a combined trait with Roundup Ready 2 Yield soybean will not likely exceed the current levels in Roundup Ready corn.

Prior to the introduction of Roundup Ready corn, the use of dicamba in corn production was greater since it was used mainly to complement the control of residua l grass herbicides by providing postemergence control of annual broa dleaf weed escapes. Dicamba-treated acres in conventional corn peaked at 29% of the corn acres in 1994 . Dicamba use in corn dropped

to 9.4% of the corn acres in 2008 followi ng the commercial intr oduction of Roundup Ready corn (Table VIII-12). Therefore, consideri ng the current use of dicamba in Roundup Ready corn (conservatively assumed to be 43 to 70%), the historical use of dicamba in conventional corn (peaking at 29% of the cornacres in 1994), and the current use of nonglyphosate herbicides in soybean (15 to 21% of growers applied a non-glyphosate herbicide in 2005), it can reasonably be concluded that dicamba-treated acres of MON 87708 will not likely exceed 50% of the total soybean acres following introduction of MON 87708.

A third factor impacting dicamba -treated soybean acreage is th e current and future need for control of glyphosate-resi stant weeds. Glyphosate-resistant weeds have been identified in multiple states (Heap, 2009). When a glyphosate -resistant weed biotype has been confirmed to be present in a geographica l area, growers in that area are recommended by Monsanto, glyphosate distributors, and university specia lists to proactively implement glyphosateresistant weed management programs to ensure effective control of the resistant weed biotype regardless of whether the weed speci es has been confirmed to be resistant on a grower’s farm. Therefore, the acreage in an area where responsiv e weed resistanceMonsanto Company 10-SY-210U 223 of 721 

### management practices are implemented is potenti ally greater than the actual acres known to be impacted by glyphosate-resistant weeds. Un iversity weed scient ists are recommending growers proactively implemen t best management practic es, including a non-glyphosate herbicide with a second mode-of-action, in their cropping systems to minimize the development and potential spread of glyphosate- resistant weeds in the future (Owen et al., 2009; Frisvold et al., 2009). Monsanto s upports this recommenda tion where glyphosateresistant biotypes are present, such that MON 87708 integrated into the Roundup Ready soybean system should be treated with gl yphosate and dicamba plus another herbicide

mode-of-action to ensure at least two effectiv e modes-of-action cover the spectrum of weeds present in a grower’s soybean field. In this case, the other herbic ide mode-of-action that will be recommended in the MON 87708 plus R oundup Ready soybean system is likely to be a soil active residual preemergence herbicide.

It is anticipated that even in locations where glyphosate-resistan t weeds are present, glyphosate will continue to be the base herbic ide applied to MON 87708 as a combined trait product with MON 89788 (Roundup Ready 2 Yiel d soybean), thereby providing broad spectrum control of grass and broadleaf weeds. MON 87708 allows th e use of dicamba in the Roundup Ready soybean system for contro l of glyphosate-resistant weeds and to improve the control of dicotyle donous weeds that are hard to control with glyphosate alone. As described in Section VIII.F.1 and Table VIII-8, numerous residual and postemergence herbicides are utilized currently to compleme nt the control of glyphosate for hard-to-control weeds and to mitigate or control glyphosate-resi stant weeds. These alternative herbicides will compete with dicamba and undoubtedly will reduce the potential dicamba use on MON 87708 integrated into the Roundup Ready s oybean system and in future combined trait products containing di camba tolerance.

An additional factor influenci ng the number of dicamba-treated soybean acres in the future will be the introduction of compe ting herbicide-tolerant traits in soybean. Currently, there are numerous herbicide-tolerant soybean pr oducts under regulatory review or recently authorized (see Appendix J, Tabl e J-4). This includes several products that ha ve tolerance to multiple herbicides with different mode s-of-action. These new biotechnology-derived herbicide-tolerant soybean products are anticipate d to be introduced in future years and will compete with Monsanto’s MON 87708 × M ON 89788 combined-trait soybean product, further reducing the ulti mate potential of dicamba applica tions in soybean. A grower may

chose to not cultivate MON 87708 or to use an alternative soybean herb icide for their weed control. Furthermore, compliance with EPA mandatory label instructions may have the practical affect of limiting the use in some soybean growing areas.

Taking into consideration the above assessm ent, the potential dicamba-treated MON 87708 acreage is estimated to be 40% of the U.S. soybean acres, and would represent approximately 30 million dicamba-treated soybean acres. Considering the acreage currently treated with dicamba (20.2 million acres of which 0.53 million acres area soybean), this would potentially result in a tota l of 50.2 million acres treated w ith dicamba. As presented previously, dicamba was used on approximately 36 million acres at its peak use in 1994. Furthermore, considering the anticipated weed control recommendations for dicamba use in MON 87708, as outlined in Section VIII.H, comb ined with current dicamba usage in other crops (2.7 million pounds), a total of 25 million pounds a.e. of dicamba is estimated (high-Monsanto Company 10-SY-210U 224 of 721 

### end) to be applied annually, compared to 9.4 million pounds a.e. of dicamba applied at its peak use in 1994.

VIII.H.3. Conclusions on Dicamba-Tolerant Soybean MON 87708

Upon integration of MON 87708 into the Roundup Ready soybean system, growers will have the ability to continue use of establishe d soybean production practices including tillage systems; the same planting and harvesting mach inery; traditional management of insects, diseases, and other pests; and many of the cu rrent herbicides used for weed control, including glyphosate with its esta blished environmental benefits . Similarly, certified seed production will continue to use well-establishe d industry practices to deliver high quality seed to growers. Due to the excellent cr op safety of MON 87708 to dicamba, growers will

have a new herbicide mode-of-action for in-cr op control of hard-to-control and herbicide

resistant broadleaf weeds that are present in U.S. soybean pr oduction. As expected with a new use of herbicide in U.S. soybean produc tion, the number of dicamba-treated soybean

acres and the total dicamba use will increase. Th e total dicamba use is expected to be about double the historical peak le vels experienced since dicamba’s introduction in 1967. Additionally, due to the expanded timing of in-crop applications to soybean, dicamba treatments may be later in the growing season than currently occurs for most labeled dicamba uses, including applications to cor n. Monsanto has requested EPA approval for the use of a low volatility dicamba (DGA salt) formulation, together with allowing only ground application on MON 87708, s ubject to certain limitations specifie d on the label. With these additional limitations, the potential for offsite movement (spray drift and volatility) onto adjacent crops due to applicat ions on MON 87708 later in the season is not expected to significantly impact adjacent crops . In addition, Monsanto will consult with U.S. EPA to identify what additional measur es, if any, are appropr iate to address any potential impact of dicamba offsite movement. In addition, to reinforce anticipated EPA label requirements, Monsanto will implement a robust stewardship program that will include a strong emphasis on grower and applicator training.

VIII.I. Crop Rotation Practices in Soybean

The well-established farming practice of crop rotation is still a key management tool for growers. The purpose of growing soybean in rota tion is to improve yield and profitability of one or both crops over time, decrease the need for nitrogen fertilizer on the crop following soybean, increase residue cover, mitigate or br eak disease, insect, and weed cycles, reduce soil erosion, increase soil organic matter, improve soil tilth and soil physical properties, and reduce runoff of nutrients, herb icides, and insecticides (Boe rma and Specht, 2004; Al-Kaisi et al., 2003). According to USDA Economic Res earch Service, 95% of the soybean-planted acreage has been in some form of a crop ro tation system since 1991 (USDA-ERS, 2001). Corn- and wheat-planted acreage has been rotate d at a slightly lower level of 75% and 70%, respectively. Although the bene fits of crop rotations can be substantial, the grower must make cropping decisions by evaluating both the agronomic and economic returns of various cropping systems. Crop rotations also afford growers the opportunity to diversify farm production in order to minimize market risks.Monsanto Company 10-SY-210U 225 of 721 

### Continuous soybean production is not a common practice in the Midwest and is discouraged by most extension soybean specialists to redu ce the risk of damage from diseases and

nematodes (Hoeft et al., 2000; Al-Kai si et al., 2003). Corn a nd soybean occupy more than 80% of the farmland in many of the Midwestern states, and the two- year cropping sequence of soybean-corn is used most extensively in this region. However, a soybean crop sometimes is grown after soybean and then ro tated to corn in a 3-year rotation sequence (soybean-soybean-corn) in the Midwest. Compared to corn, soybean shows a greater yield response to being grown after a number of year s without soybean. The yield of both corn and soybean is approximately 10% higher when grown in rotation than when either crop is grown continuously (Hoeft et al., 2000).

A combination of conservation tillage practices and crop rotation has b een shown to be very effective in improving soil physical properties. Long-term studi es in the Midwest indicate that the corn-soybean rotation improves yield potential of no-till systems compared to continuous corn production (Al-Ka isi, 2001). The reduction in yield of continuous corn production in no-till systems is attributed to low soil temperature during seed germination, which is evident on poorly draine d soils under no-till practices.

Unique to the southern porti on of the Midwest and the Southe ast regions, soybean is grown in a double-cropping system. Doub le-cropping refers to the pr actice of growing two crops in one year. This practice can improve income and reduce soil and water losses by having the soil covered with a plant canopy most of the year (Hoeft et al., 2000). In the Midwest,

winter wheat is harvested in late June or Ju ly, and then soybean is planted into the wheat residue in a no-till syst em to conserve moisture. Due to the uncertainty of double-cropping yields, growers sometimes do not plant if soils are too dry at the time of wheat harvest. Soybean typically is grown in a corn-wheat -soybean rotation sequence when soybean is grown in a double-cropping system.

Rotation practices for soybean vary from state to state. However, there are similarities among states within certain growing regions. This section provides a detailed description and quantitative assessment of the rotationa l cropping practices i mmediately following soybean production, by region and state. This assessment accounts for about 99% of total U.S. soybean acreage. This assessment is based on current agronomic practices following soybean production and current dicamba herb icide usage in labeled crops. USDA-NASS (2004, 2006, 2007b, 2008) and AgroTrak (2009) data on dicamba herbicide usage for corn, sorghum, cotton, wheat, barley, and oats were ut ilized for this assessment. For the purpose of this assessment, a 50% a doption rate in U.S. soybean production was assumed for

MON 87708. These data on rota tional patterns are presented in Tables VIII-24 through

VIII-27.

The majority of the U.S. soybean acreage (68.6 %) is rotated to corn (Table VIII-24). The second largest rotational crop following soybean is soybean. Approximately 14.5% of the soybean acreage is rotated back to soybean th e following year. Wheat follows soybean on approximately 11.2% of the U.S. soybean acreage, with cotton, rice, and sorghum the next largest rotational crops. However, these th ree crops were planted on only 4.6% of the soybean acreage. Other minor rotational crops that follow soybean production are listed in

Table VIII-24.Monsanto Company 10-SY-210U 226 of 721 

### Column J of each table provides the percentage of soybean acreage as a function of the total rotational crop acreage to indicate the level th at soybean is the primary crop preceding the rotational crops. For the entire country (Table VIII-24), this percentage is 35.3% indicating that soybean is a major crop preceding these rota tional crops. The percentage of soybean as a preceding crop varies widely in different states, which ranges from 16.8% (KS, Table VIII-25) to 95.2% (NJ, Table VII I-27). In the Midwest region where 82% of the soybean is grown, 34.6% of the rotational crop area was planted with soybean during the previous growing season.

Dicamba-tolerant soybean can be followed by dicamba-tolerant soybean or another rotational crop with a labeled ap plication of dicamba. To de termine the likelihood that the rotational crops planted after MON 87708 w ill be a rotational crop where dicamba application is possible, an assessment also ha s been provided in Column K of Tables VIII24 through VIII-27. This assessment showed that the percentage of the total rotational crop acreage that may be rotated from MON 87708 to another crop with a potential for dicamba application (Table VIII-24 - Column K) is es timated to be 4.9% in the U.S., with ranges from32.3% in Mississippi to Georgia and New Je rsey having essentia lly no rotational crop acres with dicamba usage since these states have essentially no continuous soybean acres and USDA-NASS and AgroTrak statistics show no use of di camba currently in labeled crops in these states. The percentage is 4.1% in the Midwest region, which is the largest soybean production region.Monsanto Company 10-SY-210U 227 of 721TableVIII-24. Rotational Practices in the U.S. Following Soybean ProductionA B C D E F G H I J KStateTotalSoybeanAcres1MajorCropsFollowingSoybean inRotationTotalAcreage ofRotationalCrop in theU.S.1RotationalCrop AcresFollowingSoybean2%RotationalCropFollowingSoybean3%RotationalCrop ofTotalSoybean4Acreage ofDicamba inRotationalCropOption5% DicambaUsage inRotationalCropOption6% SoybeanAcresPrecedingMajorRotations7Estimated% DicambaUsage inMajorRotations8UnitedStates75037CornSoybeanSorghumCottonWheatBarleyOatsRiceAlfalfaSugar BeetsPotatoesDry BeansDry PeasMilletFlaxOther98013075037402037673741421591995230118648303341183520250345452Total: 212601515001086684115708396419810421621443235384176155Total: 7503764.314.520.941.722.41.94.945.38.717.39.63.07.316.422.034.368.614.51.12.111.20.050.11.40.20.190.040.050.050.050.100.244315438153448Total: 104708.6%40.0%9.7%5.3%35.34.9This U.S. summary was developed by compiling the data from all three regional summaries. All acreage is expressed as 1000s ofacres.1Acreage planted of the specific crops is based on 2008 planting data (USDA-NASS, 2009a); “other” crop and newly seeded alfalfaacreages are based on 2008planting data from the Individual States data which was obtained from Quick Stat searches onhttp://www.nass.usda.gov/Data_and_Statistics/Quick_Stats/index.asp.2Column E is obtained by compiling the data from all three regional summaries.3Column F is obtained by dividing Column E by Column D.4Column G is obtained by dividing Column E by Column B.5Column H is obtained by compiling the data from all three regional summaries.6Column I is obtained by dividing Column H by Column E.7Column J is obtained by dividing Column B by Column D Total.8Column K is obtained by dividing Column H Total by Column D Total.9Various vegetables.Monsanto Company 10-SY-210U 228 of 721TableVIII-25. Rotational Practices Following Soybean Production in the Midwest RegionA B C D E F G H I J KStateTotalSoybeanAcres1Major CropsFollowingSoybean inRotationTotalAcreage ofRotationalCrop inStates1RotationalCrop AcresFollowingSoybean2%RotationalCropFollowingSoybean3%RotationalCrop ofTotalSoybean4Acreage ofDicamba inRotationalCrop Option5% DicambaUsage inRotationalCropOption6% SoybeanAcresPrecedingMajorRotations7Estimated %DicambaUsage inMajorRotations8Region 62150CornSoybeanSorghumCottonWheatBarleyOatsRiceAlfalfa9Sugar BeetsPotatoesDry BeansDry PeasMilletFlaxOther1072260621503553341320391929159020016178302781166520250345342Total: 1794104748048856707881024198182162144323538417687Total: 6215065.77.918.822.925.32.16.291.010.017.311.63.07.316.422.025.376.47.91.10.113.00.070.20.30.30.20.050.060.060.070.10.145911954313144486Total: 73269.74046.8185.515*NANANANANANA*NANA34.64.1IL 9200CornSoybeanSorghumWheat121009200801200Total: 22580855623074340Total: 9200713922893.02.50.83.7171192265Total: 2068204046*40.79.2IN 5450CornSoybeanWheat57005450580Total: 117304905273273Total: 5450865479055343109Total: 453740*46.53.9Monsanto Company 10-SY-210U 229 of 721Table VIII-25 (continued). Rotational Practices Following Soybean Production in the Midwest RegionA B C D E F G H I J KStateTotalSoybeanAcres1Major CropsFollowingSoybean inRotationTotalAcreage ofRotationalCrop inStates1RotationalCrop AcresFollowingSoybean2%RotationalCropFollowingSoybean3%RotationalCrop ofTotalSoybean4Acreage ofDicamba inRotationalCrop Option5% DicambaUsage inRotationalCrop Option6% SoybeanAcresPrecedingMajorRotations7Estimated %DicambaUsage inMajorRotations8IA 9750CornSoybeanAlfalfa9133009750125Total: 23175945819598Total: 975071278972137878Total: 456440NA42.12.0KS 3300CornSoybeanSorghumWheat3850330029009600Total: 1965016503301651155Total: 3300431061250105352971321358Total: 50018408516.82.5KY 1390CornSoybeanWheat12101390580Total: 3180118213970Total: 13909810128510510656Total: 162940*43.75.1MI 1900CornSoybeanWheat24001900730Total: 5030133095475Total: 19005556570525160385Total: 20212401637.84.0MN 7050CornSoybeanWheatSugar beetsDry BeansOther11770070501925440150203Total: 17468535821212691063571Total: 705070366242435763181.50.516978514Total: 796134016NANANA40.44.6Monsanto Company 10-SY-210U 230 of 721Table VIII-25 (continued). Rotational Practices Following Soybean Production in the Midwest RegionA B C D E F G H I J KStateTotalSoybeanAcres1Major CropsFollowingSoybean inRotationTotalAcreage ofRotationalCrop inStates1RotationalCrop AcresFollowingSoybean2%RotationalCropFollowingSoybean3%RotationalCrop ofTotalSoybean4Acreage ofDicamba inRotationalCrop Option5% DicambaUsage inRotationalCropOption6% SoybeanAcresPrecedingMajorRotations7Estimated %DicambaUsage inMajorRotations8MO 5200CornSoybeanSorghumCottonWheatRice28005200903061250200Total: 98462756156010478520182Total: 52009830116254291533021.5103.520162414Total: 8397640*18*NA52.88.5NE 4900CornSoybeanSorghumWheat880049003001750Total: 157503675490245490Total: 49004210822875105101471962215Total: 3804409331.12.4ND 3800CornSoybeanWheatSugar BeetsDry PeasFlax255038009230208520335Total: 1664311407981710383876Total: 380045211918723302145112331319274Total: 923295016NANANA22.85.6OH 4500CornSoybeanWheat330045001120Total: 89203150450900Total: 45009510807010203218018Total: 2301402550.42.6Monsanto Company 10-SY-210U 231 of 721Table VIII-25 (continued). Rotational Practices Following Soybean Production in the Midwest RegionA B C D E F G H I J KStateTotalSoybeanAcres1Major CropsFollowingSoybean inRotationTotalAcreage ofRotationalCrop inStates1RotationalCrop AcresFollowingSoybean2%RotationalCropFollowingSoybean3%RotationalCrop ofTotalSoybean4Acreage ofDicamba inRotationalCrop Option5% DicambaUsage inRotationalCropOption6% SoybeanAcresPrecedingMajorRotations7Estimated %DicambaUsage inMajorRotations8SD 4100CornSoybeanSorghumWheatBarleyOatsMillet47504100170366163220110Total: 1307429528282820418241Total: 410062248226537377222201211483313666Total: 26654016815**31.42.0WI 1610CornSoybeanWheatOatsAlfalfa9PotatoesOther123800161037327042063.5139.2Total: 66761369328116643216Total: 1610362226155112852514214113Total: 54340**NANANA24.10.8This Midwest region summary table was developed by compiling thedata from all the states within the region except where notedin footnotes. NA denotes NotApplicable. An asterisk (*) indicates no usage reported. All acreage are expressed as 1000s of acres.1Acreage planted of the specific crops is based on 2008 planting data (USDA-NASS, 2009a); “other” crop and newly seeded alfalfaacreages are based on 2008planting data from the Individual States data which were obtained from Quick Stat searches onhttp://www.nass.usda.gov/Data_and_Statistics/Quick_Stats/index.asp.2Column E for the regional summary is obtained by compiling data from all the stateswithin the region. Column E for the individual states is obtained by multiplying Column B by Column G.3Column F is obtained by dividing Column E by Column D.4Column G for the regional summary is obtained by dividing Column E by Column B. The rotational crop percentages in Column G for the individual states arebased on estimates from personal communications (2006) with individual state Extension Crop Production Specialist; Extension Agronomists – Soybean, Cornand Cotton; Extension Weed Control Specialist on Soybean and Corn; and/or Monsanto Technology Development Representatives.5Column H for the regional summary is obtained by compiling the data from all the states within the region. Column H for the individual states is obtained bymultiplying Column E by Column I.Monsanto Company 10-SY-210U 232 of 7216Column I for the regional summary is obtained by dividing Column H by Column E. Dicamba usage except for individual states forsoybean is based on mostrecent statistics from USDA-NASS (2004 -sorghum, 2006 - corn and oats, 2007b - wheat and barley, 2008 -cotton). Percentages with the superscript 6 areusage statistics from AgroTrak (2009), due to USDA-NASS data not being reported for the particular crop and state. Dicamba usage in soybean (40%) is afuture market adoption estimate.7Column J is obtained by dividing Column B by Column D Total.8Column K is obtained by dividing Column H Total by Column D Total.9Newly seeded alfalfa.10Various vegetables.11Sweet corn and green peas.12Sweet corn, green peas and onions.Monsanto Company 10-SY-210U 233 of 721Table VIII-26. Rotational Practices FollowingSoybean Production in the Southeast RegionA B C D F E G H I J KStateTotalSoybeanAcres1Major CropsFollowingSoybean inRotationTotalAcreage ofRotationalCrop inStates1RotationalCrop AcresFollowingSoybean2%RotationalCrop ofTotalSoybean3%RotationalCropFollowingSoybean4Acreage ofDicamba inRotationalCropOption5% DicambaUsage inRotationalCrop Option6% SoybeanAcresPrecedingMajorRotations7Estimated% DicambaUsage inMajorRotations8Region 10860CornSoybeanSorghumCottonWheatRiceOther*35351043024523801530163165Total: 1981622845748171146929486034Total:1086021.052.91.613.52.77.90.364.655.169.861.719.252.752.0722299139Total: 25103.240*9.4*NA54.812.7AL 360CornSoybeanCottonWheat260360290240Total: 11501261818036Total: 3603555010485621526713Total: 46216407*31.34.0AR 3300CornSoybeanSorghumWheatRice440330012510701401Total: 6336231211266231660Total: 330076427205364532247845Total: 845*40**NA52.113.3GA 430CornCotton370940Total: 131043387Total: 430109012412Total: 256*32.80.2LA 1050CornSoybeanSorghumCotton5201050120300Total: 1990105683105158Total: 105010651015206588533273Total: 2763640**52.813.9Monsanto Company 10-SY-210U 234 of 721Table VIII-26 (continued). Rotational Practices Following Soybean Production in the Southeast RegionA B C D E F G H I J KStateTotalSoybeanAcres1MajorCropsFollowingSoybean inRotationTotalAcreage ofRotationalCrop inStates1RotationalCrop AcresFollowingSoybean2% RotationalCrop ofTotalSoybean3% RotationalCropFollowingSoybean4Acreage ofDicamba inRotationalCropOption5% DicambaUsage inRotationalCropOption6% SoybeanAcresPrecedingMajorRotations7Estimated %DicambaUsage inMajorRotations8MS 2000SoybeanRice2000230Total: 22301800200Total: 200090109087720Total: 72040NA89.732.3NC 1690CornSoybeanCottonOther9900169043065Total: 308581142342334Total: 169048252529025985236169Total: 2054640*NA54.86.7TN 1490CornSoybeanCotton6901490285Total: 2465671551268Total: 14904537189737945220126Total: 35116404760.414.2SC 540CornSoybeanCottonWheat355540135220Total: 12502971625427Total: 54055301058430401265Total: 65*40**43.25.2This Mid-South region summary table was developed by compiling the data from all the states within the region except where noted otherwise in footnotes. NAdenotes not applicable. An asterisk (*) indicates no usage reported. All acreage are expressed as 1000s of acres.1Acreage planted of the specific crops is based on 2008 planting data (USDA-NASS, 2009a); “other”crop and newly seeded alfalfaacreages are based on 2008planting data from the Individual States data which were obtained from Quick Stat searches onhttp://www.nass.usda.gov/Data_and_Statistics/Quick_Stats/index.asp.2Column E for the regional summary is obtained by compiling data from all the stateswithin the region. Column E for individual states is obtained by multiplying Column B by Column G.3Column F is obtained by dividing Column E by Column D.4Column G for the regional summary is obtained by dividing Column E by Column B. The rotational crop percentages in Column G forthe individual states arebased on estimates from personal communications (2006) with individual state Extension Crop Production Specialist; Extension Agronomists – Soybean, Cornand Cotton; Extension Weed Control Specialist on Soybean and Corn ;and/or Monsanto Technology Development Representatives.5Column H for the regional summary is obtained by compiling the data from all the states within the region. Column H for the individual states is obtained bymultiplying Column E by Column I.Monsanto Company 10-SY-210U 235 of 7216Column I for the regional summary is obtained by dividing Column H by Column E. Dicamba usage except for individual sites for soybean is based on mostrecent statistics from USDA-NASS(2004 - sorghum, 2006 - corn andoats, 2007b - wheat and barley,2008 - cotton). Percentageswith the superscript 5 areusage statistics from AgroTrak (2009) becauseUSDA-NASS data was not reported for the particular crop and state. Dicamba usagein soybean (40%) is afuture market adoption estimate.7Column J is obtained by dividing Column B by Column D Total.8Column K is obtained by dividing Column H Total by Column D Total.9Cucumbers and sweet potatoes.Monsanto Company 10-SY-210U 236 of 721TableVIII-27. Rotational Practices Following Soybean Production in the East Coastal RegionA B C D E F G H I J KStateTotalSoybeanAcres1Major CropsFollowingSoybean InRotationTotalAcreage ofRotationalCrop inStates1RotationalCrop AcresFollowingSoybean2% RotationalCropFollowingSoybean3% RotationalCrop ofTotalSoybean4Acreage ofDicamba inRotationalCropOption5% DicambaUsage inRotationalCropOption6% SoybeanAcresPrecedingMajorRotations7Estimated %DicambaUsage inMajorRotations8Region 4687CornSoybeanCottonOther9361517056145Total: 542617412422321Total: 202748.214.238.045.985.911.91.11.039097Total: 48722.440*NA37.49.0DE 195CornSoybean160195Total: 35515639Total: 1959820802016Total: 16*4054.94.5MD 495CornSoybean460495Total: 95544650Total: 4959710901025820Total: 2785864051.829.1NJ 92CornOther98511.6Total: 97839Total: 9297799010Total: 0*NA95.20NY 230CornOther10109033.5Total: 112421912Total: 230203495511Total: 115*20.51.0PA 435CornSoybean1350435Total: 17854269Total: 435322982304Total: 3474024.41.9VA 580CornSoybeanCotton47058061Total: 111141214523Total: 580882538712549158Total: 14922640*52.213.4This Eastern Coastal region summary table was developed by compiling the data from all the states within the region except where noted in footnotes. NAdenotes not applicable. An asterisk (*) indicates no usage reported. All acreage are expressed as 1000s of acres.Monsanto Company 10-SY-210U 237 of 7211Acreage planted of the specific crops is based on 2008 planting data (USDA-NASS, 2009a); “other”crop and newly seeded alfalfaacreages are based on 2008planting data from the Individual States data which were obtained from Quick Stat searches onhttp://www.nass.usda.gov/Data_and_Statistics/Quick_Stats/index.asp.2Column E for the regional summary obtained by compiling data from all of the states within the region. Column E for individualstates is obtained bymultiplying Column B by Column G.3Column F is obtained by dividing Column E by Column D.4Column G for the regional summary is obtained by dividing Column E by Column B. The rotational crop percentages in Column G forthe individual states arebased on estimates from personal communications (2006) with individual state Extension Crop Production Specialist; Extension Agronomists – Soybean, Cornand Cotton; Extension Weed Control Specialist on Soybean and Corn; and/or Monsanto Technology Development Representatives.5Column H for the regionalsummary is obtained by compiling the data from all the states within the region. Column H for the individual states is obtained by multiplying Column E byColumn I.6Column I for the regional summary is obtained by dividing Column H by Column E. Dicamba usage except for individual states forsoybean is based on mostrecent statistics from USDA-NASS (2004 -sorghum, 2006 -corn and oats, 2007b-wheat and barley, 2008-cotton). Percentages withthe superscript 6 are usagestatistics from AgroTrak (2009) because USDA-NASS data was not reported for the particular crop and state. Dicamba usage in soybean (40%) is a futuremarket adoption estimate.7Column J is obtained by dividing Column B by Column D Total.8Column K is obtained by dividing Column H Total by Column D Total.9Sweet corn and other vegetables.10Sweet corn and onions.Monsanto Company 10-SY-210U 238 of 721 

### VIII.J. Soybean Volunteer Management

Volunteer soybean is defined as a plant that has germinated and emerged unintentionally in a subsequent crop. Soybean seeds can remain in a field after soybean harvest as a result of pods splitting before or during ha rvest. Soybean seeds also can remain in a field when pod placement on the plants is too close to the gr ound for the combine head to collect all the pods or when the combine is improperly adju sted for efficient ha rvesting. Volunteer soybean in rotational crops is not a concern in the Midwest region because the soybean seed is typically not viable after the winter period (Carpenter et al., 2002; OECD, 2000). In southern soybean growing areas of the U.S. where the winter temperatures are milder, it is possible for soybean seed to remain viable over the winter and germinate the following spring.

Volunteer soybean normally is not a concern in rotational crops, such as corn, cotton, rice, and small grains ( e.g. , wheat, barley, sorghum, and oats), that are the significant rotational crops following soybean due to control measures that are available for volunteer soybean when they arise (Carpenter et al., 2002; OECD, 2000). Becaus e of these control measures and field testing which conf irmed that MON 87708 has equiva lent volunteer potential as other soybean, the introduction of MON 87708 plus Roundup Ready soybean will not elevate concerns about managing volunteer so ybeans nor will it result in more dependence on preplant or in-crop tillage a nd cultivation because there are adequate alternative herbicide options. Preplant tillage and/or herbicides are the first management tool for control of emerging volunteer soybean in the spring, where this may be an issue, such as in the south. If volunteer soybean should emerge after planting, shallow cultivation and/or use of another

herbicide will control vo lunteers and effectively reduce co mpetition with the crop. Several postemergence herbicides also are available to control volun teer soybean (conventional or glyphosate-tolerant soybean, and by extension dicamba-tolerant soybean ) in each of the major soybean rotational crops. Table V III-28 provides control ratings on volunteer soybean for several herbicides used in th e major rotational crops . Additionally, 19 commonly used herbicides in agriculture , representing eight modes-of-action ( i.e. , ALS inhibitor, chloroacetamide, E PSPS, PPO inhibitor, PSI disrupti on, PSII inhibitor, synthetic auxin, and tubulin inhibitor classes) were te sted as potential substrates for the MON 87708 DMO present in MON 87708 (Section V.A). Acro ss nearly all of the herbicides tested,

MON 87708 and the conventional control were simila r in their level of to lerance, except for the three phenoxy synthetic auxin herbicides (2,4-D, 2,4-DB, and MCPA). These results

indicate that herbicides which are effective fo r the control of volunteer soybean can still be

used to control MON 87708.

To provide control of volunt eer soybean in corn, postemerg ence applications of AAtrex ®


### (atrazine), Hornet ® 

### (flumetsulam + clopyralid) and Widematch ® 

### (clopyralid + fluroxypyr) provide excellent control (Zollinger, 2009). AAtrex and Permit ® 

### (halosufuron) provide excellent volunteer control in sorghum. In small grains (wheat, barley, oats), Bronate ®


### Advanced (bromoxynil/MCPA), and Widematch ® 

### applied postemergence provide excellent control of volunteer soyb ean (Zollinger, 2009).

Volunteer soybean in cotton is normally not a concern. However, hurricanes or other extreme weather conditions can damage a soybean crop preceding cotton production in theMonsanto Company 10-SY-210U 239 of 721 

### Mid-South region states, where the unharveste d soybean seed can produce volunteer plants. Preplant applications of paraqua t or herbicide mixtures contai ning paraquat will effectively control volunteer glyphosate-to lerant soybean (Montgomery et al., 2002; Murdock et al., 2002). Recent research in Nort h Carolina indicates Envoke (t rifloxysulfuron) will provide excellent postemergence control of (voluntee r) soybean containing traits for glyphosate and sulfonylurea herbicide tole rance in Roundup Ready cotton (York et al., 2005).

Volunteer soybean in rice is rarely a concern due to the combination of preplant tillage, flooding practices, and herbicides used in produc ing rice. If volunteer plants should emerge in rice, postemergence applications of Grands tand (triclopyr), Regiment (bispyribac), Grasp (penoxsulam), and Permit (halosulfuron) typical ly used for weed control in rice will effectively alleviate competition from volunt eer soybean (Dillon et al., 2006; Bond and Walker, 2009).

Given the low potential for soybean to volunteer in subsequent crops, the availability of multiple herbicidal and cultivation methods for controlling volunteers, as well as the

demonstrated lack of differe nce in germination of MON 87708 compared to conventional soybean (see Section VII.C.1), the intr oduction of MON 87708 into the Roundup Ready soybean system is not expected to impact the management of soybean volunteer plants.Monsanto Company 10-SY-210U 240 of 721 

### Table VIII-28. Ratings for Postemergence Cont rol of Volunteer Soybean in Labeled Rotational Crops 1



### Product Rate (Product/Acre) Soybean

V2 – V3 Soybean

V4- V6

Corn 2


### AAtrex 4L (atrazine) 0.38 qts E P 0.50 qts E F Hornet WDG (flumetsulam/clopyralid) 1 – 2 oz E F-G Widematch (clopyralid/fluroxypyr) 0.25 pt E G Sorghum 2,4


### AAtrex 4L (atrazine) 0.38 qts E P 0.50 qts E F Permit (halosulfuron) 2/3 oz E E

Buctril ® 

### (bromoxynil) 1 pt Wheat, Barley & Oats 2


### Buctril (bromoxynil) 1 pt E E Widematch (clopyralid/fluroxypyr) 0.25 pt E G

Cotton 3



### Envoke ® 

### (trifloxysulfuron) 0.1 oz E E

Rice 4



### Grandstand ® 

### CA (triclopyr) 0.5 pint E E Regiment ® 

### (bispyribac) 0.4 oz E E Grasp ® 

### SC (penoxsulam) 2 oz E E Permit (halosulfuron) 2/3 oz E E

NA denotes “not applicable.”

1 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 is Zollinger (2009).

3 Source is York et al. (2005).

4 Sources are Dillon et al. (2006); Bond and Walker (2009).Monsanto Company 10-SY-210U 241 of 721 

### VIII.K. Weed Resistance to Dicamba Herbicide

The risk of weeds developing resistance and the potential impact of resistance on the usefulness of an herbicide vary greatly acro ss different herbicide 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 unders tand the proper uses and stewardship of our herbicide-tolerant soybean systems. This re search includes an evaluation of the factors that can contribute to the development of weed resistance. Detailed information regarding dicamba stewardship is presented in Appendix K.

VIII.L. Stewardship of MON 87708

Monsanto Company develops effective produc ts and technologies and is committed to assuring that its products and technol ogies are safe and environmentally responsible. Monsanto demonstrates this commitment by implementing product stewardship processes throughout the lifecycle of a product a nd by participation in the Excellence Through Stewardship SM 

### (ETS) Program 8

. 

### These policies and practices include rigorous field compliance and quali ty management systems and verification through auditing. Monsanto’s Stewardship Principles are also articulated in Technology

Use Guides 9 

### that are distributed annu ally to growers who utili ze Monsanto branded traits. Growers who purchase seeds containing Monsan to’s proprietary biotechnology traits are contractually required to comply with stew ardship measures outlined in the Technology

Use Guide.

As an integral action of fulfilling our stew ardship commitment, Monsanto will seek biotechnology regulatory approvals for MON 87708 in all key soybean import countries with a functioning regulatory sy stem to assure global compliance and support the flow of

international trade. These actions will be consistent with the Biotechnology Industry Organization (BIO) Poli cy on Product Launch. 10 

### Monsanto continue s to monitor other countries that are key importers of soybean fr om the U.S. for the de velopment of formal biotechnology approval proce sses. If new functioning regulatory processes are developed, Monsanto will make appropria te and timely regulatory submissions.

Monsanto also commits to best industry practices on seed quality assurance and control to ensure the purity and integrity of MON 87708 seed. As with all of Monsanto’s products, before commercializing MON 87708 in any country, a MON 87708 detection method will be made available to soybean producers, processors, and buyers.

Dicamba is a selective herbicide registered with the U.S. EPA for the preemergent and postemergent control of certain broadleaf w eeds in agriculture. Dicamba has a long

8 Excellence Through Stewardship Program can be found at: http://www.excellencethr oughstewardship.org/

. 9 Monsanto Technology Use Guides can be found at:http://www.monsanto.com/SiteCollectionD ocuments/Technology-Use-Guide.pdf.

10 BIO’s Product Launch guidelines can be found at: http://www.excellencethroughstewar dship.org/LinkClick.aspx?fileticket=ppgyTABguQs%3d&tabid=84.Monsanto Company 10-SY-210U 242 of 721 

### history of effective use in U.S. crop production, including co rn, soybean, cotton, sorghum, wheat, barley, oats, millet, pasture, rangeland, asparagus, sugarcane, turf, grass grown for seed, conservation reserve program s, and fallow croplands (as described in Section VIII.G). Although herb icide resistance may eventua lly occur in a weed species when an herbicide is widely used, research, education and best management practices can be utilized to potentially de lay, contain and manage resistance. The addition of dicamba tolerance to the Roundup Ready soybean syst em will provide an efficient method for incorporation of an additional herbicide m ode-of-action in the system, and reduce the potential for further resistan ce development to glyphosate and dicamba as well as other critical soybean herbicides. Current rese arch conducted by Monsanto to define the optimum weed management systems indicat e the following: 1) in the absence of glyphosate resistant populations, the recomme ndation will be to ap ply a soil active preemergence residual herbicid e followed by an in-crop po stemergence application of glyphosate plus dicamba to cont rol weed escapes, and 2) in the presence of glyphosate resistant populations, the same system w ill be recommended with a potential second application of glyphosate plus di camba if needed. In this latter case, the preemergence

herbicide to be recommended will be one with activity against the targeted glyphosate resistant species. This will ensure more th an one mode of action against the targeted species. These management systems will reduce the potential for further resistance development to glyphosate, dicamba and ot her critical soybean herbicides. In conservation tillage systems, a preplant appl ication of glyphosate plus dicamba may be

recommended in some situations in addition to the in-crop applicatio ns described above. This is not expected to increase selection pressure on either product since the preplant weed spectrum is generally differe nt from the in-crop spectrum.

Stewardship of dicamba to preserve its useful ness for growers is an important aspect of Monsanto’s stewardship commitment, as is discussed in Appendix K. Specifically, Monsanto will develop weed resistance ma nagement practices, and utilize multiple

methods to distribute technical and stewards hip information to growers, academics and grower advisors through a variety of comm unication tools. Monsanto’s Technology Use Guide (TUG) will set forth the requirements and best practices for the cultivation of MON 87708 including recommendations on weed resistance management practices. Growers purchasing products containing dicamb a tolerance will be obligated to follow all practices outlined in the TUG as required in the Monsanto Technology Stewardship Agreement (MTSA). Furthermore, Monsanto is committed to actively evaluate herbicide performance and weed efficacy on a continui ng basis, and develop additional mitigation plans as necessary to manage resistan ce development for glyphosate and dicamba. Dicamba tolerance integrated into the Roundup Ready soybean system will enable expanded use of dicamba herbicide in s oybean production when and in the manner appropriate. Monsanto is seeking regulat ory approvals with the U.S. EPA for the expanded application of dicamba herbicide, as a weed control tool, as well as establishing appropriate dicamba Maximum Residue Le vels (MRLs) for key soybean import countries, including the EU, Canada, a nd Japan, and CODEX, to support importing

countries that do not have an established re gulatory system to set MRLs including China

.

As with all U.S. EPA registered herbicides fo r agricultural use, it is possible that offsite movement during and/or following application can occur such that non-target plants mayMonsanto Company 10-SY-210U 243 of 721 

### be exposed to direct spray or to spray drift. Research has demonstrated that herbicide formulation, application equipment and appl ication procedures can be optimized to significantly reduce spray drif t potential in most circumst ances (Jordan et al, 2009; STDF, 1997).

Monsanto is addressing such issues in its a pplication to U.S. EPA to amend Registration Number 524-582. Specifically, Monsanto seek s approval of a low volatility dicamba (DGA salt) formulation that will only be applied to MON 87708 using ground application equipment. In addition, Monsanto will consul t with U.S. EPA to identify what additional measures, if any, are necessary to address any potential impact of dicamba offsite movement. To reinforce EPA’s label requir ements, Monsanto will implement a robust stewardship program that will include a strong emphasis on grower and applicator training. Such training will, for example, teach growers and applicators that dicamba herbicide spray drift can be further reduced during ground applicati on by using industry standard procedures for minimizing spray dr ift, e.g. making applications with coarse droplet size through appropriate nozzle selection, using lowe r spray pressure, applying at the lowest nozzle height that provides unifo rm coverage, and making applications when wind speeds are low and consistent in direction (SDTF, 1997).

VIII.M. Impact of the Introduction of MON 87708 on Agricultural Practices

Introduction of MON 87708 is expected to ha ve no impact on current cultivation and management practices for soybean, with the exception of expanded dicamba application timings. Monsanto recommends th e use of preemergent soil resi dual herbicides as part of

our current weed contro l programs for soybean. Dicamba has been used in corn, soybean and small grain cropping systems since 1967 . MON 87708 with it s excellent crop tolerance to dicamba allows preemergen ce applications thr ough crop emergence (cracking) and in-crop postemergence applica tions through the R1/R2 growth stage.

MON 87708 will be combined with M ON 89788 (Roundup Ready 2 Yield) utilizing traditional breeding techniques. Soybean containing both M ON 87708 and MON 89788 will allow the use of glyphosate and dicamb a herbicides in an integrated weed management program to control a broad spectru m of grasses and broadleaf weed species, and sustain and compliment the benefits an d value of the glyphosat e use in the Roundup Ready soybean system. The addition of a nother herbicid e with a different mode-ofaction for preemergence and in-crop posteme rgence control of broadleaf weeds in soybean that is compatible with glyphosate will help to preserve the growth in conservation tillage acres associated with Roundup Ready cropping systems. For an overview of the cumulative impacts on agricult ural practices (weed control, tillage and crop rotation) from deregulati on of MON 87708, see Appendix J.

MON 87708 has been shown to be no differe nt from conventional soybean in its agronomic, phenotypic, and compositional characte ristics (refer to Se ctions VI, VII, and VIII), and has the same levels of susceptibil ity to insects and diseases as commercial soybean. Like other herbicide-tolerant soybean, such as Roundup Ready and Roundup Ready 2 Yield that have been cultivated and consumed in the U.S. since 1996, dicambatolerant soybean MON 87708 will improve the cu rrent agricultural practices for U.S. soybean growers by providing another pr eemergence and in-crop postemergenceMonsanto Company 10-SY-210U 244 of 721 

### herbicide mode-of-action for the control of hard-to-cont rol and herbicide resistant broadleaf weeds, thereby improving the effici ency in the U.S. soybean production system to maximize or maintain soybean yield potential.Monsanto Company 10-SY-210U 245 of 721 

### IX. ENVIRONMENTAL CONSEQUENCES

IX.A. Introduction

This section provides a brief review and assessment of the plant pest potential of MON 87708 and its impact on current ag ronomic practices. USDA-APHIS has responsibility, under th e Plant Protection Act (7 U.S. C. § 7701-7772), to prevent the introduction and dissemination of plant pe sts into the U.S. APHIS regulation 7 CFR § 340.6 provides that an applicant may petition APHIS to evaluate submitted data to determine that a particular regulated arti cle does not present a pl ant pest risk and no longer should be regulated. If APHIS dete rmines that the regulated article does not present a plant pest risk, the petition must be granted, thereby allowing unrestricted

introduction of the article.

The definition of “plant pest” in the Plant Protection Act (PPA) in cludes living organisms that could directly or indirectly injure, da mage, or cause disease in any plant or plant product (7 U.S.C. § 7702[14]).

The regulatory endpoint under the PPA for biot echnology-derived crop products is that deregulation of the regulated article is not like ly to pose a plant pest risk. The approach used to assess the plant pest potential of MON 87708 is a weight of the evidence approach based primarily on eigh t lines of evidence: 1) in sertion of a single functional copy of the dmo expression cassette, 2) characte rization of MON 87708 DMO expressed in MON 87708, 3) safety of MON 87708 DMO, 4) compositional equivalence of harvested MON 87708 seed and forage to conventional soybean, 5) phenotypic, agronomic, and environmental interaction characteristics demonstrating no increased plant pest potential compared to conventiona l soybean, 6) negligible risk to NTOs and threatened or endangered species, 7) familiarity with soybean as a cultivated crop and the inherently low plant pest potential of s oybean, and (8) no greater likelihood to impact agronomic practices, including land use, cu ltivation practices, or the management of weeds (other than the intended benefit of di camba weed control), diseases, and insects than conventional soybean.

Using the assessment above, the data and analys is presented in this petition lead to a conclusion that MON 87708 is unlikely to be a plant pest and, therefore, should no longer be subject to regulation under 7 CFR § 340.

Under current regulations, APHIS’s noxious w eed authorities are not at issue unless a petition is filed under7 C.F.R. Part 360. Although no such i ssues are posed by a Part 340 petition for non-regulated status, the data presented in this petition nevertheless

demonstrate that MON 87708 is neither noxious nor a weed and could not be designated as such even in the appropriate procedural context.Monsanto Company 10-SY-210U 246 of 721 

### IX.B. Plant Pest Assessment of th e MON 87708 Insert and Expressed Protein

IX.B.1. Characteristics of the Geneti c Insert and the Expressed Protein

This section summarizes the details of the genetic insert, characteristics of the genetic modification, and safety and expression of the MON 87708 DMO used to evaluate the food, feed, and environmen tal safety of MON 87708.

IX.B.1.1. Genetic Insert

As described in more detail in Section III, MON 87708 was produced by Agrobacterium tumefaciens- mediated transformation of soybean with PV-GMHT4355, which is a binary vector containing two T-DNAs (Figur e III-1). T-DNA I contains the dmo expression

cassette and T-DNA II contains the cp4 epsps expression cassette. During plant transformation, both T-DNAs were inserted into the soybean genome, with the cp4 epsps expression cassette functioning as a selectable marker. Subsequently, conventional selfpollinated breeding methods and segregation we re used to isolate those plants that

contain the dmo expression cassette (T-DNA I) and do not contain the cp4 epsps expression cassette (T-DNA II), resulting in the production of marker-free MON 87708.

T-DNA I contains the dmo coding sequence under the regulation of the PC1SV promoter,

TEV leader, the RbcS targeting sequence, and the E9 3 ′ non-translated region. In addition, T-DNA I contains Left and Right Border seque nces. The promoter, leader, targeting, and border sequences of T-DNA I are not known to cau se plant disease. Furthermore, these sequences are well characterized, are noncodi ng regions, and will not cause MON 87708 to promote plant disease.

Molecular analyses demonstrat ed that MON 87708 contains one copy of the inserted T-DNA I at a single integration locus. No T-DNA II or backbone sequences from PV-GMHT4355 were detected in the genome of MON 87708. Additionally, the data confirmed the organization and sequence of the insert and demonstrated the stability of the insert over several genera tions. These data demonstrated that there are no unintended changes in the MON 87708 genome as a result of the insertion of the dmo expression cassette, and support the overall conclusion that MON 87708 is unlikely to be a plant pest.

IX.B.1.2. Protein Safety

MON 87708 exhibits tolerance to the herbicide dicamba through the insertion of a demethylase gene from Stenotrophomonas maltophilia that encodes for DMO. S. maltophilia is an environmentally ubiquitous bacterium. Infections caused by S. maltophilia are extremely uncommon and it can be found in healthy individuals without any harmful effects. DMO is a Rieske t ype non-heme iron oxygenase that catalyzes the demethylation of dicamba to the non-herb icidal compound DCSA (Section V.A). DMO is specific for dicamba (Dumitru et al., 2009).

DMO is a trimer comprised of three DMO monomers (Chakraborty et al., 2005). MON 87708 DMO is comprised of two forms of the DMO protein; denoted as DMO and DMO+27. Since DMO can be formed by DMO, DMO+27, or a combination of both, all

forms of the trimer are referred to as MON 87708 DMO (Section V). MON 87708 DMOMonsanto Company 10-SY-210U 247 of 721 

### was fully characterized confirming the N-term inal and internal sequence of both protein forms, neither of which were glycosyl ated. MON 87708 DMO is structurally and functionally similar to oxygenase homologs present in bacteria and plants, where a history of safe use is es tablished (Section V.E.2).

The MON 87708 DMO levels in MON 87708 tissu e samples ranged from 6.1 to 69 μg/g dwt in root, forage, harvested seed, and overs eason leaf, and represent a low percentage of the total protein in soybean (Section V. C). MON 87708 DMO was ra pidly digested in simulated gastric fluids, lacked homology with known toxins and allergens, and lacked acute toxicity in a mouse oral gavage study, which taken together, support the conclusion that there is no meaningful ri sk to human or animal health from dietary exposure to

MON 87708 DMO.

The low level of MON 87708 DMO expressed in MON 87708 tissues ta ken together with the safety of MON 87708 DMO support the conclusion that food and feed products containing or derived from MO N 87708 are as safe for human and animal consumption as soybean currently on the market. Theref ore, unintended environm ental effects are not anticipated from dietary exposure to MON 87708 DMO, and support the overall

conclusion that MON 87708 is unlik ely to be a plant pest.

IX.B.2. Compositional Characteristics

Detailed compositional analys es in accordance with OECD guidelines were conducted to assess whether levels of key nutrient s and anti-nutrients in MON 87708, both dicamba-treated and untreated, were comparable to levels present in the near isogenic conventional soybean control A3525 and severa l commercial reference varieties. Seed and forage were harvested from five individual sites in which MON 87708, the conventional control, and a range of commercial refere nce varieties were grown concurrently in the same field trial. The commercial reference varieties were used to establish a range of natural variability fo r the key nutrients an d anti-nutrients in commercial soybean varieties that have a history of safe consumption.

The combined-site analysis was conducted to determine statis tically significant differences (5% level of significance) between MON 87708, both dicamba-treated and untreated, and the conventional control A3525. The results from the combined-site data were reviewed using considerations releva nt to food and feed safety and nutritional quality. These considerations included the relative magnit udes of the difference in the

mean values of nutrient and anti- nutrient components of MON 87708 and the conventional control, whether the MON 87708 component mean value was within the range of natural variability of that component as represented by the 99% tolerance

interval of the commercial refe rence varieties grown concurren tly in the same field trial, and analyses of the reproduc ibility of the statistically significant combined-site component differences at individual sites.

Assessment of the analytical results confir med that the differences observed in the combined-site analysis were not meaningful to food and f eed safety or the nutritional quality of MON 87708 soybeans. In additi on, the levels of assessed components inMonsanto Company 10-SY-210U 248 of 721 

### MON 87708 were compositionally equivalent to the conventional control and within the range of variability of commercial soybeans that were grown concurrently in the same field trial. These results support the overall conclusion that MON 87708 is unlikely to be a plant pest.

IX.B 3. Phenotypic and Agronomic and En vironmental Interaction Characteristics

An extensive set of comparative plant charac terization data were used to assess whether

the introduction of the dicamba tolerance trai t and the associated treatment of dicamba herbicide altered the plant pest potential of MON 87708 compared to the conventional control (Section VII). Phenotypic, agro nomic, and environmental interaction

characteristics of MON 87708 were evaluated an d compared to those of the conventional control (Section VII.B). As described below, these assessments included: seed dormancy and germination characteristics; plant gr owth and development characteristics; observations for abiotic stress response, di sease damage, arthr opod-related damage; pollen characteristics; and arthropod abunda nce; and plant-symbiont interaction characteristics. Results from the phenotypi c, agronomic, and envi ronmental interaction

assessments demonstrated that MON 87708 doe s not possess weedy characteristics, or increased susceptibility or tolerance to specifi c diseases, insects, or abiotic stressors, or altered symbiont interacti ons compared to conventiona l soybean, and the dicamba

treatment does not alter the plant pest potential of M ON 87708. Taken together, the results of the analysis suppor t a determination that MON 87708 is no more likely to pose a plant pest risk or have a biologically mean ingful change in environmental impact than conventional soybean.

IX.B.3.1. Seed Dormancy and Germination

Seed dormancy and germination characteri zation demonstrated that MON 87708 seed had germination characteristics similar to those of the conventi onal control (Section VII.C.1). In particular, the lack of ha rd seed, a well-accepte d characteristic often associated with plants that are weeds, suppor ts a conclusion of no increased weediness or plant pest potential of MON 87708 co mpared to conventional soybean.

IX.B.3.2. Plant Growth and Development

Evaluations of plant growth and development characteristics in the field are useful for assessing potential weediness characteristics such as lodgi ng and pod shattering (Section VII.C.2.3). Of the growth and develo pment characteristics assessed between MON 87708 and the conventional control, no st atistically significant differences were detected (5% level of signi ficance) with the exception of plant height and 100 seed weight in a combined-site analysis of the data . The differences in these parameters were relatively small in magnitude, and the mean values of MON 87708 were within the range

of the commercial reference varieties for thes e characteristics. Thus, the differences in these parameters are not considered to be bi ologically meaningful in terms of increased weediness or plant pest pot ential of MON 87708 compared to conventional soybean.Monsanto Company 10-SY-210U 249 of 721 

### IX.B.3.3. Response to Abiotic Stressors

No biologically meaningful differences were observed during comparative field

observations between MON 87708 and the conven tional control and th eir response to abiotic stressors, such as drought, heat st ress, high winds, nutrient deficiency, etc. (Section VII.C.2.4). The lack of significant biologically meaningful differences in the MON 87708 response to abiotic stress support th e conclusion that the introduction of the dicamba tolerance trait is unlikely to result in increased weediness or plant pest potential compared to conventional soybean.

IX.B.3.4. Pollen Morphology and Viability

Evaluations of pollen morphology and viabili ty from field-grown plants provide information useful in a plant pest assessment as it relates to the potential for gene flow to, and introgression of, the biotec hnology-derived trait into othe r soybean varieties and wild relatives (Section VII.C.3). Pollen morphol ogy and viability evaluations demonstrated no statistically signifi cant differences between MON 87708 a nd the conventional control. Taken together, these comparative assessments indicate that MON 87708 is not likely to

have increased weediness or plant pest potential compar ed to conventional soybean.

IX.B.3.5. Interactions with Non-Target Organisms Includi ng Threatened and Endangered Species

Evaluation of MON 87708 for potential adverse impacts on NTOs is a component of the plant pest risk assessment. Since MON 87708 is a product with no pesticidal activity, all organisms that interact with MON 87708 are considered to be NTOs. In a 2008 U.S. phenotypic and agronomic assessment, observati onal data on environmental interactions

were collected for MON 87708 and the conven tional control. In addition, multiple

commercial reference varieties were included in the analysis to establish a range of natural variability for each characteristic. The environmental interactions assessment (Section VII.C.2.4) included data collect ed on plant-arthropod and plant-disease

interactions. The results of this assessment indicated that the presence of the dicamba tolerance trait did not alter plant-arthropod in teractions, including beneficial arthropods and arthropod pests, nor did it alter disease susceptibility of MON 87708 compared to conventional soybean. The lack of differen ces in disease damage , arthropod-related damage, and pest and beneficial arthropod ab undance demonstrate th at the introduction of the dicamba tolerance trait is unlikely to be biologically meaningful in terms of increased plant pest potential.

In the field, soybean forms a complex sy mbiotic relationship with members of the bacterial family Rhizobiaceae and Bradyrhizobiaceae . This symbiosis results in the

formation of root nodules in which the b acteria reduce or fix atmospheric nitrogenproducing ammonia that can be used by the plant. MON 87708 was assessed for changes in the symbiotic relationship with B. japonicum relative to the conve ntional control by evaluating shoot total nitrogen, nodule number, and nodule, root, and shoot dry weights (Section VII.C.4). No statis tically significant differenc es were detected between

MON 87708 and the conventional control for the parameters measured, indicating noMonsanto Company 10-SY-210U 250 of 721 

### impact on either the symbiotic relationship or the symbiotic nitrogen-fixing bacteria. These data support a conclusion of no change in plant pest potential and no expected impact to cultivation practices relative to nitrogen inputs for MON 87708 compared to conventional soybean.

The potential for MON 87708 to harm NTOs was evaluated using a combination of biochemical information and experimental data. The biochemical information and experimental data included molecular characterization, the MON 87708 DMO safety assessments, the history of environmental exposure to mono-oxygenases (the class of enzymes to which DMO belongs), results from the environmental assessment described above, and the demonstration of compositiona l, agronomic and phenotypic equivalence to conventional soybean. Taken together, th ese data support the conclusion that MON 87708 is unlikely to adversely affect NTOs , or pose an additional risk to threatened and endangered species above those posed by th e cultivation of conventional soybean.

Furthermore, according to APHIS, the only list ed threatened or endangered animal that occupies a habitat where it is likely to incl ude soybean fields, and that might feed on soybean, is the federally endangered Delmarva Peninsula Fox Squirrel, ( Sciurus niger

cinereus ), found in areas of the mid-Atlantic Ea stern seaboard (USDA-APHIS, 2007). It is known to utilize certain agricultural lands readily, but its diet includes acorns; nuts/seeds of hickory, beech, walnut, and loblol ly pine; buds and flowers of trees; fungi; insects; fruit; and an occasional bird e gg (NatureServe, 2007). The safety of the MON 87708 DMO, the compositional, agro nomic and phenotypic equivalence of MON 87708 to conventional soybean, and the dive rsity of the Fox Squirrel diet, support a conclusion that no biologically significant chan ges to the habitat or diet of the Delmarva Peninsula Fox Squirrel are e xpected. Consequently, the planting of MON 87708 is not expected to affect the Delmar va Peninsula Fox Squirrel.

IX.B.3.6. Effects of dicamba reaction products

MON 87708 DMO rapidly demethylates di camba rendering it inactive, thereby conferring tolerance to dicamba in MON 87708. In dicamba-treated MON 87708 the demethylation of dicamba produces 3,6-dichlo rosalicylic acid (DCS A), a known soybean, soil, and livestock metabolite whose safety has been evaluated by the Environmental Protection Agency (U.S. EPA), and formal dehyde. In the ab sence of a dicamba treatment on MON 87708, DCSA and formaldehyde would not be produced. DCSA is structurally similar to salicylic acid (SA). Numerous studies have reported on the stress defense activities of SA, although most studies have looked at the protective effects of exogenously applied SA (Janda et al, 2007). Formaldehyde has a potential linkage to apoptosis in plants (Szende and Tyihak, 2010), and formaldehyde concentrations in plants have been found to in crease under certain stress cond itions (Szabo et al, 2003). The National Toxicology Program (NTP) 12 th 

### Report on Carcinogens has reclassified formaldehyde as a known human carcinogen (US HHS-NTP, 2011). The relevant route of exposure for this health risk is from repeated inhalation at levels a ssociated with indoor or occupational environments, which are ge nerally higher than outdoor environments (USHHS-NTP, 2011). Formaldehyde is presen t in food and in the human body naturally,Monsanto Company 10-SY-210U 251 of 721 

### and there is no evidence to suggest that di etary intake of formaldehyde is important (USHHS-NTP, 2011).

A full discussion on DCSA, and the safety of th is metabolite, is provided in Appendix M. DCSA as well as other dicamba metabolite pr oducts were measured in the residue study provided to the EPA to demonstrate that dicamba and dicamba metabolite residues are well below the current MRL set for dicamba in soybeans in the U.S.

Formaldehyde is ubiquitous in the environm ent and present in pl ants and animals. Formaldehyde was not considered a relevant metabolite in the demethylation of dicamba by the EPA. According to the guidelines published by Office of Prevention, Pesticides and Toxic Substances, United States Envir onmental Protection Agency (US EPA OPPTS 860.1300), the methoxy sidechain that is cleav ed from dicamba to form formaldehyde would specifically not be chosen to be labe led in a metabolism study (U.S. EPA, 1996). This is because it is not metabolically stab le and would not be c onsidered a significant moiety as it would be readily metabolized a nd incorporated into the 1-carbon pool of the plant through known pathways. Therefore, formaldehyde was not measured in the residue study when dicamb a was applied to MON 87708.

Data from both dicamba-treated and non-tr eated MON 87708 compared to a conventional control are available from multiple sites acr oss the U.S. where agronomic, phenotypic

and observational environmental interaction data were collected. The results of this assessment demonstrate no biologically m eaningful difference between MON 87708 treated with dicamba, or MON 87708 not treated with dicamba compared to the conventional control, and support a conclu sion that the formation of DCSA and formaldehyde does not alter the weedy characte ristics, or increased susceptibility and tolerance to diseases, insects or abiotic stresses. Therefor e, MON 87708, as cultivated, is no more likely to be a plant pest risk or have a biologically meaningful change in environmental impact than conventional soybean.

Further, the metabolism of formaldehyde in plants is well underst ood, and there are a number of natural occurring sources of form aldehyde in plants. For example, it is produced during the process of photorespi ration (Oliver, 1994) and during oxidative demethylation of DNA (Zhu, 2009), which supports why it is not c onsidered a byproduct of interest as dicamba is demethylate d. It is well known and understood that formaldehyde is rapidly metabolized in plan ts through two basic routes: 1) it can be incorporated into the one-carbon folate pool via spontaneous or enzyme-mediated formation of methylene tetrahydrofol ate (Hanson and Roje, 2001); or 2) it can be oxidized to formate by a detoxification pathway that be gins with spontaneous formation of the glutathione adduct S -hydroxymethylglutathione (Hanson and Roje, 2001). In each case, formaldehyde is further metabolized to carbon di oxide or entered into the 1C folate pool (Hanson and Roje, 2001; Giese et al., 1994). The maximum theoretical production of formaldehyde produced from dicamba-treated MON 87708 is estimated to be 16.7 and 37.5 mg/kg 11 

### . This is well within the range of formaldehyde concentrations measured for

11 Calculation based on an assumption that the entire 0.56 kg/ha (0.5 lb/acre a.e.) application of dicamba to MON 87708 soybean at the V3 growth stage is interc epted by the soybean plants, is instantaneously andMonsanto Company 10-SY-210U 252 of 721 

### a variety of agricultural commodities – up to 60 mg/kg in fruits and vegetables (WHOIPCS, 1989). Plants have a large capaci ty to metabolize formaldehyde naturally produced from internal processes (A. Hans on (2011), C.V. Griffin, Sr. Eminent Scholar, Horticulture Department, University of Florida, Personal Communication), and any additional amount of formaldehyde that could be theoretically produ ced in the plant by

dicamba treatment in MON 87708 would be meta bolized very quickly. Additionally, as dicamba would not be instantaneously ab sorbed and metabolized, the incremental increase in formaldehyde over and above the levels already presumed to be present in the soybean plant would be small and transient. Fu rther, since current li terature supports that formaldehyde is only emitted from foliage under certain conditions (Nemecek-Marshall et al., 1995; Cojocariu et al., 2004; Cojocariu et al., 2005) and that emission rates are low (Nemecek-Marshall et al., 1995), little opportunity exists for formaldehyde to be released

from MON 87708 after dicamba treatment.

In addition to formaldehyde production in pl ants, plants and animals are constantly exposed to low levels of formaldehyde. Formaldehyde is already present in the environment and the atmosphere from a variety of biogenic (e.g. plant and animal) and anthropogenic (e.g. automotive or industria l emissions) sources. Additionally, formaldehyde degrades rapidly in environmenta l compartments (air, soil, and water). In water, formaldehyde dissipates through biodegr adation to low levels in a few days (USHHS-ATSDR, 1999). Aerobic biodegradation half-lives are estimated to be 1-7 days for surface water and 2-14 days for ground water (US EPA, 2008). The half-life of formaldehyde in air is dependent on a number of factors (light inte nsity, temperature and location). Through reaction with hydroxyl radi cal, the half-life of formaldehyde in air varies from 7 to 70 hours (US EPA, 2008). The photolytic half-lif e of formaldehyde in air (e.g., in the presence of sunlight) is estimated to be 1.6-6 hours (US EPA, 2008; USHHS-ATSDR, 1999). The rapid degradati on of formaldehyde in the environment

combined with the understandi ng that formaldehyde is wide ly used by living organisms as a 1C source, support a conclusion that any environmental e ffects of formaldehyde, including effects on other plants and NTO’ s, resulting from dicamba-treated MON 87708 would be negligible.

Humans are also constantly exposed to low levels of fo rmaldehyde. Human exposure to formaldehyde is primarily due to indoor air exposures (USHHS-ATSDR, 1999). Formaldehyde is also found in a variety of consumer products such as cosmetics and paints, often as an antimicrobial agent, a nd is used extensively in urea-formaldehyde

“slow-release” fertilizer fo rmulations and adhesives (USHHS-ATSDR, 1999). Indoor formaldehyde air concentrations are gene rally significantly hi gher than outdoor air concentrations (USHHS-ATSDR, 1999) as a result of combustion (cooking, heating, tobacco use) and the emission of formaldehyde from a variety of construction materials (e.g., particle board, plywood or foam insula tion) as well as permanent press fabrics (clothing or draperies) (US CPSC, 1997). Formaldehyde pres ent in outdoor air results

completely absorbed, and then instantaneously metabolized by the DMO enzyme (Complete demethylation of 560 g (2.5 mol)/ ha dicamba would yield 2.5 mol/ha formaldehyde). Above ground biomass of V3 plants is estimated at 2 metric tons/ha, and results in 37.5 mg/kg formaldehyde in planta. For dicamba applications at R1 growth stage, the crop biomass is estimated to be 4.5 metric tons/ha, and level of formaldehyde produced in planta is 16.7 mg/kg.Monsanto Company 10-SY-210U 253 of 721 

### from a number of sources, and levels of fo rmaldehyde are generally higher in urban areas than in rural areas (WHO-IPCS, 1989). Dire ct contributions of formaldehyde to the atmosphere (i.e., those in the form of form aldehyde itself) from man-made sources are present, but are generally considered to be small relative to natural sources or indirect production of formaldehyde in the atmosphe re (WHO, 2002). Formaldehyde is rapidly consumed in the atmosphere through direct photolysis or by oxida tion with hydroxyl or nitrate radicals (USHHS-ATSDR, 1999).

The National Toxicology Program (NTP) 12 th 

### Report on Carcinogens has reclassified formaldehyde as a known human carcinogen (USHHS-NTP, 2011). However, the relevant route of exposure for this health risk is from repeated inha lation of concentrated levels associated with indoor or occupationa l environments. As previously discussed, formaldehyde may only be released by plants in very small quanti ties and under certain conditions. Any incremental exposure to form aldehyde associated with the application of dicamba to MON 87708 would occur outdoors, w ould be minimal, and also transient in nature. Therefore human safety concerns of formaldehyde released from dicamba treated MON 87708 are considered to be negligible. USHHS-NTP (2011) has already stated that there is no evidence to suggest that dietary intake of formaldehyde is important. In addition, commodity soybean seed is not di rectly consumed by humans, and would be processed into food products, limiting potential exposure to humans to any formaldehyde

in dicamba-treated MON 87708 seed.

IX.C. Weediness Potential of MON 87708

The commercial Glycine species in the U.S. ( Glycine max L .) does not exhibit weedy

characteristics and is not effective in invadi ng established ecosystems. Soybean is not listed as a weed in the major weed references (Crockett, 1977; Holm et al., 1979; Holm et al., 1997), nor is it present on the lists of noxi ous weed species distributed by the federal government (7 CFR Part 360). Soybean does not possess any of the attributes commonly associated with weeds (Baker, 1965), such as long persisten ce of seed in the soil, the ability to disperse, invade, and become a dom inant species in new or diverse landscapes or the ability to compete well with native ve getation. Due to the lack of dormancy, which

is a trait that has been removed from soybean through commercial breeding, soybean seed can germinate quickly under adequate temperature a nd moisture conditions, and potentially grow as volunteer plants. Howeve r, volunteer plants likely would be killed by frost during autumn or winter of the year they were produced. If they did become established, volunteer plants would not comp ete well with the succeeding crop, and could be controlled readily by eith er mechanical or chemical me ans (Carpenter et al., 2002; Dillon et al., 2006; OECD, 2000; York et al ., 2005; Zollinger, 2009). In addition, since wild populations of Glycine species are not known to exist in the U.S., the potential does

not exist for MON 87708 to outcross to wild or weedy relatives and to alter their weediness potential.

In comparative studies between MON 87708 a nd the conventional control, phenotypic, agronomic, and environmental interaction data were evaluated (Section VII) for changes that would impact the plant pest potential and, in particular, plant weediness potential.Monsanto Company 10-SY-210U 254 of 721 

### Results of these evaluations show that th ere is no biologically meaningful difference

between MON 87708 and the conve ntional control for char acteristics potentially associated with weediness. Furthermore, comparative field observations between

MON 87708 and its conventional control in thei r response to abiotic stressors, such as drought, heat stress, and high wi nds, indicated no biologically meaningful differences and, therefore, no increased weediness potential. Data on environmental interactions also

indicate that MON 87708 does not confer any biologically meaningful increased susceptibility or tolerance to specific diseases or insect pests. Collectively, these findings support the conclusion that MON 87708 has no in creased weediness potential compared to conventional soybean.

Because MON 87708 has the same limited weedin ess potential as conventional soybean, it is similarly unlikely to survive as a volunt eer plant. Under 7 C.F. R. Part 340, no “noxious weed” issues are reviewed by APHIS. Nevertheless, even if APHIS’s Part 360

noxious weed authorities were at issue here, MON 87708 could pose no “noxious weed”

risks under the Plant Protection Act becau se it would not be “noxious” as APHIS ordinarily interprets that term. APHIS ha s made clear that “noxious” weeds refer to weeds that are “likely to be aggressively i nvasive, have significant negative impacts,” and are “extremely difficult to manage or control once established.” 73 Fed. Reg. 60,008, 60,013 (Oct. 9, 2008). Volunteer MON 87708— like volunteer conve ntional soybean— would compete poorly with any succeeding cr ops and soon be killed, making it extremely unlikely to have any prolonged negative eff ects. Volunteer MON 87708 would also not be “extremely difficult to manage” because it can be controlled easily with numerous

alternative herbicides and other mechanical mean s. (Carpenter et al ., 2002; Dillon et al., 2006; OECD, 2000; York et al., 2005; Zollin ger, 2009). The conclusion that MON 87708 does not pose noxious weed risks is consistent with APHIS’s historical interpretations of the Plant Protection Act, as APHIS has never before considered a genetically engineered crop to be a noxious weed.

IX.D. Potential for Pollen Mediated Gene Flow

Gene introgression is a process whereby one or more genes successfully integrate into the genome of a recipient plant population. Introgression is affected by many factors, including the frequency of the initial pollina tion event, environmental factors, sexual compatibility of pollen donor and recipient plants, pollination biology, flowering phenology, hybrid stability and fert ility, selection, and the ability to backcross repeatedly. Because gene introgression is a natural biological process, it does not constitute an environmental risk in and of itself (Sut herland and Poppy, 2005). Gene introgression

must be considered in the context of the transgene(s) inserted into the biotechnologyderived plant, and the likelihood that the presence of the transgene(s) and their subsequent transfer to recipien t plants will result in increased plant pest potential. The potential for gene introgression fr om MON 87708 is discussed below.

The assessment for gene introgression from MON 87708 with other cultivated or wild relatives of soybean, discussed in detail below, indicates that MON 87708 is no more likely to become a weed than conventional soybean, and M ON 87708 is expected to be similar to conventional soybean regarding its potential for and impacts from gene flow.Monsanto Company 10-SY-210U 255 of 721

### Soybean lacks sexually-compatible relatives in the U.S.; therefore, the only pollenmediated gene flow would be within cultivated soybean.

IX.D.1. Hybridization with Cultivated Soybean

Although soybean is largely a self-pollinate d species, low levels of natural crosspollination can occur (Caviness, 1966; OECD, 2000; Ray et al., 2003; Yoshimura et al., 2006). In studies with cultiv ated soybean, where conditions have been optimized to ensure close proximity and flowering synchr ony, natural cross-pollin ation generally has been found to be very low. Most outcros sing occurred with su rrounding plants, and cross-pollination frequencie s varied depending on growing season and genotype. Insect activity does increase the outcrossing rate, but soybean generally is not a preferred plant for pollinators (Abrams et al, 1978; Erickson 1975; Jaycox 1970a, b).

Numerous studies on soybean cross-pollina tion have been conducted, and the published results, with and without supplemental pollina tors, are summarized in Table IX-1. Under natural conditions, cross-pollination among adja cent plants in a row or among plants in adjacent rows ranged from 0 to 6.3%. In experiments where supplemental pollinators (usually bees) were added to the experimental area, cross-pollination ranged from 0.5 to 7.74% in adjacent plants or adjacent rows. However, cross-pollination does not occur at these levels over long distances. Cross-pol lination rates decrease to less than 1.5% beyond one meter from the pollen source, and rapidly decrease with greater distances from the source. The following cross-pollinat ion rates at extended distances have been reported: 0.05% at 5.4 meters (Ray et al., 2003), 0% at 6.5 meters (Abud et al., 2003), 0% at 10.5 m (Yoshimura et al., 2006) , and 0.004% at 13.7 meters of separation (Caviness, 1966).

The potential for cross-pollination in soybean is limited. This is recognized in certified seed regulations for foundation seed in th e U.S., which permit any distance between different soybean cultivars in the field as l ong as the distance is adequate to prevent mechanical mixing (USDA-APHIS, 2006).

The consequence of introgression of the di camba tolerance trait from MON 87708 into other soybean is negligible since soybean ge ne flow is naturally low; therefore the dicamba tolerance trait confers no increased pl ant pest potential to cultivated soybean.

IX.D.2. Hybridization with Wild Annual Species within Subgenus Soja

The subgenus Soja includes the cultivated soybean Glycine max and the wild annual species Glycine soja

. Glycine soja is found in China, Taiwa n, Japan, Korea, and Russia and can hybridize naturally with the cultivated soybean, G. max (Hymowitz, 2004; Lu, 2004). Hybridization between female G. soja and male G. max was less successful than hybridization in the opposite direction (D orokhov et al., 2004), wh ere frequency of spontaneous cross pollination in reciprocal combinations of G. max and G. soja varied from 0.73 ( ♀ G. soja × ♂ G. max ) to 12.8% ( ♀ G. max × ♂ G. soja ). Species relationships in the subgenus soja indicated that F 1 

### hybrids of G. max and G. soja carry similar genomes and are fertile (Singh and Hymowitz, 1989). Abe et al. (1999) note thatMonsanto Company 10-SY-210U 256 of 721 

### “natural hybrids between G. max and G. soja are rare and hybrid swarms involving both species have never been reported.” This is also supported by work from Kuroda et al. (2008) in which molecular markers we re used and no gene flow from G. max to G. soja was detected. Many barriers to natural hybridization exis t between soybean and wild relatives, including the highly se lfing nature of both plants, required proximity of wild soybean to cultivated soybean, synchrony of fl owering, and presence of pollinators. As such, it is highly unlikely that naturally occurring, pollen-mediated gene flow and transgene introgression into wild soybean relatives from incidentally released biotechnology-derived soybean will occur at any meaningful frequency.

The subgenus Soja also contains an unofficial species, G. gracilis (Hymowitz, 2004). Glycine gracilis is known only from Northeast China, and is considered to be a weedy or

semi-wild form of G. max , with some phenotypic characteristics intermediate to those of

G. max and G. soja

. Glycine gracilis may be a hybrid between G. soja and G. max (Hymowitz, 1970; Lu, 2004). Interspecific fert ile hybrids formed by intentional crosses

between G. max and G. soja and between G. max and G. gracilis have been easily obtained (Dorokhov et al., 2004; Singh and Hy mowitz, 1989). Although hybridization

between G. max and members of the subgenus Soja can take place, G. soja is not found in North or South America, and it is highly un likely that gene transfer will occur.

IX.D.3. Hybridization with the Wild Perennial Species of Glycine Subgenus

Wild perennial species of the Glycine subgenus occur in Austra lia; West, Central and South Pacific Islands; China; Papua New Guin ea; Philippines; and Taiwan (Hymowitz et al., 1992). Therefore, the onl y opportunities for inter-subg eneric hybridization would occur in areas where those species are en demic. Nonetheless, the likelihood of interspecific hybridization between G. max and the wild perennial Glycine species is extremely low because they are genomically dissimilar (Hymowitz, 2004; Lu, 2004) and pod abortion is common. From time to time, immature seeds of the crosses have been germinated aseptically in vitro , but the resulting F1 hybrids are slow-growing, morphologically weak, and completely sterile. Their sterility is due to poor chromosome pairing. Furthermore, species distantly rela ted usually produce nonvi able F1 seeds that either have premature death of the germinating seedlings or suffer from seedling and vegetative lethality (Kollipar a et al., 1993). In North an d South America, it is not possible for gene transfer to occur between cu ltivated soybean and wild perennial species

of Glycine subgenera because thes e wild species do not exist in these regions.

IX.D.4. Transfer of Genetic Information to Species with Which Soybean Cannot Interbreed (Horizontal Gene Flow)

Monsanto is unaware of any reports regardi ng the unaided transfer of genetic material from soybean species to other sexually-inc ompatible plant species. The likelihood for horizontal gene flow to occur is exceedingly small. Therefore, potential ecological risk associated with horizontal gene flow fr om MON 87708 due to the presence of the dicamba tolerance trait is not expected. The consequence of horizontal gene flow of the dicamba tolerance trait into other plants that are sexually-incompatible is negligible since, as data presented in this petition confirm, th e gene and trait confer no increased plant pestMonsanto Company 10-SY-210U 257 of 721 

### potential to soybean. Thus in the highly unlik ely event that horizontal gene transfer were to occur, the presence of the dicamba toleran ce trait would not be ex pected to increase pest potential in the recipient species.

IX.E. Potential Impact on Soybean Agronomic Practices

An assessment of current soybean agronom ic practices was conducted to determine

whether the cultivation of MON 87708 has the potential to impact current soybean and weed management practices (Section VIII). Soybean fields are typically highly managed agricultural areas that are dedicated to crop production. MON 87708 is likely to be used in common rotations on land pr eviously used for agricultura l purposes. Certified seed production will continue to use well-established industry practices to deliver high quality seed containing MON 87708 to growers. Cu ltivation of MON 87708 is not expected to differ from typical soybean cu ltivation, with the exception of an expanded window of dicamba applications. Due to the excelle nt crop safety of MON 87708 to dicamba, growers will have a new herbicide mode-o f-action for in-crop control of glyphosate’s

hard-to-control and resistant broadleaf weeds th at are present in U.S. soybean production.

As a result of cultivation of MON 87708 in tegrated into the Roundup Ready soybean system, the number of dicamba-treated soybean acres is expected to double from historic peak levels. Additionally, due to the expande d timing of in-crop applications to soybean, dicamba treatments will be later in the grow ing season than most current labeled dicamba uses. These later applications of dicamba to MON 87708 are not expected to impact dicamba-sensitive crops from drift (spray or vo latility) because, in its application to U.S. EPA, Monsanto is registering the use on MON 87708 on a low volatility dicamba (DGA salt) formulation, not allowing aerial app lication on MON 87708, and will consult with U.S. EPA to develop any necessary additional meas ures to protect against offsite impacts.

MON 87708 is similar to conventional soybean in its agronomic, phenotypic, ecological, and compositional characteristics and has levels of resistance to insects and diseases comparable to conventional soybean. Ther efore, no significant impacts on current cultivation and management practices for soybean are expected following the

introduction of MON 87708. Based on this assessment, the introduction of MON 87708 will not impact current U.S. soybean cultivatio n practices or weed management practices,

other than intended w eed control benefits.

IX.F. Summary of Plant Pest Assessments

Plant pests are defined in th e Plant Protection Act as cert ain living organisms that can directly or indirectly injure , cause damage to, or cause di sease to any plant or plant product (7 U.S.C. § 7702[14]). Characterization data presen ted in Sections III through VII of this petition confirm that although MON 87708 contains th e dicamba-tole rant trait, it is not different from conventional soybean in terms of pest poten tial in its phenotypic, agronomic, and environmental interaction charac teristics. Monsanto is not aware of any study results or observations associated with MON 87708 that would suggest an increased plant pest risk woul d result from its introduction.Monsanto Company 10-SY-210U 258 of 721 

### The plant pest assessment was based on multiple lines of evidence developed from a detailed characterization of MON 87708 comp ared to conventional soybean, followed by

a risk assessment on detected differences. Th e risk assessment considered various factors including: 1) insertion of a single functional copy of the dmo expression cassette, 2) characterization of the MON 87708 DMO, 3) safety of the MON 87708 DMO, 4) compositional equivalence of harvested MON 87708 soybean seed and forage as compared to conventional soybean, 5) phe notypic, agronomic, and environmental

interaction characteristics dem onstrating no increased plant pest potential compared to conventional soybean, 6) negligib le risk to NTOs and threat ened or endangered species, 7) familiarity with soybean as a cultivated crop and the inhere ntly low plant pest potential of soybean, and 8) no greater likelihood to impact agronomic practices, including land use, cultivation practices, or the manageme nt of weeds, diseases, and insects than conventional soybean, with the exception of the expanded window of dicamba application.

Based on the data and information presented in this petition, it is concluded that, like conventional soybean and currently dere gulated biotechnology-derived soybean, MON 87708 is highly unlikely to be a plant pest. Therefore, Monsanto Company requests a determination from APHIS th at MON 87708 and any progeny derived from

crosses between MON 87708 and other commer cial soybean be granted nonregulated

status under 7 CFR Part 340.Monsanto Company 10-SY-210U 259 of 721 

### Table IX-1. Summary of Published Literature on Soybean Cross Pollination

Distance from

Pollen Source (meters) CrossPollination (%) Comments Reference 0.3 0.04 (estimated per pod) Interspaced plants within a row. Experiment conducted in a single year. Single male and female parental varieties. Percent outcrossing calculated per pod rather than per seed. (Woodworth, 1922)

0.8 0.07 to 0.18 Adjacent rows. Experiment conducted over two years. Several male and female parental varieties. (Garber and Odland, 1926) 0.1 0.38 to 2.43 Adjacent plants within a row. Experiment conducted in a single year. Several male and female parental varieties. (Cutler, 1934)

0.1 0.2 to 1.2 Adjacent plants within a row. Experiment conducted in single year at two locations. Several male and female parental varieties. (Weber and Hanson, 1961)

0.9

2.7–4.6

6.4–8.2

10–15.5 0.03 to 0.44

0.007 to 0.06

0 to 0.02

0 to 0.01 Frequency by distance was investigated. Experiment conducted over three years. Single male and female parental varieties. (Caviness, 1966)

0.8 m 0.3 to 3.62 Various arrangements within and among adjacent rows. Experiment conducted over three years. Several male and female parental varieties. (Beard and Knowles, 1971)

One row (undefined) 1.15 to 7.74 Bee pollination of si ngle-row, small-plots of pollen receptor surrounded by large fields (several acres) of pollen donor soybean. Soybean is not a preferred flower for alfalfa leafcutting bees. (Abrams et al., 1978)

0.1–0.6 0.5 to 1.03 (depending on planting design) Bee pollination of soybean grown in various spatial arrangements. Experiment conducted over four years. Several soybean cultivars. (Chiang and Kiang, 1987)

1.0 0.09 to 1.63 Adjacent rows. Experiment conducted over two years. Several male and female parental varieties. (Ahrent and Caviness, 1994)

0.5

1.0

6.5 0.44 to 0.45

0.04 to 0.14

none detected Frequency by distance was investigated. Experiment conducted in a single year. Single male and female parental varieties. (Abud et al., 2003)

0.9

5.4 0.29 to 0.41

0.03 to 0.05 Frequency by distance was investigated. Experiment conducted in a single year. Single male and female parental varieties. (Ray et al., 2003)

0.15 0.65 to 6.32 (avg. 1.8) Interspaced plants within a row. Experiment conducted in a single year. Single male and female parental varieties. (Ray et al., 2003)

0.7

1.4

2.1

2.8

3.5

7.0

10.5 0 to 0.19

0 to 0.04

0 to 0.05

0 to 0.08

0 to 0.04

0 to 0.04

0 Interspaced plants within a row arranged in small plots. Experiment conducted in a four year period. Single male and two female parental varieties. (Yoshimura et al., 2006)Monsanto Company 10-SY-210U 260 of 721 

### X. ADVERSE CONSEQUENCES OF INTRODUCTION

Monsanto knows of no resu lts or observations associ ated with MON 87708 or the MON 87708 DMO indicating that there would be an adverse environmental consequence

from the introduction of MON 87708. M ON 87708 contains DMO that renders the soybean plant tolerant to the herbicide dicam ba. As demonstrated by field results and laboratory tests, the only phenotypic di fference between MON 87708 and conventional soybean is tolerance to dicamba.

The data and information presented in th is petition demonstrate that MON 87708 is unlikely to pose an increased plant pest risk or to have an adverse environmental consequence compared to conventional soybean. This conclusion is based on multiple lines of evidence developed fr om a detailed characterizatio n of the product compared to conventional soybean, followed by risk a ssessment on detected differences: 1)

characterization evaluations included molecula r analyses, which confirmed the insertion of a single functional copy of the dmo expression cassette at a single locus within the soybean genome; 2) measurement of the MON 87708 DMO levels in various soybean tissues; 3) characterization of the MON 87708 DMO confirming it is not novel and is structurally and functionally similar to oxyge nase homologs widely present in bacteria and plants, where a history of sa fe use is established; and 4) extensive characterization of the plant phenotype, including compositional anal ysis of key nutrients and antinutrients, and environmental interactions. Therefore, based on the lack of increased pest potential or adverse environmental consequences comp ared to conventional soybean, the risks for humans, animals, and other NTOs from M ON 87708 are negligible under the conditions of use. Additionally the introducti on of MON 87708 will not adversely impact cultivation practices or the management of weeds, diseases, and insects in soybean production systems, other than the use of dicamba postemergence in soybean.

Successful integration of MON 87708 into the Roundup Ready soybean system will provide growers with an opportunity for an ef ficient, effective weed management system for the management of glyphosate’s hard-to- control and resistant broadleaf weeds; provide an easy system for in clusion of a second herbicid e mode-of-action in soybean production practices as reco mmended by weed science e xperts to manage weed resistance development; and continue to pr ovide soybean growers with effective weed control systems necessary for production yiel ds to meet the growing needs of the food,

feed and industrial markets.Monsanto Company 10-SY-210U 261 of 721 

### REFERENCES

Abe, J., A. Hasegawa, H. Fukushi, T. Mikami, M. Ohara and Y. Shimamoto. 1999. Introgression between w ild and cultivated soybeans of Ja pan revealed by RFLP analysis for chloroplast DNAs. Economic Botany 53:285-291.

Abrams, R.I., C.R. Edwards and T. Harri s. 1978. Yields and cross-pollination of soybeans as affected by honey bees and alfa lfa leafcutting bees. American Bee Journal

118:555-558.

Abud, S., P.I. Mello de Souza, C.T. Moreir a, S.R.M. Andrade, A.V. Ulbrich, G.R. Vianna, E.L. Rech and F.J. Lima Aragão. 2003. Gene flow in transgenic soybean in the Cerrado region, Brazil. Pesquisa Agropecuária Brasileira 38:1229-1235.

Adrian-Romero, M., G. Blunden, B.G. Carpenter and E. Tyihák. 1999. HPLC quantification of formaldehyde, as formaldemethone, in plants and plant-like organisms. Chromatographia 50:160-166.

Agranova. 2010. Herbicide synopsis - A chronolog ical listing of all herbicide, safener and PGR compounds to 1995. Agranova, Kent, United Kingdom. http://www.agranova.co.uk/herbhist.asp Accessed June 17, 2010

.

AgroTrak. 2009. Unpublished 2008 agricultural survey data. Gfk Kynetec marketing Research, Inc., Berkshire, United Kingdom.

Ahrens, W.H. 1994. Dicamba. 3,6-dichloro-2-methoxybenzoic acid. Pages 91-94 in

Herbicide Handbook. Seventh Edition. Weed Sc ience Society of America, Champaign,

Illinois.

Ahrent, D.K. and C.E. Caviness. 1994. Natu ral cross-pollination of twelve soybean cultivars in Arkansas. Crop Science 34:376-378.

Alexander, M.P. 1980. A versatile stain fo r pollen fungi, yeast and bacteria. Stain Technology 55:13-18.

Al-Kaisi, M. 2001. Value of crop rotation in ni trogen management. Iowa State University, Ames, Iowa. http://www.ipm.iastate.edu/ipm/icm/2001/4-23-2001/valuen.html Accessed May 12, 2010

.

Al-Kaisi, M., M. Hanna and M. Tidma n. 2003. Crop rotation considerations for 2004 management season rotation. Iowa State University, Ames, Iowa. http://www.ipm.iastate.edu/ipm/icm/2003/12-15-2003/croprotation.html Accessed June
19, 2006

.

Anderson, W.P. 1996. Weed ecology. Pages 27-38 in Weed Science: Principles and Applications. Third Edition. West Pub lishing Company, St. Paul, Minnesota.Monsanto Company 10-SY-210U 262 of 721 

### AOSA. 2000. Tetrazolium testing handbook. Contribu tion no. 29. Association of Official Seed Analysts, Lincoln, Nebraska.

AOSA. 2007. Rules for testing seeds. Associatio n of Official Seed Analysis, Ithaca, New

York.

AOSCA. 2009. Seed certification handbook: In cluding genetic and crop standards, procedures, and AOSCA service programs. A ssociation of Offici al Seed Certifying Agencies, Moline, Illinois.

Appunu, C. and B. Dhar. 2006. Differential symbio tic response of pha ge-typed strains of Bradyrhizobium japonicum with soybean cultivars. Journal of Microbiology 44:363-368.

Aref, S. and D.R. Pike. 1998. Midwest farmer s’ perceptions of crop pest infestation. Agronomy Journal 90:819-825.

ASA. 2009. Soybean exports. American Soybean Association, St. Louis, Missouri. http://www.soystats.com Accessed March 15, 2010

.

ASA. 2011. Soy stats 2011. American Soybean Association, St. Louis, Missouri. http://www.soystats.com/2011/Default-fr ames.htm [Accessed September 22, 2011].

Baker, H.G. 1965. Characteristics and modes of origin of weeds. Pages 147-172 in The Genetics of Colonizing Species. H.G. Baker a nd G.L. Stebbins (eds.). Academic Press, New York, New York.

Barker, R.F., K.B. Idler, D.V. Thompson and J.D. Kemp. 1983. Nucleotide sequence of the T-DNA region from the Agrobacterium tumefaciens octopine Ti plasmid pTi15955. Plant Molecular Biology 2:335-350.

Barry, G.F., G.M. Kishore, S.R. Padgette and W.C. Stallings. 1997. Glyphosate-tolerant 5-enolpyruvylshikimate-3-phosphate synthase s. Patent 5,633,435, U.S. Patent Office, Washington, D.C.

Beard, B.H. and P.F. Knowles. 1971. Frequenc y of cross-pollination of soybeans after seed irradiation. Crop Science 11:489-492.

Beckie, H.J. 2006. Herbicide-resistant weeds: Management tactics and practices. Weed Technology 20:793-814.

Beckie, H.J. and X. Reboud. 2009. Selecting for weed resistance: Herbicide rotation and mixture. Weed Technology 23:363-370.Monsanto Company 10-SY-210U 263 of 721 

### Behrens, M.R., N. Mutlu, S. Chakraborty, R. Dumitru, W.Z. Jiang, B.J. LaVallee, P.L. Herman, T.E. Clemente and D.P. Weeks. 2007. Dicamba resistance: Enlarging and preserving biotechnology-based weed management strategies. Science 316:1185-1188.

Berg, G., P. Marten and G. Ballin. 1996. Stenotrophomonas maltophilia in the rhizosphere of oilseed rape - Occurrence, characteriza tion and interaction with phytopathogenic fungi. Mi crobiological Research 151:19-27.

Berg, G., N. Roskot and K. Smalla. 1999. Genotypic and phenotypic relationships

between clinical and envi ronmental isolates of Stenotrophomonas maltophilia . Journal of Clinical Microbiology 37:3594-3600.

Berg, G., N. Roskot, A. Steidle, L. Eberl, A. Zock and K. Smalla. 2002. Plant-dependent genotypic and phenotypic diversit y of antagonistic rhizobacteria isolated from different

Verticillium host plants. Applied and Environmental Microbiology 68:3328-3338.

Berg, R.D. 1996. The indigenous gastrointest inal microflora. Trends in Microbiology

4:430-435.

Berman, K.H., G.G. Harrigan, S.G. Riordan, M.A. Nemeth, C. Hanson, M. Smith, R. Sorbet, E. Zhu and W.P. Ri dley. 2009. Compositions of s eed, forage, and processed fractions from insect-protected soybean MON 87701 are equivalent to those of conventional soybean. Journal of Agricultural and Food Chemistry 57:11360-11369.

Boerma, H.R. and J.E. Specht. 2004. Worl d distribution and trade of soybean and managing inputs for peak production; soybean production in the U.S.A. Pages 3, 502, 507 and 522 in Soybeans: Improvement, Production, a nd Uses. Boerma H.R. and J.E. Specht, eds. American Society of Agronomy, Inc., Cr op Science Society of America, Inc., Soil Science Society of America, Inc., Madison, Wisconsin.

Bond, J.A. and T.W. Walker. 2009. Control of volunteer glyphosate-re sistant soybean in rice. Weed Technology 23:225-230.

Bonny, S. 2008. Genetically modified glyphosate- tolerant soybean in the USA: Adoption factors, impacts and prospects. A review . Agronomy for Sustainable Development 28:21-

32.

Bradford, K.J. 2006. Methods to maintain gene tic purity of seed stocks. Publication 8189. University of Californi a, Oakland, California.

Breyton, C. 2000. The cytochrome b 6 

### f

complex: Structural st udies and comparison with

the bc 1 

### complex. Biochimica et Biophysica Acta 1459:467-474.

Britz, S.J., D.F. Kremer and W.J. Kenwor thy. 2008. Tocopherols in soybean seeds:

Genetic variation and envir onmental effects in field-gr own crops. Journal of the American Oil Chemists' Society 85:931-936.Monsanto Company 10-SY-210U 264 of 721 

### Brookes, G. and P. Barfoot. 2009. GM crops: Global socio-economic and environmental impacts 1996-2007. PG Economics, Ltd, Dorchester, United Kingdom. www.pgeconomics.co.uk/pdf /2009globalimpactstudy.pdf Accessed May 12, 2010

.

Carpenter, J.E. and L.P. Gianessi. 2001. Agricultural biotechnology: Updated benefits

estimates. National Center for Food and Agricultural Policy, Washington, D.C.

Carpenter, J., A. Felsot, T. Goode, M. Hammig, D. Onstad, and S. Sankula. 2002. Comparative environmental impacts of biot echnology-derived and traditional soybean, corn, and cotton crops. Council for Agricultur al Science and Technology, Ames, Iowa.

CAST. 2007. Implications of gene flow in the scale-up and commercial use of biotechnology-derived crops: Economic and policy considerations. Issue paper 37. Council for Agricultural Science and Technology, Ames, Iowa.

Caviness, C.E. 1966. Estimates of natural cr oss-pollination in Jackson soybeans in Arkansas. Crop Science 6:211-212.

Chakraborty, S., M. Behrens, P.L. Herman, A.F. Arendsen, W.R. Hagen, D.L. Carlson, X.-Z. Wang and D.P. Weeks. 2005. A three-component dicamba O -demethylase from Pseudomonas maltophilia , strain DI-6: Purification and characterization. Archives of Biochemistry and Biophysics 437:20-28.

Chiang, Y.C. and Y.T. Kiang. 1987. Geometric position of genotypes, honeybee foraging patterns and outcrossing in soybean. Bo tanical Bulletin of Academia Sinica 28:1-11.

Chung, G. and R.J. Singh. 2008. Broadening the genetic base of soybean: A multidisciplinary approach. Criti cal Reviews in Plant Sciences 27:295-341.

Clemente, T.E. and E.B. Cahoon. 2009. Soybean oil: Genetic approaches for modification of functionality and total content. Plant Physiology 151:1030-1040.

Codex Alimentarius. 2003. Guideline for the con duct of food safety assessment of foods derived from recombinant-DNA plants. CAC/GL 45-2003. Codex Alimentarius Commission, Joint FAO/WHO Food Standards Programme, Food and Agriculture Organisation, Rome, Italy. ftp://ftp.fao.org/codex/Publications /Booklets/Biotech/Biotech_2003e.pdf Accessed May
12, 2010

.

Cojocariu, C., J. Kreuzwieser and H. Re nnenberg. 2004. Correlation of short-chained carbonyls emitted from Picea abies with physio logical and environmental parameters. New Phytologist 162: 717-727.

Cojocariu, C., P. Escher, K.-H. Häberle, R. Matyssek, H. Rennenberg and J. Kreuszeiser.

2005. The effect of ozone on the emission of carbonyls from leaves of adult Fagus sylvatica. Plant, Cell and Environment 28: 603-611.Monsanto Company 10-SY-210U 265 of 721 

### Comai, L., N. Larson-Kelly, J. Kiser, C.J. D. Mau, A.R. Pokalsky, C.K. Shewmaker, K. McBride, A. Jones and D.M. Stalker. 1988. Chloroplast transport of a ribulose bisphosphate carboxylase small subunit-5-e nolpyruvyl 3-phosphoshikimate synthase chimeric protein requires part of the matu re small subunit in addition to the transit peptide. The Journal of Biological Chemistry 263:15104-15109.

Coruzzi, G., R. Broglie, C. Edwards and N.-H. Chua. 1984. Tissue-specific and lightregulated expression of a pea nuclear gene encoding the small subunit of ribulose-1, 5- bisphosphate carboxylase. EMBO Journal 3:1671-1679.

Crockett, L. 1977. Contents. Pages vii-ix in Wildly Successful Plants: A Handbook of

North American Weeds. Macmillan Pub lishing Co., Inc., New York, New York.

CTIC. 2004. National survey shows more farmers choose conservation. Conservation Technology Information Center, West Lafayette , Indiana. http://www. ctic.purdue.edu/
Accessed November 9, 2004

.

CTIC. 2007. 2007 amendment to the national crop residue management survey summary. Conservation Technology Information Center, West Lafayette, Indiana. http://www.conservationinformation.org/pdf /National_Summary_2007_Amendment.pdf
Accessed June 14, 2010.

CTIC. 2011. Top 10 conservation tillage benefits : Conservation for agriculture's future. Conservation Technology Information Center, West Lafayette, Indiana. http://www.ctic.purdue.edu/resourcedisplay/293/ Accessed September 21, 2011

.

Cunha, B.A. 2010. Stenotrophomonas maltophilia . WebMD, LLC, New York, New York. http://www.emedicine.com/med/topic3457.htm Accessed January 2, 2010

.

Cutler, G.H. 1934. A simple method for making soybean hybrids. Journal of the American Society of Agronomy 26:252-254.

Dalley, C.D., K.A. Renner and J.J. Ke lls. 2001. Weed competition in Roundup Ready soybean and corn. Michigan State University, Department of Crop and Soil Science, East Lansing, Michigan. http://www.ipm.msu.e du/cat06field/pdf/weedfactsheet.pdf Accessed
June 15, 2010

.

Darrouzet, E., J.W. Cooley and F. Daldal. 2004. The cytochrome bc 1 

### complex and its homologue the b 6 

### f

complex: Similarities and differences. Photosynthesis Research 79:25-

44.

Davis, A., K. Renner, C. Sprague, L. Dyer and D. Mutch. 2005. Integrated weed management: One year's seeding. Extension Bulletin E-2931. Michigan State University, East Lansing, Michigan.Monsanto Company 10-SY-210U 266 of 721 

### Denton, M., N.J. Todd, K.G. Kerr, P.M. Hawkey and J.M. Littlewood. 1998. Molecular epidemiology of Stenotrophomonas maltophilia isolated from clinical specimens from patients with cystic fibrosis and associated environmental samples. Journal of Clinical Microbiology 36:1953-1958.

Depicker, A., S. Stachel, P. Dhaese, P. Zambryski and H.M. Goodman. 1982. Nopaline synthase: Transcript mapping and DNA sequen ce. Journal of Molecular and Applied

Genetics 1:561-573.

Dillon, T.W., R.C. Scott, N.D. Pearrow and K. A. Meins. 2006. Effect of sulfonylurea rice herbicides on soybeans. Page 1 in Southe rn Weed Science Society Proceedings, Little Rock, Arkansas.

D'Ordine, R.L., T.J. Rydel, M.J. Storek, E.J. Sturman, F. Moshiri, R.K. Bartlett, G.R. Brown, R.J. Eilers, C. Dart, Y. Qi, S. Flasinski and S.J. Franklin. 2009. Dicamba monooxygenase: Structural insigh ts into a dynamic Rieske oxygenase that catalyzes an exocyclic monooxygenation. Journal of Molecular Biology 392:481-497.

Dorokhov, D., A. Ignatov, E. Deineko, A. Serjapin, A. Ala and K. Skryabin. 2004. Potential for gene flow from herbicide-resistan t GM soybeans to wild soya in the Russian Far East. Pages 151-161 in Introgression from Genetically Modified Plants into Wild Relatives. H.C.M. den Nijs, D. Bartsch, a nd J. Sweet (eds.). CAB International, Wallingford, United Kingdom.

Dorrance, A.E., M.A. Draper and D. Hershm an. 2007. Using foliar fungicides to manage soybean rust. The Ohio State University, Colombus, Ohio.

Dumitru, R., W.Z. Jiang, D.P. Weeks and M.A. Wilson. 2009. Crystal structure of dicamba monooxygenase: A Rieske nonheme ox ygenase that catalyzes oxidative demethylation. Journal of Molecular Biology 392:498-510.

Echemendia, Y. 2010. Microor ganism of the month: Stenotrophomonas maltophilia

. Environmental Microbiology Laborator y, Inc, Cherry Hill, New Jersey. http://www.emlab.com/s/sampling/env-report-07-2007.html Accessed January 2, 2007

.

Erickson, E.H. 1975. Honey bees and soybeans. American Bee Journal 115:351-353.

European Commission. 2008. Review report for the active substance dicamba, Finalised in the Standing Committee on the Food Chain and Animal Health at its meeting on 14

March 2008 in view of the inclusion of di camba in Annex I of Directive 91/414/EEC. European Commission, Health and Consumer Protection Directorate-General, Paris,

France.

Fehr, W.R. 2007. Breeding for modified fatty acid composition in soybean. Crop Science

47:S72-S87.Monsanto Company 10-SY-210U 267 of 721 

### Ferraro, D.J., L. Gakhar and S. Ramaswamy. 2005. Rieske business : Structure-function of Rieske non-heme oxygenases. Bioc hemical and Biophysical Research and

Communications 338:175-190.

Fling, M.E., J. Kopf and C. Richards. 1985. Nucleotide sequence of the transposon Tn 7 gene encoding an aminoglycoside-modifying enzyme, 3"(9)- O -nucleotidyltransferase.

Nucleic Acids Research 13:7095-7106.

Fluhr, R., P. Moses, G. Morelli, G. Co ruzzi and N.-H. Chua. 1986. Expression dynamics of the pea rbcS multigene family and organ distribution of the transcripts. EMBO Journal

5:2063-2071.

Frisvold, G.B., T.M. Hurley and P.D. Mitc hell. 2009. Adoption of best management practices to control weed re sistance by corn, cotton, and soybean growers. AgBioForum

12:370-381.

Gage, D.J. 2004. Infection and invasion of r oots by symbiotic, nitrogen-fixing Rhizobia during nodulation of temperate legumes. Microbiology and Molecular Biology Reviews

68:280-300.

Garber, R.J. and T.E. Odland. 1926. Natural crossing in soybeans. Journal of the American Society of Agronomy 18:967-970.

Gianessi, L.P., C.S. Silvers, S. Sankula and J.E. Carpenter. 2002. Plant biotechnology: Current and potential impact for improving pe st management in U.S. agriculture. An analysis of 40 case studies. National Center for Food & Agricultural Policy, Washington,

D.C.

Giese, M., U. Bauer-Doranth, C. Langebart els and H. Sanderma nn. 1994. Detoxification of formaldehyde by the spider plant (C hlorophytum comosum L.) and by soybean (Glycine max L.) cell-suspension cu ltures. Plant Physiology 104: 1301-1309

Givens, W.A., D.R. Shaw, W. G. Johnson, S.C. Weller, B.G. Young, R.G. Wilson, M.D.K. Owen and D. Jordan. 2009. A grower survey of herbicide use patterns in glyphosateresistant cropping syst ems. Weed Technology 23:156-161.

Giza, P.E. and R.C.C. Huang. 1989. A self -inducing runaway-replication plasmid expression system utilizing the Rop protein. Gene 78:73-84.

Gray, J., E. Wardzala, M. Yang, S. Reinbot he, S. Haller and F. Pauli. 2004. A small family of LLS1-related non-heme oxygenases in plants with an origin amongst oxygenic photosynthesizers. Plan t Molecular Biology 54:39-54.

Gupta, M., W. Nirunsuksiri, G. Schulenberg, T. Hartl, S. Novak, J. Bryan, N. Vanopdorp, J. Bing and S. Thompson. 2008. A non-PCR-based Invader ® 

### assay quantitatively detects single-copy genes in complex pl ant genomes. Molecular Breeding 21:173-181.Monsanto Company 10-SY-210U 268 of 721 

### Hanson, A.D. and S. Roje. 2001. One-carbon metabolism in higher plants. Annual Review of Plant Physiology and Plant Molecular Biology 52: 119-137.

Harayama, S., M. Kok and E.L. Neidle. 1992. Functional and evolutionary relationships among diverse oxygenases. Annual Review of Microbiology 46:565-601.

Harrigan, G.G., D. Lundry, S. Drury, K. Berman, S.G. Riordan, M.A. Nemeth, W.P. Ridley and K.C. Glenn. 2010. Natural variati on in crop composition and the impact of transgenesis. Nature Biotechnology 28:402-404.

Heap, I. 2009. International survey of herbicide resistant weeds. Weed Science Society of America, Corvallis, Oregon. http: //www.weedscience.org/in.asp Accessed November 11,

2009

.

Heap, I. 2011. Herbicide resistant weeds summar y table. WeedScience.com, International Survey of Herbicide Resistan t Weeds, Corvallis, Oregon. http://www.weedscience.org/summary/MOASummary.asp Accessed May 26, 2011.

Heatherly, L.G., and H.F. Hodges. 1999. Soybean production in the Midsouth. CRC Press, Boca Raton, Florida.

Helsel, Z.R. and H.C. Minor. 1993. G4410: Soyb ean production in Missouri. University of Missouri Extension, Columbia, Missouri. http://extension.missouri.edu/e xplore/agguides/crops/g04410.htm Accessed November
11, 2009

.

Herman, P.L., M. Behrens, S. Chakraborty, B. M. Chrastil, J. Barycki and D.P. Weeks. 2005. A three-component dicamba  -demethylase from Pseudomonas maltophilia , strain DI-6. Journal of Biological Chemistry 280:24759-24767.

Herrmann, K.M. 1995. The shikimate pathway: Early steps in the biosynthesis of aromatic compounds. Plant Cell 7:907-919.

Hibino, T., R. Waditee, E. Araki, H. Ishika wa, K. Aoki, Y. Tanaka and T. Takabe. 2002.

Functional characterization of choline monooxygenase, an enzyme for betaine synthesis in plants. Journal of Biological Chemistry 277:41352-41360.

Higley, L.G. 1994. Insect injury to soybean. Pages 11-13 in Handbook of Soybean Insect Pests. L.G. Higley and D.J. Boethel (eds.) . Entomological Society of America, Lanham, Maryland.

Hock, S.M., S.Z. Knezevic, A.R. Martin and J.L. Lindquist. 2006. Soybean row spacing and weed emergence time influence weed competitiveness and competitive indices.

Weed Science 54: 38-46.Monsanto Company 10-SY-210U 269 of 721 

### Hoeft, R.G., E.D. Nafziger, R.R. Johnson and S.R. Aldrich. 2000. Soybean as a crop. Pages 31, 36, 38, 39, 40, 41, 47, 86, 87, 89, 90, 93, 94, 96, 97,120,121, 208, 209 in Modern Corn and Soybean Production. MC SP Publications, Champaign, Illinois.

Holm, L., J.V. Pancho, J.P. Herberger, and D.L. Plucknett. 1979. A geographical atlas of world weeds. John Wiley and S ons., Inc., New York, New York.

Holm, L., J. Doll, E. Holm, J.V. Pancho, and J.P. Herberger. 1997. World weeds: natural

histories and distribution. John Wiley and Sons., Inc ., New York, New York.

HRAC. 2009. Classification of herbicides according to mode of action. Herbicide Resistance Action Committee, Corvallis, Oregon. http://www.hracglobal.com/Publications/Cla ssificationofHerbicideModeofAction/tabid/2 22/Default.aspx Accessed June 26, 2010

.

Hurley, T.M., P.D. Mitchell and G.B. Fris vold. 2009. Weed management costs, weed best management practices, and the Roundup Ready weed management program. AgBioForum 12:281-290.

Hymowitz, T. 1970. On the domesticati on of the soybean. Economic Botany 24:408-421.

Hymowitz, T. 2004. Speciation and cytoge netics. Pages 97-136 in Soybeans: Improvement, Production, and Uses. Third Editi on. H.R. Boerma and J.E. Specht (eds.). American Society of Agronomy, Inc., Crop Science Society of America, Inc., Soil Science Society of America, Inc., Madison, Wisconsin.

Hymowitz, T., R.G. Palmer and R.J. Singh. 1992. Cytogenetics of the genus Glycine

. Pages 53-63 in Chromosome Engineering in Pl ants: Genetics, Breedi ng and Evolution. T. Tsuchiya and P.K. Gupta (eds.). Elsevier Sc ience Publishers, Amsterdam, Netherlands.

ILSI. 2006. Crop Composition Database, Vers ion 3.0. International Life Sciences Institute, Washington, D.C. http://www.cropcomposition.org/

. Accessed October 1,

2009

.

ISO. 2009. Selection and use of the ISO 9000 family of standards. International Standards Organization, Geneva, Switzerland.

ISU. 2004. Soybean growth and development. Iowa State University, Ames, Iowa.

Israel, D.W., J.N. Mathis, W.M. Barbour and G.H. Elkan. 1986. Symbiotic effectiveness

and host-strain interactions of Rhizobium fredii USDA 191 on different soybean cultivars. Applied and Environmental Microbiology 51:898-903.

Janas, K.M., M. Cvikrová, A. Pa ą giewicz and J. Eder. 2000. Alterations in phenylpropanoid content in soybean roots dur ing low temperature acclimation. Plant Physiology and Biochemistry 38:587-593.Monsanto Company 10-SY-210U 270 of 721 

### Janda, T., E. Horvath, G. Szalai and E. Paldi. 2007. Role of salicylic acid in the induction of abiotic stress tolerance. Pages 91-150 in Salicylic Acid: A Plant Hormone. S. Hayat and A. Ahmad (eds.). Springer, Dordrecht, The Netherlands.

Jaycox, E.R. 1970a. Ecological relationships between honey bees and soybeans: II the plant factors. American Bee Journal 110:343-345.

Jaycox, E.R. 1970b. Ecological relationships be tween honey bees and soybeans: III the honey-bee factors. American Bee Journal 110:383-385.

Johnson, W.G., M.D.K. Owen, G.R. Kruger, B. G. Young, D.R. Shaw, R.G. Wilson, J.W. Wilcut, D.L. Jordan and S.C. Weller. 2009. US farmer awareness of glyphosate-resistant weeds and resistance management strategies. Weed Technology 23:308-312.

Johnson, B., B. Young, J. Matthews, P. Marqua rdt, C. Slack, K. Bradley, A. York, S. Culpepper, A. Hager, K. Al-Khatib, L. St eckel, M. Moechnig, M. Loux, M. Bernards and

R. Smeda. 2010. Weed control in dicamb a-resistant soybeans. Plant Management Network, St. Paul, Minnesota. http://www.plantmanagementnetwork.org/sub/cm/research/2010/dicamba/ Accessed September 20, 2010

.

Jordan, N., D.A. Mortensen, D.M. Prenzlow and K.C. Cox. 1995. Simu lation analysis of crop rotation effects on weed seedbanks. American Journal of Botany 82:390-398.

Jordan, T., G. Nice, B. Johnson and T. Bauman. 2009. Reducing spray drift from glyphosate and growth regulator herbicide dr ift caution. Purdue Extension, Purdue University, West Lafayette, Indiana. http://www.ag.purdue.edu/btny/weedscienc e/Documents/ReducingDrift09.pdf Accessed
December 18, 2009

.

Juhnke, M.E. and E. des Jardin. 1989. Selective medium for isolation of Xanthomonas maltophilia from soil and rhizosphere envir onments. Applied and Environmental Microbiology 55:747-750.

Juhnke, M.E., D.E. Mathre and D.C. Sands. 1987. Identification and characterization of rhizosphere-competent bacteria of wheat . Applied and Environmental Microbiology

53:2793-2799.

Klee, H.J., Y.M. Muskopf and C.S. Gasser. 1987. Cloning of an Arabidopsis thaliana gene encoding 5-enolpyruvylshikimate-3-phos phate synthase: Sequence analysis and manipulation to obtain glyphosate-tolerant plants. Molecular a nd General Genetics

210:437-442.Monsanto Company 10-SY-210U 271 of 721 

### Klingman, G.C., F.M. Ashton and L.J. Noor dhoff. 1975. Methods of weed control. Pages 13-28 in Weed Science: Principles and Pr actices. John Wiley & Sons, Inc., New York,

New York.

Kollipara, K.P., R.J. Singh and T. Hymow itz. 1993. Genomic diversity in aneudiploid (2n=38) and diploid (2n=40) Glycine tomentella revealed by cytogenetic and biochemical

methods. Genome 36:391-396.

Krueger, J.P., R.G. Butz, Y.H. Atallah and D.J. Cork. 1989. Isolation and identification of microorganisms for the degradation of dicamba. Journal of Ag ricultural and Food Chemistry 37:534-538.

Kruger, G.R., W.G. Johnson, S.C. Weller, M. D.K. Owen, D.R. Shaw, J.W. Wilcut, D.L. Jordan, R.G. Wilson, M.L. Bernards a nd B.G. Young. 2009. U.S. grower views on problematic weeds and changes in weed pre ssure in glyphosate-resi stant corn, cotton, and soybean cropping systems. Weed Technology 23:162-166.

Kuroda, Y., A. Kaga, N. Tomooka and D. A. Vaughan. 2008. Gene flow and genetic structure of wild soybean ( Glycine soja ) in Japan. Crop Science 48:1071-1079.

Lambert, B., F. Leyns, L. Van Rooyen, F. Gossele, Y. Papon and J. Swings. 1987. Rhizobacteria of maize and their antifunga l activities. Applie d and Environmental Microbiology 53:1866-1871.

Lingenfelter, D.D. and N.L. Hartwig. 2003. In troduction to weeds and herbicides. Penn State University Agricultural Research a nd Cooperative Extension, University Park, Pennsylvania.

Loux, M.M., D. Doohan, A.F. Dobbels, W.G. Johnson, G.R.W. Nice, T.N. Jordan, and T.T. Bauman. 2009. Weed control guide for Oh io and Indiana. Purdue University and Ohio State. Publication WS16. (http ://www.btny.purdue.edu/Pubs/ws/ws-16/) Accessed
on June 14, 2010

.

LSU. 2009. Asian soybean rust: Background. L ouisiana State University, Baton Rouge,

Louisiana. http://www.agctr.lsu.edu/en/crops_livesto ck/crops/soybeans/Asian_Soybean_Rust/
Accessed June 17, 2010

.

Lu, B.-R. 2004. Conserving biodiversity of s oybean gene pool in the biotechnology era. Plant Species Biology 19:115-125.

Lundry, D.R., W.P. Ridley, J.J. Meyer, S.G. Riordan, M.A. Nemeth, W.A. Trujillo, M.L. Breeze and R. Sorbet. 2008. Composition of grai n, forage, and processed fractions from second-generation glyphosate-tolerant soybea n, MON 89788, is equivalent to that ofMonsanto Company 10-SY-210U 272 of 721 

### conventional soybean ( Glycine max L.). Journal of Agricu ltural and Food Chemistry

56:4611-4622.

Maiti, I.B. and R.J. Shepherd. 1998. Isolation and expression analysis of peanut chlorotic streak caulimovirus (PCISV) full-length transcri pt (FLt) promoter in transgenic plants. Biochemical and Biophysical Research Communications 244:440-444.

Martinell, B.J., L.S. Julson, C.A. Emler, Y. Huang, D.E. McCabe and E.J. Williams. 2002. Soybean agrobacterium transforma tion method. Patent 6,384,301, U.S. Patent Office, Washington, D.C.

Montgomery, R.F., R.M. Hayes, C.H. Tingle and J.A. Kendig. 2002. Control of glyphosate-tolerant soybeans ( Glycine max ) in no-till Roundup Ready ® 

### cotton ( Gossypium hirsutum L.). Proceedings of the Beltwide Cotton Conference, National Cotton Council, Atlanta, Georgia.

MSU. 2010. 2010 weed control guidelines for Mi ssissippi. Mississippi State University Extension Service, Mississippi State, Mississippi. http://msucares.com/pubs/publications/p1532.pdf Accessed June 14, 2010

.

Mullin, W.J. and W. Xu. 2001. Study of soyb ean seed coat co mponents and their relationship to water absorption. Journa l of Agricultural and Food Chemistry 49:5331-

5335.

Murdock, E.C., M.A. Jones and R.F. Gr aham. 2002. Control of volunteer glyphosate (Roundup)-tolerant cotton and soybean in Roundup Ready cotton. Proceedings of the Beltwide Cotton Conference, National Cotton Council, Atlanta, Georgia.

Naeve, S. 2005. Minnesota soybean production – growth and development. Department of Agronomy, University of Minnesota. http://www.soybeans.umn.edu/crop/growth/index.htm Accessed December 17, 2009

.

Nam, J.-W., H. Nojiri, T. Yoshida, H. Habe, H. Yamane and T. Omori. 2001. New classification system for oxygenase co mponents involved in ring-hydroxylating oxygenations. Bioscience, Biot echnology, and Biochemistry 65:254-263.

NatureServe. 2007. NatureServe explorer: An online encyclopedia of life. Version 6.2. NatureServe, Arlington, Virginia. http ://www.natureserve.org/explorer. Accessed July
6, 2007

.

NDSU. 2002. Soybean production field guide for North Dakota and northwestern Minnesota. North Dakota State University Ex tension Service and North Dakota Soybean Council, Fargo, North Dakota. http://www.a g.ndsu.edu/pubs/plantsci/rowcrops/a1172.pdf
Accessed November 11, 2009

.Monsanto Company 10-SY-210U 273 of 721 

### Nemecek-Marshall, M., R.C. Macdonald, J.J. Franzen, C.L. Wojciechowski and R. Fall. 1995. Methanol emission from leaves: Enzymatic detection of gas-phase methanol and

relation of methanol fluxes to stomatal conductance and leaf development. Plant Physiology 108: 1359-1368.

Nice, G. and B. Johnson. 2005. Indiana's top ten most problematic weeds. Purdue Extension, West Lafayette, Indiana. http://www.btny.purdue.edu/WeedScience/2005/topten05.pdf Accessed November 11,

2009

.

Nielsen, R.L. 2008. Use thermal time to predict leaf stage development in corn. Purdue University, West Lafayette, Indiana. http://www.agry.purdue.edu/ext/corn/new s/timeless/vstageprediction.html Accessed
December 18, 2009

.

Niepel, M. and D.R. Gallie. 1999. Identificati on and characterization of the functional

elements within the tobacco etch virus 5' l eader required for cap-independent translation. Journal of Virology 73:9080-9088.

NOAA-ESRL. 2011. Average mean temperatur e index by month: Climatology by state

based on climate division data: 1971- 2000. National Oceanic and Atmospheric Administration, Earth System Res earch Laboratory, Washington, D.C. http://www.esrl.noaa.gov/psd/data /usclimate/tmp.state.19712000.climo Accessed September 28, 2011

.

NOAA-NCDC. 2011. Data through 2009. Natio nal Oceanic and Atmospheric Administration, National Climatic Data Center, Washington, D.C. http://www.ncdc.noaa.gov/oa/climat e/online/ccd/relhum.txt Accessed September 28,

2011

.

Norsworthy, J.K. and L.R. Oliver. 2002. He mp sesbania interference in drill-seeded glyphosate-resistant soybean. Weed Science 50: 34-41.

Nunes, F.V. and I.S. de Melo. 2006. Isolati on and characterization of endophytic bacteria of coffee plants and their potential in ca ffeine degradation. Environmental Toxicology

10:293-297.

OECD. 1993. Safety considerations for biotechnology: Scale-up of crop plants. Organisation for Economic Co-operati on and Development, Paris, France.

OECD. 2000. Consensus document on the biology of Glycine max (L.) merr. (soybean). ENV/JM/MONO(2000)9. Series on harmonizat ion of regulatory oversight in biotechnology No.15. Organisation for Economic Co-operation and Development, Paris,

France.Monsanto Company 10-SY-210U 274 of 721 

### OECD. 2001. Consensus document on compositiona l considerations fo r new varieties of soybean: Key food and feed nutrients and anti-nutrients. ENV/JM/MONO(2001)15. Series on the Safety of Novel Foods a nd Feeds No.2. Organisation for Economic Cooperation and Development, Paris, France.

Oliver, D.J. 1994. The glycine decarboxylase complex from plant mitochondria. Annual Review of Plant Physiology and Plant Molecular Biology 45: 323-337.

Owen, M.D.K., B.G. Young, D.R. Shaw, R.G. Wilson, D.L. Jordan, S.C. Weller and P. Dixon. 2009. Benchmark study: Perspectives on glyphosate-resistant crops and the sustainability of chemical weed manageme nt. Page 38 in 2009 North Central Weed Science Society Proceedings, Las Cruces, New Mexico.

Padgette, S.R., D.B. Re, G.F. Barry, D.E. Ei chholtz, X. Delannay, R.L. Fuchs, G.M. Kishore and R.T. Fraley. 1996a. New weed control opportunities: Development of soybeans with a Roundup Ready TM 

### gene. Pages 53-84 in Herbicide-Resistant Crops: Agricultural, Environmental, Economic, Regul atory and Technical Aspects. S.O. Duke (ed.). CRC Press, Inc., Boca Raton, Florida.

Padgette, S.R., N.B. Taylor, D.L. Nida, M.R. Bailey, J. MacDonald, L.R. Holden and R.L. Fuchs. 1996b. The composition of glyphosate-tolerant soybean seeds is equivalent to that of conventional soybeans. Journal of Nutrition 126:702-716.

Palleroni, N.J. and J.F. Bradbury. 1993. Stenotrophomonas , a new bacterial genus for Xanthomonas maltophilia (Hugh 1980) Swings et al. 1983. International Journal of Systematic Bacteriology 43:606-609.

Pedersen, P. 2008a. Soybean production. Iowa State University Extension, Ames, Iowa. http://extension.agron.ia state.edu/soybean Accessed November 11, 2009

.

Pedersen, P. 2008b. Soybean - insects and dise ases. Iowa State University Extension, Ames, Iowa. http://extension.a gron.iastate.edu/soybean Accessed November 11, 2009

.

Pedersen, P. 2008c. Managing soybean cyst ne matode. Iowa State University Extension, Ames, Iowa. http://extension.agron.iastat e.edu/soybean/documents/SCN.pdf Accessed
November 11, 2008

.

Pimentel, D. 1991. Soybean pest manageme nt. Pages 341-343 in CRC Handbook of Pest Management in Agriculture. Volume 3. Second Edition. CRC Press, Inc., Boca Raton,

Florida.

PMRA. 2008. Pest Management Regulatory Agency Re-evaluation – dicamba. Pest Management Regulatory Authority, Health Canada. Ottawa, Canada.

Powles, S.B., C. Preston, I.B. Bryan and A. R. Jutsum. 1996. Herbicid e resistance: Impact and management. Pages 57-93 in Advances in Agronomy. Volume 58. D.L. Sparks (ed.). Academic Press, San Diego, California.Monsanto Company 10-SY-210U 275 of 721 

### Powles, S.B. 2008. Evolved glyphosate-resistant weeds around the world: Lessons to be learnt. Pest Management Science 64:360-365.

Prather, T.S., J.M. Ditomaso and J.S. Ho lt. 2000. Herbicide resistance: Definition and management strategies. University of Calif ornia, Division of Ag riculture and Natural Resources, Oakland, California.

Price, A.J., K.S. Balkcom, S.A. Culpepper, J.A. Kelton, R.L. Nic hols and H. Schomberg. 2011. Glyphosate-resistant Palmer amaranth: A thr eat to conservation tillage. Journal of

Soil and Water Conservation 66:265-275.

Rathinasabapathi, B., M. Burnet, B.L. Russell, D.A. Gage, P.-C. Liao, G.J. Nye, P. Scott,

J.H. Golbeck and A.D. Hanson. 1997. Cholin e monooxygenase, an unusual iron-sulfur enzyme catalyzing the first step of Glycine betaine synthesis in plants : Prosthetic group characterization and cDNA cloning. Proceedings of the National Academy of Sciences of

the United States of America 94:3454-3458.

Ray, J.D., T.C. Kilen, C.A. Abel and R.L. Paris. 2003. Soybean natu ral cross- pollination

rates under field conditions. E nvironmental Biosafety Research 2:133-138.

Rensing, S.A. and U.-G. Maier. 1994. Phyloge netic analysis of the stress-70 protein family. Journal of Molecular Evolution 39:80-86.

Reynolds, T.L., M.A. Nemeth, K.C. Glenn, W.P. Ridley and J.D. Astwood. 2005. Natural variability of metabolites in maize grain: Differences due to genetic background. Journal of Agricultural and Food Chemistry 53:10061-10067.

Ridley, W.P., R.D. Shillito, I. Coats, H.-Y. Steiner, M. Shawgo, A. Phillips, P. Dussold and L. Kurtyka. 2004. Development of the Inte rnational Life Sciences Institute Crop Composition Database. Journal of Food Composition and Analysis 17:423-438.

Rodoni, S., W. Mühlecker, M. Anderl, B. Kräutler, D. Moser, H. Thomas, P. Matile and S. Hörtensteiner. 1997. Chlorophyll breakdown in senescent chloroplasts (cleavage of pheophorbide a in two enzymic steps). Plant Physiology 115:669-676.

Rogers, S.G. 2000. Promoter for transgenic plants. Patent 6,018,100, U.S. Patent Office, Washington, D.C.

Rosche, B., B. Tshisuaka, B. Hauer, F. Lingens and S. Fetzner. 1997. 2-oxo-1,2- dihydroquinoline 8-monooxygenase: Phylogenetic relationship to other multicomponent nonheme iron oxygenases. Journal of Bacteriology 179:3549-3554.

Russell, B.L., B. Rathinasabapathi and A.D. Hanson. 1998. Osmotic stress induces expression of choline monooxygenase in sugar beet and amaranth. Plant Physiology

116:859-865.Monsanto Company 10-SY-210U 276 of 721 

### Ryan, R.P., S. Monchy, M. Cardinale, S. Ta ghavi, L. Crossman, M.B. Avison, G. Berg,

D. van der Lelie and J.M. Dow. 2009. The vers atility and adaptation of bacteria from the

genus Stenotrophomonas . Nature Reviews Microbiology 7:514-525.

Salomon, S. and H. Puchta. 1998. Capture of genomic and T-DNA sequences during double-strand break repair in soma tic plant cells. EMBO Journal 17:6086-6095.

Schmidt, C.L. and L. Shaw. 2001. A comprehe nsive phylogenetic analysis of Rieske and Rieske-type iron-sulfur pr oteins. Journal of Bioenergetics and Biomembranes 33:9-26.

Scott, W.O. and S.R. Aldrich. 1970. Modern soybean production. The Farm Quarterly:

16-152.

SDTF. 1997. A summary of ground a pplication studies. Spray Drif t Task Force, St. Louis, Missouri. http://www.agdrift.com/PDF_FILES/Ground.pdf Accessed June 7, 2010

.

Seguin, P., P. Turcotte, G. Tremblay, D. Pageau and W. Liu. 2009. Tocopherols concentration and stability in early maturing soybean genotypes. Agronomy Journal

101:1153-1159.

Shurtleff, J.L. and H.D. Coble. 1985. Interferen ce of certain broadleaf weed species in soybeans ( Glycine max ). Weed Science 33: 654-657.

Singh, R.J. and T. Hymowitz. 1989. The genomic relationships among Glycine soja Sieb. and Zucc., G. max (L.) Merr. and ' G. gracilis ' Skvortz. Plant Breeding 103:171-173.

Singh, R.J. and T. Hymowitz. 1999. Soybean ge netic resources and crop improvement.

Genome 42:605-616.

Soyatech. 2010. Statistics and reference. Pages 314-317 in Soya and Oilseed Bluebook. Soyatech, Inc., Southwest Harbor, Maine.

Stalker, D.M., C.M. Thomas and D.R. Helinski. 1981. Nucleotide sequence of the region of the origin of replicatio n of the broad host range plasmid RK2. Molecular and General

Genetics 181:8-12.

Steffen, D. 2004. Soybean SN70025 PVPC Number 200400321. U.S. Department of Agriculture, Plant Variety Prot ection Office, Washington, D.C.

Sundstrom, F.J., J. Williams, A.V. Deynze and K.J. Bradford. 2002. Identity preservation of agricultural commodities. Publication 8077. University of California, Division of Agricultural and Natural Re sources, Oakland, California.Monsanto Company 10-SY-210U 277 of 721 

### Sutcliffe, J.G. 1979. Complete nucleotide sequence of the Escherichia coli plasmid pBR322. Pages 77-90 in Cold Spring Harbor Symposia on Quantitative Biology, Cold Spring Harbor, New York.

Sutherland, J.P. and G.M. Poppy. 2005. Quantif ying exposure. Pages 186-212 in Gene Flow from GM Plants. G.M. Poppy and M.J. Wilkinson (eds.). Blackwell Publishing, Ames, Iowa.

Swings, J., P. De Vos, M. Van den M ooter and J. De Ley. 1983. Transfer of Pseudomonas maltophilia Hugh 1981 to the genus Xanthomonas as Xanthomonas maltophilia (Hugh 1981) comb. nov. International J ournal of Systematic Bacteriology

33:409-413.

Szabó, B., E. Tyihák, L.G. Szabó and L. Botz. 2003. Mycotoxin and drought stress induced change of alkaloid content of Pa paver somniferum plantlets. Acta Botanica Hungarica 45: 409-417.

Szende, B. and E. Tyihák. 2010. Effect of formaldehyde on cell proliferation and death. Cell Biology Internat ional 34: 1273-1282.

University of Tennessee. 2010. 2010 weed cont rol manual for Tennessee. University of Tennessee, Institute of Agriculture, Knoxville, Tennessee.

U.S. EPA. 1993. Reregistration eligibili ty decision (RED): Glyphosate. U.S. Environmental Protection Ag ency, Washington, D.C.

U.S. EPA. 1996. Residue chemistry test guid elines - Nature of the residue - Plants,

livestock. OPPTS 860.1300. U.S. Environm ental Protection Agency, Office of Prevention, Pesticides and Toxic Substances, Washington, D.C.

U.S. EPA. 2008. Reregistration eligib ility decision for formaldehyde and paraformaldehyde. EPA 739-R-08-004. U.S. Envi ronmental Protection Agency, Office of Pesticide Programs Washington, D.C.

U.S. EPA. 2009. Reregistration eligibility decision for dicamba and associated salts (Amended). U.S. Environmental Protection Ag ency, Office of Prevention, Pesticides, and Toxic Substances, Washington, D.C.

U.S. FDA. 1992. Statement of policy: Foods derived from new plant varieties. Federal Register: 22984-23005.

USDA-APHIS. 2006. Soybean. U.S. Department of Agriculture, Animal and Plant Health Inspection Service, Washington, D.C. http ://www.aphis.usda.gov/br/soybean.html Accessed January 20, 2006

.

USDA-APHIS. 2007. Environmental assessment , in response to Monsanto petition 06- 178-01p seeking a determination of non-regul ated status for Roundup Ready 2 YieldMonsanto Company 10-SY-210U 278 of 721 

### soybean MON 89788. U.S. Department of Ag riculture, Animal and Plant Health Inspection Service, Washington, D.C. http://www.aphis.usda.gov/brs/aphisdocs2/06_17801p_com.pdf Accessed May 28,

2010

.

U.S. CPSC. 1997. An update on formalde hyde. U.S. Consumer Product Safety Commission, Wash ington, D.C.

USDA-ERS. 2001. How much U.S. cropland acr eage is under crop residue management. Briefing Room. U.S. Department of Agri culture, Economic Research Service, Washington, D.C. http://www.ers.usda.gov/Br iefing/AgChemicals/soilmangement.htm
Accessed November 14, 2009

.

USDA-ERS. 2008. Data sets: U.S. and regional co st and return estimates in soybean for 2008. U.S. Department of Agriculture, Econom ic Research Service, Washington, D.C. http://www.ers.usda.gov/data/Cost sandReturns/testpick.htm Accessed January 18, 2010

.

USDA-NASS. 2001. Agricultural chemical usage - 2000 field crops summary. U.S. Department of Agriculture, National Agricult ural Statistics Service, Washington, D.C.

USDA-NASS. 2004. Agricultural chemical usage - 2003 field crops summary. U.S. Department of Agriculture, National Agricult ural Statistics Service, Washington, D.C.

USDA-NASS. 2006. Agricultural chemical usage - 2005 field crops summary. U.S. Department of Agriculture, National Agricultur al Statistics Service, Washington, D.C.

USDA-NASS. 2007a. Acreage - June 2006. U.S. Department of Agriculture, National Agricultural Statistics Service, Washington, D.C.

USDA-NASS. 2007b. Agricultural chemical usage 2006 field crops summary. U.S. Department of Agriculture, National Agricult ural Statistics Service, Washington, D.C.

USDA-NASS. 2008. Agricultural chemical usage - 2007 field crops summary. U.S. Department of Agriculture, National Agricult ural Statistics Service, Washington, D.C.

USDA-NASS. 2009a. Crop production: 2008 summar y. U.S. Department of Agriculture, National Agricultural Statistic s Service, Washington, D.C.

USDA-NASS. 2009b. Crop values: 2008 summar y. U.S. Department of Agriculture, National Agricultural Statistic s Service, Washington, D.C.

USDA-NASS. 2009c. Acreage - June 2009. U.S. Department of Agriculture, National Agricultural Statistics Service, Washington, D.C.Monsanto Company 10-SY-210U 279 of 721 

### USDA-NASS. 2009d. Crop progress reports. U.S. Department of Agriculture, National Agricultural Statistics Service, Washington, D.C.

USDA-NASS. 2010. Charts and maps: Soybeans: Planted acreage by country. U.S. Department of Agriculture, National Agricultur al Statistics Service, Washington, D.C.

USDA-NRCS. 2010. The PLANTS database. U.S. Department of Agriculture, Natural Resources Conservation Service, Washington, D.C. http://www.plants.usda.gov/index.html Accessed March 23, 2010

USGS. 2004. Pesticide national synthesis project: Pesticide use. United States Geological Survey, Washington, D.C. http://water.usgs.gov/nawqa/pnsp/usage/maps/ Accessed May
28, 2010

.

USHHS-ATSDR. 1999. Toxicological profile for formaldehyde. U.S. Department of Health and Human Services, Agency for Toxic Substances and Disease Registry, Washington, D.C.

USHHS-NTP. 2011. Report on carcinogens. U.S. Department of Health and Human Services, National Toxicology Program, Washington, D.C.

Vail, G.D. and L.R. Oliver. 1993. Barnyardgrass (Echinochloa crus-galli) interference in soybeans (Glycine max). Weed Technology 7: 220-225.

Wang, X.-Z., B. Li, P.L. Herman and D.P. Weeks. 1997. A three-component enzyme system catalyzes the O demethylation of the herbicide dicamba in Pseudomonas maltophilia DI-6. Applied and Envi ronmental Microbiology 63:1623-1626.

Weber, C.R. and W.D. Hanson. 1961. Natural hybridization with and without ionizing radiation in soybeans. Crop Science 1:389-392.

Webster, T.M., M. Patterson, J. Everest, J. Ferrell, B. Brecke, A.S. Culpepper, E.P. Prostko, J.D. Green, J.R. Martin, E. Webster, S. Kelly, J. Griffin, D. Sanders, J. Byrd, A. Kendig, A. York, D. Jordan, L. Fisher, C. Medlin, D. Murray, J. Norsworthy, J. Chapin, L. Nelson, and L. Steckel. 2005. Weed survey: Southern states 2005: Broadleaf crops section (cotton, peanut, soybean, tobacco, a nd forestry). Pages 291-306, Southern Weed Science Society Proceedings, Las Cruces, New Mexico.

Webster, T.M., M. Patterson, J. Everest, K. Sm ith, J. Norsworthy, B. Scott, C. Fristoe, D. Oliver, N. Burgos, J. Boyd, J. Ferrell, P. Minogue, B. Becke, A.S. Culpepper, D. Moorhead, E. Prostko, J.M. Moore, J.D. Gree n, J.R. Martin, B. Miller, D. Miller, J. Griffin, D. Stephenson, D. Sanders, K. Bradley, A. York, L. Fisher, S. Jordan, D. Murray, M. Marshall, S. Hagood, and C. Johnson. 2009. Weed survey: Southern states 2009: Broadleaf crops subsection (cotton, peanut, soybean, tobacco, and forestry). Pages 517- 520 in Southern Weed Science Society Proceedings, Las Cruces, New Mexico.Monsanto Company 10-SY-210U 280 of 721 

### Werlen, C., H.-P.E. Kohler and J.R. van der Meer. 1996. The broad substrate chlorobenzene dioxygenase and cis -chlorobenzene dihydrod iol dehydrogenase of

Pseudomonas sp. strain P51 are linked evolutiona rily to the enzymes for benzene and toluene degradation. Journal of Biological Chemistry 271:4009-4016.

WHO. 2002. Concise International Chemical Assessment Document 40: Formaldehyde. World Health Organization, Geneva.

WHO-IPCS. 1989. Environmental health crit eria 89: Formaldehyde. World Health Organization, Internat ional Programme on Chemical Safety, Geneva, Switzerland. http://www.inchem.org/documents/ehc/ehc/ehc89.htm Accessed February 2, 2011

.

Wishart, D.S. 2010. Human Metabolome Database. www.hmdb.ca Accessed June, 2

2010

.

Wishart, D.S., C. Knox, A.C. Guo, R. Ei sner, N. Young, B. Gautam, D.D. Hau, N. Psychogios, E. Dong, S. Bouatra, R. Mandal, I. Sinelnikov, J. Xia, L. Jia, J.A. Cruz, E. Lim, C.A. Sobsey, S. Shrivastava, P. Hua ng, P. Liu, L. Fang, J. Peng, R. Fradette, D. Cheng, D. Tzur, M. Clements, A. Lewis, A. De Souza, A. Zuniga, M. Dawe, Y. Xiong, D. Clive, R. Greiner, A. Nazyrova, R. Shaykhut dinov, L. Li, H.J. Vogel and I. Forsythe. 2009. HMDB: A knowledgebase for the human metabolome. Nucleic Acids Research

37:D603-D610.

Woodworth, C.M. 1922. The extent of natural cross-pollination in s oybeans. Journal of the American Society of Agronomy 14:278-283.

Wrather, J.A., W.C. Stienstra and S.R. Koenning. 2001. Soybean disease loss estimates

for the United States from 1996 to 1998. Cana dian Journal of Plant Pathology-Revue Canadienne De Phytopathologie 23:122-131.

Wrather, J.A. and S. Koenning. 2011. Soybean disease loss estimates for the United States, 1996-2010. University of Missouri Delta Research Cent er, Agricultural Experiment Station, Portageville, Missouri. http://aes.missouri.edu/delta/research/soyloss.stm Accessed February 22, 2012

Wrather, J.A., W.C. Stienstra and S.R. Koenning. 2001. Soybean disease loss estimates

for the United States from 1996 to 1998. Cana dian Journal of Plant Pathology-Revue Canadienne De Phytopathologie 23:122-131.

WSSA. 1992. Tables. Pages 88, 103, 107, 118, 123, 130, 145, 393-401 in Crop Losses

Due to Weeds in the United States. D.C. Bri dges (ed.). Weed Science Society of America, Champaign, Illinois.

WSSA. 2010. WSSA supports NRC findings on w eed control. Weed Science Society of America, Champaign, Illinois. Accessed June 9, 2010

.Monsanto Company 10-SY-210U 281 of 721 

### Yang, M., E. Wardzala, G.S. Johal and J. Gray. 2004. The wound-inducible Lls1 gene from maize is an orthologue of the Arabidopsis Acd1 gene, and the LLS1 protein is present in non-photosynthetic ti ssues. Plant Molecular Biology 54:175-191.

York, A.C., J.B. Beam and A.S. Culpe pper. 2005. Control of volunteer glyphosateresistant soybean in cotton. Journal of Cotton Science 9:102-109.

Yoshimura, Y., K. Matsuo and K. Yas uda. 2006. Gene flow from GM glyphosatetolerant to conventional s oybeans under field conditions in Japan. Environmental Biosafety Research 5:169-173.

Zambryski, P., A. Depicker, K. Kruger and H.M. Goodman. 1 982. Tumor induction by Agrobacterium tumefaciens : Analysis of the boundaries of T-DNA. Journal of Molecular and Applied Genetics 1:361-370.

Zhang, L.X., J. Zhang, S. Kye-Boahen and C.E. Watson. 2004. Redefining zones of adaptation of soybean matur ity groups in the US. 2004 A nnual Meeting Abstracts, CDROM. American Society of Agronomy, Inc., Crop Science Society of America, Inc., Soil Science Society of America, Inc., Madison, Wisconsin.

Zhu, J.-K. 2009. Active DNA demethylation me diated by DNA glycosylases. Annual

Review of Genetics 43: 143-166.

Zollinger, R.K. 2000. Survey of weeds in No rth Dakota. North Dakota State University, Extension Weed Specialist, Fargo, North Dakota. http://www.ag.ndsu.nodak.edu/weeds/ER83/ER83.htm Accessed November 11, 2009

.

Zollinger, R.K. 2009. 2009 North Dakota weed control guide – relative herbicide

effectiveness on weeds-volunt eer Roundup Ready crops. Circular W-253, North Dakota State University, Extension Weed Specialist, Fargo, North Dakota http://www.ag.ndsu.edu/weeds/w253/w253-4d.htm Accessed November 11, 2009

.Monsanto Company 10-SY-210U 282 of 721 

### APPENDICESMonsanto Company 10-SY-210U 283 of 721 

### Appendix A: USDA Notifications

Field trials of MON 87708 have been conduc ted in the U.S. since 2005. The protocols

for these trials included fi eld performance, breeding and observation, agronomics, and generation of field materials and data necessary for this petition. In addition to MON 87708 phenotypic assessment data, observati onal data on pest and disease stressors were collected from these product development trials. The majority of the final reports have been submitted to the USDA. However, some final reports, mainly from the 2008-2009 seasons, 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.Monsanto Company 10-SY-210U 284 of 721 

### Table A-1. USDA Notifica tions Approved for MON 87708 and Status of Trials

Conducted under These Notifications and Permits

USDA Number Effective Date Release Site (State) Trial Status

2005

05-269-02n 11/16/2005 PR Submitted to USDA

2006 06-045-15n 5/18/2006 HI(5) Submitted to USDA 06-045-17n 5/18/2006 PR(3) Submitted to USDA 06-052-01n 3/20/2006 IL(7), KS(5) Submitted to USDA 06-052-02n 4/24/2006 IA(7), IL(5), IN(2) Submitted to USDA 06-052-09n 4/24/2006 IA(2), IL(6), IN(2) Submitted to USDA 06-067-05n 4/24/2006 IL(2) Submitted to USDA 06-090-03n 5/5/2006 IL(2) Submitted to USDA

06-275-102n 11/14/2006 PR Submitted to USDA 06-345-101n 1/10/2007 PR(3) Submitted to USDA

2007 07-018-103n 2/17/2007 IL(10), IN(3), MO, PR Submitted to USDA 07-018-106n 2/17/2007 IA(7), KS(6) Submitted to USDA 07-018-109n 2/17/2007 IA, IL(10), IN(3), MO Submitted to USDA 07-024-101n 3/18/2007 IA(7), KS(6) Submitted to USDA

07-039-101n 3/18/2007 IA(4), IL(5), IN(3), KS(3) Submitted to USDA 07-043-102n 4/10/2007 IA, IL(2), KS, MD, WI Submitted to USDA
07-050-107n 4/9/2007 IA, IL (2), IN, KS, KY, MN, NE, SD Submitted to USDA

07-057-109n 4/6/2007 AL, IA (3), IL, IN, LA, MN, MO(2), MS, NE, SD, TN Submitted to USDA

07-094-104n 5/4/2007 IA(2) Submitted to USDA

07-094-116n 5/4/2007 MN Submitted to USDA 07-113-103n 6/4/2007 PR(2) Submitted to USDA

07-241-103n 9/28/2007 PR Submitted to USDA 07-250-102n 10/7/2007 PR(2) Submitted to USDA

07-261-101n 10/18/2007 PR Submitted to USDA 07-271-101n 10/28/2007 PR(2) Submitted to USDA

07-312-101n 12/5/2007 PR Submitted to USDAMonsanto Company 10-SY-210U 285 of 721 

### Table A-1 (continued). US DA Notifications Approved for MON 87708 and Status of

Trials Conducted under These Notifications and Permits USDA Number Effective Date Release Site (State) Trial Status

2008 07-352-101rm 3/26/2008 IA(8), IL(16), IN(4), KS(7),

MO Submitted to USDA

08-030-103n 2/29/2008 PR(2) Submitted to USDA 08-031-105n 3/13/2008 IA(5), IL(4), KS(5) Submitted to USDA 08-031-106n 3/1/2008 IA(2), IL(5), IN(3) Submitted to USDA 08-039-107n 3/9/2008 IA(5), IL, IN(3), KS(5), MO Submitted to USDA 08-043-107n 3/13/2008 IA(3), IL(10), IN, OH Submitted to USDA
08-049-101n 3/19/2008 IL, MD, WI Submitted to USDA 08-058-101n 3/28/2008 IA(3), IL(2), IN, MO, PA, WI(2) Submitted to USDA

08-059-109n 3/29/2008 IA Submitted to USDA

08-059-110n 3/29/2008 IL Submitted to USDA

08-059-112n 3/29/2008 IN Submitted to USDA

08-060-103n 4/2/2008 MN Submitted to USDA 08-063-112n 4/2/2008 IA(4), IL(2), IN, MI, MO, NE(2) Submitted to USDA

08-063-113n 4/4/2008 MN(2), ND, SD(5), WI(5) Submitted to USDA 08-065-101n 4/4/2008 IL(2), IN Submitted to USDA

08-064-102n 4/3/2008 PA Submitted to USDA

08-064-103n 4/3/2008 IL Submitted to USDA 08-064-104n 4/3/2008 AR, GA, KS(5), LA, MO, SC Submitted to USDA 08-064-105n 4/3/2008 AR, IL(2), IN, KS(3), MD, MN(3), NC, SD, WI, ND Submitted to USDA

08-072-110n 4/25/2008 AR, IA, IN(3), KS, MI, MO(2),

NE Submitted to USDA

08-079-101n 4/17/2008 IA(3) Submitted to USDA
08-084-102n 4/24/2008 IA, NE Submitted to USDA 08-182-101n 8/1/2008 PR(2) Submitted to USDA

08-219-101n 9/5/2008 PR Submitted to USDA

08-263-101n 10/19/2008 AR, IA, IL, MO Submitted to USDA

08-266-105n 10/22/2008 PR Submitted to USDA

08-323-101n 12/18/2008 PR Submitted to USDA

08-352-108n 1/26/2009 PR Submitted to USDA 08-357-101rm 3/17/2009 IA(8), IL(7), IN(3), KS(5), NE Submitted to USDAMonsanto Company 10-SY-210U 286 of 721 

### Table A-1 (continued). US DA Notifications Approved for MON 87708 and Status of

Trials Conducted under These Notifications and Permits

USDA Number Effective Date Release Site (State) Trial Status

2009

09-007-106n 2/25/2009 PR Submitted to USDA 09-036-103n 3/7/2009 IA(2), IL(2), IN, NE Submitted to USDA

09-042-103n 3/19/2009 MS Submitted to USDA

09-049-110n 3/20/2009 IA Submitted to USDA 09-050-136n 4/3/2009 IA(2), IL(2), IN(2), MD, MN, OH, PR, SD Submitted to USDA

09-061-108n 4/1/2009 AR, IA, IL(3), KS Submitted to USDA 09-061-117n 4/1/2009 IL, MO(2) Submitted to USDA 09-068-111n 4/8/2009 IL(2), IN(2), MS, NE(2), OH Submitted to USDA 09-071-102n 4/11/2009 NE, SD, TN(2) Submitted to USDA 09-082-103n 4/22/2009 IN(2) Submitted to USDA

09-091-103n 5/1/2009 AR Submitted to USDA

09-093-120n 5/3/2009 AR Submitted to USDA

09-124-102n 6/3/2009 PR Submitted to USDA

09-124-105n 5/13/2009 IA Submitted to USDA

09-135-104n 6/14/2009 IL Submitted to USDA

09-162-105n 7/11/2009 PR Submitted to USDA

09-162-106n 7/11/2009 PR Submitted to USDA 09-222-101n 9/9/2009 PR(2) Submitted to USDA

09-237-104n 9/24/2009 PR Submitted to USDA

09-247-101rm 11/17/2009 PR Submitted to USDAMonsanto Company 10-SY-210U 287 of 721 

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

MON 87708

B.1. Materials

The genomic DNA used in molecular analyses was isolated from le af tissue harvested from MON 87708 and the near isogenic conven tional soybean contro l A3525 (seed lot: GLP-0707-18882-S, and GLP-0707-18884-S, respect ively). Additional DNA extracted from leaf tissue of various MON 87708 genera tions was used in generational stability analyses. The control was conventional soybean variety A3525 that has a similar genetic background as MON 87708. Plasmid vector PV -GMHT4355 (Figure III-1) was used as a positive hybridization control in Southern blot analyses. Probe templates generated from PV-GMHT4355 were used as additional positiv e hybridization controls. As additional reference standards, the 1 kb DNA extension ladder and λ DNA/ Hind III segments from Invitrogen (Carlsbad, CA) were used for size estimations on Southern blots and agarose gels. The GeneRulerTM 1 kb Plus DNA ladder from Fermentas (Hanover, MD) was used for size estimations on agarose gels for polymerase chain reaction (PCR) analyses.

B.2. Characterization of the Materials

The identity of the leaf material from MON 87708 and the conventional control was verified by event-specific PCR analysis to confirm the presence or absence of the dmo expression cassette. The stability of the genomic DNA was confirmed in each Southern blot analysis by observation of the digested DNA sample on an ethidium bromide-stained agarose gel, and/or interpreta ble signals on Southern blots, and/or produced specific PCR products.

B.3. DNA Isolation for South ern Blot and PCR Analyses

Genomic DNA from MON 87708 and the conventi onal control was isolated from leaf tissue. The leaf tissue was gr ound to a fine powder in liquid nitrogen using a mortar and pestle. DNA was extracted using a hexadecyltrimethylammonium bromide (CTAB)-based method. Briefly, 20 ml of CTAB buffer (1.5% w/v CTAB, 75 mM Tris HCl, 100 mM EDTA, 1.05 M NaCl, and 0.75% w/v PVP) and 10 mg RNase A were added to approximately 4 ml of ground l eaf tissue and incubated at 60-70°C for 40-50 minutes with intermittent mixing. Twen ty ml of chloroform was added to the samples and mixed by hand for 2-3 minutes, then centrifuged at 10,300 × g for 8-10 minutes. The upper aqueous phase was put into a clean tube and the chloroform step was repeated twice. After the last chlo roform step, the aqueous phase was put into a clean tube and the DNA was precipitated with 20 ml of 100% ethanol. The sample was centrifuged for one minute to condense th e pellet, and then the precipitated DNA was hooked out and put into a tube with 4-6 ml of 70% ethanol to wash the DNA pellet. The samples were centrifuged at 5,100 × gravity for 5 minutes to pellet the DNA. DNA pellets were air dried, then resuspended in 300 μl of TE buffer (10 mM Tris HCl, 1 mM EDTA, pH8.0). All extracted DNA was stored in a 4  C refrigerator or a -20  C freezer.Monsanto Company 10-SY-210U 288 of 721 

### B.4. Quantification of Genomic DNA

Extracted genomic DNA was quantified using a Hoefer DyNA Quant 200 Fluorometer. Molecular size marker IX (Roche, Indianapolis , IN) was used as the calibration standard.

B.5. Restriction Enzyme Digestion of Genomic DNA

Approximately 10 μg of genomic DNA extr acted from MON 87708 and the conventional control was digested with appropriate combinations of restriction enzymes Bsp1286 I/ Pvu II or Hpa I/ Kpn I (New England Biolabs, Ipswich, MA). All digests were conducted in 1 × NEBuffer 4 (New England Biolabs) plus 1 × BSA (New England Biolabs) at 37  C in a total volume of ~500 μl with ~ 50 units of each restriction enzyme. For the purpose of running positive hybridizat ion controls, ~10 μg of genomic DNA

extracted from the conventional control was digested with the restriction enzyme

combination Bsp1286 I/ Pvu II or Hpa I/ Kpn I and the appropriate positive hybridization control(s) were added to these digests.

B.6. Agarose Gel Electrophoresis

Digested genomic DNA was resolved on ~0.8% (w/v) agarose gels. Individual digests of MON 87708 and the 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 the detection of small molecular weight DNA. For the insert stability an alysis, individual dige sts of genomic DNA extracted from leaf tissue across multiple generations were loaded on the agarose gel in a single short-run format. The positive hybridiza tion controls were only run in the shortrun format.

B.7. DNA Probe Preparation fo r Southern Blot Analyses

Probe templates were prepared by PCR amplification from plasmid vector PV-GMHT4355. Approximately 25 ng of each pr obe template were radiolabeled with

32 

### P-deoxycytidine triphosphate (dCTP) (6000 Ci/mmol) or 32 

### P-deoxyadenosine triphosphate (dATP) (6000 Ci/mmol) us ing the RadPrime DNA Labeling System (Invitrogen). Probe locations relative to the genetic elements in plasmid vector PV-GMHT4355 are depicted in Figure III-1.

B.8. Southern Blot Analyses of Genomic DNA

Digested genomic DNA isol ated from MON 87708 and the conventional control was evaluated using Southern blot analyses . The plasmid vector PV-GMHT4355 DNA digested with the enzyme combination Aat II/ Nde I was added to the conventional control genomic DNA previously digested with the enzyme combination Bsp1286 I/ Pvu II or Hpa I/ Kpn I to serve as a positiv e hybridization control. When multiple probes were hybridized simultaneously to one Southern blot, the appropria te probe templates generated from PV-GMHT4355 were mixed w ith previously digested conventional control genomic DNA to serve as additiona l positive hybridizat ion controls. The digested DNA was then separated by agarose gel electrophoresis a nd transferred onto aMonsanto Company 10-SY-210U 289 of 721 

### nylon membrane. Southern blots were hybr idized and washed at 55, 60, or 65°C, depending on the melting temperature (Tm) of the probes. Table B-1 lists the radiolabeling conditions and hybrid ization temperatures of the probes used in this study. Multiple exposures of each blot were then generated using Kodak Biomax MS film in conjunction with one or two Kodak Biomax MS intensifying screen(s) in a -80  C freezer.

Table B-1. Hybridization Conditions of Utilized Probes

Probe DNA Probe Element Sequence Spanned by DNA Probe Probe labeled

with dNTP ( 32 

### P) Hybridization Temperature (  C) 1 Backbone Probe Backbone sequence dCTP 60 2 Backbone Probe Backbone sequence dCTP 60 3 Backbone Probe Backbone sequence dCTP 60

4 T-DNA II Probe T- E9, and CS- cp4 epsps (portion) dATP 55

5 T-DNA II Probe CS- cp4 epsps (portion)

, and TS- CTP2 (portion) dCTP 60

6 T-DNA II Probe TS- CTP2 (portion),

L- DnaK , P- FMV dATP 55

7 Backbone Probe Backbone sequence dCTP 60

8 T-DNA I Probe B-Right Border, P- PC1SV

, L- TEV, TS- RbcS dATP 60

9 T-DNA I Probe TS- RbcS (portion),

CS- dmo , T- E9 (portion) dCTP 65

10 T-DNA I Probe T- E9 , and B-Left Border dATP 55Monsanto Company 10-SY-210U 290 of 721 

### B.9. DNA Sequence Analyses of the Insert

Overlapping PCR products that span the insert and adjacent 5 ′ and 3 ′ flanking DNA sequences in MON 87708 (Figure B-1) were ge nerated. These products were sequenced

to determine the nucleotide sequence of the insert in MON 87708 as well as the nucleotide sequence of the DNA flanking the 5 ′ and 3 ′ ends of the insert.

The PCR analyses were conducted using 50 ng of genomic DNA template in a 25 μl reaction volume containing a final concen tration of 1 M betaine, 1 mM MgSO 4 

### , 0.8 μM of each primer, 0.2 mM of each dNTP, and 0.5 units of KOD Hot Start DNA polymerase (Novagen, Madison, WI). The amplification of Product A (Figure B-1) was performed under the following cycling conditions: one cycle at 94  C for 2 minutes; 35 cycles at

94  C for 45 seconds, 60.2  C for 45 seconds, 72  C for 5 minutes; one cycle at 72  C for 10 minutes. The amplification of Product B (Figure B-1) was performed under the following cycling conditions: one cycle at 94  C for 2 minutes; 35 cycles at 94  C for
45 seconds, 60.8  C for 45 seconds, 72  C for 5 minutes; one cycle at 72  C for 10 minutes.

Following PCR amplification, exonuclease I (Exo; US Biochemicals, Cleveland, OH)/ shrimp alkaline phosphatase (SAP; US Bioche micals) purification of the PCR products used for sequencing was performed in a 21 μl reaction volume containing 15 μl of the PCR product and a final concentration of 0.1 units/μl of Exo and 0.1 units/μl of SAP.

The reaction was incubated at 37oC for 15 minutes, followed by 80oC for an additional

15 minutes.

Prior to sequencing, aliquots of untreated and Exo/SAP treated PCR product were separated on 0.8% (w/v) agarose E-gels (Invi trogen) and visualized by ethidium bromide staining to verify that the pr oducts were of the expected size. The PCR products were sequenced using multiple primers including primers used for PCR amplification and primers designed internal to the amplified sequences. All sequencing was performed by the Monsanto Genomics Sequencing Center using BigDye terminator chemistry (ABI, Foster City, CA).

B.10. PCR and DNA Sequence Analysis to Examine the MON 87708 Insertion Site

To examine the insertion s ite of conventional soybean and MON 87708, PCR analysis was performed on genomic DNA from both MON 87708 and the conventional control (Figure B-2). The primers used in this an alysis were designed from the genomic DNA sequences flanking the insert in MON 87708. One primer designed from the genomic DNA sequence flanking the 5 ′ end of the insert was paired with a second primer located in the genomic DNA sequence flanking the 3 ′ end of the insert.

The PCR analysis was conducted using appr oximately 50 ng of genomic DNA template in a 25 μl reaction volume containing a final concentration of 1 M betaine, 1 mM MgSO 4 

### , 0.8 μM of each primer, 0.2 mM of each dN TP, and 0.5 units of KOD Hot Start DNA polymerase (Novagen). The amplificati on of the product was performed under the following cycling conditions: one cycle at 94  C for 2 minutes; 35 cycles at 94  C for
45 seconds, 60.2  C for 45 seconds, 72  C for 5 minutes; one cycle at 72  C for 10 minutes.Monsanto Company 10-SY-210U 291 of 721 

### Following PCR amplification, Exo/SAP purif ication of the PCR products used for sequencing was performed in a 21 μl r eaction volume containing 15 μl of the PCR product and a final concentration of 0.1 units /μl of Exo and 0.1 units/μl of SAP (U.S. Biochemicals). The reaction was incubated at 37oC for 15 minutes, followed by 80oC for

an additional 15 minutes.

Prior to sequencing, aliquots of untreated and Exo/SAP treated PCR product were separated on 0.8 % (w/v) agarose E-gels (Invi trogen) and visualized by ethidium bromide staining to verify that the pr oducts were of the expected si ze prior to sequencing. The PCR products were sequenced using multiple primers, including primers used for PCR amplification and primers designed internal to the amplified sequences. All sequencing was performed by the Monsanto Genomics Se quencing Center using BigDye terminator chemistry.Monsanto Company 10-SY-210U 292 of 721 

### Figure B-1. Overlapping PCR Analys is Across the Insert in MON 87708 PCR analyses were performed on MON 87708 ge nomic DNA extracted from leaf (Lanes 3 and 6). Lanes 2 and 5 contain reactions with c onventional control DNA while lanes 4 and 7 are reactions containing no template DNA. Lanes 1 and 8 contain Fermentas GeneRulerTM 1 kb Plus DNA Ladder. Lanes are marked to show which pr oduct has been loaded and is visualized on the agarose gel. The expected product size for each amplicon is provided in the illustration of the insert in MON 87708 that appears at the bottom of the figure. Five microliters of each of the PCR products was loaded on the gel. This figure is representative of the data generated; however, the specific bands from this gel were not excised and sequenced.

Lane:

1. GeneRulerTM 1 kb Plus DNA Ladde r 5. Conventional control DNA 2. Conventional control DNA 6. MON 87708 genomic DNA 3. MON 87708 genomic DNA 7. No template DNA control 4. No template DNA control 8. GeneRulerTM 1 kb Plus DNA Ladder

Arrows denote sizes of DNA, in kilobase pairs, obtained from molecular weight markers on ethidium bromide stained gel. 1 2 3 4 5 6 7 8

5.0 4.0

3.0

2.0 5.0
4.0

3.0

2.0B-Right BorderP-PC1SVCS-dmoT-E9B-Left BorderTS-RbcS 5 ′ Flank 3 ′ Flank Product B: ~ 3.0 kb Product A: ~ 3.2 kb Product A Product BL-TEVMonsanto Company 10-SY-210U 293 of 721 

### Figure B-2. PCR Amplification of the MON 87708 Insertion Site Depiction of the MON 87708 insertion locus in conventional control and MON 87708. PCR amplification was performed using Primer A in the 5 ′ flanking sequence and Primer B in the 3 ′ flanking sequence of the insert in MON 87708 to verify to examine the insertion site in conventional soybean and MON 87708.

Lane Description

1 GeneRulerTM 1 kb Plus DNA Ladder

2 Conventional control DNA 3 MON 87708 genomic DNA 4 No template DNA control

5 GeneRulerTM 1 kb Plus DNA Ladder

Arrows denote sizes of DNA, in kilobase pairs, obtained from molecular weight markers on ethidium bromide stained gel.Monsanto Company 10-SY-210U 294 of 721 

### Appendix C: Materials, Methods, and Results for the Characterization of

MON 87708 DMO and Substrat e Specificity of DMO

C.1. Characterization of MON 87708 DMO

C.1.1. Materials

MON 87708 DMO (lot 11261646) was purified from defatted soybean flour as described in C.3. As described in Section V, MON 87708 produces two forms of DMO. In MON 87708, the DMO trimer can be comprise d of DMO, DMO+27, or a combination of both. Therefore, this document will refer to both forms of the protein and all forms of the

trimer as the MON 87708 DMO. The iden tity of the MON 87708 harvested seed processed to make the defatted soybean flour was confirmed by event-specific polymerase chain reaction (PCR) an d a copy of the verification of identity is archived in the Monsanto Regulatory archives with the records documenting protein isolation. The purified MON 87708 DMO was stored in a -80 oC freezer in a buffer solution containing 50 mM potassium phosphate, pH 8.0, 100 mM NaCl, 1 mM dithiothreitol (DTT) and 5% glycerol. The records describing the purification of the MON 87708 DMO are archived in the Monsanto Regulat ory archives under Orion lot 11261646.

C.1.2. Description of Assay Control

Protein molecular weight standards (SeeBlue ® 

### Plus2 Pre-stained, Invitrogen,) were used to calibrate SDS-PAGE gels and verify pr otein transfer to polyvinylidene difluoride (PVDF) membranes. The broad range SDS-PA GE molecular weight standards (Bio-Rad, Hercules, CA) were used to determine the appa rent molecular weight of both forms of the MON 87708 DMO protein. A peptide mixtur e (SequazymeTM Peptide Mass Standards kit, Applied Biosystems, Foster City, CA) was used to calibrat e the MALDI-TOF mass spectrometer for tryptic mass and intact mass an alysis. Transferrin provided with the kit (GE Healthcare, Piscataway, NJ) was used as a positive control for glycosylation analysis.

C.1.3. MON 87708 DMO Purification

MON 87708 DMO was purified from defatted flour processed from harvested seed of MON 87708. MON 87708 DMO was purified us ing a combination of extraction, filtration and diafiltration, and various chromatographic separations. A brief description of the purification process is below.

Defatting of seed from MON 87708 was comp leted at Pilot Plant Corporation in Saskatoon, Canada. The seed was cracked, dehulled, and gr ound to meal in the presence of dry ice. The meal was then solvent ex tracted, dried, and shi pped to Monsanto and

stored in a -20°C cold room.

Aliquots of the defatted flour we re used as starting material in the purification process. Approximately 7.5 kg of defatted MON 87708 flour were extracted with 75 liters (L) of extraction buffer (25 mM potassium phos phate, pH 7.2, 10 mM MgCl2, 1 mM DTT,Monsanto Company 10-SY-210U 295 of 721 

### 1 mM benzamidine-HCl, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 μ M E-64, and

0.1 μ M bestatin). The extraction was conducte d at room temperature (RT) for 2 hours using a Lightnin ® 

### mixer with slow stirring (Graham Transmissions Inc, Menomonee Falls, WI). The resulting slurry was filtere d using an Ertel Alsop filter press (Kingston, NY) with Die 42 micro media filter pads and a Cuno filter (Hagedorn and Gannon Co., Inc) after the addition of 7.5 kg of diatomaceous earth (5.6 kg fine hy-flo (Celite Corporation, Lompoc, CA) and 1.9 kg Celite 560 coarse (Sigma-Aldrich, St Louis, MO)). The pads on the press were pr e-coated with 1.8 kg of fine hy- flo prior to the filtration of the extract. After washing the press with an additional volume of extraction buffer, the filtrate was collected (f inal volume: 150 L).

The filtrate was then concentrated at RT to 75 L using a hollow fiber cartridge with a 30,000 kDa molecular weight cutoff (MWCO) (G E Healthcare) at RT to remove small

molecules. Solid KCl was added to a final concentration of 0.15 M. The concentrated

filtrate was dialfiltered with four exch anges of 25 L each of a phenyl sepharose equilibration buffer (25 mM potassium phosphate, pH 8.0, 1 mM DTT, 1 mM benzamidine-HCl, 1 mM PMSF, 1 μ M E-64, 0.1 μ M bestatin, and 0.15 M KCl).

The first chromatographic step was perfor med at RT. A 30 L phenyl sepharose (GE Healthcare) column equilibrated with phenyl sepharose equilibration buffer was charged

with the dialfiltered extract and then washed with three column volumes (CV) of the phenyl sepharose equilibration buffer. A single CV of elution buffer (50 mM triethanolamine, pH 8.0, 1 mM D TT, 1 mM benzamidine-HCl, 1 mM PMSF,

1 μ M E-64, 0.1 μ M bestatin, and 100 μ M dicamba) was loaded ont o the column, the flow stopped and the column incubated for 1 hour. Th e released proteins we re eluted with an

additional CV of elution bu ffer and stored at 4°C.

Solid potassium phosphate was added to the phenyl column elution to a final concentration of 25 mM and the pH adjusted to 8.0, followed by the addition of 1 mM DTT, 1 mM benzamidine-HCl, 1 mM PMSF, 1 μ M E-64, and 0.1 μ M bestatin. A 3 L ceramic hydroxyapatite co lumn (CHT) (Bio-Rad) was packed at 4 °C and equilibrated in a buffer containing 25 mM potassium phosphate, pH 8.0, 1 mM DTT, 1 mM benzamidine-HCl, 1 mM PMSF, 1 μ M E-64, 0.1 μ M bestatin, and

100 μ M dicamba. Half of the adjusted phenyl elution was charged on the CHT column.

The column was washed with two CV of the CHT equilibration buffer. The bound proteins were then eluted with 400 mM potassium phosphate, pH 8.0. The flow-through containing the MON 87708 DMO, de tected by immunoblot analys is, was collected. The

eluted fractions without the MON 87708 DMO were discarded and the column was reequilibrated. The second half of the phenyl elution was processed with the CHT column

in the same manner as the first half. Th e flow-through collected from each CHT column

run was combined in to a single pool.

Before charging onto the next column, fresh, solid DTT and protease inhibitors were added to the CHT column flow-through pool. The flow-through pool from the CHT step was then charged on a 5 L DEAE macroprep (Bio-Rad) column at 4°C and equilibrated in a buffer containing 25 mM Tris-HCl, pH 8.0, 1 mM DTT, 1 mM benzamidine-HCl, 1 mM PMSF, 1 μ M E-64, 0.1 μ M bestatin, and 100 μ M dicamba. The DEAE columnMonsanto Company 10-SY-210U 296 of 721 

### was then washed with five CV of the DEAE equilibration buffer followed by five CV of the equilibration buffer plus 100 mM NaCl . The MON 87708 DMO was eluted with a 20 CV linear NaCl gradient from 100 mM to 350 mM in the equi libration buffer. The fractions collected throughout the gradient were analyzed by immunoblot and those fractions containing the M ON 87708 DMO were pooled.

To concentrate the DEAE macroprep pool , it was first diluted with the DEAE equilibration buffer (to reduce the conductiv ity) and then charged onto a 1 L DEAE macroprep column. After charging, the co lumn was washed with three CV of equilibration buffer and then eluted with mini mal volume of the equilibration buffer plus

1 M NaCl. This concentrated the DEAE macroprep pool from 16 to 1.6 L.

The concentrated DEAE macroprep pool was mi xed with 1 L of concanavalin A (Con A) sepharose 4B (Sigma-Aldrich) that was previously equilibrated with the DEAE equilibration buffer with fresh, solid DTT and protease inhibitors added. The purification step was run in batch mode at RT and was intended to remove contaminants that bind to Con A, while not binding MON 87708 DMO. The concentrated DEAE macroprep pool was stirred Con A resin for 1 hour, the resin was filtered out using a Büchner funnel and

Whatman ® 

### filter paper (GE Healthcare). The resin was washed with 3 L of equilibration buffer. All filtrates containing MON 87708 DMO were combined.

The Con A filtrate pool was concentrated on ice for approximately 4 hours using a tangential flow membrane (Sartorius-Stedim, Goe ttingen, Germany) with a

100 kDa MWCO. After a 10× c oncentration step, the rete ntate containing MON 87708

DMO was dialfiltered with 10 volume exch anges of DEAE macr oprep equilibration buffer containing 1 mM DTT, 1 mM benzamidine-HCl, 1 mM PMSF, 1 μ M E-64, and

0.1 μ M bestatin.

The concentrated and dialfiltered Con A pool was further purified on CHT at RT, this time in a binding mode where MON 87708 DM O was bound to the resin. This is achieved in the complete absence of phosphate where MON 87708 DMO binds to the

CHT column and is then eluted. A 1 L CHT column was packed and equilibrated with the DEAE macroprep equilibration buffer with fresh DTT and protease inhibitors. The column was washed with three CV of equilibra tion buffer. The protein was eluted with a linear phosphate gradient using an elution buffer (400 mM potassium phosphate, pH 8.0, 1 mM DTT, 1 mM benzamidine-HCl, 1 mM PMSF, 1 μ M E-64, 0.1 μ M bestatin, and

100 μ M dicamba) increasing from 0% to 50% ov er 10 CV. The fractions were collected and analyzed by SDS-PAGE. Those contai ning at least 80% pure MON 87708 DMO as estimated by gel densitometry were pooled.

This entire purification pro cedure was repeated with two additional batches of 7.5 kg of defatted flour from MON 87708. After analysis , all final CHT pools were combined into a single final pool that was concentrated on i ce for approximately 2 hours to 370 ml with a tangential flow membrane with a 30 kDa MW CO. The concentrated pool was dialyzed against enzyme storage buffer (50 mM potassium phosphate, pH 8.0, 100 mM NaCl, 5% glycerol, and 1 mM DTT). Four liters of storage buffer were used and exchangedMonsanto Company 10-SY-210U 297 of 721 

### twice over two days and the di alysis was conducted at 4°C. The dialysate was aliquoted, assigned APS lot 11261646 and stored at -80°C.

C.1.4. Molecular Weight and Purity Estimation using SDS-PAGE Method

SDS-PAGE analysis was performed to dete rmine the molecular weight and purity of

MON 87708 DMO.

An aliquot of MON 87708 DMO was mixed with 5 × loading buffer (LB) to a final total protein concentration of 0.09 μg /μl and heated at 99°C for three minutes. A molecular weight marker (Broad Range MW Marker, Bio- Rad) was diluted to a final total protein concentration of 0.9 μg/μl. MON 87708 DM O was loaded in duplicate at 0.5, 1.0 and 1.5 μg of total protein per lane onto a pr e-cast Tris glycine 4-20% polyacrylamide gradient 10-well gel (Invitrogen). The molecular weight markers were loaded in parallel at 4.5 μg protein per lane. Electrophoresis was performed at a constant 125 V for 90 minutes. Proteins were fixed by placing th e gel in a solution of 40% (v/v) methanol and 7% (v/v) acetic acid for 30 minutes, stained for 16 hours with Brilliant Blue G Colloidal stain (Sigma-Aldrich), destaine d 30 seconds with a solution containing 10% (v/v) acetic acid and 25% (v/v) methanol, and finally destaine d with 25% (v/v) methanol for 6 hours. Analysis of the gel was performed using a Bio-Rad GS-800 densitometer with the supplied Quantity One ® 

### software (version 4.4.0). Molecular weight markers were used to estimate the apparent molecula r weight of each obser ved band. All visible bands within each lane were quantified using Quantity One software. Apparent molecular weight was obtained for both fo rms of the MON 87708 DMO protein while the purity was calculated based on the addition of the average purity of both proteins. The results were reported as an average of a ll six samples loaded onto the gel containing

MON 87708 DMO.

C.1.5. Immunoblot Analysis Method

Immunoblot analysis was performed to c onfirm the identity of MON 87708 DMO.

An aliquot of MON 87708 DMO was diluted with water and mixed with 5 × LB (312 mM Tris-HCl, 20% (v/v) 2-mercap toethanol, 10% (w/v) SDS, 0.025% (w/v) bromophenol blue, and 50% (v/v) glycerol, pH 6.8), heated at 99°C for 3 minutes, and applied on a pre-cast Tris glycine 4- 20% polyacrylamide gradient 10-well gel (Invitrogen). Three amounts (20, 30, and 40 ng) of MON 87708 DMO were loaded in duplicate on the gel. Electrophoresis was pe rformed at a constant 125 V for 90 minutes. Pre-stained molecular weight markers (SeeBlue ® 

### Plus2 Pre-stained, Invitrogen) were loaded in parallel to verify electrotransfer of the proteins to the membrane and estimate

the size of the immunoreactive bands observe d. Electrotransfer to a 0.45 μm PVDF membrane (Invitrogen) was performed for 90 minutes at a constant 25 V.

For immunodetection, the membrane was bl ocked for 1 hour with 10% (w/v) Non-Fat Dried Milk (NFDM) in 1× Phosphate Buff ered Saline containing 0.05% (v/v) Tween-20 (PBST). The membrane was then probed with a 1:3,000 dilution of goat anti-DMO antibody, which is specific for both forms of the MON 87708 DMO protein, in 5% (w/v)Monsanto Company 10-SY-210U 298 of 721 

### NFDM in PBST for one hour. Excess anti body was removed using three 10 minutes washes with PBST. Finally, the membrane was probed with horseradish peroxidase (HRP)-conjugated rabbit anti-goat IgG (Therm o, Rockford, IL) at a dilution of 1:10,000 in 5% (w/v) NFDM in PBST for 1 hour. Excess HRP-conjugate was removed using three

10 minutes washes with PBST. All incubati ons were performed at RT. Immunoreactive bands were visualized using the ECL (Enha nced Chemiluminescence) detection system (GE Healthcare) and exposed to Amersham Hy perfilm (GE, Healthcare). The film was developed using a Konica SRX-101A automate d film processor (Tokyo, Japan). Three exposures (20, 30, and 60 seconds) were take n and the 20 second exposure was scanned using a Bio-Rad GS-800 densitometer with th e supplied Quantity One software (version 4.4.0).

C.1.6. MALDI-TOF Tryptic Mass Map Analysis Method

MALDI-TOF mass spectrometry was used to co nfirm the identity of both forms of the MON 87708 DMO proteins. The proteins were first separated by SDS-PAGE prior to trypsinization.

An aliquot (89.5 μ l) of MON 87708 DMO was mixed with 22.5 μ l of 5 × LB, heated at 99°C for 3 minutes and loaded in four lanes (three lanes each loaded with 4.2 μ g and one

lane with 3.1 μ g of total protein) onto a pre-cast Tris glycine 4-20% polyacrylamide gradient 10-well gel (Invitrogen). Pre-st ained MW markers (SeeBlue Plus2 Pre-stained, Invitrogen) were loaded in parallel to estim ate the size of the stained bands observed. Electrophoresis was carried out at a c onstant 150 V for 80 minutes. Following electrophoresis, the gel was staine d with Brilliant Blue G Collo idal (Sigma-Aldrich). The bands corresponding to DMO or DMO+27 were excised from four lanes of the gel, destained, reduced, and alkylated. Each gel band was destaine d for 30 minutes by

incubation in 100 μ l of destain solution (40% metha nol, 50% water, and 10% glacial acetic acid) in a microfuge tube. This step was repeated twice for 60 minutes each, removing all visible Brilliant Blue G Colloidal stain. Follo wing destaining, the gel bands

were incubated in 100 μ l per band of 100 mM ammonium bicarbonate buffer for 16 hours at RT. The protein was reduced in 100 μ l of 10 mM DTT solution for two hours at 37 °C. After removing the reducing solution, the protein in the gel was alkylated by incubating in 100 μ l of 20 mM iodoacetic acid. The alkylation reaction was allowed to proceed at RT for 20 minutes in the dark. The gel containing the protein band was

incubated in 200 μ l of 25 mM ammonium bicarbonate buffer for 15 minutes at RT. This step was repeated two additional times for 15 minutes each; then the gel band was dried using a Savant Speed Vac concentrator (H olbrook, NY). Each gel band was rehydrated

with 20 μ l of 0.02 μ g/ μ l trypsin in 25 mM ammonium bicarbonate and 10% acetonitrile, and the incubated for approxi mately one hour at RT. Fo llowing incubation, the excess solution was removed and the gel/trypsin re action mixture was incubated overnight at

37°C in 40 μ l of 25 mM ammonium bicarbonate a nd 10% acetonitrile. The following day, the sample was sonicated for 5 minutes, and the supernatant transferred to a new tube and dried using a Speed Vac concentr ator (Extract 1). The gel band(s) was resuspended in 30 μ l of a solution consisting of 60% acetonitrile, 0.1% trifluoroacetic acid (TFA), and 0.1% octyl- β -D-glucopyranoside, and then sonicated for 5 minutes. After transfer of the supernatant to a new mi crocentrifuge tube, this step was repeatedMonsanto Company 10-SY-210U 299 of 721 

### once and the combined supernatants were dried using the Speed Vac concentrator (Extract 2). Extracts 1 and 2 were separately dissolved in 20 μ l 0.1% TFA and then dried using a Speed Vac concentrator. Fi nally, Extract 1 was dissolved in 5 μ l of 50% acetonitrile/0.1% TFA, while Extract 2 was dissolved in 10 μ l of the same solution. To maximize the solubilization, each sample was sonicated for 5 minutes.

Mass calibration of the mass spectrometer wa s performed using an external peptide mixture (SequazymeTM Peptide Mass Standard s Kit, Calibration Mixture 2, Applied Biosystems). The samples Extract 1 and Extract 2 (0.3 μ l) were co-crystallized with

0.75 μ l each of the following matrix solutions: dihydroxybenzoic acid (DHB), α -cyano-4- hydroxy cinnamic acid ( α -cyano), and 3,5 dimethoxy-4 hydroxycinnamic acid (sinapinic acid) at separate locations on the analysis plate. The samples in DHB matrix were analyzed in the 300 to 5,000 Dalton (Da) range. The samples in α -cyano matrix were analyzed in the 500 to 5,000 Da range. The samples in sinapinic acid matrix were analyzed in the 500 to 7,000 Da range. Protonated (MH+) peptide masses were observed monoisotopically in reflector mode (Aebersold, 1993), except above 3,000 Da, where mass-averaged values were used. GPMAW32 ® 

### software (Lighthous e data, Denmark) was used to generate a theoretical trypsin digestion of the deduced DMO and DMO+27 amino acid sequences. Masses were calculated for each theoretical peptide and compared to the raw experimental mass data. Belo w 1000 Da, experimental masses (MH+) were assigned to peaks when two or more isotopic ally resolved peaks we re observed. Above 1000 Da, experimental masses (MH+) were a ssigned to peaks when three or more isotopically resolved peaks were observed. P eaks were not assessed if the peak heights were less than approximately twice the ba seline noise, or when a mass could not be assigned due to overlap with a stronger mass signal. Known autocatalytic segments from trypsin digestion were identified in the raw data. The list of experimental masses was compared to the theoretical list from the GPMAW software. Those experimental masses

within 1 Da of a theoretical mass were matc hed. All matching masses were tallied and a coverage map was generated. The trypt ic mass map coverage was considered acceptable if ≥ 40% of the protein sequence was id entified by matching experimental masses observed for the tryptic peptide se gments to the expected masses for the

segments.

C.1.7. N-Terminal Sequencing Method

N-terminal sequencing using automated Edma n degradation chemis try (Hunkapiller et al., 1983) was used to confirm the iden tity of both forms of the MON 87708 DMO proteins.

Ninety microliters of MON 87708 DMO were mixed with 22.5 μ l of 5× LB, heated at

99°C for 3 minutes and load ed in four lanes (10 μ l/lane) onto a pre-cast Tris glycine 4-20% polyacrylamide gradient 10-well gel (Invitrogen). El ectrophoresis was carried out at a constant voltage of 150 V for 80 minutes. Proteins in the gel were electrotransferred to a PVDF (Invitrogen) membrane for 90 mi nutes in a buffer containing 10 mM CAPS, pH 11 and 10% methanol at a constant voltage of 25 V. Pre-stained molecular weight markers (SeeBlue Plus2 Pre-stai ned, Invitrogen) were loaded in parallel to verify theMonsanto Company 10-SY-210U 300 of 721 

### electrotransfer of protein to the membrane and estimate the size of the stained bands observed. The blot was stained with Ponceau S (Sigma-Aldrich).

Following electrotransfer and staining, th e bands corresponding to DMO and DMO+27 were excised based on apparent molecular weig ht from the blot and N-terminal sequence analyses were performed for 15 cycles us ing automated Edman degradation chemistry (Hunkapiller et al., 1983). An Applied Bi osystems 494 ProciseTM Protein Sequencing System with 140C Microgradient HPLC pump, ABI 785A Programmable Absorbance Detector and ProciseTM Control Software (ver sion 2.1) were used. Chromatographic data were collected using AtlasTM 2003 software (Thermo Fisher Scientific Inc, Waltham, MA). A PTH (Phenylthiohydantoin) -amino aci d standard mixture (A pplied Biosystems)

was used as the calibration standard in th e chromatographic analysis. This mixture served to verify system suitability criteria such as percent peak resolution and relative amino acid chromatographic retention times. A control protein, 10 pmol β lactoglobulin (Applied Biosystems), was analyzed before an d after the analysis to verify that the sequencer met performance criteria for repetitive yield and sequence identity.

C.1.8. Glycosylation Analysis Method

Glycosylation analysis was used to determ ine whether either form of the MON 87708 DMO proteins were post-translationally m odified with covale ntly bound carbohydrate

moieties.

An aliquot of MON 87708 DMO and the positive control, tr ansferrin (GE Healthcare)

were each diluted with water and mixed with 5 × LB. These samples were heated at 101.0 °C for three minutes, cooled, and loaded on a Tris glycine 4-20% polyacrylamide gradient 10-well mini-gel (Invitrogen). Three amounts of transferrin (50, 100, and 200 ng) and two amounts (100 and 200 ng) of the purity-corrected DMO enzyme was loaded in the gel. SeeBlue ® 

### Plus2 Pre-stained protein molecular weight markers (Invitrogen) were loaded to verify electrot ransfer of the proteins to the membrane. Electrophoresis was performed at a constant 150 V for 87 minutes. Electrotransfer to a 0.45 μm PVDF membrane (Invitrogen) was perfor med for 60 minutes at a constant 25 V, followed by 30 minutes at 30 V.

Carbohydrate detection was performed direct ly on the PVDF membrane using the GE Healthcare Glycosylation De tection Module (Cat. No. RPN 2190). The manufacturer’s protocol was followed and all the reagents except phosphate buffered saline (PBS) were provided with the kit. All steps were perf ormed at RT. Following electrotransfer to PVDF membrane, the blot was incubated in 30 ml of PBS for 10 minutes, followed by

incubation with 20 ml of 10 mM NaIO4 for 20 minutes in the dark. The membrane was

rinsed twice with PBS and washed three times with 20 ml PBS for 10 minutes each. The membrane was incubated with 20 ml solution consisting of 0.125 mM biotin-hydrazide, 100 mM acetate, pH 5.5 for 60 minutes followed by two PBS rinses and three 10 minute

washes with PBS. The membrane was bloc ked for 60 minutes using 5% blocking reagent in PBS followed by two PBS rinses and th ree 10 minute washes with PBS. The membrane was incubated with strepavidin- HRP at 1:6000 dilution for 30 minutes. After

two PBS rinses and three 10 minute washes w ith PBS, the membrane was developed withMonsanto Company 10-SY-210U 301 of 721 

### ECL detection reagents by mixing 1 ml of Reagent 1 and 1 ml of Reagent 2. After one minute incubation, the excess detection solution was removed by blotting with paper towels and the blot was exposed to Hyperf ilm ECL (GE Healthcare ). The film was developed using a Konica SRX-101A automate d film processor (Tokyo, Japan). Three exposures (30 seconds, 1 and 2 minutes) were performed. The image was captured using a Bio-Rad GS-800 densitometer with the supplied Quantity One ® 

### software (version 4.4.0).

C.1.9. Functional Activity Analysis Method

The specific activity of MON 87708 DMO was determined by quantifying the conversion of 3,6-dichloro-2-methoxybenzoic acid (dicamba) to 3,6 dichlorosalicylic acid (DCSA) via HPLC (Agilent Technologies 1100 series, Santa Clara, CA) separation and fluorescence detection (Agilent Technologies 1200 series, G1321A ). The standard assays

were conducted in 200 μ l solutions consisting of 25 mM potassium phosphate, pH 7.2,

3.4 μ g Ferredoxin, 3.4 μ g Reductase, 0.5 mM FeSO4, 10 mM MgCl2, 0.7 mM NADH, 0.3 mM dicamba, 2 μ l (42.48 U/ml) of formaldehyde dehydrogenase and either 2

 μ g MON 87708 DMO or 1 μ g HIS-DMO as an assay positive c ontrol. The reactions were performed in PCR tubes (Sorenson, Salt Lake City, UT) and incubated at 30°C for 15 minutes. Reactions were initiated by the addition of dicamba and quenched with the

addition of 50 μ l of 5% H 2 

### SO 4 

### . Reactions were then filt ered using Whatman Anotop 10 filters (0.2 μ m, GE healthcare), and 40 μ l was transferred to a HPLC sample vial (200 μ l, Agilent) for analysis. Twenty-five microliters of the filtered reaction was injected onto a

Phenomenex ® 

### Synergi 4 μ m C18/ODS Hydro-RP column (150 x 4.6 mm ID, Torrance, CA). The mobile phase consisted of solv ent A (21.5 mM phosphoric acid) and solvent B (100% acetonitrile) running at 1.5 ml/min. DC SA was eluted from the column using a linear gradient from 90% to 40% solvent A fo r the first 14 minutes, followed by a step to 10% solvent A for 1 minute and then re-equi libration at 90% solvent A for 10 minutes before the next injection. DCSA was mon itored by the detection of fluorescent emission at 424 nm (excitation 306 nm) and quantifie d relative to a standa rd curve of DCSA generated using 0.1, 0.3, 0.6, 0.9, 1.2, 2.4, and 4.8 nmol/250 μ l. Chromatographic data were collected using Atlas TM 

### 2003 software (Thermo Fisher Scientific Inc). The specific activity was calculated based on the amount of purity corrected MON 87708 DMO added to the reaction mixture and expressed as nm ol of DCSA produced per minute per mg of MON 87708 DMO (nmol/min/mg).

C.1.10. Results of MON 87708 DMO Proteins Molecular Weight and Purity

The apparent molecular weights of the M ON 87708 DMO proteins were determined by using SDS-PAGE and the gel stained using Brilliant Blue G Colloidal stain (SigmaAldrich). Purity and apparent molecular weight of DMO and DM O+27 were determined using densitometric analysis of the gel (F igure C-1). As summarized in Table C-1, apparent molecular weight values were av eraged from duplicated loads of 0.5, 1.0, and

1.5 μ g of total protein (Figure C-1, lanes 2- 7). The predominant bands identified as DMO and DMO+27 were estimated to have ap parent molecular weights of 39.8 kDa and 42.0 kDa, respectively. The average purity of the combined DMO proteins was 81%.Monsanto Company 10-SY-210U 302 of 721 

### Table C-1. Molecular Weight and Pu rity of the MON 87708-Produced DMO

Proteins

Total Protein

Loaded Apparent Molecular Weight (kDa) Purity (%)

DMO DMO+27 DMO DMO+27 DMO

Proteins

0.5  g in lane 3

0.5  g in lane 4

1.0  g in lane 5

1.0  g in lane 6

1.5  g in lane 7

1.5  g in lane 8

Average 











 











 











 











 

The apparent molecular weight and the purity of DMO and DMO+27 were determined by densitometric analysis of SDS PAGE shown in Figure C-1. Final molecular weight was rounded to one decimal place and purity was re ported as a whole number percentage. Purity of the MON 87708 DMO proteins equa ls the average purity of DMO plus the average purity of DMO+27.Monsanto Company 10-SY-210U 303 of 721 

### Figure C-1. Molecular Weight and Puri ty Analysis of MON 87708 DMO Proteins An aliquot of MON 87708 DMO was separated on a 4 - 20% Tris glycine polyacrylamide gradient gel and then staine d with Brilliant Blue G Collo idal stain. Approximate apparent molecular weights (kDa) are shown on the left and correspond to the markers loaded in Lanes 1 and 8. Empty lanes were cropped.

Lane Sample Amount (  g) 1 Broad Range MW markers 4.5 2 MON 87708 DMO proteins 0.5 3 MON 87708 DMO proteins 0.5 4 MON 87708 DMO proteins 1.0 5 MON 87708 DMO proteins 1.0 6 MON 87708 DMO proteins 1.5 7 MON 87708 DMO proteins 1.5 8 Broad Range MW markers 4.5Monsanto Company 10-SY-210U 304 of 721 

### C.11. Results of Immunoblot Analysis

On the immunoblot, the goat anti-DMO antibod y recognized two bands migrating at the expected apparent molecular weights of approximately 39.8 kDa (DMO) and 42.0 kDa (DMO+27), respectively (Figure C-2). As e xpected, the intensity of the immunoreactive bands increased with increasing amount of to tal protein loaded. No additional bands

were observed. This immunoblot analysis confirmed the identity of both forms of the MON 87708 DMO protein.

Figure C-2. Immunoblot Analys is of MON 87708 DMO Proteins An aliquot of MON 87708 DMO and molecula r weight markers were separated by SDSPAGE and electrotransferred to a PVDF memb rane. The membrane was incubated with goat anti-DMO antibody and immunoreactive bands were visualized using an ECL system. Approximate MWs (kDa) are shown on the left and correspond to the markers

loaded in Lane 1. The 20 second exposure is shown.

Lane Sample Amount (ng)

1 See Blue Plus2 Pre-Stained MW markers

 2 empty 3 MON 87708 DMO proteins 20 4 MON 87708 DMO proteins 20 5 empty 6 MON 87708 DMO proteins 30 7 MON 87708 DMO proteins 30 8 empty 9 MON 87708 DMO proteins 40 10 MON 87708 DMO proteins 40Monsanto Company 10-SY-210U 305 of 721 

### C.12. Results of MALDI-TOF Tryptic Mass Map Analysis

The identity of both forms of the MON 87708 DMO proteins was confirmed by tryptic mapping using MALDI-TOF MS analysis of the fragments. Prior to analysis, DMO and DMO+27 were separated by SDS-PAGE, reduced, alkylated and digested with trypsin. The ability to identify a protein using this method is dependent upon matching a sufficient number of observed tryptic pept ide fragment masses w ith predicted tryptic peptide fragment masses. In general, prot ein 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 pep tides (Jensen et al., 1997).

There were 26 unique peptides identified fr om DMO that corresponded to the expected masses of the DMO trypsin-digested peptid es while 29 fragments from DMO+27 were found to match the expected pe ptides (Tables C-2 and C-3, respectively). The identified peptides were used to assemble a coverage map indicating the matched peptide sequences for the entire DMO and DMO+27 protein sequences, resulting in 77.4% (263/340) and 82.0% (301/367) coverage of the amino acid sequence, respectively (Figures C-3 and C-4). N-terminal peptides of both DMO and DMO+27 were identified and consistent with the N-terminal sequencing data that determined the N-terminal methionine was missing in DMO and methylated in DMO+27 (Tables C-2 and C-3; Figures C-3 and C-4). These results confirm the identity of both forms of the MON 87708 DMO proteins.Monsanto Company 10-SY-210U 306 of 721Table C-2. Summary of the Tryptic Masses Identifiedfor the MON 87708 DMO Protein Using MALDI-TOF MSMatrixExpectedMass1Difference2AAposition3Fragment-CyanoDHB Sinapinic acidExt.1Ext.2 Ext.1 Ext.2 Ext.1 Ext.2593.61720.67833.78856.77914.891030.961108.931276.171287.141429.181507.271578.241745.421762.481994.652143.782294.972398.862581.872700.08593.61720.68833.80856.78914.911030.971108.951276.201429.201507.271578.271745.511762.481994.672143.842398.77391.34435.38593.51720.60833.74856.72914.841030.921108.891171.081276.191287.191429.231502.351507.321578.321745.591762.621994.762143.94331.20435.38593.53720.60833.77856.75914.881030.921108.941276.211429.261502.371578.331276.191429.201502.341578.301745.561762.541994.782143.962294.932399.152582.222700.314218.4742143.95331.22391.18435.27593.34720.37833.45856.43914.531030.571108.501170.631275.731286.701428.691501.791506.731577.731744.931761.901994.032143.122294.092398.082582.342699.254217.7740.020.160.110.270.300.330.340.360.390.430.450.440.440.490.560.540.510.490.580.620.660.880.780.470.830.70304-305293-295206-2082-6131-13699-105242-248296-303284-292167-176194-20526-36293-303209-221180-193167-179270-283225-24137-52150-1667-25306-326249-269225-248106-13099-136RREDKFLRATFVRVDPAYRSFPVVEREQSIHSRVVVEAIERSWQAQALVKANAQTDAFDRIPGGTPSVLMAKTILDTPLALYREDKVVVEAIERGANTPVDAWNDIREVIVGDGEIQALMKANAQTDAFDRLERNFGIDDPEMDGVLRVSAMLNFIAVAPEGTPKQPDGVVAALLDICPHRLLVDNLMDLGHAQYVHRNAWYVAALPEELSEKPLGRAYVEANGIRPAMLSCDEAAVRGTHILTPETEASCHYFFGSSRVSAMLNFIAVAPEGTPKEQSIHSRDALIWICPGDPALADPGAIPDFGCRSFPVVERDALIWICPGDPALADPGAIPDFGCRVDPAYR1Only experimental masses that matched expected masses are listed in the table.2The number represents the difference between the expected mass and the first column, which has the corresponding numbers.3AA position refers to amino acid position within the predicted DMO sequence as depicted in Figure C-3.4Mass average.Monsanto Company 10-SY-210U 307 of 721Table C-3. Summary of the Tryptic Masses Identifiedfor the MON 87708 DMO+27 Protein Using MALDI-TOF MSMatrixExpectedMass1Difference2AAposition3Fragment-CyanoDHB Sinapinic acidExt.1Ext.2 Ext.1 Ext.2 Ext.1 Ext.2720.55795.61833.65856.64914.741030.801069.781108.751275.971286.951428.951507.011565.131578.021693.261745.171762.171994.342143.46720.64795.71833.73856.721030.891069.921108.901276.121429.101507.181565.301578.141693.381745.361762.371994.552143.632398.72435.30720.47795.54833.59856.58914.691030.751108.711275.981286.971429.001470.931507.031565.221578.061693.291745.281762.291994.482143.572398.492699.73331.19720.55795.66833.69856.641170.981276.141429.261565.341578.291745.512143.981275.971428.931469.941502.101578.041745.221994.422143.572294.622398.522581.782699.844215.70331.22435.27720.37795.42833.45856.43914.531030.571069.571108.501170.661275.731286.701428.691470.631501.791506.731564.871577.731692.971744.931761.901994.032143.122294.092398.082582.342699.254215.030.030.030.180.190.200.210.210.230.210.250.320.240.250.260.300.310.280.260.290.290.240.270.310.340.530.640.560.480.67331-332233-235158-16327-33126-132269-275323-330311-3191-9194-203221-23253-63320-330236-248164-176207-220194-20611-23297-31010-23252-26864-79177-19334-52333-353276-296252-275133-157126-163RRFLRVDPAYRAMATFVRSFPVVEREQSIHSRVVVEAIERSWQAQALVKM*QVWPPIGKANAQTDAFDRIPGGTPSVLMAKTILDTPLALYREDKVVVEAIERGANTPVDAWNDIRTVGGYGHVDCNYKEVIVGDGEIQALMKANAQTDAFDRLERKFETLSYLPPLTRNFGIDDPEMDGVLRKKFETLSYLPPLTRVSAMLNFIAVAPEGTPKQPDGVVAALLDICPHRLLVDNLMDLGHAQYVHRNAWYVAALPEELSEKPLGRAYVEANGIRPAMLSCDEAAVRGTHILTPETEASCHYFFGSSRVSAMLNFIAVAPEGTPKEQSIHSRDALIWICPGDPALADPGAIPDFGCRSFPVVERDALIWICPGDPALADPGAIPDFGCRVDPAYR1Only experimental masses that matched expected masses are listed in the table.2The number represents the difference betweenthe expected mass and the first column, which has the corresponding numbers.3AA position refers to amino acid position within the predicted DMO+27 sequence as depicted in Figure C-4.*Methylated methionine.Monsanto Company 10-SY-210U 308 of 721 

### Figure C-3. MALDI-TOF MS Coverage Map of the MON 87708 DMO Protein The amino acid sequence of DMO was deduced from the dmo coding region present in MON 87708. Boxed regions correspond to tryptic peptides that were identified from DMO using MALDI-TOF MS. In total, 77.4% (263 of 340 total amino acids) of the expected protein sequence was identified.



### Figure C-4. MALDI-TOF MS Coverage Map of the MON 87708 DMO+27 Protein The amino acid sequence of DMO+27 was deduced from the dmo coding region and RbcS present in MON 87708. Boxed regions correspond to tryptic peptides that were identified from DMO+27 using MALDI-TOF MS. In total, 82.0% (301 of 367 total amino acids) of the expected protein sequence was identified.



### 001 MATFVRNAWY VAALPEELSE KPLGRTILDT PLALYRQPDG VVAALLDICP

051 HRFAPLSDGI LVNGHLQCPY HGLEFDGGGQ CVHNPHGNGA RPASLNVRSF

101 PVVERDALIW ICPGDPALAD PGAIPDFGCR VDPAYRTVGG YGHVDCNYKL

151 LVDNLMDLGH AQYVHRANAQ TDAFDRLERE VIVGDGEIQA LMKIPGGTPS

201 VLMAKFLRGA NTPVDAWNDI RWNKVSAMLN FIAVAPEGTP KEQSIHSRGT

251 HILTPETEAS CHYFFGSSRN FGIDDPEMDG VLRSWQAQAL VKEDKVVVEA

301 IERRRAYVEA NGIRPAMLSC DEAAVRVSRE IEKLEQLEAA



### 001 MQVWPPIGKK KFETLSYLPP LTRDSRAMAT FVRNAWYVAA LPEELSEKPL

051 GRTILDTPLA LYRQPDGVVA ALLDICPHRF APLSDGILVN GHLQCPYHGL

101 EFDGGGQCVH NPHGNGARPA SLNVRSFPVV ERDALIWICP GDPALADPGA

151 IPDFGCRVDP AYRTVGGYGH VDCNYKLLVD NLMDLGHAQY VHRANAQTDA

201 FDRLEREVIV GDGEIQALMK IPGGTPSVLM AKFLRGANTP VDAWNDIRWN

251 KVSAMLNFIA VAPEGTPKEQ SIHSRGTHIL TPETEASCHY FFGSSRNFGI

301 DDPEMDGVLR SWQAQALVKE DKVVVEAIER RRAYVEANGI RPAMLSCDEA

351 AVRVSREIEK LEQLEAAMonsanto Company 10-SY-210U 309 of 721 

### C.13. Results of N-terminal Sequencing

N-terminal sequence analysis of the two major protein bands on the PVDF membrane (Figure C-2) returned a seque nce of 15 amino acids per band that matched the expected N-terminal sequences of DMO and DMO+27 (F igures C-5 and C-6, respectively), which

were deduced from the dmo coding region present in the seed of MON 87708 (Section IV). The N-terminal methionine residue in DMO was not observed, indicating that it was removed during pos ttranslational processing of the protein. Removal of the N-terminal methionine occurs through meth ionine aminopeptidase (Arfin and Bradshaw, 1988; Bradshaw et al., 1998; Polevoda and Sherman, 2000) and is common in many organisms.

In the case of DMO+27, the first cycle of N-terminal sequence analysis resulted in a PTH-amino acid derivative that corresponds to a methylated modification of the

N-terminal methionine. It is well-known th at the amino-terminal methionine of the Rubisco small subunit is pos t-translationally modified to N-methyl-methionine in vivo in pea and other plant species (Grimm et al., 1997; Whitney and Andrews, 2001).

The N-terminal sequencing results clearly c onfirm the identity of the DMO and DMO+27

isolated from the seed of MON 87708.Monsanto Company 10-SY-210U 310 of 721 

### Figure C-5. N-Terminal Sequence of the MON 87708 DMO Protein The expected amino acid sequence of the N-terminus of DMO was deduced from the dmo coding region present in MON 87708. The experimental sequence obtained from DMO was compared to the expected sequence. (-) indicates the residue was not observed.



### Figure C-6. N-Terminal Sequence of the MON 87708 DMO+27 Protein The expected amino acid sequence of the N-terminus of DMO+27 was deduced from the dmo coding region present in MON 87708. The expe rimental sequence obtained from DMO+27 was compared to the expected sequen ce. M*, methylated methionine. 

### Amino acid

residue # from

the N-terminus → 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 

### Expected Sequence M A T F V R N A W Y V A A L P E | | | | | | | | | | | | | | | |
Experimental Sequence

- A T F V R N A W Y V A A L P E

Amino acid

residue # from

the N-terminus → 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 

### Expected Sequence M Q V W P P I G K K K F E T L | | | | | | | | | | | | | | |
Experimental Sequence M * Q V W P P I G K K K F E T LMonsanto Company 10-SY-210U 311 of 721 

### C.1.14. Results of Glycosylation Analysis

To test whether DMO or DM O+27 was glycosylated when expressed in the seed of MON 87708, the MON 87708 DMO prot eins were analyzed for glycosylation using a GE Glycoprotein Detection Module (GE healthcare). Transferrin, a naturally glycosylated protein, was used as a positive control in the assay. The results of this analysis are presented in Figure C-7. The positive control was clearly detected at the expected molecular weight and the bands increase d with increasing protein concentration (Figure C-7, lanes 2-4). No bands were obs erved for DMO or DMO+27 at their expected molecular weight positions (39.8 and 42.0 kDa) (Figure C-7, lanes 5 and 6) indicating

that both forms of the DMO protein are not glycosylated.

Figure C-7. Glycosylation Analysis of the MON 87708 DMO Proteins Molecular weight markers, transferrin (positive control) and an aliquot of the MON 87708 DMO were separated by SDS-PAGE (4 - 20%) and el ectrotransferred to a PVDF membrane. The image was captured using a Bio-Rad GS800 with Quantity One software (version 4.4.0). The 30 second exposure is shown.



### Lane Sample Amount (ng)

1 See Blue Plus2 Pre-Stained MW markers

 2 Transferrin 50

3 Transferrin 100

4 Transferrin 200 5 MON 87708 DMO proteins 100 6 MON 87708 DMO proteins 200 7 Empty lane 8 Empty lane Lane # 1 2 3 4 5 6 7 8

kDa

250

148

98

64

50

36

22

16

6

4 Expected position

for DMO+27/DMO 

### TransferrinMonsanto Company 10-SY-210U 312 of 721 

### C.15. Results of Functional Activity

MON 87708 DMO activity was determined by measuring the production of DCSA. The specific activity was 62.21 nmol/min/mg of MON 87708 DMO (Table C-4). The value represents an average of three independent assays. This result demonstrates that

MON 87708 DMO isolated from the seed of MON 87708 is f unctionally active.

Table C-4. MON 87708 DMO Functional Activity Assay

Assay# Specific activity (DCSA nmol/min/mg) Average (nmol/min/mg)

±Standard Deviation

1

2

3 61.92

51.33

73.39 62.21 ± 11.03

C.2. Substrate Specificity of DMO

C.2.1. Materials

MON 87708 (lot 11225299-114) and the near isogenic conventional soybean control A3525 (lot 11225301-104) were used for the exogenous specificity greenhouse tests conducted in 2009 and 2010. At the V2-V3 growth stage, MON 87708 and the conventional control plants were sprayed with different herbicides. The herbicides tested

are listed in Table C-5.

The DMO protein used in the e ndogenous and exogenous specificity in vitro experiments was generated in Escherichia coli with a histidine-tag at the N-terminus and has an identical amino acid sequence to MON 87708 DMO with the exception of a single amino acid change at position 112 (W112C) and the l ack of alanine at the second position. The compounds tested and standards used in the in vitro experiments are listed in Table C-6.Monsanto Company 10-SY-210U 313 of 721 

### Table C-5. Herbicides Tested in Exoge nous Specificity Herbicide Tolerance

Greenhouse Trials

Manufacturer/

Retailer Chemistry/ Compound Formulation Lot Number

Agriliance MCPA MCPA L. V.Ester 4 RUD-0102-11027-F

Albaugh 2,4-DB Butyrac ® 200 HPR-0404-14987-F

BASF dicamba Clarity ® KIH-0702-18134-F

BASF imazapyr Arsenal ® KIH-0408-15423-F

Dow picloram Tordon ® 22K ABR-9912-99121-F

Dow clopyralid Stinger ® AGD-0104-11295

Dow triclopyr Garlon ® 3A AGD-0205-12641

Dow atrazine Atrazine ® AGT-0804-19336-F

Dow trifluralin Treflan ® MB231656T7

Dow oxyfluorfen Goal ® 2XL EWP-0107-11628-F

DuPont chlorimuron Classic ® MPO-9304-19319

DuPont chlorsulfuron Glean ® MPO-9910-9869

DuPont linuron Lorox ® NIF-0103-1173-F

Helena 2,4-D 2,4-D Amine 4 RUD-0502-15805-F

Monsanto alachlor Lasso ® MUS-0905-19849-F

Monsanto acetochlor Harness ® MUS-0704-18520-F

Monsanto glyphosate Roundup WeatherMax ® MUS-0905-19887-F

Monsanto halosulfuron Permit ® MUS-0405-15154-F

Syngenta paraquat Gramoxone ® GTA-0606-17421

Valent lactofen Cobra ® LVT-0905-19884-F

® Roundup WeatherMax is a trademark of Monsanto Technology LLC. All other trademarks are the property of their respective owners.Monsanto Company 10-SY-210U 314 of 721 

### Table C-6. Compounds Used in Specificity In Vitro Experiments

C.2.2. Exogenous Specificity Herb icide Tolerance Greenhouse Method

MON 87708 and the conventional control were planted in pots containing Redi-earth ®



### and Osmocote ® 

### 14-14-14 slow release fertilizer or Peters ® 

### 20-20-20 fertilizer. There were ten replicate pots each (MON 87708 and th e conventional control) with one plant per replicate. The pots were placed in a greenhouse and grown under normal agronomic conditions for soybean (relative hu midity 10-70%, temperature 21-29  ̊ C, 14 hour photoperiod, and watering as needed). When th e plants were at the V2-V3 growth stage, the replicates were sprayed with an he rbicide (10 replicates of MON 87708 and the conventional control per each treatment of ea ch tested herbicide; Table C-5). Two different application rates of each herbicide were applied to different replicate sets. Twenty to 21 days after treatment, all plants were rated for percent of injury. Ratings

were based on visual assessment of chloro sis, necrosis, malformation, stunting, and biomass reduction with 100% equaling comple tely dead and 0% equaling no visual adverse effects. All ten repl icate ratings were averaged.

C.2.3. In Vitro Specificity Experiments Enzyma tic Reaction Mixture Method

The reaction of E. coli -produced DMO with different compounds evaluated as potential

substrates was carried out using similar reaction co nditions described in the characterization portion of this appendix (Appendix C.1.9.). The compounds (Table C-6)

were combined with E. coli -produced DMO at 0.2 and/or 0.012 mM. The concentrations tested ensured adequate reaction conditions in terms of the substrate for the detection of product formation or disappearance of substrate. Manufacturer/

Retailer Compound Common

Name Lot/Product

Number

Compounds Tested:

Aldrich 2-methoxybenzoic acid o-anisic acid A0230443

Chem Service 3,6-dichloro-2-methoxybenzoic acid dicamba 341-9143

Sigma 2,4-dichlorophenoxyacetic acid 2,4-D D7299-100G

Sigma 3-(4-hydroxy-3,5- dimethoxyphenyl)prop-2-enoic acid sinapic acid D7927-1G

Fluka 3,5-dimethoxy-4-hydroxybenzoic acid syringic acid 86230

Fluka 4-hydroxy-3-methoxybenzoic acid vanillic acid 94770

Fluka 3-(4-hydroxy-3-methoxy-phenyl)prop2-enoic acid ferulic acid 46278

Compounds Used as Standards:

Monsanto 3,6-dichlorosalicylic acid DCSA GLP-0603-16959-T

Riedel-de Haen 2,4-dichlorophenol 2,4-DCP 35811Monsanto Company 10-SY-210U 315 of 721 

### C.2.4. In Vitro Experiments Liquid Chromatography Separation Method

The reaction mixture was separated by U ltra Performance Liquid Chromatography (UPLC) using an ACQUITY UPLC BEH C18 Column containing 1.7 μm Bridged Ethyl Hybrid (BEH) particles and an ACQUITY BEH C18 VanGuard Pre-column. The

column was heated to 40°C. The tested s ubstrates and potential oxidative by-products were monitored by ACQUITY UPLC photodiode array (PDA) with wavelength range

from 200nm to 320nm with 1.2nm resolution. The chromatography was performed at

0.25ml/min and directed to th e mass spectrometer following the separation. Both mobile phase A (water) and solvent B (acetonitrile) contained 0.1% v/v formic acid. Gradients used were substrate specific:

 The gradient for dicamba was run from 40 to 50% solvent B in 3min, 50 to 100% solvent B in 0.1 min and then kept at 100% solvent B for 1min before returning to

40% solvent B in 0.1 min.

 The gradient for 2,4-D was run from 40 to 45% solvent B in 6min, held at 45% solvent B for 1min, 45 to 100% solvent B in 0.1 min, and then held at 100% solvent B for 0.5 min before returning to 40% solvent B in 0.1 min.

 The gradient for ferulic acid, o-anisic aci d, sinapic acid, syringic acid, and vanillic

acid were run from 0 to 100% solvent B in 4 min and then held at 100% solvent B for 1 min before returning to 0% solvent B in 0.1 min.

Five microliters injection of each sample was used for UPLC analysis where the disappearance of the potential s ubstrate was monitored, and a 50 μ l injection was used for UPLC analysis where formation of potent ial oxidative by-products was monitored.

C.2.5. In Vitro Experiments Mass Spectrometry Detection Method

Elution from the UPLC column (C.2.4) flowed directly to a Wate rs Micro Q-TOF mass spectrometer. The parameters used for th e mass determination were: negative mode, capillary voltage of 2800 V, sample cone vol tage of 26 V for all analytes with the exception of 2,4-D and 2,4-DCP, which was 10 V. The extraction cone was 1.5 V. The source temperature was 150 °C and the de solvation temperature was 390 °C. The desolvation gas flow was 500 L/hour. Scan time was 0.76 seconds a nd inter scan delay was 0.1 seconds. The m/z range used was specific to each substrate and product. The m/z range for dicamba and DCSA was from 16 0 to 225 from 0 to 4 minutes. The m/z at 175, which is the fragment ion of dicamba, wa s used as a detection method for dicamba. This fragment ion of dicamba gave better sens itivity, than the parent ion. The m/z at 205

or 207was used to detect DCSA. The m/z range for 2,4-D and 2,4-DCP was from 160- 164 or 160-225 dependent on the specific experi ment from 0 to 6 minutes. The m/z range for all other acids is fr om 120 to 230 within 4 minutes.Monsanto Company 10-SY-210U 316 of 721 

### C.2.6. Results of Herbicide Tolerance Greenhouse Trials

MON 87708 plants demonstrated similar levels of tolerance as th e conventional control

for 16 of the 19 herbicides te sted (Table C-7). When 2,4-D, 2,4-DB, and MCPA were applied, the MON 87708 plants showed higher to lerance (expressed as injury rating in Table C-8) than the conventional control. Based on these results 2,4-D was further examined as a potential substrate for DMO in in vitro experiments. 2,4-D was selected as representative of the three a uxin herbicides (2,4-D, 2,4-DB, and MCPA) as it is the most structurally similar to dicamba.

C.2.7. Results of In Vitro Experiments with 2,4-D

The reaction of dicamba with E. coli -produced DMO has been well characterized utilizing an in vitro enzymatic assay that monitors the formation of DCSA by LC-MS, which allows for the detection of the produc t with high sensitivity. Both the substrate and reaction products can be detected by LC-UV and LC-MS after separation by UPLC (Figure C-8).

The same enzymatic assay using LC-MS as the detection method was used to look for the potential conversion of 2,4-D to 2,4-DCP by E. coli -produced DMO. 2,4-DCP is predicted to be the product formed from the possible oxidative reaction of 2,4-D and DMO, based on the mechanism of action fo r dicamba conversion to DCSA by DMO. Both the amount of substrate and product were monitored in reactions using dicamba and 2,4-D as the substrate (Figure C-9). Using dicamba as a substrate, the formation of DCSA is clearly observe d in the presence of E. coli -produced DMO (Figure C-9A). When 2,4-D is utilized as a possible substr ate, there is no signi ficant formation of product, as minimal amounts of 2,4-D CP are observed with and without E. coli -produced

DMO as well as in a substrat e only control (Figure C-9B).

C.2.8. Results of In Vitro Experiments with Endogenous Soybean Compounds

Compounds structurally similar to dicamba a nd found in soybean were used as potential substrates to determine if these compounds could be metabolized by DMO (Table C-6). The compounds tested were: syringic acid, o- anisic acid, vanillic acid, ferulic acid, and sinapic acid. Mass spectrometry scans were taken from 120 m/z to 250 m/z to cover the range of all potential oxidatio n products formed by DMO. Standard reaction conditions

of dicamba with E. coli -produced DMO were used as a positive control. LC-MS data demonstrated that there are no additional p eaks formed when reactions of each compound

incubated with E. coli -produced DMO and without E. coli -produced DMO are compared (Figure C-10) (dicamba m/z 205, 2, 4-D m/z 163, ferulic acid m/z 175, o-anisic acid m/z 137, sinapic acid m/z 209, syringic acid m/z 183, and vanillic acid m/z 153). There were no peaks observed at the resp ective masses for the predicte d reaction products of each compound incubated with E. coli -produced DMO, indicating these compounds are not catabolized by DMO.Monsanto Company 10-SY-210U 317 of 721Table C-7. Herbicide Tolerance Trials Injury RatingsInjuryObservations(days afterapplication)Injury ratings (%)2Formulation ManufacturerActiveIngredientLabeled Rate Range(g/ha)1Rates Applied(g/ha)1Control3Average(Range)MON 877084Average(Range)Clarity BASF dicamba 0.13-2.00 (a.e.)0.50 (a.e.)1.00 (a.e.) 20100a100a0a1.8 (0-5)2,4-D Amine 4 Helena 2,4-D 0.13-2.00 (a.e.)0.25 (a.e.)0.50 (a.e.) 2096 (90-100)98 (90-100)59 (50-70)70 (60-80)Butyrac 200 Albaugh 2,4-DB 0.12-1.50 (a.e.)0.25 (a.e.)0.50 (a.e.) 2092 (85-100)93 (85-100)52 (40-65)61 (50-70)MCPA L.V.Ester 4 Agriliance MCPA 0.12-1.50 (a.e.)0.25 (a.e.)0.50 (a.e.) 2095 (90-100)97 (95-100)61 (30-80)82 (65-95)Stinger Dow clopyralid 0.09-0.49 (a.e.)0.25 (a.e.)0.50 (a.e.) 2098 (95-100)100a95 (90-100)100aGarlon 3A Dow triclopyr 0.25-9.01 (a.e.)0.25 (a.e.)0.50 (a.e.) 2097 (90-100)99 (95-100)98 (95-100)99 (95-100)Tordon 22K Dow picloram 0.06-1.00 (a.e.)0.25 (a.e.)0.50 (a.e.) 2098 (95-100)100 (95-100)98 (90-100)100 (99-100Classic DuPont chlorimuron 0.02-0.08 (a.i.)0.07 (a.i.)0.18 (a.i.) 2088 (85-98)90 (85-98)88 (85-90)89 (85-90)Glean DuPont chlorsulfuron 0.008-0.046 (a.i.)0.07 (a.i.)0.18 (a.i.) 2089 (85-95)94 (90-98)89 (85-95)93 (85-98)Gramoxone Syngenta paraquat 0.25-1.00 (a.e.)0.50 (a.e.)0.75 (a.e.) 2098 (95-99)99 (98-99)97 (95-98)98 (95-99)Lasso Monsanto alachlor 0.15-4.00 (a.i.)4.00 (a.i.)6.01 (a.i.) 2121 (10-35)45 (30-60)19 (15-30)34 (20-50)Harness Monsanto acetochlor 0.83-4.00 (a.i.)4.00 (a.i.)6.01 (a.i.) 2171 (50-85)92 (80-98)88 (60-98)97 (90-100)Monsanto Company 10-SY-210U 318 of 721Table C-7 (continued). HerbicideTolerance Trials Injury RatingsFormulation ManufacturerActiveIngredientLabeled Rate Range(lb/acre)1Rates Applied(lb/acre)1InjuryObservations(days afterapplication)Injury ratings (%)2Control3Average(Range)MON 877084Average(Range)Atrazine®Dow atrazine 0.98-3.39 (a.i.)1.50 (a.i.)3.00 (a.i.) 2180 (50-100)97 (85-100)83 (40-100)98 (90-100)Lorox®DuPont linuron 0.25-2.50 (a.i.)4.00 (a.i.)6.01 (a.i.) 2158 (30-100)70 (45-100)55 (35-100)79 (40-100)Treflan®Dow trifluralin 0.50-2.00 (a.i.)4.00 (a.i.)6.01 (a.i.) 219 (5-15)11 (5-15)8 (5-15)10 (5-15)RoundupWeatherMax®Monsanto glyphosate 0.25-3.72 (a.e.)0.07 (a.e.)0.21 (a.e.) 2147 (40-55)64 (60-70)48 (35-55)62 (55-70)Goal®2XL Dow oxyfluorfen 0.25-2.00 (a.i.)0.50 (a.i.)0.75 (a.i.) 2154 (40-70)67 (50-80)56 (40-70)69 (65-80)Cobra® Valent lactofen 0.06-0.39 (a.i.)0.50 (a.i.)0.75 (a.i.) 2133 (25-50)41 (25-50)34 (25-50)45 (35-60)Arsenal® BASF imazapyr0.21-1.50 (a.i.)0.07 (a.i.)0.18 (a.i.) 2158 (50-65)67 (60-75)60 (55-70)66 (55-75)Permit® Monsanto halosulfuron 0.032-0.13 (a.i.)0.07 (a.i.)0.18 (a.i.) 2143 (30-50)52 (40-60)50 (45-60)55 (45-60)1a.e. = acid equivalent; a.i. = active ingredient. Each herbicidecontains the active ingredient directly or the salt form of the active ingredient. Whendetermining the rate ofapplication, the salt form is calculated back to the acid that is the active ingredient and therefore called acid equivalent.Each labeled rate is for cerealor/and broad acre row cropssince these herbicides are not labeled to be sprayed on soybeansor are labeled for soybeans only as a pre-plant treatment. Based on the labeled rates, the rates for the experimentswere chosen and then adjusted for use in-crop on soybeans and for the optimal growingconditions in the greenhouse.2Injury ratings were determined by visualinspection of each plant. Ratings were based on visual assessment of chlorosis, necrosis, malformation, stunting,and biomass reduction.100 percent = completely dead and 0 percent = no visual adverse effects.3Control plants were near isogenic conventional soybean control A3525. Reported average and range of 10 replicate plants.4Reported average and range of 10 replicate plants.Monsanto Company 10-SY-210U 319 of 721Figure C-8. UPLC separation of dicamba (DCB) and DCSADicamba and DCSA were separated by UPLC and detected by UV absorbance using a Photo Diode Array (PDA) and mass spectrometry (MS).Monsanto Company 10-SY-210U 320 of 721Figure C-9.E. coli-produced DMO Conversion of Dicamba (DCB) toDCSA and 2,4-D to 2,4-Dichlorophenol (2,4-DCP)The formation of DCSA from dicamba (Panel A) and 2,4-DCP from 2,4-D (Panel B) due toE. coli-producedDMO were determined. The scaleon the lefthand side of each graph represents the concentrationof the potential substrate, and the scale on the righthand sideof each graphrepresents the concentration of the predicted oxidative product.Each bar represents a replicate assay. Substrate only refersto either DCB or2,4-D in the 25 mM KPi, 10 mM MgCl2 buffer. The concentration of the dicamba or 2,4-D used in each experiment was 12μM.Monsanto Company 10-SY-210U 321 of 721Figure C-10.E. coli-produced DMO Conversion of Endogenous SubstratesEndogenous substrates, as well as dicamba and 2,4-D, were incubated withE. coli-produced DMO and the formation of products anddisappearance of substrate was monitored byLC-MS (top two chromatograms) and LC-UV(bottom to chromatograms) for a positive control(dicamba (a)), (2,4-D (b)) and each endogenouscompound:, sinapic acid(c), ferulic acid (d), anisic acid (e), syringic acid (f), and vanillic acid (g).For each experiment the reaction mixture was made with (+E. coli-produced DMO, upper) and without (-E. coli-produced DMO, lower). The redline indicates the migration of the substrates (and DCSA in the case of dicamba) in each chromatogram.8,107,108 G4 Vanillic2.50 2.75 3.00 3.25 3.50 3.75

%01002.50 2.75 3.00 3.25 3.50 3.75

%0100Dicamba-02-12-09-11261: TOF MS ES1533.14e33.733.572.772.483.443.283.172.96Dicamba-02-12-09-11301: TOF MS ES1533.66e32.483.783.613.492.772.99Time2.50 2.75 3.00 3.25 3.50 3.75

AU0.02.0e+22.50 2.75 3.00 3.25 3.50 3.75

AU0.02.0e+2Dicamba-02-12-09-11263: Diode ArrayRange: 2.491e+22.68Dicamba-02-12-09-11303: Diode ArrayRange: 2.586e+22.682.38+DMO+DMO-DMO-DMO8,107,108 G5 Syringic2.50 2.75 3.00 3.25 3.50 3.75

%01002.50 2.75 3.00 3.25 3.50 3.75

%0100Dicamba-02-12-09-11751: TOF MS ES1831.24e32.762.512.683.033.47Dicamba-02-12-09-11791: TOF MS ES1831.05e32.772.512.302.683.013.52Time2.50 2.75 3.00 3.25 3.50 3.75

AU0.02.0e+22.50 2.75 3.00 3.25 3.50 3.75

AU0.02.0e+2Dicamba-02-12-09-11753: Diode ArrayRange: 2.417e+22.672.78Dicamba-02-12-09-11793: Diode ArrayRange: 2.419e+22.672.422.78+DMO+DMO-DMO-DMO8,107,108 G8 Anistic2.50 2.75 3.00 3.25 3.50 3.75

%01002.50 2.75 3.00 3.25 3.50 3.75

%0100Dicamba-02-12-09-11481: TOF MS ES1371.54e33.453.192.783.333.71Dicamba-02-12-09-11521: TOF MS ES1372.12e33.453.192.653.32Time2.50 2.75 3.00 3.25 3.50 3.75

AU0.01.0e+22.0e+22.50 2.75 3.00 3.25 3.50 3.75

AU0.01.0e+2Dicamba-02-12-09-11483: Diode ArrayRange: 1.661e+23.092.442.94Dicamba-02-12-09-11523: Diode ArrayRange: 2.097e+23.092.44+DMO+DMO-DMO-DMO8,107,108 G7 Ferrulic2.50 2.75 3.00 3.25 3.50 3.75

%01002.50 2.75 3.00 3.25 3.50 3.75

%0100Dicamba-02-12-09-11031: TOF MS ES1791.89e32.703.022.86Dicamba-02-12-09-11071: TOF MS ES1792.20e32.703.00Di b 02 12 09 11033 Di d ATime2.50 2.75 3.00 3.25 3.50 3.75

AU0.02.0e+22.50 2.75 3.00 3.25 3.50 3.75

AU0.02.0e+2Dicamba-02-12-09-11033: Diode ArrayRange: 2.866e+2.912.99Dicamba-02-12-09-11073: Diode ArrayRange: 2.978e+2.912.392.99+DMO+DMO-DMO-DMO8,107,108 B2 DCB2.00 2.50 3.00

%01002.00 2.50 3.00

%0100D icam ba-02-12-09-10231: TO F M S ES2054.56e32.54D icam ba-02-12-09-10271: TO F M S ES2059 92.642.042.382.812.873.383.09Tim e2.00 2.50 3.00

AU0.01.0e+12.00 2.50 3.00

AU0.01.0e+12.0e+1D icam ba-02-12-09-10233: D iode ArrayRange: 2.288e+12.662.44D icam ba-02-12-09-10273: D iode ArrayRange: 1.837e+12.65+DMO-DMO+DMO-DMO3,6‐DCSA3,5‐DCSADCBT im e3.00 4.00 5.00

AU0.05.0e+13.00 4.00 5.00

AU0.05.0e+1Range:9.014e+ 1x1 63.7 33.384.33D ic a m b a -0 2 -1 2 -0 9 -1 0 7 03 : D io d e A rra yR ange: 8.997e+1x163.733.384.338,107,108 C 3 2,4-D3.00 4.00 5.00

%01003.00 4.00 5.00

%0100D ic a m b a -0 2 -1 2 -0 9 -1 0 7 01: T O F M S E S -1639 43.833.553.494.433.965.264.51D ic a m b a -0 2 -1 2 -0 9 -1 0 7 41: T O F M S E S -1638 93.863.073.303.904.435.235.12+DMO+DMO-DMO-DMO2,4‐DCP8,107,108 H6 Sinapic2.00 2.50 3.00 3.50

%01002.00 2.50 3.00 3.50

%0100Dicamba-02-12-09-11971: TOF MS ES209472.382.192.131.932.492.713.173.123.54Dicamba-02-12-09-12021: TOF MS ES209432.522.362.292.261.941.832.783.253.063.45Time2.00 2.50 3.00 3.50

AU0.02.0e+22.00 2.50 3.00 3.50

AU0.02.0e+2Dicamba-02-12-09-11973: Diode ArrayRange: 2.36e+22.151.692.822.32Dicamba-02-12-09-12023: Diode ArrayRange: 2.501e+22.151.782.822.332.663.103.20+DMO+DMO-DMO-DMOea. Dicamba (DCB)b. 2,4-Dc. Sinapic Acidd. Ferulic Acide. Anisic Acidf. Syringic Acidg. Vanillic AcidMonsanto Company 10-SY-210U 322 of 721 

### References to Appendix C

Aebersold, R. 1993. Mass spectr ometry of proteins and peptides in biotechnology. Currrent Opinion in Biotechnology 4:412-419.

Arfin, S.M., and R.A. Bradshaw. 1988. Cotran slational processing and protein turnover in eukaryotic cells. Biochemistry 27:7979-7984.

Bradshaw, R.A., W.W. Bricke y, and K.W. Walker. 1998. N- terminal processing: the methionine aminopeptidase and N alpha-acetyl transferase families. Trends in

Biochemical Sciences 23:263-267.

Grimm, R., M. Grimm, C. Eckerskorn, K. Pohlmeyer, T. Rohl, and J. Soll. 1997. Postimport methylation of the small subunit of ribulose-1,5-bisphosphate carboxylase in chloroplasts. FEBS Letters 408:350-354.

Hunkapiller, M.W., R.M. Hewick, W.J. Dr eyer, and L.E. Hood. 1983. High-sensitivity sequencing with a gas-phase sequenato r. Methods in Enzymology 91:399-413.

Jensen, O.N., A.V. Podtelejnikov, and M. Mann. 1997. Identificati on of the components of simple protein mixtures by high-accuracy peptide mass mapping and database searching. Analytical Chemistry 69:4741-4750.

Polevoda, B., and F. Sherman. 2000. N alpha term inal acetylation of eukaryotic proteins. The Journal of Biological Chemistry 275:36479-36482.

Whitney, S.M., and T.J. Andrews. 2001. The gene for the ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) small subunit relocated to the plastid genome of tobacco directs the synthesis of small subun its that assemble into Rubisco. Plant Cell

13:193-205.Monsanto Company 10-SY-210U 323 of 721 

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

MON 87708 DMO

D.1. Materials

Over-season leaf, root, forage, and seed tissue samples from MON 87708 and the near isogenic conventional soybean control A3525 were harvested from five field sites in the U.S. during 2008 from plants grown fr om starting seed lot 10001256 and 10001257, respectively. An E. coli -produced DMO protein (lot 11247247) was used as the analytical reference standard.

The identities of MON 87708 and the convent ional control samples were confirmed by verifying the chain of custody documentation prior to analysis . To further confirm the

identities of MON 87708 and the conventi onal control samples, event specific polymerase chain reaction (PCR) analyses were conducted on the seed tissue samples from each site to confirm the presence or absence of the dmo expression cassette. The PCR analyses and the resulting verification of identities were archiv ed under the starting

seed lot numbers.

D.2. Field Design and Tissue Collection

Field trials were initiate d during the 2008 planting season to generate MON 87708 and the conventional control samples at the followi ng locations in the U.S.: Jefferson County, Iowa; Stark County, Illinois; Clinton County, Illinois; Park e County, Indiana; and Berks County, Pennsylvania. The fiel d sites were representative of soybean producing regions suitable for commercial soybean production. At each site, three replicated plots containing MON 87708, as well as the conventional c ontrol, were planted using a randomized complete block field design. Over -season leaf (OSL), r oot, forage, and seed samples were collected from each replicated plot at each field site (except for the conventional control from Berks County, Penns ylvania where only two replicates were tested).

The OSL tissue samples were collected from the youngest set of fully expanded trifoliate leaves at the following growth stages: OS L-1 at V3-V4; OSL-2 at V5-V8; OSL-3 at R2-V12; and OSL-4 at R5-V16. The root and forage tissue samples were collected at approximately the R6 growth stage. Seed ti ssue samples were collected at the R8 growth

stage.

D.3. Tissue Processing and Protein Extraction

All tissue samples harvested were shipped to Monsanto’s processing facility and were prepared by the Monsanto Sample Management Team. The prepared tissue samples were

stored in a -80°C freezer until transferred on dry ice to the analytical facility.

MON 87708 DMO was extracted from the seed ti ssue samples at a tissue to buffer ratio of 1:100 with a Tris-borate buffer (0.1 M Tris, 0.1 M Na2B4O7 • 10H2O, 0.01 M MgCl2, 0.05% (v/v) Tween-20 at pH 7.8). MON 87708 DMO was extracted from OSL, forage,Monsanto Company 10-SY-210U 324 of 721 

### and root tissues samples at a tissue to bu ffer ratio of 1:100, 1:50, and 1:50, respectively, using phosphate buffered saline (PBS) with Tween 20 and 0.5% (w/v) bovine serum albumin (BSA) (1 × PBST with 0.5% (w/v) BSA). Extractions were done using 8 1/4 ′′ chrome-steel beads, and shaking in a Harbil mixer (Fluid Management, Wheeling, Illinois). Insoluble material was removed fr om all tissue extracts using a serum filter (Fisher Scientific, Pittsburgh, PA). The extracts were aliq uotted and stored frozen in a -80°C freezer until enzy me-linked immunosorbent a ssay (ELISA) analysis.

D.4. DMO Antibodies

Goat polyclonal anti-DMO antibodies were pu rified using Protein-G Agarose affinity chromatography. The concentration of the purified IgG was determined to be 8.1 mg/ml by spectrophotometric methods. The purified antibody was stored in 1× PBS, pH 7.4. The purified anti-DMO antibodies were coup led with biotin (Pierce, Rockford, IL) according to the manufacturer’s instructions . The detection reagent was NeutrAvidin (Pierce, Rockford, IL) conjuga ted to horseradish peroxidase (HRP). The goat polyclonal

anti-DMO antibodies react with the DM O and DMO+27 present in the MON 87708 DMO and were used as capture an tibodies for the DMO ELISA method.

D.5. DMO ELISA Method

Goat polyclonal anti-DMO antibodies were diluted in coating buffer (15 mM Na2CO3, 35 mM NaHCO3, and 150 mM NaCl, pH 9.6) to a final concentration of 5.0 μg/ml and

then immobilized onto 96-well microtiter plates followed by incubation in a 4 °C refrigerator for ≥ 8 hours. Plates were wash ed with 1 × PBS containing 0.05% (v/v) Tween-20 (1 × PBST). The plates were blocked using 10% Casein in tris buffered saline (TBS) blocking buffer (Pierce, Rockford, IL) at 200 μl per well for 1 hour at room temperature. The blocking buffe r was aspirated and DM O standard or tissue sample extract was added at 100 μl per well an d incubated for 1 hour at 37°C. Prior to the addition of biotinylat ed antibody, NuetrAvidin-HRP and 3,3',5,5' tetramethylbenzidine (TMB; Kirkegaard & Perry, Gaithers burg, MD) reagents, plates were washed with 1 × PBST. The captured MON 87708 DMO was detected by the addition of 100 μl per well of biotinylated goat anti-DMO antibod ies and NeutrAvidin-HRP. Plates were developed by adding 100 μl per well of TMB. The enzymatic reaction was terminated by the addition of 100 μl per well of 3 M H3 PO4. Quantification of MON 87708 DMO was accomplished by interpolation on a DMO standard curve that ranged from 0.313-20 ng/ml.

D.6. Moisture Analysis

Tissue moisture content was determined us ing an IR-200 Moisture Analyzer (Denver Instrument Company, Arvada, CO). A homogeneous site and tissue-specific pool (TSSP) was prepared using MON 87708 and the conventional control samples from each tissue type grown at each site. The average per cent moisture for each TSSP was calculated from triplicate analyses. A TSSP Dry Weight Conversion Fa ctor (DWCF) was calculated

as follows:Monsanto Company 10-SY-210U 325 of 721 

##     100 Moisture TSSP % Mean 1 DWCF

 



### The DWCF was used to convert protein levels from a μg/g fresh weight (fwt) basis into a μg/g dry weight (dwt) basis us ing the following calculation:



##     DWCF Weight Fresh Level Protein Weight Dry in Level Protein 



### The protein levels that were reported on a fwt ba sis to be less than or equal to the limit of detection or less than the limit of quantifica tion (LOQ) were not reported on a dwt basis.

D.7. Data Analyses

All MON 87708 DMO ELISA plates were analyzed on a SPECTRAmax Plus 384 (Molecular Devices, Sunnyvale, CA) micr oplate spectrophotometer, using a dual wavelength detection method. All protein con centrations were determined by optical absorbance at a wavelength of 450 nm w ith a simultaneous reference reading of 620-650 nm. Data reduction analyses were performed using Molecular Devices

SOFTmax PRO GxP version 5.0.1 software. Ab sorbance readings and protein standard concentrations were fitted with a four para meter logistic curve fit. Following the interpolation from the standard curve, the amount of protein (ng/ml) in the tissue was reported on a μg/g fwt basis for data that were greater than or equal to the LOQ. For MON 87708 DMO, this conversion utilized a sample dilution factor and a tissue-tobuffer ratio. The enzyme values in μg/g fwt were converted to μ g/g dwt by applying the DWCF. Microsoft Excel 2007 (Versi on 12.0.6514.5000 SP2 Microsoft, Redmond, WA)

was used to calculate MON 87708 DMO levels in tissue sa mples. The sample mean, standard deviations, and ra nges were also calculated using Microsoft Excel 2007.Monsanto Company 10-SY-210U 326 of 721 

### Appendix E: Materials, Methods, and In dividual-Site Results for Compositional Analysis of MON 87708 Soybean Seed and Forage

Compositional comparisons between MON 877 08 and the near isogenic conventional soybean control A3525 were performed using th e principles and analytes outlined in the OECD consensus documents for soybean composition (OECD, 2001). These principles are accepted globally and have been employed previously in assessments of soybean products derived through biotechnology. Th e compositional assessment was conducted on seed and forage samples harvested from a single growing season c onducted in the U.S. during 2008 under typical agronomic practices.

The materials and methods for compositional an alysis, as well as the individual-site results (Tables E-4 to E-18), are provided below.

E.1. Materials

Forage and harvested seed from MON 87708 , a near isogenic conventional soybean control A3525 that has similar geneti c background to that of MON 87708, and commercial reference varieties were comp ositionally assessed. The commercial

reference varieties are listed in Table E-1.

Table E-1. Commercial Reference Varieties

Material Name Seed Lot# Field Site Code

CST3461 10000890 IARL

Wilken 3316 10001505 IARL

Midland 363 10001570 IARL

Stine 3300-0 10001312 IARL Croplan HT3596STS 10001450 ILWY

FS 3591 10001448 ILWY

Garst 3585N 10000883 ILWY

Pioneer 93M52 10001311 ILWY

Stine 3608-0 10001392 ILCY Quality Plus 365C 10001608 ILCY

Crows C37003N 10001508 ILCY

NK S38-T8 10001509 ILCY

Lewis 372 10001475 INRC

Pioneer 93M50 10000888 INRC

Dekalb DKB34-51 10000889 INRC

Stewart SB3454 10000887 INRC

Dekalb DKB31-51 10001285 PAHM

NK 32Z3 10001607 PAHM Hoegemeyer 333 10001590 PAHM

Pioneer 93B15 10001304 PAHMMonsanto Company 10-SY-210U 327 of 721 

### E.2. Characterization of the Materials

The identities of the forage and seed samp les from MON 87708, the conventional control,

and the commercial reference varieties were verified by the Study Director prior to the study by confirming the chain-of-custody docum entation supplied with the forage and

seed harvested from the field sites. The seed of MON 87708, the conventional control,

and the commercial reference varieties were characterized by event-specific polymerase chain reaction (PCR) analysis to confirm the presence or absence of the dmo expression

cassette.

E.3. Field Production of the Samples

Harvested seed and forage of MON 87708, the conventional control, and the commercial

reference varieties were collected from five re plicated sites in the U.S. during the 2008 growing season. These sites are Jefferson County, Iowa (IARL); Stark County, Illinois (ILWY); Clinton County, Illinois (ILCY); Parke County, Indiana (INRC); and Berks County, Pennsylvania (PAHM). Starting seeds were planted in a randomized complete block design with three plots for each of MON 87708, the conventional control, and the commercial reference varieties. The pr oduction was conducted under normal agronomic field conditions. All soybean plants incl uding MON 87708, the convent ional control, and commercial reference varieties were treated with maintenance pesticides as necessary throughout the growing season. In addition, MON 87708 plots were treated at the V2-V3 growth stage with dicamba herbic ide at the target label rate (0.5 lb/Acre a.e.). Seed and forage samples were harvested from all pl ots and shipped on dry ice (forage) or at ambient temperature (harvested seed) to Monsanto Company, St. Louis, MO. A subsample for compositional analysis was obt ained for each tissue sample collected. These subsamples were ground and stored in a freezer set to maintain -20°C until their shipment on dry ice to Covance Laboratori es Inc. (Madison, WI) for analysis.

E.4. Summary of Analytical Methods

Nutrients assessed in this study included proximates (ash, carbohydrates by calculation, moisture, protein, and fat), fiber, amino acids (18 components), fatty acids (FA, C8-C22), and vitamin E ( α -tochopherol) in seed, and proxima tes (ash, carbohydrates by calculation, moisture, protein, and fat) and fiber in forage . Anti-nutrients assessed in seed included raffinose, stachyose, lectin, phytic acid, tryps in inhibitors, and isoflavones (daidzein, genistein, and glycitein).

All compositional analyses were performed at Covance Laboratories, Inc. (Madison, WI). Methods for analysis were based on internat ionally-recognized procedures and literature publications. Brief descriptions of the methods utilized for the analyses are described

below.Monsanto Company 10-SY-210U 328 of 721 

### E.5. Analytical Method Summaries and Reference Standards

E.5.1. Acid Detergent Fiber

The sample was placed in a fritted vessel and washed with an acidic boiling detergent solution that dissolved the protein, carbohydrat e, and ash. An acetone wash removed the fats and pigments. The lignocellulose fracti on was collected on the frit and determined gravimetrically (Goering and Van Soest, 1970) . The limit of quantitation (LOQ) for this analysis was 0.100%.

E.5.2. Amino Acid Composition

The sample was assayed by three methods to obtain the full profile. Tryptophan required a base hydrolysis with sodium hydroxide. Th e sulfur-containing ami no acids required an oxidation with performic acid prior to hydrolysis with hydrochloric acid. Analysis of the samples for the remaining amino acids was accomplished through direct acid hydrolysis with hydrochloric acid. Once hydrolyzed, the individual amino acids were then quantitated using an automated amino acid analyzer (AOAC-Internat ional, 2005a). The LOQ for this analysis was 0.100 mg/g.

Reference Standards:

 ThermoScientific K18, 2.5  mol/mL per constituent except cystine (1.25  mol/mL), Lot Number JK126327

 Sigma, L-Tryptophan, 100%, Lot Number 076K0075

 Sigma/BioChemika, L-Cysteic Acid M onohydrate, 99.5% (used as 100%), Lot

Number 1305674

 Sigma, L-Methionine Sulfone, 100%, Lot Number 047K1321

E.5.3. Ash

The sample was placed in an electric furnace at 550  C and ignited to drive off all volatile organic matter. The nonvolat ile matter remaining was quan titated gravimetrically and calculated to determine percent ash (AOAC -International, 2005b). The LOQ for this analysis was 0.100%.

E.5.4. Carbohydrates

The total carbohydrate level was calculated by difference using the fwt-derived data and the following equation (USDA, 1973):

% carbohydrates = 100 % - (% protein + % fat + % moisture + % ash)

The LOQ for this analysis was 0.100%.Monsanto Company 10-SY-210U 329 of 721 

### E.5.5. Crude Fiber

Crude fiber was quantitated as the loss on ignition of dr ied residue remaining after digestion of the sample with 1.25% sulfuric acid and 1.25% sodium hydroxide solutions under specific conditions (AOAC -International, 2005c). The lim it of quantitation for this study was 0.100%.

E.5.6. Fat by Acid Hydrolysis

The sample was hydrolyzed with hydrochloric ac id at an elevated temperature. The fat

was extracted with ether and hexane. The extract was evaporated on a steambath, redissolved in hexane and filtered through a s odium sulfate column. The hexane extract was then evaporated again on a steambat h under nitrogen, dried, and weighed (AOACInternational, 2005d). The LOQ for this analysis was 0.100%.

E.5.7. Fat by Soxhlet Extraction

The sample was weighed into a cellulose thim ble containing sodium sulfate and dried to remove excess moisture. Pentane was dripped through the sample to re move the fat. The extract was then evaporated, dried, and weighed (AOAC-International, 2005e). The LOQ for this analysis was 0.100%.

E.5.8. Fatty Acids

The lipid was extracted and saponified with 0.5 N sodium hydroxide in methanol. The saponification mixture was methylated with 14% boron trifluoride in methanol. The resulting methyl esters were extracted with heptane containing an internal standard. The methyl esters of the fatty acids were an alyzed by gas chromatography using external standards for quantitation ( AOAC-International, 2005f; AOCS, 1997a, c). The limit of quantitation was 0.0200%.

Reference Standards:

 Nu Chek Prep GLC Reference Standard Hazleton No. 1, *, Lot Number AU18-S

 Nu Chek Prep GLC Reference Standard Hazleton No. 2, *, Lot Number M13-O

 Nu Chek Prep GLC Reference Standard Hazleton No. 3, *, Lot Number MA18-S

 Nu Chek Prep GLC Reference Standard Hazleton No. 4, *, Lot Number JA16-T

 Nu Chek Prep Methyl Gamma Linolenat e, used as 100%, Lot Number U-63MJY12-R

 Nu Chek Prep Methyl Tridecanoate, us ed as 100%, Lot Number N-13M-JA16-T
* Overall purity of the sum of the mixt ure of components is used as 100%.



### E.5.9. Isoflavones

The sample was extracted using a solution of hydrochloric acid and reagent alcohol heated on steam baths or hot plates. The ex tract was brought to volume, diluted, and centrifuged. An aliquot of the supernatant was placed onto a C18 solid-phase extraction column. Unwanted components of the matrix were rinsed off with 20% methanol and

then the isoflavones were eluted with 80% methanol. The sample was analyzed on aMonsanto Company 10-SY-210U 330 of 721 

### high-performance liquid chromatography syst em with ultraviole t detection and was compared to an external st andard curve of known standard s for quantitation (Pettersson and Kiessling, 1984; Seo and Morr, 1984). The LOQ for each component was 10.0 ppm ( μ g/g).

Reference Standards:

 Chromadex, Daidzein, 96.5%, Lot Number 04007-120

 Chromadex, Glycitein, 96.3%, Lot Number 07344-571

 Indofine, Genistein,  99% (100% used in calcul ations), Lot Number 0309074

E.5.10. Lectin

The sample was suspended in phosphate buffered saline (PBS), shaken, and filtered. An aliquot of the resulting extract was serially diluted in 10 cuvettes c ontaining PBS. A 10% hematocrit of lyophilized rabbit blood in PB S was added to each dilution. After 2.5 hours, the absorbance of each dilution of th e sample and lectin control was measured on a spectrophotometer at 620 nm, using PBS to zero the instrument. One hemagglutinating unit (H.U.) was defined as the level that caused 50% of the standard cell suspension to sediment in 2.5 hours (K lurfeld and Kritchevsky, 1987; Liener, 1955). The LOQ for this anal ysis was 0.10 H.U./mg.

Reference Standard:

 Sigma-Aldrich, Red Blood Cells, Rabbit, Product #R1629, Lot Number

105K6042

E.5.11. 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 (AOACInternational, 2005g). The LOQ for this analysis was 0.100%.

E.5.12. Neutral Detergent Fiber, Enzyme Method

The sample was placed in a fritted vessel and washed with a neutral boiling detergent solution that dissolved the protein, carbohydr ate, enzyme, and ash. An acetone wash removed the fats and pigments. Hemicellu lose, cellulose, and li gnin fractions were

collected on the frit and determined gravimetrically (AACC, 1998; Goering and Van Soest, 1970). The LOQ for this analysis was 0.100%.

E.5.13. Phytic Acid

The sample was extracted using 0.5 M HCl with ultrasonication. Purification and concentration were accomplished on a silica- based anion-exchange column. The sample was analyzed on a polymer high-performan ce liquid chromatography column PRP-1, 5 μm (150 × 4.1 mm) with a refractive index detector (Lehrfeld, 1989; Lehrfeld, 1994). The LOQ for this analysis was 0.100%.Monsanto Company 10-SY-210U 331 of 721 

### Reference Standard:

 Aldrich, Phytic Acid, Dodecasodium Salt Hydrate, 98%, Lot Number 068K0755

E.5.14. Protein

Nitrogenous compounds in the sample were redu ced in the presence of boiling sulfuric acid and a mercury catalyst mixture to form am monia. 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-International, 2005h; Bradstreet, 1965; Kolthoff and Sandell, 1948). The LOQ for this analysis was 0.100%.

E.5.15. Raffinose and Stachyose

The sample was extracted with deionize d water and the extract treated with a hydroxylamine hydrochloride solution in pyridine, containing phenyl- β -D-glucoside as an internal standard. The resulting oxime s were converted to silyl derivatives by treatment with hexamethyldisilazane and trifluoracetic acid and analyzed by gas chromatography using a flame ionization de tector (Brobst, 1972; Mason and Slover, 1971). The LOQ for this analysis was 0.0500%.

Reference Standards:

 Sigma, D-(+)-Raffinose Pentahydrate, 95.5% after correction for degree of hydration, Lot Number 037K1059

 Sigma, Stachyose, 97.1% after correcti on for degree of hydration, Lot Number

078K3802

E.5.16. Trypsin Inhibitor

The sample was ground and defatted with petr oleum ether. A sample of matrix was extracted with 0.01 N sodium hydroxide. Vary ing aliquots of the sample suspension were exposed to a known amount of trypsin and benzoy1-DL-arginine~p~nitroanilide hydrochloride. The sample was allowed to react for 10 minutes at 37°C. After 10 minutes, the reaction was halted by the a ddition of acetic acid. The solution was centrifuged and then the absorbance was determined at 410 nm. Trypsin inhibitor activity was determined by photometrically meas uring the inhibition of trypsin’s reaction with benzoyl-DL-arginine~p~nitroanilide hydrochloride (AOCS, 1997b). The LOQ for this analysis was 1.00 Trypsin Inhibitor Units (TIU)/mg.

E.5.17. 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 quantitated by hi gh-performance liquid chromatography using a silica column (Cort et al., 1983; McMurray et al., 1980; Speek et al., 1985). The LOQ for this analysis was 0.500 mg/100g.Monsanto Company 10-SY-210U 332 of 721 

### Reference Standard:

 USP, Alpha Tocopherol, 100%, Lot Number M

E.6. Data Processing and Statistical Analysis

After compositional analyses were performed, data spreadsheets were forwarded to Monsanto Company. The data were reviewed, fo rmatted, and sent to Ce rtus International, Inc. for statistical analysis.

The following formulas were used for re-e xpression of soybean composition data for statistical analysis (Table E-2):

Table E-2. Re-Expression Formulas for St atistical Analysis of Composition Data

Component From (X) To Formula 1

Proximates (excluding Moisture), Fiber, Phytic Acid, Raffinose, Stachyose % fwt % dwt X/d

Isoflavones μ g/g fwt μ g/g dwt X/d

Lectin H.U./fwt H.U./dwt X/d Trypsin Inhibitor TIU/mg fwt TIU/mg dwt X/d

Vitamin E mg/100g fwt mg/100g dwt X/d Amino Acids (AA) mg/g fwt % dwt X/(10d)

Fatty Acids (FA) % fwt % Total FA (100)X j /  X, for

each FA j where

 X is over all

the FA

1 ‘X’ is the individual sample value; ‘d’ is th e fraction of the sample that is dry matter.



### In order to complete a statistical analysis fo r a compositional analyte, at least 50% of the values for an analyte had to be greater than the assay LOQ. The following 14 analytes

with more than 50% of observations below th e assay LOQ were excluded from statistical analysis: 8:0 caprylic acid, 10:0 capric aci d, 12:0 lauric acid, 14:0 myristic acid, 14:1 myristoleic acid, 15:0 pent adecanoic acid, 15:1 pentadec enoic acid, 16:1 palmitoleic acid, 17:0 heptadecanoic acid, 17:1 hept adecenoic acid, 18:3 ga mma-linolenic acid, 20:3 eicosatrienoic acid, 20:2 eicosadienoic acid, a nd 20:4 arachidonic acid.

If less than 50% of the observations for a component we re below the LOQ, individual analyses that were below the LOQ were assign ed a value equal to one-half the LOQ. The following analyte was assigned a value (Table E-3):

Table E-3. Component with Observations Below the Assay Limit of Quantitation Not Excluded from Statistical Analysis

Obs. Below LOQ Total N LOQ Value Assigned Component Units N (%)

Seed Fatty Acid

20:1 Eicosenoic % fwt 45 42.9 105 0.020 0.010Monsanto Company 10-SY-210U 333 of 721 

### The data were assessed for potential outli ers using a studentized PRESS residuals

calculation. A PRESS residual is the difference between any value and its predicted

value 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 va lues are expected to be between  3. Extreme data points that are also outside of the  6 studentized PRESS resi dual range are considered for exclusion, as outliers, from the final analyses. No results had PRESS residual values

outside of the  6 range.

All soybean compositional components were st atistically analyzed using a mixed model analysis of variance. The five replicated sites were analyzed both separately and combined. Individual replicated site analyses used model (1)

.

(1) Y ij 

### = U + T i 

### + B j 

### + e ij 

### ,

where Y ij 

### = unique individual observa tion, U = overall mean, T i 

### = substance effect,

B j 

### = random block effect, and e ij 

### = residual error.

Combined-site analyses used model (2)

.

(2) Y ijk 

### = U + T i 

### + L j 

### + B(L) jk



### + LT ij 

### + e ijk,



### where Y ijk 

### = unique individua l observation, U = overall mean, T i 

### = substance effect,

L

j 

### = random site effect, B(L) jk



### = random block within site effect,

LT ij 

### = random site by substance interaction effect, and e ijk 

### = residual error.

A range of observed values from the refe rence varieties was determined for each analytical component. Additionally, data fr om the reference varieties were used to develop tolerance intervals. A tolerance interval is an interval that one can claim, with a specified degree of confidence, contains at least a specifi ed proportion, p, of an entire sampled population for the parameter measured.

For each compositional component, 99% tolera nce intervals were calculated that are expected to contain, with 95% confidence, 99% of the quantitie s expressed in the population of commercial refe rence varieties. Because negative quantities are not possible, negative calculated lower to lerance bounds were set to zero.

SAS ® 

### (Version 9.2) software was used to genera te 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 10-SY-210U 334 of 721Table E-4. Statistical Summary of SiteIARL Soybean Seed Nutrients for MON87708 (Treated) vs. Conventional ControlDifference (MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Proximate (% dwt)Ash5.30 (0.070)5.29 (0.070)0.0063 (0.098)-0.27, 0.280.9524.74, 6.01(5.20 - 5.37)(5.19 - 5.47)(-0.27 - 0.17)(4.93 - 5.88)Carbohydrates38.55 (0.56)37.97 (0.56)0.57 (0.55)-0.96, 2.110.35832.07, 40.08(38.03 - 39.11)(37.17 - 38.45)(-0.27 - 1.33)(33.82 - 39.26)Moisture (% fwt)7.13 (0.28)6.07 (0.28)1.06 (0.39)-0.033, 2.150.0544.27, 9.58(6.92 - 7.27)(5.84 - 6.36)(0.56 - 1.43)(5.50 - 9.23)Protein40.92 (0.27)41.09 (0.27)-0.18 (0.16)-0.63, 0.280.34235.50, 45.19(40.40 - 41.41)(40.69 - 41.74)(-0.33 - 0.088)(37.06 - 43.42)Total Fat15.22 (0.47)15.61 (0.47)-0.40 (0.66)-2.23, 1.430.57912.33, 24.10(14.77 - 15.62)(15.38 - 15.82)(-1.05 - -0.017)(15.47 - 21.34)Fiber (% dwt)Acid Detergent Fiber13.03 (0.38)12.60 (0.38)0.43 (0.54)-1.08, 1.940.47210.06, 18.04(12.68 - 13.58)(11.92 - 13.17)(-0.34 - 1.66)(12.07 - 17.46)Monsanto Company 10-SY-210U 335 of 721Table E-4 (continued). Statistical Summaryof Site IARL Soybean Seed Nutrients for MON 87708 (Treated) vs. ConventionalControlDifference (MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Fiber (% dwt)Crude Fiber7.71 (0.33)7.41 (0.33)0.30 (0.46)-0.98, 1.580.5455.76, 10.76(7.26 - 8.48)(7.17 - 7.60)(-0.34 - 1.03)(6.35 - 11.31)Neutral Detergent Fiber14.25 (0.89)13.27 (0.89)0.98 (1.25)-2.51, 4.460.47911.36, 19.38(13.11 - 16.38)(11.81 - 14.42)(-1.31 - 4.57)(11.66 - 19.45)Amino Acid (% dwt)Alanine1.72 (0.026)1.74 (0.026)-0.027 (0.036)-0.13, 0.0740.5021.56, 1.91(1.66 - 1.76)(1.69 - 1.77)(-0.11 - 0.042)(1.59 - 1.86)Arginine3.28 (0.055)3.45 (0.055)-0.17 (0.061)-0.33, 0.00400.0532.55, 3.83(3.26 - 3.30)(3.27 - 3.56)(-0.26 - -0.013)(2.88 - 3.74)Aspartic Acid4.55 (0.060)4.63 (0.060)-0.076 (0.084)-0.31, 0.160.4164.04, 5.13(4.44 - 4.63)(4.46 - 4.74)(-0.29 - 0.12)(4.22 - 4.94)Cystine0.61 (0.0094)0.59 (0.0094)0.018 (0.013)-0.019, 0.0540.2570.50, 0.68(0.60 - 0.62)(0.56 - 0.62)(-0.0056 - 0.053)(0.53 - 0.66)Monsanto Company 10-SY-210U 336 of 721Table E-4 (continued). Statistical Summaryof Site IARL Soybean Seed Nutrients for MON 87708 (Treated) vs. ConventionalControlDifference (MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Amino Acid (% dwt)Glutamic Acid7.24 (0.10)7.41 (0.10)-0.16 (0.15)-0.57, 0.250.3326.28, 8.30(7.05 - 7.38)(7.12 - 7.58)(-0.53 - 0.17)(6.69 - 7.92)Glycine1.72 (0.024)1.76 (0.024)-0.037 (0.034)-0.13, 0.0570.3311.53, 1.92(1.67 - 1.76)(1.70 - 1.79)(-0.12 - 0.042)(1.58 - 1.84)Histidine1.04 (0.014)1.06 (0.014)-0.018 (0.019)-0.071, 0.0360.4080.93, 1.16(1.02 - 1.06)(1.02 - 1.08)(-0.066 - 0.031)(0.95 - 1.13)Isoleucine1.84 (0.038)1.88 (0.038)-0.036 (0.054)-0.19, 0.110.5401.65, 2.06(1.75 - 1.90)(1.79 - 1.94)(-0.16 - 0.11)(1.68 - 2.02)Leucine3.01 (0.041)3.07 (0.041)-0.055 (0.058)-0.22, 0.110.4012.72, 3.39(2.93 - 3.07)(2.96 - 3.14)(-0.21 - 0.072)(2.80 - 3.27)Lysine2.60 (0.030)2.62 (0.030)-0.017 (0.042)-0.13, 0.100.7102.33, 2.84(2.53 - 2.64)(2.54 - 2.66)(-0.12 - 0.090)(2.38 - 2.74)Monsanto Company 10-SY-210U 337 of 721Table E-4 (continued). Statistical Summaryof Site IARL Soybean Seed Nutrients for MON 87708 (Treated) vs. ConventionalControlDifference (MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Amino Acid (% dwt)Methionine0.58 (0.013)0.56 (0.013)0.016 (0.019)-0.037, 0.0680.4520.50, 0.64(0.56 - 0.60)(0.53 - 0.60)(-0.022 - 0.071)(0.52 - 0.63)Phenylalanine1.98 (0.036)2.03 (0.036)-0.052 (0.050)-0.19, 0.0870.3571.80, 2.30(1.92 - 2.04)(1.95 - 2.09)(-0.17 - 0.023)(1.85 - 2.21)Proline1.92 (0.035)1.98 (0.035)-0.063 (0.050)-0.20, 0.0760.2741.65, 2.26(1.90 - 1.96)(1.89 - 2.07)(-0.17 - 0.065)(1.74 - 2.16)Serine1.98 (0.044)2.00 (0.044)-0.024 (0.036)-0.12, 0.0760.5451.78, 2.27(1.92 - 2.03)(1.95 - 2.08)(-0.087 - 0.047)(1.90 - 2.18)Threonine1.53 (0.023)1.54 (0.023)-0.011 (0.023)-0.075, 0.0530.6531.40, 1.69(1.48 - 1.56)(1.51 - 1.58)(-0.051 - 0.052)(1.47 - 1.64)Tryptophan0.45 (0.0092)0.45 (0.0092)0.00085 (0.013)-0.035, 0.0370.9500.38, 0.52(0.45 - 0.46)(0.44 - 0.47)(-0.0079 - 0.0069)(0.39 - 0.50)Monsanto Company 10-SY-210U 338 of 721Table E-4 (continued). Statistical Summaryof Site IARL Soybean Seed Nutrients for MON 87708 (Treated) vs. ConventionalControlDifference (MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Amino Acid (% dwt)Tyrosine1.34 (0.013)1.38 (0.013)-0.042 (0.016)-0.087, 0.00350.0621.24, 1.50(1.32 - 1.37)(1.37 - 1.42)(-0.079 - -0.00044)(1.26 - 1.49)Valine1.95 (0.046)1.99 (0.046)-0.042 (0.065)-0.22, 0.140.5521.72, 2.20(1.82 - 2.03)(1.90 - 2.05)(-0.20 - 0.13)(1.73 - 2.13)Fatty Acid (% Total FA)16:0 Palmitic11.49 (0.051)11.00 (0.051)0.49 (0.062)0.32, 0.660.0018.44, 12.56(11.44 - 11.54)(10.92 - 11.08)(0.39 - 0.62)(9.40 - 11.54)18:0 Stearic4.06 (0.067)4.00 (0.067)0.059 (0.095)-0.21, 0.320.5682.90, 5.19(3.99 - 4.19)(3.99 - 4.01)(-0.016 - 0.20)(3.24 - 4.67)18:1 Oleic19.38 (0.20)21.67 (0.20)-2.29 (0.28)-3.07, -1.520.00115.73, 27.19(19.07 - 19.73)(21.48 - 21.78)(-2.71 - -1.75)(17.88 - 25.31)18:2 Linoleic53.85 (0.33)52.70 (0.33)1.16 (0.46)-0.13, 2.440.06648.61, 59.37(53.42 - 54.07)(52.66 - 52.73)(0.68 - 1.41)(50.95 - 56.68)Monsanto Company 10-SY-210U 339 of 721Table E-4 (continued). Statistical Summaryof Site IARL Soybean Seed Nutrients for MON 87708 (Treated) vs. ConventionalControlDifference (MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Fatty Acid (% Total FA)18:3 Linolenic10.64 (0.13)10.04 (0.13)0.60 (0.19)0.074, 1.120.0336.01, 12.58(10.58 - 10.74)(10.00 - 10.12)(0.46 - 0.74)(7.43 - 11.37)20:0 Arachidic0.25 (0.0038)0.25 (0.0038)0.0016 (0.0054)-0.013, 0.0170.7770.19, 0.34(0.25 - 0.26)(0.25 - 0.26)(-0.0021 - 0.0080)(0.20 - 0.30)20:1 Eicosenoic0.073 (0.0020)0.070 (0.0020)0.0030 (0.0028)-0.0048, 0.0110.3480.022, 0.24(0.071 - 0.075)(0.069 - 0.071)(0.0011 - 0.0062)(0.065 - 0.17)22:0 Behenic0.26 (0.0035)0.28 (0.0035)-0.015 (0.0042)-0.027, -0.00360.0220.24, 0.40(0.25 - 0.27)(0.27 - 0.28)(-0.023 - -0.0038)(0.28 - 0.36)Vitamin (mg/100g dwt)Vitamin E1.15 (0.058)0.94 (0.058)0.21 (0.066)0.027, 0.390.0330, 3.49(1.10 - 1.22)(0.89 - 0.97)(0.18 - 0.24)(0.69 - 2.91)1dwt = dry weight; fwt = fresh weight; FA = fatty acid.2MON 87708 was treated with dicamba.3Mean (S.E.) = least-square mean (standard error).4Control refers to the near isogenic conventional soybean control A3525.5With 95% confidence, interval contains 99% of the valuesexpressed in the population of commercial reference soybeanvarieties. Negative limits set to zero.Monsanto Company 10-SY-210U 340 of 721Table E-5. Statistical Summary of SiteIARL Soybean Seed Anti-nutrients for MON 87708 (Treated) vs. Conventional ControlDifference (MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Anti-nutrient (% dwt)Lectin (H.U./mg dwt)2.04 (0.64)1.53 (0.64)0.52 (0.90)-1.98, 3.020.5950, 7.73(0.80 - 3.44)(0.46 - 2.70)(-0.81 - 2.98)(0.68 - 8.34)Phytic Acid1.36 (0.032)1.53 (0.032)-0.17 (0.045)-0.30, -0.0480.0180.77, 1.91(1.33 - 1.38)(1.47 - 1.62)(-0.29 - -0.10)(1.00 - 1.64)Raffinose0.38 (0.022)0.43 (0.022)-0.051 (0.020)-0.11, 0.00340.0590.13, 0.70(0.34 - 0.42)(0.40 - 0.45)(-0.10 - -0.018)(0.26 - 0.59)Stachyose3.61 (0.20)3.89 (0.20)-0.29 (0.28)-1.06, 0.490.3652.30, 4.07(3.15 - 4.02)(3.76 - 4.15)(-1.00 - 0.26)(2.50 - 3.94)Trypsin Inhibitor (TIU/mg dwt)33.59 (2.63)29.67 (2.63)3.92 (3.19)-4.94, 12.790.28622.05, 41.12(29.66 - 37.60)(25.64 - 32.71)(-3.05 - 7.87)(22.81 - 44.56)Isoflavone (μg/g dwt)Daidzein1489.23 (145.92)1447.97 (145.92)41.27 (206.36)-531.67, 614.200.8510, 2271.38(1175.46 - 1654.49)(1404.40 - 1505.77)(-258.27 - 233.35)(451.33 - 2033.05)Monsanto Company 10-SY-210U 341 of 721Table E-5 (continued). Statistical Summary of Site IARL Soybean Seed Anti-nutrients for MON 87708 (Treated) vs.Conventional ControlDifference (MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Isoflavone (μg/g dwt)Genistein1050.87 (83.29)954.99 (83.29)95.88 (117.79)-231.16, 422.920.46178.36, 1869.48(1019.09 - 1070.05)(900.10 - 984.62)(38.84 - 163.37)(533.88 - 1726.03)Glycitein108.39 (5.35)106.40 (5.35)1.98 (2.24)-4.23, 8.200.42531.24, 233.60(97.06 - 117.44)(97.49 - 111.71)(-0.44 - 5.73)(73.61 - 231.75)1dwt= dry weight; H.U. = Hemagglutinating Units; TIU = Trypsin Inhibitor Units.2MON 87708 was treated with dicamba.3Mean (S.E.) = least-square mean (standard error).4Control refers to the near isogenic conventional soybean control A35255With 95% confidence, interval contains 99% of the values expressed in the population of commercial reference soybean varieties.Negative limitsset to zero.Monsanto Company 10-SY-210U 342 of 721Table E-6. Statistical Summary of SiteIARL Soybean Forage Nutrients for MON 87708 (Treated) vs. Conventional ControlDifference (MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Proximate (% dwt)Ash9.44 (0.62)8.87 (0.62)0.57 (0.52)-0.88, 2.030.3343.36, 10.84(9.14 - 9.65)(7.58 - 10.46)(-0.81 - 1.56)(5.20 - 9.81)Carbohydrates63.02 (0.80)65.57 (0.80)-2.55 (1.13)-5.69, 0.600.08760.69, 73.46(62.21 - 63.79)(63.58 - 67.74)(-3.95 - -1.37)(62.73 - 71.72)Moisture (% fwt)82.60 (0.33)81.97 (0.33)0.63 (0.44)-0.59, 1.860.22362.08, 89.80(82.40 - 82.80)(81.40 - 82.70)(0.10 - 1.20)(70.40 - 84.10)Protein25.21 (0.54)23.00 (0.54)2.21 (0.76)0.098, 4.330.04315.69, 26.63(24.71 - 25.52)(22.15 - 24.07)(1.33 - 2.75)(18.50 - 25.86)Total Fat2.30 (0.32)2.52 (0.32)-0.22 (0.46)-1.49, 1.060.6620, 10.04(2.00 - 2.59)(2.01 - 3.27)(-0.68 - 0.042)(1.57 - 7.99)Fiber (% dwt)Acid Detergent Fiber40.04 (2.83)32.62 (2.83)7.42 (3.30)-1.75, 16.590.08816.54, 41.80(32.90 - 45.11)(28.87 - 38.15)(2.07 - 16.24)(20.98 - 39.23)Monsanto Company 10-SY-210U 343 of 721Table E-6 (continued). Statistical Summary of Site IARL Soybean Forage Nutrients for MON 87708 (Treated) vs.Conventional ControlDifference (MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Fiber (% dwt)Neutral Detergent Fiber35.16 (2.66)35.01 (2.66)0.15 (3.76)-10.30, 10.600.96920.28, 44.03(30.00 - 38.51)(27.47 - 39.42)(-8.13 - 11.03)(24.81 - 42.80)1dwt = dry weight; fwt = fresh weight.2MON 87708 was treated with dicamba.3Mean (S.E.) = least-square mean (standard error).4Control refers to the near isogenic conventional soybean control A3525.5With 95% confidence, interval contains 99% of the values expressed in the population of commercial reference soybean varieties.Negative limits set to zero.Monsanto Company 10-SY-210U 344 of 721Table E-7. Statistical Summary of SiteILCY Soybean Seed Nutrients for MON87708 (Treated) vs. Conventional ControlDifference (MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Proximate (% dwt)Ash5.10 (0.080)5.05 (0.080)0.050 (0.089)-0.20, 0.300.6024.74, 6.01(5.07 - 5.13)(4.88 - 5.22)(-0.14 - 0.23)(4.93 - 5.88)Carbohydrates36.35 (0.68)35.77 (0.68)0.58 (0.97)-2.11, 3.260.58332.07, 40.08(35.65 - 36.91)(34.11 - 37.16)(-0.38 - 2.37)(33.82 - 39.26)Moisture (% fwt)5.73 (0.20)6.44 (0.20)-0.71 (0.28)-1.49, 0.0620.0624.27, 9.58(5.17 - 6.25)(6.20 - 6.63)(-1.46 - -0.24)(5.50 - 9.23)Protein40.17 (0.40)41.72 (0.40)-1.55 (0.55)-3.08, -0.0240.04735.50, 45.19(39.44 - 40.96)(40.81 - 42.67)(-2.56 - -0.73)(37.06 - 43.42)Total Fat18.39 (0.45)17.49 (0.45)0.89 (0.63)-0.87, 2.650.23112.33, 24.10(18.25 - 18.56)(16.81 - 18.39)(-0.047 - 1.74)(15.47 - 21.34)Fiber (% dwt)Acid Detergent Fiber15.13 (0.45)14.04 (0.45)1.10 (0.61)-0.59, 2.780.14410.06, 18.04(14.86 - 15.57)(13.47 - 14.57)(0.41 - 2.10)(12.07 - 17.46)Monsanto Company 10-SY-210U 345 of 721Table E-7 (continued). Statistical Summary of Site ILCY Soybean Seed Nutrients for MON 87708 (Treated) vs. ConventionalControlDifference (MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Fiber (% dwt)Crude Fiber9.13 (0.29)8.38 (0.29)0.76 (0.41)-0.38, 1.900.1395.76, 10.76(8.84 - 9.39)(8.20 - 8.64)(0.55 - 0.97)(6.35 - 11.31)Neutral Detergent Fiber16.62 (0.63)16.85 (0.63)-0.23 (0.73)-2.27, 1.810.76611.36, 19.38(16.34 - 16.77)(15.19 - 17.99)(-1.23 - 1.56)(11.66 - 19.45)Amino Acid (% dwt)Alanine1.80 (0.014)1.81 (0.014)-0.0030 (0.020)-0.060, 0.0540.8911.56, 1.91(1.77 - 1.82)(1.78 - 1.84)(-0.028 - 0.034)(1.59 - 1.86)Arginine3.22 (0.053)3.30 (0.053)-0.081 (0.049)-0.22, 0.0560.1742.55, 3.83(3.14 - 3.28)(3.19 - 3.43)(-0.20 - 0.0059)(2.88 - 3.74)Aspartic Acid4.67 (0.037)4.76 (0.037)-0.089 (0.034)-0.18, 0.00510.0584.04, 5.13(4.59 - 4.75)(4.73 - 4.82)(-0.15 - -0.043)(4.22 - 4.94)Cystine0.60 (0.0078)0.59 (0.0078)0.010 (0.011)-0.021, 0.0410.4160.50, 0.68(0.58 - 0.62)(0.58 - 0.60)(-0.0071 - 0.034)(0.53 - 0.66)Monsanto Company 10-SY-210U 346 of 721Table E-7 (continued). Statistical Summary of Site ILCY Soybean Seed Nutrients for MON 87708 (Treated) vs. ConventionalControlDifference (MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Amino Acid (% dwt)Glutamic Acid7.43 (0.073)7.61 (0.073)-0.18 (0.056)-0.34, -0.0240.0326.28, 8.30(7.27 - 7.54)(7.52 - 7.76)(-0.25 - -0.067)(6.69 - 7.92)Glycine1.79 (0.013)1.81 (0.013)-0.021 (0.014)-0.059, 0.0180.2131.53, 1.92(1.75 - 1.81)(1.79 - 1.83)(-0.049 - 0.0022)(1.58 - 1.84)Histidine1.06 (0.0071)1.08 (0.0071)-0.020 (0.0055)-0.035, -0.00460.0220.93, 1.16(1.04 - 1.07)(1.07 - 1.09)(-0.030 - -0.0050)(0.95 - 1.13)Isoleucine1.89 (0.013)1.97 (0.013)-0.078 (0.017)-0.13, -0.0310.0101.65, 2.06(1.87 - 1.93)(1.97 - 1.97)(-0.10 - -0.037)(1.68 - 2.02)Leucine3.09 (0.022)3.17 (0.022)-0.077 (0.012)-0.11, -0.0440.0022.72, 3.39(3.04 - 3.14)(3.14 - 3.19)(-0.10 - -0.051)(2.80 - 3.27)Lysine2.66 (0.017)2.67 (0.017)-0.013 (0.013)-0.048, 0.0220.3662.33, 2.84(2.62 - 2.69)(2.65 - 2.69)(-0.041 - 0.0088)(2.38 - 2.74)Monsanto Company 10-SY-210U 347 of 721Table E-7 (continued). Statistical Summary of Site ILCY Soybean Seed Nutrients for MON 87708 (Treated) vs. ConventionalControlDifference (MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Amino Acid (% dwt)Methionine0.55 (0.012)0.57 (0.012)-0.017 (0.017)-0.064, 0.0300.3700.50, 0.64(0.53 - 0.59)(0.56 - 0.58)(-0.039 - 0.024)(0.52 - 0.63)Phenylalanine2.11 (0.022)2.13 (0.022)-0.016 (0.018)-0.067, 0.0350.4291.80, 2.30(2.08 - 2.14)(2.09 - 2.19)(-0.048 - 0.011)(1.85 - 2.21)Proline2.02 (0.019)2.06 (0.019)-0.044 (0.019)-0.096, 0.00820.0791.65, 2.26(1.98 - 2.04)(2.04 - 2.09)(-0.063 - -0.016)(1.74 - 2.16)Serine2.10 (0.027)2.08 (0.027)0.023 (0.025)-0.046, 0.0930.4041.78, 2.27(2.06 - 2.12)(2.01 - 2.15)(-0.027 - 0.054)(1.90 - 2.18)Threonine1.59 (0.015)1.59 (0.015)0.0058 (0.019)-0.046, 0.0580.7721.40, 1.69(1.55 - 1.62)(1.58 - 1.59)(-0.035 - 0.046)(1.47 - 1.64)Tryptophan0.51 (0.0090)0.48 (0.0090)0.030 (0.0084)0.0064, 0.0530.0240.38, 0.52(0.49 - 0.53)(0.47 - 0.50)(0.015 - 0.045)(0.39 - 0.50)Monsanto Company 10-SY-210U 348 of 721Table E-7 (continued). Statistical Summary of Site ILCY Soybean Seed Nutrients for MON 87708 (Treated) vs. ConventionalControlDifference (MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Amino Acid (% dwt)Tyrosine1.44 (0.033)1.43 (0.033)0.014 (0.045)-0.11, 0.140.7731.24, 1.50(1.43 - 1.46)(1.39 - 1.51)(-0.079 - 0.078)(1.26 - 1.49)Valine1.96 (0.015)2.05 (0.015)-0.090 (0.021)-0.15, -0.0310.0131.72, 2.20(1.94 - 2.01)(2.05 - 2.06)(-0.12 - -0.048)(1.73 - 2.13)Fatty Acid (% Total FA)16:0 Palmitic12.05 (0.073)11.95 (0.073)0.096 (0.089)-0.15, 0.340.3408.44, 12.56(11.95 - 12.16)(11.73 - 12.08)(-0.082 - 0.29)(9.40 - 11.54)18:0 Stearic3.91 (0.044)3.93 (0.044)-0.025 (0.062)-0.20, 0.150.7052.90, 5.19(3.88 - 3.93)(3.86 - 4.02)(-0.086 - 0.018)(3.24 - 4.67)18:1 Oleic19.74 (0.29)21.57 (0.29)-1.83 (0.41)-2.96, -0.690.01115.73, 27.19(19.44 - 19.94)(21.07 - 22.44)(-2.51 - -1.21)(17.88 - 25.31)18:2 Linoleic54.54 (0.25)53.26 (0.25)1.28 (0.35)0.31, 2.260.02148.61, 59.37(54.45 - 54.70)(52.77 - 53.74)(0.73 - 1.68)(50.95 - 56.68)Monsanto Company 10-SY-210U 349 of 721Table E-7 (continued). Statistical Summary of Site ILCY Soybean Seed Nutrients for MON 87708 (Treated) vs. ConventionalControlDifference (MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range))Fatty Acid (% Total FA)18:3 Linolenic9.07 (0.074)8.58 (0.074)0.50 (0.099)0.22, 0.770.0076.01, 12.58(8.99 - 9.16)(8.42 - 8.71)(0.36 - 0.56)(7.43 - 11.37)20:0 Arachidic0.26 (0.0035)0.26 (0.0035)-0.0048 (0.0050)-0.019, 0.00910.3930.19, 0.34(0.26 - 0.26)(0.26 - 0.27)(-0.012 - 0.00087)(0.20 - 0.30)20:1 Eicosenoic0.16 (0.0016)0.16 (0.0016)-0.0064 (0.0023)-0.013, 0.000050.0510.022, 0.24(0.16 - 0.16)(0.16 - 0.17)(-0.010 - -0.0034)(0.065 - 0.17)22:0 Behenic0.28 (0.0030)0.29 (0.0030)-0.011 (0.0042)-0.023, 0.000330.0540.24, 0.40(0.27 - 0.28)(0.28 - 0.30)(-0.019 - -0.0066)(0.28 - 0.36)Vitamin (mg/100g dwt)Vitamin E2.13 (0.077)1.86 (0.077)0.27 (0.089)0.022, 0.520.0380, 3.49(2.10 - 2.17)(1.71 - 2.11)(0.059 - 0.42)(0.69 - 2.91)1dwt = dry weight; fwt = fresh weight; FA = fatty acid.2MON 87708 was treated with dicamba.3Mean (S.E.) = least-square mean (standard error).4Control refers to the near isogenic conventional soybean control A3525.5With 95% confidence, interval contains 99% of the values expressed in the population of commercial reference soybean varieties.Negative limits set to zero.Monsanto Company 10-SY-210U 350 of 721Table E-8. Statistical Summary of SiteILCY Soybean Seed Anti-nutrients for MON 87708 (Treated) vs. Conventional ControlDifference (MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Anti-nutrient (% dwt)Lectin (H.U./mg dwt)6.89 (1.81)5.54 (1.81)1.34 (2.55)-5.75, 8.430.6260, 7.73(4.28 - 10.27)(2.14 - 10.38)(-4.27 - 8.13)(0.68 - 8.34)Phytic Acid1.12 (0.032)1.16 (0.032)-0.037 (0.045)-0.16, 0.0880.4560.77, 1.91(1.08 - 1.20)(1.10 - 1.22)(-0.14 - 0.10)(1.00 - 1.64)Raffinose0.58 (0.013)0.57 (0.013)0.011 (0.018)-0.039, 0.0610.5750.13, 0.70(0.57 - 0.59)(0.54 - 0.60)(-0.027 - 0.046)(0.26 - 0.59)Stachyose3.33 (0.10)3.64 (0.10)-0.31 (0.14)-0.70, 0.0850.0952.30, 4.07(3.25 - 3.46)(3.43 - 3.83)(-0.58 - 0.023)(2.50 - 3.94)Trypsin Inhibitor (TIU/mg dwt)31.75 (1.10)32.78 (1.10)-1.02 (1.56)-5.35, 3.300.54622.05, 41.12(30.61 - 33.32)(31.06 - 34.22)(-2.43 - 0.26)(22.81 - 44.56)Isoflavone (μg/g dwt)Daidzein925.54 (72.66)922.21 (72.66)3.32 (102.76)-281.99, 288.630.9750, 2271.38(899.83 - 974.38)(762.49 - 1098.08)(-198.25 - 139.91)(451.33 - 2033.05)Monsanto Company 10-SY-210U 351 of 721Table E-8 (continued). Statistical Summary of Site ILCY Soybean Seed Anti-nutrients for MON 87708 (Treated) vs.Conventional ControlDifference (MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Isoflavone (μg/g dwt)Genistein655.78 (45.60)653.27 (45.60)2.51 (64.49)-176.54, 181.560.97078.36, 1869.48(594.13 - 712.01)(588.17 - 770.79)(-58.78 - 60.35)(533.88 - 1726.03)Glycitein98.02 (8.11)113.29 (8.11)-15.27 (11.47)-47.13, 16.590.25431.24, 233.60(77.67 - 112.00)(96.25 - 122.09)(-43.86 - 15.75)(73.61 - 231.75)1dwt = dry weight; H.U. = Hemagglutinating Units; TIU = Trypsin Inhibitor Units.2MON 87708 was treated with dicamba.3Mean (S.E.) = least-square mean (standard error).4Control refers to the near isogenic conventional soybean control A3525.5With 95% confidence, interval contains 99% of the valuesexpressed in the population of commercial reference soybeanvarieties. Negative limits set to zero.Monsanto Company 10-SY-210U 352 of 721Table E-9. Statistical Summary of SiteILCY Soybean Forage Nutrients for MON87708 (Treated) vs. Conventional ControlDifference (MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Proximate (% dwt)Ash6.45 (0.22)6.27 (0.22)0.18 (0.32)-0.70, 1.050.6013.36, 10.84(6.12 - 6.70)(6.10 - 6.49)(-0.37 - 0.48)(5.20 - 9.81)Carbohydrates65.26 (0.61)66.38 (0.61)-1.12 (0.87)-3.53, 1.290.26560.69, 73.46(64.42 - 65.78)(65.53 - 67.94)(-3.52 - 0.11)(62.73 - 71.72)Moisture (% fwt)73.13 (0.25)73.53 (0.25)-0.40 (0.35)-1.37, 0.570.31662.08, 89.80(72.40 - 73.70)(73.20 - 73.80)(-1.20 - 0.50)(70.40 - 84.10)Protein22.20 (0.51)21.64 (0.51)0.56 (0.68)-1.33, 2.460.45415.69, 26.63(21.25 - 23.11)(20.88 - 23.03)(-0.78 - 2.23)(18.50 - 25.86)Total Fat6.11 (0.38)5.77 (0.38)0.34 (0.52)-1.09, 1.770.5490, 10.04(5.62 - 6.88)(5.15 - 6.72)(0.17 - 0.47)(1.57 - 7.99)Fiber (% dwt)Acid Detergent Fiber30.24 (1.09)28.46 (1.09)1.78 (1.54)-2.49, 6.050.31016.54, 41.80(28.75 - 32.14)(27.05 - 30.90)(-2.15 - 5.09)(20.98 - 39.23)Monsanto Company 10-SY-210U 353 of 721Table E-9 (continued). StatisticalSummary of Site ILCY Soybean ForageNutrients for MON 87708 (Treated) vs.Conventional ControlDifference (MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Fiber (% dwt)Neutral Detergent Fiber27.72 (1.65)28.06 (1.65)-0.35 (2.33)-6.82, 6.130.88920.28, 44.03(27.34 - 27.98)(23.66 - 32.73)(-4.90 - 3.68)(24.81 - 42.80)1dwt = dry weight; fwt = fresh weight.2MON 87708 was treated with dicamba.3Mean (S.E.) = least-square mean (standard error).4Control refers to the near isogenic conventional soybean control A3525.5With 95% confidence, interval contains 99% of the valuesexpressed in the population of commercial reference soybeanvarieties. Negative limits set to zero.Monsanto Company 10-SY-210U 354 of 721Table E-10. Statistical Summary of Site ILWY Soybean SeedNutrients for MON 87708 (Treated) vs. Conventional ControlDifference (MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Proximate (% dwt)Ash5.43 (0.084)5.23 (0.084)0.20 (0.084)-0.031, 0.440.0734.74, 6.01(5.24 - 5.69)(5.13 - 5.29)(0.097 - 0.40)(4.93 - 5.88)Carbohydrates38.84 (0.44)38.29 (0.44)0.54 (0.45)-0.71, 1.790.29232.07, 40.08(38.13 - 39.21)(38.19 - 38.42)(-0.064 - 0.96)(33.82 - 39.26)Moisture (% fwt)6.96 (0.16)6.16 (0.16)0.80 (0.22)0.18, 1.420.0224.27, 9.58(6.80 - 7.17)(5.79 - 6.41)(0.63 - 1.01)(5.50 - 9.23)Protein40.88 (0.28)41.99 (0.28)-1.11 (0.38)-2.15, -0.0620.04235.50, 45.19(40.56 - 41.37)(41.72 - 42.25)(-1.54 - -0.63)(37.06 - 43.42)Total Fat14.83 (0.38)14.49 (0.38)0.34 (0.44)-0.89, 1.570.48712.33, 24.10(14.00 - 15.90)(14.40 - 14.54)(-0.53 - 1.49)(15.47 - 21.34)Fiber (% dwt)Acid Detergent Fiber13.43 (0.43)12.96 (0.43)0.47 (0.61)-1.21, 2.150.48010.06, 18.04(12.71 - 14.61)(12.48 - 13.69)(-0.71 - 2.13)(12.07 - 17.46)Monsanto Company 10-SY-210U 355 of 721Table E-10 (continued). Statistical Summaryof Site ILWY Soybean Seed Nutrients for MON 87708 (Treated) vs. ConventionalControlDifference (MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Fiber (% dwt)Crude Fiber8.22 (0.52)7.58 (0.52)0.64 (0.74)-1.40, 2.690.4305.76, 10.76(7.39 - 9.07)(7.39 - 7.82)(-0.14 - 1.68)(6.35 - 11.31)Neutral Detergent Fiber15.62 (0.72)13.78 (0.72)1.84 (0.94)-0.77, 4.450.12211.36, 19.38(13.84 - 17.83)(13.44 - 14.00)(-0.064 - 4.39)(11.66 - 19.45)Amino Acid (% dwt)Alanine1.74 (0.0076)1.75 (0.0076)-0.014 (0.0093)-0.039, 0.0120.2191.56, 1.91(1.73 - 1.76)(1.75 - 1.76)(-0.024 - -0.0071)(1.59 - 1.86)Arginine3.30 (0.037)3.57 (0.037)-0.27 (0.040)-0.38, -0.160.0022.55, 3.83(3.24 - 3.33)(3.55 - 3.60)(-0.32 - -0.23)(2.88 - 3.74)Aspartic Acid4.59 (0.017)4.67 (0.017)-0.089 (0.020)-0.14, -0.0340.0114.04, 5.13(4.55 - 4.61)(4.67 - 4.68)(-0.13 - -0.065)(4.22 - 4.94)Cystine0.62 (0.0090)0.61 (0.0090)0.014 (0.0092)-0.012, 0.0390.2050.50, 0.68(0.62 - 0.63)(0.58 - 0.62)(-0.0029 - 0.036)(0.53 - 0.66)Monsanto Company 10-SY-210U 356 of 721Table E-10 (continued). Statistical Summaryof Site ILWY Soybean Seed Nutrients for MON 87708 (Treated) vs. ConventionalControlDifference (MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Amino Acid (% dwt)Glutamic Acid7.29 (0.030)7.51 (0.030)-0.21 (0.031)-0.30, -0.130.0026.28, 8.30(7.20 - 7.35)(7.49 - 7.53)(-0.29 - -0.16)(6.69 - 7.92)Glycine1.75 (0.0053)1.77 (0.0053)-0.021 (0.0075)-0.042, 0.000330.0521.53, 1.92(1.74 - 1.76)(1.76 - 1.77)(-0.034 - -0.0096)(1.58 - 1.84)Histidine1.05 (0.0032)1.07 (0.0032)-0.017 (0.0046)-0.030, -0.00460.0190.93, 1.16(1.05 - 1.05)(1.06 - 1.07)(-0.027 - -0.0057)(0.95 - 1.13)Isoleucine1.87 (0.012)1.90 (0.012)-0.023 (0.0040)-0.034, -0.0120.0041.65, 2.06(1.85 - 1.89)(1.88 - 1.91)(-0.033 - -0.017)(1.68 - 2.02)Leucine3.02 (0.011)3.10 (0.011)-0.077 (0.0062)-0.094, -0.060<0.0012.72, 3.39(3.00 - 3.04)(3.09 - 3.13)(-0.086 - -0.061)(2.80 - 3.27)Lysine2.63 (0.0062)2.63 (0.0062)0.0011 (0.0088)-0.023, 0.0260.9042.33, 2.84(2.63 - 2.64)(2.62 - 2.64)(-0.011 - 0.0071)(2.38 - 2.74)Monsanto Company 10-SY-210U 357 of 721Table E-10 (continued). Statistical Summaryof Site ILWY Soybean Seed Nutrients for MON 87708 (Treated) vs. ConventionalControlDifference (MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Amino Acid (% dwt)Methionine0.59 (0.0074)0.58 (0.0074)0.0079 (0.010)-0.021, 0.0370.4920.50, 0.64(0.57 - 0.60)(0.56 - 0.59)(-0.016 - 0.029)(0.52 - 0.63)Phenylalanine2.01 (0.019)2.07 (0.019)-0.061 (0.021)-0.12, -0.00150.0461.80, 2.30(1.96 - 2.06)(2.05 - 2.10)(-0.085 - -0.047)(1.85 - 2.21)Proline1.94 (0.020)2.05 (0.020)-0.10 (0.028)-0.18, -0.0270.0201.65, 2.26(1.93 - 1.96)(2.01 - 2.09)(-0.16 - -0.048)(1.74 - 2.16)Serine1.98 (0.013)2.06 (0.013)-0.079 (0.013)-0.11, -0.0440.0031.78, 2.27(1.97 - 2.00)(2.02 - 2.09)(-0.095 - -0.048)(1.90 - 2.18)Threonine1.52 (0.0042)1.55 (0.0042)-0.026 (0.0049)-0.040, -0.0130.0051.40, 1.69(1.51 - 1.53)(1.54 - 1.55)(-0.030 - -0.022)(1.47 - 1.64)Tryptophan0.44 (0.0083)0.47 (0.0083)-0.025 (0.011)-0.055, 0.00610.0890.38, 0.52(0.44 - 0.45)(0.45 - 0.48)(-0.035 - -0.015)(0.39 - 0.50)Monsanto Company 10-SY-210U 358 of 721Table E-10 (continued). Statistical Summaryof Site ILWY Soybean Seed Nutrients for MON 87708 (Treated) vs. ConventionalControlDifference (MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Amino Acid (% dwt)Tyrosine1.35 (0.024)1.36 (0.024)-0.013 (0.027)-0.090, 0.0630.6511.24, 1.50(1.32 - 1.41)(1.34 - 1.40)(-0.032 - 0.0095)(1.26 - 1.49)Valine1.99 (0.013)2.01 (0.013)-0.022 (0.012)-0.057, 0.0120.1491.72, 2.20(1.95 - 2.01)(2.01 - 2.02)(-0.053 - 0.0028)(1.73 - 2.13)Fatty Acid (% Total FA)16:0 Palmitic11.26 (0.041)11.04 (0.041)0.22 (0.057)0.063, 0.380.0178.44, 12.56(11.25 - 11.27)(10.97 - 11.12)(0.15 - 0.28)(9.40 - 11.54)18:0 Stearic4.32 (0.063)4.25 (0.063)0.076 (0.089)-0.17, 0.320.4392.90, 5.19(4.23 - 4.40)(4.16 - 4.31)(0.067 - 0.085)(3.24 - 4.67)18:1 Oleic19.52 (0.25)21.14 (0.25)-1.62 (0.36)-2.62, -0.620.01015.73, 27.19(19.34 - 19.64)(20.78 - 21.55)(-1.97 - -1.43)(17.88 - 25.31)18:2 Linoleic53.74 (0.32)52.90 (0.32)0.85 (0.46)-0.43, 2.120.13948.61, 59.37(53.55 - 54.06)(52.33 - 53.20)(0.47 - 1.22)(50.95 - 56.68)Monsanto Company 10-SY-210U 359 of 721Table E-10 (continued). Statistical Summaryof Site ILWY Soybean Seed Nutrients for MON 87708 (Treated) vs. ConventionalControlDifference (MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Fatty Acid (% Total FA)18:3 Linolenic10.54 (0.10)10.05 (0.10)0.49 (0.14)0.095, 0.890.0266.01, 12.58(10.51 - 10.59)(9.89 - 10.14)(0.39 - 0.65)(7.43 - 11.37)20:0 Arachidic0.27 (0.0041)0.27 (0.0041)-0.00093 (0.0058)-0.017, 0.0150.8790.19, 0.34(0.26 - 0.27)(0.26 - 0.27)(-0.0057 - 0.0027)(0.20 - 0.30)20:1 Eicosenoic0.075 (0.0020)0.076 (0.0020)-0.0014 (0.0023)-0.0076, 0.00490.5830.022, 0.24(0.070 - 0.079)(0.075 - 0.077)(-0.0071 - 0.0019)(0.065 - 0.17)22:0 Behenic0.26 (0.0032)0.28 (0.0032)-0.019 (0.0040)-0.030, -0.00790.0080.24, 0.40(0.26 - 0.27)(0.28 - 0.29)(-0.021 - -0.017)(0.28 - 0.36)Vitamin (mg/100g dwt)Vitamin E1.18 (0.037)0.94 (0.037)0.23 (0.053)0.086, 0.380.0110, 3.49(1.08 - 1.26)(0.89 - 0.99)(0.19 - 0.31)(0.69 - 2.91)1dwt = dry weight; fwt = fresh weight; FA = fatty acid.2MON 87708 was treated with dicamba.3Mean (S.E.) = least-square mean (standard error).4Control refers to the near isogenic conventional soybean control A3525.5With 95% confidence, interval contains 99% of the valuesexpressed in the population of commercial reference soybeanvarieties. Negative limits set to zero.Monsanto Company 10-SY-210U 360 of 721Table E-11. Statistical Summary of SiteILWY Soybean Seed Anti-nutrientsfor MON 87708 (Treated) vs. ConventionalControlDifference (MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Anti-nutrient (% dwt)Lectin (H.U./mg dwt)1.10 (0.44)2.33 (0.44)-1.23 (0.43)-2.43, -0.0350.0450, 7.73(0.59 - 1.51)(1.34 - 3.68)(-2.17 - -0.75)(0.68 - 8.34)Phytic Acid1.40 (0.033)1.55 (0.033)-0.14 (0.044)-0.27, -0.0230.0300.77, 1.91(1.33 - 1.46)(1.47 - 1.61)(-0.22 - -0.054)(1.00 - 1.64)Raffinose0.37 (0.026)0.41 (0.026)-0.037 (0.036)-0.14, 0.0650.3710.13, 0.70(0.32 - 0.45)(0.41 - 0.41)(-0.086 - 0.040)(0.26 - 0.59)Stachyose3.44 (0.19)3.76 (0.19)-0.33 (0.27)-1.08, 0.430.2942.30, 4.07(3.07 - 4.02)(3.68 - 3.85)(-0.78 - 0.34)(2.50 - 3.94)Trypsin Inhibitor (TIU/mg dwt)34.32 (2.07)29.73 (2.07)4.59 (2.89)-3.43, 12.620.18722.05, 41.12(29.54 - 39.27)(25.43 - 32.22)(-2.68 - 8.72)(22.81 - 44.56)Isoflavone (μg/g dwt)Daidzein1458.08 (35.08)1271.60 (35.08)186.48 (31.48)99.09, 273.880.0040, 2271.38(1416.31 - 1535.98)(1196.71 - 1354.96)(153.17 - 225.25)(451.33 - 2033.05)Monsanto Company 10-SY-210U 361 of 721Table E-11 (continued). Statistical Summary of Site ILWYSoybean Seed Anti-nutrients for MON 87708 (Treated) vs.Conventional ControlDifference (MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Isoflavone (μg/g dwt)Genistein898.89 (28.15)860.58 (28.15)38.31 (39.82)-72.23, 148.860.39078.36, 1869.48(873.39 - 913.50)(784.27 - 913.26)(-10.80 - 129.23)(533.88 - 1726.03)Glycitein111.77 (2.15)79.70 (2.15)32.07 (2.23)25.87, 38.27<0.00131.24, 233.60(109.88 - 113.86)(77.14 - 81.62)(31.24 - 32.73)(73.61 - 231.75)1dwt = dry weight; H.U. = Hemagglutinating Units; TIU = Trypsin Inhibitor Units.2MON 87708 was treated with dicamba.3Mean (S.E.) = least-square mean (standard error).4Control refers to the near isogenic conventional soybean control A3525.5With 95% confidence, interval contains 99% of the valuesexpressed in the population of commercial reference soybeanvarieties. Negative limits set to zero.Monsanto Company 10-SY-210U 362 of 721Table E-12. Statistical Summary of Site ILWY Soybean Forage Nutrients for MON 87708 (Treated) vs. Conventional ControlDifference (MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Proximate (% dwt)Ash6.61 (0.23)6.88 (0.23)-0.27 (0.30)-1.10, 0.560.4163.36, 10.84(6.02 - 7.21)(6.82 - 6.99)(-0.79 - 0.37)(5.20 - 9.81)Carbohydrates67.99 (0.77)66.26 (0.77)1.73 (1.08)-1.28, 4.740.18560.69, 73.46(66.09 - 69.50)(65.71 - 66.67)(-0.30 - 3.78)(62.73 - 71.72)Moisture (% fwt)75.17 (0.55)75.40 (0.55)-0.23 (0.65)-2.04, 1.570.73762.08, 89.80(74.10 - 76.70)(75.10 - 75.60)(-1.40 - 1.10)(70.40 - 84.10)Protein20.93 (0.58)21.97 (0.58)-1.03 (0.82)-3.32, 1.250.27815.69, 26.63(19.27 - 22.70)(21.81 - 22.13)(-2.69 - 0.57)(18.50 - 25.86)Total Fat4.44 (0.52)4.91 (0.52)-0.47 (0.35)-1.44, 0.500.2520, 10.04(3.92 - 5.10)(4.50 - 5.63)(-0.67 - -0.19)(1.57 - 7.99)Fiber (% dwt)Acid Detergent Fiber29.18 (1.73)27.77 (1.73)1.41 (2.44)-5.36, 8.190.59316.54, 41.80(26.72 - 31.00)(25.12 - 31.00)(-4.28 - 4.71)(20.98 - 39.23)Monsanto Company 10-SY-210U 363 of 721Table E-12 (continued). Statistical Summary of Site ILWY Soybean ForageNutrients for MON 87708 (Treated) vs.Conventional ControlDifference (MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Fiber (% dwt)Neutral Detergent Fiber28.64 (1.83)33.25 (1.83)-4.61 (2.59)-11.80, 2.580.14920.28, 44.03(27.22 - 31.26)(31.89 - 34.06)(-6.63 - -0.62)(24.81 - 42.80)1dwt = dry weight; fwt = fresh weight.2MON 87708 was treated with dicamba.3Mean (S.E.) = least-square mean (standard error).4Control refers to the near isogenic conventional soybean control A3525.5With 95% confidence, interval contains 99% of the valuesexpressed in the population of commercial reference soybeanvarieties. Negative limits set to zero.Monsanto Company 10-SY-210U 364 of 721Table E-13. Statistical Summary of SiteINRC Soybean Seed Nutrients for MON87708 (Treated) vs. Conventional ControlDifference (MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Proximate (% dwt)Ash5.03 (0.11)4.95 (0.11)0.075 (0.15)-0.35, 0.500.6444.74, 6.01(4.94 - 5.18)(4.73 - 5.23)(-0.28 - 0.45)(4.93 - 5.88)Carbohydrates37.60 (0.78)35.95 (0.78)1.65 (1.10)-1.41, 4.710.20832.07, 40.08(37.15 - 38.07)(35.27 - 37.04)(1.03 - 2.05)(33.82 - 39.26)Moisture (% fwt)6.74 (0.20)6.53 (0.20)0.21 (0.29)-0.59, 1.010.5134.27, 9.58(6.48 - 7.13)(6.32 - 6.84)(-0.23 - 0.69)(5.50 - 9.23)Protein42.11 (0.53)43.58 (0.53)-1.48 (0.76)-3.57, 0.620.12235.50, 45.19(41.33 - 42.53)(43.50 - 43.69)(-2.36 - -0.97)(37.06 - 43.42)Total Fat15.30 (0.45)15.55 (0.45)-0.25 (0.56)-1.82, 1.320.67812.33, 24.10(14.54 - 15.95)(14.52 - 16.10)(-0.63 - 0.025)(15.47 - 21.34)Fiber (% dwt)Acid Detergent Fiber13.22 (0.35)12.77 (0.35)0.46 (0.46)-0.81, 1.730.37410.06, 18.04(12.64 - 13.79)(12.28 - 13.25)(-0.14 - 1.52)(12.07 - 17.46)Monsanto Company 10-SY-210U 365 of 721Table E-13 (continued). Statistical Summary of Site INRC Soybean Seed Nutrients for MON 87708 (Treated) vs. ConventionalControlDifference (MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Fiber (% dwt)Crude Fiber8.06 (0.18)6.89 (0.18)1.17 (0.25)0.47, 1.880.0095.76, 10.76(7.76 - 8.47)(6.59 - 7.12)(0.64 - 1.51)(6.35 - 11.31)Neutral Detergent Fiber15.98 (0.67)14.66 (0.67)1.32 (0.95)-1.32, 3.960.23711.36, 19.38(14.97 - 16.49)(13.24 - 15.56)(0.93 - 1.73)(11.66 - 19.45)Amino Acid (% dwt)Alanine1.81 (0.021)1.84 (0.021)-0.032 (0.022)-0.093, 0.0290.2231.56, 1.91(1.80 - 1.83)(1.80 - 1.87)(-0.072 - 0.013)(1.59 - 1.86)Arginine3.44 (0.044)3.72 (0.044)-0.27 (0.062)-0.45, -0.100.0112.55, 3.83(3.39 - 3.50)(3.64 - 3.81)(-0.38 - -0.15)(2.88 - 3.74)Aspartic Acid4.76 (0.062)4.89 (0.062)-0.13 (0.078)-0.34, 0.0870.1734.04, 5.13(4.70 - 4.80)(4.80 - 4.95)(-0.23 - 0.0034)(4.22 - 4.94)Cystine0.61 (0.0067)0.59 (0.0067)0.020 (0.0094)-0.0067, 0.0460.1070.50, 0.68(0.61 - 0.61)(0.59 - 0.60)(0.0087 - 0.029)(0.53 - 0.66)Monsanto Company 10-SY-210U 366 of 721Table E-13 (continued). Statistical Summary of Site INRC Soybean Seed Nutrients for MON 87708 (Treated) vs. ConventionalControlDifference (MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Amino Acid (% dwt)Glutamic Acid7.66 (0.11)7.93 (0.11)-0.26 (0.14)-0.66, 0.130.1386.28, 8.30(7.57 - 7.73)(7.75 - 8.02)(-0.45 - -0.018)(6.69 - 7.92)Glycine1.82 (0.020)1.85 (0.020)-0.035 (0.027)-0.11, 0.0390.2571.53, 1.92(1.80 - 1.83)(1.82 - 1.87)(-0.072 - 0.013)(1.58 - 1.84)Histidine1.09 (0.012)1.11 (0.012)-0.028 (0.016)-0.073, 0.0180.1660.93, 1.16(1.07 - 1.10)(1.10 - 1.12)(-0.056 - -0.0026)(0.95 - 1.13)Isoleucine1.94 (0.042)2.00 (0.042)-0.060 (0.057)-0.22, 0.0990.3541.65, 2.06(1.90 - 1.97)(1.95 - 2.03)(-0.11 - 0.025)(1.68 - 2.02)Leucine3.16 (0.041)3.24 (0.041)-0.082 (0.052)-0.23, 0.0610.1862.72, 3.39(3.11 - 3.19)(3.20 - 3.26)(-0.14 - -0.0086)(2.80 - 3.27)Lysine2.70 (0.030)2.72 (0.030)-0.030 (0.035)-0.13, 0.0680.4462.33, 2.84(2.66 - 2.71)(2.68 - 2.76)(-0.070 - 0.031)(2.38 - 2.74)Monsanto Company 10-SY-210U 367 of 721Table E-13 (continued). Statistical Summary of Site INRC Soybean Seed Nutrients for MON 87708 (Treated) vs. ConventionalControlDifference (MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Amino Acid (% dwt)Methionine0.58 (0.0086)0.59 (0.0086)-0.0055 (0.012)-0.039, 0.0280.6740.50, 0.64(0.58 - 0.59)(0.58 - 0.60)(-0.017 - 0.00070)(0.52 - 0.63)Phenylalanine2.15 (0.030)2.20 (0.030)-0.049 (0.040)-0.16, 0.0620.2891.80, 2.30(2.12 - 2.18)(2.13 - 2.23)(-0.11 - 0.048)(1.85 - 2.21)Proline2.07 (0.010)2.06 (0.010)0.0045 (0.015)-0.036, 0.0450.7711.65, 2.26(2.06 - 2.09)(2.05 - 2.07)(-0.0071 - 0.016)(1.74 - 2.16)Serine2.09 (0.028)2.13 (0.028)-0.038 (0.029)-0.12, 0.0430.2601.78, 2.27(2.06 - 2.12)(2.13 - 2.14)(-0.070 - -0.016)(1.90 - 2.18)Threonine1.60 (0.019)1.61 (0.019)-0.014 (0.027)-0.089, 0.0600.6221.40, 1.69(1.57 - 1.62)(1.60 - 1.62)(-0.040 - 0.012)(1.47 - 1.64)Tryptophan0.47 (0.013)0.45 (0.013)0.019 (0.018)-0.032, 0.0690.3650.38, 0.52(0.44 - 0.50)(0.43 - 0.46)(-0.015 - 0.064)(0.39 - 0.50)Monsanto Company 10-SY-210U 368 of 721Table E-13 (continued). Statistical Summary of Site INRC Soybean Seed Nutrients for MON 87708 (Treated) vs. ConventionalControlDifference (MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Amino Acid (% dwt)Tyrosine1.38 (0.024)1.44 (0.024)-0.065 (0.022)-0.13, -0.00240.0441.24, 1.50(1.35 - 1.43)(1.38 - 1.47)(-0.12 - -0.036)(1.26 - 1.49)Valine2.05 (0.045)2.12 (0.045)-0.074 (0.063)-0.25, 0.100.3051.72, 2.20(2.01 - 2.09)(2.06 - 2.16)(-0.14 - 0.026)(1.73 - 2.13)Fatty Acid (% Total FA)16:0 Palmitic11.42 (0.068)11.19 (0.068)0.23 (0.097)-0.036, 0.500.0748.44, 12.56(11.39 - 11.46)(11.14 - 11.21)(0.19 - 0.26)(9.40 - 11.54)18:0 Stearic4.18 (0.047)4.25 (0.047)-0.064 (0.066)-0.25, 0.120.3872.90, 5.19(4.11 - 4.27)(4.16 - 4.31)(-0.19 - 0.11)(3.24 - 4.67)18:1 Oleic18.78 (0.085)20.19 (0.085)-1.41 (0.093)-1.66, -1.15<0.00115.73, 27.19(18.58 - 18.95)(20.12 - 20.23)(-1.64 - -1.27)(17.88 - 25.31)18:2 Linoleic54.98 (0.10)54.43 (0.10)0.54 (0.14)0.15, 0.940.01948.61, 59.37(54.80 - 55.14)(54.32 - 54.64)(0.16 - 0.82)(50.95 - 56.68)Monsanto Company 10-SY-210U 369 of 721Table E-13 (continued). Statistical Summary of Site INRC Soybean Seed Nutrients for MON 87708 (Treated) vs. ConventionalControlDifference (MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Fatty Acid (% Total FA)18:3 Linolenic10.03 (0.050)9.31 (0.050)0.71 (0.070)0.52, 0.91<0.0016.01, 12.58(9.89 - 10.10)(9.26 - 9.40)(0.60 - 0.84)(7.43 - 11.37)20:0 Arachidic0.26 (0.0029)0.27 (0.0029)-0.0053 (0.0041)-0.017, 0.00620.2700.19, 0.34(0.26 - 0.27)(0.26 - 0.27)(-0.013 - 0.0051)(0.20 - 0.30)20:1 Eicosenoic0.072 (0.0020)0.071 (0.0020)0.0010 (0.0025)-0.0059, 0.00790.7010.022, 0.24(0.069 - 0.076)(0.069 - 0.076)(-0.00013 -0.0025)(0.065 - 0.17)22:0 Behenic0.28 (0.0033)0.29 (0.0033)-0.014 (0.0047)-0.027, -0.00120.0380.24, 0.40(0.27 - 0.29)(0.29 - 0.30)(-0.023 - 0.00015)(0.28 - 0.36)Vitamin (mg/100g dwt)Vitamin E1.28 (0.054)1.16 (0.054)0.12 (0.067)-0.065, 0.310.1460, 3.49(1.25 - 1.30)(1.10 - 1.23)(0.018 - 0.18)(0.69 - 2.91)1dwt = dry weight; fwt = fresh weight; FA = fatty acid.2MON 87708 was treated with dicamba.3Mean (S.E.) = least-square mean (standard error).4Control refers to the near isogenic conventional soybean control A3525.5With 95% confidence, interval contains 99% of the values expressed in the population of commercial referencevarieties. Negative limits set to zero.Monsanto Company 10-SY-210U 370 of 721Table E-14. Statistical Summary of Site INRC Soybean SeedAnti-nutrients for MON 87708 (Treated) vs. ConventionalControlDifference (MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Anti-nutrient (% dwt)Lectin (H.U./mg dwt)3.03 (0.32)2.56 (0.32)0.47 (0.45)-0.79, 1.740.3570, 7.73(2.75 - 3.45)(2.33 - 3.02)(-0.26 - 1.13)(0.68 - 8.34)Phytic Acid1.27 (0.071)1.19 (0.071)0.078 (0.087)-0.16, 0.320.4210.77, 1.91(1.22 - 1.34)(1.09 - 1.36)(-0.022 - 0.15)(1.00 - 1.64)Raffinose0.37 (0.023)0.40 (0.023)-0.032 (0.033)-0.12, 0.0600.3840.13, 0.70(0.33 - 0.43)(0.36 - 0.43)(-0.098 - 0.069)(0.26 - 0.59)Stachyose3.14 (0.077)3.46 (0.077)-0.32 (0.11)-0.62, -0.0150.0432.30, 4.07(3.12 - 3.17)(3.33 - 3.67)(-0.51 - -0.21)(2.50 - 3.94)Trypsin Inhibitor (TIU/mg dwt)29.17 (1.79)29.28 (1.79)-0.12 (1.83)-5.21, 4.970.95222.05, 41.12(26.09 - 33.09)(25.22 - 31.77)(-4.76 - 3.09)(22.81 - 44.56)Isoflavone (μg/g dwt)Daidzein1683.50 (67.03)1419.40 (67.03)264.10 (94.79)0.92, 527.280.0490, 2271.38(1593.24 - 1777.49)(1416.92 - 1421.55)(173.52 - 360.58)(451.33 - 2033.05)Monsanto Company 10-SY-210U 371 of 721Table E-14 (continued). Statistical Summary of Site INRC Soybean Seed Anti-nutrients for MON 87708 (Treated) vs.Conventional ControlDifference (MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Isoflavone (μg/g dwt)Genistein1033.37 (50.59)862.03 (50.59)171.34 (71.54)-27.29, 369.970.07478.36, 1869.48(963.43 - 1092.19)(840.10 - 890.94)(108.39 - 204.37)(533.88 - 1726.03)Glycitein111.51 (3.23)98.42 (3.23)13.10 (3.31)3.91, 22.290.01631.24, 233.60(110.91 - 112.28)(89.42 - 103.14)(7.77 - 21.94)(73.61 - 231.75)1dwt = dry weight; H.U. = Hemagglutinating Units; TIU = Trypsin Inhibitor Units.2MON 87708 was treated with dicamba.3Mean (S.E.) = least-square mean (standard error).4Control refers to the near isogenic conventional soybean control A3525.5With 95% confidence, interval contains 99% of the values expressed in the population of commercial reference soybean varieties.Negative limits set to zero.Monsanto Company 10-SY-210U 372 of 721Table E-15. Statistical Summary of SiteINRC Soybean Forage Nutrients for MON87708 (Treated) vs. Conventional ControlDifference (MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Proximate (% dwt)Ash6.45 (0.24)6.95 (0.24)-0.50 (0.26)-1.23, 0.230.1283.36, 10.84(5.94 - 7.05)(6.84 - 7.07)(-0.89 - 0.11)(5.20 - 9.81)Carbohydrates65.19 (0.69)63.82 (0.69)1.38 (0.98)-1.34, 4.100.23260.69, 73.46(63.10 - 66.54)(62.91 - 64.44)(-1.34 - 3.03)(62.73 - 71.72)Moisture (% fwt)73.00 (0.32)72.27 (0.32)0.73 (0.39)-0.34, 1.800.12962.08, 89.80(72.40 - 73.70)(71.60 - 72.70)(-0.10 - 1.30)(70.40 - 84.10)Protein21.78 (0.41)23.33 (0.41)-1.55 (0.41)-2.69, -0.410.01915.69, 26.63(20.99 - 22.51)(22.64 - 24.11)(-2.26 - -0.73)(18.50 - 25.86)Total Fat6.54 (0.27)5.88 (0.27)0.66 (0.39)-0.42, 1.730.1640, 10.04(6.12 - 7.34)(5.39 - 6.19)(-0.069 - 1.96)(1.57 - 7.99)Fiber (% dwt)Acid Detergent Fiber26.46 (1.50)23.83 (1.50)2.63 (2.13)-3.27, 8.540.28316.54, 41.80(23.30 - 31.06)(22.93 - 25.53)(-0.51 - 8.13)(20.98 - 39.23)Monsanto Company 10-SY-210U 373 of 721Table E-15 (continued). Statistical Summary of Site INRC Soybean ForageNutrients for MON 87708 Treated) vs.Conventional ControlDifference (MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Fiber (% dwt)Neutral Detergent Fiber27.20 (1.62)26.11 (1.62)1.08 (2.04)-4.59, 6.760.62320.28, 44.03(24.21 - 31.27)(23.91 - 29.42)(0.30 - 1.85)(24.81 - 42.80)1dwt = dry weight; fwt = fresh weight.2MON 87708 was treated with dicamba.3Mean (S.E.) = least-square mean (standard error).4Control refers to the near isogenic conventional soybean control A3525.5With 95% confidence, interval contains 99% of the valuesexpressed in the population of commercial reference soybeanvarieties. Negative limits set to zero.Monsanto Company 10-SY-210U 374 of 721Table E-16. Statistical Summary of SitePAHM Soybean Seed Nutrients for MON87708 (Treated) vs. Conventional ControlDifference (MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Proximate (% dwt)Ash5.33 (0.11)5.05 (0.11)0.28 (0.16)-0.16, 0.720.1484.74, 6.01(5.27 - 5.39)(4.96 - 5.17)(0.15 - 0.44)(4.93 - 5.88)Carbohydrates38.30 (0.51)35.23 (0.51)3.07 (0.64)1.29, 4.840.00832.07, 40.08(37.69 - 38.65)(34.49 - 35.75)(2.23 - 4.07)(33.82 - 39.26)Moisture (% fwt)7.84 (0.22)10.50 (0.22)-2.66 (0.22)-3.27, -2.04<0.0014.27, 9.58(7.38 - 8.47)(10.40 - 10.60)(-3.12 - -2.13)(5.50 - 9.23)Protein40.25 (0.41)43.69 (0.41)-3.43 (0.51)-4.86, -2.010.00235.50, 45.19(39.00 - 41.05)(43.46 - 43.85)(-4.84 - -2.70)(37.06 - 43.42)Total Fat16.10 (0.63)16.05 (0.63)0.050 (0.89)-2.41, 2.510.95712.33, 24.10(14.95 - 18.03)(15.64 - 16.85)(-1.90 - 2.37)(15.47 - 21.34)Fiber (% dwt)Acid Detergent Fiber12.91 (0.35)11.96 (0.35)0.96 (0.43)-0.24, 2.150.08910.06, 18.04(12.45 - 13.21)(11.62 - 12.17)(0.37 - 1.44)(12.07 - 17.46)Monsanto Company 10-SY-210U 375 of 721Table E-16 (continued). StatisticalSummary of Site PAHM Soybean SeedNutrients for MON 87708 (Treated) vs.Conventional ControlDifference (MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Fiber (% dwt)Crude Fiber8.30 (0.63)6.61 (0.63)1.68 (0.71)-0.30, 3.660.0775.76, 10.76(6.23 - 9.65)(6.05 - 6.98)(0.18 - 2.67)(6.35 - 11.31)Neutral Detergent Fiber13.97 (0.36)13.11 (0.36)0.86 (0.50)-0.54, 2.250.16411.36, 19.38(13.43 - 14.97)(12.63 - 13.62)(-0.18 - 1.88)(11.66 - 19.45)Amino Acid (% dwt)Alanine1.75 (0.017)1.86 (0.017)-0.11 (0.024)-0.17, -0.0420.0101.56, 1.91(1.74 - 1.77)(1.82 - 1.90)(-0.16 - -0.054)(1.59 - 1.86)Arginine3.25 (0.053)3.88 (0.053)-0.63 (0.074)-0.83, -0.420.0012.55, 3.83(3.09 - 3.36)(3.83 - 3.93)(-0.83 - -0.47)(2.88 - 3.74)Aspartic Acid4.56 (0.041)4.94 (0.041)-0.38 (0.057)-0.54, -0.220.0024.04, 5.13(4.45 - 4.63)(4.90 - 5.01)(-0.56 - -0.27)(4.22 - 4.94)Cystine0.62 (0.0062)0.59 (0.0062)0.028 (0.0081)0.0061, 0.0510.0240.50, 0.68(0.60 - 0.63)(0.59 - 0.60)(0.0092 - 0.039)(0.53 - 0.66)Monsanto Company 10-SY-210U 376 of 721Table E-16 (continued). StatisticalSummary of Site PAHM Soybean SeedNutrients for MON 87708 (Treated) vs.Conventional ControlDifference (MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Amino Acid (% dwt)Glutamic Acid7.28 (0.081)8.00 (0.081)-0.73 (0.11)-1.05, -0.410.0036.28, 8.30(7.06 - 7.40)(7.91 - 8.14)(-1.09 - -0.54)(6.69 - 7.92)Glycine1.73 (0.015)1.86 (0.015)-0.13 (0.022)-0.19, -0.0660.0041.53, 1.92(1.69 - 1.75)(1.83 - 1.89)(-0.20 - -0.076)(1.58 - 1.84)Histidine1.05 (0.0089)1.13 (0.0089)-0.085 (0.013)-0.12, -0.0500.0020.93, 1.16(1.02 - 1.06)(1.11 - 1.14)(-0.12 - -0.053)(0.95 - 1.13)Isoleucine1.85 (0.026)2.00 (0.026)-0.15 (0.036)-0.25, -0.0510.0141.65, 2.06(1.79 - 1.90)(1.94 - 2.04)(-0.24 - -0.046)(1.68 - 2.02)Leucine3.03 (0.027)3.28 (0.027)-0.24 (0.038)-0.35, -0.140.0022.72, 3.39(2.96 - 3.09)(3.24 - 3.32)(-0.36 - -0.15)(2.80 - 3.27)Lysine2.60 (0.023)2.75 (0.023)-0.15 (0.032)-0.24, -0.0600.0092.33, 2.84(2.53 - 2.65)(2.71 - 2.77)(-0.23 - -0.091)(2.38 - 2.74)Monsanto Company 10-SY-210U 377 of 721Table E-16 (continued). StatisticalSummary of Site PAHM Soybean SeedNutrients for MON 87708 (Treated) vs.Conventional ControlDifference (MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Amino Acid (% dwt)Methionine0.58 (0.0090)0.58 (0.0090)-0.00056 (0.0072)-0.021, 0.0200.9420.50, 0.64(0.57 – 0.60)(0.57 – 0.60)(-0.0027 –0.00085)(0.52 – 0.63)Phenylalanine2.04 (0.027)2.21 (0.027)-0.18 (0.038)-0.28, -0.0690.0101.80, 2.30(2.00 – 2.07)(2.16 – 2.27)(-0.27 - -0.083)(1.85 – 2.21)Proline1.98 (0.024)2.10 (0.024)-0.13 (0.032)-0.21, -0.0380.0161.65, 2.26(1.94 – 2.00)(2.08 – 2.13)(-0.14 - -0.11)(1.74 – 2.16)Serine2.04 (0.035)2.16 (0.035)-0.12 (0.050)-0.26, 0.0180.0731.78, 2.27(2.01 – 2.06)(2.06 – 2.21)(-0.19 – 0.0063)(1.90 – 2.18)Threonine1.55 (0.015)1.62 (0.015)-0.069 (0.021)-0.13, -0.0110.0291.40, 1.69(1.52 – 1.57)(1.60 – 1.64)(-0.10 - -0.032)(1.47 – 1.64)Tryptophan0.47 (0.012)0.46 (0.012)0.010 (0.017)-0.036, 0.0570.5670.38, 0.52(0.44 – 0.48)(0.45 – 0.46)(-0.0098 – 0.023)(0.39 – 0.50)Monsanto Company 10-SY-210U 378 of 721Table E-16 (continued). StatisticalSummary of Site PAHM Soybean SeedNutrients for MON 87708 (Treated) vs.Conventional ControlDifference (MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Amino Acid (% dwt)Tyrosine1.35 (0.027)1.49 (0.027)-0.14 (0.032)-0.23, -0.0520.0111.24, 1.50(1.28 – 1.43)(1.48 – 1.52)(-0.20 - -0.088)(1.26 – 1.49)Valine1.95 (0.029)2.13 (0.029)-0.17 (0.041)-0.29, -0.0610.0121.72, 2.20(1.89 – 2.00)(2.05 – 2.17)(-0.27 - -0.050)(1.73 – 2.13)Fatty Acid (% Total FA)16:0 Palmitic11.74 (0.12)11.49 (0.12)0.25 (0.15)-0.17, 0.670.1698.44, 12.56(11.39 – 12.07)(11.38 – 11.55)(-0.15 – 0.52)(9.40 – 11.54)18:0 Stearic3.85 (0.12)3.76 (0.12)0.093 (0.14)-0.30, 0.480.5442.90, 5.19(3.60 – 4.12)(3.67 – 3.91)(-0.078 – 0.42)(3.24 – 4.67)18:1 Oleic18.58 (0.31)20.01 (0.31)-1.43 (0.35)-2.40, -0.450.01515.73, 27.19(17.85 – 19.42)(19.60 – 20.32)(-1.74 - -0.90)(17.88 – 25.31)18:2 Linoleic54.89 (0.41)54.68 (0.41)0.21 (0.53)-1.25, 1.670.70848.61, 59.37(53.59 – 55.67)(54.18 – 54.99)(-0.59 – 0.68)(50.95 – 56.68)Monsanto Company 10-SY-210U 379 of 721Table E-16 (continued). StatisticalSummary of Site PAHM Soybean SeedNutrients for MON 87708 (Treated) vs.Conventional ControlDifference (MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Fatty Acid (% Total FA)18:3 Linolenic10.33 (0.21)9.47 (0.21)0.85 (0.16)0.40, 1.310.0066.01, 12.58(9.91 - 10.88)(9.13 - 9.68)(0.59 - 1.20)(7.43 - 11.37)20:0 Arachidic0.25 (0.0063)0.24 (0.0063)0.0034 (0.0071)-0.016, 0.0230.6520.19, 0.34(0.23 - 0.26)(0.24 - 0.25)(-0.0079 - 0.020)(0.20 - 0.30)20:1 Eicosenoic0.091 (0.015)0.072 (0.015)0.018 (0.022)-0.042, 0.0780.4450.022, 0.24(0.073 - 0.12)(0.068 - 0.075)(-0.0014 - 0.050)(0.065 - 0.17)22:0 Behenic0.27 (0.0046)0.27 (0.0046)-0.0067 (0.0034)-0.016, 0.00280.1210.24, 0.40(0.26 - 0.28)(0.27 - 0.28)(-0.012 - 0.0024)(0.28 - 0.36)Vitamin (mg/100g dwt)Vitamin E1.32 (0.10)1.23 (0.10)0.097 (0.022)0.037, 0.160.0100, 3.49(1.21 - 1.54)(1.11 - 1.40)(0.049 - 0.14)(0.69 - 2.91)1dwt = dry weight; fwt = fresh weight; FA = fatty acid.2MON 87708 was treated with dicamba.3Mean (S.E.) = least-square mean (standard error)4Control refers to the near isogenic conventional soybean control A3525.5With 95% confidence, interval contains 99% of the valuesexpressed in the population of commercial reference soybeanvarieties. Negative limits set to zero.Monsanto Company 10-SY-210U 380 of 721Table E-17. Statistical Summary of Site PAHM Soybean Seed Anti-nutrients for MON 87708 (Treated) vs. ConventionalControlDifference (MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Anti-nutrient (% dwt)Lectin (H.U./mg dwt)2.81 (0.80)3.85 (0.80)-1.03 (1.03)-3.88, 1.810.3700, 7.73(2.08 - 3.34)(3.28 - 4.45)(-1.73 - 0.051)(0.68 - 8.34)Phytic Acid1.35 (0.077)1.50 (0.077)-0.15 (0.092)-0.40, 0.110.1790.77, 1.91(1.13 - 1.51)(1.41 - 1.62)(-0.28 - -0.054)(1.00 - 1.64)Raffinose0.47 (0.047)0.54 (0.047)-0.072 (0.066)-0.26, 0.110.3390.13, 0.70(0.32 - 0.55)(0.49 - 0.57)(-0.24 - 0.058)(0.26 - 0.59)Stachyose3.29 (0.18)3.36 (0.18)-0.075 (0.25)-0.77, 0.620.7772.30, 4.07(3.19 - 3.47)(3.07 - 3.90)(-0.69 - 0.40)(2.50 - 3.94)Trypsin Inhibitor (TIU/mg dwt)32.53 (4.33)30.39 (4.33)2.14 (5.59)-13.38, 17.660.72122.05, 41.12(27.64 - 36.16)(26.59 - 33.33)(1.05 - 2.83)(22.81 - 44.56)Isoflavone (μg/g dwt)Daidzein1918.51 (144.27)1642.38 (144.27)276.14 (204.03)-290.35, 842.620.2470, 2271.38(1565.54 - 2305.26)(1510.07 - 1729.91)(-121.61 - 795.19)(451.33 - 2033.05)Monsanto Company 10-SY-210U 381 of 721Table E-17 (continued). Statistical Summary of Site PAHM Soybean Seed Anti-nutrients for MON 87708 (Treated) vs.Conventional ControlDifference (MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Isoflavone (μg/g dwt)Genistein1196.16 (107.80)1101.98 (107.80)94.18 (152.45)-329.09, 517.450.57078.36, 1869.48(976.03 - 1496.78)(983.22 - 1162.01)(-185.98 - 513.56)(533.88 - 1726.03)Glycitein110.37 (7.95)81.44 (7.95)28.93 (11.25)-2.30, 60.160.06131.24, 233.60(93.26 - 119.09)(68.68 - 90.51)(2.75 - 50.41)(73.61 - 231.75)1dwt = dry weight; H.U. = Hemagglutinating Units; TIU = Trypsin Inhibitor Units.2 MON 87708 was treated with dicamba.3Mean (S.E.) = least-square mean (standard error).4Control refers to the near isogenic conventional soybean control A3525.5With 95% confidence, interval contains 99% of the valuesexpressed in the population of commercial reference soybeanvarieties. Negative limits set to zero.Monsanto Company 10-SY-210U 382 of 721Table E-18. Statistical Summary of SitePAHM Soybean Forage Nutrients for MON 87708 (Treated) vs. Conventional ControlDifference (MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Proximate (% dwt)Ash7.51 (0.26)7.88 (0.32)-0.37 (0.41)-1.67, 0.940.4383.36, 10.84(7.18 – 8.13)(7.67 – 8.09)(-0.86 - -0.49)(5.20 – 9.81)Carbohydrates70.95 (1.04)65.81 (1.16)5.14 (1.03)1.85, 8.430.01560.69, 73.46(69.23 – 73.31)(65.74 – 66.41)(3.49 – 6.90)(62.73 – 71.72)Moisture (% fwt)74.27 (0.63)74.91 (0.63)-0.64 (0.15)-1.11, -0.180.02162.08, 89.80(73.40 – 75.40)(73.80 – 74.90)(-0.90 - -0.40)(70.40 – 84.10)Protein17.47 (1.26)21.96 (1.45)-4.49 (1.45)-9.10, 0.120.05315.69, 26.63(15.23 – 19.58)(21.49 – 21.91)(-6.26 - -2.34)(18.50 – 25.86)Total Fat3.97 (0.26)4.18 (0.31)-0.20 (0.31)-1.19, 0.780.5530, 10.04(3.82 – 4.21)(4.30 – 4.35)(-0.42 - -0.14)(1.57 – 7.99)Fiber (% dwt)Acid Detergent Fiber26.97 (1.95)26.02 (2.39)0.96 (3.08)-8.86, 10.770.77616.54, 41.80(25.46 – 29.89)(21.79 – 30.24)(-4.78 – 8.09)(20.98 – 39.23)Monsanto Company 10-SY-210U 383 of 721Table E-18 (continued). Statistical Summary of Site PAHM Soybean ForageNutrients for MON 87708 (Treated) vs.Conventional ControlDifference (MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)CommercialTolerance Interval5(Range)Fiber (% dwt)Neutral Detergent Fiber29.45 (1.01)30.20 (1.24)-0.75 (1.60)-5.83, 4.340.67220.28, 44.03(27.00 - 32.07)(30.00 - 30.40)(-3.40 - -0.71)(24.81 - 42.80)1dwt = dry weight; fwt = fresh weight.2 MON 87708 was treated with dicamba.3Mean (S.E.) = least-square mean (standard error)4Control refers to the near isogenic conventional soybean control A3525.5With 95% confidence, interval contains 99% of the valuesexpressed in the population of commercial reference soybeanvarieties. Negative limits set to zero.Monsanto Company 10-SY-210U 384 of 721Table E-19. Statistical Summary of SiteIARL Soybean Seed Nutrients for MON87708 (Untreated) vs. Conventional ControlDifference (MON 87708minus Control)Analytical Component (Units)1MON 877082Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)ConventionalTolerance Interval5(Range)Proximate (% dw)Ash5.35 (0.070)5.29 (0.070)0.058 (0.098)-0.22, 0.330.5894.74, 6.01(5.24 - 5.46)(5.19 - 5.47)(-0.0028 - 0.16)(4.93 - 5.88)Carbohydrates37.68 (0.56)37.97 (0.56)-0.29 (0.55)-1.83, 1.250.62832.07, 40.08(36.27 - 39.10)(37.17 - 38.45)(-0.90 - 0.65)(33.82 - 39.26)Moisture (% fw)7.84 (0.28)6.07 (0.28)1.77 (0.39)0.68, 2.860.0104.27, 9.58(7.39 - 8.73)(5.84 - 6.36)(1.05 - 2.89)(5.50 - 9.23)Protein40.95 (0.27)41.09 (0.27)-0.15 (0.16)-0.60, 0.310.42035.50, 45.19(40.72 - 41.20)(40.69 - 41.74)(-0.54 - 0.069)(37.06 - 43.42)Total Fat16.03 (0.47)15.61 (0.47)0.42 (0.66)-1.41, 2.250.56212.33, 24.10(14.69 - 17.31)(15.38 - 15.82)(-0.69 - 1.49)(15.47 - 21.34)Fiber (% dw)Acid Detergent Fiber13.78 (0.38)12.60 (0.38)1.18 (0.54)-0.33, 2.690.09610.06, 18.04(12.82 - 14.36)(11.92 - 13.17)(-0.35 - 2.45)(12.07 - 17.46)Monsanto Company 10-SY-210U 385 of 721Table E-19 (continued). Statistical Summary of Site IARL Soybean Seed Nutrients for MON 87708 (Untreated) vs. ConventionalControlDifference (MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)ConventionalTolerance Interval5(Range)Fiber (% dw)Crude Fiber7.63 (0.33)7.41 (0.33)0.23 (0.46)-1.05, 1.500.6505.76, 10.76(6.85 - 8.07)(7.17 - 7.60)(-0.60 - 0.90)(6.35 - 11.31)Neutral Detergent Fiber14.50 (0.89)13.27 (0.89)1.22 (1.25)-2.26, 4.710.38411.36, 19.38(13.26 - 15.98)(11.81 - 14.42)(-0.34 - 4.17)(11.66 - 19.45)Amino Acid (% dw)Alanine1.75 (0.026)1.74 (0.026)0.0049 (0.036)-0.096, 0.110.9001.56, 1.91(1.72 - 1.79)(1.69 - 1.77)(-0.028 - 0.030)(1.59 - 1.86)Arginine3.35 (0.055)3.45 (0.055)-0.099 (0.061)-0.27, 0.0700.1772.55, 3.83(3.30 - 3.41)(3.27 - 3.56)(-0.19 - 0.037)(2.88 - 3.74)Aspartic Acid4.60 (0.060)4.63 (0.060)-0.033 (0.084)-0.27, 0.200.7114.04, 5.13(4.57 - 4.62)(4.46 - 4.74)(-0.11 - 0.10)(4.22 - 4.94)Cystine0.61 (0.0094)0.59 (0.0094)0.018 (0.013)-0.019, 0.0540.2560.50, 0.68(0.61 - 0.61)(0.56 - 0.62)(-0.0068 - 0.044)(0.53 - 0.66)Monsanto Company 10-SY-210U 386 of 721Table E-19 (continued). Statistical Summary of Site IARL Soybean Seed Nutrients for MON 87708 (Untreated) vs. ConventionalControlDifference (MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)ConventionalTolerance Interval5(Range)Amino Acid (% dw)Glutamic Acid7.33 (0.10)7.41 (0.10)-0.074 (0.15)-0.48, 0.340.6446.28, 8.30(7.25 - 7.43)(7.12 - 7.58)(-0.19 - 0.12)(6.69 - 7.92)Glycine1.75 (0.024)1.76 (0.024)-0.013 (0.034)-0.11, 0.0810.7201.53, 1.92(1.74 - 1.76)(1.70 - 1.79)(-0.049 - 0.041)(1.58 - 1.84)Histidine1.05 (0.014)1.06 (0.014)-0.0055 (0.019)-0.059, 0.0480.7860.93, 1.16(1.05 - 1.06)(1.02 - 1.08)(-0.026 - 0.029)(0.95 - 1.13)Isoleucine1.87 (0.038)1.88 (0.038)-0.011 (0.054)-0.16, 0.140.8511.65, 2.06(1.86 - 1.88)(1.79 - 1.94)(-0.068 - 0.085)(1.68 - 2.02)Leucine3.05 (0.041)3.07 (0.041)-0.020 (0.058)-0.18, 0.140.7462.72, 3.39(3.01 - 3.08)(2.96 - 3.14)(-0.065 - 0.055)(2.80 - 3.27)Lysine2.62 (0.030)2.62 (0.030)-0.00013 (0.042)-0.12, 0.120.9972.33, 2.84(2.61 - 2.62)(2.54 - 2.66)(-0.047 - 0.083)(2.38 - 2.74)Monsanto Company 10-SY-210U 387 of 721Table E-19 (continued). Statistical Summary of Site IARL Soybean Seed Nutrients for MON 87708 (Untreated) vs. ConventionalControlDifference (MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)ConventionalTolerance Interval5(Range)Amino Acid (% dw)Methionine0.58 (0.013)0.56 (0.013)0.019 (0.019)-0.034, 0.0720.3760.50, 0.64(0.57 - 0.59)(0.53 - 0.60)(-0.0030 - 0.056)(0.52 - 0.63)Phenylalanine2.03 (0.036)2.03 (0.036)-0.00029 (0.050)-0.14, 0.140.9951.80, 2.30(1.98 - 2.09)(1.95 - 2.09)(-0.024 - 0.022)(1.85 - 2.21)Proline1.94 (0.035)1.98 (0.035)-0.048 (0.050)-0.19, 0.0910.3901.65, 2.26(1.89 - 1.98)(1.89 - 2.07)(-0.10 - 0.043)(1.74 - 2.16)Serine2.02 (0.044)2.00 (0.044)0.017 (0.036)-0.082, 0.120.6531.78, 2.27(1.91 - 2.10)(1.95 - 2.08)(-0.043 - 0.073)(1.90 - 2.18)Threonine1.53 (0.023)1.54 (0.023)-0.0028 (0.023)-0.067, 0.0610.9091.40, 1.69(1.49 - 1.57)(1.51 - 1.58)(-0.026 - 0.033)(1.47 - 1.64)Tryptophan0.44 (0.0092)0.45 (0.0092)-0.012 (0.013)-0.048, 0.0240.4050.38, 0.52(0.41 - 0.46)(0.44 - 0.47)(-0.053 - 0.018)(0.39 - 0.50)Monsanto Company 10-SY-210U 388 of 721Table E-19 (continued). Statistical Summary of Site IARL Soybean Seed Nutrients for MON 87708 (Untreated) vs. ConventionalControlDifference (MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)ConventionalTolerance Interval5(Range)Amino Acid (% dw)Tyrosine1.40 (0.013)1.38 (0.013)0.012 (0.016)-0.033, 0.0570.4941.24, 1.50(1.38 - 1.40)(1.37 - 1.42)(-0.011 - 0.032)(1.26 - 1.49)Valine1.98 (0.046)1.99 (0.046)-0.0086 (0.065)-0.19, 0.170.9001.72, 2.20(1.97 - 2.00)(1.90 - 2.05)(-0.077 - 0.097)(1.73 - 2.13)Fatty Acid (% Total FA)16:0 Palmitic11.51 (0.051)11.00 (0.051)0.51 (0.062)0.34, 0.680.0018.44, 12.56(11.39 - 11.63)(10.92 - 11.08)(0.41 - 0.59)(9.40 - 11.54)18:0 Stearic3.95 (0.067)4.00 (0.067)-0.052 (0.095)-0.32, 0.210.6132.90, 5.19(3.75 - 4.06)(3.99 - 4.01)(-0.23 - 0.044)(3.24 - 4.67)18:1 Oleic19.28 (0.20)21.67 (0.20)-2.39 (0.28)-3.17, -1.610.00115.73, 27.19(18.77 - 19.68)(21.48 - 21.78)(-2.71 - -2.07)(17.88 - 25.31)18:2 Linoleic54.30 (0.33)52.70 (0.33)1.60 (0.46)0.32, 2.890.02548.61, 59.37(53.70 - 55.34)(52.66 - 52.73)(1.04 - 2.61)(50.95 - 56.68)Monsanto Company 10-SY-210U 389 of 721Table E-19 (continued). Statistical Summary of Site IARL Soybean Seed Nutrients for MON 87708 (Untreated) vs. ConventionalControlDifference (MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)ConventionalTolerance Interval5(Range)Fatty Acid (% Total FA)18:3 Linolenic10.38 (0.13)10.04 (0.13)0.34 (0.19)-0.18, 0.860.1446.01, 12.58(9.94 - 10.60)(10.00 - 10.12)(-0.18 - 0.61)(7.43 - 11.37)20:0 Arachidic0.25 (0.0038)0.25 (0.0038)-0.0024 (0.0054)-0.017, 0.0130.6800.19, 0.34(0.24 - 0.26)(0.25 - 0.26)(-0.014 - 0.0033)(0.20 - 0.30)20:1 Eicosenoic0.071 (0.0020)0.070 (0.0020)0.00087 (0.0028)-0.0070, 0.00870.7730.022, 0.24(0.065 - 0.076)(0.069 - 0.071)(-0.0039 - 0.0043)(0.065 - 0.17)22:0 Behenic0.26 (0.0035)0.28 (0.0035)-0.014 (0.0042)-0.025, -0.00210.0300.24, 0.40(0.26 - 0.27)(0.27 - 0.28)(-0.017 - -0.010)(0.28 - 0.36)Vitamin (mg/100g dw)Vitamin E1.26 (0.058)0.94 (0.058)0.32 (0.066)0.13, 0.500.0080, 3.49(1.11 - 1.42)(0.89 - 0.97)(0.23 - 0.46)(0.69 - 2.91)1dw = dry weight; fw = fresh weight; FA = fatty acid.2MON 87708 plants were not sprayed with dicamba, but received another conventional treatment as was done for the conventional control.3Mean (S.E.) = least-square mean (standard error).4Control refers to the non-biotechnology derived, conventional control (A3525).5With 95% confidence, interval contains 99% of the values expressed in the population of conventional substances. Negative limits set to zero.Monsanto Company 10-SY-210U 390 of 721Table E-20. Statistical Summary of Site IARL Soybean Seed Anti-Nutrientsfor MON 87708 (Untreated) vs. ConventionalControlDifference(MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)ConventionalTolerance Interval5(Range)Anti-nutrientLectin (H.U./mg dw)2.98 (0.64)1.53 (0.64)1.46 (0.90)-1.04, 3.960.1800, 7.73(2.35 - 3.87)(0.46 - 2.70)(-0.35 - 2.46)(0.68 - 8.34)Phytic Acid (% dw)1.43 (0.032)1.53 (0.032)-0.10 (0.045)-0.23, 0.0240.0880.77, 1.91(1.38 - 1.47)(1.47 - 1.62)(-0.18 - -0.040)(1.00 - 1.64)Raffinose (% dw)0.41 (0.022)0.43 (0.022)-0.024 (0.020)-0.079, 0.0300.2840.13, 0.70(0.38 - 0.45)(0.40 - 0.45)(-0.063 - 0.00015)(0.26 - 0.59)Stachyose (% dw)3.30 (0.20)3.89 (0.20)-0.59 (0.28)-1.37, 0.190.1012.30, 4.07(2.94 - 3.59)(3.76 - 4.15)(-0.82 - -0.18)(2.50 - 3.94)Trypsin Inhibitor (TIU/mg dw)29.97 (2.63)29.67 (2.63)0.30 (3.19)-8.57, 9.170.92922.05, 41.12(23.32 - 33.42)(25.64 - 32.71)(-2.32 - 2.51)(22.81 - 44.56)Isoflavone (μg/g dw)Daidzein1734.44 (145.92)1447.97 (145.92)286.47 (206.36)-286.47, 859.410.2370, 2271.38(1522.84 - 2125.56)(1404.40 - 1505.77)(17.08 - 691.83)(451.33 - 2033.05)Monsanto Company 10-SY-210U 391 of 721Table E-20 (continued). Statistical Summary of Site IARLSoybean Seed Anti-Nutrients for MON 87708 (Untreated) vs.Conventional ControlDifference (MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)ConventionalTolerance Interval5(Range)Isoflavone (μg/g dw)Genistein1099.12 (83.29)954.99 (83.29)144.13 (117.79)-182.91, 471.170.28878.36, 1869.48(954.54 - 1380.52)(900.10 - 984.62)(-22.32 - 400.27)(533.88 - 1726.03)Glycitein110.70 (5.35)106.40 (5.35)4.30 (2.24)-1.92, 10.520.12731.24, 233.60(99.92 - 117.72)(97.49 - 111.71)(2.43 - 7.73)(73.61 - 231.75)1dw = dry weight; H.U. = Hemagglutinating Units; TIU = Trypsin Inhibitor Units.2 MON 87708 plants were not sprayed with dicamba, but received another conventional treatmentas was done for the conventionalcontrol.3Mean (S.E.) = least-square mean (standard error).4Control refers to the non-biotechnology derived, conventional control (A3525).5With 95% confidence, interval contains 99% of the values expressed in the population of conventional substances. Negative limits set to zero.Monsanto Company 10-SY-210U 392 of 721Table E-21. Statistical Summary of Site IARL Soybean Forage Nutrients for MON 87708 (Untreated) vs. ConventionalControlDifference (MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)ConventionalTolerance Interval5(Range)Proximate (% dw)Ash8.37 (0.62)8.87 (0.62)-0.50 (0.52)-1.95, 0.950.3953.36, 10.84(7.23 - 9.45)(7.58 - 10.46)(-1.01 - -0.13)(5.20 - 9.81)Carbohydrates62.49 (0.80)65.57 (0.80)-3.08 (1.13)-6.22, 0.0670.05360.69, 73.46(61.64 - 63.41)(63.58 - 67.74)(-6.11 - -0.17)(62.73 - 71.72)Moisture (% fw)83.47 (0.33)81.97 (0.33)1.50 (0.44)0.28, 2.720.02762.08, 89.80(82.70 - 84.10)(81.40 - 82.70)(0.90 - 2.70)(70.40 - 84.10)Protein25.76 (0.54)23.00 (0.54)2.76 (0.76)0.65, 4.880.02215.69, 26.63(24.63 - 27.04)(22.15 - 24.07)(1.54 - 4.89)(18.50 - 25.86)Total Fat3.29 (0.32)2.52 (0.32)0.77 (0.46)-0.50, 2.040.1690, 10.04(2.61 - 3.91)(2.01 - 3.27)(-0.66 - 1.63)(1.57 - 7.99)Fiber (% dw)Acid Detergent Fiber31.48 (2.83)32.62 (2.83)-1.14 (3.30)-10.31, 8.040.74816.54, 41.80(29.42 - 34.63)(28.87 - 38.15)(-3.52 - 1.51)(20.98 - 39.23)Monsanto Company 10-SY-210U 393 of 721Table E-21 (continue). Statistical Summary of Site IARL Soybean Forage Nutrients for MON 87708 (Untreated) vs. ConventionalControlDifference (MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)ConventionalTolerance Interval5(Range)Fiber (% dw)Neutral Detergent Fiber37.35 (2.66)35.01 (2.66)2.34 (3.76)-8.11, 12.790.56720.28, 44.03(37.11 - 37.80)(27.47 - 39.42)(-2.29 - 9.63)(24.81 - 42.80)1dw = dry weight; fw = fresh weight.2MON 87708 plants were not sprayed with dicamba, but received another conventional treatmentas was done for the conventionalcontrol.3Mean (S.E.) = least-square mean (standard error).4Control refers to the non-biotechnology derived, conventional control (A3525).5With 95% confidence, interval contains 99% of the values expressed in the population of conventional substances. Negative limits set to zero.Monsanto Company 10-SY-210U 394 of 721Table E-22. Statistical Summary of SiteILCY Soybean Seed Nutrients for MON87708 (Untreated) vs. Conventional ControlDifference (MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)ConventionalTolerance Interval5(Range)Proximate (% dw)Ash5.10 (0.080)5.05 (0.080)0.046 (0.089)-0.20, 0.290.6324.74, 6.01(4.94 - 5.27)(4.88 - 5.22)(0.025 - 0.059)(4.93 - 5.88)Carbohydrates35.98 (0.68)35.77 (0.68)0.21 (0.97)-2.48, 2.900.83932.07, 40.08(35.27 - 37.36)(34.11 - 37.16)(-1.89 - 3.25)(33.82 - 39.26)Moisture (% fw)5.78 (0.20)6.44 (0.20)-0.66 (0.28)-1.43, 0.110.0774.27, 9.58(5.68 - 5.88)(6.20 - 6.63)(-0.75 - -0.52)(5.50 - 9.23)Protein41.07 (0.40)41.72 (0.40)-0.65 (0.55)-2.17, 0.880.30535.50, 45.19(40.92 - 41.22)(40.81 - 42.67)(-1.60 - 0.42)(37.06 - 43.42)Total Fat17.87 (0.45)17.49 (0.45)0.37 (0.63)-1.39, 2.130.58812.33, 24.10(16.66 - 18.66)(16.81 - 18.39)(-1.73 - 1.46)(15.47 - 21.34)Fiber (% dw)Acid Detergent Fiber14.54 (0.45)14.04 (0.45)0.50 (0.61)-1.18, 2.190.45210.06, 18.04(13.36 - 15.72)(13.47 - 14.57)(-0.71 - 1.16)(12.07 - 17.46)Monsanto Company 10-SY-210U 395 of 721Table E-22 (continued). Statistical Summary of Site ILCY Soybean Seed Nutrients for MON 87708 (Untreated) vs. ConventionalControlDifference (MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)ConventionalTolerance Interval5(Range)Fiber (% dw)Crude Fiber8.08 (0.29)8.38 (0.29)-0.30 (0.41)-1.44, 0.840.5085.76, 10.76(7.30 - 8.89)(8.20 - 8.64)(-1.33 - 0.60)(6.35 - 11.31)Neutral Detergent Fiber17.05 (0.63)16.85 (0.63)0.20 (0.73)-1.84, 2.240.79711.36, 19.38(16.33 - 18.38)(15.19 - 17.99)(-1.05 - 1.27)(11.66 - 19.45)Amino Acid (% dw)Alanine1.78 (0.014)1.81 (0.014)-0.027 (0.020)-0.084, 0.0300.2591.56, 1.91(1.77 - 1.80)(1.78 - 1.84)(-0.067 - -0.0037)(1.59 - 1.86)Arginine3.21 (0.053)3.30 (0.053)-0.094 (0.049)-0.23, 0.0430.1292.55, 3.83(3.12 - 3.27)(3.19 - 3.43)(-0.21 - -0.0074)(2.88 - 3.74)Aspartic Acid4.69 (0.037)4.76 (0.037)-0.072 (0.034)-0.17, 0.0220.0994.04, 5.13(4.63 - 4.74)(4.73 - 4.82)(-0.12 - 0.0057)(4.22 - 4.94)Cystine0.60 (0.0078)0.59 (0.0078)0.013 (0.011)-0.018, 0.0430.3220.50, 0.68(0.59 - 0.61)(0.58 - 0.60)(-0.0016 - 0.030)(0.53 - 0.66)Monsanto Company 10-SY-210U 396 of 721Table E-22 (continued). Statistical Summary of Site ILCY Soybean Seed Nutrients for MON 87708 (Untreated) vs. ConventionalControlDifference (MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)ConventionalTolerance Interval5(Range)Amino Acid (% dw)Glutamic Acid7.44 (0.073)7.61 (0.073)-0.17 (0.056)-0.33, -0.0140.0396.28, 8.30(7.33 - 7.51)(7.52 - 7.76)(-0.28 - -0.042)(6.69 - 7.92)Glycine1.78 (0.013)1.81 (0.013)-0.027 (0.014)-0.066, 0.0120.1261.53, 1.92(1.77 - 1.78)(1.79 - 1.83)(-0.046 - -0.0099)(1.58 - 1.84)Histidine1.06 (0.0071)1.08 (0.0071)-0.014 (0.0055)-0.029, 0.000900.0590.93, 1.16(1.05 - 1.07)(1.07 - 1.09)(-0.019 - -0.0059)(0.95 - 1.13)Isoleucine1.94 (0.013)1.97 (0.013)-0.035 (0.017)-0.083, 0.0130.1101.65, 2.06(1.91 - 1.95)(1.97 - 1.97)(-0.058 - -0.021)(1.68 - 2.02)Leucine3.11 (0.022)3.17 (0.022)-0.058 (0.012)-0.090, -0.0250.0082.72, 3.39(3.08 - 3.13)(3.14 - 3.19)(-0.060 - -0.056)(2.80 - 3.27)Lysine2.66 (0.017)2.67 (0.017)-0.012 (0.013)-0.047, 0.0240.4132.33, 2.84(2.62 - 2.67)(2.65 - 2.69)(-0.032 - 0.017)(2.38 - 2.74)Monsanto Company 10-SY-210U 397 of 721Table E-22 (continued). Statistical Summary of Site ILCY Soybean Seed Nutrients for MON 87708 (Untreated) vs. ConventionalControlDifference (MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)ConventionalTolerance Interval5(Range)Amino Acid (% dw)Methionine0.57 (0.012)0.57 (0.012)-0.0026 (0.017)-0.049, 0.0440.8860.50, 0.64(0.56 - 0.58)(0.56 - 0.58)(-0.0052 - -0.00024)(0.52 - 0.63)Phenylalanine2.07 (0.022)2.13 (0.022)-0.064 (0.018)-0.12, -0.0140.0241.80, 2.30(2.05 - 2.08)(2.09 - 2.19)(-0.11 - -0.038)(1.85 - 2.21)Proline1.95 (0.019)2.06 (0.019)-0.11 (0.019)-0.16, -0.0580.0041.65, 2.26(1.90 - 1.98)(2.04 - 2.09)(-0.14 - -0.058)(1.74 - 2.16)Serine2.06 (0.027)2.08 (0.027)-0.015 (0.025)-0.084, 0.0550.5931.78, 2.27(2.05 - 2.08)(2.01 - 2.15)(-0.069 - 0.037)(1.90 - 2.18)Threonine1.58 (0.015)1.59 (0.015)-0.00051 (0.019)-0.052, 0.0510.9791.40, 1.69(1.56 - 1.61)(1.58 - 1.59)(-0.023 - 0.020)(1.47 - 1.64)Tryptophan0.48 (0.0090)0.48 (0.0090)-0.0026 (0.0084)-0.026, 0.0210.7700.38, 0.52(0.46 - 0.48)(0.47 - 0.50)(-0.015 - 0.017)(0.39 - 0.50)Monsanto Company 10-SY-210U 398 of 721Table E-22 (continued). Statistical Summary of Site ILCY Soybean Seed Nutrients for MON 87708 (Untreated) vs. ConventionalControlDifference (MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)ConventionalTolerance Interval5(Range)Amino Acid (% dw)Tyrosine1.42 (0.033)1.43 (0.033)-0.0065 (0.045)-0.13, 0.120.8921.24, 1.50(1.35 - 1.48)(1.39 - 1.51)(-0.075 - 0.098)(1.26 - 1.49)Valine2.01 (0.015)2.05 (0.015)-0.043 (0.021)-0.10, 0.0160.1131.72, 2.20(1.99 - 2.04)(2.05 - 2.06)(-0.070 - -0.011)(1.73 - 2.13)Fatty Acid (% Total FA)16:0 Palmitic12.12 (0.073)11.95 (0.073)0.17 (0.089)-0.076, 0.420.1278.44, 12.56(12.10 - 12.15)(11.73 - 12.08)(0.067 - 0.37)(9.40 - 11.54)18:0 Stearic3.94 (0.044)3.93 (0.044)0.0064 (0.062)-0.16, 0.180.9222.90, 5.19(3.87 - 4.05)(3.86 - 4.02)(-0.13 - 0.19)(3.24 - 4.67)18:1 Oleic19.85 (0.29)21.57 (0.29)-1.72 (0.41)-2.86, -0.580.01315.73, 27.19(19.62 - 20.22)(21.07 - 22.44)(-2.82 - -0.85)(17.88 - 25.31)18:2 Linoleic54.31 (0.25)53.26 (0.25)1.05 (0.35)0.074, 2.020.04048.61, 59.37(53.67 - 54.63)(52.77 - 53.74)(-0.064 - 1.85)(50.95 - 56.68)Monsanto Company 10-SY-210U 399 of 721Table E-22 (continued). Statistical Summary of Site ILCY Soybean Seed Nutrients for MON 87708 (Untreated) vs. ConventionalControlDifference (MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)ConventionalTolerance Interval5(Range)Fatty Acid (% Total FA)18:3 Linolenic9.09 (0.074)8.58 (0.074)0.51 (0.099)0.23, 0.790.0066.01, 12.58(8.94 - 9.23)(8.42 - 8.71)(0.23 - 0.67)(7.43 - 11.37)20:0 Arachidic0.26 (0.0035)0.26 (0.0035)-0.0013 (0.0050)-0.015, 0.0130.8020.19, 0.34(0.26 - 0.27)(0.26 - 0.27)(-0.014 - 0.014)(0.20 - 0.30)20:1 Eicosenoic0.15 (0.0016)0.16 (0.0016)-0.0078 (0.0023)-0.014, -0.00130.0280.022, 0.24(0.15 - 0.16)(0.16 - 0.17)(-0.013 - -0.0010)(0.065 - 0.17)22:0 Behenic0.28 (0.0030)0.29 (0.0030)-0.0065 (0.0042)-0.018, 0.00520.1990.24, 0.40(0.28 - 0.28)(0.28 - 0.30)(-0.019 - -0.00002)(0.28 - 0.36)Vitamin (mg/100g dw)Vitamin E2.21 (0.077)1.86 (0.077)0.35 (0.089)0.10, 0.600.0160, 3.49(2.11 - 2.27)(1.71 - 2.11)(0.16 - 0.49)(0.69 - 2.91)1dw = dry weight; fw = fresh weight; FA = fatty acid.2 MON 87708 plants were not sprayed with dicamba, but received another conventional treatmentas was done for the conventionalcontrol.3Mean (S.E.) = least-square mean (standard error).4Control refers to the non-biotechnology derived, conventional control (A3525).5With 95% confidence, interval contains 99% of the values expressed in the population of conventional substances. Negative limits set to zero.Monsanto Company 10-SY-210U 400 of 721Table E-23. Statistical Summary of Site ILCY Soybean Seed Anti-Nutrientsfor MON 87708 (Untreated) vs. ConventionalControlDifference (MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)ConventionalTolerance Interval5(Range)Anti-nutrientLectin (H.U./mg dw)3.52 (1.81)5.54 (1.81)-2.02 (2.55)-9.11, 5.070.4730, 7.73(2.56 - 4.82)(2.14 - 10.38)(-7.83 - 1.05)(0.68 - 8.34)Phytic Acid (% dw)1.08 (0.032)1.16 (0.032)-0.077 (0.045)-0.20, 0.0480.1630.77, 1.91(1.05 - 1.11)(1.10 - 1.22)(-0.17 - 0.015)(1.00 - 1.64)Raffinose (% dw)0.56 (0.013)0.57 (0.013)-0.0071 (0.018)-0.057, 0.0430.7110.13, 0.70(0.55 - 0.58)(0.54 - 0.60)(-0.045 - 0.049)(0.26 - 0.59)Stachyose (% dw)3.56 (0.10)3.64 (0.10)-0.086 (0.14)-0.48, 0.310.5762.30, 4.07(3.35 - 3.74)(3.43 - 3.83)(-0.32 - 0.30)(2.50 - 3.94)Trypsin Inhibitor (TIU/mg dw)34.71 (1.10)32.78 (1.10)1.93 (1.56)-2.39, 6.260.28222.05, 41.12(33.01 - 37.61)(31.06 - 34.22)(-0.72 - 6.55)(22.81 - 44.56)Isoflavone (μg/g dw)Daidzein1061.06 (72.66)922.21 (72.66)138.85 (102.76)-146.46, 424.160.2470, 2271.38(910.73 - 1156.87)(762.49 - 1098.08)(-187.35 - 394.38)(451.33 - 2033.05)Monsanto Company 10-SY-210U 401 of 721Table E-23 (continued). Statistical Summary of Site ILCYSoybean Seed Anti-Nutrients for MON 87708 (Untreated) vs.Conventional ControlDifference (MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)ConventionalTolerance Interval5(Range)Isoflavone (μg/g dw)Genistein721.38 (45.60)653.27 (45.60)68.12 (64.49)-110.93, 247.170.35078.36, 1869.48(654.16 - 792.83)(588.17 - 770.79)(-116.63 - 204.65)(533.88 - 1726.03)Glycitein125.94 (8.11)113.29 (8.11)12.65 (11.47)-19.20, 44.510.33231.24, 233.60(120.06 - 133.73)(96.25 - 122.09)(-2.04 - 37.48)(73.61 - 231.75)1dw = dry weight; H.U. = Hemagglutinating Units; TIU = Trypsin Inhibitor Units.2 MON 87708 plants were not sprayed with dicamba, but received another conventional treatmentas was done for the conventionalcontrol.3Mean (S.E.) = least-square mean (standard error).4Control refers to the non-biotechnology derived, conventional control (A3525).5With 95% confidence, interval contains 99% of the values expressed in the population of conventional substances. Negative limits set to zero.Monsanto Company 10-SY-210U 402 of 721Table E-24. Statistical Summary of Site ILCY Soybean Forage Nutrients for MON 87708 (Untreated) vs. ConventionalControlDifference (MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)ConventionalTolerance Interval5(Range)Proximate (% dw)Ash5.45 (0.22)6.27 (0.22)-0.82 (0.32)-1.70, 0.0520.0593.36, 10.84(4.92 - 6.04)(6.10 - 6.49)(-1.57 - -0.054)(5.20 - 9.81)Carbohydrates66.21 (0.61)66.38 (0.61)-0.17 (0.87)-2.58, 2.240.85560.69, 73.46(65.30 - 67.29)(65.53 - 67.94)(-1.89 - 1.62)(62.73 - 71.72)Moisture (% fw)73.27 (0.25)73.53 (0.25)-0.27 (0.35)-1.24, 0.700.48862.08, 89.80(73.20 - 73.40)(73.20 - 73.80)(-0.60 - 0.20)(70.40 - 84.10)Protein22.53 (0.51)21.64 (0.51)0.89 (0.68)-1.00, 2.790.26115.69, 26.63(22.29 - 22.65)(20.88 - 23.03)(-0.38 - 1.77)(18.50 - 25.86)Total Fat5.73 (0.38)5.77 (0.38)-0.040 (0.52)-1.47, 1.390.9420, 10.04(5.34 - 6.04)(5.15 - 6.72)(-1.38 - 0.67)(1.57 - 7.99)Fiber (% dw)Acid Detergent Fiber28.43 (1.09)28.46 (1.09)-0.027 (1.54)-4.30, 4.240.98616.54, 41.80(27.35 - 30.49)(27.05 - 30.90)(-0.41 - 0.31)(20.98 - 39.23)Monsanto Company 10-SY-210U 403 of 721Table E-24 (continued). Statistical Summary of Site ILCY Soybean Forage Nutrients for MON 87708 (Untreated) vs. ConventionalControlDifference (MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)ConventionalTolerance Interval5(Range)Fiber (% dw)Neutral Detergent Fiber32.83 (1.65)28.06 (1.65)4.76 (2.33)-1.72, 11.240.11020.28, 44.03(30.68 - 34.48)(23.66 - 32.73)(0.59 - 10.81)(24.81 - 42.80)1dw = dry weight; fw = fresh weight.2MON 87708 plants were not sprayed with dicamba, but received another conventional treatment as was done for the conventional control.3Mean (S.E.) = least-square mean (standard error).4Control refers to the non-biotechnology derived, conventional control (A3525).5With 95% confidence, interval contains 99% of the values expressed in the population of conventional substances. Negative limits set to zero.Monsanto Company 10-SY-210U 404 of 721Table E-25. Statistical Summary of SiteILWY Soybean Seed Nutrients for MON87708 (Untreated) vs. Conventional ControlDifference (MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)ConventionalTolerance Interval5(Range)Proximate (% dw)Ash5.30 (0.084)5.23 (0.084)0.068 (0.084)-0.17, 0.300.4654.74, 6.01(5.24 - 5.34)(5.13 - 5.29)(0.039 - 0.11)(4.93 - 5.88)Carbohydrates38.39 (0.44)38.29 (0.44)0.10 (0.45)-1.15, 1.350.83532.07, 40.08(37.03 - 39.08)(38.19 - 38.42)(-1.17 - 0.83)(33.82 - 39.26)Moisture (% fw)6.66 (0.16)6.16 (0.16)0.51 (0.22)-0.11, 1.120.0844.27, 9.58(6.34 - 6.83)(5.79 - 6.41)(-0.070 - 1.03)(5.50 - 9.23)Protein41.96 (0.28)41.99 (0.28)-0.022 (0.38)-1.07, 1.020.95635.50, 45.19(41.53 - 42.72)(41.72 - 42.25)(-0.46 - 0.47)(37.06 - 43.42)Total Fat14.36 (0.38)14.49 (0.38)-0.14 (0.44)-1.37, 1.100.77612.33, 24.10(13.95 - 15.03)(14.40 - 14.54)(-0.59 - 0.62)(15.47 - 21.34)Fiber (% dw)Acid Detergent Fiber13.71 (0.43)12.96 (0.43)0.75 (0.61)-0.93, 2.430.28310.06, 18.04(13.31 - 14.17)(12.48 - 13.69)(-0.39 - 1.68)(12.07 - 17.46)Monsanto Company 10-SY-210U 405 of 721Table E-25 (continued). Statistical Summary of Site ILWY Soybean Seed Nutrients for MON 87708 (Untreated) vs. ConventionalControlDifference (MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)ConventionalTolerance Interval5(Range)Fiber (% dw)Crude Fiber7.37 (0.52)7.58 (0.52)-0.21 (0.74)-2.25, 1.830.7875.76, 10.76(6.14 - 8.72)(7.39 - 7.82)(-1.25 - 1.19)(6.35 - 11.31)Neutral Detergent Fiber15.79 (0.72)13.78 (0.72)2.00 (0.94)-0.61, 4.620.10011.36, 19.38(15.13 - 16.53)(13.44 - 14.00)(1.23 - 3.09)(11.66 - 19.45)Amino Acid (% dw)Alanine1.77 (0.0076)1.75 (0.0076)0.020 (0.0093)-0.0057, 0.0460.0961.56, 1.91(1.76 - 1.79)(1.75 - 1.76)(0.0086 - 0.043)(1.59 - 1.86)Arginine3.52 (0.037)3.57 (0.037)-0.049 (0.040)-0.16, 0.0620.2882.55, 3.83(3.41 - 3.60)(3.55 - 3.60)(-0.14 - 0.051)(2.88 - 3.74)Aspartic Acid4.72 (0.017)4.67 (0.017)0.050 (0.020)-0.0047, 0.110.0644.04, 5.13(4.68 - 4.75)(4.67 - 4.68)(-0.0019 - 0.082)(4.22 - 4.94)Cystine0.63 (0.0090)0.61 (0.0090)0.021 (0.0092)-0.0042, 0.0470.0810.50, 0.68(0.61 - 0.64)(0.58 - 0.62)(0.0091 - 0.034)(0.53 - 0.66)Monsanto Company 10-SY-210U 406 of 721Table E-25 (continued). Statistical Summary of Site ILWY Soybean Seed Nutrients for MON 87708 (Untreated) vs. ConventionalControlDifference (MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)ConventionalTolerance Interval5(Range)Amino Acid (% dw)Glutamic Acid7.53 (0.030)7.51 (0.030)0.023 (0.031)-0.064, 0.110.5066.28, 8.30(7.49 - 7.56)(7.49 - 7.53)(-0.0030 - 0.066)(6.69 - 7.92)Glycine1.78 (0.0053)1.77 (0.0053)0.013 (0.0075)-0.0078, 0.0340.1551.53, 1.92(1.77 - 1.79)(1.76 - 1.77)(-0.0019 - 0.032)(1.58 - 1.84)Histidine1.08 (0.0032)1.07 (0.0032)0.011 (0.0046)-0.0016, 0.0240.0710.93, 1.16(1.07 - 1.08)(1.06 - 1.07)(0.00005 - 0.024)(0.95 - 1.13)Isoleucine1.90 (0.012)1.90 (0.012)0.0031 (0.0040)-0.0081, 0.0140.4861.65, 2.06(1.88 - 1.91)(1.88 - 1.91)(-0.00070 -0.0093)(1.68 - 2.02)Leucine3.12 (0.011)3.10 (0.011)0.017 (0.0062)-0.00030, 0.0340.0522.72, 3.39(3.10 - 3.13)(3.09 - 3.13)(0.0081 - 0.030)(2.80 - 3.27)Lysine2.68 (0.0062)2.63 (0.0062)0.046 (0.0088)0.022, 0.0710.0062.33, 2.84(2.66 - 2.69)(2.62 - 2.64)(0.029 - 0.069)(2.38 - 2.74)Monsanto Company 10-SY-210U 407 of 721Table E-25 (continued). Statistical Summary of Site ILWY Soybean Seed Nutrients for MON 87708 (Untreated) vs. ConventionalControlDifference (MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)ConventionalTolerance Interval5(Range)Amino Acid (% dw)Methionine0.59 (0.0074)0.58 (0.0074)0.0067 (0.010)-0.022, 0.0360.5530.50, 0.64(0.58 - 0.59)(0.56 - 0.59)(-0.0018 - 0.017)(0.52 - 0.63)Phenylalanine2.07 (0.019)2.07 (0.019)-0.0031 (0.021)-0.063, 0.0560.8921.80, 2.30(2.06 - 2.08)(2.05 - 2.10)(-0.044 - 0.023)(1.85 - 2.21)Proline2.02 (0.020)2.05 (0.020)-0.028 (0.028)-0.11, 0.0490.3681.65, 2.26(1.97 - 2.05)(2.01 - 2.09)(-0.12 - 0.041)(1.74 - 2.16)Serine2.10 (0.013)2.06 (0.013)0.043 (0.013)0.0084, 0.0780.0261.78, 2.27(2.09 - 2.11)(2.02 - 2.09)(0.023 - 0.073)(1.90 - 2.18)Threonine1.59 (0.0042)1.55 (0.0042)0.044 (0.0049)0.031, 0.058<0.0011.40, 1.69(1.58 - 1.60)(1.54 - 1.55)(0.031 - 0.052)(1.47 - 1.64)Tryptophan0.48 (0.0083)0.47 (0.0083)0.016 (0.011)-0.015, 0.0460.2270.38, 0.52(0.46 - 0.50)(0.45 - 0.48)(-0.014 - 0.032)(0.39 - 0.50)Monsanto Company 10-SY-210U 408 of 721Table E-25 (continued). Statistical Summary of Site ILWY Soybean Seed Nutrients for MON 87708 (Untreated) vs. ConventionalControlDifference (MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)ConventionalTolerance Interval5(Range)Amino Acid (% dw)Tyrosine1.40 (0.024)1.36 (0.024)0.032 (0.027)-0.044, 0.110.3051.24, 1.50(1.35 - 1.44)(1.34 - 1.40)(-0.0023 - 0.084)(1.26 - 1.49)Valine2.00 (0.013)2.01 (0.013)-0.011 (0.012)-0.045, 0.0240.4431.72, 2.20(1.97 - 2.02)(2.01 - 2.02)(-0.031 - 0.012)(1.73 - 2.13)Fatty Acid (% Total FA)16:0 Palmitic11.13 (0.041)11.04 (0.041)0.085 (0.057)-0.074, 0.240.2128.44, 12.56(11.02 - 11.21)(10.97 - 11.12)(-0.094 - 0.24)(9.40 - 11.54)18:0 Stearic4.47 (0.063)4.25 (0.063)0.23 (0.089)-0.019, 0.470.0622.90, 5.19(4.30 - 4.57)(4.16 - 4.31)(-0.010 - 0.38)(3.24 - 4.67)18:1 Oleic20.24 (0.25)21.14 (0.25)-0.89 (0.36)-1.89, 0.110.06815.73, 27.19(19.52 - 20.73)(20.78 - 21.55)(-2.03 - -0.045)(17.88 - 25.31)18:2 Linoleic52.87 (0.32)52.90 (0.32)-0.024 (0.46)-1.30, 1.250.96148.61, 59.37(52.18 - 53.74)(52.33 - 53.20)(-0.98 - 1.41)(50.95 - 56.68)Monsanto Company 10-SY-210U 409 of 721Table E-25 (continued). Statistical Summary of Site ILWY Soybean Seed Nutrients for MON 87708 (Untreated) vs. ConventionalControlDifference (MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)ConventionalTolerance Interval5(Range)Fatty Acid (% Total FA)18:3 Linolenic10.65 (0.10)10.05 (0.10)0.60 (0.14)0.20, 1.000.0146.01, 12.58(10.37 - 10.90)(9.89 - 10.14)(0.25 - 1.01)(7.43 - 11.37)20:0 Arachidic0.28 (0.0041)0.27 (0.0041)0.011 (0.0058)-0.0048, 0.0270.1230.19, 0.34(0.27 - 0.29)(0.26 - 0.27)(-0.0031 - 0.022)(0.20 - 0.30)20:1 Eicosenoic0.077 (0.0020)0.076 (0.0020)0.00030 (0.0023)-0.0060, 0.00660.9010.022, 0.24(0.073 - 0.079)(0.075 - 0.077)(-0.0037 - 0.0039)(0.065 - 0.17)22:0 Behenic0.28 (0.0032)0.28 (0.0032)-0.0029 (0.0040)-0.014, 0.00810.5030.24, 0.40(0.28 - 0.29)(0.28 - 0.29)(-0.010 - 0.0054)(0.28 - 0.36)Vitamin (mg/100g dw)Vitamin E1.15 (0.037)0.94 (0.037)0.21 (0.053)0.061, 0.350.0170, 3.49(1.11 - 1.20)(0.89 - 0.99)(0.12 - 0.31)(0.69 - 2.91)1dw = dry weight; fw = fresh weight; FA = fatty acid.2 MON 87708 plants were not sprayed with dicamba, but received another conventional treatmentas was done for the conventionalcontrol.3Mean (S.E.) = least-square mean (standard error).4Control refers to the non-biotechnology derived, conventional control (A3525).5With 95% confidence, interval contains 99% of the values expressed in the population of conventional substances. Negative limits set to zero.Monsanto Company 10-SY-210U 410 of 721Table E-26. Statistical Summary of Site ILWY Soybean Seed Anti-Nutrientsfor MON 87708 (Untreated) vs. ConventionalControlDifference (MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)ConventionalTolerance Interval5(Range)Anti-nutrientLectin (H.U./mg dw)1.36 (0.44)2.33 (0.44)-0.97 (0.43)-2.17, 0.230.0870, 7.73(1.18 - 1.63)(1.34 - 3.68)(-2.04 - -0.16)(0.68 - 8.34)Phytic Acid (% dw)1.43 (0.033)1.55 (0.033)-0.12 (0.044)-0.24, 0.00510.0560.77, 1.91(1.43 - 1.43)(1.47 - 1.61)(-0.19 - -0.045)(1.00 - 1.64)Raffinose (% dw)0.37 (0.026)0.41 (0.026)-0.038 (0.036)-0.14, 0.0640.3610.13, 0.70(0.34 - 0.41)(0.41 - 0.41)(-0.065 - 0.0072)(0.26 - 0.59)Stachyose (% dw)3.60 (0.19)3.76 (0.19)-0.17 (0.27)-0.92, 0.590.5732.30, 4.07(3.37 - 3.85)(3.68 - 3.85)(-0.39 - 0.0078)(2.50 - 3.94)Trypsin Inhibitor (TIU/mg dw)38.93 (2.07)29.73 (2.07)9.20 (2.89)1.18, 17.220.03322.05, 41.12(37.89 - 39.49)(25.43 - 32.22)(5.67 - 13.97)(22.81 - 44.56)Isoflavone (μg/g dw)Daidzein1307.12 (35.08)1271.60 (35.08)35.52 (31.48)-51.87, 122.920.3220, 2271.38(1291.91 - 1320.17)(1196.71 - 1354.96)(-34.79 - 95.20)(451.33 - 2033.05)Monsanto Company 10-SY-210U 411 of 721Table E-26 (continued). Statistical Summary of Site ILWY Soybean Seed Anti-Nutrients for MON 87708 (Untreated) vs.Conventional ControlDifference (MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)ConventionalTolerance Interval5(Range)Isoflavone (μg/g dw)Genistein832.43 (28.15)860.58 (28.15)-28.14 (39.82)-138.69, 82.400.51878.36, 1869.48(780.21 - 862.94)(784.27 - 913.26)(-103.98 - 69.88)(533.88 - 1726.03)Glycitein96.96 (2.15)79.70 (2.15)17.26 (2.23)11.06, 23.460.00131.24, 233.60(92.19 - 103.25)(77.14 - 81.62)(11.83 - 21.63)(73.61 - 231.75)1dw = dry weight; H.U. = Hemagglutinating Units; TIU = Trypsin Inhibitor Units.2MON 87708 plants were not sprayed with dicamba, but received another conventional treatment as was done for the conventional control.3Mean (S.E.) = least-square mean (standard error).4Control refers to the non-biotechnology derived, conventional control (A3525).5With 95% confidence, interval contains 99% of the values expressed in the population of conventional substances. Negative limits set to zero.Monsanto Company 10-SY-210U 412 of 721Table E-27. Statistical Summary of Site ILWY Soybean Forage Nutrients for MON 87708 (Untreated) vs. ConventionalControlDifference (MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)ConventionalTolerance Interval5(Range)Proximate (% dw)Ash6.76 (0.23)6.88 (0.23)-0.12 (0.30)-0.95, 0.710.7063.36, 10.84(6.52 - 7.14)(6.82 - 6.99)(-0.30 - 0.15)(5.20 - 9.81)Carbohydrates65.94 (0.77)66.26 (0.77)-0.31 (1.08)-3.32, 2.690.78660.69, 73.46(64.34 - 67.08)(65.71 - 66.67)(-1.37 - 0.68)(62.73 - 71.72)Moisture (% fw)75.13 (0.55)75.40 (0.55)-0.27 (0.65)-2.07, 1.540.70262.08, 89.80(74.10 - 75.70)(75.10 - 75.60)(-1.00 - 0.10)(70.40 - 84.10)Protein22.27 (0.58)21.97 (0.58)0.30 (0.82)-1.98, 2.590.73015.69, 26.63(22.05 - 22.54)(21.81 - 22.13)(0.091 - 0.58)(18.50 - 25.86)Total Fat5.01 (0.52)4.91 (0.52)0.11 (0.35)-0.86, 1.080.7730, 10.04(4.25 - 6.52)(4.50 - 5.63)(-0.31 - 0.88)(1.57 - 7.99)Fiber (% dw)Acid Detergent Fiber27.49 (1.73)27.77 (1.73)-0.28 (2.44)-7.06, 6.490.91216.54, 41.80(23.32 - 29.63)(25.12 - 31.00)(-7.68 - 4.38)(20.98 - 39.23)Monsanto Company 10-SY-210U 413 of 721Table E-27 (continued). Statistical Summary of Site ILWY Soybean Forage Nutrients for MON 87708 (Untreated) vs. ConventionalControlDifference (MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)ConventionalTolerance Interval5(Range)Fiber (% dw)Neutral Detergent Fiber29.89 (1.83)33.25 (1.83)-3.36 (2.59)-10.55, 3.830.26420.28, 44.03(26.37 - 35.43)(31.89 - 34.06)(-5.93 - 1.37)(24.81 - 42.80)1dw = dry weight; fw = fresh weight.2MON 87708 plants were not sprayed with dicamba, but received another conventional treatment as was done for the conventional control.3Mean (S.E.) = least-square mean (standard error).4Control refers to the non-biotechnology derived, conventional control (A3525).5With 95% confidence, interval contains 99% of the values expressed in the population of conventional substances. Negative limits set to zero.Monsanto Company 10-SY-210U 414 of 721Table E-28. Statistical Summary of SiteINRC Soybean Seed Nutrients for MON87708 (Untreated) vs. Conventional ControlDifference (MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)ConventionalTolerance Interval5(Range)Proximate (% dw)Ash5.13 (0.11)4.95 (0.11)0.18 (0.15)-0.24, 0.600.2954.74, 6.01(5.05 - 5.30)(4.73 - 5.23)(-0.18 - 0.41)(4.93 - 5.88)Carbohydrates37.64 (0.78)35.95 (0.78)1.69 (1.10)-1.37, 4.750.20032.07, 40.08(35.63 - 39.79)(35.27 - 37.04)(0.10 - 4.52)(33.82 - 39.26)Moisture (% fw)6.67 (0.20)6.53 (0.20)0.13 (0.29)-0.67, 0.930.6674.27, 9.58(6.26 - 7.11)(6.32 - 6.84)(-0.18 - 0.31)(5.50 - 9.23)Protein41.47 (0.53)43.58 (0.53)-2.11 (0.76)-4.21, -0.0140.04935.50, 45.19(40.22 - 43.06)(43.50 - 43.69)(-3.28 - -0.63)(37.06 - 43.42)Total Fat15.75 (0.45)15.55 (0.45)0.20 (0.56)-1.37, 1.770.73912.33, 24.10(14.93 - 16.26)(14.52 - 16.10)(-1.10 - 1.55)(15.47 - 21.34)Fiber (% dw)Acid Detergent Fiber12.89 (0.35)12.77 (0.35)0.12 (0.46)-1.15, 1.390.80010.06, 18.04(12.06 - 13.49)(12.28 - 13.25)(-0.72 - 1.22)(12.07 - 17.46)Monsanto Company 10-SY-210U 415 of 721Table E-28 (continued). Statistical Summary of Site INRC Soybean Seed Nutrients for MON 87708 (Untreated) vs. ConventionalControlDifference (MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)ConventionalTolerance Interval5(Range)Fiber (% dw)Crude Fiber7.38 (0.18)6.89 (0.18)0.49 (0.25)-0.21, 1.190.1235.76, 10.76(7.06 - 7.61)(6.59 - 7.12)(0.11 - 0.87)(6.35 - 11.31)Neutral Detergent Fiber14.43 (0.67)14.66 (0.67)-0.23 (0.95)-2.86, 2.410.82211.36, 19.38(12.91 - 15.21)(13.24 - 15.56)(-2.27 - 1.97)(11.66 - 19.45)Amino Acid (% dw)Alanine1.79 (0.021)1.84 (0.021)-0.054 (0.022)-0.12, 0.00670.0681.56, 1.91(1.76 - 1.84)(1.80 - 1.87)(-0.10 - -0.027)(1.59 - 1.86)Arginine3.40 (0.044)3.72 (0.044)-0.32 (0.062)-0.49, -0.140.0072.55, 3.83(3.35 - 3.50)(3.64 - 3.81)(-0.46 - -0.19)(2.88 - 3.74)Aspartic Acid4.72 (0.062)4.89 (0.062)-0.17 (0.078)-0.39, 0.0440.0914.04, 5.13(4.59 - 4.90)(4.80 - 4.95)(-0.34 - -0.050)(4.22 - 4.94)Cystine0.62 (0.0067)0.59 (0.0067)0.031 (0.0094)0.0050, 0.0570.0290.50, 0.68(0.60 - 0.63)(0.59 - 0.60)(0.0031 - 0.047)(0.53 - 0.66)Monsanto Company 10-SY-210U 416 of 721Table E-28 (continued). Statistical Summary of Site INRC Soybean Seed Nutrients for MON 87708 (Untreated) vs. ConventionalControlDifference (MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)ConventionalTolerance Interval5(Range)Amino Acid (% dw)Glutamic Acid7.56 (0.11)7.93 (0.11)-0.36 (0.14)-0.76, 0.0350.0646.28, 8.30(7.36 - 7.88)(7.75 - 8.02)(-0.66 - -0.14)(6.69 - 7.92)Glycine1.80 (0.020)1.85 (0.020)-0.054 (0.027)-0.13, 0.0200.1111.53, 1.92(1.76 - 1.85)(1.82 - 1.87)(-0.11 - -0.016)(1.58 - 1.84)Histidine1.07 (0.012)1.11 (0.012)-0.038 (0.016)-0.083, 0.00720.0790.93, 1.16(1.05 - 1.11)(1.10 - 1.12)(-0.072 - -0.0075)(0.95 - 1.13)Isoleucine1.92 (0.042)2.00 (0.042)-0.079 (0.057)-0.24, 0.0800.2381.65, 2.06(1.80 - 2.02)(1.95 - 2.03)(-0.22 - -0.0049)(1.68 - 2.02)Leucine3.12 (0.041)3.24 (0.041)-0.12 (0.052)-0.26, 0.0230.0802.72, 3.39(3.03 - 3.24)(3.20 - 3.26)(-0.22 - -0.023)(2.80 - 3.27)Lysine2.67 (0.030)2.72 (0.030)-0.053 (0.035)-0.15, 0.0450.2052.33, 2.84(2.60 - 2.76)(2.68 - 2.76)(-0.13 - -0.0027)(2.38 - 2.74)Monsanto Company 10-SY-210U 417 of 721Table E-28 (continued). Statistical Summary of Site INRC Soybean Seed Nutrients for MON 87708 (Untreated) vs. ConventionalControlDifference (MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)ConventionalTolerance Interval5(Range)Amino Acid (% dw)Methionine0.59 (0.0086)0.59 (0.0086)0.00055 (0.012)-0.033, 0.0340.9660.50, 0.64(0.56 - 0.61)(0.58 - 0.60)(-0.036 - 0.021)(0.52 - 0.63)Phenylalanine2.08 (0.030)2.20 (0.030)-0.12 (0.040)-0.23, -0.0110.0371.80, 2.30(2.03 - 2.14)(2.13 - 2.23)(-0.17 - -0.090)(1.85 - 2.21)Proline2.01 (0.010)2.06 (0.010)-0.054 (0.015)-0.095, -0.0140.0201.65, 2.26(1.99 - 2.04)(2.05 - 2.07)(-0.079 - -0.015)(1.74 - 2.16)Serine2.06 (0.028)2.13 (0.028)-0.072 (0.029)-0.15, 0.00940.0701.78, 2.27(1.97 - 2.11)(2.13 - 2.14)(-0.15 - -0.026)(1.90 - 2.18)Threonine1.57 (0.019)1.61 (0.019)-0.040 (0.027)-0.11, 0.0340.2061.40, 1.69(1.51 - 1.61)(1.60 - 1.62)(-0.088 - -0.0061)(1.47 - 1.64)Tryptophan0.48 (0.013)0.45 (0.013)0.026 (0.018)-0.025, 0.0760.2310.38, 0.52(0.46 - 0.50)(0.43 - 0.46)(0.0012 - 0.044)(0.39 - 0.50)Monsanto Company 10-SY-210U 418 of 721Table E-28 (continued). Statistical Summary of Site INRC Soybean Seed Nutrients for MON 87708 (Untreated) vs. ConventionalControlDifference (MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)ConventionalTolerance Interval5(Range)Amino Acid (% dw)Tyrosine1.39 (0.024)1.44 (0.024)-0.055 (0.022)-0.12, 0.00730.0701.24, 1.50(1.37 - 1.40)(1.38 - 1.47)(-0.077 - -0.018)(1.26 - 1.49)Valine2.03 (0.045)2.12 (0.045)-0.097 (0.063)-0.27, 0.0780.1981.72, 2.20(1.90 - 2.13)(2.06 - 2.16)(-0.25 - -0.015)(1.73 - 2.13)Fatty Acid (% Total FA)16:0 Palmitic11.45 (0.068)11.19 (0.068)0.27 (0.097)-0.00083, 0.540.0508.44, 12.56(11.23 - 11.57)(11.14 - 11.21)(0.014 - 0.44)(9.40 - 11.54)18:0 Stearic4.15 (0.047)4.25 (0.047)-0.096 (0.066)-0.28, 0.0880.2192.90, 5.19(4.07 - 4.24)(4.16 - 4.31)(-0.20 - -0.022)(3.24 - 4.67)18:1 Oleic18.80 (0.085)20.19 (0.085)-1.39 (0.093)-1.65, -1.14<0.00115.73, 27.19(18.63 - 18.96)(20.12 - 20.23)(-1.60 - -1.26)(17.88 - 25.31)18:2 Linoleic54.94 (0.10)54.43 (0.10)0.51 (0.14)0.11, 0.900.02448.61, 59.37(54.79 - 55.13)(54.32 - 54.64)(0.15 - 0.81)(50.95 - 56.68)Monsanto Company 10-SY-210U 419 of 721Table E-28 (continued). Statistical Summary of Site INRC Soybean Seed Nutrients for MON 87708 (Untreated) vs. ConventionalControlDifference (MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)ConventionalTolerance Interval5(Range)Fatty Acid (% Total FA)18:3 Linolenic10.05 (0.050)9.31 (0.050)0.74 (0.070)0.54, 0.93<0.0016.01, 12.58(10.00 - 10.10)(9.26 - 9.40)(0.60 - 0.84)(7.43 - 11.37)20:0 Arachidic0.26 (0.0029)0.27 (0.0029)-0.0072 (0.0041)-0.019, 0.00430.1560.19, 0.34(0.26 - 0.27)(0.26 - 0.27)(-0.014 - -0.0026)(0.20 - 0.30)20:1 Eicosenoic0.070 (0.0020)0.071 (0.0020)-0.0014 (0.0025)-0.0083, 0.00560.6150.022, 0.24(0.068 - 0.073)(0.069 - 0.076)(-0.0073 - 0.0035)(0.065 - 0.17)22:0 Behenic0.28 (0.0033)0.29 (0.0033)-0.016 (0.0047)-0.029, -0.00320.0250.24, 0.40(0.27 - 0.28)(0.29 - 0.30)(-0.023 - -0.0045)(0.28 - 0.36)Vitamin (mg/100g dw)Vitamin E1.25 (0.054)1.16 (0.054)0.091 (0.067)-0.094, 0.280.2440, 3.49(1.11 - 1.40)(1.10 - 1.23)(0.0086 - 0.17)(0.69 - 2.91)1dw = dry weight; fw = fresh weight; FA = fatty acid.2 MON 87708 plants were not sprayed with dicamba, but received another conventional treatmentas was done for the conventionalcontrol.3Mean (S.E.) = least-square mean (standard error).4Control refers to the non-biotechnology derived, conventional control (A3525).5With 95% confidence, interval contains 99% of the values expressed in the population of conventional substances. Negative limits set to zero.Monsanto Company 10-SY-210U 420 of 721Table E-29. Statistical Summary of Site INRC Soybean Seed Anti-Nutrientsfor MON 87708 (Untreated) vs. ConventionalControlDifference (MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)ConventionalTolerance Interval5(Range)Anti-nutrientLectin (H.U./mg dw)2.94 (0.32)2.56 (0.32)0.39 (0.45)-0.88, 1.650.4430, 7.73(2.24 - 3.81)(2.33 - 3.02)(-0.78 - 1.48)(0.68 - 8.34)Phytic Acid (% dw)1.24 (0.071)1.19 (0.071)0.052 (0.087)-0.19, 0.290.5850.77, 1.91(1.09 - 1.37)(1.09 - 1.36)(-0.099 - 0.26)(1.00 - 1.64)Raffinose (% dw)0.45 (0.023)0.40 (0.023)0.049 (0.033)-0.043, 0.140.2150.13, 0.70(0.42 - 0.47)(0.36 - 0.43)(0.042 - 0.056)(0.26 - 0.59)Stachyose (% dw)3.50 (0.077)3.46 (0.077)0.044 (0.11)-0.26, 0.350.7042.30, 4.07(3.35 - 3.62)(3.33 - 3.67)(-0.32 - 0.28)(2.50 - 3.94)Trypsin Inhibitor (TIU/mg dw)30.68 (1.79)29.28 (1.79)1.40 (1.83)-3.69, 6.490.48722.05, 41.12(28.59 - 32.19)(25.22 - 31.77)(0.42 - 3.37)(22.81 - 44.56)Isoflavone (μg/g dw)Daidzein1671.09 (67.03)1419.40 (67.03)251.69 (94.79)-11.48, 514.870.0560, 2271.38(1485.63 - 1842.13)(1416.92 - 1421.55)(68.71 - 422.41)(451.33 - 2033.05)Monsanto Company 10-SY-210U 421 of 721Table E-29 (continued). Statistical Summary of Site INRCSoybean Seed Anti-Nutrients for MON 87708 (Untreated) vs.Conventional ControlDifference (MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)ConventionalTolerance Interval5(Range)Isoflavone (μg/g dw)Genistein992.90 (50.59)862.03 (50.59)130.88 (71.54)-67.75, 329.510.14178.36, 1869.48(842.93 - 1103.14)(840.10 - 890.94)(-48.01 - 248.10)(533.88 - 1726.03)Glycitein119.27 (3.23)98.42 (3.23)20.86 (3.31)11.67, 30.050.00331.24, 233.60(113.04 - 124.24)(89.42 - 103.14)(17.40 - 23.62)(73.61 - 231.75)1dw = dry weight; H.U. = Hemagglutinating Units; TIU = Trypsin Inhibitor Units.2MON 87708plants were not sprayed with dicamba, but received another conventional treatment as was done for the conventional control.3Mean (S.E.) = least-square mean (standard error).4Control refers to the non-biotechnology derived, conventional control (A3525).5With 95% confidence, interval contains 99% of the values expressed in the population of conventional substances. Negative limits set to zero.Monsanto Company 10-SY-210U 422 of 721Table E-30. Statistical Summary of Site INRC Soybean Forage Nutrients for MON 87708 (Untreated) vs. ConventionalControlDifference (MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)ConventionalTolerance Interval5(Range)Proximate (% dw)Ash6.20 (0.24)6.95 (0.24)-0.74 (0.26)-1.47, -0.0150.0473.36, 10.84(5.87 - 6.72)(6.84 - 7.07)(-1.20 - -0.22)(5.20 - 9.81)Carbohydrates64.35 (0.69)63.82 (0.69)0.53 (0.98)-2.18, 3.250.61460.69, 73.46(63.90 - 64.94)(62.91 - 64.44)(-0.23 - 0.99)(62.73 - 71.72)Moisture (% fw)72.70 (0.32)72.27 (0.32)0.43 (0.39)-0.64, 1.500.32362.08, 89.80(72.30 - 72.90)(71.60 - 72.70)(-0.20 - 1.30)(70.40 - 84.10)Protein23.46 (0.41)23.33 (0.41)0.13 (0.41)-1.01, 1.280.76015.69, 26.63(23.10 - 24.15)(22.64 - 24.11)(-0.10 - 0.46)(18.50 - 25.86)Total Fat5.98 (0.27)5.88 (0.27)0.10 (0.39)-0.97, 1.170.8090, 10.04(5.92 - 6.01)(5.39 - 6.19)(-0.18 - 0.63)(1.57 - 7.99)Fiber (% dw)Acid Detergent Fiber25.59 (1.50)23.83 (1.50)1.77 (2.13)-4.14, 7.670.45216.54, 41.80(24.69 - 27.00)(22.93 - 25.53)(-0.44 - 3.99)(20.98 - 39.23)Monsanto Company 10-SY-210U 423 of 721Table E-30 (continued). Statistical Summary of Site INRC Soybean Forage Nutrients for MON 87708 (Untreated) vs. ConventionalControlDifference (MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)ConventionalTolerance Interval5(Range)Fiber (% dw)Neutral Detergent Fiber26.13 (1.62)26.11 (1.62)0.020 (2.04)-5.65, 5.690.99220.28, 44.03(25.38 - 27.64)(23.91 - 29.42)(-4.04 - 3.73)(24.81 - 42.80)1dw = dry weight; fw = fresh weight.2MON 87708 plants were not sprayed with dicamba, but received another conventional treatment as was done for the conventional control.3Mean (S.E.) = least-square mean (standard error).4Control refers to the non-biotechnology derived, conventional control (A3525).5With 95% confidence, interval contains 99% of the values expressed in the population of conventional substances. Negative limits set to zero.Monsanto Company 10-SY-210U 424 of 721Table E-31. Statistical Summary of SitePAHM Soybean Seed Nutrients for MON87708 (Untreated) vs. Conventional ControlDifference (MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)ConventionalTolerance Interval5(Range)Proximate (% dw)Ash5.20 (0.11)5.05 (0.11)0.16 (0.16)-0.28, 0.590.3784.74, 6.01(4.89 - 5.51)(4.96 - 5.17)(-0.28 - 0.55)(4.93 - 5.88)Carbohydrates36.81 (0.51)35.23 (0.51)1.58 (0.64)-0.20, 3.350.06932.07, 40.08(35.36 - 37.56)(34.49 - 35.75)(0.87 - 2.06)(33.82 - 39.26)Moisture (% fw)9.53 (0.22)10.50 (0.22)-0.97 (0.22)-1.58, -0.360.0114.27, 9.58(9.21 - 9.91)(10.40 - 10.60)(-1.19 - -0.69)(5.50 - 9.23)Protein40.38 (0.41)43.69 (0.41)-3.31 (0.51)-4.73, -1.880.00235.50, 45.19(39.96 - 40.97)(43.46 - 43.85)(-3.89 - -2.78)(37.06 - 43.42)Total Fat17.61 (0.63)16.05 (0.63)1.56 (0.89)-0.90, 4.020.15312.33, 24.10(17.32 - 18.17)(15.64 - 16.85)(1.32 - 1.70)(15.47 - 21.34)Fiber (% dw)Acid Detergent Fiber11.96 (0.35)11.96 (0.35)0.0073 (0.43)-1.18, 1.200.98710.06, 18.04(11.01 - 12.89)(11.62 - 12.17)(-0.61 - 0.72)(12.07 - 17.46)Monsanto Company 10-SY-210U 425 of 721Table E-31 (continued). Statistical Summary of Site PAHM Soybean Seed Nutrients for MON 87708 (Untreated) vs. ConventionalControlDifference (MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)ConventionalTolerance Interval5(Range)Fiber (% dw)Crude Fiber7.05 (0.63)6.61 (0.63)0.43 (0.71)-1.55, 2.410.5785.76, 10.76(6.81 - 7.28)(6.05 - 6.98)(0.064 - 0.76)(6.35 - 11.31)Neutral Detergent Fiber14.44 (0.36)13.11 (0.36)1.33 (0.50)-0.064, 2.730.05611.36, 19.38(14.21 - 14.87)(12.63 - 13.62)(1.12 - 1.62)(11.66 - 19.45)Amino Acid (% dw)Alanine1.79 (0.017)1.86 (0.017)-0.068 (0.024)-0.13, -0.00160.0461.56, 1.91(1.76 - 1.81)(1.82 - 1.90)(-0.10 - -0.043)(1.59 - 1.86)Arginine3.39 (0.053)3.88 (0.053)-0.49 (0.074)-0.70, -0.290.0022.55, 3.83(3.35 - 3.44)(3.83 - 3.93)(-0.55 - -0.45)(2.88 - 3.74)Aspartic Acid4.66 (0.041)4.94 (0.041)-0.28 (0.057)-0.44, -0.120.0084.04, 5.13(4.64 - 4.69)(4.90 - 5.01)(-0.35 - -0.22)(4.22 - 4.94)Cystine0.61 (0.0062)0.59 (0.0062)0.015 (0.0081)-0.0078, 0.0370.1440.50, 0.68(0.61 - 0.61)(0.59 - 0.60)(0.0099 - 0.021)(0.53 - 0.66)Monsanto Company 10-SY-210U 426 of 721Table E-31 (continued). Statistical Summary of Site PAHM Soybean Seed Nutrients for MON 87708 (Untreated) vs. ConventionalControlDifference (MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)ConventionalTolerance Interval5(Range)Amino Acid (% dw)Glutamic Acid7.42 (0.081)8.00 (0.081)-0.58 (0.11)-0.90, -0.260.0076.28, 8.30(7.32 - 7.50)(7.91 - 8.14)(-0.70 - -0.46)(6.69 - 7.92)Glycine1.77 (0.015)1.86 (0.015)-0.090 (0.022)-0.15, -0.0290.0141.53, 1.92(1.76 - 1.78)(1.83 - 1.89)(-0.13 - -0.068)(1.58 - 1.84)Histidine1.07 (0.0089)1.13 (0.0089)-0.061 (0.013)-0.096, -0.0260.0080.93, 1.16(1.07 - 1.08)(1.11 - 1.14)(-0.073 - -0.045)(0.95 - 1.13)Isoleucine1.89 (0.026)2.00 (0.026)-0.11 (0.036)-0.21, -0.0130.0351.65, 2.06(1.88 - 1.91)(1.94 - 2.04)(-0.16 - -0.036)(1.68 - 2.02)Leucine3.10 (0.027)3.28 (0.027)-0.18 (0.038)-0.28, -0.0740.0092.72, 3.39(3.08 - 3.11)(3.24 - 3.32)(-0.23 - -0.15)(2.80 - 3.27)Lysine2.64 (0.023)2.75 (0.023)-0.11 (0.032)-0.20, -0.0230.0252.33, 2.84(2.62 - 2.65)(2.71 - 2.77)(-0.12 - -0.091)(2.38 - 2.74)Monsanto Company 10-SY-210U 427 of 721Table E-31 (continued). Statistical Summary of Site PAHM Soybean Seed Nutrients for MON 87708 (Untreated) vs. ConventionalControlDifference (MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)ConventionalTolerance Interval5(Range)Amino Acid (% dw)Methionine0.57 (0.0090)0.58 (0.0090)-0.017 (0.0072)-0.037, 0.00310.0780.50, 0.64(0.55 - 0.59)(0.57 - 0.60)(-0.036 - -0.0056)(0.52 - 0.63)Phenylalanine2.08 (0.027)2.21 (0.027)-0.13 (0.038)-0.24, -0.0270.0251.80, 2.30(2.03 - 2.11)(2.16 - 2.27)(-0.18 - -0.047)(1.85 - 2.21)Proline2.00 (0.024)2.10 (0.024)-0.11 (0.032)-0.20, -0.0190.0281.65, 2.26(1.93 - 2.04)(2.08 - 2.13)(-0.17 - -0.060)(1.74 - 2.16)Serine2.08 (0.035)2.16 (0.035)-0.078 (0.050)-0.22, 0.0600.1911.78, 2.27(2.02 - 2.11)(2.06 - 2.21)(-0.19 - 0.054)(1.90 - 2.18)Threonine1.57 (0.015)1.62 (0.015)-0.047 (0.021)-0.10, 0.0100.0851.40, 1.69(1.55 - 1.60)(1.60 - 1.64)(-0.077 - 0.0039)(1.47 - 1.64)Tryptophan0.48 (0.012)0.46 (0.012)0.021 (0.017)-0.026, 0.0670.2870.38, 0.52(0.44 - 0.50)(0.45 - 0.46)(-0.013 - 0.049)(0.39 - 0.50)Monsanto Company 10-SY-210U 428 of 721Table E-31 (continued). Statistical Summary of Site PAHM Soybean Seed Nutrients for MON 87708 (Untreated) vs. ConventionalControlDifference (MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)ConventionalTolerance Interval5(Range)Amino Acid (% dw)Tyrosine1.44 (0.027)1.49 (0.027)-0.057 (0.032)-0.15, 0.0330.1521.24, 1.50(1.42 - 1.46)(1.48 - 1.52)(-0.086 - -0.028)(1.26 - 1.49)Valine2.00 (0.029)2.13 (0.029)-0.13 (0.041)-0.24, -0.0140.0351.72, 2.20(2.00 - 2.00)(2.05 - 2.17)(-0.17 - -0.049)(1.73 - 2.13)Fatty Acid (% Total FA)16:0 Palmitic11.79 (0.12)11.49 (0.12)0.31 (0.15)-0.11, 0.720.1128.44, 12.56(11.74 - 11.89)(11.38 - 11.55)(0.20 - 0.37)(9.40 - 11.54)18:0 Stearic3.70 (0.12)3.76 (0.12)-0.056 (0.14)-0.45, 0.330.7082.90, 5.19(3.55 - 3.93)(3.67 - 3.91)(-0.12 - 0.023)(3.24 - 4.67)18:1 Oleic18.45 (0.31)20.01 (0.31)-1.56 (0.35)-2.53, -0.580.01115.73, 27.19(18.26 - 18.80)(19.60 - 20.32)(-2.02 - -1.31)(17.88 - 25.31)18:2 Linoleic55.44 (0.41)54.68 (0.41)0.76 (0.53)-0.70, 2.220.22348.61, 59.37(55.13 - 55.62)(54.18 - 54.99)(0.26 - 1.43)(50.95 - 56.68)Monsanto Company 10-SY-210U 429 of 721Table E-31 (continued). Statistical Summary of Site PAHM Soybean Seed Nutrients for MON 87708 (Untreated) vs. ConventionalControlDifference (MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)ConventionalTolerance Interval5(Range)Fatty Acid (% Total FA)18:3 Linolenic10.03 (0.21)9.47 (0.21)0.56 (0.16)0.11, 1.010.0266.01, 12.58(9.74 - 10.18)(9.13 - 9.68)(0.49 - 0.61)(7.43 - 11.37)20:0 Arachidic0.24 (0.0063)0.24 (0.0063)-0.0067 (0.0071)-0.026, 0.0130.3960.19, 0.34(0.23 - 0.25)(0.24 - 0.25)(-0.011 - -0.0013)(0.20 - 0.30)20:1 Eicosenoic0.086 (0.015)0.072 (0.015)0.014 (0.022)-0.046, 0.0730.5650.022, 0.24(0.066 - 0.13)(0.068 - 0.075)(-0.0088 - 0.058)(0.065 - 0.17)22:0 Behenic0.26 (0.0046)0.27 (0.0046)-0.013 (0.0034)-0.022, -0.00360.0180.24, 0.40(0.25 - 0.27)(0.27 - 0.28)(-0.018 - -0.0076)(0.28 - 0.36)Vitamin (mg/100g dw)Vitamin E1.37 (0.10)1.23 (0.10)0.15 (0.022)0.087, 0.210.0020, 3.49(1.26 - 1.59)(1.11 - 1.40)(0.10 - 0.19)(0.69 - 2.91)1dw = dry weight; fw = fresh weight; FA = fatty acid.2MON 87708 plants were not sprayed with dicamba, but received another conventional treatment as was done for the conventional control.3Mean (S.E.) = least-square mean (standard error).4Control refers to the non-biotechnology derived, conventional control (A3525).5With 95% confidence, interval contains 99% of the values expressed in the population of conventional substances. Negative limits set to zero.Monsanto Company 10-SY-210U 430 of 721Table E-32. Statistical Summary of Site PAHM Soybean Seed Anti-Nutrientsfor MON 87708 (Untreated) vs. ConventionalControlDifference (MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)ConventionalTolerance Interval5(Range)Anti-nutrientLectin (H.U./mg dw)4.41 (0.80)3.85 (0.80)0.57 (1.03)-2.28, 3.410.6100, 7.73(1.96 - 6.35)(3.28 - 4.45)(-1.85 - 3.07)(0.68 - 8.34)Phytic Acid (% dw)1.44 (0.077)1.50 (0.077)-0.057 (0.092)-0.31, 0.200.5690.77, 1.91(1.41 - 1.48)(1.41 - 1.62)(-0.21 - 0.034)(1.00 - 1.64)Raffinose (% dw)0.53 (0.047)0.54 (0.047)-0.014 (0.066)-0.20, 0.170.8410.13, 0.70(0.48 - 0.55)(0.49 - 0.57)(-0.022 - -0.0042)(0.26 - 0.59)Stachyose (% dw)3.43 (0.18)3.36 (0.18)0.067 (0.25)-0.63, 0.760.8002.30, 4.07(3.26 - 3.65)(3.07 - 3.90)(-0.64 - 0.53)(2.50 - 3.94)Trypsin Inhibitor (TIU/mg dw)40.89 (4.33)30.39 (4.33)10.50 (5.59)-5.02, 26.010.13322.05, 41.12(28.31 - 51.50)(26.59 - 33.33)(-2.94 - 18.17)(22.81 - 44.56)Isoflavone (μg/g dw)Daidzein2085.21 (144.27)1642.38 (144.27)442.84 (204.03)-123.65, 1009.320.0950, 2271.38(1931.40 - 2297.58)(1510.07 - 1729.91)(296.74 - 610.43)(451.33 - 2033.05)Monsanto Company 10-SY-210U 431 of 721Table E-32 (continued). Statistical Summary of Site PAHMSoybean Seed Anti-Nutrients for MON 87708 (Untreated) vs.Conventional ControlDifference (MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)ConventionalTolerance Interval5(Range)Isoflavone (μg/g dw)Genistein1300.54 (107.80)1101.98 (107.80)198.56 (152.45)-224.71, 621.830.26278.36, 1869.48(1209.90 - 1469.13)(983.22 - 1162.01)(61.89 - 307.12)(533.88 - 1726.03)Glycitein99.12 (7.95)81.44 (7.95)17.68 (11.25)-13.55, 48.910.19131.24, 233.60(83.25 - 112.67)(68.68 - 90.51)(10.93 - 27.53)(73.61 - 231.75)1dw = dry weight; H.U. = Hemagglutinating Units; TIU = Trypsin Inhibitor Units.2MON 87708 plants were not sprayed with dicamba, but received another conventional treatment as was done for the conventional control.3Mean (S.E.) = least-square mean (standard error).4Control refers to the non-biotechnology derived, conventional control (A3525).5With 95% confidence, interval contains 99% of the values expressed in the population of conventional substances. Negative limits set to zero.Monsanto Company 10-SY-210U 432 of 721Table E-33. Statistical Summary of Site PAHM Soybean Forage Nutrients for MON 87708 (Untreated) vs. ConventionalControlDifference (MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)ConventionalTolerance Interval5(Range)Proximate (% dw)Ash8.26 (0.26)7.88 (0.32)0.38 (0.41)-0.93, 1.690.4223.36, 10.84(7.81 - 8.65)(7.67 - 8.09)(-0.28 - 0.97)(5.20 - 9.81)Carbohydrates68.76 (1.04)65.81 (1.16)2.95 (1.03)-0.35, 6.240.06560.69, 73.46(67.19 - 71.05)(65.74 - 66.41)(1.45 - 4.64)(62.73 - 71.72)Moisture (% fw)74.47 (0.63)74.91 (0.63)-0.44 (0.15)-0.91, 0.0210.05562.08, 89.80(73.40 - 75.60)(73.80 - 74.90)(-0.50 - -0.40)(70.40 - 84.10)Protein19.46 (1.26)21.96 (1.45)-2.49 (1.45)-7.10, 2.120.18315.69, 26.63(16.28 - 21.25)(21.49 - 21.91)(-5.21 - -0.66)(18.50 - 25.86)Total Fat3.46 (0.26)4.18 (0.31)-0.72 (0.31)-1.70, 0.270.1020, 10.04(2.69 - 3.87)(4.30 - 4.35)(-0.49 - -0.48)(1.57 - 7.99)Fiber (% dw)Acid Detergent Fiber25.62 (1.95)26.02 (2.39)-0.40 (3.08)-10.21, 9.420.90516.54, 41.80(24.22 - 28.01)(21.79 - 30.24)(-2.23 - 2.83)(20.98 - 39.23)Monsanto Company 10-SY-210U 433 of 721Table E-33 (continued). Statistical Summary of Site PAHM Soybean Forage Nutrients for MON 87708 (Untreated) vs. ConventionalControlDifference (MON 87708 minus Control)Analytical Component (Units)1MON 877082Mean (S.E.)3(Range)Control4Mean (S.E.)(Range)Mean (S.E.)(Range)95%Confidence IntervalSignificance(p-Value)ConventionalTolerance Interval5(Range)Fiber (% dw)Neutral Detergent Fiber28.70 (1.01)30.20 (1.24)-1.50 (1.60)-6.59, 3.580.41520.28, 44.03(27.46 - 29.45)(30.00 - 30.40)(-0.95 - -0.83)(24.81 - 42.80)1dw = dry weight; fw = fresh weight.2MON 87708 plants were not sprayed with dicamba, but received another conventional treatment as was done for the conventional control.3Mean (S.E.) = least-square mean (standard error).4Control refers to the non-biotechnology derived, conventional control (A3525).5With 95% confidence, interval contains 99% of the values expressed in the population of conventional substances. Negative limits set to zero.Monsanto Company 10-SY-210U 434 of 721 

### References for Appendix E

AACC. 1999. Official me thod 32-20. American Association of Cereal Chemists, St. Paul,

Minnesota.

AOAC-International. 2005a. AOAC offici al method 982.30. Official Methods of Analysis of AOAC International, Associati on of Analytical Communities International, Gaithersburg, Maryland.

AOAC-International. 2005b. AOAC offici al method 923.03. Official Methods of Analysis of AOAC International, Associati on of Analytical Communities International, Gaithersburg, Maryland.

AOAC-International. 2005c. AOAC offici al method 962.09. Official Methods of Analysis of AOAC International, Associati on of Analytical Communities International, Gaithersburg, Maryland.

AOAC-International. 2005d. AOAC offi cial method 922.06 and 954.02. Official Methods of Analysis of AOAC International, Association of Analytical Communities International, Gaithersburg, Maryland.

AOAC-International. 2005e. AOAC offici al method 960.39 and 948.22. Official Methods of Analysis of AOAC International, Associat ion of Analytical Communities International, Gaithersburg, Maryland.

AOAC-International. 2005f. AOAC offi cial method 983.23. Official Methods of Analysis of AOAC International, Associati on of Analytical Communities International, Gaithersburg, Maryland.

AOAC-International. 2005g. AOAC offi cial method 926.08 and 925.09. Official Methods of Analysis of AOAC Internationa l, Association of Analytical Communities International, Gaithersburg, Maryland.

AOAC-International. 2005h. AOAC offi cial method 955.04 and 979.09. Official Methods of Analysis of AOAC Internationa l, Association of Analytical Communities International, Gaithersburg, Maryland.

AOCS. 1997a. Method Ce 2-66. Official Methods and Reco mmended Practices of the American Oil Chemists Society, 5th Ed ition. American Oil Chemists Society, Champaign.

AOCS. 1997b. Method Ba 12-75. O fficial Methods and Recomm ended Practices of the American Oil Chemists Society, 5th Ed ition. American Oil Chemists Society, Champaign.Monsanto Company 10-SY-210U 435 of 721 

### AOCS. 1997c. Method Ce 1e-62. Official Methods and Reco mmended Practices of the American Oil Chemists Society, 5th Edition American Oil Chemists Society, Champaign.

Bradstreet, R.B. 1965. The Kjel dahl method for organic nitrogen. Academic Press. New York, New York.

Brobst, K.H. 1972. Gas-liquid chromatography of trimethylsilyl derivatives. Pages 3-8 in Methods in Carbohydrate Chemistry. Volume 6. Academic Press, New York, New York.

Cort, W., T.S. Vicente, E.H. Waysek and B.D. Williams. 1983. Vitamin E content of feedstuffs determined by high-performance li quid chromatographic fluorescence. Journal of Agriculture and Food Chemistry 31:1330-1333.

Goering, H.K. and P.J. Van Soest. 1970. Forage and fiber analyses (apparatus, reagents, procedures, and some applications). Agri culture Handbook No. 379. U.S. Department of Agriculture, Agricultural Res earch Service, Washington, D.C.

Klurfeld, D.M., and D. Kritchevsky. 1987. Is olation and quantitatio n of lectins from vegetable oils. Lipids 22:667-668.

Kolthoff, I.M., and E.B. Sandell. 1948. Quantit ative inorganic analysis. MacMillan, New York, New York.

Lehrfeld, J. 1989. High-performance liquid chro matography analysis of phytic acid on a pH-stable, macroporous polymer co lumn. Cereal Chemistry 66:510-515.

Lehrfeld, J. 1994. HPLC separation and quant itation of phytic acid and some inositol phosphates in foods: Problem and solutions. J ournal of Agricultural and Food Chemistry

42:2726-2731.

Liener, I.E. 1955. The photometric determina tion of the hemagglutinating activity of soyin and crude soybean extracts. Archives of Biochemistry and Biophysics 54:223-231.

Mason, B.S., and H.T. Slover. 1971. A gas chromatographic method for the determination of sugars in foods. Journa l of Agricultural and Food Chemistry 19:551-

554.

McMurray, C.H., W.J. Blanchflower, and D.A. Rice. 1980. Influence of extraction techniques on determination of alpha-tocop herol in animal feedstuffs. JournalAssociation of Official An alytical Chemists 63:1258-1261.

OECD. 2001. Consensus document on compositiona l considerations fo r new varieties of soybean: Key food and feed nutrients and anti-nutrients. ENV/JM/MONO(2001)15. Series on the Safety of Novel Foods a nd Feeds No.2. Organisation for Economic Cooperation and Development, Paris, France.Monsanto Company 10-SY-210U 436 of 721 

### Pettersson, H., and K.-H. Kiessling. 1984. Liqui d chromatographic determination of the plant estrogen coumestrol and isoflavones in animal feed. Journa l of Association of Official Analytical Chemis ts International 67:503-506.

SAS Software Release 9.2 (TS1M0). © 

### 2002-2008 by SAS Institute Inc., Cary, North

Carolina.

Seo, A., and C.V. Morr. 1984. Improved hi gh-performance liquid chromatographic analysis of phenolic acids a nd isoflavonoids from soybean protein products. Journal of Agricultural and Food Chemistry 32:530-533.

Speek, A.J., J. Schrijver, and W.H.P. Sc hreurs. 1985. Vitamin E composition of some seed oils as determined by high-performa nce liquid chromatography with fluorometric

detection. Journal of Food Science 50:121-124.

USDA. 1973. Energy value of foods. Agricultural handbook No. 74:2-11.Monsanto Company 10-SY-210U 437 of 721 

### Appendix F: Materials, Methods, and Individual Site Results for the Seed Dormancy and Germination Assessment of MON 87708

F.1. Materials

Seed dormancy and germination characterist ics were assessed on seed from MON 87708, the near isogenic conventional soybean c ontrol A3525, and commercial reference varieties produced at the Howard County, Iowa; Stark County, Illinois; and Shelby County, Missouri sites in 2008 field trials (Appendi x G). The field tria l at each site was established in a randomized complete block design with three replications. The seed from MON 87708, conventional control, and th e commercial reference varieties were harvested from all three replicated plots at each of the three fi eld sites. In order to provide a representative sample from each field site for dormancy and germination testing, the replicate samples from each field trial were po oled to produce one seed sample (lot) of MON 87708, the conventional control, and each commercial reference variety (Table F1).

Table F-1. Starting Seed of MON 87708, Conventional Control, and Commercial

Reference Varieties Used in the Dorm ancy and Germination Assessment

Production

Site 1 Material Name 2 Material Type Phenotype Seed Lot #

IA MON 87708 Test Dicamba-Tolerant 11222450-001

IA A3525 Control Conventional 11222449-001

IA Garst 3585N Reference Conventional 11222451-001

IA Pioneer 93B15 Referenc e Conventional 11222452-001

IA Crows C37003N Referenc e Conventional 11222453-001 IA NK S33-A8 Reference Roundup Ready 11222454-001

IL MON 87708 Test Dicamba-Tolerant 11222456-001

IL A3525 Control Conventional 11222455-001 IL Croplan HT3596STS Reference Conventional 11222457-001 IL NK S37-N4 Reference Roundup Ready 11222458-001

IL Stewart SB3454 Referenc e Conventional 11222459-001

IL Midland 363 Referenc e Conventional 11222460-001

MO MON 87708 Test Dicamba-Tolerant 11222462-001

MO A3525 Control Conventional 11222461-001

MO Garst 3585N Reference Conventional 11222463-001

MO Pioneer 93B15 Referenc e Conventional 11222464-001

MO Crows C37003N Referenc e Conventional 11222465-001 MO NK S33-A8 Reference Roundup Ready 11222466-001

1 IA = Howard County, IA; IL = Stark County, IL; MO = Shelby County, MO.

2 MON 87708, the conventional control, and commercial reference varieties seed used to assess dormancy and germination characteristics were all produced from re plicated field trials conducted in 2008 to assess plant phenotypic characteristics and, therefore, were not obtained from commercial sources. TheMonsanto Company 10-SY-210U 438 of 721 commercial reference varieties were all conventional so ybean varieties with the exception of NK S33-A8 and NK S37-N4, which were Roundup Ready  soybean varieties.



### F.2. Characterization of the Materials

For the MON 87708, conventional control, a nd the commercial reference varieties starting seed lots, the presence or absence of the dmo expression cassette was confirmed by event-specific polymerase chain reaction analyses.

F.3. Germination Testing Facili ty and Experimental Methods

Seed dormancy and germination evaluations we re conducted at BioDiagnostics, Inc. in River Falls, WI. The principal investigat or was qualified to conduct seed dormancy and germination testing consistent with the st andards established by the Association of Official Seed Analysts, a seed trade a ssociation (AOSA, 2000; AOSA, 2006; AOSA, 2007).

Seed lots of MON 87708, the conventional c ontrol, and four commercial reference varieties were produced from each of thre e sites and tested under six different temperature regimes. Six germination chambe rs were maintained dark under one of the following temperature regimes: constant temperature of approximately 10, 20, or 30°C or alternating temperatures of approximat ely 10/20, 10/30, or 20/30°C. The alternating temperature regimes were maintained at the lower temperature for 16 hours and the higher temperature for eight hours. The temperature inside each germination chamber was monitored and recorded every 15 minutes throughout the duration of the assessment. For each seed lot, four replic ated paper germination towels were prepared per facility SOPs for each temperature regime. Wax co ated paper was placed on a large tray followed by a water-moistened germination towe l. A target of 100 seeds per seed lot were placed on the germination towel ( i.e. , one seed lot per to wel) using a vacuum planting system. A second water-moistened germination towel was placed on top of the seed. The towels were then rolled up a nd secured with a rubbe r band. All rolled germination towels were placed into appropriate ly labeled buckets that were then covered with ventilated plastic bags attached with rubber bands. The bucke ts were arranged in the germination chambers in a split-plot de sign, where the whole-plot treatment was seed production site and the sub-plot treatment was seed material ( i.e. , MON 87708, the conventional control, or commerc ial reference varieties).

A description of each germination characterist ic evaluated and the timing of evaluations are presented in Table VII-1. The types of data collected depended on the temperature regime. Each rolled germination towel in the AOSA-recommended temperature regime ( i.e. , 20/30°C) was evaluated periodically during the study for normal germinated, abnormal germinated, hard, dead, and firm-swollen seed as defined by AOSA guidelines (AOSA, 2006; AOSA, 2007). AOSA only provid es guidelines (AOSA, 2007) for testing seed under optimal temperatures (20/30°C); however, additional temperature regimes were included to test a range of temperature conditions. Each rolled germination towel in

 Roundup Ready is a registered trademark of Monsan to Technology LLC. All other trademarks are the property of their respective owners.Monsanto Company 10-SY-210U 439 of 721 

### the additional temperature regimes ( i.e. , 10, 20, 30, 10/20, and 10/30°C) was evaluated periodically for germinated, hard, dead, and firm-swollen seed. Emergence and/or development of essential struct ures of seedlings that otherw ise would be categorized as “normal germinated” under optimal temperature conditions may not be so at non-optimal temperatures. Therefore, for the additional te mperature regimes, no distinction was made between normal and abnormal germinated seed.

F.4. Statistical Analysis

Analysis of variance was conducted according to a split-plot design with four replications.

SAS ® 

### (Version 9.2) was used to compare MON 87708 to the conventional control within each seed production site (individual-site anal yses) and in a combined-site analysis, in which the data were pooled across all sites, for the following germination characteristics: percent germinated (categorized as percent normal germinated and percent abnormal germinated for the AOSA temperature regime), percent viable hard, percent dead, and percent viable firm-swollen seed. The level of statistical significance was predetermined to be 5% ( α = 0.05). MON 87708 was not statistical ly compared to the commercial reference varieties nor were comparisons ma de across temperature regimes. For each assessed characteristic, the minimum and ma ximum means were determined from among the commercial reference varieties to provide a range of values that are representative of commercial soybean varieties. The following is a summary of the results from the individual-site analyses. Results from the co mbined-site analysis are presented in Table

VII-2.

F.5. Individual-Site Seed Dormancy and Germination Analysis

In the individual-site analys es, no statistically significan t differences were detected between MON 87708 and the conventional control for any of the measured characteristics ( i.e. , percent germinated, viab le hard, dead, or viable firm-swollen seed) in any temperature regime for seed produced at the MO site. Six stat istically significant

differences in total were detected betw een MON 87708 and the conventional control for seed produced at the IA and IL sites (Table F-2). MON 87708 had lower percent germinated seed than the conventional cont rol at 10°C for seed produced at the IA (97.5% vs. 99.3%) and IL (99.3% vs. 100.0%) site s and at 10/30°C for seed produced at the IA site (96.8% vs. 99.0%). MON 87708 had lower percent viable hard seed than the conventional control at 10/30°C for seed pro duced at the IA site (0.0% vs. 0.3%). Percent dead seed was higher for MON 87708 than the conventional control at 10°C (2.0% vs. 0.3%) and 10/30°C (3.3% vs. 0.8%) for seed produced at the IA site.

Statistically significant diffe rences between MON 87708 and the conventional control for germination characteristics in the individual-site analyses were not consistently detected across temperature regimes or seed production s ites. While some statistically significant

differences were detected in the combin ed-site analysis, the assessed dormancy and germination characteristics of MON 87708 were within the range of values expected for

the commercial reference varieties and th erefore are considered not biologically meaningful in terms of increased w eediness of MON 87708 compared to the conventional soybean.Monsanto Company 10-SY-210U 440 of 721Table F-2. Dormancy and Germination Characteristics of MON 87708 andConventional Control SeedProduced at each ofThree Field SitesTemperatureRegime Germination CategoryMean % (S.E.)1IA IL MOMON 87708 Control MON 87708 Control MON 87708 Control10°C Germinated 97.5 (1.0)* 99.3 (0.5) 99.3 (0.3)* 100.0 (0.0) 100.0 (0.0) 99.8 (0.3)Viable Hard 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0)Dead 2.0 (0.9)* 0.3 (0.3) 0.5 (0.3) 0.0 (0.0) 0.0 (0.0) 0.3 (0.3)Viable Firm-swollen 0.5 (0.3) 0.5 (0.3) 0.3 (0.3) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0)20°C Germinated 98.0 (0.9) 98.3 (0.6) 99.8 (0.3) 99.8 (0.3) 100.0 (0.0) 100.0 (0.0)Viable Hard 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.3 (0.3) 0.0 (0.0) 0.0 (0.0)Dead 2.0 (0.9) 1.8 (0.6) 0.3 (0.3) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0)Viable Firm-swollen 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0)30°C Germinated 96.3 (1.1) 98.3 (0.3) 100.0 (0.0) 100.0 (0.0) 99.8 (0.3) 99.8 (0.3)Viable Hard 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0)Dead 3.8 (1.1) 1.8 (0.3) 0.0 (0.0) 0.0 (0.0) 0.3 (0.3) 0.3 (0.3)Viable Firm-swollen 0.0 (0.0)† 0.0 (0.0) 0.0 (0.0)† 0.0 (0.0) 0.0 (0.0)† 0.0 (0.0)Monsanto Company 10-SY-210U 441 of 721Table F-2 (continued). Dormancy and Germination Characteristicsof MON 87708 and ConventionalControl Seed Producedat each of Three Field SitesTemperatureRegime Germination CategoryMean % (S.E.)1IA IL MOMON 87708 Control MON 87708 Control MON 87708 Control10/20°C Germinated 98.0 (0.4) 97.8 (0.3) 100.0 (0.0) 100.0 (0.0) 100.0 (0.0) 100.0 (0.0)Viable Hard 0.3 (0.3) 0.3 (0.3) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0)Dead 1.8 (0.3) 1.8 (0.3) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0)Viable Firm-swollen 0.0 (0.0) 0.3 (0.3) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0)10/30°C Germinated 96.8 (0.8)* 99.0 (0.4) 99.0 (0.6) 100.0 (0.0) 100.0 (0.0) 100.0 (0.0)Viable Hard 0.0 (0.0)* 0.3 (0.3) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0)Dead 3.3 (0.8)* 0.8 (0.5) 1.0 (0.6) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0)Viable Firm-swollen 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0)20/30°C2Normal Germinated 91.5 (2.1) 92.0 (0.7) 97.5 (1.0) 99.0 (0.4) 98.8 (0.5) 98.8 (0.6)Abnormal Germinated 5.8 (1.0) 6.3 (1.0) 1.8 (0.9) 0.8 (0.5) 1.3 (0.5) 1.3 (0.6)Viable Hard 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0)Dead 2.8 (1.3) 1.8 (0.8) 0.8 (0.3) 0.3 (0.3) 0.0 (0.0) 0.0 (0.0)Viable Firm-swollen 0.0 (0.0)† 0.0 (0.0) 0.0 (0.0)† 0.0 (0.0) 0.0 (0.0)† 0.0 (0.0)Note: Seed for the germination study were produced in Howard County, IA; Stark County, IL; and Shelby County, MO in 2008. Seed was arranged ingermination chambers in a split-plot design where the whole-plot treatment was seed production site and the sub-plot treatmentwas seed material (i.e.,MON 87708, the conventional control, or commercial reference soybean varieties).*Indicates a statistically significant difference between MON 87708and the conventional control (α=0.05).†No statistical comparisons were made due to lack of variability in the data.1Means based on four replicates (n = 4) of 100 seeds. Thetotal percentage of all germination characteristics of MON 87708or the conventional control in sometemperature regimes is greater than 100.0% due to numerical rounding of the means. S.E. = Standard Error2Germinated seed in the AOSA temperature regime 20/30°C were categorized as either normal germinated or abnormal germinated seed.Monsanto Company 10-SY-210U 442 of 721 

### References for Appendix F

AOSA. 2000. Tetrazolium testin g handbook. Association of Official Seed Analysts, Lincoln, Nebraska

AOSA. 2006. Seed evaluation handbook. Co ntribution No. 35. in Handbook on Seed Testing Association of Official Seed Analysts, Lincoln, Nebraska.

AOSA. 2007. Rules for testing seeds. Associatio n of Official Seed Analysis, Ithaca, New

York.

SAS Software Release 9.2 (TS1M0). © 

### 2002-2008 by SAS Institute Inc., Cary, North

Carolina.Monsanto Company 10-SY-210U 443 of 721 

### Appendix G: Materials, Methods, an d Individual-Site Results from the Phenotypic, Agronomic, and Enviro nmental Interaction Assessment of

MON 87708 under Field Conditions

G.1. Materials

The soybean materials for the phenotypic and environmental interactions assessment in

the 2008 field included untreat ed MON 87708, the near isogen ic conventional soybean control A3525, and 18 commercial reference soyb ean varieties. The references included both conventional and Roundup Ready  

### soybean varieties. The list of the soybean materials planted at each of 18 field sites is presented in Table G-1.

The soybean materials for the phenotypic and environmental interactions assessment in

the 2009 field included dicamba-treated MO N 87708, the near isogenic conventional soybean control A3525, and 14 conventional, commercial reference soybean varieties. The list of the soybean materials planted at eac h of 8 field sites is presented in Table G-2.

G.2. Characterization of the Materials

For the MON 87708 and the conven tional control starting seed lots, the presence or

absence of the dmo expression cassette was confirmed by event-specific polymerase chain reaction analyses.

G.3. Field Sites and Plot Design

Data were collected at 16 field sites in th e U.S. and two sites in Canada during 2008 (Section VII, Table VII-3). Data were coll ected at 8 field sites in the U.S. during 2009 (Section VII, Table VII-4). These 26 lo cations provided a diverse range of environmental and agronomic conditions representative of commercial soybean production areas in North America. The resear chers at each field site were familiar with the growth, production, and evaluati on of soybean characteristics.

The experiment was established at each of the 18 sites in the 2008 field trials in a randomized complete block design with three replica tions. Each plot at the IL2, IN1, MI,

and MO1 sites consisted of tw elve 30 feet long rows spaced approximately 30 inches apart. Rows # 2 and 3 were designated fo r the collection of phenotypic data. Rows # 5 and 6 were designated for the collection of abiotic stress re sponse, disease damage, and arthropod-related damage da ta. Rows # 8-10 were desi gnated for the collection of arthropod samples. Rows # 1, 4, 7, 11, and 12 were used as buffer rows. Each plot was surrounded by approximately 5-15 feet of a commercial soybean variety by planting border rows in the alleyways between blocks and around the entire perimeter of the plot area. The purpose of the planted borders wa s to create a continuous soybean stand across the plot area to ensure collection of mo re robust arthropod abundance data.

 Roundup Ready is a registered trademark of Monsan to Technology LLC. All other trademarks are the property of their respective owners.Monsanto Company 10-SY-210U 444 of 721 

### Each plot at the AR, Can1, Can2, IA1, IA 2, IA3, IL1, IN2, IN3, KS, MO2, NE, PA, and WI sites consisted of four 20 feet long ro ws spaced approximately 30 inches apart. Rows # 2 and 3 were designated for the coll ection of phenotypic, abiotic stress response, disease damage, and arthropod-related damage da ta. Rows # 1 and 4 were used as buffer rows. The entire plot area was surrounded by an approximately 10 foot wide, four-row border of a commercial soybean variety.

In the 2009 field trials the experiment was es tablished at each of th e sites in a randomized complete block design with four replications. Each plot c onsisted of eight 20 feet long rows spaced approximately 30 inches apart. Rows 1 and 2 were designated for the collection of plant tissue and ha rvested seed samples for use in other studies. Rows 4 and 5 were designated for the collection of phe notypic, abiotic stre ss response, disease damage, and arthropod damage data in this report. Rows 3 and 6 - 8 were used as buffer rows. The plots within each replicate were separated by approximately a two-row buffer of a commercially-available s oybean variety, and th e entire plot area was surrounded by approximately a four-row border of a commer cially-available soybean variety. A

minimum of a 15 foot fallow area was estab lished around the perimeter of the study area at each site to clearly isolate th e experiment from other soybean.

G.4. Planting and Field Operations

Field and planting information for the 2008 field trials are listed in Table G-3. Field and planting information for the 2009 field tria ls are listed in Table G-4. Agronomic practices used to prepare and maintain each st udy site were characteristic of those used in each respective geographic region. All ma intenance operations were performed uniformly over the entire trial area.Monsanto Company 10-SY-210U 445 of 721Table G-1. Starting Seed for the Phenotypic, Agronomic, and Environmental Interaction Assessment for 2008 Field TrialsMaterial NameMaterialTypeRelativeMaturityPhenotypeMonsantoSeed Lot # Sites1MON 87708 Test 3.5 Dicamba-Tolerant 10001256 AllA3525 Control 3.5 Conventional 10001257 AllFS 3591Reference3.5Conventional10001448 AR, IA2, IN1, MI, PAAG3505Reference3.5Roundup Ready210001281 AR, IA2, IN1, MI, PAWilken 3316Reference3.3Conventional10001505 AR, IA2, IN1, MI, PAStine 3300-0Reference3.3Conventional10001312 AR, IA2, IN1, MI, PAGarst 3585NReference3.5Conventional10000883 IA3, IN2, MO1, WICrows C37003NReference3.7Conventional10001508 IA3, IN2, MO1, WIGarst S33-A8Reference3.3Roundup Ready210001284Can1, IA3, IN2, MO1,Pioneer 93B15Reference3.1Conventional10001304Can1, IA3, IN2, MO1,Dekalb DKB28-53 Reference 2.8 Roundup Ready210001950 Can1, Can2Asgrow AG2801 Reference 2.8 Roundup Ready210001951 Can1, Can2Pioneer 93M52Reference3.5Conventional10001311 Can2, IL1, IN3, MO2NK S38-T8Reference3.8Conventional10001509 IL1, IN3, MO2Lewis 3716Reference3.7Roundup Ready210001278 IL1, IN3, MO2Hoegemeyer 333Reference3.2Conventional10001590 Can2, IL1, IN3, MO2CroplanReference3.5Conventional10001450 IA1, IL2, KS, NENK S37-N4Reference3.7Roundup Ready210001286 IA1, IL2, KS, NEStewart SB3454Reference3.4Conventional10000887 IA1, IL2, KS, NEMidland 363Reference3.3Conventional10001570 IA1, IL2, KS, NE1MON 87708 and the conventional control were planted at all fieldsites; the commercial reference varieties were site-specific.Site codes are as follows: AR =Jackson County, AR; Can1 = Norfolk, Ontario, Canada; Can2 = Kent, Ontario, Canada; IA1 = Jefferson County, IA; IA2 = Benton County, IA; IA3 = HowardCounty, IA; IL1 = Clinton County, IL; IL2 = Stark County, IL, IN1 = Boone County, IN; IN2 = Clinton County, IN; IN3 = Parke County, IN; KS = PawneeCounty, KS; MI = Ottawa County, MI; MO1 = Shelby County, MO; MO2= Macon County, MO; NE = York County, NE; PA = Berks County, PA; WI =Walworth County, WI.2Commercial Roundup Ready soybean varietyMonsanto Company 10-SY-210U 446 of 721Table G-2. Starting Seed for the Phenotypic, Agronomic, and Environmental Interaction Assessment for 2009 Field TrialsMaterial NameMaterial Type1RelativeMaturityPhenotype2MonsantoSeed Lot #Site3A3525Control3.5Conventional11225301ALLFS 3591 Reference3.5 Conventional10001448 ARNECrows C3908 Reference3.9Conventional 10001074 ARNENK S38-T8 Reference3.8Conventional 10001509 ARNECroplan HT3596STS Reference3.5Conventional 10001117IARLMidland 363 Reference3.3Conventional 10001570IARLStewart SB3454 Reference3.4Conventional 10001130IARLQuality Plus 365C Reference3.6Conventional 10001129 ILCYChannel Bio 3461 Reference3.4Conventional 10001115 ILCYPioneer 93M52 Reference3.5Conventional 10001128 ILCYFS 3591 Reference3.5Conventional 10001448 ILWYStewart SB3454 Reference3.4Conventional 10001130 ILWYNK 32Z3 Reference3.2Conventional 10001126 ILWYGarst 3585N Reference3.5Conventional 10001119 INRCChannel Bio 37002 Reference3.7Conventional 10001116 INRCQuality Plus 365C Reference3.6Conventional 10001129 INRCCroplan HT3596STS Reference3.5Conventional 10001117 INSHStewart SB3454 Reference3.4Conventional 10001130 INSHCrows C37003N Reference3.7Conventional 10001508 INSHPioneer 93M52 Reference3.5Conventional 10001128 KSLANK S38-T8 Reference3.8Conventional 10001509 KSLAQuality Plus 365C Reference3.6Conventional 10001129 KSLAGarst 3585N Reference3.5Conventional 10001119NEYOWilken 3316 Reference3.3Conventional 10001505NEYOMidland 363 Reference3.3Conventional 10001570NEYOMON 877084TestDT 11225299 ALLMonsanto Company 10-SY-210U 447 of 7211T = Test, C = Control, and R = reference.2Phenotypic abbreviations: NT = non-biotech conventional, Conventional = conventional commercial, DT = dicamba-tolerant.3Site codes are as follows: ARNE = Jackson County, AR; IARL = Jefferson County, IA; ILCY = Clinton County, IL; ILWY = Stark County, IL;INRC = Parke County, IN; INSH = Boone County, IN;KSLA = Pawnee County, KS; NEYO = York County, NE4Received a mandatory dicamba application.Monsanto Company 10-SY-210U 448 of 721Table G-3. Field and PlantingInformation for 2008 Field TrialsSitePlantingDate1PlantingRate(seeds/ft)PlantingDepth(in)PlotSize(ft)2Rows/Plot Soil Series, Organic Matter, pHCropping History2007 2006AR 05-29-088-9 0.7510 × 204 Bosket sandy loam; 1.2%; 6.4 Cotton SoybeanCan1 05-26-0891.510 × 204 Norfolk sandy loam; 1.5%; 6.6Corn SoybeanCan2 05-27-089 1.5 10 × 20 4 Thames clay loam; 3.4%; 7.4 Corn SoybeanIA1 06-07-0891.2510 × 204 Mahaska silty clay loam; 3.18%; 5.9 Sorghum SoybeanIA2 06-19-0892.010 × 204Tama Muscatine silty clay loam; 3.9%;6.1Soybean Milk thistleIA3 06-26-0891.010 × 204 Lawler loam; 7.3%; 7.6 Corn SoybeanIL1 06-19-0891.2510 × 204Cisne-Huey Complex silt loam; 1.3%;7.1Milo SoybeanIL2 06-02-0891.2530 × 3012 Plano silt loam; 3.5%; 6.4 Corn SoybeanIN1 05-28-0891.530 × 3012 Crosby silt loam; 2.5%; 7.1 Corn SoybeanIN2 05-27-0891.510 × 204 Fincastle silt loam; 2.0%; 7.0 Sweet corn SoybeanIN3 07-01-0891.010 × 204 Silty loam; 3%; 6.5 Soybean WheatKS 06-04-089 1−1.25 10 × 20 4 Farnum loam; 2.6%; 7.6 Sorghum WinterwheatMonsanto Company 10-SY-210U 449 of 7211Month-day-year.2Width × length.MI 05-27-0891.530 × 3012 Nester loam; 2.1%; 6.5 Corn SoybeanMO1 06-18-0891.2530 × 3012 Putnam silt loam; 2.1%; 6.6 Corn SoybeanMO2 06-19-0891.010 × 204 Gorin silt loam; 4.2%; 6.3 Soybean FescueNE 06-02-0891.010 × 204 Hastings silt loam; 3.0%; 6.2 Soybean SoybeanPA 06-03-0891.2510 × 204 Philo/Atkins silt loam; 2.0%; 6.2 Fallow TomatoesWI 05-29-0891.2510 × 204 Radford silt loam; 2.2%; 5.9 Corn CornMonsanto Company 10-SY-210U 450 of 721Table G-4. Field and PlantingInformation for 2009 Field Trials1Site codes are as follows: ARNE = Jackson County, AR; IARL = Jefferson County, IA; ILCY = Clinton County, IL; ILWY = Stark County, IL;INRC = Parke County, IN; INSH = Boone County, IN;KSLA = Pawnee County, KS; NEYO = York County, NE2Month-day-year.3Width × length.*Two packets were planted on 7/06/2009 toreplace two packets broken on 6/30/2009.SiteCode1Plantingdate2Plantingrate(seeds/ft)Plantingdepth(in)Plotsize (ft)3Rows/plot Soil series, organic matter, pHCropping History2008ARNE 6/20/2009 9 0.5 20 × 20 8 Bosket sandy loam, 1.0%, 6.0 CottonIARL 6/26/2009 9 1.3 20 × 20 8 Taintor/Mahaskasilty clay loam, 3.5%, 7.0 Sorghum/SoybeanILCY 6/29/2009 9 1.4 20 × 20 8 Hoyleton-Darmstadt silt loam, 2.6%, 7.3 SorghumILWY 6/24/2009 9 1.8 20 × 20 8 Flanagan silt loam, 3.8%, 6.5 CornINRC 6/30/20097/06/2009*91.0 20 × 20 8 Reeseville silt loam, 1.4%, 5.8 WheatINSH 6/19/2009 9 1.5 20 × 20 8 Crosby silt loam, 2.3%, 5.6 CornKSLA 6/23/2009 9 1.0 20 × 20 8 Siltloam, 2.6%, 7.6 Fallow/SorghumNEYO 6/24/2009 9 1.0 20 × 20 8 Hastings silt loam, 3.0%, 6.2 SorghumMonsanto Company 10-SY-210U 451 of 721 

### G.5. Phenotypic Observations

The description of the charac teristics measured and the de signated developmental stages

when observations occurred are listed in Table VII-1.

G.6. Environmental Interaction Observations

Environmental interactions ( i.e. , interactions between the cr op plants and their receiving environment) were used to characteri ze MON 87708 by evaluating plant response to abiotic stress, disease damage, arthropod-re lated damage, and pest and beneficial arthropod abundance in the pl ots using the methods described in G.7 and G.8.

G.7. Abiotic Stress Response, Disease Damage, and Arthropod-Related Damage

MON 87708 and the conventional control were ev aluated at all 26 site s for differences in plant response to abiotic stress, disease da mage, and arthropod-related damage. Three abiotic stressors, three diseases, and thr ee arthropod pests were evaluated four times during the growing season at the following intervals:

Observation 1: V2 – V4 growth stage 12


### Observation 2: R1 – R2 growth stage Observation 3: R3 – R5 growth stage Observation 4: R6 – R8 growth stage

The researcher at each field site chose abio tic stressors, diseases, and arthropod pests that were either actively causing pl ant injury in the study area or were likely to occur in soybean during the given observa tion period. Therefore, abio tic stressors, diseases, and arthropod pests assessed often varied between observations at a site and between sites.

Abiotic stress response and disease damage obs ervations were collected from each plot using a continuous 0 – 9 scale of increasing se verity. Data were collected numerically and then placed into one of the follo wing 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)

Arthropod-related damage was assessed from each plot on the upper four nodes of 10 representative plants using the arthropod-sp ecific 0 – 5 rating sc ales of increasing severity listed below.

12 For the 2009 field trials Observation 1 occurred at the V2-V3 growth stage.Monsanto Company 10-SY-210U 452 of 721 

### Defoliating arthropods ( e.g. , corn earworm, bean leaf beetle, Japanese beetle, soybean looper) Rating Severity of plant damage

0 None

1 1 – 20 % defoliation

2 21 – 40% defoliation

3 41 – 60% defoliation

4 61 – 80% defoliation

5 > 80% defoliation

Pod feeding arthropods ( e.g. , corn earworm, bean leaf beetle, stink bug, Lygus bug on reproductive plant parts) Rating Severity of plant damage

0 None 1 1 – 20 % damaged pods 2 21 – 40% damaged pods 3 41 – 60% damaged pods 4 61 – 80% damaged pods 5 > 80% damaged pods

Leafhoppers ( e.g. , potato leafhopper) Rating Severity of plant damage

0 None

1 1 – 50% of foliage with leaf yellowing; no leaf puckering or leaf margin necrosis

2 1 – 50% of foliage with leaf yellowing, leaf puckering and/or leaf margin necrosis

3 > 50% of foliage with leaf yellowing; no leaf puckering or leaf margin necrosis

4 > 50% of foliage with leaf yellowing, leaf puckering, and/or leaf margin necrosis

5 > 50% of foliage with necrotic leaves (leaves dead due to leafhopper damage)

Aphids ( e.g. , soybean aphid) Rating Severity of plant damage

0 None 1 1 – 100 aphids per plan t; no leaf puckering 2 101 – 250 aphids per plant; no leaf puckering

3 ≥ 250 aphids per plant with leaf puckering

4 ≥ 250 aphids per plant with leaf puckering and leaf yellowing and/or necrosis

5 ≥ 250 aphids per plant with plant stuntingMonsanto Company 10-SY-210U 453 of 721 

### G.8. Arthropod Abundance

Pest and beneficial arthropods were collect ed at the IL2, IN1, MI, and MO1 sites four times during the growing season at the following intervals:

Collection 1: R1 – R2 growth stage Collection 2: Approximately two weeks after collection 1 Collection 3: Approximately two weeks after collection 2 Collection 4: Approximately two weeks after collection 3

Arthropods were collected using a beat shee t sampling method (Kogan and Pitre, 1980). The beat sheet was a 36 × 42 inch white, vinyl sheet that was spread between the plants of two adjacent rows. Plants were shaken vigorously along the length of each side of the beat sheet to dislodge arthr opods from the plants. A tota l of four subsamples were collected in this way from each plot. Speci fically, two subsamples were collected from rows # 8 and 9 of each plot (subsamples 1 and 3) and two subsamples were collected from rows # 9 and 10 of each plot (subsamples 2 and 4). The subsamples collected from within each pair of rows were at least 10 feet apart and at least 3 feet from the edge of each plot. The four subsamples were combined into one pre-labeled container and placed on freezer ice packs. The samples were then sent overnight to Monsanto Company, St. Louis, MO for arthropod id entification and enumeration.

A maximum of six pest and si x beneficial arthropods were evaluated for each collection interval. These specific arth ropods were then enumerated ac ross all samples from a given

collection interval at each individual site. Three of the six pest and three of the six beneficial arthropods were predetermined prior to the collection of sa mples, namely bean leaf beetle, green cloverworm, and stink bugs for the pests and Araneae (spiders), Nabis spp. and Orius spp. for the beneficial arthropods, and were evaluated from all collections from all sites. For each specific collection in terval at each indivi dual site, up to three additional pest and three additional beneficial arthropods, which were determined to be the most abundant, were evaluated across all samples from the site in addition to the predetermined arthropods. Th e suite of pest and benefici al arthropods assessed often

varied between collections from a site and between sites due to differences in temporal activity and geographical dist ribution of arthropod taxa.

No arthropod abundance data were co llected from the 2009 field trials.

G.9. Environmental Interact ions Evaluation Criteria

For the assessments of abiotic stress re sponse and disease damage, MON 87708 and the

conventional control were considered different in susceptibility or to lerance to an abiotic stress or disease on a particular observation date at a site if th e range of injury severity to MON 87708 did not overlap with the range of injury severity to the conventional control across all three replications. These data are categorical and we re not subjected to statistical analysis. For each observation at a site, the range of injury severity across the

commercial reference varieties provided data that are repr esentative of commercialMonsanto Company 10-SY-210U 454 of 721 

### soybean varieties. Arthr opod-related damage and abundance data were quantitatively evaluated and subjected to statis tical analysis as appropriate.

G.10. Data Assessment

Experienced scientists familiar with the expe rimental design and evaluation criteria were involved in all components of data collecti on, summarization, and analysis. Personnel assessed that measurements were taken properl y, data were consistent with expectations based on experience with the cr op, and the experiment was carefully monitored. Prior to analysis, the overall dataset was evaluated fo r evidence of biologically relevant changes and for possible evidence of an unexpected plant response. Any unexpe cted observations or issues that would impact the evaluation objectives were noted. Data were then subjected to statistical anal ysis as indicated below.

G.11. Statistical Analysis

Analysis of variance was conducted according to a randomized complete block design using SAS ® 

### (Version 9.2). The level of statistical significance was predetermined to be 5% ( α =0.05). MON 87708 was compared to the conventional control within each site (individual-site analyses) and in a combined-sit e analysis, in which th e data were pooled across sites, for early stand count, seedling vi gor, days to 50% flowering, plant height, lodging, pod shattering, final stand count, se ed moisture, 100 seed weight, seed test weight, and yield. Growth st age, flower color, plant pubesc ence, abiotic stress response, and disease damage data were categorical and not statistically analyzed. Arthropodrelated damage and pest and beneficial arthropod abundance data were statistically analyzed only within individual observations/co llections and sites due to the variation in temporal activity and geographi cal distribution of the taxa.

No statistical comparisons were made between MON 87708 and the commercial reference varieties. The reference range fo r each measured phenotypic characteristic was

determined from the minimum and maxi mum mean values from among the 18 commercial reference varieties planted among the sites. The reference range for the damage from and abundance of each arthropod evaluated from a given

observation/collection and site was determin ed from the minimum and maximum mean damage or abundance values collected from th e commercial reference varieties planted at

the site.

G.12. Individual Field Site Plant Growth and Developmen t Results and Discussion

G.12.1. 2008 Untreated MON 87708 Individual Field Site Plant Growth and Development Results and Discussion

In the individual-site analys es, no statistically significan t differences were detected

between MON 87708 and the conventional cont rol for 153 out of 179 comparisons for the assessed phenotypic characte ristics (Table G-5). Lack of variability in the data precluded statistical comparisons between MON 87708 and the conventional control for seedling vigor at the IL2 site; days to 50% flow ering at the IN1 site and MI sites; lodging at the Can1, IN3, and MO1 sites; and pod sh attering at the Can1, Can2, IA1, IA2, IA3,Monsanto Company 10-SY-210U 455 of 721 

### IL1, IL2, IN1, IN2, IN3, KS, MI, and NE s ites. For these data, the means for

MON 87708 and the conventional control were the same value, indicating no biological differences (Table G-5).

A total of 26 statistically significant differe nces were detected between MON 87708 and

the conventional control in the individual-site analyses (Table G-5). These differences were distributed among nine phenotypic charac teristics. Seedlings of MON 87708 were less vigorous than the conventional control at the Can2 site (6.3 vs. 5.0 rating). MON 87708 flowered one day later than the co nventional control at the MO2 site (214 vs. 213 days after Jan. 1, 2008), but one day earlier than the conventional control at the WI site (210 vs. 211 days after Jan. 1, 2008). Plants of MON 87708 were taller than the conventional control at the AR (28.9 vs. 27.1 inches), IA1 (39.1 vs. 34.1 inches), IA2 (37.1 vs. 34.6 inches), IL1 (26.1 vs. 22.3 inch es), IN1 (33.6 vs. 30.5 in ches), IN2 (39.6 vs. 37.3 inches), MO1 (23.4 vs. 21.1 inches), and MO2 (31.4 vs. 28.7 inches) sites. MON 87708 had more lodging than the conve ntional control at the IA1 (2.0 vs. 0.7 rating), IL2 (1.0 vs. 0.3 rating), and the WI ( 1.3 vs. 0.0 rating) sites, but less lodging than the conventional control at the KS site (1.3 vs. 2.0 rating). Final stand count was higher

for MON 87708 than the conventional control at the IN3 site (330.3 vs. 298.0 plants/plot). Seed moisture was higher for MON 87708 than the conventional contro l at the WI site (11.7 vs. 11.2%). The weight of 100 seeds was lower for MON 87708 than the conventional control at the Can1 (15.9 vs. 17.1 g), Can2 (16.9 vs. 18.1 g), IL2 (14.2 vs. 15.0 g), NE (15.4 vs. 15.9 g), and PA (13.4 vs. 15.4 g) sites. Test weight was higher for

MON 87708 than the conventional control at th e AR site (55.4 vs. 54.0 lb/bu), but lower

than the conventional control at the IL1 site (54.2 vs. 55.9 lb/bu). Yield was lower for

MON 87708 than the conventional control at the AR (63.3 vs. 70.4 bu/a) and PA (53.9 vs. 65.5 bu/a) sites. Since the stat istically significant differences detected in the individualsite analyses for seedling vigor, days to 50% flowering, lodging, fi nal stand count, seed moisture, test weight, and yiel d were not detected in the combined--site analysis, this suggests these differences were not indicativ e of a consistent re sponse in the data

associated with the trait a nd are considered not biologically meaningful in terms of increased weediness of MON 87708 compared to the conventional control. While a statistically significant differe nce was detected for plant height and 100 seed weight in the combined-site analysis, the assesse d phenotypic values of MON 87708 for both characteristics in the combined-site analysis were within the range of values observed for

the commercial reference varieties.

G.12.2. 2009 Dicamba-Treated MON 87708 Indivi dual Field Site Plant Growth and Development Results and Discussion

In the individual-site analysis, a total of 88 comparisons were made (Table G-6). Of these comparisons, no numerical differences were observed for 9 comparisons for which p-values could not be genera ted due to lack of variabil ity. The eight flower color comparisons were categorical and were not st atistically analyzed; however, at each site, all plants of treated MON 87708 and the contro l had purple flowers as expected. For the remaining comparisons, a total of fourteen statistically significant differences were detected out of 71 compar isons between treated M ON 87708 and the conventionalMonsanto Company 10-SY-210U 456 of 721 

### control. These differences were dist ributed among eight out of the 11 phenotypic characteristics. Early stand count was lo wer for treated MON 87708 than the control at the ARNE site (263.0 vs. 300.5 plants/plot) a nd higher for the ILWY site (338.0 vs. 321.0 plants/plot). Plants of MON 87708 flowered earlier than the contro l at the ILCY site (215.3 vs. 217.3 days after 1 Jan. 2009), and later than the control at the NEYO site (214.0 vs. 210.0 days after 1 Jan. 2009. Plants of MON 87708 were taller than the control at the ILCY site (26.4 vs. 24.1 inch es). MON 87708 had less lodging than the control at the ARNE site ( 5.3 vs. 6.3 rating) and the NEYO site (1.3 vs. 2.0 rating). Pod shattering was higher for MON 87708 then th e control at the NEYO site (1.8 vs. 1.3 rating). Treated MON 87708 had a lower final st and count than the control at the ARNE site (254.3 vs. 290.8 plants/plot). Seed mois ture was lower for treated MON 87708 than the control at the ARNE site (10.8 vs. 13.3%). The weight of 100 seeds was lower for

MON 87708 than the control at the ARNE site (13.8 vs. 15.2 g), INSH site (15.4 vs. 16.9 g), KSLA site (17.0 vs. 17.8 g), and the NE YO site (14.0 vs. 15.3 g). Considering that

the statistical differences detected in the in dividual-site analyses for early stand count, days to 50% flowering, plant height, lodging, pod shattering, final stand count, and seed

moisture were not detected in the combined-s ite analysis, this suggests these differences

were not indicative of a cons istent plant response associated with the trait and are unlikely to be biologically meaningful in terms of increased weed potential of MON 87708 when treated with dicamba compared to th e control. While a statistical difference was detected for 100 seed weight at three si tes and in the combined-site analysis, the assessed phenotypic values of treated MON 87708 was within the expected values for commercial soybean (Section 3.8, step 4, “no” answer). Conventional cultivars seed weight ranges from 0.12 to 0.18 grams per seed (Heatherly and Elmore, 2004).Monsanto Company 10-SY-210U 457 of 721Table G-5. 2008 Individual-Site Phenotypic Comparison of Untreated MON 87708 to Conventional ControlPhenotypic Characteristic (units)Early stand count (#/plot) Seedling vigor (1-9 scale) Days to 50% flowering1Flower Color/Plantpubescence2SiteMON 87708Mean (S.E.)ControlMean (S.E.)MON 87708Mean (S.E.)ControlMean (S.E.)MON 87708Mean (S.E.)ControlMean (S.E.) MON 87708 ControlAR160.3(11.3) 142.7(13.3)6.0(0.0) 6.0(0.0)183.3(0.3)183.0 (0.0)Purple/hairyPurple/hairyCan1224.7(2.0) 237.3(15.1)3.0(0.0) 3.0(0.0)201.0(0.6)200.3 (0.3)Purple/hairyPurple/hairyCan2327.3(24.5) 363.0(13.3)6.3(0.3)* 5.0(0.0)202.0(0.0)202.7 (0.7)Purple/hairyPurple/hairyIA1281.0(11.9) 251.7(16.8)1.7(0.7) 1.7(0.3)203.7(0.7)202.3 (1.3)Purple/hairyPurple/hairyIA2142.3(23.0) 111.0(21.3)3.3(0.3) 4.0(0.6)221.7(0.7)221.7 (0.7)Purple/hairyPurple/hairyIA3328.3(13.7) 293.7(2.7)1.0(0.0) 1.0(0.0)216.0(0.0)216.0 (0.0)Purple/hairyPurple/hairyIL1317.3(4.4) 315.3(5.0)4.3(0.3) 4.3(0.3)212.0(0.0)212.0 (0.0)Purple/hairyPurple/hairyIL2299.9(3.5) 309.9(1.2)2.0(0.0) † 2.0(0.0)203.3(0.7)203.3 (0.3)Purple/hairyPurple/hairyIN1307.9(3.7) 305.0(4.2)5.7(0.3) 4.7(0.3)206.0(0.0)†206.0 (0.0)Purple/hairyPurple/hairyIN2285.7(55.4) 304.0(39.3)5.0(1.0) 4.3(0.7)202.0(0.0)202.0 (0.0)Purple/hairyPurple/hairyIN3338.0(5.1) 310.3(15.5)3.7(0.9) 3.3(0.9)220.0(0.0)220.7 (0.7)Purple/hairyPurple/hairyKS198.0(11.2) 175.3(29.2)2.7(0.3) 3.3(0.7)198.7(0.3)199.0 (0.0)Purple/hairyPurple/hairyMI333.7(3.1) 342.8(3.2)4.0(0.0) 3.7(0.3)208.0(0.0)†208.0 (0.0)Purple/hairyPurple/hairyMO1195.2(13.9) 219.4(14.6)4.7(0.3) 4.7(0.3)212.7(0.9)213.0 (0.0)Purple/hairyPurple/hairyMO2325.7(0.9) 318.0(6.0)3.3(0.3) 2.7(0.3)214.0(0.0)*213.0 (0.0)Purple/hairyPurple/hairyNE271.3(9.5) 254.7(3.3)2.3(0.3) 2.3(0.3)199.3(1.3)198.0 (0.0)Purple/hairyPurple/hairyPA266.3(14.3) 262.3(14.0)3.0(0.6) 2.7(0.3)201.3(0.3)201.0 (0.0)Purple/hairyPurple/hairyWI277.0(24.2) 254.0(0.0)1.3(0.3) 1.0(0.0)210.0(0.0)*211.0 (0.0)Purple/hairyPurple/hairyMonsanto Company 10-SY-210U 458 of 721Table G-5 (continued). 2008 Individual-SitePhenotypic Comparison of Untreated MON 87708 to Conventional ControlPhenotypic Characteristic (units)Plant height (inch)Lodging (0-9 scale) Pod shattering (0-9 scale)Final stand count (#/plot)SiteMON 87708Mean (S.E.)ControlMean (S.E.)MON 87708Mean (S.E.)Control Mean(S.E.)MON 87708Mean (S.E.)ControlMean (S.E.)MON 87708Mean (S.E.)ControlMean (S.E.)AR 28.9 (0.2)* 27.1 (0.1) 2.7 (0.3) 2.0 (0.6) 0.3 (0.3) 0.3 (0.3) 140.3 (8.7) 138.0 (16.0)Can1 32.1 (0.6) 31.7 (0.5) 0.0 (0.0)† 0.0 (0.0) 0.0 (0.0)† 0.0 (0.0) 240.3 (1.7) 248.0 (5.5)Can2 40.1 (0.7) 38.6 (0.8) 4.0 (0.6) 3.3 (0.9) 0.0 (0.0)† 0.0 (0.0) 309.3 (6.9) 287.7 (2.3)IA1 39.1 (0.5)* 34.1 (0.3) 2.0 (0.6)* 0.7 (0.3) 0.0 (0.0)† 0.0 (0.0) 242.7 (5.8) 221.3 (15.1)IA2 37.1 (0.3)* 34.6 (0.4) 2.0 (0.0) 2.0 (0.0) 0.0 (0.0)† 0.0 (0.0) 123.0 (17.7) 102.7 (17.9)IA3 24.6 (0.5) 23.5 (0.8) 0.7 (0.3) 0.7 (0.3) 0.0 (0.0)† 0.0 (0.0) 293.3 (4.7) 285.7 (5.2)IL1 26.1 (1.4)* 22.3 (0.2) 1.0 (0.0) 0.3 (0.3) 0.0 (0.0)† 0.0 (0.0) 321.7 (6.1) 317.0 (7.2)IL2 39.0 (0.3) 37.4 (0.5) 1.0 (0.0)* 0.3 (0.3) 0.0 (0.0)† 0.0 (0.0) 295.9 (4.5) 287.0 (3.5)IN1 33.6 (2.3)* 30.5 (1.9) 1.3 (0.3) 0.7 (0.3) 0.0 (0.0)† 0.0 (0.0) 223.5 (25.1) 223.9 (29.1)IN2 39.6 (0.6)* 37.3 (0.4) 1.0 (0.0) 1.0 (0.0) 0.0 (0.0)† 0.0 (0.0) 262.3 (7.0) 264.0 (39.2)IN3 25.3 (0.3) 24.3 (0.5) 0.0 (0.0)† 0.0 (0.0) 0.0 (0.0)† 0.0 (0.0) 330.3 (7.7)* 298.0 (15.9)KS 40.5 (1.5) 38.2 (1.3) 1.3 (0.3)* 2.0 (0.0) 0.0 (0.0)† 0.0 (0.0) 187.7 (10.5) 167.0 (24.0)MI 29.5 (2.6) 30.9 (1.8) 0.7 (0.3) 0.3 (0.3) 0.0 (0.0)† 0.0 (0.0) 304.2 (4.5) 308.8 (5.2)MO1 23.4 (1.0)* 21.1 (0.8) 0.0 (0.0)† 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 186.8 (3.7) 214.8 (9.0)MO2 31.4 (1.7)* 28.7 (1.1) 0.7 (0.3) 0.3 (0.3) 1.0 (0.0) 1.0 (0.0) 308.3 (1.7) 299.3 (1.9)NE 40.5 (0.9) 38.9 (1.3) 0.3 (0.3) 1.0 (0.0) 0.0 (0.0)† 0.0 (0.0) 254.0 (7.8) 233.7 (5.9)PA 32.9 (0.8) 33.3 (0.8) 1.3 (0.3) 0.7 (0.7) 0.0 (0.0) 0.0 (0.0) 260.0 (16.2) 255.0 (11.4)WI 39.5 (0.7) 37.3 (0.3) 1.3 (0.3)* 0.0 (0.0) 0.0 (0.0) 0.5 (0.5) 235.7 (20.2) 223.0 (1.0)Monsanto Company 10-SY-210U 459 of 721Table G-5 (continued). 2008 Individual-Site Phenotypic Comparison of Untreated MON 87708 to Conventional ControlPhenotypic Characteristic (units)Seed moisture (%)100 seed weight (g) Test weight (lb/bu)Yield bu/a)SiteMON 87708Mean (S.E.)ControlMean (S.E.)MON 87708Mean (S.E.)Control Mean(S.E.)MON 87708Mean (S.E.)Control Mean(S.E.)MON 87708Mean (S.E.)Control Mean(S.E.)AR 9.5 (0.1) 9.7 (0.3) 16.1 (0.3) 15.1 (1.1) 55.4 (0.4)* 54.0 (0.7) 63.3 (3.7)* 70.4 (3.3)Can1 12.4 (0.1) 12.1 (0.3) 15.9 (0.5)* 17.1 (0.0) 55.7 (0.4) 55.5 (0.5) 66.7 (3.0) 65.8 (5.0)Can2 13.3 (0.2) 13.2 (0.1) 16.9 (0.2)* 18.1 (0.2) 57.3 (0.2) 57.4 (0.2) 76.4 (0.1) 78.5 (3.3)IA1 11.4 (0.2) 11.2 (0.1) 15.0 (0.0) 14.7 (0.3) 55.6 (0.8) 54.5 (0.3) 71.7 (3.3) 75.0 (3.4)IA2 12.6 (0.1) 12.1 (0.5) 16.5 (0.3) 16.0 (0.2) 57.3 (0.3) 57.3 (0.3) 54.7 (6.7) 45.5 (2.7)IA3 7.5 (0.1) 7.5 (0.1) 12.0 (0.3) 12.5 (0.2) 58.1 (0.6) 57.2 (0.7) 20.5 (1.1) 20.4 (2.0)IL1 10.4 (0.0) 10.4 (0.1) 11.7 (0.5) 12.0 (0.5) 54.2 (0.7)* 55.9 (0.2) 46.5 (5.5) 37.2 (1.8)IL2 12.3 (0.1) 12.3 (0.3) 14.2 (0.1)* 15.0 (0.2) 54.3 (0.3) 54.3 (0.9) 55.3 (0.8) 55.9 (2.8)IN1 9.2 (0.1) 9.3 (0.1) 14.5 (0.0) 14.6 (0.0) 55.4 (0.9) 54.6 (0.9) 56.0 (2.4) 55.5 (4.1)IN2 10.9 (0.2) 10.7 (0.3) 14.9 (0.1) 15.0 (0.1) 51.0 (1.1) 50.7 (0.5) 61.9 (4.3) 66.2 (4.8)IN3 10.0 (0.1) 10.0 (0.2) 16.0 (0.2) 16.4 (0.3) 59.5 (1.3) 59.6 (1.5) 48.7 (2.6) 45.8 (0.6)KS 17.7 (0.2) 17.9 (0.3) 15.0 (0.0) 15.7 (0.3) 56.2 (0.4) 55.4 (0.3) 66.0 (2.9) 60.0 (6.0)MI 15.5 (0.0) 14.8 (0.2) 18.2 (0.7) 18.8 (0.3) 56.6 (0.3) 56.2 (0.2) 42.9 (4.7) 44.2 (2.6)MO1 11.9 (0.4) 12.1 (0.4) 14.3 (0.3) 15.0 (2.0) 55.8 (0.2) 55.7 (0.3) 35.1 (2.4) 31.6 (5.2)MO2 12.0 (0.0) 11.8 (0.2) 15.3 (0.3) 15.7 (0.3) 54.0 (0.3) 53.5 (1.2) 43.5 (1.4) 40.0 (2.2)NE 13.1 (0.1) 13.4 (0.3) 15.4 (0.2)* 15.9 (0.3) 59.2 (0.1) 59.1 (0.2) 69.3 (1.3) 71.5 (1.2)PA 10.4 (0.2) 10.5 (0.2) 13.4 (0.3)* 15.4 (0.2) 57.2 (0.3) 57.5 (0.3) 53.9 (1.3)* 65.5 (2.1)WI 11.7 (0.1)* 11.2 (0.1) 14.2 (0.2) 15.4 (0.4) 58.0 (0.6) 58.3 (0.3) 65.0 (2.7) 66.4 (1.3)Note: The experimental design at each site was a randomized complete block with three replications. S.E. = Standard Error1Calendar day number when approximately 50% ofthe plants in each plot were flowering.2Flower color and plant pubescence data were categorical and were not statistically analyzed.*Indicates a statistically significant difference between MON 87708and the conventional control (α=0.05).†No statistical comparisons were made due to lack of variability in the data.Monsanto Company 10-SY-210U 460 of 721Table G-6. 2009 Individual-Site Phenotypic Comparison of Dicamba Treated MON 87708 to Conventional ControlPhenotypic Characteristic (units)Early stand count (#/plot)Seedling vigor(1-9 scale)Days After 1 Jan 2009 to50% flowering Flower color2Site1MON 87708Mean (S.E.)ControlMean (S.E.)MON 87708Mean (S.E.)ControlMean(S.E.)MON 87708Mean (S.E.)ControlMean (S.E.)MON 87708Mean (S.E.)ControlMean(S.E.)ARNE 263.0*(13.73) 300.5 (5.42)5.8 (0.25) 5.3 (0.48)203.8 (0.25) 204.0 (0.41)Purple PurpleIARL 290.8 (3.42) 285.0 (12.88) 4.8 (1.18) 5.8 (0.48) 220.3 (0.48) 220.3 (0.25)Purple PurpleILCY337.0 (2.35) 328.3 (6.70)1.0 (0.00) 1.8 (0.25)215.3*(0.48) 217.3 (0.25)Purple PurpleILWY 338.0* (5.00) 321.0 (3.03) 1.0†(0.00) 1.0 (0.00) 216.0 (0.00) 216.0 (0.00)Purple PurpleINRC 276.5 (5.07) 293.3 (7.34) 3.3 (0.75) 2.3 (0.48) 224.3 (0.85) 225.3 (0.25)Purple PurpleINSH 300.8 (8.19) 287.8 (6.94) 2.0 (0.41) 1.5 (0.29) 217.0 (0.00) 217.0 (0.00)Purple PurpleKSLA 276.3 (9.41) 285.3 (6.80) 3.3 (0.25) 3.3 (0.25) 207.0 (0.00) 207.3 (0.25)Purple PurpleNEYO309.0 (2.35) 307.8 (4.52)3.0†(0.00) 3.0 (0.00)214.0*(0.00) 210.0 (0.00)Purple PurpleMonsanto Company 10-SY-210U 461 of 721Table G-6 (continued). 2009 Individual-Site Phenotypic Comparison of Dicamba Treated MON 87708 to ConventionalControlPhenotypic Characteristic (units)Plant Height (in)Lodging (1-9 scale)Pod Shattering(1-9 scale) Final Stand Count (#/plot)Site1MON 87708Mean (S.E.)ControlMean (S.E.)MON 87708Mean (S.E.)ControlMean (S.E.)MON 87708Mean (S.E.)ControlMean (S.E.)MON 87708Mean (S.E.)ControlMean (S.E.)ARNE32.9 (1.07) 32.3 (0.77)5.3* (0.48) 6.3 (0.25)1.0†(0.00) 1.0 (0.00)254.3*(12.02) 290.8 (5.48)IARL 33.6 (0.60) 32.7 (0.43) 3.3 (0.63) 3.5 (0.65)1.0 (0.00) 1.5 (0.50) 280.5 (1.32) 271.3 (5.92)ILCY26.4* (0.99) 24.1 (0.26)1.0†(0.00) 1.0 (0.00)1.0†(0.00) 1.0 (0.00)231.8 (37.47)242.5(11.21)ILWY 35.2 (0.18) 35.3 (0.33) 2.0 (0.00) 2.0 (0.00) 1.0†(0.00) 1.0 (0.00) 300.5 (7.71) 294.3 (8.80)INRC 23.1 (0.74) 21.7 (0.72) 1.0†(0.00) 1.0 (0.00) 1.0†(0.00) 1.0 (0.00) 226.3 (9.54) 223.3 (7.47)INSH 33.3 (0.97) 33.4 (0.56) 1.5 (0.50) 1.0 (0.00) 1.0†(0.00) 1.0 (0.00) 270.0 (15.87) 255.8 (8.61)KSLA35.1 (0.82) 35.1 (0.30)2.3 (0.25) 2.5 (0.29)1.0 (0.00) 1.0 (0.00)260.8 (5.44)257.3(10.58)NEYO 33.4 (0.42) 35.4 (1.45) 1.3* (0.25) 2.0 (0.00)1.8* (0.25) 1.3 (0.25) 295.8 (2.50) 299.0 (5.55)Monsanto Company 10-SY-210U 462 of 721Table G-6 (continued). 2009 Individual-Site Phenotypic Comparison of Dicamba Treated MON 87708 to ConventionalControlPhenotypic Characteristic (units)Seed Moisture (%)100 Seed Weight (g)Yield (bu/ac)Site1MON 87708Mean (S.E.)ControlMean (S.E.)MON 87708Mean (S.E.)ControlMean (S.E.)MON 87708Mean (S.E.)ControlMean (S.E.)ARNE 10.8* (0.38) 13.3 (0.76) 13.8* (0.23) 15.2 (0.18) 48.5 (2.96) 47.9 (2.33)IARL 11.7 (0.05) 11.5 (0.16) 13.0 (0.41) 14.0 (0.41) 37.3 (1.52) 35.6 (1.25)ILCY 12.9 (0.14) 13.1 (0.25) 14.9 (0.27) 15.7 (0.25) 52.0 (8.22) 51.6 (1.58)ILWY 17.7 (0.14) 17.8 (0.20) 15.1 (0.21) 15.4 (0.24) 49.7 (1.97) 52.9 (2.74)INRC 12.5 (0.27) 12.7 (0.31) 13.8 (0.15) 14.2 (0.30) 29.6 (2.75) 31.3 (2.00)INSH 12.6 (0.04) 12.5 (0.13) 15.4* (0.31) 16.9 (0.22) 58.2 (2.56) 57.7 (2.48)KSLA 11.9 (0.34) 11.5 (0.38) 17.0* (0.00) 17.8 (0.25) 65.5 (1.63) 66.0 (1.54)NEYO 15.0 (0.13) 15.0 (0.09) 14.0* (0.41) 15.3 (0.25) 33.2 (1.58) 31.7 (0.55)Note: The experimental design was a randomizedcomplete block with four replications.* Statistically significant differences (α=0.05) between MON 87708 and the conventional soybean control.†No statistical comparisons were madedue to lack of variability in the data.1Site codes are as follows: ARNE = Jackson County, AR; IARL = Jefferson County, IA; ILCY = Clinton County, IL; ILWY = Stark County, IL;INRC = Parke County, IN; INSH = Boone County, IN;KSLA = Pawnee County, KS; NEYO = York County, NE2Flower color data were categoricaland were not statistically analyzed.Monsanto Company 10-SY-210U 463 of 721Table G-7. Growth Stage Monitoringof Untreated MON 87708, Conventional Control, and the Commercial ReferenceVarieties from 2008 Field TrialsAssessment Date and Rangeof Growth Stages Observed2Site1MaterialObs. 1 Obs. 2 Obs. 3 Obs. 4 Obs. 5 Obs. 6 Obs. 7 Obs. 8 Obs. 9Obs. 10AR 06/18/2008 07/6/2008 07/23/2008 08/06/2008 08/23/2008 09/02/2008 9/24/2008 — — —MON 87708 V2 R2 R3 R5 R6 R6 R8 — — —Control V2 R2 R3 R5 R6 R6 R8 — — —References V2-V3 R2R3 R5 R6 R6 R8 — —Can1 06/11/2008 06/24/2008 07/08/2008 07/25/2008 08/11/2008 08/25/2008 09/08/2008 09/23/2008 10/10/200810/18/2008MON 87708 VE V1-V2 V3-V4 R2 R3-R4 R4-R5 R5-R6 R6-R7 R7-R8R8Control VE V1-V2 V3-V4 R2 R3-R4 R4-R5 R5-R6 R6-R7 R7-R8R8References VE V1-V2 V3-V4 R2 R3-R4 R4-R5 R5-R6 R6-R7 R7-R8R8Can2 06/10/2008 06/25/2008 07/10/2008 07/21/2008 08/06/2008 08/23/2008 09/03/2008 09/20/2008 10/15/2008MON 87708VEV2-V3 V5 R2 R3 R5 R6 R7 R8 —ControlVEV2-V3 V5-V6 R2 R3 R5 R6 R7 R8 —ReferencesVEV2-V3 V5-V6 R2 R3 R5 R6 R7 R8 —IA1 07/01/2008 07/16/2008 08/06/2008 08/27/2008 09/15/2008 10/1/2008MON 87708 V1-V2 V6-V7 R2 R4 R6 R7-R8 — — — —Control V2 V6 R2-R3 R4 R6 R7-R8 — — — —References V1-V2 V6-V7 R2-R3 R4 R6 R7 — — — —IA2 07/17/2008 08/05/2008 08/20/2008 09/10/2008 09/29/2008 10/15/2008 11/04/2008MON 87708 V3 R2 R3 R5 R6 R7 R8 — — —Control V3 R2 R3 R5 R6 R7 R8 — — —References V3 R2 R3 R5 R6 R7 R8 — — —Monsanto Company 10-SY-210U 464 of 721TableG-7 (continued). Growth Stage Monitoring of Untreated MON 87708, Conventional Control, and the CommercialReference Varieties from 2008 Field TrialsAssessment Date and Rangeof Growth Stages Observed2Site1MaterialObs. 1 Obs. 2 Obs. 3 Obs. 4 Obs. 5 Obs. 6 Obs. 7 Obs. 8 Obs. 9Obs. 10IA3 07/10/2008 07/20/2008 08/04/2008 08/24/2008 09/13/2008 09/30/2008MON 87708 VC-V1 V1-V2 R2 R4 R5 R6 — — — —Control V1 V2 R2 R4 R5 R6 — — — —References VC-V1 V1-V2 R2-R3 R3-R4 R4-R6 R6 — — — —IL1 07/07/2008 07/22/2008 08/12/2008 09/01/2008 09/19/2008 10/06/2008MON 87708 V1 R1 R2-R3 R6 R6 R8 — — — —Control V1 R1 R2 R5-R6 R6-R7 R8 — — — —References V1 R1 R2-R3 R5-R6 R6-R7 R8 — — — —IL2 06/25/2008 07/16/2008 08/07/2008 08/27/2008 09/17/2008 10/08/2008MON 87708 V2 V6 R3 R5 R7 R7-R8 — — — —Control V2 V6 R3 R5 R7 R7-R8 — — — —References V2 V6 R3 R5 R7 R7-R8 — — — —IN1 06/25/2008 07/14/2008 07/25/2008 08/13/2008 08/28/2008 09/18/2008 10/01/2008MON 87708 V2-V3 R1 R2 R5 R6 R7 R8 — — —Control V2-V3 R1 R2 R4-R5 R6 R7 R8 — — —References V2-V3 R1 R2R4-R5 R6 R7-R8 R8 — — —IN2 06/25/2008 07/14/2008 07/21/2008 08/11/2008 08/26/2008 09/10/2008 10/01/2008MON 87708 V3 R1 R2 R5 R6 R6 R8 — — —Control V2-V3 R1 R2 R5 R6 R6-R7 R8 — — —References V2-V3 R1 R2R4-R5 R6 R6-R7 R8 — — —Monsanto Company 10-SY-210U 465 of 721Table G-7 (continued). Growth StageMonitoring of Untreated MON 87708, Conventional Control, and the CommercialReference Varieties from 2008 Field TrialsAssessment Date and Range ofGrowth Stages Observed2Site1Material Obs. 1 Obs. 2 Obs. 3 Obs. 4 Obs. 5 Obs. 6 Obs. 7 Obs. 8 Obs. 9Obs. 10IN3 07/15/2008 08/08/2008 08/27/2008 09/11/2008 09/25/2008 10/07/2008 10/20/2008MON 87708 V2 R1-R2 R4 R5 R6 R7 R8 — — —Control V2 R1-R2 R4 R5 R6 R7 R8 — — —References V2 R1-R2 R4 R5 R6 R7 R8 — — —KS 06/27/2008 07/08/2008 07/23/2008 08/04/2008 08/18/2008 08/29/2008 09/10/2008 09/26/2008 10/10/2008MON 87708 V2 V5-V6 R2 R3 R5 R5 R6 R8 R8 —Control V2 V5-V6 R2 R3 R5 R5 R6 R8 R8 —References V2 V5-V6 R2R3 R4-R5 R5 R6 R7 R8 —MI 06/24/2008 07/08/2008 07/22/2008 08/05/2008 08/18/2008 09/02/2008 09/17/2008 10/02/2008 10/13/2008MON 87708 V2 V4-V5 V8-V9 R3 R5 R6 R6R7R8 —Control V2 V4-V5 V8-V9 R3 R5 R6 R6R7R8 —References V2 V4-V5V8-V9 R3 R5 R6 R6R7R8 —MO1 07/16/2008 07/30/2008 08/19/2008 09/09/2008 09/23/2008 10/07/2008 10/21/2008 10/31/2008MON 87708 V2-V3 V5-R2 R4 R5 R6 R8 R8 R8 — —Control V3 R1 R4 R5 R6 R8 R8 R8 — —References V2-V3 R1-R2 R4R5 R6 R7-R8 R8 R8 — —MO2 07/14/2008 08/02/2008 08/14/2008 08/20/2008 09/10/2008 09/24/2008 10/07/2008 10/31/2008MON 87708 V2 R2 R3 R4 R6 R6 R8 R8 — —Control V2 R2 R3 R4 R6 R6 R8 R8 — —References V2-V3 R1-R2 R2-R3 R4 R5-R6 R6 R7-R8 R8 — —Monsanto Company 10-SY-210U 466 of 721Table G-7 (continued). Growth StageMonitoring of Untreated MON 87708, Conventional Control, and the CommercialReference Varieties from 2008 Field TrialsAssessment Date and Range of Growth Stages Observed2Site1Material Obs. 1 Obs. 2 Obs. 3 Obs. 4 Obs. 5 Obs. 6 Obs. 7 Obs. 8 Obs. 9Obs. 10NE 06/26/2008 07/17/2008 08/07/2008 08/28/2008 09/23/2008 10/08/2008MON 87708 V2 R1-R2 R4 R6 R7 R8 — — — —Control V2 R2 R4 R6 R7 R8 — — — —References V2 R1-R2 R4 R6 R7 R8 — — — —PA 06/20/2008 07/10/2008 07/24/2008 08/12/2008 08/29/2008 09/16/2008 09/30/2008 10/15/2008MON 87708 V1 V4 R2 R4 R5-R6 R7 R8 R8 — —Control V1 V4 R2 R4 R5 R7 R7-R8 R8 — —References V1 V4-V5 R2-R3 R4-R5 R5-R6 R6-R7 R7-R8 R8 — —WI 07/01/2008 07/16/2008 08/08/2008 08/29/2008 09/16/2008 09/30/2008MON 87708 V3 V5* R3 R5 R6 R7 — — — —Control V3 V4 R3 R5 R6 R7 — — — —References V3 V4-V5R3 R5 R6 R7 — — — —*Indicates that MON 87708and the conventional control were not within the same range of plant growth stages on this observation date.1Site codes are as follows: AR = Jackson County, AR; Can1 = Norfolk, Ontario, Canada; Can2 = Kent, Ontario, Canada; IA1 = Jefferson County, IA;IA2 = Benton County, IA; IA3 = Howard County, IA; IL1 = Clinton County, IL; IL2 = Stark County, IL; IN1 = Boone County, IN; IN2= Clinton County, IN;IN3 = Parke County, IN; KS = Pawnee County, KS; MI = Ottawa County, MI; MO1 = Shelby County, MO; MO2 = Macon County, MO; NE = York County,NE; PA = Berks County, PA; WI = Walworth County, WI.2Obs. = Observation number; dates in month/day/year format.Dash (—) indicates information not available orplants had already reached full maturity (R8).Monsanto Company 10-SY-210U 467 of 721Table G-8. Growth Stage Monitoring ofDicamba Treated MON 87708, ConventionalControl, and the Commercial ReferenceVarieties from 2009 Field TrialsAssessment Date and Rangeof Growth Stages Observed1Site2MaterialObs. 1 Obs. 2 Obs. 3 Obs. 4 Obs. 5 Obs. 6 Obs. 7 Obs. 8 Obs. 9 Obs. 10 Obs. 11 Obs. 12ARNE 7/10/2009 7/25/2009 8/12/2009 9/02/2009 9/22/2009 10/08/2009MON 877083V2 R1-R2 R4-R5 R6 R6 R8Control V2 R1-R2 R5 R6 R6 R8References V2 V6-R2 R4-R5 R6 R6 R8IARL 7/17/2009 7/23/2009 7/27/2009 7/29/2009 8/12/2009 8/14/2009 8/21/2009 8/28/2009 9/7/20099/14/2009 9/28/2009 10/29/2009MON 877083V2 V3 V3 V4 R1 R2 R2-R3 R4 R5 R5 R6 R8Control V2 V3 V3 V3-V4 R1 R2 R2-R3 R4 R5 R5 R6 R8References V2 V3 V3 V3-V4 R1R2 R2-R4 R4 R5 R5 R6 R8INRC 7/30/2009 8/6/2009 8/22/2009 8/26/2009 9/3/2009 9/15/200910/5/2009 10/12/2009 10/29/2009 — — —MON 877083V2 V3 R2 R3 R5 R5 R6 R7 R8 — — —Control V2 V3 R2 R3 R5 R5 R6 R7 R8 — — —References V2 V3 R2 R2-R3 R5 R5 R6 R7 R8 — — —Monsanto Company 10-SY-210U 468 of 721Table G-8 (continued). Growth StageMonitoring of Dicamba Treated MON 87708, Conventional Control, and theCommercial Reference Varieties from 2009 Field TrialsAssessment Date and Range of Growth Stages Observed1Site2MaterialObs. 1 Obs. 2 Obs. 3 Obs. 4 Obs. 5 Obs. 6 Obs. 7 Obs. 8 Obs. 9 Obs. 10 Obs. 11 Obs. 12INSH 7/14/2009 8/11/2009 9/1/2009 10/5/2009 11/6/2009 — — — — — — —MON 877083V3 R2 R4 R7 R8 — — — — — — —Control V3 R2 R4 R7 R8 — — — — — — —References V3 V10-R2 R4 R7 R8 — — — — — — —KSLA 7/9/2009 7/27/2009 8/6/2009 8/20/2009 9/3/2009 9/16/200910/1/2009 10/16/2009 10/28/2009 — — —MON 877083V2R1 R1 R3-R4 R5 R6 R7 R8 R8 — — —ControlV2R1 R1 R4 R5 R6 R7 R8 R8 — — —ReferencesV2R1 R1 R3-R4 R4-R5 R6 R6-R7 R8 R8 — — —NEYO 7/17/2009 8/7/2009 8/26/2009 — — — — — — — — —MON 877083V3 R1 R4 — — — — — — — — —Control V3 R1 R4 — — — — — — — — —References V3 R1 R4 — — — — — — — — —Obs. = Observation number1Month-day-year.2Site codes are as follows: ARNE = Jackson County, AR; IARL = Jefferson County, IA; ILCY = Clinton County, IL; ILWY = Stark County, IL;INRC = Parke County, IN; INSH = Boone County, IN;KSLA = Pawnee County, KS; NEYO = York County, NE3Received a mandatory dicamba application.— Dashes indicate information not provided. Growth stage datafrom the ILWY and ILCY site were not collected properly and arenot presented.Monsanto Company 10-SY-210U 469 of 721 

### Table G-9. Abiotic Stress Response Evaluations of Untreated MON 87708 and

Conventional Control from 2008 Field Trials Using an Observational Severity Scale

Note: The experimental design was a randomized complete block with three replications.

Observations were conducted at four crop developmental stages: Observation 1 at V2-V4;

Observation 2 at R1-R2; Observation 3 at R3-R5; Observation 4 at R6-R8.

*Indicates a difference observed between MON 87708 (slight damage observed) and the conventional control (no damage observe d) for wind damage during Observation 4 at the WI site. Data were not subjected to statistical analysis.

1 Includes iron deficiency. Abiotic Stressor Number of

observations

across all sites Number of observations where no

differences were observed between

MON 87708 and conventional control

Total 194 193

Cold 6 6 Compaction 9 9 Crusting 1 1 Drought 27 27

Excess moisture 20 20 Flooding 18 18

Frost 3 3

Hail 30 30 Heat damage 21 21
Mineral Toxicity 1 1
Nutrient deficiency 1 28 28

Wind 30 29*Monsanto Company 10-SY-210U 470 of 721 

### Table G-10. Disease Damage Eval uations of Untreated MON 87708 and

Conventional Control from 2008 Field Trials Using an Observational Severity Scale

Disease stressor Number of

observations

across all sites Number observations where no

differences were observed between

MON 87708 and conventional control

Total 215 215 Alternaria leaf spot 7 7

Anthracnose 10 10

Asian rust 4 4 Bacterial blight 16 16

Brown stem rot 5 5 Cercospora 1 3 3

Charcoal rot 4 4 Downy mildew 7 7 Frogeye leaf spot 24 24 Phytophthora 2 13 13 Pod and stem blight 1 1 Powdery mildew 20 20 Pythium 7 7

Rhizoctonia 10 10 Septoria brown spot 3 39 39 Soybean cyst nematode 2 2 Soybean mosaic virus 1 1 Soybean rust 4 14 14

Stem canker 2 2 Sudden death syndrome 5 14 14

White mold 11 11

Yellow mosaic virus 1 1 Note: The experimental design was a randomized complete block with three replications.

Observations were conducted at four crop developmental stages: Observation 1 at V2-V4;

Observation 2 at R1-R2; Observation 3 at R3-R5; Observation 4 at R6-R8.

1 Includes Cercospora leaf blight and Cercospora leaf disease.

2 Includes Phytophthora root rot.

3 Includes Septoria.

4 Includes rust.

5 Includes sudden death.Monsanto Company 10-SY-210U 471 of 721Table G-11. Arthropod–Related Damage Evaluations ofUntreated MON 87708 and Conventional Control from 2008 FieldTrials Using an Observational Severity ScaleArthropodNumber ofobservationsacross sites1Number of observationswhere no differences weredetected betweenMON 87708 andconventional control2Statistically Significant Differences4Site3ObservationNumberArthropod-Related DamageRating (0-5 scale)MON 87708(S.E.)Control (S.E.)ReferenceRangeAphid 33 31IA2 3 0.3 (0.06) 0.5 (0.07) 0.3-0.5IA3 2 0.8 (0.03) 0.9 (0.06) 0.8-1.0Armyworm 2 2−−−−−Bean leaf beetle 48 48−−−−−Blister beetle 5 4 MO1 2 0.1 (0.03) 0.4 (0.12) 0.0-0.2Cabbage looper 1 1−−−−−Corn rootworm beetle 3 3−−−−−Cutworm 1 1−−−−−Fall armyworm 3 3−−−−−Grasshopper 29 29−−−−−Green cloverworm 10 10−−−−−Monsanto Company 10-SY-210U 472 of 721Table G-11 (continued). Arthropod-Related Damage Evaluations of UntreatedMON 87708 and Conventional Control from2008 Field Trials Using an Observational Severity ScaleArthropodNumber ofobservationsacross sites1Number of observationswhere no differences weredetected betweenMON 87708 andconventional control2Statistically Significant Differences4Site3ObservationNumberArthropod-Related DamageRating (0-5 scale)MON 87708(S.E.)Control (S.E.)ReferenceRangeJapanese beetle 27 25IN1 2 0.6 (0.03) 0.9 (0.12) 0.7-0.9PA 4 0.6 (0.07) 0.4 (0.03) 0.3-0.5Leafhopper55 4 PA 1 1.1 (0.07) 0.6 (0.23) 0.4-1.3Seedcorn maggot 1 1−−−−−Soybean looper 7 7−−−−−Spider mite 8 8−−−−−Sting bug 24 24−−−−−Thistle caterpillar 3 3−−−−−Thrips 2 2−−−−−Yellow woollybear 4 4−−−−−Note: The experimental design was a randomized complete block design with three replications. Observations were conducted at four crop developmentalstages: Observation1 = V2-V4, Observation2 = R1-R2, Observation3 = R3-R5, and Observation4 = R6-R8.1Statistical comparisons were made between MON 87708 and the conventional control for 95 of the observations. Lack of variability in the data precludedstatistical comparisons for121 of the observations.2No statistically significant difference (α= 0.05) or numerical difference.3Site codes IA2 = Benton County, IA; IA3 = Howard County, IA; IN1 = Boone County, IN; MO1 = Shelby County, MO; PA = Berks County, PA.4Means, standard errors (S.E.), and reference ranges for differences between MON 87708 and the conventional control that were statistically different (α=0.05).Reference ranges were determined from the minimum and maximummean values from among the commercial reference varieties.5The difference detected in leafhopper damage during the first observation at the PA site was for damage caused by potato leafhoppers.Dash (−) indicates no statistically significant difference between MON 87708 and the conventional control (α= 0.05).Monsanto Company 10-SY-210U 473 of 721Table G-12. Abundance of Pest Arthropods in Beat Sheet Samples Collectedfrom Untreated MON 87708, ConventionalControl, and the Commercial ReferenceVarieties from 2008 Field TrialsPest Arthropod2Aphid Bean Leaf Beetle Grape ColaspisColl. Site1MON 87708(S.E.)Control(S.E.)ReferencerangeMON 87708(S.E.)Control(S.E.)ReferencerangeMON 87708(S.E.)Control(S.E.)Referencerange1 IL2−−−13.7 (4.8) 13.7 (4.1) 6.3 - 13.3 0.7 (0.3) 1.3 (1.3) 0.3 - 2.7IN1−−−0.3 (0.3) 0.3 (0.3) 0.7 - 1.0 0.0 (0.0) 0.3 (0.3) 0.0 - 0.0MI−−−0.0 (0.0)†0.0 (0.0) 0.0 - 0.0−−−MO1−−−0.0 (0.0) 0.0 (0.0) 0.3 - 0.7−−−2 IL2−−−4.3 (2.6) 5.7 (3.5) 2.7 - 5.0−−−IN1−−−1.3 (0.9) 2.0 (0.6) 0.3 - 1.7−−−MI 816.7 (252.2) 383.3 (116.7) 600.0 - 1366.7 0.0 (0.0)†0.0 (0.0) 0.0 - 0.0−−−MO1−−−0.0 (0.0) 0.0 (0.0) 0.0 - 0.3 0.0 (0.0) 0.3 (0.3) 0.0 - 0.03 IL2−−−0.3 (0.3) 1.3 (0.9) 0.7 - 1.3−−−IN1−−−0.3 (0.3) 0.7 (0.3) 0.0 - 1.3−−−MI 93.3 (78.4) 33.3 (16.7) 3.3 - 60.0 0.0 (0.0) 0.0 (0.0) 0.0 - 0.3−−−MO1 13.3 (3.3) 16.7 (3.3) 6.7 - 33.3 1.0 (1.0) 0.0 (0.0) 0.0 - 1.3−−−4 IL2−−−14.0 (3.5) 14.3 (4.7) 5.7 - 10.3−−−IN1−−−2.0 (0.6) 2.3 (1.2) 0.3 - 2.0−−−MI−−−0.0 (0.0)†0.0 (0.0) 0.0 - 0.0−−−MO1−−−5.0 (2.6) 3.3 (0.9) 3.3 - 4.7−−−Monsanto Company 10-SY-210U 474 of 721Table G-12 (continued). Abundance of Pest Arthropods inBeat Sheet Samples Collected from Untreated MON 87708,Conventional Control, and the CommercialReference Varieties from 2008 Field TrialsPest Arthropod2Garden Flea-hopper Green Clover-worm Japanese BeetleColl. Site1MON 87708(S.E.)Control(S.E.)ReferencerangeMON 87708(S.E.)Control(S.E.)ReferencerangeMON 87708(S.E.)Control(S.E.)Referencerange1 IL2 - - - 0.0 (0.0) * 2.0 (0.0) 0.3 - 2.7 0.3 (0.3) 0.0 (0.0) 0.0 - 0.7IN1 - - - 0.0 (0.0) 0.3 (0.3) 0.0 - 1.3 9.0 (4.6) 12.0 (7.6) 0.7 - 11.7MI - - - 1.7 (0.9) * 0.0 (0.0) 0.0 - 1.7 2.3 (1.9) * 8.0 (4.2) 1.7 - 5.7MO1 - - - 2.3 (1.9) 5.0 (2.1) 1.0 - 3.7−−−2 IL2 - - - 16.0 (5.0) 14.7 (2.8) 7.3 - 17.7−−−IN1 - - - 6.3 (0.3) 3.7 (0.9) 3.7 - 7.3 1.3 (0.7) 0.7 (0.7) 0.5 - 2.7MI - - - 1.7 (0.7) 2.7 (1.7) 1.0 - 3.0 2.7 (0.3) 5.3 (2.9) 1.0 - 5.0MO1 0.3 (0.3) 1.0 (0.6) 0.0 - 1.0 7.0 (2.3) 7.7 (0.3) 0.7 - 8.3−−−3 IL2 - - - 6.3 (2.8) 6.3 (2.4) 7.7 - 15.7−−−IN1 - - - 7.0 (1.7) * 11.3 (2.2) 8.0 - 13.0 2.3 (0.3) 0.3 (0.3) 0.5 - 5.7MI - - - 1.0 (0.6) 2.0 (0.6) 0.3 - 2.3 1.3 (0.9) 5.3 (1.8) 0.7 - 4.3MO1 - - - 1.3 (0.9) 1.3 (0.9) 0.0 - 1.3−−−4 IL2 - - - 1.0 (1.0) 1.0 (0.0) 0.3 - 2.0−−−IN1 - - - 1.7 (0.9) 2.3 (1.2) 1.0 - 3.7 0.0 (0.0) 0.0 (0.0) 0.0 - 1.3MI - - - 0.0 (0.0) * 0.7 (0.3) 0.0 - 0.0 0.0 (0.0) 0.3 (0.3) 0.0 - 1.3MO1 0.3 (0.3) 0.0 (0.0) 0.0 - 0.3 0.0 (0.0) 0.7 (0.7) 0.0 - 0.7−−−Monsanto Company 10-SY-210U 475 of 721Table G-12 (continued). Abundance of Pest Arthropods inBeat Sheet Samples Collected from Untreated MON 87708,Conventional Control, and the CommercialReference Varieties from 2008 Field TrialsPest Arthropod2Potato Leafhopper Stink Bug Tarnished Plant BugColl. Site1MON 87708(S.E.)Control(S.E.)ReferencerangeMON 87708(S.E.)Control(S.E.)ReferencerangeMON 87708(S.E.)Control(S.E.)Referencerange1 IL2 13.0 (10.1) 28.7 (12.3) 15.7 - 43.3 0.0 (0.0) 0.0 (0.0) 0.0 - 2.7−−−IN1−−−0.0 (0.0) 0.3 (0.3) 0.0 - 0.3−−−MI−−−0.0 (0.0) 0.3 (0.3) 0.0 - 0.3 1.3 (1.3) 0.3 (0.3) 0.3 - 1.0MO1 0.0 (0.0) 0.3 (0.3) 0.0 - 1.0 0.3 (0.3) 1.0 (0.6) 0.0 - 0.7−−−2 IL2−−−0.3 (0.3) 0.3 (0.3) 0.3 - 1.3−−−IN1−−−0.7 (0.3) 0.0 (0.0) 0.3 - 0.7−−−MI−−−0.3 (0.3) 1.0 (0.6) 1.0 - 6.3 1.3 (1.3) 3.0 (2.5) 0.7 - 4.0MO1−−−0.3 (0.3) 0.0 (0.0) 0.0 - 1.3−−−3 IL2−−−0.0 (0.0) * 1.3 (0.9) 0.0 - 0.7 0.0 (0.0) 0.0 (0.0) 0.0 - 1.0IN1−−−2.0 (1.5) * 0.0 (0.0) 0.0 - 0.7−−−MI−−−2.0 (1.2) 2.3 (0.9) 1.7 - 3.7 6.0 (1.5) 12.3 (0.3) 6.0 – 13.0MO1−−−0.3 (0.3) 0.7 (0.7) 0.3 - 1.3−−−4 IL2−−−2.3 (0.3) 2.0 (1.5) 1.7 - 5.0 0.7 (0.7) 2.3 (0.7) 0.3 - 1.3IN1−−−1.3 (0.9) 1.0 (0.6) 1.0 - 5.0−−−MI−−−3.7 (0.7) 3.0 (0.6) 1.7 - 4.7 2.0 (1.2) 1.7 (0.3) 1.3 - 1.3MO1−−- 1.3 (0.9) 1.0 (0.6) 0.3 - 4.0−−−Monsanto Company 10-SY-210U 476 of 721Table G-12 (continued). Abundance of Pest Arthropods inBeat Sheet Samples Collected from Untreated MON 87708,Conventional Control, and the CommercialReference Varieties from 2008 Field TrialsPest Arthropod2Velvet-bean Caterpillar Wooly-bear CaterpillarColl. Site1MON 87708(S.E.)Control(S.E.)ReferencerangeMON 87708(S.E.)Control(S.E.)Referencerange1 IL2−−−−−−IN1−−−−−−MI−−−−−−MO1−−−−−−2 IL2−−−−−−IN1−−−−−−MI−−−−−−MO1−−−−−−3 IL2−−−−−−IN1 0.7 (0.7) 1.3 (0.3) 0.0 - 1.5−−−MI−−−−−−MO1−−−−−−4 IL2−−−−−−IN1−−−2.3 (0.9) 3.0 (1.2) 0.7 - 3.0MI−−−0.7 (0.3) 0.3 (0.3) 0.3 - 1.0MO1−−−0.0 (0.0) 0.0 (0.0) 0.0 - 0.7Note: The experimental design was a randomized complete block with three replications. Arthropod collection (Coll.)1 was conducted at the R1-R2 growthstage, and the three subsequent collections were conducted at approximately two week intervals thereafter.* Indicates a statistically significant difference between MON 87708and the conventional control (α=0.05).† No statistical comparisons were made due to lack of variability in the data.1Site codes are as follows: IL2 = Stark County, IL; IN1 = BooneCounty, IN; MI = Ottawa County, MI; MO1 = Shelby County, MO.2MON 87708and the conventional control values represent mean number of arthropods collected from three replications. S.E. = Standard Error Referenceranges were determined from the minimum and maximum mean values from among the commercial reference varieties at the site.Dash (−) indicates arthropod not evaluated.Monsanto Company 10-SY-210U 477 of 721Table G-13. Abundance of Beneficial Arthropods in Beat Sheet Samples Collected from Untreated MON 87708, ConventionalControl, and the Commercial ReferenceVarieties from 2008 Field TrialsBeneficial Arthropod2Araneae (spiders) Big eyed bug CarabidaeColl. Site1MON 87708(S.E.)Control(S.E.)ReferencerangeMON 87708(S.E.)Control(S.E.)ReferencerangeMON 87708(S.E.)Control(S.E.)Referencerange1 IL2 1.0 (1.0) 0.0 (0.0) 0.0 - 1.0−−−−−−IN1 0.0 (0.0) 0.0 (0.0) 0.0 - 0.7−−−−−−MI 0.0 (0.0) 0.0 (0.0) 0.0 - 0.3−−−−−−MO1 0.0 (0.0) 0.0 (0.0) 0.3 - 1.0 0.0 (0.0) 0.0 (0.0) 0.0 - 0.3−−−2 IL2 0.7 (0.3) 1.0 (0.0) 0.0 - 1.3−−−−−−IN1 0.3 (0.3) 0.7 (0.7) 0.3 - 0.7−−−−−−MI 1.3 (0.3) 0.7 (0.3) 0.0 - 1.0−−−−−−MO1 3.7 (2.7) 1.0 (0.0) 0.7 - 2.3 0.0 (0.0) 0.3 (0.3) 0.0 - 0.3−−−3 IL2 0.0 (0.0) 0.3 (0.3) 0.0 - 0.3−−−0.3 (0.3) 0.0 (0.0) 0.0 - 0.7IN1 0.0 (0.0) 0.3 (0.3) 0.0 - 0.3−−−−−−MI 0.3 (0.3) 0.3 (0.3) 0.0 - 0.0−−−−−−MO1 7.7 (0.9) 7.7 (0.9) 4.3 - 7.7−−−−−−4 IL2 0.3 (0.3) 0.7 (0.7) 0.3 - 1.3−−−−−−IN1 0.3 (0.3) 0.0 (0.0) 0.0 - 1.0−−−−−−MI 0.0 (0.0)* 3.0 (0.6) 0.7 - 1.0−−−−−−MO1 0.3 (0.3) 0.3 (0.3) 1.0 - 2.0−−−−−−Monsanto Company 10-SY-210U 478 of 721Table G-13 (continued). Abundance of Beneficial Arthropods in Beat Sheet Samples Collected from Untreated MON 87708,Conventional Control, and the CommercialReference Varieties from 2008 Field TrialsBeneficial Arthropod2Lacewings Ladybird beetles Micro-parasitic waspsColl. Site1MON 87708(S.E.)Control(S.E.)ReferencerangeMON 87708(S.E.)Control(S.E.)ReferencerangeMON 87708(S.E.)Control(S.E.)Referencerange1 IL2−−−−−−−−−IN1−−−0.0 (0.0) 0.0 (0.0) 0.0 - 0.3−−−MI 0.7 (0.7) 0.0 (0.0) 0.0 - 0.7 0.0 (0.0) 0.7 (0.3) 0.3 - 1.0−−−MO1−−−1.0 (0.6) 0.7 (0.7) 0.3 - 1.7−−−2 IL2−−−−−−−−−IN1−−−−−−−−−MI 4.7 (1.7) 5.7 (1.2) 2.3 - 8.0 0.7 (0.7) 0.7 (0.7) 1.7 - 5.0−−−MO1 1.0 (0.6) 0.7 (0.3) 0.0 - 1.7 1.7 (0.3) 2.3 (0.9) 0.0 - 1.0−−−3 IL2 0.0 (0.0) 0.0 (0.0) 0.0 - 0.3 0.0 (0.0)†0.0 (0.0) 0.0 - 0.0−−−IN1 0.0 (0.0) 0.0 (0.0) 0.0 - 0.3−−−−−−MI 8.7 (1.2) 6.0 (3.1) 5.3 - 6.0 13.7 (6.7) 5.7 (0.7) 5.3 - 14.0−−−MO1 6.3 (5.4) 2.0 (0.6) 2.0 - 6.0 8.3 (0.9) 9.7 (4.3) 4.7 - 12.7 0.7 (0.7) 1.7 (0.9) 0.0 - 2.04 IL2 0.7 (0.3) 0.7 (0.3) 0.0 - 2.0 0.0 (0.0) 0.7 (0.3) 0.0 - 0.7 0.0 (0.0) 0.0 (0.0) 0.0 - 0.3IN1−−−−−−−−−MI 4.0 (0.6) 2.0 (1.2) 1.3 - 6.0 3.7 (0.9) 4.0 (1.5) 2.7 - 7.3 1.0 (0.6) 1.3 (0.7) 0.3 - 1.3MO1 1.7 (1.7) 1.3 (0.9) 0.7 - 3.0 7.3 (0.3) 4.7 (1.2) 2.0 - 8.3−−−Monsanto Company 10-SY-210U 479 of 721Table G-13 (continued). Abundance of Beneficial Arthropods in Beat Sheet Samples Collected from Untreated MON 87708,Conventional Control, and the CommercialReference Varieties from 2008 Field TrialsBeneficial Arthropod2Nabisspp. OpilionesOriusspp.Coll. Site1MON 87708(S.E.)Control(S.E.)ReferencerangeMON 87708(S.E.)Control(S.E.)ReferencerangeMON 87708(S.E.)Control(S.E.)Referencerange1 IL2 1.7 (0.7) 1.0 (0.6) 0.7 - 2.7 0.7 (0.3) 0.3 (0.3) 0.0 - 0.3 0.7 (0.7) 2.3 (0.9) 0.7 - 3.3IN1 0.0 (0.0)†0.0 (0.0) 0.0 - 0.0 0.3 (0.3) 0.3 (0.3) 0.0 - 0.7 0.0 (0.0)†0.0 (0.0) 0.0 - 0.0MI 0.0 (0.0) 0.3 (0.3) 0.0 - 0.7−−−0.0 (0.0)†0.0 (0.0) 0.0 - 0.0MO1 0.0 (0.0) 0.0 (0.0) 0.0 - 1.0−−−0.3 (0.3) 0.7 (0.7) 0.0 - 0.72 IL2 1.3 (0.3) 1.3 (0.9) 1.0 - 2.0−−−0.0 (0.0) 0.7 (0.7) 0.0 - 1.0IN1 0.0 (0.0) 0.3 (0.3) 0.0 - 1.3 0.3 (0.3) 0.7 (0.3) 0.0 - 1.0 0.0 (0.0) 0.3 (0.3) 0.0 - 0.0MI 2.3 (1.2) 4.0 (1.5) 1.7 - 2.3−−−7.7 (1.3) 7.0 (1.5) 4.7 - 6.7MO1 0.3 (0.3) 0.0 (0.0) 0.0 - 1.0−−−1.7 (0.9) 2.0 (1.2) 1.3 - 2.03 IL2 0.3 (0.3) 0.7 (0.3) 0.3 - 1.3−−−0.0 (0.0)†0.0 (0.0) 0.0 - 0.0IN1 0.3 (0.3) 0.0 (0.0) 0.0 - 0.3 0.0 (0.0) 0.0 (0.0) 0.0 - 0.7 0.0 (0.0) 0.0 (0.0) 0.0 - 0.3MI 3.3 (0.9) 1.3 (0.7) 2.0 - 4.0−−−4.7 (1.2) 4.7 (1.8) 2.3 - 12.0MO1 1.3 (1.3) 2.0 (1.5) 0.3 - 3.3−−−4.7 (0.9) 2.3 (1.3) 3.7 - 6.74 IL2 0.0 (0.0) 1.0 (0.6) 1.0 - 2.7−−−3.3 (0.3) 1.3 (0.9) 0.3 - 3.3IN1 0.0 (0.0) 0.7 (0.7) 0.0 - 0.7 1.0 (0.0) 1.3 (0.3) 0.3 - 1.0 0.7 (0.7) 0.0 (0.0) 0.0 - 0.3MI 4.7 (0.7)* 1.7 (1.7) 2.0 - 6.3−−−1.0 (0.6) 2.7 (0.9) 0.7 - 2.0MO1 0.7 (0.7) 0.3 (0.3) 0.3 - 3.0−−−0.0 (0.0) 3.0 (1.5) 0.7 - 6.0Monsanto Company 10-SY-210U 480 of 721Table G-13 (continued). Abundance of Beneficial Arthropods in Beat Sheet Samples Collected from Untreated MON 87708,Conventional Control, and the CommercialReference Varieties from 2008 Field TrialsBeneficial Arthropod2Syrphid larvaeColl. Site1MON 87708(S.E.)Control(S.E.)Referencerange1 IL2−−−IN1−−−MI−−−MO1−−−2 IL2−−−IN1−−−MI 1.7 (1.2) 3.0 (1.0) 1.0 - 4.3MO1−−−3 IL2−−−IN1−−−MI 1.3 (0.7) 0.7 (0.7) 0.0 - 2.3MO1−−−4 IL2−−−IN1−−−MI−−−MO1−−−Note: The experimental design was a randomized complete block with three replications. Arthropod collection (Coll.)1 was conducted at the R1-R2 growthstage, and the three subsequent collections were conducted at approximately two week intervals thereafter.* Indicates a statistically significant difference between MON 87708and the conventional control (α=0.05).† No statistical comparisons were made due to lack of variability in the data.1Site codes are as follows: IL2 = Stark County, IL; IN1 = BooneCounty, IN; MI = Ottawa County, MI; MO1 = Shelby County, MO.2MON 87708and the conventional control values represent mean number of arthropods collected from three replications. S.E. = Standard Error. Referenceranges were determined from the minimum and maximum mean values from among the commercial reference varieties at the site.Dash (−) indicates arthropod not evaluated.Monsanto Company 10-SY-210U 481 of 721 

### Table G-14. Abiotic Stress Response Evaluations of Dicamba-Treated MON 87708

and Conventional Control fr om 2009 Field Trials Using an Observational Severity

Scale

Abiotic Stressor Number of

observations

across all sites 1 

### Number of observations where no

differences were observed between

MON 87708 and control

Total 89 89

Cold 1 1 Compaction 3 3 Drought 2 

### 21 21

Flood 3 

### 17 17 Frost damage 4 4 Hail damage 13 13 Nutrient deficiency 10 10 Wind damage 20 20

Note: The experimental design was a randomized complete block with four replications. Observations were made at approximately th e following four crop developmental stages: Observation 1 at V3 –V5; Observation 2 at R1 - R2; Observation 3 at R3 - R5; Observation 4

at R6 - R8. Data were not subjected to statistical analysis.

1 Site codes are as follows: ARNE = Jackson County, AR; IARL = Jefferson County, IA; ILCY = Clinton County, IL; ILWY = Stark County, IL; INRC = Parke County, IN; INSH = Boone County, IN; KSLA = Pawnee County, KS; NEYO = York County, NE

2 Includes heat and dry.

3 Includes wet soil, excess moisture.Monsanto Company 10-SY-210U 482 of 721 

### Table G-15. Disease Damage Evalua tions of Dicamba-Treated MON 87708 and

Conventional Control from 2009 Field Trials Using an Observational Severity Scale

Disease stressor Number of

observations

across all sites 1 

### Number observations where no

differences were observed between

MON 87708 and control

Total 93 92

Anthracnose 1 1 Bacterial blight 2 

### 7 7 Bacterial leaf spot 2 2

Brown stem rot 3 3 Cercospora 3 

### 5 5

Charcoal rot 1 1 Downy mildew 13 13 Frogeye leaf spot 4 

### 8 8 Leaf spot 5 

### 21 21 Phytophthora root rot 7 7 Powdery mildew 5 5 Pythium 2 2

Rhizoctonia 3 3 Soybean mosaic virus 1 1 Soybean rust 1 1

Stem canker 1 1

Sudden death 9 9

White mold 6 

### 3 2

Note: The experimental design was a randomized complete block with four replications. Observations were made at approximately th e following four crop developmental stages: Observation 1 at V3 –V5; Observation 2 at R1 - R2; Observation 3 at R3 - R5; Observation 4 at

R6 - R8.

1 Site codes are as follows: ARNE = Jackson County, AR; IARL = Jefferson County, IA; ILCY = Clinton County, IL; ILWY = Stark County, IL; INRC = Parke County, IN; INSH = Boone County, IN; KSLA = Pawnee County, KS; NEYO = York County, NE

2 Includes Pseudomonas

. 3 Includes Cercospora leaf blight and Cercospora leaf disease.

4 Includes eye spot.

5 Includes Septoria and Alternaria

. 6 Indicates a difference observed between MON 87708 and the control for white mold at the ILWY site (Observation 2; slight vs. none). The damage rating for MON 87708 was outside of the reference range (no white mold was observed in the references). Data were not subjected to statistical analysis.Monsanto Company 10-SY-210U 483 of 721Table G-16. Arthropod–Related DamageEvaluations of Dicamba-Treated MON87708 and Conventional Control from 2009Field Trials Using an Observational Severity ScaleArthropodNumber ofobservationsacross sites1Number of observationswhere no differences weredetected betweenMON 87708(N) and thecontrol2Statistically Significant Differences4Site3ObservationNumberArthropod Damage Rating (0-5 scale)MON 87708(N)(S.E.)Control(S.E.)ReferenceRangeAphids510 10−−−−−Bean leaf beetle 23 22 KSLA 3 0.00(0.00) 0.08(0.05) 0.00-0.03Blister beetle 1 1−−−−−Cabbage looper 1 1−−−−−Corn earworm 1 1−−−−−Fall armyworm 1 0−−−−−Grasshopper 18 16 INRC 3 0.45(0.09) 0.10 (0.06) 0.23-0.28KSLA 3 0.20(0.00) 0.03(0.03) 0.10-0.20Green cloverworm 7 7−−−−−Japanese beetle 10 10−−−−−Monsanto Company 10-SY-210U 484 of 721Table G-16 (continued). Arthropod–Related Damage Evaluations of Dicamba-Treated MON 87708 and Conventional Controlfrom 2009 Field Trials Using anObservational Severity ScaleArthropodNumber ofobservationsacross sites1Number of observationswhere no differences weredetected betweenMON 87708 and the control2Statistically Significant Differences4Site3ObservationNumberArthropod Damage Rating (0-5 scale)MON 87708(S.E.)Control(S.E.)ReferenceRangePotato leaf hopper 3 3−−−−−Soybean looper 2 2−−−−−Stink bugs69 9Three corneredalfalfa hopper3 3−−−−−Velvet beancaterpillar4 4−−−−−Note: The experimental design was a randomized complete block design with four replications. Observations were conducted at approximately the followingfour crop developmental stages: Observation1 = V3-V5, Observation2 = R1-R2, Observation3 = R3-R5, and Observation4 = R6-R8.1A total of 93 arthropod damage observations were made across sites. Lack of variability in the data precluded statistical comparisons for 34 of the observations.Statistical comparisons could be made between MON 87708 and control for 59 of the observations.2No statistically significant differences were detected (α=0.05) or numerical differences between MON 87708 and the conventional soybean control where pvalues could not be generated due to lack of variability in the data.3Site codes are as follows: ARNE = Jackson County, AR; IARL = Jefferson County, IA; ILCY = Clinton County, IL; ILWY = Stark County, IL; INRC = ParkeCounty, IN; INSH = Boone County, IN; KSLA =Pawnee County, KS; NEYO = York County, NE4Means, standard errors (S.E.), and reference ranges are reportedfor a statistically significant difference that was detected (α=0.05) between MON87708 and theconventional soybean control. Reference range = minimum and maximum mean values among the commercial reference varieties.5Aphids include soybean aphids.6Stink bugs include green stink bugs.−indicates that there were no statistically significant differences were detected (α=0.05) between MON 87708 and the conventional soybean control.Monsanto Company 10-SY-210U 485 of 721 

### References for Appendix G

Heatherly, L.G. and R.W. Elmore. 2004. Ma naging inputs for peak production. Pages 451-536 in Soybeans: Improvement, Production, a nd Uses. H.R. Boerma and J.E. Specht (eds.). American Society of Agronomy, Inc., Crop Science Society of America, Inc., Soil Science Society of America, Inc., Madison, Wisconsin.

Kogan, M. and H.N. Pitre. 1980. General sampling methods for above-ground populations of soybean arthropods. Pages 30-60 in Sampling Methods in Soybean Entomology. M. Kogan and D.C. Herzog (eds .). Springer-Verlag, New York, New York.

SAS Software Release 9.2 (TS1M0). © 

### 2002-2008 by SAS Institute Inc., Cary, North

Carolina.Monsanto Company 10-SY-210U 486 of 721 

### Appendix H: Materials and Methods for Pollen Morphology and Viability

Assessment

H.1. Plant Production

MON 87708, the near isogenic conventio nal soybean control A3525, and four commercial reference varieties were grown unde r similar agronomic conditions in a field trial in Clinton County, IL (Table G-1; IL1 si te). The trial was arranged in a randomized complete block design with three replicatio ns. Each plot consisted of four rows approximately 20 feet in length.

H.2. Flower Collection

When the soybean plants were flowering, twenty whole flow ers were collected from the fourth row of each plot. Four flowers were taken from each of five representative plants per plot. All flowers from a plot were placed into a sing le, clean container that was labeled with the plot number from which th e sample originated. The containers were kept on ice for ≤ 8 hours until the pollen was prepared and stained.

H.3. Pollen Sample Preparation

Pollen samples were prepared in a laboratory. Clean microscope slides were labeled with the plot number. A circle of approximately one centimeter in diameter was drawn in the center of the slide with a pap hydrophobic barr ier pen. Tweezers and a dissecting needle were used to open each of the collected flow ers from a plot and br ush the pollen into the

circle on the slide. The utensils were clea ned between extractions. Approximately 20 μl of Alexander’s stain (Alexander, 1980) was adde d to the center of the circle containing the pollen. The pollen was stained at ambient te mperature for at least ten minutes prior to examination. Pollen samples from all plots wi thin a replicate were stained and evaluated on the same day.

H.4. Data Collection

Pollen characteristics were assessed by vi ewing samples under an Olympus Provis AX70 light/fluorescence microscope equipped with an Olympus DP70 digital color camera. The microscope and camera were connected to a computer runni ng Microsoft Windows

2000 Professional and installed with asso ciated DP Controller v1.2.1.108 and DP Manager v1.2.1.107 camera software and Imag e-Pro Plus v6.2.1.491 imaging software.

H.4.1. Pollen Viability

When exposed to the staining solution, viable pollen grains stained red to purple due to the presence of living cytoplas mic content. Nonviable pollen grains stained blue to green and may have appeared round to collapsed in shape, depending on the degree of hydration. The number of viable and nonviable pollen grains in each pollen sample was

counted from a random field of view under the microscope. A minimum of 75 pollen grains were counted in each sample. Dense clusters of pol len or pollen grains adhering toMonsanto Company 10-SY-210U 487 of 721 

### flower parts were not counted because they may not have ab sorbed the staining solution uniformly.

H.4.2. Pollen Diameter

Micrographs of ten representative pollen gr ains from each plot were taken at 400× magnification and imported into the imagi ng software. The software was used to measure pollen grain diameter along two perpendicular axes for each selected pollen grain. Mean pollen diameter for each plot wa s calculated from the 20 total measurements.

H.4.3. General Pollen Morphology

General pollen morphology was observe d from micrographs of MON 87708, the conventional control, and commercial referen ce varieties that were also used for pollen

diameter measurements.

H.5. Statistical Analysis

An analysis of variance was conducted accord ing to a randomized complete block design using SAS ® 

### (Version 9.2). The level of statistical significance was predetermined to be 5% ( α =0.05). MON 87708 was compared to the c onventional control for percent viable pollen and pollen diameter. No statistical comparisons were made between MON 87708

and the commercial reference va rieties. Instead, a reference range for each measured

characteristic was determined from th e minimum and maximum mean values from among the four commercial reference va rieties. General pollen morphology was qualitative; therefore, no st atistical analysis was conduc ted on these observations.Monsanto Company 10-SY-210U 488 of 721 

### References for Appendix H

Alexander, M.P. 1980. A versat ile stain for pollen fungi, yeast and bacteria. Stain Technology 55:13-18.

SAS Software Release 9.2 (TS1M0). © 

### 2002-2008 by SAS Institute Inc., Cary, North

Carolina.Monsanto Company 10-SY-210U 489 of 721 

### Appendix I: Materials and Methods for Symbiont Assessment

I.1. Materials

The soybean materials for the symbiont in teraction assessment included MON 87708, the near isogenic conventional soybean cont rol A3525, and six commercial reference varieties (Table I-1). Nodul es, root tissue, and shoot ti ssue collected from MON 87708, the conventional control, and the commercia l reference varieties were evaluated.

Table I-1. Starting Seed of MON 87708, Conventional Control, and Commercial Reference Varieties Used in the Symbiont Assessment

Material Name Material Type Phenotype Monsanto Seed Lot #

MON 87708 Test Dicamba-tolerant 11225299

A3525 Control Conventional 10001822

A2553 Reference Conventional 10000961

LG C3884N Reference Conventional 11226859

Stewart SB3454 Reference Conventional 11242910

Garst 3585N Reference Conventional 10001517

Hartz H5218 Reference Conventional 10001410

A5560 Reference Conventional 10001114



### I.2. Characterization of the Materials

For the MON 87708 and conventional control star ting seed lots, the presence or absence

of the dmo expression cassette was confirmed by event-specific polymerase chain reaction analyses.

I.3. Greenhouse Phase and Experimental Design

Eight replicate 6-inch pots were prepared for each soybean material (MON 87708, the conventional control, and commercial referenc e varieties). The pots contained nitrogen deficient potting medium (Sunshine ® 

### Mix #2 Basic/LB2) com posed of primarily peat, vermiculite, and perlite. At planting, each seed was inoculated with approximately

1 × 10 7 

### cells of Bradyrhizobium japonicum (VAULT ® 

### NP, Becker Underwood) in phosphate-buffered saline. Three seeds were planted in each pot. The soybean plants were grown in a greenhouse where temperatur es ranged from approximately 17 to 35°C. Once pots were thinned to a single plant per pot, the pots were arranged in eight replicated blocks using a random ized complete block design.

Replicates 1, 2, and 3 were planted on Sept ember 8, 2009; replicat es 4, 5, and 6 were planted on September 9, 2009; and replicates 7 and 8 were planted on September 10, 2009. In all cases, replicate pots had a minimu m of one plant emerge within one week. A nitrogen-free nutrient solution (approximate ly 250 ml) was added weekly after plant

emergence.Monsanto Company 10-SY-210U 490 of 721 

### I.4. Plant Harvesting/Data Collection

Six weeks after emergence, plants were exci sed at the surface of the potting medium and shoot and root plus nodule mate rial were removed from the pot s. The shoot material was cut into smaller pieces and placed in labeled bags. The plant roots with nodules were separated from the potting medium by washi ng with water. Excess moisture was removed using absorbent paper towels, and the roots with nodules were placed in labeled bags. On the same day that plants were harv ested, nodules were re moved by hand from the roots of each plant, enumerated, and wei ghed to determine the fresh weight of the

nodules.

The remaining root and shoot mass were determ ined for each plant. Nodules as well as root and shoot material were placed in a dr ying oven on the same day as collected or stored for up to four days at approximatel y 4°C before being place d in the drying oven. The plant material was dried for at least 72 hours at approxi mately 65°C to determine dry weight. The shoot tissue was ground afte r drying with a Harbil 5G high-speed paint shaker prior to total nitr ogen analysis. Shoot total nitrogen was determined by combustion using a nitrogen analyzer (ra pid N cube, Elementar Americas, Inc.).

I.5. Statistical Analysis

An analysis of variance was conducted accord ing to a randomized complete block design using SAS ® 

### (Version 9.2). The level of statistical significance was predetermined to be 5% ( α =0.05). MON 87708 was compared to the conventional control for nodule number, nodule dry weight (g), shoot dry weight (g), root dry weight (g), and shoot total nitrogen (% and g/plant). No statis tical comparisons were ma de between MON 87708 and the

commercial reference varieties. Instead , a reference range for each measured

characteristic was determined from th e minimum and maximum mean values from among the six commercial reference varieties.Monsanto Company 10-SY-210U 491 of 721 

### References for Appendix I

SAS Software Release 9.2 (TS1M0). © 

### 2002-2008 by SAS Institute Inc., Cary, North

Carolina.Monsanto Company 10-SY-210U 492 of 721 

### Appendix J: Petitioner’ s Environmental Report

J.1. Background

This appendix provides information regardin g four key areas to be covered in an environmental assessment: Purpose and Need, Alternatives, the Affected Environment, and Potential Environmental Impacts. This environmental report has been prepared by Monsanto to facilitate APHIS’s compliance with the National Environmental Policy Act (NEPA), including compliance with the Council on Environmental Quality’s (CEQ) regulations that implement NEPA 13



### .

Monsanto has produced dicamba-tolerant so ybean plants (hereafter referred to as MON 87708) by inserting a gene that wa s discovered in a naturally occurring microorganism. The gene produces a dicam ba mono-oxygenase (DMO) protein (referred to as the MON 87708 DMO) in the plant that inactivates the herb icide dicamba, thus rendering the plant tolera nt to applications of the herb icide. Other broadleaf plants, including weeds in soybean fields, do not natu rally contain the gene or exhibit tolerance

to dicamba and when treated with dicamba do not survive. Numerous field trials

conducted in the U.S. under APHIS notifica tions since 2005 have included MON 87708.

Information has been collected from field tr ials, laboratory and gr eenhouse studies, and

the literature to assess whether the tolerance to dicamba through production of the MON 87708 DMO protein and/or the gene insertion proces s has altered MON 87708 in any way that would make these plants more of a plant pest compared to conventional soybean or cause significant environmental im pacts, including cumulative impacts. The purpose of this report is to provide releva nt information regarding the potential for reasonably foreseeable, signifi cant environmental impacts of widespread cultivation of

MON 87708.

MON 87708 will enable an expanded applic ation window of dicamba in soybean, allowing for preemergence application through crop emergence (cracking), and postemergence in-crop applications through the R1/R2 growth stage. The current maximum allowable dicamba use pattern a llowed on soybean, the proposed maximum allowable dicamba use pattern on MON 87708, and the maximum annual allowable

dicamba rates are summarized in Table J-1. The anticipated use rates for dicamba on MON 87708, however, are expected to be less than the proposed maximum allowabable use rates. Section VIII.H.2 of the petition desc ribes in detail the anticipated use rates and patterns for dicamba on MON 87708.

13 Title 40 of the Code of Federal Regulation (40 CFR) Parts 1500-1508.Monsanto Company 10-SY-210U 493 of 721 

### Table J-1. Summary of Dicamba Uses on Soybean

Current Approved Uses Proposed Uses on MON 87708

Application Timing Maximum Single Application

Rate (lbs a.e./acre) Maximum

Annual Application

Rate (lbs a.e./acre) Maximum Single Application

Rate (lbs a.e./acre) Maximum

Annual Application

Rate (lbs a.e./acre)

Preemergence 0.50 a



### 2.0 1.0 b



### 2.0Postemergence Not labeled 0.50 (V3)

+

0.50 (R1/R2) c



### Pre-harvest (7 days prior to harvest) 1.0 Not labeled

a 14-28 day planting interval based on product application rate

b No planting interval

c In-crop application through V3 with a sequen tial application through R1/R2 growth stage as

needed. Total of all in-crop applications fro m emergence up to R1/R2 is 1.0 lb a.e./acre.



### An analysis of the potential impact from deregulation of MON 87708 on current soybean agronomic production systems, and related acti vities such as soybean processing, food and feed uses as well as marketing of soybean and soybean products is presented in this Environmental Report. Factor s evaluated as part of th e assessment include potential impacts to:

 land use patterns, climate, water, soil a nd air quality, non-agricultural lands, farming practices, commodity and speci alty soybean production,

 marketability of soybean seed for planting and seed for specialty and commodity markets, and

 public health, non-target organisms, th reatened or endangered species, and biodiversity.

The analysis conducted considers current and reasonably foreseeable conditions, the potential for deregulation of MON 87708 to impact these conditions, and potential cumulative impacts. In most cases, there are no impacts to current conditions ( e.g. , no differences between deregulation of MON 87708 versus continuing to regulate). Where differences are noted, these differences are desc ribed and their significance evaluated.Monsanto Company 10-SY-210U 494 of 721 

### J.2. Purpose and Need for the Action

APHIS’ mission is to protect the health and value of American agriculture and natural

resources. 14 

### Under the authority of the Pl ant Protection Act (PPA), APHIS’ Biotechnology Regulatory Services (BRS) regulates the introduction (importation, interstate movement, or release into the environment) of certain genetically engineered (GE) organisms and products that may pose a risk to plant health. 15 

### An organism is no longer subject to these regulations when APHIS determines that it is unlikely to pose a plant pest risk. A GE organism is considered a regulated article if APHIS has reason to believe it could pose a plant pest risk. A person may petit ion the agency 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 as a potential plant pest. 16 

### The petitioner is required to provide information re lated to plant pest risk that the USDA may use to assist in determining wh ether the regulated article is unlikely to present a plant pest

risk. 17 

### If, based on this information, as well as other sources of information, the USDA determines that the article is unlikely to pose a plant pest risk, the article may be granted nonregulated status.

Monsanto Company (Monsanto) has submitted a petition (designated as 10-188-01p) to APHIS seeking the determination of nonr egulated status in whole for MON 87708 soybean plants genetically engineered to produce the MON 87708 DMO which is not typically found in soybean. Mo nsanto researchers have fou nd that when soybean plants produce the MON 87708 DMO, they are tolerant to the broadleaf herbicide dicamba.

Due to the unique broad spectrum herbicide properties of glyphosate, growers may use only glyphosate for their total weed management. As a resu lt, in certain cropping and cultural systems (e.g., common corn and-soybean rotations), an increa se in the number of crop acres with glyphosate resi stant weed populations has occu rred over the last decade and, recently, the number of growers incorpor ating other weed management practices hasalso grown over time. A prominent strategy to delay the development of herbicideresistant weeds is to increase the diversity of weed management options in agriculture, including use of herbicides w ith different modes-of-action

, 

### in a grower’s weed management program (Duke and Powles, 2009). MON 87708 has been developed to provide an effective and efficient method to incorporate another herbicide mode-of-action into the Roundup Ready soybean system (dicamba tolerance will be integrated into the Roundup Ready soybean system). This combin ation of herbicide-to lerance traits will allow the preemergence (including pre-plan t) and postemergence use of both dicamba and glyphosate herbicides in an integrated weed management program to control a broad spectrum of grass and broadleaf weed species (Johnson et al., 2010). Increasing postemergence herbicide options is importa nt, especially in conservation tillage situations, where the performance and cons istency of postemergen ce herbicides has generally been greater than that of soil active residual products. Dicamba will improve

the control of broadleaf weeds that are hard-to- control with glyphosate ( e.g. , common

14 Souce is website: http://www.aphis.usda.gov/index.shtml.

15 Source is 7 U.S.C. § 7701–7772, 7 CFR Part 340.

16 Source is 7 CFR § 340.6 “Petition for De termination of Nonregulated Status”.

17 Source is 7 CFR § 340.6(c)(4).Monsanto Company 10-SY-210U 495 of 721 

### lambsquarters, hemp sesbania, morni ngglory species, nightshade, Pennsylvania smartweed, prickly sida ̧ and wild buckwheat) and also offer an effective control option for glyphosate-resistant broadleaf weed speci es, namely marestail, common ragweed, giant ragweed, palmer pigweed, and wate rhemp (Johnson et al., 2010). Dicamba will also offer an effective control option for broadleaf species resi stant to ALS and PPO

chemistries. In the case of PPO herbicides which are currently being heavily relied upon for control of glyphosate resistant amaranthus species, a primary dicamba benefit will be to provide options for dela ying the further evolution a nd spread of PPO resistant

amaranthus species (University of Tennessee, 2010)

.



### Upon integration of MON 87708 into the R oundup Ready soybean system, growers will have the ability to continue to use estab lished soybean production practices including crop rotation, tillage systems, labeled herbicid es, and other cultural practices such as row spacing, thereby using the same planting and ha rvesting machinery currently being used. Growers will also continue to have the flexibility and simplicity in weed control provided by glyphosate that will allow growers to conti nue to reap the environmental benefits

associated with the use of c onservation-tillage that is fac ilitated by the use of glyphosate for postemergence weed control in the R oundup Ready soybean system (CTIC, 2011; CTIC, 2004).

Monsanto has requested that APHIS make a determination that these soybean plants and their progeny will no longer be considered regulated articles under 7 CFR Part 340.

APHIS’ action in this case is the determin ation whether or not to grant nonregulated

status to MON 87708. APHIS’ purpose and need in making this determination is to fulfill its responsibilities under the PPA as defined under 7 CFR Part 340.

While APHIS must consider impacts associated with the use of dicamba on the quality of the human environment as part of its dete rmination to grant nonregulated status of MON 87708, the use of the pesticide is regulated at the federal level by the U.S. Environmental Protection Agency (U.S. EPA), not APHIS. APHIS’s authority under the Plant Protection Act does not allow it to specify conditions for the use of herbicides. Instead, EPA specifically approves labeling for any herbicide use including uses on agricultural crops. Before any dicamba product could be used over MON 87708 it must first be approved by the EPA as required by the Federal Insect icide Fungicide and Rodenticide Act (FIFRA); EPA must approve the pesticide (herbi cide) product labeling for that specific use. Nevertheless, this environmental report examines the potential impact of dicamba use on MON 87708 on the human environment.

J.3. Affected Environment

This section describes the affected environment as it relates to soybean, Glycine max (L.) Merr., its uses and where soybean is grown. Related agricultural practices such as tillage, crop rotation, pesticide use, weed management, irrigation practices, and non-agricultural lands are also considered part of the aff ected environment, as is specialty soybean production, including organic soybean producti on and seed production. The information in this section, with minor ex ceptions as noted, is based upo n information from SectionsMonsanto Company 10-SY-210U 496 of 721 

### II through IX of the petition, with those sectio ns more specifically referenced throughout

this section.

J.3.1. Commercial Soybean Production and Uses

Commercial soybean production an d uses are discussed in Sec tion VIII.B of the petition and summarized here; refer to the petition for more detail. Soybean is grown as a commercial crop in over 35 countries and is one of the most valued agricultural commodities because of its high protein and oil content. In 2008, soybean represented 56% of world oilseed production, and appr oximately 33% of those soybean were produced in the U.S. In 2008, the U.S. expor ted 1.16 billion bushels (31.6 million metric tons) of soybean, which accounted for 40% of the world's soybean exports and was valued at $15.5 billion (ASA, 2009).

Approximately 94% of the world’s soybean seed supply was crushe d to produce soybean meal and oil in 2008 (Soyatec h, 2010), and the majority wa s used to supply the feed industry for livestock use or the food indus try for edible vegetable oil and soybean protein isolates.

The U.S. soybean acreage in the past 10 year s has varied from a pproximately 64.7 to 75.7 million acres. Average soybean yields have varied from 33.9 to 43.3 bushels per acre over this same time period. According to data from USDA-NASS (2009a), soybean was planted on approximately 75.7 million acres in the U.S. in 2008, producing 2.96 billion bushels of soybean with a value of $27.4 billion (USDA-NASS, 2009b,c).

In the U.S., soybean production occurs in three major soybean growing regions accounting for 99.1% of the soyb ean acreage: Midwest region (IL, IN, IA, KS, KY, MI, MN, MO, NE, ND, OH, SD, and WI), South east region (AL, AR, GA, LA, MS, NC, SC, and TN) and the Eastern Coastal region (D E, MD, NJ, NY, PA, and VA). Table J-1 shows the relative productivity of the three regions in 2008.

Table J-2. 2008 Soybean Productivity by Region

Region % 2008 U.S. Soybean Acreage 2008 Average Yield (bushels per acre) Range of Average State Yields (bushels per acre)

Midwest/Great Plains 82.1 38.6 28.0 – 47.0

Southeast 14.3 34.4 30.0 – 40.0

Eastern Coastal 2.7 34.1 27.5 – 46.0

Source: USDA-NASS (2009a,b).



### J.3.2. Specialty Soybean Production

Specialty soybean are grown on fewer acres than commercial or commodity soybean and are typically grown, harvested and handled differently than commodity soybean, and premiums and incentives are paid for delive ring a product that meet s purity and qualityMonsanto Company 10-SY-210U 497 of 721 

### standards for the soybean variety. This category includes certified seed, organic production, soybean with specialty oil prof iles, food grade soybean, and others.

J.3.2.1. Certified Seed Production

Certified seed production is discussed in Se ction VIII.B.2 of petition #10-SY-210U and summarized here; refer to the petition for mo re detail. Standardized seed production practices are responsible for maintaining high- quality seed stocks, an essential basis for U.S. agriculture. The value of seed quali ty (including genetic purity, vigor, and minimizing presence of weed seed, seed-borne diseases, and inert materials, such as dirt) has been identified as a major fact or in determining crop yields.

Soybean seed has four classes: 1) breeder, 2) foundation, 3) register ed, and 4) certified (Association of Official Seed Certifying Ag encies (AOSCA), 2009). Breeder seed is seed directly controlled by the originating or sponsoring plant br eeding organization or firm. Foundation seed is first-generation seed increased from breeder seed and is handled to maintain purity and identity of a specific variety. Registered s eed is the progeny of

foundation seed that is handled to maintain satisfactory variety purity and identity. Certified seed is the progeny of breeder, foundation or registered seed, and is typically two generations from foundation seed. Not all s oybean seed sold is officially certified; however, commercial soybean seed sold and planted for commodity soybean production typically meets or exceeds certified seed standards.

Seed certification programs were initiated in th e early 1900s in the U.S. to preserve the genetic identity and variety purity of seed. There are special land requirements, seed stock eligibility requirements, field inspec tions and seed labelin g standards for seed

certification. Seed certification services are available through va rious state agencies

affiliated with the AOSCA.

Soybean seed is produced throughout most of the U.S. soybean-growing regions by companies that produce and sell seed, and by toll seed producers, or tollers, which are companies that produce certified seed for othe r companies pursuant to a contract. Seed companies and tollers in turn contract acr eage with growers to produce the required amount of soybean seed. Production or proces sing plants at these seed companies clean, condition, and bag the harvested soybean seed as well as monitor and inspect all the processes at the plant.

The entire seed production process at the majority of the seed companies and tollers operates under standards established by the International Organization for Standardization (ISO), and in cludes internal and external audits (ISO, 2009). Field inspections are conducted on seed production fields throughout the soybean growing season to evaluate variety pur ity and ensure soyb ean plants are developing properly. Management practices are designed with the intent to keep the fields free of weeds, insects, and diseases, and to prevent mechan ical mixing with other soybean varieties. The fields are also mapped to ensure th e seed field has the minimum isolation requirement to prevent potential outcrossing. Isolation dist ances are agreed to by theMonsanto Company 10-SY-210U 498 of 721 

### seed companies based on topography, varieties, volunteers, weeds, insects and weather patterns (AOSCA, 2009; ASTA, 2011).

The field operations and management practices for producing soybean seed are similar to normal soybean production. However, special attention is needed in certain production practices to produce seed w ith high quality, high germination rates, and high genetic purity (Helsel and Minor, 1993).

J.3.2.2. Organic Soybean Production

Organic soybean production occurred on 125,600 acres in 2008 ac ross Midwest and Southeast regions. Iowa, Minne sota, Michigan and Arkansas combined for over half the U.S. organic soybean acres. Over the past decade, organic soybean production peaked at 174,500 acres in 2001 and ranged from 100,000 to 125,600 acres since 2000 (USDAERS, 2008a). Organic farming operations as described by the Nationa l Organic Program, which is administered by USDA’s Agricult ural Marketing Service (AMS), requires organic production operations to have distin ct, 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 producti on management plan. Organic production operations must also develop and maintain an or ganic production system plan approved by an accredited certifying agent. This plan enables the production operation to achieve and document compliance with the Na tional Organic Standa rds, including the prohibition of the use of excluded methods. 18 

### Excluded methods include a variety of methods used to genetically engineer or ganisms or influence their growth and development by means that are not possible und er natural conditions or processes. The use of biotechnology such as that used to produce MON 87708 is an excluded method under the National Organic Program. 19



### Organic certification involves oversight by an accredited certifying ag ent of the materials and practices used to produce or handle an or ganic agricultural produc t. This oversight

includes an annual review of the certified operation’s organic syst em plan and on-site inspections of the certified operation and its records. Although the National Organic Standards prohibit the use of excluded methods , they do not require testing of inputs or products for the presence of excluded methods. The presence of a detectable residue of a product of excluded methods alone does not n ecessarily constitute a violation of the National Organic Standards (USDA-AMS, 2011) . The unintentional presence of the products of excluded methods will not affect the status of an organic pr oduct or operation when the operation has not used excluded me thods and has taken reasonable steps to avoid contact with the products of excluded me thods as detailed in an approved organic system plan. Organic certification indi cates that organic production and handling processes have been followed, not that the pr oduct itself is “free” from any particular

substance.

18 Title 7 of the Code of Federal Regulation (7 CFR) Part 205.

19 Source is 7 CFR § 205.2.Monsanto Company 10-SY-210U 499 of 721 

### Organic soybean producers use production prac tices designed to prevent commingling of their crop with neighboring cr ops treated with herbicides and other pesticides (spray drift), or that may be using plant vari eties produced by excluded methods (pollen movement). These well established practices in clude isolation zones, use of buffer rows surrounding the organic crop, adjusted pl anting dates, and varietal selection. 20 

### The implementation of management practices to avoid pollen from a biotechnology-derived crop in organic or conventiona l soybean production operations is facilitated by the nature of soybean pollination. Soybean is a highly se lf-pollinated species and exhibits a very low level of outcrossing (see Sec tion IX.D). Outcrossing is the genetic transmission of a

defined heritable characterist ic from one group of indivi duals (population, crop variety) to another. Outcrossing most commonly result s from cross-pollination. Since soybean is highly self-pollinating, organic or conve ntional soybean producers can and have effectively implemented practices ( e.g. , isolation during the growing season, equipment cleaning during harvest, and post-harvest separation of harv ested seed) that allow them to reasonably avoid biotechnology-de rived soybean and maintain organic or conventional production status (Brookes a nd Barfoot, 2004). Information about the National Organic Program, organic standards, and practices can be viewed on line (USDA-AMS, 2011).

J.3.3. Agricultural Practices for Soybean

J.3.3.1. Tillage

Tillage is performed to prepare the soil for seed bed preparation and also serves as a weed control method because it uproots established plants and smothers them. Tillage in soybean production is discussed in Sections VIII.C.1 and VIII.C.2 of the petition and summarized here; refer to the petition for mo re detail. The bene fits of conservation tillage or no-till syst ems relative to conventional tillage are well documented and include reduced soil erosion, reduced fuel and la bor costs, conservation of soil moisture, improvement of soil structure, reduction of soil compaction and improvement of soil organic matter content. In 2007, approxima tely 27.5 million acres (39.6%) of soybean were planted in a no-till sy stem (CTIC, 2007). In 2011 over 65% of U.S. soybean acres used some form of conservation tillage (USB , 2011a). The decision to plant soybean in a conservation tillage or no-till system is made long before planting as it may require special equipment. In addition, this deci sion is usually a l ong-term commitment, provided the system is successful.

Slow soybean emergence and growth, plus lowe r yields, have been some of the concerns associated with adoption of c onservation tillage systems in s oybean, especially no-till.

Research in Wisconsin and Minnesota shows that soil temperatures ca n be four to five degrees colder in no-till systems than in conventional tillage systems, which can slow emergence, but have little effect on soybean yield (Pedersen, 2008). Improved planters for establishment of good soybean populations and planting Roundup Ready ® 

### soybean varieties to effectively control weeds in no-ti ll fields have made no-till a more viable

20 Source is at website: http://attra.ncat.org/attra-pub/PDF/cropsfarmplan.pdf [Accessed on May 26, 2010].

® Roundup Ready is a trademark of Monsanto Technolo gy LLC. All other trademar ks are the property of their respective owners.Monsanto Company 10-SY-210U 500 of 721 

### production system for soybean. In 1995, before the introduction of Roundup Ready soybean, approximately 27% of the U.S. soyb ean acres used no-till production. In 2004, nine years after the introduction of Roundup Ready soybean system , no-till acreage increased to 36% of the total soybean acres (Sankula, 2006). The most recent surveys indicate that 39% of the soybean acres are produced using no-till methods (CTIC, 2007). Researchers still recommend some spring tillage on fine-textured a nd poorly drained soils for proper seedbed preparation (Pedersen, 2008).

J.3.3.2. Crop Rotation

The use of crop rotation in soybean production is discussed in Section VIII.I of petition #10-SY-210U and summarized here; refer to the petition for more detail. The wellestablished farming practice of crop rotation is still a key management tool for soybean growers. The purposes of growi ng soybean in rotation include:

 improving yield and profitability of one or both crops over time;

 decreasing the need for nitrogen fe rtilizer on the crop following soybean;

 mitigating or breaking disease, insect, and weed cycles;

 improving soil tilth and soil physical properties;

 increasing residue cover;

 reducing soil erosion;

 increasing soil organic matter; and

 reducing runoff of nutrients, herbicides, a nd insecticides (Boerma and Specht, 2004; Al-Kaisi et al., 2003).

According to USDA Economic Research Serv ice, 95% of the soybean-planted acreage has been in some form of a crop rotation system since 1991 (USDA-ERS, 2001). Cornand wheat-planted acreage has been rotated at a slightly lower level of 75% and 70%, respectively. Although the benef its of crop rotations can be s ubstantial, the grower must make cropping decisions by evaluating both the agronomic and economic returns of various cropping systems. Crop rotations also afford growers the opportunity to diversify farm production in order to minimize market risks.

Agronomic practices such as rotation patterns for soybean vary from stat e to state. However, there are similarities among states within certain growing regions. The majority of the U.S. soybean acreage (68.6 %) is rotated to corn with approximately 14.5% of the soybean acreage rotated back to soybean the following year. Wheat follows soybean on approximately 11.2% of the U.S. soybean acreage (see Table VIII-22 in the petition).Monsanto Company 10-SY-210U 501 of 721 

### Continuous soybean production is uncommon in the Midwest. Soybean extension specialists encourage growers to avoid the pract ice as a way to reduce the risk of damage

from diseases and nematodes (Hoeft et al., 2000; Al-Kaisi et al., 2003). Corn and soybean occupy more than 80% of the farmla nd in many of the Midwestern states, and the two-year cropping sequence of soybean-corn is used most extensively in this region. However, a soybean crop sometimes is grown af ter soybean and then rotated to corn in a 3-year rotation sequence (soybean-soybean-corn) in the Midwest. Compared to corn, soybean shows a greater yield response to being grown after a number of years without soybean. The yield of both corn and soybean is approximately 10% higher when grown in rotation than when either crop is gr own continuously (Hoeft et al., 2000).

A combination of conservation tillage practi ces and crop rotation has been shown to be very effective in improving soil physical prope rties. Long-term studies in the Midwest indicate that the corn-soybean rotation im proves yield potential of no-till systems compared to continuous corn production (Al-Kaisi, 2001).

J.3.3.3. Irrigation

The use of irrigation in soybean production is discussed in Section VIII.B of the petition and summarized here; refer to the petition for more detail. The productivity of soybean is highly dependent upon soil and cl imatic conditions. In the U.S., the soil and climatic requirements for growing soybean are very sim ilar to corn. The soils and climate in the Midwestern, Eastern and portions of the Gr eat Plains regions of the U.S. provide sufficient water under normal climatic conditions to produce a soybean crop. The general water requirement for a high-yielding soybean crop is approximately 20 inches of water during the growing season (Hoe ft et al., 2000). Soil text ure and struct ure are key components determining water availability in soils, wher e medium-textured soils hold more available water, allowing soybean root s to penetrate deeper in medium-textured soils than in clay soils. Irrigation is used on approximately 9% of the soybean acreage in the U.S. to supplement the water supply duri ng dry periods in the Western and Southern soybean growing regions (USDA-ERS, 2008b).

J.3.3.4. Management of Insects

The management of insects in soybean producti on is discussed in Section VIII.D of the petition and summarized here; refer to the pe tition for more detail. Although insects are rated as less problematic than weeds in U. S. soybean production, management of insect pests during the growth and development of soybean is important fo r protecting the yield of soybean (Aref and Pike, 1998). Understa nding the impact of insects on soybean growth is essential for proper management (Hig ley and Boethel, 1994). It is important to understand the way that insects injure soybean as well as how the soybean plant responds to insect injury. Insect injury can impact yield, plant maturity, or seed quality. The ultimate impact of injury is damage, as a measurable reduction in plant growth development or reproduction. Insect injury in soybean seldom reaches levels to cause an economic loss in the primary soybean prod uction areas, as indicated by the low percentage (16%) of soybean acreage that receives an insecticide treatment (USDANASS, 2007b).Monsanto Company 10-SY-210U 502 of 721 

### Characterizing soybean responses to insect in jury is essential in establishing economic injury levels (Higley and Boethel, 1994). Most often, soybean insects pests are categorized or defined by the plant parts th ey injure, namely r oot-feeding, stem-feeding, leaf-feeding, or pod-feeding in sects. The root- and stem-f eeding insect groups are often the hardest to scout and typically are not de tected until after they have caused their damage. The leaf-feeding insects comprise the biggest group of soybean insect pests, but not necessarily the most economically damagi ng insects. Recent research on defoliation has determined that a major effect of leaf injury is to reduce light interception by the soybean canopy which in turn can have a signif icant effect on yield (Higley and Boethel, 1994). Soybean has an extraordinary capacity to withstand considerable defoliation early in the season without significant yield loss. By contrast, defoliati on during the flowering and pod filling stages poses a greater threat to yield, because the soybean plant has less time to compensate for injury compared to ot her growth stages. Re search indicates that the soybean plant can sustain a 35% leaf lo ss prior to the pre-bloom period without lowering yield (NDSU, 2002). However, from p od-set to maturity, the plant can tolerate only a 20% defoliation level befo re yield is impacted.

J.3.3.5. Management of Diseases and Other Pests

The management of diseases and other pe sts in soybean producti on is discussed in Section VIII.E of the petition and summarized here ; refer to the petition for more detail. More than 100 pathogens are known to affect soybean, of which 35 are considered to be of economic importance (Heatherly and Hodge s, 1999). The estimated yield losses to soybean diseases in the U.S. were 12.5, 13.2, and 13.0 million metric tons in 2008, 2009, and 2010, respectively (Wrather and Koe nning, 2011), which equates to 15.5%, 14.4% and 14.4% losses of total soybean production, respectively (ASA, 2011). Pathogens can affect all parts of the soybean plant, resulting in reduced quality and yi eld. The extent of losses depends upon the pathogen , the state of plant deve lopment and health when infection occurs, the severity of the disease on individual plants, and the number of plants affected (Heatherly and Hodges, 1999).

According to field surveys conducted in fift een soybean-producing states during 1996 to 2010, soybean cyst nematode ( Heterodera glycines ) caused the greatest soybean yield losses (Wrather and Koenning, 2011). Phytophthora root and stem rot ( Phytophthora
sojae ), brown spot ( Septoria glycines ), charcoal rot ( Macrophomina phaseolina ), sclerotinia stem rot ( Sclerotinia sclerotiorum ), seedling diseases, and sudden death syndrome ( Fusarium solani f.sp. glycines ) followed in economical importance. As expected, yield losses vary by regi on (Wrather et al., 2001).

Selecting resistant varieties is the primar y tool growers have for disease control (Heatherly and Hodges, 1999). Resistant varieties may have morphological or physiological characteristics that provide immunity, resistance, toleran ce or avoidance to certain pathogens. Cultural practices can al so play an important role in disease management by reducing initial inoculums or reducing the rate of disease development (Heatherly and Hodges, 1999).Monsanto Company 10-SY-210U 503 of 721 

### Preplant tillage can bury crop residue, wh ich encourages the decomposition of fungalresting structures. Crop rotation is ofte n recommended as a disease-management strategy. Rotating crops in terrupts the disease cycle and allows time for the decomposition of inoculums. One exception is Rhizoctonia , a soil-inhab itant pathogen that grows on a wide variety of crops and can survive sufficiently in the soil to make crop rotation an impractical means of control ling this pest. Row spacing, plant population, and planting date can also be ch anged to manage soybean diseases.

Soybean cyst nematode (SCN), Heterodera glycines , is one of the most damaging pathogens of soybean throughout the soybean growing regions of the U.S. with losses estimated to be about $1.5 billion (Pederse n, 2008). The simplest, least expensive method to reduce populations of this pest is to rotate soybean with a non-host crop such as corn, small grains, or sorghum. However, pl anting resistant varieties is regarded as the best and most effective management practice to prevent losses from this pest (Wrather and Mitchum, 2010).

High-quality seed is essential for control ling seedling diseases. The most important seedling diseases in soybean are Phytophthora

, Pythium

, Rhizoctonia , and Fusarium (Pedersen, 2008). Many soyb ean varieties demonstrate resi stance to spec ific taxonomic

races of Phytophthora . Treating soybean seed with a fungicide ( e.g. , metalaxyl or mefenoxam) is effective against damping-o ff disease (seedling blight) caused by common soil fungi, such as Phytophthora and Pythium . Fungicide seed treatments are recommended where there is a history of these seedling diseases.

Asian soybean rust is a foliar fungal dise ase that typically infests soybean during reproductive stages of development and can cause defoliation and reduce yields significantly in geographies su ch as Brazil (Dorrance et al., 2007). Soybean rust is caused by the fungus Phakopsora pachyrhizi . This disease in the U.S. was first detected in Louisiana in 2004 (LSU, 2009). At this ti me, foliar application of fungicides is the standard disease-management practice to lim it yield losses due to soybean rust.

J.3.3.6. Management of Weeds

The management of weeds in soybean produ ction is discussed in Section VIII.F of petition #10-SY-210U and summarized here; refer to the petition for mo re detail. Annual weeds are perceived to be th e greatest pest problem in soybean production, followed by perennial weeds (Aref and Pike, 1998). Weed control in soybean is essential to optimizing yields because weeds compete with soybean for light, nutrients, and soil

moisture. Weeds can also harbor insects and diseases, and also can interfere with harvest, causing extra wear on harv est equipment (Pedersen, 2008).

Foxtail spp. ( Setaria spp

. ), pigweed ( Amaranthus spp.), velvetleaf ( Abutilon theophrasti ), lambsquarters ( Chenopodium album ), and cocklebur ( Xanthium strumarium ) are common weeds in Midwest corn and soybean fields. However, growers consider giant ragweed ( Ambrosia artemisiifolia ), lambsquarters, Canada thistle ( Cirsium arvense) , cocklebur, and velvetleaf to be the top five most probl ematic weeds in corn and soybean because of the difficulty to control thes e weeds (Nice and Johnson, 2005).Monsanto Company 10-SY-210U 504 of 721 

### The primary weed competition factors that aff ect a potential yield loss in soybean from weed competition are the weed species, weed density, and the duration of the competition. When weeds are left to compete with soybean for the entire growing season, yield losses can exceed 75% (Dalley et al., 2001). Generally, the competition between crops and weeds increases with incr easing weed density. The time period that weeds compete with the soybean crop influences the level of yield loss. In general, the later the weeds emerge, the less impact the weeds will have on yield. Soybean plants withstand early-season weed competition l onger than corn, and the canopy generally closes earlier in soybean than corn ( i.e. , plants in adjacent rows grow to a sufficient size such that their foliage touches between the ro ws blocking the sunlight from reaching the ground). In addition, canopy closure is much s ooner when soybean is planted in narrow

rows.

The most effective weed management programs in soybeans use a combination of cultural, mechanical, and/or herbicide control practices, hereafter called diversified weed management practices. 

### Herbicide practices include the use of several herbicides with different modes-of-action, either w ithin or across seasons, applying he rbicides at the labeled rate at the correct timing, and proper application of the herbicide. Cultural and mechanical weed control practices can also be important components of an effective diversified weed management program (Loux et al., 2009). Cultural practices such as crop rotation, narrow row spacing and planting date are a few of the crop ma nagement practices that are implemented to provide the crop with a co mpetitive edge over weeds. Mechanical

methods of weed control includi ng tillage have been used fo r centuries to control weeds in crop production. Spring or fall preplant tillage and in-crop shallow cultivation can effectively reduce the competitive ability of weeds by burying the plants, disturbing or weakening their root systems, or causing suffi cient physical injury to kill the plants. A consequence of in-crop cultivation for weed control is that it can injure crop roots and cause moisture loss. The planting of winter cover crops is another cultural practice that can also be utilized. The planting of cove r crops, such as grasses, legumes or small grains, can protect and improve soil quality, he lp reduce erosion, and can serve as surface mulch in no-till cropping prac tices (Mannering et al, 2007). However the planting of a cover crop incurs additional cost s to the grower and therefor e cover crops are not a major weed management practice in major soybean growing areas (Iowa State University, 2006).

The use of herbicides has become an importa nt part of managing weeds in soybean. In 2006, approximately 98% of the soybean acrea ge received an herbicide application (USDA-NASS, 2007b). Over 35 different herbic ide active ingredients are registered and available for use by soybean growers to control weeds (Tables VIII-7 and VIII-8). Herbicide weed control programs in conve ntional soybean consist of preemergence

herbicides used alone or in mixtures with other preemergen ce herbicides. Mixtures of two preemergence herbicides are used to broa den the spectrum of control to both grasses and broadleaf weed species. Preemergen ce herbicides are followed by postemergence applications to control weeds that emerge later in the crop. Total postemergence weed control programs were seldom used in c onventional soybean prio r to 1995. Prior to Roundup Ready soybean, soybean planted in a no- till system would receive a preplant burndown herbicide application fo r broad-spectrum control of existing weeds at time ofMonsanto Company 10-SY-210U 505 of 721 

### planting, followed by different multiple soil re sidual herbicides at planting and possibly still other herbicides applied postemergence to the crop and the weeds. In conventional soybeans the typical herbicide program consisted of multiple soil residual herbicides applied preemergence to the crop and weed s and, possibly, other herbicides applied postemergence to the crop and weeds. Therefore, multiple herbicides and/or multiple applications were generally made in conventional and no-till non-Roundup Ready soybean. The average number of herbicide app lications per acre in soybean rose from 1.5 in 1990 to 1.7 applications in 1995 reflecting the use of at-plant and postemergence applications or two postemergence a pplications (Giane ssi et al., 2002).

Selective herbicides are designed to kill specif ic types of plants, usually grasses or broad leaf weeds, and have proved effective to reduc e in-crop tillage or cu ltivation to control weeds in soybean production. The developmen t of selective herbic ides has progressed

since the introduction of the fi rst herbicide (2,4-D) for weed control in corn in early 1940s. Although the primary purpose of tillage is for seedbed preparat ion, tillage still is used to supplement weed cont rol with selective herbicides in soyb ean production.

The availability of herbicide-tolerant soyb ean products are an important aspect of weed management in U.S. soybean production, as disc ussed in Section VIII of the petition and summarized here; refer to the petition for more detail. He rbicide-tolerant soybean was introduced to provide growers with add itional options by improving crop safety (no herbicide damage to the crop) and improving weed control. In 2006, herbicide-tolerant soybean (glyphosate-tolerant) was planted on 89% (67.2 million acres) of the of soybean acres (USDA-NASS, 2007a). Th e percentage of herbicide-tolerant soybean subsequently increased to 91% in 2009. With the high per centage of glyphosate-tolerant soybean and the use of glyphosate for preplant burndown a nd postemergence in-crop applications, it is not surprising that glyphosate was used on 97% of the total soybean acres in 2006.

Because of the unique broad spectrum herbic ide activity of glyphosate, it is possible for growers to choose to use onl y glyphosate for their total weed management. Some growers made this choice, choosing not to ut ilize diversified weed management practices and instead relying only on glyphos ate for total weed manageme nt. As a result of these practices, there has been an in crease in the number of crop acr es with glyphosate resistant weed populations over the last decade. Ho wever, recently the number of growers incorporating other weed mana gement practices along with th e use of glyphosate, such as

other herbicide modes-of-action or cultivati on practices, has grown over time in part in response to the evolution of glyphosate re sistant weeds and based upon recommendations from Monsanto, academics, and the pestic ide industry. Academics and university

extension services recommend growers use diversified weed management practices, as described above, as the guiding principle for managing resist ance and shifts in weed population. The specific pract ices to be recommended by ar ea or by farm will be communicated by local experts versed in the be st methods for both proactive and reactive management of resistance. Since many soyb ean farmers practice c onservation tillage and some may chose to plant soybeans repeated ly on the same land, the use of multiple herbicide modes-of-action with overlapping eff ectiveness on the targeted weed spectrum will be the primary method employed. Studies have demonstrated that using the same combination of herbicides with multiple modes-of-action and overlapping effectivenessMonsanto Company 10-SY-210U 506 of 721 

### over multiple seasons is an effective way to proactively manage resistance (Beckie and Reboud, 2009).

The incorporation of additional weed manage ment practices has been most pronounced in Roundup Ready corn and Roundup Ready cotton but a growing trend in Roundup Ready soybeans has also occurred. In a 2005 growersurvey, 15 to 21% of growers applied nonglyphosate herbicides in addition to glyphosat e for weed control in glyphosate-tolerant soybean (Givens et al., 2009). These non-glyp hosate herbicides were applied prior to planting, at planting and/or postemergence in soybean (see Section VIII.F.1 of the petition). In another grower survey conduc ted at the end of the 2009 growing season, the percent of growers applying non-glyphosate herbicides rose to 33% for those growing continuous Roundup Ready soybeans, and to 33% and 52% for growers growing Roundup Ready soybeans in rotation with Roundup Ready corn and Roundup Ready cotton, respectively (personnel communication from Dr. David Shaw, December 2011).

These data indicate a trend towards increas ed diversification of weed management practices in glyphosate- tolerant crops.

Further evidence of increased adoption of diversified weed management practices, including incorporation of multiple herb icide modes-of-acti on, across Roundup Ready corn, cotton and soybeans is presented by Prin ce et. al, (2011). This study reported that between 46% and 54% of surveyed Roundup Re ady growers (corn, cotton and soybeans) who responded that they did not have gl yphosate-resistant weeds on their farm used either a non-glyphosate residual and/or pos temergence herbicide in the 2009 growing season. For growers indicating they have on-farm herbicid e-resistant weed populations to other herbicides, the percen tage of growers was higher at 72% to 75%. Furthermore, researchers report that approximately 40 to 50% of the grower s utilizing glyphosatetolerant crops indicate that rotating herbicides or ta nk mixing glyphosate with other herbicides is an effective management practice to minimize the development of glyphosate resistance (Powles et al., 1996; Diggle et al., 2003; Beckie, 2006; Beckie and Reboud, 2009). Indeed, as described in detail below, a prominent strategy to delay the development of herbicide-re sistant weeds is to increase the diversity of weed management options in agricultu re, including use of herbicid es with different modes-ofaction in a grower’s weed manageme nt program (Duke and Powles, 2009).

Herbicide-resistant Weeds

The use of any herbicide results in the potential for the selection of weeds resistant to that herbicide particularly when the herbicide is not used as part of a diversified weed management program. Within a weed speci es, individuals may possess an inherent ability to withstand the effects of a particular herbicide. Repeated use of that herbicide will expose the weed population to a "selection pressure," which may lead to an increase in the number of surviving resistant indi viduals in the population (HRAC, 2010). The increased and repeated use of glyphosate over glyphosate-tolerant crops without incorporation of diversifie d weed management practices , such as crop rotation, cultivation or use of multiple herbicide modes-of-action, has resulted in the selection of glyphosate-resistant weeds. Glyphosate-resistan t weed biotypes found in soybean fields include Palmer pigweed ( Amaranthus palmer i), tall waterhemp ( AmaranthusMonsanto Company 10-SY-210U 507 of 721 

### tuberculatus ), common ragweed ( Ambrosia artemisiifolia ), giant ragweed ( Ambrosia trifida ), hairy fleabane ( Conyza bonariensis ), horseweed ( Conyza canadensis ), kochia ( Kochia scoparia ), goosegrass ( Eleusine indica ), Italian ryegrass ( Lolium multifloru ), rigid ryegrass ( Lolium rigidum ), Johnsongrass ( Sorghum halepe ) (Heap, 2011). As with

other herbicide resistant weeds that have developed, the emergence of glyphosate resistant weed biotypes over the past decade re presents a need for growers to adapt and implement improved weed management strategies.

Weed resistance is common in the majo r non-glyphosate soybean herbicide groups.

Table J-2 and Table J-3 summarize known resistance among the major weed species present in soybean within each of the key s oybean herbicide groups and herbicide classes active on broadleaf weeds (Heap, 2011). Resi stance to the ALS group of herbicides is present in most of the major broadleaf w eed species commonly found in soybeans. For common ragweed and waterhemp there is known resistance to at least one member for several of the major soybean herbicide chemistr y classes. While there are still effective options for managing common ragweed, wate rhemp, palmer pigweed and other key broadleaf weeds, there is a need for addi tional herbicide modes- of-action to combat future resistance in soybeans and continued management of existing herbicide-resistant weed populations. Similarly, there has been an increase in the detection of weed populations with multiple resistance (i.e., re sistance to multiple herbicide modes-ofaction) in some weed species, for example, Amaranthus spp. (Tranel et al, 2010). The emergence of these resistant biotypes and continued need to utilize diverse weed management practices supports the need fo r additional herbicide modes-of-action in major crops such as soybeans.

Monsanto and academics recommend the use of multiple herbicide modes-of-action in the Roundup Ready soybean system regardle ss of whether glyphosate-resistant or hard-to-control broadleaf weeds are present. Monsanto specifically recommends the use

of a soil residual as part of the weed management syst em. Growers may also choose to switch to other weed management systems in their soybean. APHIS has approved other herbicide-tolerant soybean including phosphinot hricin-tolerant and ALS-tolerant soybean events (Table J-4). For growers who choos e to use the Roundup R eady soybean system, Monsanto and university extension agents provide recommended control options for glyphosate-resistant weeds. These options include the use of residual and postemergent herbicides such as synthetic auxins (2,4-D) , ACCase inhibitors (clethodim, sethoxydim), PPO inhibitors (lactofen, fomesafen) , and ALS inhibitors (cloransulam). 22 

### These

herbicides alone or combinations of these herb icides as well as traditional tillage methods

are and will continue to be used to c ontrol glyphosate-resistan t or hard-to-control

broadleaf weeds.

22 Monsanto Technology Use Guide; www.weedresistancemanagement.com.Monsanto Company 10-SY-210U 508 of 721Table J-3. Known Weed Resistance in the Southern U.S.1MostCommonBroadleafWeeds(# stateswherelisted as atop weed)ResistanceGroup2ALS(Group 2)PPO(Group 14)PS II(Group 5)Glycine(Group 9)Phenoxy(Group 4)ChemistryClass2SulfonylUreaImidazolinonesTriazolesDiphenyletherN-phenylthalimideTriazinones-PhenoxyBenzoicacidExamplechlorimuronimazapyrchloransulamlactofenfomesafenflumioxazinmetribuzinglyphosate2,4 DdicambaMorning glory (5)Sida (prickly sida (5) XSicklepod (4)Hemp sesbania (3)Pigweed spp.3(3) X X X XPalmer pigweed (2) X X X XCocklebur (1) X X XHorseweed (marestail)(1)X X X1Source: www.weedscience.org2Cross resistance is possible within a resistance group and/orchemistry class3Includes redroot pigweed and smooth pigweedMonsanto Company 10-SY-210U 509 of 721Table J-4. Known Weed Resistance in the Midwest U.S.1MostCommonBroadleafWeeds(# stateswherelisted as atop weed)ResistanceGroup2ALS(Group 2)PPO(Group 14)PS II(Group 5)Glycine(Group 9)Phenoxy(Group 4)ChemistryClass2SulfonylUreaImidazolinonesTriazolesDiphenyletherN-phenylthalimideTriazinones-PhenoxyBenzoicacidExamplechlorimuronimazapyrchloransulamlactofenfomesafenflumioxazinmetribuzinglyphosate2,4 DdicambaPigweed spp.3 (12)X X X XVelvetleaf (11)Lambsquarters (10) X X XCocklebur (9) X X XCommon ragweed (7) X X X X X XSmartweed spp. (6)Morning glory (5)Waterhemp (5) X X X X X XHoresweed (marestail)(3)X X XGiant ragweed (3) X X X XKochia (2) X X X XMonsanto Company 10-SY-210U 510 of 7211Source: www.weedscience.org2Cross resistance is possible within a resistance group and/orchemistry class3Includes redroot pigweed and smooth pigweedMonsanto Company 10-SY-210U 511 of 721 

### J.3.3.7. Dicamba Herbicide Use in the U.S.

Dicamba use in the U.S. is discussed in Se ction VIII.G of the pe tition and summarized here; refer to the petition for more detail. Dicamba is a broadleaf selective herbicide that was approved by the Environmental Protec tion Agency (U.S. EPA) for agricultural application uses in 1967 (U.S . EPA, 2009b). Dicamba herbicid e is currently labeled for weed control in corn, soybean, cotton, sorghu m, wheat, barley, oats, millet, pasture, rangeland, asparagus, sugarcane, and turf, gr ass grown for seed, conservation reserve programs, and fallow croplands. Dicamba treated acreage has ranged from 17.4 to 36.3 million acres between 1990 and 2008. Usage of dicamba peaked during the period of 1994 through 1997, where 1994 was the peak year when 36 million acres were treated with 9.4 million pounds of dicamba. Sin ce 1994, the use of dicamba has steadily

declined to 20.2 million trea ted acres with 2.67 million pounds applied in 2008. The decline is due to the competitive market introductions of sulfonylurea herbicides (chlorsulfuron, metsulfuron-methyl, and thif ensulfuron-methyl) in wheat, new broadleaf herbicide active ingredie nts in corn, and glyphosate-tolerant corn. Usage in cotton is one exception, where dicamba-treate d acres (preplant applicati ons) have increased from 140,000 to 590,000 acres from 2004 to 2008. Dicamba is formulated as a standalone herbicide product and marketed by several co mpanies under various trade names such as

Banvel ® 

### , Clarity ® 

### , Diablo ® 

### , Rifle ® 

### , Sterling ® 

### , and Vision. These dicamba products can

also be tank mixed with one or more activ e ingredients depending on the treated crop. For example, Clarity can be tank mixed with over 75 herbicide products in labeled crops. Additionally, dicamba is formulated as a premix product with one or more other herbicide active ingredients such as gl yphosate, 2,4-D, difluf enzopyr, atrazine, nicosulfuron, metsulfuron, pr imsulfuron, triazulfuron, rims ulfuron and halosulfuron.

Based on USDA-NASS (2004, 2006b, 2007b, 2008) stat istics, dicamba application rates ranged from 0.03 to 0.25 pounds per acre with the average number of applications ranging from 1 to 1.2 applications per croppin g season. Dicamba rates are the lowest in barley, wheat, and oats, where typically more than one application is made in these crops per cropping season. The average application rate in corn is 0.19 pounds of dicamba per acre with slightly over one application per season.

Dicamba is currently labeled for use in conventional or Roundup Ready soybean, although dicamba use is extremely limited because applications are restricted to preplant and/or preharvest applications due to cr op tolerance concerns. The dicamba-treated acreage in 2008 soybean production was appr oximately 530,000 acres representing 0.7% of the total soybean acreage.

J.3.4. Human Health and Worker Safety

Soybean is a highly versatile crop which can be processed into a wide variety of food products. Soybean protein is used to enhance nutrition in a wide va riety of food products, such as breakfast cereals and pasta. Soybean protein is also an important component in baked goods, alternative meat products, soups, energy bars, nutritional beverages infant formula and dairy replacement products (USB, 2011b). Soybean oil constitutes the majority (68%) of consumed edible fats and oi ls in the U.S. (ASA, 2011) . It is present inMonsanto Company 10-SY-210U 512 of 721 

### numerous food products including cooking oils, shortening, margarine, mayonnaise, salad dressings and a wide variety of fat or oil-based products (USB, 2011b).

Humans consume soybean and have done so for thousands of years. Soybean improved with new traits produced by biotechnology pose no unique risks relative to other soybean developed using traditional breeding met hods. Biotechnology-derived soybean is evaluated extensively prior to commercia l introduction. All biotechnology-derived soybean products on the market today ha ve satisfactorily completed the FDA consultation process establishe d to review the safety of foods and feeds derived from biotechnology-derived crops for human and animal consumption. Biotechnology-derived soybean crop varieties that have been de regulated or are under consideration for deregulation are shown in Table J-5.

Pesticides have been used extensively in th e production of soybean (see Section J.3.3.6). The use of these pesticides is regulated by the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA). FIFRA was amende d in 1988 to accelerate the reregistration of products with active ingred ients registered prior to N ovember 1, 1984. The amended Act calls for the development and submission of data to support the reregistration of the active ingredient, as well as a review of all data submitted to the EPA. During the reregistration process, EPA thoroughly revi ews the scientific database underlying a pesticide's registration. The purpose of the Ag ency's review is to reassess the potential risks arising from the currently registered uses of a pesticide, to determine the need for additional data on health and environmental effects, and to determine whether or not the pesticide continues to meet the "no unreasonabl e adverse effects" crit eria of FIFRA.

In order to comply with FIFRA, EPA eval uates potential risks to humans and the environment, and may require applicants to submit more than 100 different scientific studies conducted according to EPA guidelines. The data required by EPA are used to evaluate whether a pesticide has the poten tial to cause adverse effects on humans (including chronic, reproductive and cancer risk), wildlife, fi sh, and plants (including endangered species and othe r non-target organisms, i.e. , organisms that the pesticide is not intended to act against). If the pesticid e may be used on food or feed crops, EPA also sets tolerances (maximum pes ticide residue levels) for the am ount of the pesticide residue that can legally remain in or on foods. EPA undertakes this analysis under the authority of the Federal Food, Drug, and Cosmetic Act (FFDCA), and must conclude that such tolerances will be safe, meaning that there is a reasonable certainty that no harm will result from aggregate (food, water and n on-occupational reside ntial/recreational) exposure to the pesticide residues.

EPA has evaluated dicamba and concluded th at is has a complete and comprehensive regulatory database (toxicity, environmental fa te, and ecological toxicity) that has been evaluated by the EPA. EPA completed th e reregistration process for dicamba and a Registration Eligibility Decisi on (RED) was issued in 2006 a nd subsequently amended in 2008 and 2009 (U.S. EPA, 2009b). EPA concluded the available data submitted for dicamba are complete and adequate to s upport the continued registration of dicamba products and uses. EPA also considered toxicity data and av ailable information concerning the variability of sensitive subpopulations, includ ing infants and children.Monsanto Company 10-SY-210U 513 of 721 

### The EPA concluded there is reasonable certainty that no harm will result to the general population, or to infants and children, as a result of aggregate exposure to dicamba residues. Thus, all then-current dicamba uses were eligible for reregistration (U.S. EPA, 2009b). See Appendix M, Section M.2 and M .3 for additional details about the EPA registration process and the reregistraiton of dicamba.

In the agricultural production of soybean, growers and workers may be exposed to pesticides applied to soybean by mixing, loading, or applying chemicals



### , or by entering a previously treated site. 

### EPA conducts a comprehensive occupational worker safety evaluation and risk assessment of pesticides to assess the risk to agricultural workers during mixing, loading, and applying . EPA evaluated occupational risk to workers as a part of the dicamba RED and concluded that worker exposure to dicamba for all registered agricultural uses, including exposures associated with the current preemergence and postemergence pre-harvest soybean uses, 

### meet the "no unreasonable adverse effects"

criteria of FIFRA (U.S. EPA, 2009b). 

### The use of dicamba on MON 87708 does not pose any new exposure considerations for work ers. Therefore the use of dicamba on MON 87708 will not pos e a risk to agricu ltural workers. See Appenidx M, Section M.2.2 for additional details on applicator exposure.



### In addition, the Worker Protection Standard (WPS) provides additi onal protections to agricultural workers and pesticide applicat ors. The WPS contains requirements for pesticide safety training, notification of pes ticide applications, use of personal protective equipment (PPE), restricted-entry interv als (REI) after pest icide application, decontamination supplies, and emergency me dical assistance. Under the WPS, EPA requires the pesticide label to specify PPE and REI, based on the properties of the pesticide product, that will provide an appropriate level of protection 23



### .

23 www.epa.gov/pesticides/health/worker.htmMonsanto Company 10-SY-210U 514 of 721 

### Table J-5. Deregulated or Submitted Bi otechnology-derived Soybean Products

Phenotype Event Institution Date Deregulated
High Oleic Acid, Low

Saturated Fat MON 87705 Monsanto December 16,

2011 Omega 3 Fatty Acid MON 87769 Monsanto Submitted

Lepidopteran Resistant MON 87701 Monsanto June 28, 2011

Herbicide-tolerant ( 2,4-D ) DAS-68416-4 Dow AgroSciences Submitted

Herbicide-tolerant ( Glyphosate / Isoxaflutole ) FG72 Bayer Crop Sciences Submitted

Herbicide-tolerant ( Imidazolinone ) BPS-CV127-9 BASF

Plant Science Submitted

High Oleic Acid DP-3Ø5423-1 Pioneer June, 2010

Glyphosate- and ALStolerant DP-356043-5 Pioneer July, 2008

Glyphosate-tolerant MON 89788 Monsanto February, 2007

Phosphinothricin-tolerant GU 262 AgrEvo October, 1998 Phosphinothricin-tolerant A5547-127 AgrEvo May, 1998Altered Oil Profile G94-1, G94-19, G168 DuPont May, 1997

Phosphinothricin-tol erant W62, W98, A2704- 12, A2704-21,

A5547-35 AgrEvo August, 1996

Glyphosate-tolerant 40-3-2 Monsanto May, 1994

Source is website: http://www.aphis.usda.gov/brs/not_reg.htmlMonsanto Company 10-SY-210U 515 of 721 

### J.3.5. Animal and Plant Communities, Soil Microorganisms and Biodiversity

J.3.5.1. Animal Communities

Soybean production systems in agriculture ar e host to many animal species including deer, groundhogs, rabbits, raccoons, geese and small rodents (USDA-APHIS, 2011b). Mammals and birds, including migratory ma mmals and birds, may seasonally consume grain (Galle et al., 2009), a nd invertebrates can feed on the plant during the entire growing season.

Animals that feed primarily on soybean are seed-feeding insects a nd rodents found in agricultural fields. Crop pest insects are considered less problematic than weeds in U.S. soybean production as indicated by the low percentage (14%) of soybean acreage that receives insecticide treatment (USDA-NASS, 2006). Management of insects in soybean production fields is discussed in Section VIII.D of the petition. Some rodents, such as mice or squirrels, may seasonally feed exclusiv ely on soybean seeds. Thus, these animals may have a diet containing signi ficant amounts of soybean seeds. Deer may also browse in soybean fields on the forage and on seed left after harvest.

Animals that feed outside soybean fields ar e also considered in this section. The environment surrounding a soybean field, which may vary in plant composition depending on the region, may serve as a food so urce and habitat for mammals, birds, fish and insects. In certain areas, soybean fiel ds may be bordered by other soybean, corn, or other crops; soybean fields may also be su rrounded by woods and/or pasture/grassland areas, as well as aquatic environments. Th erefore, the types of vegetation, including weeds, around a soybean field depend on th e area where the soybean is planted.

Fertilizers and/or water contai ning pesticides may run off into adjacent lands, pesticides may also move outside of the agroecosyst em from drift and offsite movement. Regardless of the agricultura l operation, animals and insects outside the field may be impacted directly from the use of fertil izers, pesticides, and erosion caused from agricultural operations, or indi rectly, both positively and negatively, from effects on the plant community outside the soybean field.

J.3.5.2. Plant Communities

The affected environment for growing soybean plants can generally be considered the agroecosystem (managed agricultural fields ) plus some area extending beyond the intended plantings that might be affected by ag ricultural operations. Plants, extraneous to the crop, which grow in planted fields can be considered weeds and are dealt with in

section J.3.3.6 of this document.

Plants growing outside soybean fields are considered in th is section. The environment surrounding a soybean field varies in plant composition depending on the region. In certain areas, soybean fields may be borde red by other soybean, corn, or other crops; fields may also be surrounded by woods and/or pasture/grassland areas , as well as aquatic environments. Therefore, the types of vegetation, including weeds, around a soybean field depend on the area where the soybean is planted. A variety of weeds dwell in andMonsanto Company 10-SY-210U 516 of 721 

### around soybean fields; those species may also vary depending on the region where the soybean is planted. These plants may be found in ditches, hedge rows, fence rows, wind breaks, yards, and other unculti vated areas, and may be annuals , biennials or perennials. Regardless of the agricultura l operation, these plants may be impacted, both positively and negatively, by agricultural operations. Fertilizers and/ or water may run off into adjacent lands, resulting in increased plant growth outside the agroecosystem. Negative impacts on plants adjacent to production fields can occur from herbicide runoff and drift.

Finally, soybeans infrequently occur as a volunteer when soybean seeds remain in a field following harvesting and may be considered a weed in the subsequent crop. Volunteer soybean in rotational crops is not a concer n in the Midwest region because the soybean seed is typically not viable after the winter period (Carpe nter et al., 2002; OECD, 2000). In southern soybean growing ar eas of the U.S. where the winter temperatures are milder, it is possible for soybean seed to remain viable over the winter and germinate the following spring. If volunteer soybean should emerge after planting, shallow cultivation and/or use of another herbicide will control volunteers and effectively reduce competition with the crop. Several postemergence herbicides are also available to control volunteer soybean (conventional or glyphosate-tolerant soybean, and by extension dicamba-tolerant soybean) in each of the major soybean rotati onal crops (Section VIII. J. of petition #10- SY-210U). Therefore, volunteer soybean norma lly is not a concern in rotational crops, such as corn, cotton, rice and small grains (e.g., wheat, barley, sorghum and oats), which are the primary rotational crops following soyb ean due to the availability of adequate control measures for volunteer soybean (Carpenter et al, 2002; OECD, 2000).

J.3.5.3. Soil Microorganisms

Soil microbial communities are highly comp lex and are often characterized by high microbial diversity (Tiedje et al., 1999). The occurren ce and abundance of soil microorganisms are affected by 1) soil characte ristics like tilth, or ganic matter, nutrient content, and moisture capacity, 2) typical phys ico-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, colonizat ion, and associated biochemical processes (Buckley and Schmidt, 2001; 2003). Conseque ntly, significant variation in microbial populations is expected in agricultural fields.

Members of the bacterial family Rhizobiaceae and Bradyrhizobiaceae form a highly complex and specific symbiotic relationship with leguminous plants, including soybean (Gage, 2004). The nitrogen-fixing plant-micr obe symbiosis results in the formation of root nodules, which provide an environment in which differentiate d bacteria called bacteroids are capable of re ducing or “fixing” atmospheri c nitrogen. The product of nitrogen fixation, ammonia, can then be utilized by the plant. As a result of this relationship, nitrogen inputs ar e typically not necessary fo r agricultural production of soybeans.Monsanto Company 10-SY-210U 517 of 721 

### J.3.6. Biodiversity

Biodiversity refers to all plan ts, animals, and microorganisms interacting in an ecosystem. Among other benefits, biodiversity provide s valuable genetic resources for crop improvement (Harlan, 1975) and also provide s other functions beyond food, fiber, fuel, and income. These include pollination, genetic introgression, biologica l control, nutrient recycling, competition against natural enemies, soil structure, soil and water conservation, disease suppression, control of local microclimat e, control of local hyd rological processes,

and detoxification of noxious chemicals (Altieri, 1999). The loss of biodiversity results in a need for costly external inputs in order to provide these functions to the crop (Altieri, 1999). Agricultural land subject to intensive farming practices, such as that used in crop production, generally has low levels of biodiversity compared with adjacent natural areas.

The use of broad spectrum insecticides an d herbicides is one of the most severe constraints for biological diversity in crops. Tillage, seed bed preparation, planting of a monoculture crop, pestic ide use, fertilizer use, and harves t may all limit the diversity of plants and animals (Lovett, Price, & Lovett, 200 3). Herbicide use in agricultural fields is likely to indirectly impact biodiversity by decreasing weed species present in the field and those insects, birds and mamma ls that utilize these weeds.

Conservation tillage practices can have a pos itive impact on wildlife, including beneficial arthropods (Altieri, 1999; Landis et al., 2005; Towery and Werblow, 2010). Conservation tillage practices benefit biodiversity due to de creased soil erosion improves surface water quality, retention of vegetative cover, crop re sidues serve as a food source,; and increased populations invertebrates wh ich can serve as food sources to other organisms (Landis et al. 2005; Sharpe, 2010)

J.3.7. Physical Environment

J.3.7.1. Land Use

In 2008, soybean was grown as a commercial crop on over 75 million acres in at least 27 states in the U.S. (USDA-NASS, 2009a). Soyb ean acreage in the past five years has been relatively stable varying from 64.7 million to 75.7 million acres with a 10-year average of 73.3 million acres (Table VIII-1). Soybean fields are typically highly managed agricultural areas that ca n be expected to be dedicated to crop production for many years. Fluctuations in soybean acreage are due to environmental, agronomic and economic factors, as well as government programs such as the conservation reserve program (CRP).

Currently, biotechnology-derived herbicide tolerant soybean is planted on 91% of the soybean acreage (USDA-NASS, 2009a) and the Roundup Ready soybean system has become the standard weed control program in U.S. soybean production. There is no

indication that the introducti on and widespread adoption of biotechnology-derived crops in general has resulted in a significant change to the total U.S. acreage devoted to agricultural production. The cumulative land area in the U.S. planted to principal crops, which include corn, sorghum, oats, barley, winter wheat, ry e, durum, spring wheat, rice, soybean, peanuts, sunflower, cotton, dry edible beans, potatoes, canola, proso millet, andMonsanto Company 10-SY-210U 518 of 721 

### sugar beets, has remained relatively constant over the past 27 years. From 1982 to 1995, the average yearly acreage of principal crops was 323 million. This average is essentially unchanged at 326 million acres since the introd uction of biotechnology-derived crops in 1996 (USDA-NASS, 1984, 1988, 1990, 1992, 1995, 1998, 2000, 2003, 2006a, 2009a).

J.3.7.2. Water Resources

The soils and climate in the Midwestern, East ern and portions of the Great Plains regions of the U.S. provide sufficient water under normal climatic conditions to produce a soybean crop. Irrigation is used on approximate ly 9% of the soybean acreage in the U.S. to supplement the water supply during dry peri ods in the Western and Southern soybean growing regions (USDA-ERS, 2008b). More information on water management in soybean production can be found in Secti ons VIII.B and J.3.3.3 of the petition.

Groundwater may be impacted from soybean production by the moveme nt of pesticides and fertilizers vertically through soil. Surf ace water may also be impacted from soybean production by runoff from soybean fields that ca rries soil particles and herbicides or other pesticides to streams, rivers, lakes, wetland s 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 th an is the pesticide component.

Tillage causes widespread soil disturbance. Thus, erosion, topsoil loss and the resulting sedimentation and turbidity in streams are likel y to increase with increased tillage. In 2009, based on the states’ water quality repo rts, EPA identified sedimentation and turbidity as two of the top 10 causes of im pairment to surface water in the U.S. in general; in 2007, EPA identified sedimentation/ siltation as a leading cause of impairment to rivers and streams in particular (U.S. EPA, 2009a; EPA, 2007a). EPA has projected conservation tillage to be “the major soil protection method and candidate best management practice for improving surface water quality” (U.S. EPA, 2002). EPA identifies conservation tillage as the first of its CORE4 agricultural management practices for water quality protection (U.S. EPA, 2008a).

Based on the states’ wate r quality reports to EPA, which 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 tu rbidity (U.S. EPA, 2008b). Pest icides accounted for less than one percent of reported ca uses of surface water impairment in all but four of the 17 leading U.S. soybean-producing states. In thos e four states, pesticid es accounted for two to eight percent of reported causes of impairment . Of the pesticides that were reported as contributing to impairment among the 17 leadin g soybean-producing states, almost all are highly persistent chemicals that are no longer registered for use in the U.S. (U.S. EPA, 2008b). Dicamba is not in cluded on this list.

Water resources (Ground and surface water) may also be impacted from the use herbicides used to control weeds. Dicamba has been widely used in agriculture over the last four decades with dicamba’s peak us e occurring in 1994 (see Section VIII.G of the petition). In the dicamba Reregistration Eligibility De cision (RED) document, EPAMonsanto Company 10-SY-210U 519 of 721 

### considered potential risks associated with dicamba use, and its degradate DCSA when appropriate, due to surface or ground water using screening level (high-end exposure)

models to estimate environmental concentr ations. The EPA then compared these exposure estimates to appropr iate endpoints from mammalia n and aquatic animal and plant ecotoxicity studies to determine potential impacts on human health and the

environment. The EPA used the models PRZM/EXAMS and SCIGROW to estimate levels of dicamba in surface and ground water, respectively, using the physical, chemical, and environmental fate properties, and a pproved high-end use patterns of dicamba (see Appendix M, Section M.3.2 of the petition).

For drinking water resources, estimated surface water concentrations using the simulated sugarcane crop scenario for bot h ground and aerial applications of 2.8 lbs a.e./acre ranged from 9.7 to 13 μg/L dicamba a.e. and 0.66 to 0.81 μg/L for DCSA (U.S. EPA, 2005a). For environmental-ecological water quality, esti mated surface water concentrations for a simulated soybean crop scenario for ground and aerial applications of 2.0 lbs a.e./acre ranged from 33.3 to 36.1 μg/L dicamba a.e. The U.S. Geological Survey National Water Quality Assessment (NAWQA) monitoring program also analyzed surface water in a 1993-2003 survey of surface waters of the United States, which included geographical areas where dicamba use has historically been most intense. Dicamba had a low incidence of detections (approximately 3% of samples) and the highest levels detected were approximately 2 μg/L (U.S. EPA, 2005a).

For groundwater, estimated concentrations us ing the simulated sugarcane crop scenario for both ground and aerial applications of 2.8 lbs dicamba a.e. per acre were 0.016 μg/L dicamba and 0.008 μg/L for DCSA, dicamba’s major environmental degradate (U.S. EPA, 2005a). The EPA also analyzed gr oundwater in a 1971-1991 national monitoring study. The frequency of dicamba detection was less than 2.5% based on 3172 samples analyzed, and dicamba was detected at levels from less than 0.01 to 44μg/L (U.S. EPA, 1992), and peak dicamba detections occurred between 1985 and 1990. Dicamba also had a low incidence of detections (less 3% of samples) in the 1993-2003 NAWQA monitoring program where the highest detect ions were approximately 2.5 μg/L (U.S. EPA, 2005a).

J.3.7.3. Soil Quality

Soybean is cultivated across a wide variety of soils in the U.S. Cultivation and tillage practices can directly impact th e attributes of soil, includin g its physical and biological properties. Microbial populations and associated bioche mical processes are critical to maintaining soil health and quality. Additiona lly, maintaining soil pH in the range of 6.0 to 7.0 will enhance the availability of inhe rent and fertilizer nutrients, reduce the availability of toxic elements, particul arly aluminum and manganese, and enhance microbial activity (Hoeft, 2000). The increased microbial activit y that is associated with the optimum pH level results in oxidation of organic matter and increased release of nutrients from the organic matter.

Conservation tillage and no-till systems enhance soil quality relative to conventional tillage. Benefits of conser vation tillage are we ll documented and in clude reduced soilMonsanto Company 10-SY-210U 520 of 721 

### erosion, conservation of soil moisture, improvement of soil structure, reduction of soil compaction and improvement of soil organic matter content. In 2010, approximately 27.5 million acres (39.6%) of soybean were plante d in a no-till system (CTIC, 2010). In 2011 over 65% of U.S. soybean acres used so me form of conservation tillage (USB, 2011a).

J.3.7.4. Air Quality and Climate

Many agricultural activities affect air quality including tillage, farm equipment, and

nitrous oxide emissions from the use of nitr ogen fertilizer. These agricultural activities individually have potential a dverse environmental impacts on air quality and climate and may be impacted, positively or negatively, by cha nges in agricultural practices. Issues of concern include, but are not necessarily limited to, atmospheric emission of carbon dioxide, nitrogen oxide, sulfur oxi de, and particulate matter. Agricultural practices have the potential to directly and indirectly imp act air quality and contribute emissions which could lead to climate change.

Tillage contributes to the release of gree nhouse gases (GHG) because of the loss of carbon dioxide to the atmosphere and the expo sure and oxidation of soil organic matter (Baker, et al., 2005). Emissi ons released from agricultu ral equipment (e.g., irrigation pumps and tractors) include ca rbon monoxide, nitrogen oxide s, particulat e matter, and sulfur oxides. Nitrous oxide may also be rel eased following the use of nitrogen fertilizer. Agriculture, including land-use changes for farm ing, is responsible for an estimated 17 to

32% of all human-induced GHG emissions (USDA-APHIS, 2011b). Generation of GHGs may have long term impacts on climate ch ange as they function as retainers of solar radiation. The U.S. agri cultural sector has been identified as the second largest

contributor to GHG emissions (USDA-APHIS, 2011b). Herro (2008) proposes that if agriculture practices were modified, signifi cant reductions in the release of GHGs could

be achieved.

J.3.8. Adjacent-Agricultural Crop and Non-Agricultural Plants

Soybean is widely grown throughout the U.S. on land devoted to agricultural use. The biology of soybean is discussed in Section II of the petition and summarized here; refer to the petition for more detail. Soybean does not have any related wild relatives in the U.S. with which it can hybridize. Soybean does not survive outside of cultivati on and is not invasive or weedy. Soybean is grown adjacent to large acre crops such as corn, wheat, and alfalfa, near vegetables, orchards, past ures, and adjacent to non-agricultural lands, such as forests, grasslands, streams, lakes, rivers and occasionally near urban lands.

Herbicides are commonly used in the production of soybean and a detailed discussion of

their use can be found in Section VIII.F.1 of the petition; refer to the petition for more detail. Impacts on adjacent agricultural cr ops and non-agricultural pl ants can occur from offsite movement of any herbicide, and these impacts are actively managed by farmers and applicators specially trained to use such products consistent wi th product labels and other state or local restrictions. Dependi ng upon the herbicide, factors for managing the potential for drift and offsite movement incl ude the selectivity and sensitivity of theMonsanto Company 10-SY-210U 521 of 721 

### herbicide, local weather conditions at the time of application (wind, temperature, humidity, inversion potential) , droplet size distribution, a pplication volume, boom height (height of the applic ation equipment above the crop canopy ), sprayer speed, and distance from the edge of the application area (SDT F, 1997; Felsot et al ., 2010). A variety of measures can be employed to control the pote ntial for spray drift and offsite movement, including nozzle selection and applic ation techniques and restrictions.

The potential for offsite movement of an herbic ide is regulated at a federal level by EPA. EPA considers possible effects from offsite move ment as part of the pesticide registration process required under FIFRA. Specifically, in order to ap prove the use of a pesticide (herbicide), EPA must conclude that no unreasonable adverse effects on non-target vegetation will result from potential offsite movement when the pesticide is used according to the product label. When pestic ides are applied in accordance with label instructions, offsite impacts can be avoide d. EPA reassessed th e potential risks to nontarget plants in its analysis in the dicamba RED, concluding that no specific additional drift mitigations were needed to support the continued registration of all dicamba uses, including use in conventional soybean with ap plications at early preemergence prior to planting and late postemergence prior to harvest (U.S. EPA, 2009b). A detailed

discussion of the use of dicamba herbicide in the U.S. can be found in Section VIII.G of the petition.

J.3.9. Animal Feed

Soybean meal is the most valuable co mponent obtained from processing soybean, accounting for roughly 50-75% of its overall value (USDA-ERS, 2005). The majority of soybean meal is used by the animal feed industry as a cost-effective protein and amino acid source to animal diets. Soybean meal can serve as a excellent protein source that can complement the limited amino acid profile of feeds derived from corn (Kerley and Allee, 2003). In 2009, approximately 36 million tons of soybean meal was produced, 27 million tons of which was marketed for animal feed with the largest volumes consumed by poultry (48%), swine (26%), and beef (12%) (ASA, 2010).

Dairy and livestock producers use soybean fo rage as feed. Soybean forage is an inexpensive, readily available, on-farm s ource of high-quality, high-protein forage adapted to growth during the summer months when other forage legume species typically are restricted in growth (US DA-ARS, 2006). Soybean forage can be used as hay or to produce silage (MAFRI, 2004). An additional us e of soybean for feed can be full-fat (whole) soybean for dairy cattle and swine, but for swine it is limited due to the high oil

content to a maximum of 20% of the total diet (Yacentiuk, 2008).

Monsanto has completed the biotechnology cons ultation process with FDA for the safety

and nutritional assessment of food and feed derived from MON 87708 soybean on October 11, 2011 (BNF No. 00125, Monsanto, 2011). As a part of its evaluation, FDA

reviewed information on the identity, function, and charact erization of the genes, including expression of the ge ne products in MON 87708 soybean, as well as information on the safety of the MON 87708 DMO a nd MON 87708 including a dietary risk

assessment.Monsanto Company 10-SY-210U 522 of 721 

### EPA has responsibility to regulate the use of pe sticides (herbicides) that may be used on feed crops, and must establish pesticide tole rances (maximum pesticide residue levels) for the amount of pes ticide residue that can legally rema in in or on th e feed crop. EPA undertakes this analysis under the authority of the Federa l Food, Drug, and Cosmetic Act (FFDCA), and must conclude that such tolera nces will be safe, meaning that there is a reasonable certainty that no harm to human health will result from the use of the pesticide. EPA reassessed all dicamba pestic ide tolerances (food and feed tolerances) as part of the dicamba RED, including the 10 ppm soybean seed tolerance supporting the existing use in conventional soybean for ea rly postemergence applications up to 0.5 pound a.e. per acre and late postemergence prior to harvest up to 1.0 pound a.e. per acre (U.S. EPA, 2009b). A complete listing of dica mba feed tolerances can be found at 40 CFR § 180.227. Monsanto has also petitioned (Pesticide Petition # 0F7725) the EPA to establish new feed tolerances on soybean fo rage (45 ppm) and soybean hay (70 ppm). Tolerances for soybean forage and hay for cu rrent dicamba uses in conventional soybean were not previously established because the cu rrent preharvest application is made past the stage where the crop would be useful as forage or hay.

J.4. Alternatives

The action 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 Agency actions in response to the petition:

 No action

MON 87708 would remain a regulated article

 Approval in part

MON 87708 would be granted deregulated status with some restrictions or conditions ( e.g. , geographic)

 Approval in whole

MON 87708 would be granted full deregulated status

J.4.1. No Action Alternative

Under the “no action” alternative, MON 87708 would remain a regulated article under 7 CFR Part 340. MON 87708 could be grown under USDA notification or permit and

confined release conditions. MON 87708 would not have unrestricted availability to commercial soybean growers. Under this altern ative, growers will li kely continue to use biotechnology-derived soybean th at are commercially availabl e and herbicides to control weeds in soybean fields. In 2009, 91% of th e U.S. soybean was produced with herbicidetolerant biotechnology-derived soybean. In 2006, 98% of soybean fields received an herbicide application for weed control. Over 35 different herbicide active ingredients are available for use by soybean growers (Table s VIII-7 and VIII-8) an d it is reasonably foreseeable that new herbicide active ingredients may be discovered or existingMonsanto Company 10-SY-210U 523 of 721 

### herbicides used in other crops may be adapte d for use on soybean in the future under this alternative. Growers may c ontinue to use agronomic prac tices they currently use for production of soybean, including the R oundup Ready soybean system. Residual

herbicides will continue to be recomm ended for use in the Roundup Ready soybean system to provide a second herbicide mode-of-action for weed resistance management. In areas where glyphosate-resistant or hard -to-control broadleaf weeds are present, growers may continue to choose to us e the Roundup Ready soybean system and incorporate herbicides with other modes- of-action, including residual herbicides. Recommended control options for glyphosate-re sistant broadleaf w eeds include the use of residual herbicides, preemer gent herbicides such as sy nthetic auxins (2,4-D), and postemergent herbicides such ACCase inhibi tors (clethodim, sethoxydim), PPO inhibitors (lactofen, fomesafen), and AL S inhibitors (cloransulam). 24 

### Under the no action alternative, these herbicides alone and combina tions of these herbicides, as well as tillage, could be used to control glyphosate-resistan t and hard-to-control broadleaf weeds. Because of existing resistance to currently available soybean herbicides and in the absence of new herbicide options, the numbe r of weed populations and species with multiple resistance in soybean production ar eas may continue to increase as well as resistance to herbicide classes that are currently being relied on to manage current levels of resistance to glyphosate a nd other herbicides (e.g., use of PPO herbicides to manage glyphosate resistant palmer pigweed and waterh emp). In the event new resistant biotypes to key herbicides in the PPO group evolve and increase, th ere will be fewer options

available for herbicide control of key weed species such as in the Amaranthus genus.

Under the no action alternativ e, growers may choose to us e other biotechnology-derived herbicide-tolerant soybean products that ha ve been deregulated by APHIS, including glyphosate-tolerant, ALS-tolerant (DP 356043-5), or phosphinothr icin-tolerant ( e.g. , A2704-12 and other events) soybean. Three ot her herbicide-tolera nt soybean products are currently under consideration for dere gulation by APHIS including: 2,4-D-tolerant (DAS-68416-4), glyphosate-tolerant and isoxaflutole-tolerant (FG72), and imidazolinone-tolerant (BPS-CV127-9). Thus, it is reasonably foreseeable that growers may choose these options and use of their companion herbicides under the no action

alternative.

Currently, 91% of the U.S. soybean crop is prod uced with herbicide-tolerant (principally glyphosate-tolerant) soybean (USDA-NASS, 2009a), indicating growers are using nonglyphosate based herbicides and/ or traditional tillage methods for weed control on about 10% of soybean acreage. Growers may c hoose to use the non-glyphosate based weed control systems and/or tradit ional tillage methods under the no action alternative.

J.4.2. Alternatives Considered but Elim inated from Detailed Evaluation

Approval in part based on plant pest risk

24 Monsanto Technology Use Guides can be found at: http://www.monsanto.com/SiteCollecti onDocuments/Technology-Use-Guide.pdf

.Monsanto Company 10-SY-210U 524 of 721 

### Based on APHIS’ regulatory authority, MON 87708 could be granted “approval in part” dependent upon a finding of a plant pest risk in certain geographies or under certain conditions. APHIS may impose restrictions upon the cultivation or use of MON 87708 in specific geographies or circumstances to mitigate an identified plant pest risk. MON 87708 has been thoroughly characterized, and extensive information presented in Sections I through IX of petition #10-SY- 210U demonstrates that MON 87708 does not present a plant pest risk unde r any circumstance. Therefor e, from a plan t pest risk perspective, there is no basis for impos ing geographic or other restrictions on

MON 87708.

The safe use of dicamba for agricultural pu rposes was first esta blished in 1967, and EPA recently reaffirmed its human and environmen tal safety for agricultural uses, including soybean production, with the reregistration in 2006 (U.S. EPA, 2009b). The decisions regarding the safe use of dicamba belong so lely with EPA. AP HIS has no regulatory authority to restrict the use of pesticides or impose measures to mitigate their risk. Monsanto has applied for a label for us e of dicamba on MON 87708, and requested the

establishment of new feed tolerances for s oybean forage and hay. EPA will review the proposed label amendment and assess if the re quested use pattern a nd use instructions meet its statutory standard of no unreasonable adverse effects.

On the basis of this analysis demonstrating that there is no plant pest risk consideration or

other risks that would lead USDA to consid er approval in part, and because EPA has authority to regulate the herbic ide dicamba, these alternatives were not further considered in this report.

J.4.3. Approval in Whole Alternative

Under the “approval in whole” alternative, MON 87708 would no long er be a regulated

article under 7 CFR Part 340 and would be widely available fo r planting. Growers would have the option of treating their soybean crop with dicamba using an expanded window of herbicide application. MON 87708 will allow for preemergent applications up to crop emergence (cracking) and in-c rop postemergent applications up to R1/R2 growth stage, compared to existing uses which permit only early preemergence applications 14-28-days prior to planting and late poste mergence applications prior to harvest. With the approval by the EPA for the use of dicamba on MON 87708 and the integration of MON 87708 into the Roundup Ready soybean system thr ough traditional breeding, the combined crop-tolerances to both glyphosate and dicamba would allow growers to utilize glyphosate and dicamba herbicides in their w eed management systems, as well as other herbicides currently registered for use in s oybean. Monsanto is re questing approval in whole or full deregulated st atus for MON 87708. Informati on and assessments presented throughout this environmental report dem onstrate that MON 87708 does not present a significant environmental impact when approved in whole.

J.5. Potential Environmental Impacts

The Council on Environmental Quality (CEQ) re gulations require that significance be evaluated in terms of context (affected enviro nment) and intensity (the severity of theMonsanto Company 10-SY-210U 525 of 721 

### impact). 25 

### Analysis of these factors considered the “no action” a nd the “approval in

whole” alternatives. The diffe rences between the two alternatives address the relevant issues in considering whether deregulation of MON 87708 results in a significant impact to the quality of the human e nvironment. In most cases, there are no differences between

the two alternatives. Where differences ar e noted, these differences are described and their significance evaluated. Factors evaluated as part of the assessment of significance include: potential impacts to land use patter ns, farming practices, specialty and organic soybean production; potential im pacts to non-agricultu ral lands; potential impacts to the marketability of soybean seed for planting and harvested seed for commodity markets; potential impacts to public health; and potential impacts to non-target organisms and threatened or endangered sp ecies, and biodiversity. Fi nally, cumulative impacts are considered in light of this action combined with past and reasonably foreseeable future

actions.

J.5.1. Methodology and Assumptions

MON 87708 is intended to be a trait that will have utilit y across all of the acreage upon which soybean is currently grown and widely available in soybean varieties sold to growers. It is impossible to determine the exact amount of acreage on which MON 87708 may be grown if deregulated. Howe ver, for the purposes of this assessment, it is assumed that MON 87708 will occupy 40% of the U.S. soybean acreage at peak penetration (see Section VIII.H .2 of the petition). Over the past decade, glyphosatetolerant soybean varieties have been widely ad opted in the marketplace. In most cases, glyphosate was applied to control narrowleaf an d broadleaf weeds in U.S. soybean fields, where glyphosate provided excellent control of most weeds. In recent years, the development of glyphosate-resistant weeds and shifts in broadleaf weed populations to species that are inherently more tolerant to glyphosate have increased the use of additional herbicides that work through a different mode-of-action to achieve an acceptable level of weed control. As a result of the ongoing need to control the majority of weed species present in soybean fields , additional herbicides are being used, and multiple herbicide-tolerance traits are being developed to provide growers with additional weed control options that will compete w ith MON 87708. These herb icides and traits will likely be available at the time MON 87708 is introduced to th e marketplace; thus, MON 87708 will compete for market share with established products, like Roundup Ready soybean 40-3-2 first introduced in 1996, and new herbicide-tole rance traits that

will be available in the foreseeable future. Growers will ultimately select weed control systems that fit the needs for their individual farmi ng operation, such that some proportion of growers will choose to use M ON 87708 integrated into the Roundup Ready soybean system.

It is EPA’s regulatory authority under the Fede ral Insecticide, Fungi cide and Rodenticide Act (FIFRA) to register pesticide products fo r their intended uses, see Section J.3.4 for additional detail. Potential impacts of dicamba use associated with MON 87708 are a connected activity under the dere gulation in whole alternative, and while pesticide use is regulated by EPA, dicamba use will also be di scussed in order to assess potential impacts

25 CEQ regulations are available in 40 CFR §1508.27.Monsanto Company 10-SY-210U 526 of 721 

### to the quality of the human environment. Monsanto has submitted to EPA an application to amend EPA Reg. No. 524-582 to register a new use pattern fo r dicamba. On the basis of Monsanto’s assessment of EPA regist ration decisions and approved labels for alternative herbicides, the use of dicamba on MON 87708 pos es less risk potential to

human health and the environment than some existing alternative non-glyphosate herbicides (see Appendix L). The primary basi s for this assertion is that the introduction

of MON 87708 and the associated use ofdicamba when compared to other nonglyphosate herbicides registered for use on soybean will: 1) reduce human health risk potential for applicators, bystanders, and cons umers; 2) reduce risk potential to aquatic organisms in the environment; 3) positively impact the sustainability of soybean production in the U.S. due to the addition of a second mode-of-action into the Roundup Ready soybean system, thereby potentially delaying further development of glyphosateresistant broadleaf weed populations, and to broa dleaf weed herbicides in general; and 4) support the continued use of conservation tillage with its well known environmental

benefits.

J.5.2. Physical Impacts; Land Use, Water Qu ality, Climate, Soil Quality and Air Quality

J.5.2.1. Land Use Impacts

Approval in whole alternative

The difference between the approval in whole an d the no action alternative is expected to be integration of MON 87708 into the Roundup Ready soybean system using traditional breeding techniques. For those acres wher e glyphosate resistant weeds may already be present, where application of an herbicide with a different mode-of-action would aid in weed control, or for grower implementation of weed resistant management practices, the cultivation of soybean containing the MON 87708 trait would be an option for growers.

Herbicide-tolerant soybean has been dere gulated and grown in the U.S. since 1996. Roundup Ready soybean currently occupies great er than 90% of total soybean acres. Fluctuations in total soybean acreage before and after herbicide-tolerant soybean was commercialized (USDA-NASS, 2011) indicates th at factors unrelated to the availability of the herbicide-tolerant trait play a role in total soybean acres pl anted. Agricultural land use, and consequently crop production is dictat ed by many factors, the most significant of which are commodity prices. Accordingly, growers may increase acres dedicated to soybean production to meet increased demand, but they do so in response to commodity prices and market demand, not in response to availability or adoption of biotechnologyderived traits.

With the exception of tolerance to di camba, MON 87708 is phenotypically and agronomically unchanged from conventi onal soybean. Phenotypic and agronomic

information collected from field trials c onducted in 2008 using the same agricultural inputs showed no meaningful changes betw een MON 87708 and the conventional control (see Section VII.D of the petition). Informa tion presented in the petition demonstrates that compared to conventional soyb ean, MON 87708 does not display increasedMonsanto Company 10-SY-210U 527 of 721 

### susceptibility to pests or diseases, and is not changed regarding crop emergence, growth development or yield. Additional laborator y and greenhouse-based experiments reached the same conclusion; MON 87708 was unchanged compared to the conventional control for seed germination and symbiotic relati onship parameters. Therefore, production management practices ( e.g. , planting and harvest timing, fertilizer inputs, and pesticide use other than dicamba) are not expected to change with the in troduction of MON 87708. Similarly, because there are no changes in gr owth and development or yield, there is no expectation that the introduction of MON 87708 and its use in development of soybean varieties will significantly alte r the geographical range of co mmercial soybean cultivation. Thus, the introduction of MON 87708 is not an ticipated to facilitate production of soybean in areas where it is not currently grown or have significant impact on total soybean production acres. Therefore, the a pproval in whole and no action alternatives are the same regarding their potential impact on land use.

No action alternative

Under the no action alternative MON 87708 and its progeny would continue to be regulated articles and would not be widely grown. Under this alternative dicamba herbicide would not become integrated into the Roundup Ready soybean system and dicamba use would likely remain similar to today’s use pattern in soybean. As discussed above, full deregulation of MON 87708 is not exp ected to result in changes to land use. Similarly, land use changes would not be expe cted with the no actio n alternative. As discussed, relatively minor fluctuations in s oybean acreage would be e xpected with either alternative, resulting from environmental, agronomic, economic and governmental influences. Therefore, the no action and approval in whol e alternatives are the same regarding their potentia l impact on land use.

J.5.2.2. Water Quality Impacts

Approval in whole alternative

The difference between the approval in whole an d the no action alternative is expected to be integration of MON 87708 into the Roundup Ready soybean system using traditional breeding techniques. For those acres wher e glyphosate resistant weeds may already be present, where application of an herbicide with a different mode-of-action would aid in weed control, or for grower implementation of weed resistant management practices, the cultivation of soybean containing the MON 87708 trait would be an option for growers.

Impacts associated with MON 87708

Water quality could be impacted either directly by MON 87708 via plant material impacts on water resources, or in directly via impacts from the use of dicamba or tillage practices associated with th e planting of MON 87708. Conserva tion tillage, a system that leaves 30% or more of the previous crop re sidue covering the soil when planting another crop has been increasingly employed in co mmercial soybean acres, and helps minimize any impacts of soybean production on water quality by reducing soil erosion.Monsanto Company 10-SY-210U 528 of 721 

### In terms of potential direct impacts on wa ter quality, MON 87708 has been shown to be compositionally, agronomically and phenotypica lly equivalent to conventional soybean and is therefore unlikely to have any signi ficant impact on surface water quality. The DMO protein contained in M ON 87708 is a member of the la rger family of oxygenase 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 DMO protein itself was derived from a common soil bacterium ( Stenotrophomonas maltophilia ).

MON 87708 DMO has been shown to have a high level of substrate specificity, and characterization data provided in Section V of petition #10-SY-210U demonstrate the safety of the DMO protein. Therefore, it is unlikely that the presen ce of DMO protein in MON 87708 will have a signifi cant impact on water quality.

Under full deregulation of MON 87708, there will be a decreased need for farmers employing conventional tillage pr actices in order to manage certain weed situations. There is a potential impact to soil conservati on in those situations where tillage has been employed to manage resistant weeds (CAS T, 2011). Dicamba’s complementary and supplementary postemergence activity to glyphosate will provide improved postemergence weed management options and thus support more sustainable conservation tillage practices because postermergence herbicide options are generally preferred by growers (Fawcett and Tower y, 2002). Tillage causes widespread soil disturbance causing erosion and topsoil loss, im pacting the sedimenta tion and turbidity of

streams. EPA identified sedimentation and turbidity as two of the top 10 causes of impairment to surface water in the U.S.; similarly in 2007, EPA identified sedimentation/siltation as a leading cause of impairment to rivers and streams in particular (U.S. EPA, 2007a; 2009a;). EPA has projected conservation tillage to be “the major soil protection method and candidate best management practice for improving surface water quality” (U.S. EPA, 2002). EPA id entifies conservation tillage as the first of its CORE4 agricultural management practic es for water quality protection (U.S. EPA, 2008a). Therefore, the approval in whole and no action alternatives are not significantly different regarding the impact of the cu ltivation of MON 87708 on water quality.

Impacts from use of dicamba on MON 87708

Under full deregulation, dicamba would be an additional weed management tool for managing hard-to-control and herbicide-re sistant broadleaf weeds found in soybean fields. The use of dicamba on soybean would be expected to increase relative to current and historical levels of use, up to 2.6 times the maximum hist orical annual level in 1994. However, potential impacts associated with any increased use of dicamba from the cultivation of MON 87708 have been adequately assessed by EPA as part of the dicamba Reregistration Eligibility Decision (RED), ther efore it is reasonably foreseeable that EPA will register this specific us e of dicamba under FIFRA. EP A considered potential risks associated with dicamba use, including its degradate DCSA when appropriate, on surface or ground water using scr eening level (high-end expo sure) models to estimate environmental concentrations. The EPA then compared these exposure estimates to appropriate endpoints fro m mammalian, aquatic animal and plant ecotoxicity studies, and

concluded dicamba meets the FIFRA standa rd for no unreasonable adverse effects on human health and the environment (see Section J.3.7.2 and Appendix M.5.2 of theMonsanto Company 10-SY-210U 529 of 721 

### petition for additional detail on the EPA an alysis). The EPA analysis, based on use patterns that exceed the propos ed single and annual maximum use rates for dicamba on MON 87708, does not take into account normal variation in environmental concentrations that can occur, and assumes th at greater than 85% of the water shed is

treated with the herbic ide at the maximum labeled rate on the same day. In addition, the

EPA examined and considered available monito ring data as part the dicamba RED, where concentrations of dicamba in ground and surface water were detected at levels up to 44 μg/L and 1.76 μg/L, respectively. Furt hermore, potential impacts on ground and surface water from dicamba use on MON 87708 will be considered by EPA as part of Monsanto’s pending application to register the use of dicamba on MON 87708, and must

meet the FIFRA standard for no unreasonabl e adverse effects on human health or the environment prior to approval.

It is foreseeable that the frequency of di camba detections in ground and surface water

could increase as a result of the cultivati on of MON 87708, however levels of dicamba in water are not expected to increase above the levels already evaluated and considered by EPA. Existing monitoring data provides additi onal support that wate r resources will not be impacted from any potential increase in dicamba use. Monsanto has compiled publicly available surface and ground water monitoring data from across the United States from 1990 through 2010, including sampling sites in areas wher e soybean and corn are grown (Upper Midwest, see Figure VIII-1 of th e petition) and where dicamba use has historically been most intense (S ee Figure VIII-2 of the petition). Maximum labeled use rates during most of this timeframe (2.8 lb a. e. per acre single maximum and 7.7 lb a.e. per acre annual maximum) were much higher th an presently allowed rates (1.0 lb a.e. per acre single maximum and 2.0 lb a.e. per acre annual maximum) and the rates proposed on

Monsanto’s dicamba label for use on M ON 87708. Therefore, an examination of available surface and groundwater monitoring data in these areas during the mid-1990s

would be indicative of the antic ipated levels of dicamba that may occur from the use on

MON 87708.

An evaluation of the compiled surface water data from 1994 through 1998 for the major soybean areas during the primary dicamba a pplication months of April through July

indicates that detected levels of dicamba (90 th 

### percentile concentration for all samples

where dicamba was detected 26 

### ) were less than 1 μg/L. Monitoring data from April through July were evaluated because these are the months where the majority of dicamba applications are made to soybean (preemerge nce) and corn (pre- and postemergence), and

when surface water concentratio ns from with these applica tions would be expected to peak. The maximum level of dicamba in su rface water during this same timeframe was 9.4 μg/L. Similarly, the evaluation of the groundwater data for major soybean growing areas from 1994 through 1998 indicates that detected levels of dicamba (90 th 

### percentile concentration of all samples where dicamba was detected) were 0.25 μg/L or less. The maximum level of dicamba in groundwater during this same timeframe was 2.2 μg/L.

Considering the available monitoring data for ground and surface water during the period of dicamba’s most intensive use and when app lication rates were significantly higher than

26 EPA uses the 90 th percentile as the relevant high-end endpoint when analyzing water monitoring dataMonsanto Company 10-SY-210U 530 of 721 

### the rates proposed for us on MON 87708, it is re asonable to assume that levels in ground and surface water that may result from the use of dicamba on MON 87708 would be below the levels (high-end exposure modeli ng and monitoring data) considered by the EPA in the dicamba RED, and where EP A concluded would no unreasonable adverse

effects on human health or the environment. Therefore, the a pproval in whole and no action alternatives are similar regarding their potential impact on surface and ground water quality from the use of dicamba on MON 87708.

No action alternative

Surface water may be impacted from soybean production by runoff from soybean fields that carries soil particles and herbicides or other pesticides to streams, rivers, lakes,

wetlands and other water bodies. As discu ssed above, based on exis ting data, the soil component of runoff is a much more important contributor to surface water impacts than is the pesticide component. Similarly, ground water may be impacted from soybean production due to the use of herbicides or other pesticides fo r weed management.

Under full deregulation, growers’ use of dicamba for managing hard-to-control and

herbicide-resistant broa dleaf weeds in soybean fields would be expected to increase. Under the no action alternative, growers woul d need to use other practices for dealing

with hard-to-control and herb icide-resistant broadleaf weed s. These practices would likely consist of some combination of herb icide use and traditional tillage method. However, the specific combination of herbicides used would likely be different than with full deregulation, as dicamba would not be able to be used late preemergence or postemergence with MON 87708. Growers w ould likely use some combination of herbicides currently in use for soybean (d iscussed in Section V III of the petition).

If the no action alternative resulted in increas ed use of conventional tillage practices for weed control, overall adverse surface water im pacts may be greater with the no action

alternative than under full de regulation alternativ e. Tillage cause s widespread soil disturbance. Thus, erosion, topsoil loss and the resulting sedimenta tion and turbidity in streams are likely to increase with increased tillage. Based on the states’ water quality reports to EPA, which EPA makes availabl e through its National A ssessment Database, pesticides in general and herbicides in part icular are a relatively minor contributor to impairment of surface water in the U.S., compared to sedimentation/siltation and turbidity (U.S. EPA, 2008b). Pesticides account ed for less than one percent of reported causes of surface water impairment in all but four of the 17 leading U.S. soybeanproducing states. In those four states, pest icides accounted for 2% to 8% of reported causes of impairment. Of the pesticides that were reported as contributing to impairment among the 17 leading soybean-producing stat es, almost all are highly persistent chemicals that are no longer registered for use in the U.S. Only one currently used herbicide, atrazine, was re ported (U.S. EPA, 2008b).

In summary, based on EPA data, herbicides in general are minor contributors to surface water impairment in the U.S., while sedimentation/siltation and turbidity are major

contributors. The no action alternative, compared to full deregulation of MON 87708, would likely result in a differe nt combination of alternativ e herbicides being used andMonsanto Company 10-SY-210U 531 of 721 

### may result in increased tillage to obtain effec tive weed control. Weed management is a primary reason for tillage and reduced herbicide options due to existing herbicide resistance, in some cases, may increase the need for tillage (CAST, 2011). Increased tillage could contribute to a dverse surface water impacts thro ugh increased runoff of soil particles to surface water bodies. Theref ore, the no action and approval in whole alternatives are not significantly different regarding their impact on water quality from

the cultivation of MON 87708 and associated use of dicamba.

J.5.2.3. Air Quality and Climate Impacts

Approval in whole alternative

The difference between the approval in whole an d the no action alternative is expected to be the integration of MON 87708 into the Roundup Ready soybean system using traditional breeding techniques. For those acres where glyphosate resistant weeds may already be present, where ap plication of an herbicide wi th a different mode-of-action would aid in weed control, or for grower implementation of weed resistant management practices, the cultivation of soybean containing the MON 87708 trait would be an option for growers.

Agricultural activities have the potential to impact air quality. These activities include emissions from farming equipment, burning, ni trous oxide associated with the use of nitrogen fertilizer, and pestic ide applications (Aneja et al., 2009; USDA-NRCS, 2006). Mechanical tillage practices also have th e potential to impact air quality through the suspension of soil particulates in the air (USDA-NRCS, 2005; CTIC, 2011; Baker et al, 2005).

Agricultural practices are not expected to ch ange significantly with the introduction of MON 87708. A discussion of the agricultura l practices associat ed with soybean production in the U.S. is provided in Section VIII of the petition, and includes discussion of cultural, mechanical and herbicide practices for weed management. Deregulation of MON 87708 is expected to facilitate the tre nd toward increased a doption of conservation tillage methods by soybean growers because c onservation tillage (spe cifically no-till) relies on the use of herbicides to control weeds that emerge in a field prior to or after planting the soybean seed into the previous crop stubble, thus avoidi ng disturbance of the soil. MON 87708 would help to maintain ex isting conservation tillage practices and facilitate the adopti on of conservation tilla ge practices by simp lifying weed control options for growers utilizing a non-glyphosate herbicide or where there are glyphosateresistant or hard-to-control broadleaf weeds present. Soybean represents the greatest number of acres of the major field crops ut ilizing conservation ti llage and the highest percentage of total crop acres devoted to conservation tillage pr actices (CTIC, 2007).

Considerable benefits to the physical environment, including those related to air quality,

are obtained from use of conservation till age methods including (CTIC, 2011; USDANRCS, 2005):

 Dramatic reduction in soil erosion from wind and water;Monsanto Company 10-SY-210U 532 of 721 

###  Less herbicide, water, and so il runoff from soils improving the quality of streams and lakes;

 Overall healthier soils;

 Increased carbon sequestration lead ing to reduced greenhouse gases;

 Decreased fuel emissions due to reduced use of tractors to plow fields;

 Reduced nitrogen applications (much of which is made from fossil fuels); and

 Less overall water usage for agricultural purposes.

While approval in whole of MON 87708 may f acilitate some trend towards increasing conservation tillage, it is not expected to significantly impact climate or air quality. Therefore approval in whole and no action al ternatives are not significantly different regarding their impacts on climat e and air quality.

No action alternative

As discussed above, compared with full deregulation of MON 87708, the no action alternative may result in increased tillage, and decreases in cons ervation tillage. EPA reports conservation tillage as an agricultu ral practice that “inc reases carbon storage through enhanced soil sequestration” a nd that “may reduce energy-related CO 2 

### emissions from farm equipment” (U.S. EPA, 2010). When carbon is stored, it is not available to be emitted in the form of carbon dioxide (CO 2 

### ), a greenhouse gas. Thus, the no action alternative may result in increased tillage, wh ich could cause some a dverse, but probably not significant climate and air quality im pacts compared with full deregulation.

Under the no action alternative growers would still likely practice conservation tillage, and in certain situations they would rely on tillage and/or other soybean herbicides. Other herbicide-tolerant soyb ean events have been deregu lated by APHIS or have been submitted to APHIS for deregulation. These events and their companion herbicides may be used to promote conservation tillage practices under the no ac tion alternative. Therefore, the no action and approval in whole alternatives are not significantly different regarding their potentia l impact on air quality.

J.5.2.4. Soil Quality Impacts

Approval in whole alternative

The difference between the approval in whole an d the no action alternative is expected to be integration of MON 87708 into the Roundup Ready soybean system using traditional breeding techniques. For those acres wher e glyphosate resistant weeds may already be present, where application of an herbicide with a different mode-of-action would aid in weed control, or for grower implementation of weed resistant management practices, the cultivation of soybean containing the MON 87708 trait would be an option for growers.Monsanto Company 10-SY-210U 533 of 721 

### Other than changes associated with he rbicide use, MON 87708 will not alter the agronomic practices typically utilized in the cultivation of soybean. MON 87708 has been found to be compositionally, agronomi cally and phenotypically equivalent to conventional soybean. Therefore microbial populations and asso ciated biochemical processes in soil are not exp ected to change with the introduction of MON 87708. The MON 87708-produced protein DMO demonstrates a high level of s ubstrate specificity and is not expected to pers ist in the environment (see S ection V.E in the petition). Studies have shown no impact to the symbi ont interactions of MON 87708 (see Section VII.C.4 of the petition), or to NTOs such as beneficial and pest arthropods when exposed to MON 87708 DMO in the field (see Section VII.C.2.4). Based on these data, the cultivation of MON 87708 is not expected to impact microbial populations and associated biochemical processes.

Multiple herbicides are already used in soybean production. Agricultural fields are purposefully managed to be weed-free resu lting in greater economic benefit to the grower. A discussion of weed management pr actices is provided in Section VIII.F.1 of the petition. In the U.S., 98% of soybean acr eage was treated with an herbicide in 2006 (USDA-NASS, 2007b). Therefore, introduc tion of MON 87708 and treatment with dicamba is unlikely to affect soil quality in commercial soybean production systems differently than those herbicides already used in soybean. Dicamba has been registered by the EPA for use on a wide range of agri cultural uses since 1967 (see Appendix M, Section M.1 of the petition). The EPA evaluated the environmental safety of dicamba and its metabolites as part of the RED (U.S. EPA, 2005b), and concluded that dicamba may accumulate with frequent and intensive use (2.0 and 2.8 lb per acre a.e. single application and 7.7 lb per acre a.e. annually). The EPA manda ted reductions in dicamba use rates as part of dicamba’ s continued registration to e ffect these and other potential impacts (U.S. EPA, 2009b). Based on the reduced application rates (1.0 lb per acre a.e. with a maximum annual rate of 2.0 lb a.e. per acre) dicamba is unlikely accumulate or persist in the environment. In addition, resu lts of standardized tests with dicamba and dicamba formulations indicate no long-term effects on functional processes of soil microorganisms (carbon respiration and nitrog en transformation) at rates proposed for dicamba on MON 87708 (European Commission, 2007a). Based on this analysis, the approval in whole and no action alternatives are not significan tly different regarding their impact on soil quality.

No action alternative

Under the no action alternative agronomic practi ces currently utilized in the cultivation of soybean would not be altered. However, some combination of herbicid es already used in soybean production acres and possibly increased tillage may be used more frequently to control problematic weeds. Under the no-action alternative, an increase in tillage may occur, and would negate many of the benefits of conservation tillage to soil including improvement of soil structure, reduction of soil compaction, conservation of soil moisture, reduction of soil erosion and improvement of so il organic matter content. Overall, the no action and approval in whole al ternatives are similar regarding the potential impacts on soil quality.Monsanto Company 10-SY-210U 534 of 721 

### J.5.3. Potential Impacts to Agricultural Practices

Approval in whole alternative

The difference between the approval in whole an d the no action alternative is expected to be integration of MON 87708 into the Roundup Ready soybean system using traditional breeding techniques. For those acres wher e glyphosate resistant weeds may already be present, where application of an herbicide with a different mode-of-action would aid in weed control, or for grower implementation of weed resistant management practices, the cultivation of soybean containing the MON 87708 trait would be an option for growers.

MON 87708 has been shown to be no differe nt from conventional soybean in its agronomic and ecological characteristics (s ee Sections VII, VIII, IX and X of the petition), and has the same leve ls of tolerance to insects and diseases as conventional soybean. A summary of agronomic practices currently used for soybean production is presented in Section VIII of the petition. Except for weed management relative to dicamba use, the majority of agricultural pr actices, including tillage, insect and disease management, crop rotation practices, irriga tion and volunteer management, will not change under full deregulation.

For weed management under full deregulation growers would have the option of a wider window for treating their soybean crop with dicamba and the number of acres upon which dicamba is used will likely increase un der this alternative. An estimate of the

increase in treated acres can be made using the following assumptions. If dicambatreated acres reach 40% of th e 75 million U.S. soybean acres, approximately 30 million acres of soybean would be treated with di camba. Currently dicamba is used on 20.2 million acres in all crops including soybean (soybean accounts for 0.53 million acres) and has historically been used on up to 36.3 million acres across all uses at its peak (see Table VIII-11 of the petition). The potential us e of dicamba on MON 87708 would result in a

total of 50.2 million acres trea ted with dicamba. Similarly, the total amount of dicamba applied in overall agriculture would also increase. Based upon an upper-end estimation of the anticipated commercial use pattern for dicamba in MON 87708 as described in Section VIII.H, an additional 22 million pounds of dicamba is estimated (high-end) to be added to U.S. soybean fields each season. According to NASS statistics (USDA-NASS, 2007b), approximately 103 million pounds of herbic ides were used on soybean in 2006 (Table VIII-8 of the petition), and recen tly the trend has been towards increasing herbicide use for the management of probl ematic weeds including resistant populations as well as the incorporation of diversifie d weed management practices in soybean growing areas (see Section J.3.3.6 for additional details regarding recent trends). As discussed previously, dicamba will displace in part the use of some existing soybean

herbicides.

Dicamba use presents a relative reduction of ri sk potential in comparison to some of the alternative non-glyphosate herbic ides currently available to soybean growers. The rationale and supporting information for the comp arative alternative an alysis is provided in Appendix L and summarized below:Monsanto Company 10-SY-210U 535 of 721 

### • Dicamba has a more favorable toxicity profile and poses a lower health risk potential to applicators and consumers compared to some alternative herbicides (Appendix L, Table L-25);

• Dicamba has lower toxicity to aquatic animals and plants and poses lower risk potential to aquatic organisms compared to some alternative herbicides (Appendix L, Table L-25);

• Dicamba when used in conjunction w ith MON 87708 integrated into the Roundup Ready soybean system provides growers w ith a more flexible and reliable weed management system (Peterson et al., 2011).

Dicamba tolerance would allow growers to ut ilize an expanded application window of dicamba in their weed management systems, while still allowing alternative herbicides currently registered for use in soybean to be applied. Breeding MON 87708 with glyphosate-tolerant soybean, such as Roundup Ready 2 Yield soybean, would allow for in-season application of gl yphosate and dicamba, thereby im proving the sustainability of weed efficacy for glyphosate, dicamba and other soybean herbicides.

A reasonably foreseeable impact under full deregulation is the delay in the evolution and development of glyphosate- and dicamba-resist ant broadleaf weeds as well as weeds resistant to other soybean he rbicide classes, such as PPO herbicides, in soybean producing areas. This is because growers will likely use dicamba together with glyphosate on the combined dicamba- and glyphosate-tolerant soybean product because of the excellent crop tolerance and compatibility of the two he rbicides. In addition, other herbicides will be recommended and used by growers especially in cases where the grower is managing a weed population already resistant to glyphosate. This will further assist in delaying resistance to dicamba and other herbicides used in the MON87708

system.

Dicamba is an excellent option to delay resistan ce to other herbicides because of its broad activity on broadleaf weeds and low level of weed resistance, specifically on the summer spectrum of weeds known to infest soybean acres. A prominent strategy to delay the evolution and development of herbicide-resist ant weeds is to increase the diversity of weed management practices used in a particular cropping system. D iversified weed management practices use a combination of cultural (e.g., crop rotation), mechanical (e.g., cultivation), and herbicide control practices, 

### including use of herbicides with different modes-of-action (Duke and Powles, 2009). See Section J.3.3.6 of the petition for

additional details on diversified weed management practices. Thus, MON 87708 integrated into the Roundup Ready soybean sy stem provides the opportunity to increase the diversity of in-crop herb icide control options for grow ers and, in turn, supports the long term sustainability of the Roundup Rea dy soybean system w ith its established

benefits.

In summary, no significant changes in common agricultural practices are anticipated from

to the cultivation of MON 87708 and the associated use of di camba. Shifts in herbicide use will occur, with dicamba displacing some of the alternative herbicides. A decrease inMonsanto Company 10-SY-210U 536 of 721 

### tillage practices may occur in some circum stances, and a delay in the development of glyphosate- and dicamba-resistant broadleaf weeds may be possible. Overall, the approval in whole and no acti on alternatives are similar regarding their impact on soybean agricultural practices.

No action alternative

Under the no action alternative MON 87708 and its progeny would continue to be regulated articles and would not be widely grown. Under this alternative dicamba herbicide would not become integrated into the Roundup Ready soybean system and dicamba use would likely remain similar to today’s use pattern in soybean. Growers would continue to use the Roundup Ready s oybean system for broad spectrum weed control, other registered al ternative herbicides alone or in combination with other herbicide-tolerant soybean for targeted hard-to-control weeds, and/or incorporate tillage into their practices.

Integration of MON 87708 into the Roundup Ready soybean syst em has the potential to delay or prevent development of dicamba- and glyphosate-resistant weeds (see Appendix K of petition for additional detail). For soyb ean, the use of dicamba in conjunction with glyphosate provides growers with an herbicide sy stem with two different modes of action. Thus, it is foreseeable that under the no action alternative, the inability to integrate MON 87708 into the Roundup Ready soybean system could increase the potential for glyphosate resistant weed populations to evolve and spread in soybean producing areas. In addition, the potential for resistance to evol ve and spread for othe r soybean herbicides could also increase in these areas where grow ers do not use multiple modes-of-action.

Also under the no actio n alternative, increased use of other non-glyphosa te alternative herbicides, some with higher risk potenti al for human health and environmental characteristics compared to dicamba, a nd reduced flexibility for the grower ( e.g. , restricted plant-back intervals, rotational cr op restrictions) would be expected. A number of weeds commonly found in th e Midwestern and southern por tions of the U.S. already display resistance to many of these alternativ e herbicides (Tables J- 3 and J-4), while only four broadleaf weed species have been confir med to be resistant to dicamba in the U.S., even though dicamba has been widely in use for over 40 years. Increasing the number of weed management options available to s oybean growers, includ ing other herbicidetolerant traits pending deregulation (see Table J-5), is an important element to delay and prevent further development of resistant weed populations. Given these observations, the no action alternative and corresponding lack of effective alternative modes of action may

lead to an increase in weed resistant populations for thes e alternative herbicides.

Herbicides are a critical element of c onservation tillage pract ices. Since weed management is a primary reason for tillage, he rbicides are the primary tool to replace tillage and thus are critical to the sustaina bility of conservation tillage practices. Under

the no action alternative increased use of trad itional tillage methods for the control of problematic weeds may occur in some situations and result in the potential loss of many of the benefits of conservation tillage.Monsanto Company 10-SY-210U 537 of 721 

### Overall herbicides are widely used in production of a soybean crop. Information presented in Section VIII.F.1 of the petition highlights the dynamics of herbicide use in soybean. Considerable shifts in use patterns of herbicid es occur over time based upon many factors including availabi lity of new herbicides and herbicide-tolerant crops, herbicide efficacy, convenience and economics. Yet, herbicides in general remain a critical element of soybean production.

From this analysis it is concluded that ove rall use of herbicides in soybean production is unlikely to be significantly different betw een the approval in whole and the no action alternatives although shifts in use patterns of herbicides may occur based on the decision to deregulate MON 87708. Additionally, an in crease in tillage ma y occur in some

circumstances under the no action alternative. Therefore, the no action and approval in whole alternatives are similar regarding potential impacts on soybean agricultural practices.

J.5.3.1. Potential Impacts on Weed Resistance

Approval in whole alternative

The difference between the approval in whole an d the no action alternative is expected to be integration of MON 87708 into the Roundup Ready soybean system using traditional breeding techniques. For those acres wher e glyphosate resistant weeds may already be present, where application of an herbicide with a different mode-of-action would aid in weed control, or for grower implementation of weed resistant management practices, the cultivation of soybean containing the MON 87708 trait would be an option for growers.

A potential impact with the increased use of any herbicide is the development of herbicide-resistant weeds. A comprehens ive discussion concerning the potential development of dicamba-resistant weeds ma y be found in Appendix K of the petition. The potential for the development of weed resi stance to an herbicide is a function of the duration and frequency of herbicide use in the absence of other methods of weed control. Initially, resistant weed popula tions are localized at the field level, however resistant populations can become more widespread and effect larger areas as a result of the development of additional resistant populations occurring from selection pressure, or from gene flow and/or seed movement from existing resistant popula tions combined with inadequate management practices. In e ither case, the use of diversified weed management practices are essential to effectively manage weed resistance.

Dicamba has been used for over 40 years and some resistant weeds have developed due to use of the herbicide and the selection pr essure applied with continued use of this

herbicide. The introduction of MON 87708 will likely result in limited additional selection pressure for the development of di camba-resistant weeds for several reasons: 1) dicamba, as a broadleaf herbicide, would primarily be used in combination with glyphosate on MON 87708 integrated into th e Roundup Ready soybean system because

dicamba does not control narrowleaf weeds a nd broad spectrum control of broadleaf and narrowleaf weeds is the objective of all weed control systems; 2) the use of glyphosate plus dicamba would provide multiple mode s-of-action on key broadleaf weeds whichMonsanto Company 10-SY-210U 538 of 721 

### would diminish the chance for selection of di camba-resistant broadleaf weeds; 3) in cases where glyphosate plus dicamba will be applie d to soybean fields with a known presence of glyphosate-resistant broadl eaf weeds, a third herbicid e mode of action will be recommended to growers that also has activit y on the glyphosate-resis tant broadleaf weed thereby providing two effective modes-of-ac tion to control the gl yphosate-resistant weed; and 4) the proposed dicamba herbicid e label for MON 87708, existing glyphosate herbicide labels and separate Monsanto weed management recommendations (e.g.,

Monsanto’s annual TUG 27 

### and publically available websites 28 

### ) will specify the effective rate and timing of dicamba and glyphosate applications for optimal weed control, thereby reducing selection pressure for di camba as well as glyphosate.

Furthermore, in the unlikely case that broadl eaf weeds were to evolve or develop with resistance to dicamba, existi ng cultivation and alternative he rbicide tools (see Section VIII.F.1 for description of alternative herb icides) would remain potential options to provide effective control. Additionally, as discussed above in Section J.5.3, it is reasonably foreseeable that under full deregula tion a delay in development and spread of glyphosate- and dicamba-resistant broadleaf weed s, as well as weeds resistant to other soybean herbicide classes such as PPO herbicides, will occur due to the likely use of dicamba and glyphosate on fields planted w ith the combined dicamba and glyphosate tolerance product.

In summary, there is a low probability that additional dicamba resist ant weed species or populations will evolve or develop as a resu lt of the cultivation of MON 87708 and the use of dicamba and glyphosate in the weed management systems, and a delay in the development of glyphosate- and dicamba-resist ant broadleaf weeds may be possible. Overall, the approval in whole and no action alternatives are similar regarding their impact on herbicide weed resistance.

No action alternative

Under the no action alternative MON 87708 and its progeny would continue to be regulated articles and would not be widely grown. Under this alternative dicamba herbicide would not become integrated into the Roundup Ready soybean system and dicamba use would likely remain similar to today’s use pattern in soybean. Growers would continue to use the Roundup Ready s oybean system for broad spectrum weed control, other registered al ternative herbicides alone or in combination with other herbicide-tolerant soybean, and/or incorporate tillage into their practices. There is a potential for glyphosate-resistant weed populati ons to increase and spread in soybean cultivation areas, as well as the potential fo r the development and spread of resistant populations to other altern ative soybean herbicides.

Integration of MON 87708 into the Roundup Ready soybean syst em has the potential to delay or prevent development of dicamba- and glyphosate-resistant weeds in soybean cultivation areas (Appendix K of petition). For soybean, the use of dicamba in

27 http://www.monsanto.com/SiteCollectionDocuments/Technology-Use-Guide.pdf

28 http://www.monsanto.com/weedmanagement/Pages/default.aspxMonsanto Company 10-SY-210U 539 of 721 

### conjunction with glyphosate prov ides growers with an efficient, effective and flexible herbicide system with two different herbicid e modes-of-action with overlapping activity on many broadleaf weeds. Thus, it is foreseeable, under the no action alternative and the inability to integrate MON 87708 into the Roundup Ready soybean system, that there is an increased potential for glyphosate resist ant weed populations in soybean growing areas to evolve and spread, as well as an incr eased potential for resistance to evolve and spread for other soybean herbicides wher e growers do not effectively use multiple modes-of-action. However, exis ting alternative soybean herb icides will continue to be available. Therefore, the no action and ap proval in whole alternatives are similar regarding potential impa cts on weed resistance.

J.5.4. Potential Impact to Commercial Soybean Production

Approval in whole alternative

Soybean is a globally traded commodity with the U.S. be ing the top global producer (Soyatech, 2010). An overview of U.S. soyb ean production is provided in Section VIII.B.1 of the petition. Biotechnology-derive d crops are subject to regulation in many countries. In order to support free trade in soybean, Monsanto will seek regulatory approval for MON 87708 and stack ed products (i.e., products co mbined using traditional breeding techniques) with other biotechnology -derived soybean, where required, in all key soybean import countries with a functioning regulatory system to support the flow of

international trade. As desc ribed in Section VIII.L of th e petition, Monsanto adheres to the BIO Product Launch Policy 30 

### including: 1) conducti ng a market and trade assessment, 2) securing regulatory approvals in key export countr ies prior to full commercial launch, 3) following generally accep ted 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 compa ny’s product launch stewardship policies. These actions protect against adverse impacts to trade of soybean due to the introduction of a new biotechnology-derived soybean.

The difference between the approval in whole an d the no action alternative is expected to be integration of MON 87708 into the Roundup Ready soybean system using traditional breeding techniques. For those acres wher e glyphosate resistant weeds may already be present or where application of an herbicide with a different mode of action would aid in weed control or the implementation of w eed resistant management practices, the cultivation of soybean containing the MON 87708 trait would be an option for growers.

Soybean is primarily a self-pollinated crop wi th minimal gene movement (see Section IX of the petition). Due to the biology of soybean flowers which results in low cross pollination potential, pollen movement between soybean fields is minimal. Thus the

introduction of MON 87708 is not expected to e ffect commodity s oybean production, due to low potential gene movement to neighbouring soybean crops. In 2009, 91% of the

30 BIO’s Product Launch guidelines can be found at: http://www.excellencethroughstewar dship.org/LinkClick.aspx?fileticket=ppgyTABguQs%3d&tabid=84.Monsanto Company 10-SY-210U 540 of 721 

### U.S. soybean acres were planted to biot echnology-derived herbicid e-tolerant soybean (USDA-NASS, 2009a). Thus, growers are accu stomed to the presence of biotechnologyderived soybean in proximity to production fiel ds and have developed practices to allow for production of a crop to meet customer expe ctations. Therefore, the approval in whole

and no action alternatives are similar regarding potential impacts on commercial soybean production.

No action alternative

Under the no action alternative, MON 87708 wo uld remain a regulated article and would not be available to growers. Growers c ould continue to use the Roundup Ready soybean system for broad spectrum weed control. Therefore, the no action and approval in whole alternatives are not different in their imp act to commercial soybean production, because the majority of soybean grown in the U.S. is already biotechnology-derived and due to company stewardship policies accompa nying the introduction of new biotechnologyderived products, as described above. T echnology providers main tain a dialogue with stakeholders to communicate global approval status for biotechnology-derived soybean to avoid potential market disr uption. Commodity and spec ialty soybean growers and handlers are accustomed to a diversity of soybean types in the marketplace, including soybean with modified oil pr ofiles, food grade, clear hilu m, and others. They have demonstrated an ability to provide soybean that meet their customers’ expectations. MON 87708 represents another biotechnology -derived soybean in the marketplace adding to the diversity of soybean available to growers and the market. Therefore the no action and approval in whole alte rnatives are expected to be the same regarding potential impacts on commercial soybean production.

J.5.5. Potential Impact to Certified Seed Production

Approval in whole alternative

Certified seed production is a carefully mana ged process (see Secti on VIII.B.2 of petition for additional detail). MON 87708 is not expe cted to impact certified seed production practices or production of specialty soybean seed for reasons described in this section.

If MON 87708 is deregulated, seed producti on would occur within production systems already developed by seed producers for ce rtified soybean seed. MON 87708 has been thoroughly characterized and (w ith the exception of its tolerance to dicamba) is not agronomically or phenotypica lly different from commerci al soybean. The difference between the approval in whole and the no action alternative is expected to be integration of MON 87708 into the Roundup Ready soybean system using traditional breeding techniques. For those acres where glyphosate resistant weeds may already be present or where application of an herbicide with a di fferent mode of action would aid in weed control or the implementation of weed resist ant management practices, the cultivation of soybean containing the MON 87708 trait would be an option fo r growers. However, the implementation of management practices to avoid pollen from a biotechnology-derived soybean in organic, specia lty or conventiona l soybean seed or commodity seed production operations is dictated by the na ture of soybean pollination (see SectionMonsanto Company 10-SY-210U 541 of 721 

### VIII.B.2), and MON 87708 has been shown in fi eld testing to not differ from commercial soybean varieties regarding pollen characterist ics. Soybean is a highly self-pollinated species that exhibits very low levels of outcrossing. Numerous evaluations on soybean cross-pollination have been conducted, and the published results are summarized in Table IX-1 of the petition. Under typical soybean planting and cultivat ion conditions, crosspollination among adjacent plants in a row or among plants in adjacent rows ranged from 0 to 6.3%. In experiments where supplemental pollinators (usually bees) were added to the experimental area, cross-pollination ra nged from 0.5 to 7.74% in adjacent soybeanplants or adjacent rows (Abrams et al., 1978; Chiang and Kiang, 1987). However, crosspollination even at the low levels observed in these controlled trials does not occur over long distances. Cross-pollination rates decr ease to less than 1.5% beyond one meter from the pollen source, and rapidly decrease with greater distances from the source. The following cross-pollination rates at extended di stances have been reported: 0.02% at 8.2 m of separation (Caviness, 1966), 0.05% at 5.4 m (Ray et al., 2003), and 0% at 6.5 m (Abud et al., 2003).

The low potential for cross pollination in soybean is recognized in certified seed regulations for foundation seed in the U.S., which require no measured isolation between different soybean cultivars in the field as long as there is adequate separation between the fields to prevent mechanical mixing (US DA-APHIS, 2006). Hence, certified soybean seed producers can and have eff ectively implemented practices ( e.g. , isolation distances during the growing season, equipment cleaning during harvest, and post-harvest separation of harvested seed) that allow them to maintain commercially acceptable levels of varietal purity. Because MON 87708 ha s been shown to be no different from conventional soybean relative to pollen mo rphology and viability, the cultivation of MON 87708 will not impact the ability to im plement production practices required for the production of certified seed. Therefor e, the approval in whole and no action alternatives are the same regarding the potential impact on certified seed production practices.

No action alternative

Under the no action alternative, MON 87708 w ould not be propagated to any extent by seed producers because there would be no commercial demand for seed containing MON 87708. Seed production practices would be the same as those described above for the deregulation in whole alternative and in Section VIII.B.2 of the petition. Seed producers already produce numerous varieties of soybean, the vast majority of which include a biotechnology-derived trait. Therefore, the no action and approval in whole

alternatives are the same re garding the potential impacts on certified seed production.

J.5.6. Potential Impacts to Organic Soybean Production

Approval in whole alternative

Organic soybean production is a carefully ma naged and regulated process. MON 87708 and biotechnology-derived soybean in gene ral, including those currently grown commercially, are not allowed for use in or ganic production systems because they wereMonsanto Company 10-SY-210U 542 of 721 

### developed through the use of excluded met hods as defined by the National Organic Production (NOP) program standards (7 CFR § 205.2). With the exception of its tolerance to dicamba, MON 87708 has been show n to be no different from conventional soybean in its agronomic and ecological char acteristics including ch aracteristics of the pollen produced by MON 87708. Thus, MON 87708 is expected to be no different from other soybean in its ability to cross pollin ate with other soybean and, therefore, no additional means beyond those already used to produce biotechnology-derived and organic soybean will be needed if MON 87708 were grown commercially.

Similar to the exclusion of biotechnology-de rived products in orga nic production, the use of synthetic herbicides, such as dicamba, are also excluded under the NOP. Dicamba is presently registered for early preemergence (early pre-plant) and late postemergence (pre-harvest) applications in soybean, and in other crops (corn, sorghum and wheat) commonly grown in rotation with soybean. It is likely that dicamba herbicide will be applied to MON 87708. As mentioned previous ly, herbicides are used extensively for production of soybean and the potential in creased use of dicamba, along with itsconcurrent replacement of some alternative herbicides used to control glyphosate’s hardto-control and resistan t weeds, is minimal considering the amount of herbicides (103M lbs and trending higher, see Table VIII-8 and discussion in J.5.3 of the petition) currently applied in commodity soybean production each year. For these reasons, production systems and practices in place now (discussed below) are sufficient to mitigate any impacts from the introduction of M ON 87708 to organic soybean production.

Currently, biotechnology-derived herbicide-tolerant soybean is planted on over 90% of the soybean acreage (USDA-NASS, 2009a). Despite the high adoption rates of biotechnology-derived soybean by grower s, organic and conventional soybean production remains an option for growers w ho choose to produce soybean using these production practices. The decisi on to grow organic, conve ntional, or biotechnologyderived soybean is typically an economi c one based on market dynamics. Organic soybean producers and those growing c onventional soybean for non-biotechnology markets typically receive a market premiu m offsetting the addi tional production and record-keeping costs. While the widesp read demand for Roundup Ready soybean has

reduced the number of conventi onal soybean varietie s that seed compan ies choose to sell, conventional and organically produced soyb ean seed is currently available from numerous seed suppliers (Table J-6). Addi tional information on orga nic seed sources is provided though the U.S. Agricu ltural Marketing Serv ice (AMS). Thus, growers have a choice in the soybean they plant, and this is not expected to change with the introduction

of MON 87708.

Production systems designed prior to the intr oduction of MON 87708 or even prior to the introduction of biotechnology-derived soybean have allowed for production of soybean to

meet varied customer demands. In addition to the market segments that produce organic or conventional soybean, distinct identity-preserv ed specialty soybean with such traits as clear hilum or high protein have also been grown and successfully marketed for specific food uses in domestic and export markets fo r many years (Cui et al., 2004). The NOP requires organic soybean producers utilize prod uction practices designe d to specifically avoid the presence of soybean products using conventional herbicide or other pesticideMonsanto Company 10-SY-210U 543 of 721 

### treatments, as well as avoi ding the use of biotechnology-d erived crops. These well established practices to avoi d the use of “excluded methods ” will continue with the commercial introduction of MON 87708 integr ated into the Roundup Ready system. They include isolation zones, use of buffe r rows surrounding the organic crop, adjusted planting dates and varietal selection. 31 

### The implementation of management practices to avoid pollen from a biotechnology-derived crop in organic or conventional soybean production operations is facil itated by the nature of soyb ean pollination. As noted previously in the petition, soybean is a highly self-pollinated species and exhibits a very low level of outcrossing. Hence, organic or conventional soybean producers can and have effectively implemented practices ( e.g. , isolation during the growing season, equipment cleaning during harvest, and post-ha rvest separation of harvested seed) that allow them to avoid the presence of biotec hnology-derived soybean and maintain organic or conventional production status. 32



### The difference between the approval in whole an d the no action alternative is expected to be integration of MON 87708 into the Roundup Ready soybean system using traditional breeding techniques. There is potential fo r cultivation of soybean containing the MON 87708 trait to provide weed contro l solutions for problematic weeds and proactively manage and prevent the developmen t of herbicide resistant weeds. However, the presence of a biotechnology-derived pr oduct like MON 87708 is unlikely in instances where producers utilize production practices designed to avoid biotechnology-derived products. Although the USDA’s National Organic Standards pr ohibit the use of excluded methods, the presence of products of an excluded method alone does not necessarily constitute a violation of the Standard or a loss of organic certification (USDA-AMS, 2011). While the NOP does not set specific th resholds for the allowable presence of products of excluded methods, there may be some specifications in buyer allowances that permit between 0.1 to 5% biotechnol ogy-derived soybean in organic soybean. 33


### Similarly, international regulatory authoritie s have set allowable tolerances for the presence of biotechnology-derived material in conventional produc ts to support food labeling and traceability laws. These tolerances allow from 0.9% (European Union) up to 5% (Japan) of the food to be biotechno logy-derived in products considered

“conventional.” Levels above the thre shold may trigger special labeling.

Given that allowances for minor amounts of biotechnology derived ma terial are allowed in soybean exported to key markets and that systems are in place for production of soybean that meet buyer expectations a nd the prevalence of biotechnology-derived soybean already on the market, the introducti on of a new biotechnology-derived soybean product is unlikely to have an effect on or ganic soybean production. This conclusion is supported by USDA-ERS (2010) data that show the organic soybean market share was 0.17% in 1995, the year prior to the intr oduction of Roundup Ready soybean, and was at 0.2% in 2008; indicating availability of biotechnology-derived soybean has not significantly impacted the organic soybean i ndustry. Therefore, the approval in whole

31 Information on isolation methods can be found at: www.attra.ncat.org [Accessed on June 2, 2010].

32 Source is website: http://attra.ncat.org/attra- pub/PDF/cropsfarmplan.pdf [Accessed on June 2, 2010].

33 Source is website: http://attra.ncat.org/attra-pub/P DF/marketingorganicgrains.pdf [Accessed on May 27, 2010].Monsanto Company 10-SY-210U 544 of 721 

### and no action alternatives are the same rega rding the potential impact on organic soybean production.

No action alternative

The majority of soybean is currently pr oduced using biotechnology-derived soybean. Growers have come to rely on biotechnology- derived soybean and th eir benefits. Given that several biotechnology-derived soybean have previously been deregulated by USDA

and other events are under consideration for deregulatio n, the use of biotechnologyderived soybean is expected to remain relati vely constant for the foreseeable future. Similarly, herbicide use is also expected to remain a key agronomic practice in production of commodity soybean. Thus, organi c growers will continue to manage their production fields to avoid excluded methods an d drift of pesticides under the no action alternative, and growers would continue to utilize the same practices they use now to produce their organic crop. Thus, the no action and approval in whole alternatives are the same regarding the potential imp act to organic soybean production.

Table J-6. Organic and Conventional Soybean Seed Sources

Organic Soybean Seed Sources 1 

### : Conventional Soybean Seed Sources Albert Lea Seed House AgVenture Seeds (modified oil)

Blue River Hybrids Campbell Seed (modified oil)

Golden Grains Becks Hybrids (food grade)

Great Harvest Organics Monsanto (Asgrow)

Greis Seed Farm Schillinger Seed

Lancaster Ag Products Pioneer

Lawler Farm Center Soy Genetics

Prairie Gold Seeds Stewart Seed (modified oil)

Superior Organic Grains, Ltd Stine Seed

Walter Seed and Honey Co Syngenta - multiple brands

Terral Seed

Various State Crop Improvement

Or g anizations

1 Source is: 

### www.organicgrains.ncsu.edu

.

J.5.7. Potential for Adjacent Agricultural Crop and Non-agricultural Impacts

Approval in whole alternative

Soybean ( Glycine max ) does not grow in the wild in North America (Hymowitz, 1987). Soybean does not grow and persist in unmanage d habitats and would not be expected to invade and/or persist in the na tural environment, including str eams, lakes, oceans or other aquatic environments. With the exceptio n of the production of MON 87708 DMO thatMonsanto Company 10-SY-210U 545 of 721 

### confers tolerance to dicamba, MON 87708 is similar to other commercial soybean currently grown in the U.S. It is expected that under the approval in whole alternative MON 87708 will be integrated into the Roundup Ready soybean system using traditional breeding techniques. For those acres wher e glyphosate resistant weeds may already be present or where application of an herbicide with a different mode of action would aid in weed control or the implementation of w eed resistant management practices, the cultivation of soybean containing the MON 87708 trait would be an option for growers. MON 87708 displays no altered plant pest characteristics compared to conventional soybean and is no more susceptible to insects or diseases that commonly infest soybean. On the basis of the information presente d in petition #10-SY-210U on phenotypic and agronomic characteristics of MON 87708, an d based upon the biology of soybean, MON 87708 is unlikely to effect adjacent vege tation. Therefore, the approval in whole and no action alternatives are the same regarding potential impacts on adjacent agricultural crops and non-agricultural areas.

Herbicides are extensively used in U.S. soybean production, a nd their historic and current

uses are described in Secti on VIII.F.1 of the petition. Herbicide drift and offsite movement are regulated by the U.S. EP A and actively managed by farmers and applicators specially trained to use such produc ts consistent with product labels and other state or local restrictions. Depending upon th e herbicide employed, relevant factors in managing the potential for spray drift include the selectivity and sensitivity of the herbicide, local weather conditions at the time of application (wind, temperature, humidity, inversion potential) , droplet size distribution, a pplication volume, boom height (height of the applic ation equipment above the crop canopy ), sprayer speed, and distance from the edge of the application area (SDT F, 1997; Felsot et al ., 2010). A variety of measures can be employed to control the pote ntial for spray drift and offsite movement, including nozzle selection and application techniques and restrictions.

The approval in whole alternative would result in an increase in dicamba use in soybean compared to its current use. The U.S. EPA considers possible effects from offsite movement as part of the pe sticide registration process. APHIS’s authority under the Plant Protection Act, on the other hand, does no t authorize it to spec ify conditions or in any way regulate the use of herbicides. As a result, before any dicamba formulation could be employed over MON 87708 , EPA’s approval of a FIFRA label for that specific use would be required. Such a label would address not only applic ation rates, but as appropriate, could also include other measur es to address the potential for offsite movement. EPA considers possible effects from offsite movement as part of the pesticide registration process. Specifically, in order to approve the use of a pesticide (herbicide) under FIFRA, EPA must conclude that no unrea sonable adverse effects on non-target vegetation will result from offsite movement when the pesticide is used according to the product label, and when herb icides are applied ac cording to the label requirements, offsite impacts can be avoided. EPA employed this analysis of offsite impacts in the dicamba RED, and concluded th at no specific additional drift mitigations were needed to support the continued registra tion of all dicamba uses (U.S. EPA, 2009b). Since the proposed use pattern for dicamba on MON 87708 is consistent with use patterns evaluated in the dicamba RED, it woul d be reasonable to c onclude that dicamba use on MON 87708 meets existing FIFRA standards re lated to drift and o ffsite movement.Monsanto Company 10-SY-210U 546 of 721 

### The use of dicamba on MON 87708 in accord ance with the FIFRA approved label does not pose any greater risk to non-target vegeta tion over existing dicamba agricultural uses approved by EPA. Nevertheless, Monsanto ha s already taken additi onal steps to manage dicamba offsite movement even though such steps are not required by EPA as stated in the RED. In the pending application to EP A, Monsanto requested the use of dicamba on MON 87708 on the low volatility DGA salt formulation (U.S. EPA Reg. No. 524-582) and limited dicamba application to ground application equipment as additional stewardship measures. Monsanto plans to further address the use of dicamba on MON 87708 with US EPA to evaluate whether any additional measures may be appropriate to further address poten tial drift and offsite movement.

Monsanto will also implement a robust stew ardship program to reinforce the EPA label requirements, including a strong emphasis on gr ower and applicator training by working

with American Association of Pesticid e Safety Educators (AAPSE) and other stakeholders in applicator training to further facilitate pr oper use of dicamba. Furthermore, as part of Monsanto’s stewardship program, Monsanto will encourage growers and applicators to consult with availa ble sensitive crop regist ries prior to making dicamba applications to MON 87708. Many stat e lead agriculture ag encies (IA, IL, IN, KS, MI, MN, MO, NE, OK, WI) have devel oped tools and resources to assist the applicator in the location of sensitive area s, such as vegetable or organic production fields, in an effort to minimize commercial impacts associated w ith herbicide offsite

movement.

Herbicides are widely used now in s oybean production, and gr owers and commercial herbicide applicators have over 40 years expe rience in making dicamba applications in numerous crops including preemergence applications on conventional soybean.

Alternative herbicides will con tinue to be used, as needed, for weed management or to implement weed resistance management practic es, however it is anticipated that dicamba will displace in part the use of some alternative herbicides. EPA regulates the use of

herbicides and has concluded that dicamba o ffsite movement from uses consistent with those proposed for MON 87708 does not pose unr easonable adverse effects to non-target vegetation. Furthermore, as indicated above, when herbicides are applied in accordance with the FIFRA label application use in structions, potential impacts on adjacent agricultural crops and non-agricult ural plants can be avoided. Thus, potential impacts to these adjacent areas due to deregulation of MON 87708 and use of dicamba are similar when compared to the no action alternative (discussed below). Therefore, the approval in

whole and no action alternatives are simila r regarding potential impacts on adjacent agricultural and non-ag ricultural areas.

No action alternative

Under the no action alternative, MON 87708 woul d not be widely available and growers would continue to use other practices for c ontrol of weeds in soybean fields. These practices would likely consist of some comb ination of herbicide use and tillage. However, the combination of herbicides used would likely be different than with full deregulation, as dicamba would only have a limited use pattern on soybean. Growers would likely use some combin ation of the herbicides cu rrently in use for soybeanMonsanto Company 10-SY-210U 547 of 721 

### (discussed in Section VIII.F.1 of the petition) . It is reasonably foreseeable that new herbicides will be invented, or that existing herbicides will be adopted for use on soybean

or herbicides associat ed with soybean events under cons ideration for deregulation may be used ( e.g. , other auxin type herbicides like 2,4- D) (Table J-5). Offsite impacts to adjacent plants from these alternative herbicides would vary, depending on active ingredients, weather conditions, formulations, applicatio n methods and other factors. Because herbicides are currently used extensively in soybean production and will be for the foreseeable future, it is lik ely that the effect s to adjacent crops and non agricultural plants would be similar under the no acti on and approval in w hole alternatives.

J.5.8. Potential Impacts to Raw or Pro cessed Agricultural Commodities

Approval in whole alternative

It is expected that under the approval in whole alternative MON 87708 will be integrated into the Roundup Ready soybean system using traditional breeding techniques. For those acres where glyphosate resistant weeds may already be present or where application of an

herbicide with a different mode of ac tion would aid in weed control or the implementation of weed resistant manageme nt practices, the cult ivation of soybean containing the MON 87708 trait would be an option for growers.

Within petition #10-SY-210U, extensive data have been presented relating to plant growth parameters, disease susceptibility, insect susceptibility, and forage and seed composition of MON 87708 compared to conventi onal soybean. These da ta indicate that there are no biologically relevant diffe rences between MON 87708 and conventional soybean, except for its toleran ce to dicamba. Biotechnology-d erived crops used for food or feed undergo a voluntary f ood and feed consultation pro cess with the FDA prior to release on the market. Monsanto will complete this consultation process with FDA prior

to commercial introduction of MON 87708.

Soybean compositional data on MON 87708 and conventional soybean are presented in Section VI of the petition. Information from this evaluation shows that soybean produced from MON 87708 is of comparable quality to soybean commercia lly produced in the U.S. for commodity markets. Dicamba residu e levels in soybean seed harvested from MON 87708 treated with dicamba (1.0 lb a.e. per acre preemergent followed by two, sequential 0.5 lb a.e. per acre postemergent applications) at mo re than twice the anticipated commercial in-crop application rate were low, less than 0.1 ppm, and well below the established 10 ppm pesticide residue tolerance 34 

### supporting dicamba use on commercial soybean (see Appendix M, Sectio n M.4.1.1 of the petition). Monsanto is also petitioning the agency for the establis hment of new tolerances on forage (45 ppm) and hay (70 ppm).

Due the established safety of dicamba and a llowable residues in food and feed, there is no evidence that the deregulation of MON 87708 wo uld cause any impacts on either raw or

34 EPA established tolerances for dicamba can be found at 40 CFR § 180.227. Dicamba residue is defined as dicamba, DCSA and 5-hydroxy dicamba. Analysis of MON 87708 included quantification of these defined dicamba residues, in addition to the minor metabolite DCGA.Monsanto Company 10-SY-210U 548 of 721 

### processed soybean commodities resulting fr om the deregulation of MON 87708. EPA will evaluate any potential risks associated with the pending application for the new use

of dicamba on MON 87708 and the establishm ent of new feed tolerances on soybean forage and hay as a part thei r review, and will conclude a reasonable certainty of no harm to human health when it approve s the pending new use application.

No action alternative

MON 87708 would not be widely available or grown commercially under this alternative. Under this alternative, other herbicid es and other biotec hnology-derived soybean products or traditional tillage practices w ould be used instead of MON 87708 with its

associated dicamba treatment. Therefor e, the no action a nd approval in whole alternatives are similar regarding their impacts to raw and processed agricultural

commodities.

J. 5.9. Potential Impacts to Human Health and Safety

It is expected that under the approval in whole alternative MON 87708 will be integrated into the Roundup Ready soybean system using traditional breeding techniques. For those acres where glyphosate resistant weeds may already be present or where application of an

herbicide with a different mode of ac tion would aid in weed control or the implementation of weed resistant manageme nt practices, the cult ivation of soybean containing the MON 87708 trait would be an option for growers.

Prior to the introduction of a biotechnology- derived crop product into the marketplace,

Monsanto conducts tests to assu re that the products are safe for their intended use and are appropriately labeled. For MON 87708, impact s on human health are considered for exposure to the biotechnology-derived s oybean (MON 87708) and the associated dicamba herbicide. As mentioned previous ly, biotechnology-derived crops for food and feed use undergo a voluntary consultation proc ess with the FDA prio r to release onto the market. Although a voluntary process, Monsanto routinely completes a consultation with the FDA prior to placing a new biotechnology- derived crop product on the market. The consultation process with FDA on M ON 87708 (BNF No. 125) was completed on October 11, 2011 (see Section I.C.I of the petition).

Herbicide use and resulting residues associat ed with herbicide-to lerant crops improved through biotechnology are regulated under the Federal Food, Drug, and Cosmetic Act (FFDCA) by the EPA. Under the FFDCA (21 USC 301 et seq.), pesticide residues in or on raw agricultural commodities or processed f oods are considered to be safe after a tolerance or exemption from tolerance has been established. The FDA enforces the tolerances set by the EPA. Currently, a pestic ide residue tolerance ex ists for residues of dicamba on soybean seed at 10 ppm (40 CFR § 180.227), which is based on the approved late season postemergence (prior to harvest) use of dicamba on commercial soybean. As previously mentioned, Monsanto has submitted an application to EPA to register a new use pattern for dicamba on MON 87708 that w ill allow for preemergence and up to two postemergence in-crop applica tions of dicamba. Residue data generated on MON 87708 to support this new dicamba use pattern using maximum label rates, show low levels ofMonsanto Company 10-SY-210U 549 of 721 

### dicamba residues on soybean seed (< 0.1 ppm dicamba a.e.; see Appendix M, Section M.4.1.1 of the petition) and confirm the curren t soybean seed tolerance is sufficient to support the new use on MON 87708. Monsanto ha s also petitioned the EPA to establish new tolerances for dicamba on soybean fora ge (45 ppm) and hay (70 ppm) to allow soybean commodities to be fed to or grazed by livestock. Existing dicamba tolerances for animal foodstuffs ( e.g. , meat, milk) are sufficient to address potential incremental dietary exposure to livestock from dicam ba-treated MON 87708 forage and hay, as

discussed in Section J.5.9.2.

J.5.9.1. Human Health (MON 87708)

Approval in whole alternative

Under full deregulation, MON 87708 could be grown broadly across the U.S. Soybean and forage produced from MON 87708 would en ter the food and feed chain and would be consumed by humans and animals. Ag ricultural workers would be exposed to MON 87708 and its associated agricultural practices. The potential human health impacts associated with th e introduction of MON 87708 and increased applications of dicamba are separately discussed below.

MON 87708 was developed through Agrobacterium -mediated transformation of soybean meristem tissue using the binary transf ormation plasmid PV-GMHT4355 (Section III; Figure III-1 and Table III-1). MON 87708 cont ains one copy of the insert at a single integration locus. No additional genetic el ements from the transformation vector were detected in the genome of MON 87708, incl uding backbone sequence from plasmid PVGMHT4355. Additionally the data confirm th e organization and sequence of the insert, and demonstrate the stability of the insert ove r several generations. On the basis of these data, it is concluded that only the MON 87708 DMO is produced from the inserted DNA.

For MON 87708, the available data demonstrat e that harvested seed is as safe as conventional soybean for food and feed us es; thus it is safe and wholesome for consumption. To assess the impact of MON 87708 DMO on food and feed safety, bioinformatic analyses were used to esta blish the lack of bot h structurally and immunologically-relevant sim ilarities between MON 87708 and allergens or toxins, based on the amino acid sequence of MON 87 708 DMO. Furthermore, digestive fate experiments conducted with MON 87708 DMO dem onstrate rapid digestion in simulated gastric fluid (SGF), a characteristic shared among many proteins with a history of safe consumption. Rapid digestion of MON 87708 DMO in SGF indicates that it is highly unlikely that MON 87708 DMO will reach absorptive cells of the intestinal mucosa. This, combined with the history of safe cons umption of mono-oxygenases (the class of enzymes to which MON 87708 DMO belongs) and the lack of homology of the amino acid sequence to known allergens and toxi ns, supports a conclusion that MON 87708 DMO has low allergenic and toxic potentia l. Finally, a high dose of MON 87708 DMO in a mouse acute oral toxicity evaluation dem onstrated that it is not acutely toxic, and does not cause any adverse effect. The safe ty assessment supports the conclusion that exposure to MON 87708 DMO poses no meaningful risk to human or animal health.Monsanto Company 10-SY-210U 550 of 721 

### Extensive analysis of the composition of MO N 87708 seed and forage demonstrated that no biologically relevant changes were dete ctable. A detailed compositional assessment of soybean harvested seed and forage is pr esented in Section VI of petition #10-SY-210U. The levels of key nutrients, anti-nutrien ts, and other components in MON 87708 were examined and compared to that of the nea r-isogenic conventional s oybean control, A3525, a conventional soybean variety with backgr ound genetics representative of MON 87708, but without the genetic modification. Add itionally, tolerance intervals representing 99% of the values of each analyte for a commer cial soybean population were established. Results demonstrate that the levels of key nut rients, anti-nutrients, and other components of MON 87708 are compositionally equivalent to the conventional control and within the range of variability of commercial soybean that were grow n concurrently in the same trial. Furthermore, FDA completed its consultati on on the food, feed and nutritional safety assessment on MON 87708 on October 11, 2011, c onfirming Monsanto’s conclusion on the safety of MON87708-derived food and feed.

On the basis of the characteristics of MON 87708 DMO and the extensive compositional characterization of MON 87708 harvested seed, no impacts to human health are expected from the approval in whole alternative. Thus, the approval in whole and no action alternatives are similar regarding the pot ential impacts of MON 87708 on human health.

No Action Alternative

Under the no action alternative, MON 87708 would continue to be a regulated article. Human exposure to existing conventional an d GE soybean would remain unchanged. Growers and consumers exposed to M ON 87708 would be limited to individuals

involved in the cultivation of MON 87708 under the conditions of regulation. Therefore, the no action and approval in whole alternat ives are similar regarding the potential impacts of MON 87708 to human health.

J.5.9.2. Human Health (Dicamba)

Approval in whole alternative

The toxicology or safety profile of dicamba has been extensively reviewed (U.S. EPA, 2009b). Dicamba does not pose any unusual toxicological concerns and is not carcinogenic (U.S. EPA, 2009b; Durkin and Bosch, 2004; PMRA, 2008; European Commission, 2007b). EPA comp leted the reregistration of dicamba in 2006. The Reregistration Eligibility Deci sion (RED) document for dicamba and its associated salts concluded a high level of confidence exists for the dicamba hazard data base and the reliability of these data necessary to support the required safety finding for continued registration, including the pre-harvest us e on commercial soybean. The dicamba RED document, and the related Health Effects Divi sion (HED) of the EPA chapter (U.S. EPA, 2005a), provide a detailed overview of the toxicological properties of dicamba. A summary of dicamba’s toxicity profile is pr esented in Section L.3.1 of Appendix L of the petition.Monsanto Company 10-SY-210U 551 of 721 

### EPA evaluated the potential risks to humans fr om the use of dicamba as a part of the dicamba RED, concluding that aggregate expo sure to dicamba, defined as dietary (food and water) and non-occupational (residential and recreationa l) exposures, meet the

FIFRA determination of no unreasonable adverse effects and the FFDCA determination for reasonable certainty of no harm to human he alth (see Section M.4.1 of the petition for more detail). EPA has conducted acute and chronic dietary (food and water) risk

assessments for dicamba based on a theoretica l worst case exposure estimate. For food, this estimate assumes that dicamba 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 and feed crops and derived animal food commodities (e.g., meat and milk) are at the le vel of the legally es tablished tolerance (i.e., the maximum allowable pe sticide residue level). Resi dues of dicamba are defined as dicamba and its metabolites 5-hydroxy dica mba and 3,6-dichlorosali cylic acid (DCSA) in soybean commodities, and as dicamba a nd DCSA in animal food commodities, as currently regulated in 40 CFR § 180.227. For water, EPA assumed that dicamba could potentially move offsite to ad jacent surface water bodies as a result of drift or runoff, or move through soil to groundwater. Since the estimated concentrations in groundwater were significantly lower compared to surface wa ter, surface water estimates were used in the worse case dietary assessments (see Sec tion J.5.2.2 and Section M.5.2 of the petition

for details on dicamba levels in water resour ces). Based on the worst-case assumptions outlined above, acute and chronic dietary exposure was well below the Agency’s level of concern to satisfy the FIFRA and FFDCA standards (U.S. EPA, 2009b).

Characterization of the nature of dicamba residues in MON 87708 confirms no additional

residues of concern are created in MON 87708 , and the current soybean seed dicamba residue definition is applicable for MON 87708. Residue levels in soybean seed

harvested from MON 87708 treated with di camba at the proposed maximum allowable application use pattern (1.0 pound a.e. per acre preemergence and two 0.5 pound a.e. per acre postemergence applications) were less than 0.1 ppm, well belo w the established 10 ppm tolerance supporting the current use of dicamba on conventional soybean. These

dicamba residue data for MON 87708 were s ubmitted to the U.S. EPA on April 28, 2010 (OPP Decision Number: D-432753, Regist ration Number 524-582), along with a proposed label for the use of dicamba on MON 87708.

Presently, dicamba is applied to less than 1% of soybean acres us ing pre-plant and preharvest burn down applications (see Table VIII-12 in petition #10-SY-210U). Under the approval in whole alternative, dicamba will be used on more soybean acres and a higher percentage of soybean and soybean-deriv ed foods will contain dicamba residues; however, dicamba residue levels in MON 87708 harvested seed or processed foods will be significantly lower compared to levels originating from the current pre-harvest soybean use (approximately 100-fold lower, based on <0.1 ppm. residue in MON 87708 seed compared to established 10 ppm tole rance (see Appendix M, Section M.4.1.1 for residue levels in seed). It is difficult to determine the exact impact on actual dietary exposure from the expanded use of dicamba on MON 87708; however, the EPA concluded that residues of dicamba up to 10 ppm in soybean seed are safe (reasonable certainty of no harm as defined by FFDCA) for human and animal consumption, based on the EPA’s Tier 1 dietary and aggregate (d ietary plus other non- occupational) exposureMonsanto Company 10-SY-210U 552 of 721 

### assessments which assume 100% of soybean foods contain dicamba residues at the 10 ppm tolerance level.

Additionally, Monsanto has pe titioned the EPA to establish new feed tolerances for soybean forage and hay to allow MON 87708 fo rage and hay to be fed to livestock, a practice that is presently prohibited for dicamba-treated commercial soybean (see Appendix M, Section M.4.1.1 for residue levels in forage and hay). This practice is presently not allowed because the current preh arvest application is made past the stage where the crop would be useful as forage or hay. Thus, there ha s been no reason for pursuing these tolerances until earlier in-crop applications of dicamba were possible, as with MON 87708. Dietary exposure to live stock from the feeding of MON 87708 soybean forage and hay does not result in an exceedance of the livestock maximumtheoretical dietary burden established by the EPA, which is used to establish animal byproduct commodity tolerances ( e.g. , meat and milk). Therefore, the approval of soybean forage and hay tolerances does not result in a change to the current animal by-product commodity (food) tolerances and as a result does not increase poten tial dietary exposure

of dicamba.

Since no changes to the current dicamba food tolerances (soybean seed and animal byproducts) are needed, the dietary and aggreg ate risk assessments conducted in the RED are inclusive of the incremental exposur e resulting from the use of dicamba on MON 87708. Therefore, the deregulation of MON 87708 would not present a significant impact to human health, and the approval in whole and no action alternatives are the similar regarding potential im pacts on human health.

No Action Alternative

Under the no action alternative, MON 87708 wo uld remain a regulated article and would not be commercially available to growers. It is likely that grower s will continue to use herbicides in soybean production, and use the Roundup Ready soybean system. Growers

will continue to use additional herbicides where needed or recommended to control hardto-control or glyphosate-resistant weeds. Growers may also c hoose to use other herbicide-tolerant soybean events, use a combin ation of alternative herbicides registered for use in soybean, or use trad itional tillage practices to control weeds. In addition,

dicamba will continue to be used on a sma ll number of soybean acres (refer to Section VIII.G in the petition). Consumers will be exposed to residues of dicamba and the

alternative herbicides thr ough consumption of soybean a nd soybean-derived foods, and from residues on other food crops such as corn and wheat. On the basis of the analysis above, the approval in whole and no action a lternatives involve the continued use of herbicides for production of soybean. T hus, the no action and approval in whole alternatives are similar regarding po tential impacts to human health.

J.5.9.3. Worker Safety

Approval in whole alternativeMonsanto Company 10-SY-210U 553 of 721 

### It is expected that under the approval in whole alternative MON 87708 will be integrated into the Roundup Ready soybean system using traditional breeding techniques. For those acres where glyphosate resistant weeds may already be present or where application of an

herbicide with a different mode of ac tion would aid in weed control or the implementation of weed resistant manageme nt practices, the cult ivation of soybean containing the MON 87708 trait would be an option for growers.

There is no notable worker sa fety issue related directly to exposur e to MON 87708. As described elsewhere, MON 87708 is as safe as conventional soybean for use as food or feed. MON 87708 DMO has no safety concerns th at would result in adverse effects to workers exposed to MON 87708 plant tissues.

Agricultural workers can be exposed to di camba during the herbic ide application or upon re-entry into treated MON 87708 fields. Un der the proposed label, dicamba can be applied as a preemergent treatment on MON 87708 at rates up to 1 lb a.e. per acre and then again in two sequential 0.5 lb a.e. pe r acre postemergent treat ments up to the R1/R2 growth stage using a ground application me thod. The EPA conducted a comprehensive occupational exposure assessment as part of the dicamba reregistration in 2006 and concluded no unreasonable risk to agricultu ral workers from ground and aerial dicamba applications to soybean at rates up to 2 lb a.e. per acre when required personal protective equipment is worn (U.S. EPA, 2009b). See Section M.4.2 of the petition for additional detail. The application scenario evaluate d by EPA encompasses the application method and rates for dicamba applied to MON 87708. Therefore, the deregulation of MON 87708 does not present a significant impact to the health of agricultural workers.

No Action Alternative

Under the no action alternative, MON 87708 wo uld remain a regulated article and would not be commercially available to growers. It is likely that grower s will continue to use herbicides in soybean produc tion, and use the Ro undup Ready soybean system and utilize

additional herbicides where needed or re commended to control hard-to-control or glyphosate-resistant weeds. Growers may also choose to use other herbicide-tolerant soybean events, use a combination of alternativ e herbicides registered for use in soybean, or use traditional tillage practices. The con tinued use of dicamba on a small number of soybean acres would also be expected. Therefore, agricultural workers will be exposed

to residues of dicamba and the alternative herb icides. On the basis of the analysis above, the deregulation in whole and no action al ternatives involve the continued use of herbicides for production of soybe an. Thus the impacts to ag ricultural worker health for

either alternative are not considered different.

J.5.10. Potential Impacts to Plant and An imal Communities Including Threatened or Endangered Species, Soil Microorganisms and Biodiversity

Under the approval in whole a lternative it is expected that MON 87708 will be integrated into the Roundup Ready soybean system using traditional breeding techniques. For those acres where glyphosate resistant weeds may already be present or where application of an

herbicide with a different mode of ac tion would aid in weed control or theMonsanto Company 10-SY-210U 554 of 721 

### implementation of weed resistant manageme nt practices, the cult ivation of soybean containing the MON 87708 trait would be an option for growers.

The potential for MON 87708 to harm plant, animal and microbial communities as well as threatened and endangered species (TES) was evaluated by considering the biology of soybean, biochemical information and experi mental data. Soybean does not have any sexually-compatible relatives in the U.S. Th erefore, any effect due to movement of the

dicamba tolerance trait is confined to soybean ( Glycine max ). The biochemical information and experimental data incl uded product characterization information, information from the MON 87708 DMO safety a ssessments, the history of environmental exposure to mono-oxygenases (the cla ss of enzymes to which MON 87708 DMO belongs), results from the environmental a ssessment described in the petition, and the demonstration of compositional, agronomic and phenotypic equivalence to conventional soybean. An analysis of the effects of MON 87708 on plant, animal and microbial

communities as well as threatened an d endangered species is found below.

J.5.10.1. Gene Movement

Approval in whole alternative

In assessing the potential impact to plant a nd animal communities, th e potential for gene movement and introgression from MON 87708 was evaluated because movement and establishment of the gene and trait to related species could have indi rect impacts to plant and animal communities that extend beyond the original recipient organism. Additional discussion of the potential environmental impact due to gene movement may be found in Section IX.D of the pe tition. Monsanto considered two primary issues: 1) the potential for gene flow and introgression, and 2) the potential impact of introgression. The genus Glycine has approximately nine specie s, with commercial soybean ( G. max ) being placed in the subgenus Soja along with one other species, G. soja

. G. max is sexually compatible only with G. soja and no other Glycine species. G. max is the only Glycine species located in the United States. Ther efore, the probability of gene flow and introgression of MON 87708 into other species in the U.S. is essentia lly zero (Stewart et al., 2003); thus, the potential im pact of MON 87708 introgressi on to sexually compatible relatives on plant and animal communities is nonexistent if MON 87708 were approved in whole. The approval in whole and no action alternatives are the same regarding potential impacts on gene movement.

No action alternative

As discussed above under the approval in whole alternative, soybean is not sexually compatible with any other plant species in th e U.S. Therefore, the no action and approval in whole alternatives are the same rega rding potential impact s on gene movement.

J.5.10.2. Animal Communities

Approval in whole alternativeMonsanto Company 10-SY-210U 555 of 721 

### Soybean production systems in agriculture ar e host to many animal species. Mammals and birds, including migratory mammals and bi rds, may seasonally consume grain in the field, and invertebrates can feed on the plan t during the entire growing season. Animals that feed primarily on soybean are seed-feed ing insects and rodents found in agricultural fields. Rodents, such as mice or squirrels , may seasonally feed exclusively on soybean seeds. Thus, these animals may have a di et containing significant amounts of soybean seeds. Deer may also forage in soybean fields on forage and seed left after harvest (see Section IX.B.3.5 of the petition).

Under the approval in whole al ternative, the cultivation of MON 87708 is not expected to impact soybean agronomic practices, with the exception of a change in dicamba use pattern. Cultivation of MON 87708 would not alter agronomic inputs or the number of soybean acres under cultivation, and may ha ve a small positive effect on tillage cultivation practices. Potential impacts to animals from widespread cultivation of MON 87708 would be primarily based on th e possible effects of the introduced MON 87708 DMO that provides tolerance to dicamba and a broadened application window of dicamba to MON 87708. For instance, if MON 87708 is deregulated, MON 87708 and MON 87708 DMO will be present in soybean consumed by animals. As discussed previously, there is no meaningf ul risk to animal or human health from dietary exposure to MON 87708 DMO. There are no known toxic properties associated with MON 87708 DMO. Furthermore, the composition of the seed and forage produced by MON 87708 is compositionally equivalent to conventional soybean. This information on the safety of MON 87708 DMO and com position of MON 87708 soybean seed, as detailed in petition, indicate that there would be no negativ e effects to mammals that consume MON 87708 seed and forage. Similarly, it is expected that there would be no impact to birds or other animals, includi ng migratory birds and animals, that may consume soybean forage or soybean seed from MON 87708. During field trials with MON 87708, no biologically releva nt changes in insect feed ing damage were observed (see Section VII of the petition) indicating similar insect susceptibility for MON 87708 compared to conventional soybean. As M ON 87708 exhibits no toxic effects on animals or insects, it is concluded that they will not be affected. In addition, the cultivation of MON 87708 does not impact the nutritional quality, safety or availability of animal feed (see Section J.5.11 of the petition).

Dicamba is currently registered for early s eason preemergence (early preplant) and late season postemergence (pre-harvest) soybean applications. Upon deregulation, dicamba could also be applied late preemergence ( up to cracking) and in -season postemergence applications up to R1/R2 growth stage. Theref ore, with the exception of late preemergent and in-season applications of dicamba, th e agronomic practices used to produce MON 87708 will be the same as those used to produce commercially available soybean. Animals may be affected by dicamba runoff a nd/or herbicide spray drift. However, a comprehensive safety evaluation and risk as sessment conducted by EPA concluded that dicamba has low toxicity to mammals, is not a carcinogen, does not adversely affect reproduction and development, and does not bioaccumulate in mammals (U.S. EPA, 2009b). An ecotoxicological risk assessment concluded that the use of dicamba does not pose an unreasonable risk of adverse effects to non-target species, such as birds and fish, when used according to label directions, nor does it pose an unreasonable risk of adverseMonsanto Company 10-SY-210U 556 of 721 

### effects to insects outs ide of the application area (U.S. EPA, 2009b). Furthermore, outside the cultivated soybean field, dicamba is unlikel y to affect forbs and beneficial arthropods that are dependent on plants for survival. On the basis of this anal ysis, approval in whole of MON 87708 will not result in significant impacts on animals, including insects and beneficial arthropods that live in or near soybean fields containing MON 87708, and the approval in whole and no action alternatives are the same regarding potential impacts on

animals and animal communities.

No action alternative

Under the no action alternative MON 87708 and its progeny would continue to be regulated articles and would not be widely grown. Under this alternative dicamba tolerance trait would not become integrated into the Roundup Rea dy soybean system and dicamba use would likely remain similar to today’s use pattern in soybean. Adding other

herbicides with different modes-of-action into the Roundup Ready system to mitigate development of glyphosate resistant weeds a nd control glyphosate re sistant weeds would continue to remain an option. Additionall y, conventional tillage may increase in some

instances as an additional means to control problematic weeds.

In order to manage weeds, growers would cont inue to use available herbicides, some of which may pose greater potential risks to animal s or insects than dicamba; and cultivation practices may increase. Therefore, the no act ion and approval in w hole alternatives are similar regarding their effects to animals and animal communities.

J.5.10.3. Plant Communities

Approval in whole alternative

Soybean production systems in agriculture are ho st to many plant species. Likewise, the environment surrounding a soybean field varies in plant composition depending on the region. In certain areas, soybean fields may be bordered by other soybean, corn or other crops; fields may also be surrounded by woode d and/or pasture/gras sland areas, as well as aquatic environments. Therefore, the t ypes of vegetation, incl uding weeds, around a soybean field depend on the area where the soyb ean is planted. A va riety of weeds dwell in and around soybean fields; those species will also vary depe nding on the region where the soybean is planted.

If MON 87708 is approved in whole, it will not compete with plants found outside of agricultural production, because, like commer cially available soybean, MON 87708 does

not exhibit characteristics associated with weedy growth. Weeds within fields of MON 87708 will be managed using existing ag ricultural practices, including mechanical, cultural, and chemical control measures. This will include in-season (up to R1/R2 growth stage) applica tions of dicamba.

Plants on adjacent land have the potential to be affected by dicamba runoff and offsite movement (spray drift and volat ility), but when herbicides are applied according to the FIFRA label application instructions, offsite impacts can be avoided. EPA concluded in the dicamba RED (U.S. EPA, 2009b) that ex isting label language to mitigate offsiteMonsanto Company 10-SY-210U 557 of 721 

### movement was sufficient to reduce the potential risk of damage to adjacent vegetation. Since the proposed use pattern for dicamba on MON 87708 is consistent with the uses evaluated by EPA as part of the RED and the proposed label contains the offsite movement mitigation language, it is reasonabl e to conclude that the use of dicamba on

MON 87708 also meets the FIFRA no unreasonable effects standard fo r drift and offsite movement. Nevertheless, Monsanto has taken additional steps to manage dicamba offsite movement as stewardship measures. In the pending application to EPA, Monsanto requested the use of dicamba on MON 87708 onthe low volatility DGA salt formulation (U.S. EPA Reg. No. 524-582) and limited dica mba applications to ground application equipment. Monsanto also plans to furt her address the specifi c use of dicamba on

MON 87708 with US EPA to evaluate whethe r additional measures, may be appropriate to address potential drift and offsite movement.

Therefore, the presence of the dicamba to lerance trait in MON 87708 and the use of dicamba is not expected to have a significant impact on surrounding plant communities, and the approval in whole and no action alte rnatives are similar regarding potential impacts on plant communities.

No action alternative

Under the no action alternative MON 87708 and its progeny would continue to be regulated articles and would not be widely grown. Under this alternative dicamba tolerance trait would not become integrated into the Roundup Rea dy soybean system and dicamba use would likely remain similar to today’s use pattern in soybean. Adding other

herbicides with different modes-of-action into the Roundup Ready system to mitigate development of glyphosate resistant weeds a nd control glyphosate re sistant weeds would continue to remain an option. Growers will continue to use existi ng soybean herbicides to control glyphosate-resistant or hard-to-cont rol weeds in soybean production fields. In addition, approximately 60% of soybean acres in the U.S. do not use conservation tillage practices, and it is anticipated that conventiona l tillage may increase in some instances as

an additional means to control hard-to-contro l or resistant weeds. Therefore, the no action and approval in whole alte rnatives are similar regarding potential impacts to plants and plant communities.

J.5.10.4. Threatened and Endangered Species

Congress passed the Endangered Species Act (ESA) of 1973, as amended, to prevent extinctions facing many species of fish, wildlife, and plants. The purpose of the ESA is to conserve endangered and threatened species and the ecosystems on which they depend as key components of America‘s heritage. To implement the ESA, the U.S. Fish and Wildlife Service (USFWS) works in cooperation with the National Marine Fisheries Service (NMFS); other Federal, State, a nd local agencies; Tribes; non-governmental organizations; and private citizens. Before a plant or animal species can receive the protection provided by the ESA, it must first be a dded to the Federal li st of threatened and endangered wildlif e and plants.Monsanto Company 10-SY-210U 558 of 721 

### A species is added to the list when it is determined by the USFWS/NMFS to be endangered or threatened because of any of the following factors:

 The present or threatened de struction, modification, or curt ailment of its habitat or

range;

 Overutilization for commercial, recreational, scientific, or educational purposes;

 Disease or predation;

 The inadequacy of existing regulatory mechanisms; and

 The natural or manmade factor s affecting its survival.

Once an animal or plant is added to the list, in accordance with the ESA, protective measures apply to the species and its habitat. These measures include protection from adverse effects of Federal activities. Section 7 (a)(2) of the ESA requires that Federal agencies, in consultation with USFWS and/or the NMFS, en sure that any action they authorize, fund, or carry out is not likely to je opardize the continued existence of a listed species or result in the destruction or advers e modification of designa ted critical habitat. It is the responsibility of the Federal agency taking the action to assess the effects of their

action and to consult with the USFWS and NMFS if it is determined that the action “may affect” listed species or critical habitat. To facilitate APHIS’ ESA consultation process,

APHIS has met with the USFWS from 1999 to 2003 to discuss factors re levant to APHIS’ regulatory authority and effects analysis for petitions for nonregulated status, and developed a process for conducting an effects determination consistent with the Plant Protection Act of 2000 (Title IV of Public La w 106-224). This process is described in a decision tree document, which have been in cluded in recent Envi ronmental Assessments (USDA-APHIS, 2011a) . APHIS has used this process to help fulfill its obligations and responsibilities under Secti on 7 of the ESA for biotec hnology regulatory actions.

The potential impact of MON 87708 on fede rally listed Threatened or Endangered Species (TES) and species proposed for listing ha s been evaluated. In this analysis, the biology of MON 87708 and the agricultural prac tices associated with the cultivation of MON 87708 have been considered for potentia l adverse impact on TES and their critical habitats. Additionally, the potenti al indirect effects of dicamb a applications due to either

the introduction or non-introduction of MON 87708 on TES are discussed. While APHIS does not have statutory authority to authoriz e or regulate the use of herbicides, APHIS considers the evaluation of potential impacts on TES from the application of dicamba, as conducted by the EPA, as part of its e nvironmental analysis under NEPA. APHIS’ regulatory authority over GE organisms under th e PPA is limited to those GE organisms for which it has reason to believe it may be a plant pest or those for which APHIS does not have sufficient information to determine that the GE organism is unlikely to pose a plant pest risk (7 CFR § 340.1).

Approval in whole alternative

It is expected that under the approval in whole alternative MON 87708 will be integrated into the Roundup Ready soybean system using traditional breeding techniques. For thoseMonsanto Company 10-SY-210U 559 of 721 

### acres where glyphosate resistant weeds may already be present or where application of an

herbicide with a different mode-of-actio n would aid in weed control or the implementation of weed resistant manageme nt practices, the cult ivation of soybean containing the MON 87708 trait would be an op tion for growers. Monsanto intends to make MON 87708 available for cultivation across all soybean growing regions in the

U.S. The cultivation of MON 87708 is not ex pected to change the agronomic practices or expand soybean production acres beyo nd levels and geographies of current commercial soybean varieties. Several line s of evidence can be used to assess the potential for MON 87708 to have adverse eff ects on TES. The first line of evidence is

based on the characteristics and evaluati on of MON 87708 DMO and natural constituents present in MON 87708. The second line of ev idence is the potential for TES species to

interact with MON 87708. The third line of evidence is based on the biology and competitiveness of MON 87708 compared to conventional soybean. Finally, as the introduction of MON 87708 may result in incr eased use of dicamba in soybean (see Section VIII.H of the petition for additional de tails), potential indir ect effects of dicamba on TES are also discussed, although it is the EPA’s statutory obligation to comply with the ESA in the registration of this use.

J.5.10.4.1. Evaluation of MON 87708 and MON 87708 DMO

As previously noted, (see Section V.F.), bioinformatics analysis determined that

MON 87708 DMO does not share amino aci d sequence similarities with known allergens, gliadins, glutenins, or protein toxins. MON 87708 DMO was rapidly digested

in in vitro assays using simulated gastric and in testinal fluids and did not show any

adverse effects when administered to mice via or al gavage at levels far in excess of that reasonably expected to be consumed by humans or animals. Compared to conventional soybean, MON 87708 does not express any additi onal proteins or natural toxicants that are known to directly or indire ctly affect a listed TES or species propos ed for listing by

the U.S. Fish and Wildlife Service. Com positional analysis of MON 87708 for nutrients

and anti-nutrients indicated that the harv ested seed and forage from MON 87708 were compositionally equivalent to conventiona l soybean. Thus, MON 87708 would not be expected to have any impacts on TES that would differ from conventional soybean.

J.5.10.4.2. Potential Interactions of TES with MON 87708

The only TES animal listed that occupies habita t that is likely to in clude soybean fields and that might feed on soybean is the Fe derally Endangered Delmarva Peninsula Fox Squirrel, Sciurus niger cinereus , found in areas of the mid-Atlantic Eastern seaboard. 35 

### It is known to utilize certain agricultural la nds readily, and its diet includes acorns, nuts/seeds of hickory, beech, walnut, and loblo lly pine, buds and flowers of trees, fungi, insects, fruit, and an occasional bird egg. Gi ven its varied diet, the safety of MON 87708 DMO, and the demonstrated compositional, agronomic and phenotypic equivalence of MON 87708 to conventional soybean, it is co ncluded that no biologically significant changes to the habitat or diet of th e Delmarva Peninsula Fox Squirrel are

35 Source is from website: http://ecos.fws.gov/tess_public/SpeciesReport.do ; [Accessed May 14, 2009].Monsanto Company 10-SY-210U 560 of 721 

### expected. Consequently, the planting of MON 87708 is not expected to impact the Delmarva Peninsula Fox Squirrel.

Additionally, MON 87708 has demonstrated no characteristics that would allow it to expand agricultural production into new natural areas where other TES animal species could be found. Consequently, the planting of MON 87708 is not expected to affect any TES animal species.

J.5.10.4.3. Potential Hybridization or Competition of MON 87708 with TES

Soybean is not native to the U.S., and M ON 87708 is not sexually compatible with any federally listed TES or a species pr oposed for listing. Like other G. max , MON 87708 will likely be a poor competitor with native ve getation and will not survive outside of cultivation (Baker, 1965). T hus, MON 87708 is not expected to interbreed w ith any plant species or displace natural vegetation in the U.S.

J.5.10.5. Potential Impact of Dicamba on TES

As a result of joint discu ssions between USFWS and APHI S officials on June 15, 2011, the Agencies have agreed that it is not ne cessary for APHIS to perform an ESA effects analysis for the herbicide use associated with a biotechnology-derived crop. This is because EPA has both regulatory authority over the labeling of pesticides and the necessary technical expe rtise to assess pestic ide effects on the environment under FIFRA. Similarly for MON 87708, since APHIS has no re gulatory jurisdiction to authorize or regulate the use of dicamba by growers and the EPA has an application pending for the use of dicamba on MON 87708, the EPA will also have the obligation to conduct an ESA effects analysis for the us e of dicamba on MON 87708. However, APHIS does consider the evaluation of potential impacts on TES from the application of dica mba as part of its environmental analysis.

In order to register a pesticide under FIFRA, the EPA must reach a conclusion that the pesticide will not cause an unreasonable advers e effect when used as intended. Thus, the current and future proposed uses of dicamba on soybean must meet this standard to be registered by the EPA. Monsanto has re quested approval of th e use of dicamba on MON 87708 by amending the label for EPA Re gistration Number 524-582 (as described in Appendix L, Section L.2.4). In the EPA’s re view of this registra tion request, they will apply the same statutory-base d regulatory requirements as they do to all pesticides.

In the 2006 dicamba RED (U.S. EPA, 2009b), EPA discussed the changes in the registered use pattern of dicamba that were required to assure th at dicamba meets the FIFRA regulatory standards of no unreasonable a dverse effects for pesticides in the U.S. Accordingly, EPA reduced the maximum single application rate to 1.0 lb a.e. per acre with a maximum annual rate of 2.0 lb a.e. pe r acre (reduced from 2.8 lb a.e. per acre single and 7.7 lb a.e. per acre maximum annual ra te, respectively). EPA went further in the RED to state that no specific additional m itigations for offsite movement were needed (U.S. EPA, 2009b).Monsanto Company 10-SY-210U 561 of 721 

### The EPA’s new application rates, among other measures implement by EPA as part of the dicamba RED, provide protection to non-ta rget organisms. Because the proposed use

of dicamba on MON 87708 falls within the use rate limits established by EPA in the 2006 RED, it can be concluded that risks to non-ta rget organisms from th e use of dicamba on

MON 87708 have been assessed.

Furthermore, Monsanto has conducted a speci es-specific analysis on threatened and endangered (TE) birds, mammals, amphibians, reptiles, terrestrial invertebrates, and nonmoncotyledonous terrestrial and semi-aquatic pl ants. This refined analysis utilized dicamba- and species-specific information, in accordance with guid ance and procedures

outlined in the Overview of the Ecological Risk A ssessment Process in the Office of Pesticide Programs, U.S. Environmental Pr otection Agency, Endangered and Threatened Species Effects Determinations , published in 2004 (U.S. EPA, 2004), and methods

utilized in more recent threatened and endangered species effects determinations conducted by the EPA (U.S. EPA, 2007b; 2008c). This analysis concluded that TE birds, mammals, amphibians, reptiles, terrestrial invertebrates, and m onocotyledonous plants will not be adversely affected from envi ronmental exposure to dicamba at levels anticipated from the use on MON 87708. A summ ary of the refined species analysis may be found in Appendix N of the petition. The summary and supporting detailed analyses have been provided to EPA (Frank and Kemman, 2011 and 2012; Honegger, 2012; Mueth and Foster, 2010; Schuler et al., 2012; Wright et al., 2012).

For the remaining federally listed TE non-m onocotyledonous terrestria l plants, less than one percent of potential soybean production acre s (defined as all cu ltivated crop acres

listed in the 2001 National Land Coverage database in st ates with reported soybean production 36 

### are conservatively estimated to be in proximity to a TE nonmonocotyledonous terrestrial plant species. 37 

### For the limited areas where a federally listed TE non-moncotyledonous terrestrial plan t is in proximity to a MON 87708 field where dicamba may be applied, Monsanto has already undertaken steps to manage the offsite movement of dicamba (described in Section J.5.7) to further reduce potential impacts on these plant species. These measures include registering the use on MON 87708 on the low volatility DGA salt form ulation of dicamba and limiting dicamba use on MON 87708 to ground application only. Monsanto will also consult with U.S. EPA in coming months to evaluate whether additional measures may be appropriate to address potential drift and offsite moveme nt. Finally, upon commercialization of MON 87708, Monsanto will implement a we b-based endangered species mitigation system, similar to PreServe.org, to further mitigate potential impacts of dicamba on TE non-monocotyledonous terrestrial plants. Theref ore, the approval in whole and no action alternatives are similar regarding potential impacts on threatened or endangered species

from dicamba use on MON 87708.

36 Potential soybean production acres include all land of Class 81 (cultivated crops) and Class 82 (pasture/hay) from the 2001 National Land Cover Database, in counties reporting soybean production in the 1997, 2002, and 2007 Ag Census.

37 Based on species location information available from the FIFRA Endangeres Species Task Force (FESTF) Multi-Jurisdictional Database (MJD) and other state and federal information sources.Monsanto Company 10-SY-210U 562 of 721 

### No action alternative

Under the no action alternative MON 87708 and its progeny would continue to be regulated articles and would not be widely grown. Under this alternative MON 87708 would not become integrated into the Roundup Ready soybean system and dicamba use would likely remain similar to today’s use in soybean. Adding other herbicides with different modes-of-action into the Roundup R eady system to mitigate development of glyphosate resistant weeds and to control glypho sate resistant weeds would continue to remain an option. Alternatively, tillage may increase in some instances as an additional means to control problematic weeds.

Since other herbicides are currently availabl e and used in soybean production, and would likely be used instead of dicamba, tillage prac tices would likely continue as a method of weed control. Therefore, the approval in w hole and the no action alte rnatives are similar regarding their effects to TES.

J.5.10.6. Soil Microorganisms

Approval in whole alternative

Potential impacts to soil microorganisms can arise from the exposure to the introduced gene and expressed protein in the GE crop product. In addition, agricultural practices such as pesticide applications and tillage are known to impact so il microbial populations, species composition, colonization, and a ssociated biochemical processes.

Soil microorganisms in general and symbiotic microbes associated with soybean roots are discussed in Section VII.C.4 and Appendi x J, Section J.3.5.3 of the petition, and

summarized here. The difference between the approval in whole and the no action alternative is expected to be integrat ion of MON 87708 into the Roundup Ready soybean system using traditional breeding techniques. MON 87708 is not e xpected to alter the current agronomic practices for soybean cultivation. No adverse effects on soil microorganisms are associated with MON 87708 or its cultivation, nor do the characteristics of the MON 87708 DMO pose any concern to soil microorganisms. The B. japonicum -soybean symbiosis of MON 87708 was not changed either as a result of the

introduction of the dicamba tolerance tra it or as a result of the MON 87708-produced

DMO.

Dicamba was registered as an agricultural herbicide in 1967 (U.S. EPA, 2009b) and has a long history of use. Impacts on soil microorganisms have not been raised as a significant concern, and results of standard ized tests with dicamba and dicamba formulations did not indicate any long term effects on soil microorganisms (see Section M.5.6.3 of the petition). Results of standardized tests w ith dicamba and dicamba formulations indicate no long-term effects on functional processes of soil microorganisms (carbon respiration and nitrogen transformation) at rates proposed for dicamba on MON 87708 (European Commission, 2007a).

On the basis of these observations and in conjunction with related phenotypic measurements for MON 87708, no impact on soil microorganisms is expected from theMonsanto Company 10-SY-210U 563 of 721 

### cultivation of MON 87708. Therefore, the a pproval in whole and no action alternatives are the same regarding potentia l impacts on soil microorganisms.

No action alternative

Under the no action alternative MON 87708 and its progeny would continue to be regulated articles, woul d not be commercially grown and use of dicamba herbicide would likely remain similar to today’s use pattern in soybean.

Adding other herbicides with different modes-of-acti on into the Roundup Ready system to mitigate development of glyphosate resist ant weeds and control glyphosate resistant weeds would continue to remain an opti on. Additionally, conve ntional tillage may

increase in some instances as an additi onal means to control problematic weeds.

Agricultural practices such as pesticide app lications and tillage ar e known to impact soil microbial populations, species composition, colonization, and associated biochemical processes. However, alternative herbicides ar e already available and are used in soybean, and would likely be used instead of dicamba under the no action alternative. Similarly, approximately 40% of the U.S. soybean acre s use conservation tilla ge and would likely continue to do so. Therefore, the no action a nd approval in whole alternatives are similar regarding potential impa cts to microorganisms.

J.5.10.7. Biodiversity

Approval in whole alternative

The difference between the approval in whole an d the no action alternative is expected to be integration of MON 87708 into the Roundup Ready soybean system with the potential for cultivation of soybean seed containing the MON 87708.

As confirmed in trials to evaluate plant growth and phenotypic characteristics,

MON 87708 exhibits no traits that would cause increased w eediness of soybean. Since soybean is not sexually compatible with any other plant species in the U.S., its

unconfined cultivation would not lead to increased weediness of other sexually compatible relatives. Therefore, it is unl ikely to have effects on non-target organisms common to agricultural ecosystems or TES recognized by the U.S. Fish and Wildlife

Service and National Marine Fisheries Servi ce, or to colonize ad jacent non-agricultural ecosystems thereby compromising their biodiversity.

MON 87708 DMO originates from S. maltophilia , an organism that is ubiquitous in the

environment. MON 87708 DMO shares se quence identity and ma ny catalytic and

domain structural similarities with a wide variety of oxygena ses present in bacteria and plants currently widely consumed, establis hing that animals and humans are extensively exposed to these types of enzymes. Bioinf ormatics analysis determined that MON 87708 DMO does not share amino acid sequence sim ilarities with known allergens, gliadins, glutenins, or protein toxins. M ON 87708 DMO was rapidly digested in in vitro assays using simulated gastric and inte stinal fluids and did not show any ad verse effects when administered to mice via oral gavage at levels that resulted in large margins of exposure.Monsanto Company 10-SY-210U 564 of 721 

### MON 87708 DMO demonstrates a high level of substrate specificity. Additionally, studies have shown no impact to NTOs such as beneficial and pest arthropods when exposed to MON 87708 DMO in the field. Taken together these data indicate low probability that MON 87708 DMO will have a si gnificant impact on biodiversity.

The use of herbicides in agricultural fields is likely to indirectly impact biodiversity by decreasing weed species present in the fiel d. Agricultural fields are purposefully managed to be weed-free result ing in greater economic benefit to the grower. In the U.S., 98% of soybean acreage was treated with an herbicide in 2006 (USDA-NASS, 2007b). Therefore, introduction of MON 87708 and treatment with dicam ba is unlikely to affect the animal or plant communities found in commercial soybean production systems differently than those already occurring due to the use of herbicides in soybean fields. Dicamba has an established history of safe us e in agriculture. For an overview of the environmental safety of dicamba, see Appendix M of the petition. Ba sed on this analysis, it is concluded that the potential eff ect of approval in whole of MON 87708 on biodiversity would not differ fr om impacts associated with current agricultural practices used for production of soybean. Therefor e, the approval in whole and no action alternatives are similar regarding potential impacts on biodiversity.

No action alternative

Under the no action alternative MON 87708 and its progeny would continue to be regulated articles and would not be commer cially grown. Use of dicamba herbicide would likely remain similar to today’s use pattern in soybean, and growers would likely

continue to use alternative herbicides or in corporate more tillage in soybean production to manage hard to control weeds. Thus, the a pproval in whole and the no action alternatives are similar regarding poten tial impacts on biodiversity.

J.5.11. Animal Feed

The majority of the soybean cultivated in the U.S. is grown for animal feed and primarily fed as soybean meal. Soybean forage may al so be used as feed for dairy cattle and livestock. Biotechnology-der ived products may undergo a vol untary consultation process with the FDA prior to commercialization. Monsanto has completed the biotechnology consultation process with FDA for the safety and nutritional assessment of food and feed derived from MON 87708 soybean on Oc tober 11, 2011 (BNF No. 00125, Monsanto, 2011). As a part of its eval uation, FDA reviewed informa tion on the identity, function, and characterization of the genes, includ ing expression of the gene products in MON 87708 soybean, as well as informati on on the safety of the MON 87708 DMO and MON 87708 soybean.

Approval in whole alternative

The difference between the approval in whole an d the no action alternative is expected to be integration of MON 87708 into the Roundup Ready soybean system using traditional breeding techniques. Under th e approval in whole alternativ e, there is potential for cultivation of soybean seed containing th e MON 87708 trait to proactively manage andMonsanto Company 10-SY-210U 565 of 721 

### prevent the development of he rbicide resistant weeds. Fo r those acres where glyphosate resistant weeds may already be present or where application of an herbicide with a

different mode-of-action would aid in weed control or the implementation of weed resistant management practices, the cultiva tion of soybean cont aining the MON 87708 trait would be an option for growers.

Potential impacts to the safety of animal feed from widespread cultivation of MON 87708 would be primarily based on th e possible effects of the introduced MON 87708 DMO that provides tolerance to dicamba and a broadened application

window of dicamba to MON 87708. Upon deregulation, MON 87708 containing MON 87708 DMO and dicamba residues from the application of dicamba will be present in animal feed. As discussed previously, th ere is no meaningful risk to animal health from dietary exposure to MON 87708 DMO. There are no t oxic properties associated with MON 87708 DMO. Furthermore, the composition of the seed and forage produced by MON 87708 is equivalent to conventional soyb ean. This information on the safety of MON 87708 DMO and composition of MON 87708 s oybean seed as detailed in petition

#10-SY-210U indicate that there would be no ne gative impact on the safety or nutritional quality of animal feed from the cultiv ation of MON 87708. The cultivation of MON 87708 will not change the number of so ybean acres cultivated in the U.S., the extent in which soybean acres are cultivat ed will continue to be based on the same market-based drivers that exist today

As discussed previously, dicamba is presently applied to less than 1% of soybean acres using pre-plant and pre-harvest burn down a pplications. Under the approval in whole alternative, dicamba will be used on more soybean acres and a higher percentage of soybean-derived animal feeds will contain dicamba residues. However, dicamba residue levels in MON 87708 harvested seed or proc essed meal will be significantly lower compared to levels originating from the cu rrent pre-harvest soybean use (approximately 100-fold lower, based on <0.1 ppm residue in MON 87708 seed compared to established 10 ppm tolerance - see Appendix M, Section M.4.1.1 for residue le vels in seed). As part of the pesticide tolerance setting pro cess for dicamba (40 CFR 180.227), the EPA concluded that residues of dicamba up to 10 ppm in soybean seed are safe (reasonable certainty of no harm as defined by FFDCA) for animal consumption. Additionally, as

discussed in detail in Secti on J.5.9.2, Monsanto has petitioned the EPA to establish new feed tolerances for soybean fo rage and hay to allow MON 87708 forage and hay to be fed to livestock. Dietary exposure to livest ock from the feeding of MON 87708 soybean forage and hay does not result in an increase in the maxi mum dietary (feed) exposure of dicamba evaluated and deemed safe by EPA.

MON 87708, MON 87708 DMO and dicamba re sidues in MON 87708-derived feed components do not effect on the safety or nutriti onal quality of animal feed, therefore the deregulation of MON 87708 will not result in a significant impact on animal feed, and consequently to animal health. Therefor e, the approval in w hole and the no action alternatives are the same regardi ng their effects an animal feed.

No action alternativeMonsanto Company 10-SY-210U 566 of 721 

### The no action alternative would not allow the widespread planting of MON 87708, and

the use of dicamba herbicide would rema in unchanged including the pre-harvest

treatments which result dicamba residues in soybean seed up to the 10 ppm tolerance.

Other herbicides are available and would be us ed in soybean as need to control weeds in conventional and herbicide-tolerant soybean. The availability of safe and nutritional animal feed from existing soybean crops, in cluding crops containing dicamba or other herbicide residues from currently establishe d uses, will continue . Therefore, the no action and approval in whole alternatives are similar regarding their potential impacts on

animal feed.

J.6. Socioeconomic Effects

Approval in whole alternative

The decision to deregulate MON 87708 would allow for breeding of this product with conventional and previously deregulated biotechnology-derived soybean products of diverse genetic backgrounds. These variet ies will include soybean with glyphosate tolerance (MON 89788; Roundup Ready 2 Yield soybean), to deliv er products that enhance growers ability to manage weeds. It is expected that br eeders and certified seed producers would use MON 87708 to develop vari eties and to supply seed for planned commercial markets in the U.S. Monsanto anticipates that commercial use of MON 87708 will include export of soybean and soybean products, and has described import approval submission plans in Secti on I of petition #10-SY-210U. Monsanto’s pre-launch stewardship plan for this product is de scribed in Section VIII-L of the petition.

An extensive analysis of the socioeconomic impacts of herbicide-tolerant crops can be

found in a recent series of articles published in the on-line journal AgBioForum. 38 

### Much

of the information below has been summar ized from this series of articles.

The adoption of herbicide-tolerant (Roundup Ready) soybean has been unprecedented

since its commercialization in 1996. The t echnology was rapidly adopted by growers. Four years after being intr oduced, approximately 60% of U.S. soybean acreage was planted to Roundup Ready soybean; by 2005 over 80% of the soybean acres were planted to Roundup Ready soybean, and most recentl y in 2009 approximately 91% of soybean acres were planted to Roundup R eady varieties. Herbicide- tolerant crops have had a profound impact on agricultura l production globally and si milar adoption profiles have occurred in other geographies where herbic ide-tolerant crops have been introduced ( e.g. , Argentina, Brazil, Canada). Early efforts to understa nd the reason Roundup Ready soybean and other herbicide-tolerant crops achieved such rapid adoption focused on profitability, yield and costs (Carpenter a nd Gianessi, 2001). Information from recent grower surveys cite other advantages such as simplicity, convenience, flexibility and safety as some of the primary reasons for using Roundup Ready crops (Hurley et al., 2009). One of the most significant advant ages of Roundup Ready cropping systems has

38 The source of information can be found at: http://www.agbioforum.org/v12n34/v12n34a00-frisvold.htm [Accessed May 19, 2010].Monsanto Company 10-SY-210U 567 of 721 

### been the reduction in labor. The reduction in labor allowe d growers more free time to pursue other activities and fr eed up farm management time for non-farm income.

As with all herbicides, the intensive use of glyphosate in the absence of diverse weed management practices (i.e., other herbicide m odes-of-action, mechanical cultivation, crop rotation or other cultural practices) has led to increased selection pressure and has

contributed to weed shifts for the hard -to-control broadlea f weeds and/or the development of glyphosate-resistant weeds. Diversified weed management is recognized by Monsanto, academics and other weed scien ce experts as the guiding principle for managing resistance and shifts in weed populations. Diversif ied weed management is the cornerstone of our stewardship messaging and weed management recommendations for Monsanto’s product portfolio, including MON 87708 soybean. It is well recognized that

in situations where there is diversity in weed control met hods, few to no resistant weeds develop (Dukes and Powles, 2009; Beckie and Reboud, 2009). One strategy recommended by experts to prevent or de lay the development of resistant weed populations is to diversify herb icide weed control methods.

MON 87708 was developed to provide soybean growers with a simple and flexible option to manage existing glyphos ate-resistant weeds and bette r control of other hard-tocontrol weeds as well as delay or prevent further development of glyphosate-resistant broadleaf weeds and preserve the use of glyphosate in soybean production. Through traditional breeding methods, integrati on of MON 87708 into the Roundup Ready soybean system will allow for effective weed control through the application of glyphosate and dicamba. While either herb icide can be applied independently, it is envisioned that both glyphosate and dicamba will be tank mixe d to simplify the application. Growers may also choose to treat or not treat with either herbicide depending on the unique situation for each fa rm and their overall diversified weed management plan.

Monsanto considered the properties of herb icides available for use in soybean in developing a second herbicide-tolerance tra it to complement the glyphosate tolerance trait. Dicamba was selected because it is very complementary to glyphosate in terms of the spectrum of broadleaf weeds controlled, its postemergence activity and because very few weeds have developed resistance to di camba over many decades of use. Dicamba adds another postemergence herbicide mode-of- action for growers to use in their weed control system, thus diversifying the spect rum of weed control options and providing better overall weed control supe rior to current practices.

The socio-economic benefits of MON 87708 ar e expected to complement and help maintain the benefits growers have real ized using the Roundup Ready system. These include time and labor savings to growers th rough the simplicity and flexibility of the dicamba/glyphosate weed control system over al ternative herbicides that are authorized for use in soybean production. In addition, the ability to use dicamba in combination with glyphosate will further preserve the benefits the Roundup Ready system has provided in the form of increasing adoption of conservation tillage ac res. Monsanto and

academics recommend the use of a third herb icide mode-of-action with soil residual activity as part of a comprehensive weed re sistance management program to assure thatMonsanto Company 10-SY-210U 568 of 721 

### at least two effective modes-of -action are always used in the cultivated soybean field. The cultivation of MON 87708 provides an efficient and effective method to delay or prevent the development of glyphosate- or dicamba-resistant weeds, as well as weeds

resistant to other herbicide cl ass of chemistries, and is e xpected to outweigh additional costs of controlling resistan t weeds through the dicamba/gl yphosate weed control system.

No action alternative

Under the no action alternativ e, MON 87708 would not be available to commercial growers. Alternative herbicid es would remain available for control of broadleaf weeds in soybean fields, and these herbicides will likely be used instead of dicamba. Monsanto, university extension services and other w eed science organizations such as HRAC promote the use of integrated, diversifie d weed management systems with soybean growers; these recommendations include the rotation of herbicides with different

modes-of-action or herbicide mixtures contai ning multiple modes-of-action in their weed management program. Growers have incr easingly adopted thes e diversified weed management practices, specifically use of mu ltiple modes-of-action, in major corn and soybean geographies of the U.S. (see Section J .3.3.6). Thus it is anticipated that the cost of the weed management to the soybean grow er will not significantly change as a result of the cultivation of MON 87708. Technology pr oviders are developi ng other herbicidetolerant crops that will likely be combined with glyphosate-tolerant crops to simplify weed control which may provide similar soci oeconomic benefits to growers as those

described above for MON 87708. On the basis of the above analysis , the approval in

whole and no action alternatives are simila r regarding potential increased costs and complexity in weed control for soybean growers. However, the no action alternative would not provide as many options to preven t or delay the development of herbicideresistant weeds, and not take advantage of MON 87708 to help sustain the long-term agronomic, environmental and socioeconomic value and benefits of the Roundup Ready soybean system.

J.7. Cumulative Effects

A cumulative impact may be an effect on the environment which results from the incremental impact of the proposed action when added to other past, present, and reasonably foreseeable future actions.

J.7.1. Conventional Breeding with Other Biotechnology-derived or Conventional Soybean

The potential effects associated with a de termination of nonregulated status of a biotechnology-derived crop in combination wi th the future producti on of crop varieties

with different GE traits that are no longer subject to the regulatory requ irements of 7 CFR part 340 (i.e., “stacked” traits) woul d be considered a cumulative impact.

Approval in whole alternative

As previously mentioned, several biotechnolo gy-derived soybean products have been deregulated or are under consideration fo r deregulation, and a list of the eventsMonsanto Company 10-SY-210U 569 of 721 

### deregulated by USDA is presen ted in Table J-4. Once deregulated, MON 87708 may be bred with these deregulate d biotechnology-derived soybean products as well as with conventional soybean, creating new improved vari eties. APHIS has de termined that none of the individual biotechnology- derived soybean products it has previously deregulated displays increased plant pest characteristic s or creates potentially significant impacts on

the human environment. APHIS has also concluded that any progeny derived from crosses of these deregulated biotechnology-de rived soybean products with conventional or previously deregulated biotechnology-de rived soybean are unlikely to exhibit new plant pest properties. This presumption th at combined trait biotechnology products are unlikely to exhibit new character istics that would pose new pl ant pest risks or potential environmental impacts not observed in the si ngle event biotech pr oduct is based upon several facts. Namely 1) stability of the gene tic inserts is confirmed in each single event product across multiple generations; 2) stabi lity of each of the introduced traits is continually and repeatedly assessed as new co mbined trait varieties are created by plant breeders and tested over multiple seasons prior to commercial release; 3) combined trait products are developed using th e well established process of conventional breeding that has been safely used for thous ands of years to generate ne w varieties; and 4) in both principle and practical application in the fi eld, it has been shown that two unrelated biotechnology traits combined together by conventional br eeding, do not display new characteristics or properties distinct from those present in the single event biotech products.

Demonstrated Genetic and Phenotypic Stability

An assessment of the stability of the genetic inse rt in MON 87708 is discussed in Section IV of petition #10-SY-210U and summarized here ; refer to the petition for more detail. Generational stability analysis was assessed by Southern blot analysis and demonstrated that the genetic insert present in MON 87708 was maintained across the five breeding generations evaluated. These data demonstr ate that MON 87708 is stable in its progeny. Having established that the genetic material is stable and inherited in a Mendelian fashion, and based on experience with MON 87708 in Monsanto’s plant breeding program over the course of many ge nerations and many field seasons 39 

### , it is concluded that the phenotype of MON 87708 is likewise stable. There are numerous examples in

the literature that confirm that GE events and their associated phenotypes and overall characteristics are stably inherited across generations and across genetic backgrounds, including when they are combined by convent ional breeding to produce combined trait products (McCann et al., 2005, Ridl ey et al., 2011, Zhou et al., 2011).

Combined Trait Product Performan ce in Principle and in Practice

Conventional plant breeding is routinely used to improve crop performance and is specifically employed to develop plant variet ies that fit particular environments and production practices ( Powell et al., 2003 ). The same biological and selective principles used for conventional and single GE event variety development are used to combine previously approved GE events. When c onventional breeding is used to generate varieties with combined GE events, these va rieties are screened over multiple generations

39 Field trials have been conducted with MON 87708 since 2005.Monsanto Company 10-SY-210U 570 of 721 

### and across diverse growing environments . Typically, product performance and agronomic features are evaluated and cr op characteristics such as yield, field performance, and disease resistance are measured and tested to ensure that the traits are stable, heritable, and expre ss the desired phenotype under a wi de range of environmental conditions. The phenotypic characteristics evaluated during the screening of new

combined GE event candidate va rieties are the result of the plant’s genotype and are the culmination of the complex metabolic path ways that are activated in response to environmental conditions. The evaluation of phenotypic characteristics allows breeders to assess and screen for potential unintended effects produced as a result of the various traits (both GE traits and other inherent trai ts) combined in the va riety. Selection during the conventional breeding proces s is valuable in removing unde sirable characteristics and thereby helps to maintain the safety and quality of the food a nd/or feed product ( Cellini et al., 2004; NRC, 2004; WHO, 1995 ).

When assessing the safety of combined GE events, it is important to consider international guidance regarding convent ional breeding and assessments of the substantial equivalence of GE events combined by conventional breeding. For example, the World Health Organization concludes that a substantial equivalence conclusion can

be maintained in a combined GE trait va riety if substantial equivalence had been demonstrated for each of the single event pa rents. Specifically, they argue that ‘‘...if substantial equivalence has been demonstrated both for a [genetically engineered] tomato with a gene producing a delaye d ripening phenotype and for a [genetically engineered] tomato with a gene for herbicide resistance, then crossing these two varieties would result in a new variety that would be expected to be substantially equivalent to the parents’’ ( WHO, 1995 ). Additional international groups, including the Food and Agriculture Organization/World Health Organization ( FAO, 1996 ), International Seed Federation ( ISF, 2005 ), and CropLife Internatio nal (CLI, 2005) similarly advocate basing the safety of combined GE events on the safety of the pa rental GE events. The FDA has stated that ‘‘narrow crosses are unlikely to result in unint ended changes to foods that raise safety or other regulatory questions’’ – ‘‘including narrow crosses between different rDNAmodified [GE] lines’’ (U.S. FDA, 2001 ).

Thus, the data packages that are developed for single GE events are useful in establishing the safety of the combined GE event product. Accordingly, single GE events previously

assessed to be as safe as thei r conventional counterparts should continue to be safe when combined through conventional plant breeding. As a result, the rigorous safety assessments and plant pest ri sk assessments conducted on si ngle GE events, which were deemed to pose no more risk than their conv entional counterparts, also can be used to predict the safety and potentia l risk of the combined GE event product. As described below, this assertion is supported by dir ect experience gained through the commercial planting and utilization of combined GE even t products as well as a body of information collected to support authoriza tions where combined GE even t products require additional

assessment.

There are many examples of combined trait biotech products that have been produced commercially on millions of acres without inci dent over the past decade in both the USMonsanto Company 10-SY-210U 571 of 721 

### and in other countries 40 

### . In the US, there are over 30 co mbined trait products that have been made commercially available. In 2011, 49% of the total corn acres planted (92 M acres) and 58% of total cotton planted (13M acres) contained multiple (2 or more) GE events (USDA-ERS, 2011) 41 

### . Combined trait biotech pr oducts were planted on 40 million hectares or 25% of the global bi otech area of 160 million hectares in 2011 42



### . The safety of commercially available co mbined trait biotech products has been well-demonstrated in multiple independent reports that document the continually increasing acceptance and use of th ese products by farmers globally ( Brookes and Barfoot, 2009; James, 2009; Lemaux, 2008; Sankula, 2006 ).

In addition, regulatory agencies in some c ountries request additiona l characterization of combined GE events and comparisons to singl e event parental controls and conventional comparators. These additional studies may include analysis of phenotype, molecular characteristics, protein characteristics, morphology and compositional evaluation. Analyses of combined GE events compared to parental controls have consistently revealed the following: no phenotypic differe nces from parental events; molecular characteristics that are the same as parental ev ents with all events inherited stably; levels of introduced proteins comparable to th e single event parent s; no morphological differences compared to parental events; compositional equivalence based on nutrient and anti-nutrient evaluations, and no pleiotropic or toxic effects compared to the conventional non-GE crop (Pilacinski et al., 2011).

Summary

Traditional soybean breeding has an establ ished history of safe use, and use of MON 87708 in breeding programs is expected to behave in a manner similar to other conventional traits and biotechnology-derived tr aits. Given that there have been no plant pest characteristics associated with M ON 87708, or with any of the previously deregulated events listed in Table J-5, no significant imp acts are expected to other soybean through the use of MON 87708 in br eeding programs and in combination with any of the previously deregulated bi otechnology-derived soybean products.

All biotechnology-derived soybean products on the market today have satisfactorily completed the FDA consultation process estab lished to review the safety of foods and feeds derived from biotechnology-derived crops for human and animal consumption (see Table J-5). As mentioned previously, M ON 87708 is intended to be combined with MON 89788 (Roundup Ready 2 Yield soybean ) through traditi onal breeding. MON 87708 is expected to be utilized broa dly in future additional combined trait soybean products, and thus will likely be bred with other biotechnology-derived soybean products that have been deregul ated or have deregulation pe titions pending before APHIS ( e.g. , MON 87701, MON 87705, MON 87769; see Table J-4 for current status). No impacts to public health ( e.g. , food or feed safety) are e xpected due to combination of these events through conventional breeding beca use the deregulated events have or will

40 See http:// www.biotradestatus.com for list of combined trait products that have previously been

commercialized.

41 See http://www.ers.usda.gov/data/biotechcrops/adoption.htm

42 http://www.isaaa.org/resource s/publications/briefs/43/ex ecutivesummary/default.aspMonsanto Company 10-SY-210U 572 of 721 

### have completed a safety consultation with FDA and on the basis of knowledge of the type of modifications made to each of the deregu lated events, and to th e events under review, the biochemical pathways are unrelated to the trait introduced in MON 87708 and therefore not expected to interact or re sult in the production of novel properties or

constituents.

The decision to approve in whole MON 87708 would also allow for breeding of this product with conventional soybean of diverse genetic background. No impacts to public health ( e.g. , food or feed safety) or environmental safety are expected due to the breeding of MON 87708 with these other soybean beca use these varieties ha ve an established history of safe use.

Based on the above analysis, the approval in whole and no action alte rnatives are similar regarding potential cumulative impacts asso ciated with the conventional breeding of

MON 87708 with other conventional or bi otechnology-derived soybean products.

No action alternative

Under the no action alternative, MON 87708 wo uld not be used to produce new soybean

varieties or in the commercia l production of soybean beca use the trait would remain regulated. There are no effects that ha ve been identified from combining MON 87708

with other Monsanto biotec hnology-derived soybean that have been deregulated by USDA; therefore, the no acti on and approval in whole alternatives are similar regarding their potential cumulative impacts associat ed with conventional breeding with other soybean products.

J.7.2. Conservation Tillage

Approval in whole alternative

The single most damaging effect on land due to agriculture is loss of soil caused by tillage. Tillage is primarily performed for seed bed preparat ion and has the added benefit as a weed control measure. Roundup Rea dy crops, with Roundup Re ady soybean being the most widely adopted, have enabled signi ficant implementation/adoption of no-till or reduced tillage methods for weed c ontrol (Duke and Powles, 2009).

In 1995, before the introduc tion of Roundup Ready soybean, approximately 27% of the U.S. soybean acres used no-till production. By 2004, no-till acreage increased to 36% of the total soybean acres (Sankula, 2006). The most recent surveys indicate that 39% of the soybean acres are produced using no-till methods (CTIC, 2007). A few states provide statistics on the adoption of no-till acres in thei r state. In 2006 in Illinois, no-till farming was used on 51% of the soybean acres. The University of Illinois Extension Service attributed that figure to the fact that 90% of the state’s soybean acres were planted to Roundup Ready soybean varieties, along with other factors, such as high fuel prices, improved equipment, higher yields using no-til l practices, and better grower awareness of the advantages to soil and water quality from no-till farming. Approval in whole would allow for continued adoption and preservation of conservation tillage methods resultingMonsanto Company 10-SY-210U 573 of 721 

### in positive cumulative environmental impacts. Therefore, the appr oval in whole and no action alternative are similar regarding the potential cumulative impacts on conservation tillage practices

No action alternative

Under this alternative, MON 87708 would not be grown commercially and growers

would not be able to utilize dicamba to the same extent as they would if APHIS granted approval in whole to MON 87708. This al ternative would limit grower’s options regarding the selection of herbicides available for use in conservation tillage. This option could potentially result in an increased use of tillage over time and potential loss of no-till acres in some soybean fields, thus decreasi ng the use of conservation tillage methods and

environmental benefits associ ated with conservation tillage. Overall, the no-action and approval in whole alternatives are similar re garding the potential cumulative impacts on conservation tillage practices.

J.7.3. Resistant Weeds

Approval in whole alternative

Another cumulative effect includes the potential for development of herbicide-resistant weeds, including weed populations with multip le resistance. Integration of MON 87708 into the Roundup Ready soybean system ha s the potential to delay or prevent development of dicamba- and glyphosate-resist ant weeds, as well as resistance to other

herbicide classes such as PPO herbicides. These issues have been discussed in Section J.5.3.1 and in Appendix K of petition #10-SY- 210U, but warrant additional discussion

under the assessment of cumulative effects. The development of resistant weed populations occurs over a period of time (Hea p, 2009), and is an foreseeable consequence

of herbicide use in the absence of diverse w eed management practices as is discussed in

additional detail in Section J.3.3.6. One of the strategies recommended by experts to prevent or delay the development of resist ant weed populations is to diversify weed

control methods. The use of herbicides such as dicamba in conjunction with glyphosate provides growers with an herbicide system w ith two different modes-of-action. Thus, it is foreseeable that integration of MON 87708 into the Roundup Ready soybean system with the use of dicamba and glyphosate to gether would have a low probability for increases in new weed populati ons developing resistance to eith er glyphosate or dicamba. In addition there would be the potential fo r fewer new species to evolve or develop resistance for either herbicide compared to a glyphosate or dicamba herbicide system alone. In the MON 87708 weed management system the use of glyphosate is not expected to increase. Even thought there will be an increase in the use of dicamba, it is

clear that in situations where there is diversity in weed control methods, there will be a low probability for resistant weeds to deve lop (Dukes and Powles, 2009; Beckie and Reboud, 2009). In the unlikely case that broadleaf weeds were to develop with resistance

to dicamba or with accumul ated resistance to both glypho sate and dicamba, existing

cultivation and alternative herbicide tools (see Section VIII.F.1 for description of alternative herbicides) would remain potential options to provide effective control.Monsanto Company 10-SY-210U 574 of 721 

### Therefore, the approval in w hole and no action alte rnatives are similar regarding potential cumulative impacts on weed resistance.

No action alternative

Other herbicide options for weed resistance management are av ailable to growers. Under the no action alternative, grower’s options regarding the selection of herbicides available for use to control weeds in soybean and speci fically herbicides with different modes-ofaction would be more limited than the approval in whole alternative as a result of the inability to use dicamba in the weed management system for soybean. Other soybean herbicides are available to growers to use e ither with or without the dicamba-glyphosate tolerant soybean system, and will remain available to growers in the future. However, herbicide weed management systems under the no action alternative are not as efficient and effective for managing resistant weeds a nd incorporating additional herbicide modesof-action. Thus, it is reasonable foreseeable that some increase in the development of

additional herbicide-resistant w eed populations or developmen t of new herbicide resistant weed species compared to the approval in whol e alternative will result. Therefore, the no action and approval in whole al ternatives are similar regarding the potential cumulative

effects on weed resistance.

J.8. Summary

MON 87708 has been thoroughly characterized and the extensive body of information presented in Sections I through IX of the petition demonstrate that MON 87708 does not present a plant pest risk, has no significant impact on threatened and endangered species or biodiversity, and will not impact the commerc ial interests of soybean growers or those involved in the marketing and sale of soybean and soybean products. The amount of land devoted to farming (specifically to soybean ) has not changed with the introduction of biotechnology-derived crops. Si milarly, no significant change in the use of agricultural land or amount of land devoted to farming would be expected to occur with the commercial introduction of MON 87708 integr ated into the Roundup Ready soybean system. MON 87708 integrated into the Roundup Ready soybean system will offer growers the opportunity to expand the use of dicamba to late preemergence (up to cracking) and postemergence in -crop applications and, ther eby, potentially delay or prevent the development of dicamba- and glyphosa te-resistant weeds as well as resistance to other soybean herbicides in U.S. soybean fields. Other cultivation practices typically employed for production of commodity soybean , and management practices will remain unchanged. Growers are accustomed to the use of herbicides in their soybean production systems. For these reasons, the proposed action to grant nonregulated status to MON 87708 does not represent a signifi cant impact to the environment.Monsanto Company 10-SY-210U 575 of 721 

### References for Appendix J

Abrams, R.I., C.R. Edwards, and T. Ha rris. 1978. Yields and cross-pollination of soybeans as affected by honey bees and alfalf a leafcutting bees. American Bee Journal

118:555-560.

Abud, S., P.I. Mello de Souza, C.T. Moreir a, S.R.M. Andrade, A.V. Ulbrich, G.R. Vianna, E.L. Rech, and F.J. Lima Aragao. 2003. Pollen dispersal in transgenic soybean plants in the Verrado region. Pesquisa Agropecuária Brasileira 38:1229-1235.

Al-Kaisi, M. 2001. Value of crop rotation in ni trogen management. Iowa State University. http://www.ipm.iastate.edu/ipm /icm/2001/4-23-2001/valuen.html.

Al-Kaisi, M., M.H. Hanna, and M. Tidma n. 2003. Crop rotation c onsiderations for 2004 management season rotation. Department of Entomology, Iowa State University. http://www.ent.iastate.edu/ipm/icm/2003/croprotation.html.

Altieri, M.A. 1999. The ecological role of biodiversity in agroecosystems. Agriculture, Ecosystems and Environment 74: 19-31.

Aneja, V. P., Schlesinger, W. H., & Erisma n, J. W. (2009). Effect s of agriculture upon the air quality and climate: Research, polic y, and regulations. Environmental Science & Technology, 43, 4234–4240

AOSCA. 2009. Seed certification handbook. Associ ation of Official Seed Certifying Agencies, Moline, Illinois.

Aref, S. and D.R. Pike. 1998. Midwest farmers’ perceptions of crop pest infestations. Agronomy Journal 90:819-825.

ASA. 2009. Soy Stats. American Soybean Association, St. Louis, Missouri.

ASA. 2010. Domestic utilization, U.S. soybean use by livestock 2009. American Soybean Association, St. Louis, Missouri. http://www.soystats.com/2010/Defaultframes.htm

. Accessed July 26, 2010

.

ASA. 2011. Soy stats 2011. American Soybean Association, St. Louis, Missouri. http://www.soystats.com/2011/Default-frames.htm

. Accessed February 10, 2012

.

ASTA. 2011. Existing U.S. industry producti on practices that address coexistence.

American Seed Trade Associ ation, Alexandria, Virginia.

Baker, H.G. 1965. Characteristics and modes of origin of weeds. Pages 147-172 in The Genetics of Colonizing Species. H.G. Baker a nd G.L. Stebbins (eds.). Academic Press, New York, New York.Monsanto Company 10-SY-210U 576 of 721 

### Baker, J., Southard, R., & Mitchell, J. ( 2005). Agricultural dust production in standard andconservation tillage systems in the San Joaquin Valley. Journal of Environmental Quality, 34 , 1260-1269. doi: 10.2134/jeq2003.034

Beckie, H.J. 2006. Herbicide-resistance: Ma nagement tactics an d practices. Weed Technology 20:793-814.

Beckie, H.J. and X. Reboud. 2009. Selecting for weed resistance: Herbicide rotation and mixture. Weed Technology 23:363-370.

Boerma, H.R. and J.E. Specht. 2004. World distribution and trade of soybean and managing inputs for peak production; soybean production in the U.S.A. Pages 3, 502, 507 and 522, In Soybeans: Improvement, production, a nd uses. Boerma H.R. and J.E. Specht (eds.). ASA, CSSA-SSSA, Madison, Wisconsin.

Brookes, G. and P. Barfoot. 2004. Co-exi stence case study of North America: Widespread GM cropping with non GM and organic crops. PG Economics Ltd., Dorchester, United Kingdom.

Brookes, G. and P. Barfoot. 2009. GM crops: Global socio-economic and environmental impacts 1996-2007. PG Economics, Ltd, Dorchester, United Kingdom. www.pgeconomics.co.uk/pdf /2009globalimpactstudy.pdf Accessed May 12, 2010

.

Buckley, D.H. and T.M. Schmidt. 2001. The structure of microbial communities in soil and the lasting impact of cultiv ation. Microbial Ecology 42: 11-21.

Buckley, D.H. and T.M. Schmidt. 2003. Diversity and dynamics of microbial communities in soils from agro-ecosystems. Environmental Microbiology 5: 441-452.

Carpenter, J.E. and L.P. Gianessi. 2001. W hy U.S. farmers have adopted genetically modified crops and the impact on U.S. agriculture. AgBiotechNet 3:1-4.

Carpenter, J., A. Felsot, T. Goode, M. Hammig, D. Onstad and S. Sankula. 2002. Comparative environmental impacts of biot echnology-derived and traditional soybean, corn, and cotton crops. Council for Agricult ural Science and Technology, Ames, Iowa.

CAST. 2011. Herbicide-resistant weeds thre aten soil conservation gains: Finding a balance for soil and farm sustainability. C ouncil for Agricultural Science and Technology, Ames, Iowa.

Caviness, C.E. 1966. Estimates of natural cross-pollination in Jackson soybeans in Arkansas. Crop Science 6:211-212.

Cellini, F., Chesson, A., Colquhoun, I., Constable, A., Davies, H.V., Engel, K.H., Gatehouse, A.M.R., Karenlampi, S., Kok, E.J., Leguay, J.J., Lehesranta, S., Noteborn, H., Pedersen, J., Smith, M., 2004. Unintended eff ects and their detec tion in genetically modified crops. Food and Chemical Toxicology 42, 1089–1125.Monsanto Company 10-SY-210U 577 of 721 

### Chiang, Y.C., and Y.T. Kiang. 1987. Geomet ric position of genotypes, honeybee foraging patterns and outcrossing in soybea n. Botanical Bulletin of Academia Sinica

28:1-11.

CLI. 2005. Position paper: Regulation of plan t biotechnology products containing two or more traits combined by conventional plant br eeding. Crop Life International, Brussels, Belgium.

CTIC. 2004. National survey shows more farmers choose conservation. Conservation Technology Information Center, West Lafayette, Indiana. http://www.ctic.purdue.edu/ctic/ctic.html [Accessed November 9, 2004].

CTIC. 2007. Amendment to the national crop residue management survey summary. www.conservationinformation.org/pdf/Na tional_Summary_2007_Amendment.pdf
Accessed on June 14, 2010

.

CTIC. 2010. 2010 Amendment to the national crop residue management survey summary. www.conservationinformation.org/pdf/N ational_Summary_2007_Amendment.pdf
Accessed June 14, 2010

.

CTIC. 2011. Top 10 conservation tillage benefits : Conservation for agriculture's future. Conservation Technology Information Center, West Lafayette, Indiana. http://www.ctic.purdue.edu/resourcedisplay/293/ Accessed September 21, 2011

Cui, Z., A.T. James, S. Miyazaki, R.K. Wilson and T.E. Carter. 2004. The breeding of specialty soybeans for traditional and new s oyfoods. Pages 264-322 in Proceeding of the American Oil Chemists' Society, Urbana, Illinois.

Dalley, C.D., K.A. Renner, and J.J. Kells . 2001. Weed competition in Roundup Ready soybean and corn. Michigan State Univer sity, Dept of Crop and Soil Science. http://www.ipm.msu.edu/CAT05_fld/pdf/5-26FactSheet.pdf Accessed on June 15, 2010

.

Diggle, A.J., P.B. Neve and F.P. Smith. 2003. Herbicides used in combination can reduce the probability of herbicide resistance in finite weed populations. Weed Research

43:371–382.

Dorrance, A.E., M.A. Draper, and D. Hers hman. 2007. Using foliar fungicides to manage soybean rust. Ohio State Un iversity, Columbus, Ohio.

Duke, S.O., and S.B. Powles. 2009. Glyphosate -resistant crops and weeds: Now and in the future. AgBioForum 12:346-357.

Durkin, P. and S. Bosch. 2004. Dicamba - huma n health and ecological risk assessment - final report. Prepared for USDA, Fo rest Service. SERA TR 04-43-17-06d. http://www.fs.fed.us/foresthea lth/pesticide/pdfs/112404_dicamba.pdf Accessed May 26,

2010

.Monsanto Company 10-SY-210U 578 of 721 

### European Commission. 2007a. Annex B9. Ecotoxicology. Draft Assessment Report (DAR): Initial Risk Assessment Provided by the Rapporteur Member State Denmark for the Existing Active Substance Dicamba of the Third Stage (part B) of the Review Programme Referred to in Article 8(2) of the Council Directive 91/414/EEC. European Commission, Brussels, Belgium.

European Commission. 2007b. Annex B6. Toxicology and metabolism. Draft Assessment Report (DAR): Initial Risk Asse ssment Provided by the Rapporteur Member State Denmark for the Existing Active Substanc e Dicamba of the Third Stage (part B) of the Review Programme Referred to in Article 8(2) of the Council Directive 91/414/EEC. European Commission, Brussels, Belgium.

FAO. 1996. Joint FAO/WHO expert consul tation on biotechnology and food safety. Paper 61. Food and Agriculture Organization, World Health Organization, Rome, Italy.

Fawcett, R. and D. Towery. 2002. Conser vation tillage and plant biotechnology: How new technologies can improve the envi ronment by reducing the need to plow. Conservation Technology Informa tion Center, Purdue, Indiana.

Felsot, A.S., J.B. Unsworth, J.B.H.J. Linders , G. Roberts, D. Rautman, C. Harris and E. Carazo. 2010. Agrochemical spray drift; A ssessment and mitigation—A review. Journal

of Environmental Science a nd Health, Part B 46: 1-23.

Frank AR, Kemman R. 2011. Informa tion to Support an Endangered Species

Assessment for Dicamba Use in Dicamba-Tole rant Soybeans: A County-Level Analysis for Animal Taxa. Monsanto Technical Report MSL-22547. EPA MRID 48644204.

Frank AR, Kemman R. 2012. Information to Support an Endangered Species Assessment for Dicamba Use in Dicamba-Tolerant Soybeans: A County-Level Analysis for Plant Taxa. Monsanto Technical Report MSL- 2 3442. EPA MRID 48644206

Gage, D.J. 2004. Infection and invasion of r oots by symbiotic, nitrogen-fixing rhizobia during nodulation of temperate legumes. Microbiology and Molecular Biology Reviews

68:280-300.

Galle, A.M., G.M. Linz, H.J. Homan and W.J. Bleier. 2009. Avian use of harvested crop fields in North Dakota during spring migra tion. University of Nebraska-Lincoln, USDA National Wildlife Research Center, Lincoln, Nebraska. Gianessi, L.P., C.S. Silvers, S. Sankula and J.E. Carpenter. 2002. Plant biotechnology: Current and potential impact for improving pest management in U.S. Agriculture, an analysis of 40 case studies. National Cent er for Food and Agricultural Policy. Washington, DC.

Givens, W.A., D.R. Shaw, W. G. Johnson, S.C. Weller, B.G. Young, R.G. Wilson, M.D.K. Owen, and D. Jordan. 2009. A grower survey of herbicide use patterns in glyphosateresistant cropping systems. Weed Technology 23:156-161.

Harlan, J.R. 1975. Our vanishing genetic resources. Science 188: 617-621.Monsanto Company 10-SY-210U 579 of 721 

### Heap, I. 2009. International survey of herbicide resistant weeds. http://www.weedscience.org/in.asp Accessed November 11, 2009

.

Heap, I. 2011. International survey of herbicide resistant weeds. http://www.weedscience.org/in.asp Accessed December 11, 2011 .Heatherly, L.G., and H.F. Hodges. 1999. Soybean production in the Midsouth. CRC Press, Boca Raton,

Florida.

Helsel, Z.R. and H.C. Minor. 1993. Soybean production in Missouri. University of Missouri, Department of Ag ronomy. Publication G4410. http://extension.missouri.edu/e xplore/agguides/crops/g04410.htm Accessed November
11, 2009

.

Herro, A. 2008. Adjustments to agricultu re may help mitigate global warming. Worldwatch Institute, Washington, D. C. http://www.worldwatch.org/node/5580 [Accessed April 7, 2011].

Higley, L.G., and D.J. Boethel. 1994. Handbook of soybean insect pests. The Entomological Society of America, Lanham, Maryland.

Hoeft, R.G., E.D. Nafziger, R.R. Johns on, and S.R. Aldrich. 2000. Soybean as a crop. Pages 31, 36, 38, 39, 41, 43, 47, 86, 89, 93, 9 6, 120, and 208, In Modern Corn and Soybean. Production MCSP Publications, Champaign, Illinois.

Honegger, JL. 2012. Information to Support an Endangered Species Assessment for Dicamba Use in Dicamba-Tolerant Soybeans: Overview of an Evaluation of Potential Exposure and Biological Eff ects. Monsanto Technical Report MSL-22547. EPA MRID

48644201

HRAC. 2010. Herbicide Resistance Action Co mmittee. Publications: Guideline to the management of herbicide resistance. http://www.hracglobal.com/Publications/Man agementofHerbicideResistance/tabid/225/D efault.aspx Accessed June 29, 2010

.

Hurley, T.M., P.D. Mitchell, and G.B. Fris vold. 2009. Characteristics of herbicides and weed-management programs most important to corn, cotton and soybean growers. AgBioForum 12:269-280.

Hymowitz, T. 1987. Introduction of the soybean to Illinois. Economic Botany 41:28-32.

Iowa State University. 2006. Cover crops in the corn belt: survey finds underused potential as conservation tool. Leopold Center for Sustainabl e Agriculture. Winter 2006

Letter. http://www.leopold.iastate.e du/news/leopold-letter/2006/ winter/cover-crops-cornbelt-survey-finds-underuse d-potential-conservation Accessed February 12, 2012.

ISF. 2005. Genetically modified crops and plan t breeding. Internationa l Seed Federation, Nyon, Switzerland.Monsanto Company 10-SY-210U 580 of 721 

### ISO. 2009. International Organi zation for Standardization. Se lection and use of the ISO 9000 family of standards. http://www.iso.org Accessed Jan. 9, 2009

.

James, C., 2009. Global status of commerc ialized biotech/GM cr ops: 2009. ISAAA Brief 41. International Service for the Acquisition of Agri-Biotech App lications, Ithaca, New

York.

Johnson, B., B. Young, J. Matthews, P. Marqua rdt, C. Slack, K. Bradley, A. York, S. Culpepper, A. Hager, K. Al-Khatib, L. St eckel, M. Moechnig, M. Loux, M. Bernards and

R. Smeda. 2010. Weed control in dicamba -resistant soybeans. Plant Management Network, St. Paul, Minnesota. http://www.plantmanagementnetwork.org/sub/cm/research/2010/dicamba/ Accessed September 20, 2010

.

Kerley, M.S. and G.L. Allee. 2003. Modifi cations in soybean seed composition to enhance animal feed use and value: Moving from a dietary ingred ient to a functional dietary component. AgBioForum 6:14-17.

Landis, D.A., F.D. Menalled, A.C. Costama gna and T.K. Wilkinson. 2005. Manipulating plant resources to enhance be neficial arthropods in agricu ltural landscapes. Weed Science

53:902-908.

Lemaux, P.G., 2008. Genetically e ngineered plants and foods: A scientist’s analysis of the issues (part I). Annual Re view of Plant Biology 59: 771–812.

Loux, M.M., D. Doohan, A.F. Dobbels, W.G. Johnson, G.R.W. Nice, T.N. Jordan, and T.T. Bauman. 2009. Weed control guide for Ohio and Indiana. Purdue University and Ohio State. Publication WS16. http://www .btny.purdue.edu/Pubs/ws/ws-16/ Accessed on June 14, 2010.

Lovett, S., P. Price and J. Lovett. 2003. Mana ging riparian lands in the cotton industry. Cotton Research and Development Co rporation, Narrabri, Australia.

LSU. 2009. Asian soybean rust: Background. L ouisiana State University, Baton Rouge,

Louisiana. http://www.agctr.lsu.edu/en/crops_lives tock/crops/soybeans/Asian_Soybean_Rust/
Accessed June 17, 2010

.

MAFRI. 2004. Salvaging immature and frosted crops for silage. Manitoba Agriculture, Winnipeg, Manitoba. http://www.gov.mb.ca/ag riculture/news/topics Accessed May 12,

2010.

Mannering, J.V., D.R. Griffith and K.D. J ohnson. 2007. Winter cover crops - Their value and management. AY-247. Purdue University Cooperative Extension Service, West Lafayette, Indiana.Monsanto Company 10-SY-210U 581 of 721 

### McCann, M.C., K. Liu, W.A. Trujillo and R.C. Dobert. 2005 Glyphosate-Tolerant Soybeans Remain Compositionally Equivalent to Conventional Soybeans ( Glycine max L.) during Three Years of Field Testing. J ournal of Agricultural and Food Chemistry

53:5331-5335.

Monsanto Company. 2011. Letter of completion of biotechnology consultaions on BNF 00125. Food & Drug Administration, Department of Health & Human Services, Center for Food Safety and Applied Nutrition, Office of Food Additive Safety. October 11, 2011. http://www.fda.gov/Food/Biotec hnology/Submissions/ucm282993.htm

Mueth, MG and JE Foster. 2010. Determinatio n of Dicamba Residue Decline in Forage after Application to Dicamba-Tolerant Soybean MON 87708 × MON 89788. Monsanto Technical Report MSL-22493. EPA MRID 48644205

Nice, G. and B. Johnson. 2005. Indiana's top ten most problematic weeds. Purdue Extension Weed Science. http://www .btny.purdue.edu/WeedScience/2005/topten05.pdf
Accessed November 11, 2009

.

NDSU. 2002. Soybean production guide for Nort h Dakota and Northwestern Minnesota. North Dakota State University Extension Service and North Dakota Soybean Council, Fargo, North Dakota. http://www.ag.nds u.edu/pubs/plantsci/rowcrops/a1172.pdf
Accessed November 11, 2009

.

NRC. 2004. New approaches for identifying unintended changes in food composition. Pages 73-102 in Safety of Genetically E ngineered Foods: Approaches to Assessing

Unintended Health Effects. Institute of Medicine and National Research Council, National Academies Press, Washington, D.C.

OECD. 2000. Consensus document on the biology of Glycine max (L.) merr. (soybean). ENV/JM/MONO(2000)9. Series on Harmoniza tion of Regulatory Oversight in Biotechnology No.15. Organisation for Economic Co-operation and Development, Paris,

France.

Pedersen, P. 2008. Soybean production. Io wa State University Extension. http://extension.agron.ia state.edu/soybean Accessed November 11, 2009

.

Peterson, D., C. Mayo and S. Seifert-Higgins. 2011. Weed c ontrol in dicamba tolerent soybeans in Kansas. North Central Weed Science Society 66 th Annual Meeting, Milwaukee, Wisconsin.

Pilacinski, W., A. Craw ford, R. Downey, B. Harvey, S. Huber, P. Hunst, L.K. Lahman, S. Macintosh, M. Pohl, C. Rickard, L. Tagliani and N. Weber. 2011. Plants with genetically modified events combined by conventional breeding: An assessment of the need for additional regulatory data. Food a nd Chemical Toxicology 49: 1-7.

PMRA. 2008. Pest Management Regulatory Agency Re-evaluation – dicamba. Pest Management Regulatory Authority, Health Canada. Ottawa, Canada.Monsanto Company 10-SY-210U 582 of 721 

### Powell, W.,R. Waugh, J. Bradshaw, J. Russell, L. Ramsay and B.P. Forster. 2003. Introduction to classical genetics and pl ant breeding. Pages 3-29 in Handbook of Plant Biotechnology. Volume 1. P. Christou and H. Klee (eds.). John Wiley and Sons, Inc., Hoboken, New Jersey.

Powles, S.B., C. Preston, I.B. Bryan and A. R. Jutsum. 1996. Herbicid e resistance: Impact and management. Pages 57-93 in Advances in Agronomy. Volume 58. D.L. Sparks (ed.). Academic Press, San Diego, California.

Prince, J.M., D.R. Shaw, W.A. Givens, M. D.K. Owen, S.C. Welle r, B.G. Young, R.G. Wilson and D.L. Jordan. 2011. Benchmark study: IV. Survey of grower practices for managing glyphosate-resistant weed populations . Weed Science Society of America.
Accessed December 15, 2011

.

Ray, J.D., T.C. Kilen, C.A. Abel, and R.L. Paris. 2003. Soybean natu ral cross-pollination

rates under field condit ions. Environmental Biosafety Research 2:133-138.

Ridley, W.P., G.G. Harrigan, M.L. Breeze, M. A. Nemeth, R.S. Sidhu and K.C. Glenn. 2011. Evaluation of compositional equivale nce for multitrait biotechnology crops. Journal of Agricultural and Food Chemistry 59:5865-5876.

Sankula, S. 2006. Quantification of the imp acts on U.S. agricultu re of biotechnologyderived crops planted in 2005. National Ce nter for Food and Agriculture Policy, Washington, D.C.

Schuler LJ, Leopold VA, Mortensen SR. 2011. Information to Support an Endangered Species Assessment for Dicamba Use in Dicam ba-Tolerant Soybeans: Refined Analysis

for Terrestrial Animals. Monsanto Technical Report MSL-22546. EPA MRID

48644203.

SDTF. 1997. A summary of ground a pplication studies. Spray Drif t Task Force, St. Louis,

Missouri. http://www.agdrift.com/PDF_FILES/Ground.pdf Accessed August 10, 2010

.

Sharpe, T. 2010. Tarheel wildlife - A guide for managing wildlife on private lands in

North Carolilna. North Carolina Wildlif e Resources Commission, Raleigh, North

Carolina.

Soyatech. 2010. Soya and oilseed bluebook: St atistics and reference. Southwest Habor,

Maine.

Stewart, C.N., M.D. Halfhill and S.I. Warwick. 2003. Transgene introgression from genetically modified crops to their w ild relatives. Nature Reviews Genetics 4:806-817.

Tranel, P.J., C.W. Riggins, M.S. Bell and A.G. Hager. 2010. Herbicide resistances in

Amaranthus tuberculatus: A call for new options. Journal of Agricultural and Food Chemistry 59: 5808-5812. Tiedje, J.M., S. As uming-Brempong, K. Nusslein, T.L. Marsh and S.J. Flynn. 1999. Opening the black box of soil microbial diversity. Applied Soil Ecology 13: 109-122.Monsanto Company 10-SY-210U 583 of 721 

### Towery, D. and S. Werblow. 2010. Facilita ting conservation farming practices and enhancing environmental sustainability with agricultural biotechnology: Exective summary. Conservation Technology Informati on Center, West Lafayette, Indiana. http://www.ctic.org/Bio techSustainability. Accessed January 27

, 2012.

USB. 2011a. U.S. soybean production: A comp arison of sustainabl e production systems for conventional, biotech, and organic soybeans. United Soybean Board, Chesterfield,

Missouri.

USB. 2011b. Soyfoods Guide 2011. United Soyb ean Board, Chesterfield, Missouri. http://www.soyconnection.com/soyfoods/pdf/soyfoods_guide.pdf Accessed on January

27 , 2012 U.S. EPA. 1992. Pesticides in groundw ater database: A compilation of monitoring studies: 1971-1991. National Summary [EPA 734-12-92-001]. U.S. Environmental Protection Agency, Office of Pr evention Pesticides and Toxic Substances, Washington, D.C.

U.S. EPA. 2002. Environmental implications of conservation tillage: A systems approach. Office of Research and Develo pment, National Exposure Research Lab.

Document: EPA/600/D- 85/147 (NTIS PB85237089). http://cfpub.epa.gov/si/si _public_record_report.cfm?di rEntryId=39100&fed_org_id=770 &SIType=PR&TIMSType=&showCriteria=0&a ddress=nerl&view=citation&keyword=P esticides&sortBy=pubDat eYear&count=100&dateBeginPublishedPresented Accessed on May 19, 2010

.

U.S. EPA. 2004. Overview of the ecological ri sk assessment process in the Office of Pesticide Programs. U.S. Environmental Pr otection Agency, Endangered and Threatened Species Effects Determinations, Washington, D.C.

U.S. EPA. 2005a. Dicamba: HED chapter of the reregistration eligibility decision document, phase 1. PC Code 029801; DP Barc ode: D317720 (Memorandum, C. Olinger and Y. Yang, September 13). Health Effect s Division, Office of Prevention, Pesticides and Toxic Substances, U.S. Environmen tal Protection Agency, Washington, D.C.

U.S. EPA. 2005b. Environmental Fate and Ecological Risk Assessment for the Reregistration of Dicamba and Dicamba Sodium, Potassium, Diglycoamine, Dimethylamine and Isopropylamine Salts (M emorandum, W. Erickson, I. Abdel-Saheb and S. Borges, August 31). Environmental Fate Effects Division, Office of Prevention, Pesticides and Toxic Substances, U.S. E nvironmental Protection Agency, Washington,

DC.

U.S. EPA. 2007a. National water quality i nventory: Report to Congress, 2002 reporting cycle: Findings. http://www.epa.gov/305b/2002report/ Accessed on May 19, 2010

.

U.S. EPA. 2007b. Risks of atrazine use to fe derally listed endangered Pallid Sturgeon Scaphirhyncus albus. http://www.epa.gov/oppfead1/endanger/liststa tus/effects/pallid_sturgeon_eff_deter_08- 31-07.pdf. Accessed January 1, 2008

.Monsanto Company 10-SY-210U 584 of 721 

### U.S. EPA 2008a. Agricultural management practices for water quality protection. http://www.epa.gov/watertrain/agmodule/ Accessed on May 19, 2010

.

U.S. EPA. 2008b. National assessment data base. http://www.epa.gov/waters/ir/ Accessed on May 19, 2010

.

U.S. EPA. 2008c. Risks of glyphosate use to the Federally threatened California Reglegged frog. . http://www.ep a.gov/owow/305b/2004report/ Accessed May 19, 2010

.

U.S. EPA. 2009a. National water quality inventory: Report to Congress, 2004 reporting cycle: Findings. http://www. epa.gov/owow/305b/2004report/ Accessed on May 19, 2010

.

U.S. EPA. 2009b. Reregistration eligibility decision for dica mba and associated salts. U.S. Environmental Protection Agency, O ffice of Prevention, Pesticides, and Toxic Substances, June 8, 2006, as amended June 17, 2009.

U.S. EPA. 2010. Agricultural practices that se quester carbon and/or reduce emissions of other greenhouse gases. http:// www.epa.gov/sequestration/ag.html Accessed on May 19,

2010 .U.S. FDA. 2001. Premarket notice con cerning bioengineered foods. Federal Register 66: 4706-4738.

University of Tennessee. 2010. 2010 weed cont rol manual for Tennessee. University of Tennessee, Institute of Agriculture, Knoxville, Tennessee.

USDA-AMS. 2011. Clarification of existing regulations regarding the use of genetically modified organisms in organic producti on and handling. Policy Memo 11-13. U.S. Department of Agriculture , Agricultural Marketing Se rvice, Washington, D.C.

USDA-APHIS. 2006. Soybean. U.S. Department of Agriculture, Washington, D.C. http://www.aphis.usda.gov/br/soybean.html Accessed January 20, 2006 .USDA-APHIS. 2011a. Determination of nonregulated status of insect resistant and glufosinate ammonium-tolerant (TwinLink) cotton, Gossypium hirsutum , events T304-40 x GHB119. U.S. Department of Agriculture, Animal & Plant Health Inspecti on Service, Washington,

D.C.

USDA-APHIS. 2011b. Monsanto petition (0 9-SY-2000U) for determination of nonregulated status of (MON 87769) stearidon ic acid soybean: Dr aft environmental assessment. U.S. Department of Agriculture, Animal and Plant Health Inspection Service, Washington, D.C.

USDA-ARS. 2006. Forage soybean cultivars: A source of high protein livestock feed. U.S. Department of Agriculture, Agricult ural Research Service, Washington, D.C. http://www.ars.usda.gov/Res earch/docs.htm?docid=8813 Accessed April 24, 2006

.

USDA-ERS. 2001. How much U.S. cropland acreage is under crop residue management. Briefing Room. U.S. Department of Agri culture, Economic Research Service. http://www.ers.usda.gov/Briefing/ AgChemicals/soilmangement.htm Accessed November
14, 2009

.Monsanto Company 10-SY-210U 585 of 721 

### USDA-ERS. 2005. USDA soybean baseline 2005- 14. United States Department of Agriculture, Economic Research Service, Washington, D.C. http://www.ers.usda.gov/briefing/ soybeansoilcrops/2005baseline.htm Accessed June 10,

2006

USDA-ERS. 2008a. Data sets: Organic producti on table 7. U.S. certif ied organic beans, by state, 2008. U.S. Department of Agri culture, Economic Research Service. http://www.ers.usda.gov/Data/ Organic/Data/Beans.xls Accessed January 17, 2012

.

USDA-ERS. 2008b. Data sets: U.S. and regional co st and return estimates in soybean for 2008. U.S. Department of Agriculture, Economic Research Service. http://www.ers.usda.gov/data/Co stsandReturns/testpick.htm Accessed January 18, 2010

.

USDA-ERS. 2010. Certified organic and total U.S. acreage, selected cr ops and livestock, 1995-2008. U.S. Department of Agriculture, Economic Research Service, Washington,

D.C. http://www.ers.usda.gov/Data/organic/Data/Ce rtifiedandtotalUSacreageselectedcrop

slivestock.xls

USDA-ERS. 2011. Data sets - Adoption of genetic ally engineered crops in the U.S. U.S. Department of Agriculture, Economic Re search Service, Washington, D.C. http://www.ers.usda.gov/Data/BiotechCrops/

USDA-NASS. 1984. Acreage - June 1984. U.S. Department of Agriculture, National Agricultural Statistics Service, Washington, D.C.

USDA-NASS. 1988. Crop production – 1987 Summar y. U.S. Department of Agriculture, National Agricultural Statistic s Service, Washington, D.C.

USDA-NASS. 1990. Acreage - June 1990. U.S. Department of Agriculture, National Agricultural Statistics Service, Washington, D.C.

USDA-NASS. 1992. Acreage - June 1992. U.S. Department of Agriculture, National Agricultural Statistics Service, Washington, D.C.

USDA-NASS. 1995. Acreage - June 1995. U.S. Department of Agriculture, National Agricultural Statistics Service, Washington, D.C.

USDA-NASS. 1998. Acreage - June 1998. U.S. Department of Agriculture, National Agricultural Statistics Service, Washington, D.C.

USDA-NASS. 2000. Acreage - June 2000. U.S. Department of Agriculture, National Agricultural Statistics Service, Washington, D.C.

USDA-NASS. 2003. Acreage - June 2003. U.S. Department of Agriculture, National Agricultural Statistics Service, Washington, D.C.

USDA-NASS. 2004. Agricultural chemical usage 2003 field crops summary. U.S. Department of Agriculture, National Agricult ural Statistics Service, Washington, D.C.Monsanto Company 10-SY-210U 586 of 721 

### USDA-NASS. 2006a. Acreage - June 2006. U.S. Department of Agriculture, National Agricultural Statistics Service, Washington, D.C.

USDA-NASS. 2006b. Agricultural chemical usage 2005 field cr ops summary. U.S. Department of Agriculture, National Agricult ural Statistics Service, Washington, D.C.

USDA-NASS. 2007a. Acreage - June 2006. U.S. Department of Agriculture, National Agricultural Statistics Service, Washington, D.C.

USDA-NASS. 2007b. Agricultural chemical usage 2006 field cr ops summary. U.S. Department of Agriculture, National Agricult ural Statistics Service, Washington, D.C.

USDA-NASS. 2008. Agricultural chemical usage 2007 field crops summary. U.S. Department of Agriculture, National Agricult ural Statistics Service, Washington, D.C.

USDA-NASS. 2009a. Acreage - June 2009. U.S. Department of Agriculture, National Agricultural Statistics Service, Washington, D.C.

USDA-NASS. 2009b. Crop production 2008 summary. U.S. Department of Agriculture, National Agricultural Statistic s Service, Washington, D.C.

USDA-NASS. 2009c. Crop values 2008 summar y. U.S. Department of Agriculture, National Agricultural Statistic s Service, Washington, D.C.

USDA-NASS. 2011. Quick stats: Soybeans - Acres planted. U.S. Department of Agriculture, National Agricultural Statistics Service, Washington, D.C. http://quickstats.nass.usda.gov/data /printable/4D84E77B-F692-32F3-B9AD8CDBDC688D03 Accessed December 20, 2011

.

USDA-NRCS. 2005. Residue and tillage manageme nt, no till/strip till/direct seed. U.S. Department of Agriculture, Natural Resour ces Conservation Service, Washington, D.C.

USDA-NRCS. 2006. Conservation resource br ief: air quality, number 0605 Retrieved November 9, 2010. http://www.nrcs.usda.gov/Intern et/FSE_DOCUMENTS/nrcs143_023301.pdf

WHO. 1995. Application of the principles of substantial equivalence to the safety

evaluation of foods or food components from plants de rived by modern biotechnology. World Health Organization, Rome, Italy.

Wrather, J.A., W.C. Stienstra and S.R. Koenning. 2001. Soybean disease loss estimates

for the United States from 1996 to 1998. Cana dian Journal of Plant Pathology-Revue Canadienne De Phytopathologie 23:122-131.

Wrather, J.A. and S. Koenning. 2011. Soybean disease loss estimates for the United States, 1996-2010. University of Missouri Delta Research Cent er, Agricultural Experiment Station, Portageville, Missouri. http://aes.missouri.edu/delta/research/soyloss.stm Accessed February 22, 2012Monsanto Company 10-SY-210U 587 of 721 

### Wrather, JA and M Mitchum. 2010. Soybean cy st nematode: Diagnosis and management (G-4450). University of Missouri Exte nsion Service. Columbia, Missouri. http://extension.missouri.edu/publ ications/DisplayPub.aspx?P=G4450 Accessed February 10, 2012

Wright, JP, LJ Schuler, KH Carr, TB Orr, JL Honegger. 2012. Information to Support an Endangered Species Assessment for Dicamba Use in Dicamba-Tolerant Soybeans: An Evaluation of Potential Exposure and Biologi cal Effects. Monsanto Technical Report

MSL- MSL-22545. EPA MRID 48644202

Yacentiuk, M. 2008. Full fat soybeans in swine rations. Manitoba Agriculture, Food and Rural Initiatives, Winnipeg, Manitoba. http://www.gov.mb.ca/agricultu re/liestock/pork/bab02s57.html Accessed November 11,

2008

.

Zhou et al. (2011) Stability in the Compos ition Equivalence of Grain from InsectProtected Maize and Seed from Glyphosate-Tolerant Soybean to Conventional Counterparts over Multiple Seasons, Locations, and Breeding Germplasms. J. Agric.

Food Chem. , 59:8822–8828.Monsanto Company 10-SY-210U 588 of 721 

### Appendix K: Herb icide Resistance

K.1. Introduction

Based upon theory of natural selection, plant populations can devel op resistance to an

herbicide due to the selection of individuals that carry specif ic genes that can render those individuals unaffected by the typical lethal effect s of an herbicide. The application of an herbicide to the plant does not , itself, cause a mutation in su bsequent generations. Rather, over time, those few plant biotypes containing resistant gene(s) become dominant in the population with repeated use of the herbicid e in the absence of other control methods,

such as use of other herbicides and/or use of cultural control methods. The development of resistant populations is common to all herb icides. The probability for resistance to develop is a function of: fre quency of resistant allele(s) 43 

### , mechanism of resistance,

dominance or recessive nature of the resistan t allele(s), relative f itness of the resistant biotype, and frequency or duration of herbic ide use in the absence of other control methods (Beckie, 2006; Jasieniuk, et al.,1996; Sammons et al., 2007). 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 othe r herbicides ( e.g. auxin class , glyphosate, dinitroa nilines class).

Herbicide resistance can become a limiting factor in crop production if the resistant weed population cannot be controlled with other herb icides or cultural practices. In general, this has not been the case for any herbicide. In 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 and are be ing actively promoted by the public and private sectors (HRAC, 2010) and are being implemented by growers.

Monsanto considers product st ewardship to be a fundament al component of customer service and business practices. Stewardship of the dicamba herbicide to preserve its usefulness for growers is an important aspect of Monsanto’s stewardship commitment. Although herbicide resistance may eventually occur in weed sp ecies when an herbicide is widely used, resistance can be postpone d, contained and managed through research, education and good management practices. Th ese are the key elements of Monsanto’s approach to providing stewardship of dica mba used on MON 87708 integrated into the Roundup Ready soybean system. Monsanto will invest in research, and grower/retailer education and training programs to provide information on best practices to manage dicamba weed resistance in soybean production. This appendix provides an overview of Monsanto’s approach to the development of best management practices to mitigate

dicamba weed resistance. Monsanto works clos ely with weed scientists in academia and with other companies to research and de velop best management practices and to uniformly communicate such practices to growers. Evidence of this cooperative effort is the recent development and posting of herbic ide resistant training modules on the WSSA website ( www.wssa.net ) and the publication of guidelines by the Herbicide Resistance Action Committee (HRAC) on their website ( www.hracglobal.com ).

43 An allele is any of several forms of a gene, usually arising through mutation, that are responsible for hereditary variation.Monsanto Company 10-SY-210U 589 of 721 

### K.2. The Herbicide Dicamba

Dicamba (3,6-dichloro-2-methoxybenzoic acid) is classified as a benzoic acid herbicide belonging to the synthetic auxin group of herb icides (HRAC, 2010). The herbicides in this group act as growth regulators similar to endogenous indole acetic acid (IAA) but are structurally diverse. The synthetic auxin gr oup includes five chemical families (benzoic acid, pyridine-carboxylic acid, quinoline car boxylic acid, phenoxy-car boxylic acid and a separate class which includes one herbicide, benazolin ethyl) . In addition to dicamba, specific herbicides in this group include 2,4-D, 2,4-DB, mecopr op, MCPA, clopyralid, picloram, quinclorac and several other active ingredients. Dicamba and other synthetic

auxin herbicides are classified in Herbic ide Group 4 by the Weed Science Society of America (HRAC, 2009). Most herbicides in this group are active on broadleaf weeds only, but a few have significant activity on grasses, e.g. , quinclorac. The specific site of action among the different chemistry families may be different. Dicamba provides preemergence and postemergence control of ov er 95 annual and bienni al broadleaf weed species and control or suppr ession of over 100 perennial broadleaf and woody species 44



### . Dicamba is not active on grass weeds and is often used in combination with other herbicides to provide broa d spectrum weed control.

Dicamba herbicide was commercialized in the U.S. for agricultural use in 1967 and is currently labeled for preemergence and/or pos temergence weed control in corn, soybean, cotton, sorghum, small grains (wheat, barley and oats), millet, pasture, rangeland, asparagus, sugarcane, turf, grass grown for s eed, conservation reserve program land, and fallow cropland (U.S. EPA, 2009). Dicamba is sold as standalone formulation which can be tank mixed with one or more active ingr edients depending upon the crop and the weed spectrum. Dicamba is also sold as a prem ix formulation with other herbicides.

Dicamba kills plants by mimicking naturally-occurring plant growth hormones called auxins, thereby destroying tissue through uncont rolled cell division a nd growth (Ahrens, 1994). Ahrens (1994) further states that dicamba has been found to affect cell wall integrity and nucleic acid metabolism whereas in other cases it has been found to increase cell wall permeability, leading to cell enlargem ent. At low concentrations, dicamba has been found to increase synthesis of DNA, RN A, and proteins, resulting in altered cell division and growth. At high c oncentrations, inhibition of cell division and growth occur. In general dicamba and other synthetic auxi n herbicides have been found to affect multiple plant physiological systems. The mo lecular mechanism of auxin action is still not known in detail nor completely understo od (Devine et al., 1993, Jugulam et al. 2011). However, Grossmann (2010 ) in a review of auxin herb icides, outlined a proposed

mechanism and mode of action for auxin herb icides and IAA at supraoptimal endogenous concentrations in dicot plant species. Th e proposal was based upon recent identification of receptors for auxins and hormone inter action in signaling between auxin, ethylene and the upregulations of abscisic acid biosynthe sis which would account for a large part of the various auxin-herbicide-mediated responses that are seen in sensitive dicots. In

addition research has indicated that there is a high level of redundancy in auxin receptors

44 Clarity product label http://www.cdms.net/LDat/ld797002.pdfMonsanto Company 10-SY-210U 590 of 721 

### which may account for the lack of development of widespread resistance to this herbicide group (Walsh et al., 2006).

Dicamba is taken up by plants through the root s, stems, and foliage (Ahrens, 1994; NPIC, 2002). Dicamba translocates to all plant tissu es but accumulates in growing tissues. Translocation of dicamba is typically slower in tolerant plants such as grasses compared to broadleaf plants. Dicamba has a rela tively low soil-bindi ng coefficient.

K.3. Herbicide-Resistant Weeds and Resistance Management Strategies

The development of herbicide-resistant weed s is not a new phenomen a and resistance is

not limited to certain select he rbicides. In 1957, the first U.S. herbicide-resistant weed, a spreading dayflower biotype resistant to 2,4 -D, was identified in Hawaii (Heap, 2010). Currently, there are 73 indivi dual weed species that ha ve known herbicide-resistant biotypes to one or more herbicides in the U. S. For example, there are 42 weed species resistant to ALS herbicides, 15 to ACCase i nhibitors, 24 to photosys tem II inhibitors, and 10 to glycine herbicides (Heap, 2010). Grow ers have been managi ng herbicide-resistant

weeds for decades with the use of alternative herbicides and/or cultural methods such as tillage or crop rotation.

The occurrence of an herbicide- resistant weed biotype does no t end the useful lifespan or preclude the effective use of the herbicide in question as part of an overall diversified weed management system. The three herb icide classes with th e highest number of resistant species, ALS, ACCase and triazine herbicides, are still effectively used by growers today.

It is important to distinguish herbicide resistance from herbicide tolerance. A herbicidetolerant weed species is one that is naturally tolerant to a herbicide, for example a grass species is not killed by the application of a broadleaf herbicide. Furthermore, certain weed species, while neither resistant nor tolera nt, are inherently diffi cult to control with a particular herbicide, requiring more caref ul herbicide use and weed management practices.

Since the first confirmed cases of herbicide resistance, research has been directed at determining which practices are best for ma naging existing resistan ce situations and how best to reduce the development of herbicide resistance. Resistant management practices most often recommended by University/C ooperative Extension Service (CES) and industry are: 1) use of multiple herbicide mo des-of-action in mixture, sequence, or in rotation, 2) crop rota tion, 3) use of cultu ral control measures such as tillage and time of planting, and 4) use of the labeled herb icide rate at the recommended timing of application (Gressel and Sege l, 1990; Beckie, 2006). Recent research by Beckie and Reboud (2009) indicates that in some cases he rbicide mixtures offer a better management option than rotating herbicides. Simultaneous ly using two herbicides with different modes-of-action significantly reduce the probability of weeds developing resistance to either or both herbicides (B eckie and Reboud, 2009). Crop rota tion is also an effective method for resistance management due to the fact that it fosters the use of additional herbicide modes- of-action and, potentially, use of additional cultural practices to manageMonsanto Company 10-SY-210U 591 of 721 

### weeds over time. The use of multiple methods of weed control in a single location is the technical basis for developing management programs to delay the development of resistance. This general concept has been re ferred to as applying “diversity” within a crop or across a crop rotati on (Beckie, 2006; Powles, 2008).

It is generally accepted that conservation tillage practices (minimum-till and no-till)

create environments where herbicide resistan ce is more likely to develop (Beckie, 2006). This is probably due to selection pressu re put on weeds by herb icides in these nondiverse environments and the absence of tillage as a cultural weed management practice to supplement herbicide use. However, this is not always the case. Legere et al. (2000)

found that an increase in the use of ACCase in hibitors in a conserva tion tillage system ( e.g. aryloxyphenosy propionates and phenylpyrazolines herbicide families) did not result

in an increased incidence of wild oat populations resistant to ACCase inhibitors. In conclusion, conservation tillage practices should not be cons idered a primary contributing factor to the development of resistance in all cases

K.4. Characteristics of Herbicides a nd Herbicide Use Influencing Resistance

While the incidence of weed resistance is ofte n associated with repe ated applications of
an herbicide, the actual probability for the de velopment of resistant populations is related,
in part, to the specific herbicide active ingredient, chemical family and the herbicide
group. Some herbicides are more prone to th e development of resistance than others
(Heap, 2010). The graph in Figure K-1 illustrates the global instances of weed resistance
to various herbicide groups. The different sl opes of observed resist ance are largely due

to the factors described above, which relate to the specific herbicide active ingredient as
well as to the group and herbicid e family and its function.Monsanto Company 10-SY-210U 592 of 721 

### Figure K-1. Weed Resistance to Various Herbicide Families 1

As can be seen in Figure K-1, weed resistance to the synthetic auxin group of herbicides has been slower to develop than for other herbicide gr oups even though these were the first synthetic herbicides discovered and used commercially. Po ssible reasons for this are discussed below



### .

1 Global number of resistant biotypes



### K.5. Mechanisms of Resistance and Inheritance of Resistance

To date, the three known basic mechanisms by which weed species develop resistance to a herbicide have been identified: 1) target site alteration (targe t site), 2) enhanced metabolism of the herbicides (metabolis m), 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 (exclu sion) (Sammons et al., 2007).

Herbicide resistance via target site alteration is the mostcommon resistance mechanism among the various herbicide groups and chemical families. 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 other herbicide groups , such as glyphosate (Powles and Yu, 2010). The most common type of target s ite alteration is one where amino acid substitution(s) in the protein that is the ta rget of the herbicide occurs such that the alteration prevents the binding of the herbicide to the protein and as a result the activity of the targeted protein is not alte red and the plant grows normally.

In the case of synthetic auxi n herbicides, resistance has be en speculated to be due to mutation(s) in genes encoding an auxin-bi nding protein causing reduced herbicide binding (Zheng and Hall, 2001; Goss and Dyer, 2003). In several studies, differentialMonsanto Company 10-SY-210U 593 of 721 

### herbicide absorption, translocation, and metabolism were ruled out as possible mechanisms of resistance in kochia (Cranst on et al., 2001) and in wild mustard (Zheng and Hall, 2001). However, cu rrent research has not presen ted convincing evidence for a single mechanism of resistance and this inability to elucidate the mech anism of resistance may be due to a lack of thorough understanding of au xin mechanism of activity (Jasieniuk et al., 1996). Walsh et al. (2006) identified seven a lleles at two di stinct genetic loci that conferred significant resistan ce to picolinate auxins (picloram) in Arabidopsis

, yet had minimal cross-resistance to 2,4 D and IAA.

Multiple mechanisms for inheritance of dicam ba resistance have been reported in the

literature. Jasieniuk et al. (1995) reported results indicating that inheritance of dicamba resistance in wild mustard is determined by a single, completely dominant nuclear allele. However, Cranston et al. (2001) reported results indicating th at dicamba resistance in kochia is determined by a quantitativ e trait (two or more genes).

In summary, the slow development of weed resistance to synthetic auxin herbicides may in part be due to their proposed multiple site s of physiological action in plants (Jasieniuk et al., 1996) and to the possibility that inheritance, at least in some sp ecies, is determined by a quantitative trait (Cra nston et al., 2001).

K.6. Weeds Resistant to Dicamba

As noted earlier, like other he rbicides, the use of dicamba ma y lead to the development of dicamba-resistant weed species. To date there are four species with known resistant biotypes to dicamba in the U.S./Canada afte r over 40 years of use; common hempnettle ( Galeopsis tetrahit ), kochia ( Kochia scoparia ), prickly lettuce ( Lactula serriola ) and wild mustard ( Sinapis arvensis ) (Heap, 2010). Additionally, a population of lambsquarters ( Chenopodium album ) has been confirmed to be resist ant in New Zealand for a total of five species worldwide with confirmed resistant biotypes to dicamba. For the synthetic auxin group of herbicides th ere is a total of 29 specie s globally with biotypes having

confirmed resistance to at least one member of this group but only eight species in the U.S. and four species in Ca nada (Heap, 2010). All of thes e populations are, but for two (wild carrot in OH and MI and waterhemp in Nebraska), found in western states or western Canadian provinces. In some sp ecies, cross-resistance between different

herbicides within the auxin group has been confirmed (cross-resistance is a plant’s

resistance to another herbicide as a result of exposure to a similarly acting herbicide). Therefore, consideration has to be given to the possibility that dicamba resistance could

extend to some of the other br oadleaf species listed as resistant to other synthetic auxin herbicides (Miller et al., 2001; Jasieniuk et al., 1995; Cranston et al., 2001). However,

because of differences in sites of ac tion among chemistries within this group ( i.e.

, benzoic acids compared to pyridine-carboxyl ic acids) cross resi stance between the herbicide groups is not a cer tainty (Monaco et al., 2002).

With the introduction of MON 87708 into the Roundup Ready soybean system, where dicamba will be applied in combination with glyphosate, it is impor tant to note that kochia is the only broadleaf spec ies with resistant biotypes to either synthetic auxins or glyphosate. There are no known kochia biotypes resistant to both of these herbicides.Monsanto Company 10-SY-210U 594 of 721 

### The development of a dicamba-glyphosate biotype is unlikely because both dicamba and glyphosate, each with two distin ct modes-of-action, will likely be applied in the same season to MON 87708 in the Roundup Ready soybean system. If populations with resistance to multiple herbicides were to occur, there are other herbicide options for managing the weed in soybean ( e.g. , clomazone and flumioxazin) and in its rotational crops ( e.g. , atrazine and isoxaflutole in corn) (Tab le K-1). In addition, kochia is a weed found primarily in the western soybean growi ng regions where soybean would be rotated with corn and wheat for which there are multiple options for kochia control (Casey, 2009). The glyphosate-resistant kochia biotype is also found in western soybean growing areas, but it is isolated to small areas where soybean is grow n in limited acreage.

K.7. Sustainable Use of Dicamba as a Weed Management Option in Soybean

Dicamba is a broadleaf herbicide that does not provide control of grass weeds. For that reason, MON 87708 will be sold only in soybean varieties that also contain other herbicide-tolerant traits, such as with the Roundup Ready soybean system ( e.g. , MON 89788). Soybean varieties contai ning both MON 87708 and MON 89788 will enable dicamba to be applied with glyphos ate or other soybean herbicides in an integrated weed management program, ideally as a mixture, to control a broad spectrum of grass and broadleaf weed species. Di camba applications on MON 87708 will provide effective control of glyphosat e-resistant broadleaf weeds and improve the control of annual and perennial broadleaf weed species, so me of which are difficult to control with glyphosate. Dicamba will also help delay development and/or combat existing weed

resistance issues that can limit the use of the PPO- and ALS-inhibiting herbicide groups by providing an additional mode of action fo r management of certain broadleaf species that are known to be prone to resistance to many of the current options for weed management (i.e. Amarathus spp.). MON 87708 will foster the adoption of Integrated Pest Management (IPM) practices in soyb eans by allowing growers to continue to primarily focus on postemergence in-crop weed control, as they have practiced with the Roundup Ready soybean system. This will allow growers to delay some herbicide treatments until field scouting indicates a ne ed for additional postemergence weed control which is consistent with the principles of IPM. Increasing postemergence herbicide options in soybeans is important, especially in conservation tilla ge situations, where consistency of postemergence herbicides has generally been greater than that of soil active residual products and thus a driving fa ctor in the adoption of conservation tillage systems in the U.S.

Upon the inclusion and integration of MON 87708 into the Roundup Ready soybean system and approval of the use of dicam ba on MON 87708 (Monsanto has submitted to EPA an application to amend Registration Nu mber 524-582 to register a new use pattern for dicamba on MON 87708), preplant/preemer gence applications of dicamba can be made up to 1.0 lb a.e./A up through crop emer gence (cracking) followed by two in-crop postemergence applications up to 0.5 lb a. e./A through the R1/R2 growth stage in soybean. However, the majority of w eed control scenarios in MON 87708 will not require the use of the maximum labeled rate, and the anticipated commercial preplant/preemergence and in-cr op use rates are between 0.25 to 0.375 lb a.e./A (based on established weed control rates for soybean we eds), with an average application rate ofMonsanto Company 10-SY-210U 595 of 721 

### 0.35 lb a.e./A as desc ribed in Section VIII.H). Residu al herbicides also will be recommended for use, in addition to glyphos ate and dicamba, to provide early season weed control and to supplement dicamba activity on certain hard-to-control and glyphosate-resistant weed biotypes, such as glyphosate-resistant palmer amaranth where weed populations can be very substantia l. See section K.8.1 for specific weed management recommendations.

Dicamba, as a complementary herbicide to gl yphosate, will provide new weed control options in soybean that streng then the utility and sustainabi lity of glyphosate as a weed control tool in the Roundup Ready soybean system. Likewise, glyphosate, as a complementary herbicide to dicamba, would st rengthen the utility and sustainability of

dicamba as a weed control tool for MON 87708 to be integrated into the Roundup Ready soybean system.

In the event there is known or suspected pr esence of a dicamba-resistant weed, it will be possible to provide the affected grower(s ) with options for managing the dicambaresistant biotype. There are multiple preemergence (including soil residuals) and postemergent herbicide options for managi ng broadleaf weed popul ations that are resistant or may potentially develop resistan ce to dicamba in soybean as well for crops grown in rotation with soybean. Thes e options are noted in Table K-1.Monsanto Company 10-SY-210U 596 of 721Table K-1. Management Recommendations for Control of Dicamba- and Other Synthetic Auxin-Resistant WeedsWeed Species Primary Crop SoybeanRotational Crops / Other UsesCorn Wheat Pastures/Roadsides RiceKochiaSaflufenacilaAtrazineaSaflufenacila(Kochia scoparia)ClomazoneaSaflufenacilaGlyphosateaFlumioxazinaIsoxaflutoleaBromoxynil/MCPAaGlyphosateaMesotrioneaParaquataGlyphosateaPrickly LettuceSaflufenacilaSaflufenacilaSaflufenacila(Lactuca serriola)Chlorimuron/metribuzinaAtrazineaTriasulfuronaGlyphosate + imazethapyraCarfentrazone +atrazineaMetsulfuron +thifensulfuronaIsoxaflutole +atrazineaWild CarrotGlyphosatecGlyphosatec(Daucus carota)ChlorimuroncAtrazinecChlorimuron/metribuzincPrimisulfuroncNicosulfurondHalosulfurondField BindweedGlyphosateaGlyphosateaGlyphosatea(Convolvulusarvensis)Glyphosate +imazethapyraGlyphosate +ImazamoxaMonsanto Company 10-SY-210U 597 of 721Table K-1 (continued). Management Recommendations for Control of Dicamba- andOther Synthetic Auxin- Resistant WeedsWeed Primary Crop SoybeanRotational Crops / Other UsesCorn Wheat Pastures/Roadsides RiceYellow Starthistle(Centaureasolstitialis)ChlorsulfuronAminopyralideSpreadingDayflower(Commelinadiffusa)BentazonhalosulfuronpenoxsulambispyribacfLambsquartersMetribuzinbIsoxaflutoleaBromoxynila(ChenopodiumCloransulambAtrazineaChlorsulfuron/Metsulfuronaalbum)gSaflufenacilaSaflufenacilaGlyphosateaImazamoxbMesotrioneaSaflufenacilaGlyphosatebBromoxynilbaBernards et al., 2010.bLoux et al., 2010.cMichigan State University Extension, 2010.dKells and Stachler, 1997.ePNWE, 2010.fUniversity of Arkansas CES, 2010.gResistance to lambsquarters has only been confirmed in New Zealand.Monsanto Company 10-SY-210U 598 of 721 

### K.8. Stewardship of Dicamba Use on MON 87708

In order to steward the use of agricultural herbicides and herbicide-tolerant cropping systems such as MON 87708 integrated into the Roundup Ready soybe an, Monsanto has conducted investigations and worked extens ively with academics and other herbicide

manufacturers to understand and recommend best practices to manage herbicide resistance. These investigations have de monstrated that one of the major factors contributing to the development of resistan t weed biotypes has been poor weed control management practices. The l ack of adequate management includes: 1) application of

herbicides at rates below thos e indicated on the product label fo r the weed species, and 2) sole reliance on a particular herbicide for weed control without the us e of other herbicides or cultural control methods (Beck ie, 2006; Peterson et al., 2007).

K.8.1. Weed Control Recommendations

The pending label for dicamba use on M ON 87708 is based on the maximum allowable use rates and patterns. Prior to laun ch of MON 87708 in the Roundup Ready soybean system, Monsanto, in cooperation with acad emics, will conduct tria ls to confirm the optimum rate and timing for dicamba alone a nd in combination with glyphosate and other herbicides. Recommendations to growers w ill be developed from this information and will be provided in herbicide product labe ls, Monsanto’s Technology Use Guide (TUG),

and in other education and tr aining materials to be broa dly distributed. Specifically, current research conducted by Monsanto to define the optimum weed management systems indicate the following: 1) in the absence of glyphosate resistant populations, the recommendation will be to apply a soil active residual herbicide followed by a postemergence application of glyphosate plus di camba to control weed escapes, and 2) in the presence of glyphosate resistant populati ons, the same system will be recommended with a potential second application of glyphosate plus dica mba if needed. In this latter case, the preemergence herbicide to be recomm ended will be one with activity against the targeted glyphosate resistant species. This will ensure more than one mechanism of action against the targeted species, which is a fundamental component of a good weed resistance management program. These management systems will reduce the potential for further resistance development to glyphosate, dicamba as well as other critical soybean herbicides. In conservation tillage systems, a preplant application of glyphosate plus dicamba may be recommended in some situatio ns in addition to the in-crop applications

described above. This is no t expected to increase select ion pressure on either product since the preplant weed spectrum is generally different from the in-crop spectrum.

K.8.2. Dispersal of Technical and Stewardship Information

Monsanto will use multiple methods to dist ribute technical and stewardship information to growers, academics and grower advisors. Monsanto’s TUG will set forth the requirements and best practices for the cultivation of MON 87708 including recommendations on weed resistance manage ment practices. Gr owers who purchase varieties containing MON 87708 will be required to enter into a limited use license withMonsanto Company 10-SY-210U 599 of 721 

### Monsanto and must sign and comply with the Monsanto Tec hnology Stewardship Agreement (MTSA), which requires th e grower to follow the TUG.

The weed resistance management practices that will be articulated in the TUG will also be broadly communicated to growers and retail ers in order to minimize the potential for the development of resistant weeds. Thes e practices will be communicated through a variety of means, including di rect mailings to each grower purchasing a soybean variety containing MON 87708, a public website 45 

### , and reports in farm media publications. The overall weed resistance management program will be reinforced through collaborations with U.S. academics who will provide their recommendations for appropriate stewardship of dicamba in soybean production, as well as by collaboration with crop commodity groups who have launched web-based weed resistance educational modules. Finally, Monsanto will urge growers to report any incidence of repeated non-performance of dicamba on weeds in fields planted with MON 87708, and Monsanto will investigate cases of unsatisfactory weed control to de termine the cause as defined in K.9.

The EPA is the U.S. federal regulatory agen cy that administers th e federal law governing pesticide sale and use under the Federal In secticide, Fungicide and Rodenticide Act (FIFRA). EPA encourages pesticide manufact urers to provide grow ers with information regarding an herbicide’s mode-of-action to aid growers in planning herbicide use practices and to foster the a doption of effective weed resist ance management practices as specified by EPA in Pesticide Registration (PR) Notice 2001-5 (U.S. EPA, 2001). In that

document EPA states that “thi s approach to resistance ma nagement is sound and would be highly beneficial to pest icide manufacturers and pesticid e users.” EPA approves all pesticide label use instructions based on its evaluation of supporting data supplied by the pesticide registrant or manuf acturer. By approving a label, EPA has concluded that the product will not cause unreasonable adverse eff ects to the environment when used in

accordance with the label’s dire ctions. After EPA approves a pesticide label, it is a violation of federal law to use the pesticid e for a use or in a manner not in accordance

with the label directions. Monsanto in corporates EPA’s guidelines for pesticide resistance management labeling on its agricultural herbicide labels, and will continue to do so in the future. Monsanto will adopt a similar approach to pesticide resistance management guidance on its dicamba product labels.

In summary, Monsanto will require weed re sistance management practices through the MTSA and TUG for its biotechnology-derive d herbicide-tolerant products, such as MON 87708 integrated into the Roundup Rea dy soybean system, and to promote these practices through product labeling and educationa l outreach efforts as an effective means to manage weed resistance developm ent for both dicamba and glyphosate.

K.8.3. Weed Resistance Management Practices

Monsanto will provide information to grower s and grower advisors on best management practices to delay the devel opment of resistance to dicamba. The weed resistance management recommendations for the use of dicamba in conjunction with soybean

45 http://www.monsanto.com/weedmanagement/Pages/default.aspxMonsanto Company 10-SY-210U 600 of 721 

### varieties containing MON 87708 will be consiste nt with the Herbicide Resistance Action Committee’s guidelines for prevention and mana gement of herbicid e resistance (HRAC, 2010) 46 

### . These guidelines recommend an integr ated approach to weed resistance management including crop management ( i.e. , row spacing, etc), cu ltivation techniques, and the use of multiple herb icide modes-of-action to ma nage a weed population.

In cases where resistance is confirmed for dicamba in soybean producing areas, Monsanto and University/Cooperative Exte nsion Service (CES) personnel will provide

recommendations for alternative herbicid e control methods to growers. These

recommendations would be made availabl e through Monsanto supplemental labels, Monsanto and university publications, and internet sites to growers, consultants, retailers

and distributors. For all ex isting cases of dicamba-resist ant weeds in the U.S. and globally today, alternative herb icides and cultural methods ar e available to growers to effectively control these biot ypes. Examples of recomme nded alternative herbicides from University/CES personnel that are appli cable to weed species known to be resistant to dicamba and other synthetic auxin herbicides are found in Table K-1. It is important to note that there are many alternative options in each situation.

K.9. Monsanto Weed Performance Evalua tion and Weed Resistance Management

Plan

An important part of a weed resistance mana gement plan is the timely acquisition of information regarding product performance. M onsanto has an extens ive technical, sales and marketing presence in the soybean markets where MON 87708 will be grown. Through our relationships with farm advi sors, key University/CES personnel, and growers using our seeds and tr aits products, Monsanto will acquire important and timely information regarding product performance. This will allow the timely recognition of performance issues that could arise related to weed resistance or other means. Field employees and hired consultants are traine d and provided processes for responding to product performance inquiries. Individual perf ormance issues that could be related to potential resistance are promptly handled. In addition performance inquiries are periodically reviewed by Mons anto for trends that could indicate the need for follow up

action on a broad scale.

If resistance is confirmed, the scientific and grower communities will be notified and a weed resistance mitigation plan will be im plemented by Monsanto in cooperation with the University/CES. The mitigation plan will be designed to manage the resistant biotype through effective and economical weed mana gement 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) importan ce of the weed in the agricultural system, 3)

resistance status of the weed to other herb icides with alternate modes-of-action, and 4) availability of alternative c ontrol options. These factors are analyzed by Monsanto and

46 The Herbicide Resistance Action Committee (HRAC) is an international body founded by the agrochemical industry for the purpose of suppor ting a cooperative approach to the management of

herbicide resistance and the establishment of a worldwide herbicide resistance database.Monsanto Company 10-SY-210U 601 of 721 

### 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 w eed and incorporates practical management strategies that can be im plemented by the grower.

After a mitigation plan is developed, Monsan to communicates the plan to the grower community through the use of supplemental labeling (labeling which includes newly approved use directions, or othe r instructions), informational fact sheets, retailer training programs, agriculture media and/or other means, as appropriate.

In addition to the grower inquiry initiated pr ocess, Monsanto, alone and/or in cooperation with University/CES, will conduct field st udies to understand the potential for weed

resistance and weed shifts as the resu lt of various weed management programs implemented for MON 87708 integrated into the Roundup Ready soybean system. These

studies will allow researchers to better tr ack specific factors that can influence the development of resistance to specific weeds.

K.10. Summary

Development of weed resistance is a complex process that can be difficult to accurately predict. Multiple methods for managing we ed resistance are available and no single option is best. No single agronomic practice will mitigate resistance for all herbicides or all weeds. As a result, weed resistance need s to be managed on a case-by-case basis and tailored for the particular herbicide and weed in order to meet grow er needs. Using good weed management principles, built upon achie ving high levels of control through proper application rate, choice of cultural practic es, and appropriate companion weed control products will allow dicamba herbicides to conti nue to be used effectively. In cases where weed populations have developed resistance to dicamba, effective management options are available and experience has shown that gr owers will continue to find value in using dicamba in their weed control programs.

The key principles for effective stewardship of dicamba use, includi ng the integration of MON 87708 in the Roundup Ready soybean system, include: 1) basing weed management and weed resistance management practices on local needs and using the tools necessary to optimize crop yield, 2) usi ng 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 unsatisfact ory weed control, and 5) providing up-todate weed management and weed resistance management training.

Overall, there is a low potential for dicamba-re sistant broadleaf weed populations to arise from the use of dicamba applied to M ON 87708 integrated into the Roundup Ready soybean system. The reasons are as follows:

Dicamba will be used in combination with gl yphosate in a majority of cropping situations,

and weed recommendations will also include the concurrent use of residual herbicides for complementary weed control and different m odes-of-action. These use patterns meanMonsanto Company 10-SY-210U 602 of 721 

### that there will be multiple modes-of-action ag ainst the major broadleaf species present in soybean production. This is a primary way to delay the development of resistance.

The development of resistance to auxin herbicides has been found to be relatively slow. This observation is hypothesized to be due to multiple sites of acti on within plants and evidence suggesting that resistance is determ ined by multiple genes (quantitative traits), at least in some species.

Only four broadleaf weed species have been confirmed to be resistant to dicamba in the U.S., and relatively low numbers of broadleaf species have been confirmed to be resistant to synthetic auxin herbicides even though di camba has been widely in use for over 40

years.

Known resistant broadleaf populations to dicamba and other auxin herbicides are primarily found in the western U.S. and, t hus, are not present in the major soybean geographies. In addition, the known dicam ba-resistant biotypes are not major weed species present in the U.S. soybean crop.Monsanto Company 10-SY-210U 603 of 721 

### References for Appendix K

Ahrens, W.H. 1994. Dicamba. 3,6-dichloro-2-methoxybenzoic acid. Pages 91-94 in

Herbicide Handbook. Seventh Edition. Weed Sc ience Society of America, Champaign,

Illinois. Beckie, H.J., and X. Reboud. 2009. Selecting for weed resistance: He rbicide rotation and mixture. Weed Technology 23:363-370.

Beckie, H.J. 2006. Herbicide-resistant weeds: Management tactics and practices. Weed Technology 20:793-814.

Bernards, M.L., R. E.Gaussoin, R.N. Hliei n, S.Z. Knezevic, D.J. Lyon, L.D. Sandell and R.G Wilson. 2010. Guide for weed management in Nebraska (EC130). University of Nebraska Extension, Lincoln, Nebraska

Casey, P.A. 2009. Plant guide for kochia ( Kochia scoparia ). United States Department of Agriculture, Natural Resources Conservation Se rvice, Manhattan, Ka nsas. http://plantmaterials.nrcs.usda.gov/technical/pub lications/newpubli cations/pg_kosc.pdf Accessed
June 1, 2010

.

Cranston, H. J., A. J. Kern, J. L. Hackett, E. D. Maxwell, B. D. Maxwell, and W. E. Dyer.

2001. Dicamba resistance in kochi a. Weed Science 49:164-170.

Devine, M., S.O. Duke, and C. Fedtke. 1993. Physiology of herbicide action. PTR Prentice Hall, Inc. Englewood Cliffs, NJ. pp. 295-309.

Goss, G.A., and W. E. Dyer. 2003. Physiologi cal characterization ofauxinic herbicideresistant biotypes of Kochia ( Kochia scoparia ). Weed Science 51:839-844.

Gressel, J. and L. A. Segel. 1990. Modelling the effectiveness of herbicide rotations and mixtures as strategies to delay or preclude resistan ce. Weed Technology 4:186-198.

Grossmann, K. 2010. Auxin herbicid es: current status of mech anism and mode of action. Pest Management Science 66: 113-120.

Heap, I. 2010. International survey of herbicide resistant weeds. http://www.weedscience.org/in.asp Accessed July 1, 2010

.

HRAC. 2009. Classification of herbicides according to mode of action. Herbicide Resistance Action Committee, Corvallis, Oregon. http://www.hracglobal.com/Publications/Cla ssificationofHerbicideModeofAction/tabid/2 22/Default.aspx Accessed June 29, 2009

.

HRAC. 2010. Guideline to the management of herbicide resistance. Herbicide Resistance Action Committee, Calgary, Alberta. http://www.hracglobal.com Accessed April 1, 2010

.Monsanto Company 10-SY-210U 604 of 721 

### Jasieniuk, M., I.N. Morrison, and A.L. Br ûlé-Babel. 1995. Inhe ritance of dicamba resistance in wild mustard ( Brassica kaber ). Weed Science 43:192-195.

Jasieniuk, M., A.L. Brûlé-Babel, and I.N. Morrison. 1996. The evolution and genetics of

herbicide resistance in weeds. Weed Science 44:176-193.

Jugulam, M., C. Hall, W.G. Johnson, K.B. Ke lley and D.E. Riechers. 2011. Evolution of

resistance to auxinic herbicides: Historical perspectives, mechanisms of resistance, and implications for broadleaf weed manage ment in agronomic crops. Weed Science Manuscript: 1-45

Legere, A., H.J. Beckie, F.C. Stevenson, a nd A.G. Thomas. 2000. Survey of management practices affecting the o ccurrence of wild oat ( Avena fatua ) resistant to acetyl-coA carboxylase inhibitors. Weed Technology 14:366-376.

Loux, M.M., D. Doohan, A.F. Dobbels, W.G. Johnson, G.R.W. Nice, T.N. Jordan and T.T. Bauman. 2010. Weed control guide for Ohio and Indiana. Bulletin 789. Purdue University and Ohio State University Extension, West Lafayette, Indiana.

Kells, J.J. and J.M. Stachler. 1997. Controlling wild carrot, as published in Michigan State University Extension Soybean Fact s Winter 1997 (FACT9707). Michigan State University Extension. http://web1.ms ue.msu.edu/imp/mods1/fact9707.html Accessed: May 19, 2010

.

Michigan State University Extens ion. Controlling Wild Carrot. http://fieldcrop.msu.edu/documents /controlling%20wild%20carrot.pdf Accessed: May 19,

2010

.

Miller, T.W., S.L. Shinn, and D.C. Thill. 2001. Cross-resistance in and chemical control of auxinic herbicide-resistant yellow starthistle ( Centaurea solstitialis ). Weed Technology 15:293-299.

Monaco, T.J., S.C. Weller and F.M. Asht on. 2002. Growth regulator herbicides. Pages 291-310 in Weed Science: Principles and Pr actices. Fourth Edition. John Wiley & Sons, Inc., New York, New York.

NPIC. 2002. Dicamba technical fact sheet. National Pesticide Information Center, Corvallis, Oregon. http://npic.orst .edu/factsheets/dicamba_tech.pdf Accessed April 30,

2010

.

PNWE. 2010. Control of problem weeds - Starthistle. Pacific Northwest weed management handbook. Pacific Northwest Extension Service, Portland, Oregon. http://uspest.org/pnw/weeds Accessed May 25, 2010

.

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 St ation and Cooperative Extension Service, Manhattan, Kansas.Monsanto Company 10-SY-210U 605 of 721 

### Powles, S.B. 2008. Evolved glyphosate-resist ant weeds around the world: lessons to be learnt. Pest Manageme nt Science 64:360-365.

Powles, S.B. and Qin Yu. 2010. Evolution in action: Plants resistant to herbicides. Annual Review of Plant Biology 61:317-347.

Sammons, R.D., D.C. Heeri ng, N. DiNicola, H. Glick, and G.A. Elmore. 2007. Sustainability and stewardship of glyphos ate and glyphosate-resistant crops. Weed Technology 21:347-354.

U.S. EPA. 2001. Pesticide Registration (PR) Notice 2001-5, Guidance for Pesticide Registrants on Pesticide Resist ance Management Labeling. http://www.epa.gov/opppmsd1/ PR_Notices/pr2001-5.pdf.

U.S. EPA. 2009. Reregistration Eligibility Decision (RED) for Dicamba and Associated

Salts. Case No. 0065. U.S. Environmental Protection Agency Office of Prevention, Pesticides and Toxic Substances, Speci al Review and Reregistration Division, Washington, D.C.

University of Arkansas Cooperative Exte nsion Service (CES). 2010. Recommended chemicals for weed and brush control (MP-44). Page 78. University of Arkansas, U.S. Department of Agriculture and County Governments Cooperating.

Walsh, T.A., R. Neal, A.O. Merlo, M. Honma, G.R. Hicks, K. Wolff, W. Matsumura, and

J.P. Davies. 2006. Mutations in an auxin re ceptor homolog AFB5 and in SGT1b confer resistance to synthetic pico linate auxins and not to 2,4- dichlorophenoxyacetic acid or

indole-3-acetic acid in Arabidopsis . Plant Physiology 142:542-552.

Zheng, H. and J.C. Hall. 2001. Understandi ng auxinic herbicide resistance in wild mustard: physiological, biochemical, and mo lecular genetic approaches. Weed Science

49:276-281.Monsanto Company 10-SY-210U 606 of 721 

### Appendix L: Comparative Analysis of Dicamba and Alternative Soybean

Herbicides

L.1. Introduction

Dicamba is a selective broadleaf herbicide belo nging to the auxin agonist class, the oldest known class of synthetic herbicides, and is a member of the benz oic acids sub-group. Dicamba mimics the action of the plant ho rmone indole acetic acid and causes rapid uncontrolled cell division, and growth leadin g to plant death. Dicamba has been registered for agricultural us es in the U.S. since 1967 and has been widely used in agricultural production for over fo rty years. Dicamba is pr esently approved for use on

asparagus, corn, cotton, gra ss seed production, pasture an d rangeland grasses, small cereals including barley, oats, rye, and wheat, sorghum, soybean, and sugarcane.

Dicamba is also used for indus trial vegetation management ( e.g. , forestry and roadsides), professional turf management ( e.g. , golf courses, sports complexes), and residential turf (U.S. EPA, 2009b).

Monsanto Company has developed biotec hnology-derived soybean MON 87708 that is tolerant to dicamba (3,6-dichloro-2-methoxybe nzoic acid) herbicide. MON 87708 offers growers expanded use of dicamba in soybean production. The excelle nt crop tolerance of

MON 87708 to dicamba facilitate s a wider window of applica tion in soybean, allowing a preemergence application up to emergen ce (cracking) and in-crop postemergence applications through the R1/R2 stage of grow th. Dicamba provides effective control of over 95 annual and biennial weed species, and control or suppression of over 100 perennial broadleaf and woody plant speci es. MON 87708 will be combined with MON 89788 (Roundup Ready 2 Yield  

### soybean) utilizing tr aditional breeding techniques. The combination of herbicide-tolerance traits a llows the use of dicamba and glyphosate herbicides in an integrated weed management program to control a broad spectrum of grass and broadleaf weed species, su ch that the two herbicides can be used in sequence or tank-mixed. Monsanto has submitt ed an application to the Environmental Protection Agency (U.S. EPA) to register this new use patt ern for dicamba on MON 87708 OPP Decision Number D-432752).

The availability of MON 87708 integrated into the Roundup Ready soybean system will result in a simple and effective dual mode-of-action herbicide system that will control hard-to-control broadleaf weeds, assist in th e management of resistant broadleaf weeds, including glyphosate-resistan t biotypes, and subsequen tly displace some soybean herbicides currently in use today (also re ferred to as alternative herbicides).

The intent of this comparative analysis is to define current herbicide use in U.S. soybean production, and to compare dicamba’s human health and environm ental properties to herbicides currently used by growers for weed control. In order for a pesticide (herbicide) to be registered by EPA it must meet the FIFRA and FFDCA standards for

 Roundup Ready 2 Yield is a registered trademark of Monsanto Technology LLC. All other trademarks are the property of their respective owners.Monsanto Company 10-SY-210U 607 of 721 

### safety to human health and the environment. The EPA must conclude that the herbicide when used according to the label does not pose an unreasonable risk to humans or the environment, and, in order to establish a tolerance for the use of a he rbicide on a food or feed crop, find there is a reasonable certain ty of no harm to human health from nonoccupational (food, water and re sidential/recreational) expos ures to the herbicide. Consequently all alternative s oybean herbicides can be used safely, and do not pose a risk

to humans or the environment.

Nonetheless, in some instances dicamba offers a reduction in risk potential compared to some alternative herbicides, in the same ri sk category (e.g., acute human risk, aquatic plant risks). In other instances dicamba presen ts a similar risk potential compared to the some alternatives. In some instances, dicam ba presents a greater risk potential compared

to some alternatives.

Overall, the use of dicamba on MON 87708 incorporated into the Roundup Ready soybean system will offer a benefit compared to alternative soybean herbicides. For human health, aquatic plants and animals, and preemergence appl ication flexibility. dicamba use on MON 87708 provides an overall reduction of potential risk (combination of “Yes” and “No” entries in Table L-17) compared to the alternatives except for flumiclorac-pentyl. Specifically,:

 Formulations based on the diglycolamine sa lt of dicamba, such as Clarity® or M1691 herbicide, have favorable acute toxicity profiles that reduce the risk of acute adverse effects for applicators, agricultural workers and bystanders compared to six alternative herbicide pr oducts (Section L.5.1.1).

 The active ingredient (a.i.) dicamba has favorable chronic, reproduction, carcinogenicity and developmental toxicity ch aracteristics, which reduce the potential for risks from exposure to applicators, agri cultural workers and consumers, compared to exposure from eight alternative herbicide products (Section L.5.1.2).

 Dicamba has very low toxicity to fish a nd aquatic invertebrate s, and based on EPA high end exposure screening level assessmen t methodology will not affect listed and non-listed aquatic plants and animals, which reduces the potential risk to aquatic organisms as compared to seven alte rnative products(Section L.5.2.1 and L.5.2.2).

 Dicamba use on MON 87708 will strengthen and extend the benefits of glyphosatebased weed control in soybean, which has many well-known and recognized

environmental and human health benefits as acknowledged in previous EPA Office of Pesticide Programs (OPP) decisions to grant reduced risk status to multiple glyphosate-tolerant crop uses, i.e. , corn, canola, and sugar beet. 47 

### Additionally, the use of glyphosate-based weed control in s oybean has provided growers with more profit opportunities than conventional soyb ean by reducing input costs (Gianessi, 2005). By utilizing both active ingredients, which have different herbicidal modes-of-

47 http://www.epa.gov/opprd001/workplan/completionsportrait.pdfMonsanto Company 10-SY-210U 608 of 721 

### action, the risk to soybean production posed by weeds that are ha rd-to-control with glyphosate alone, or have deve loped resistant to glyphosat e, is reduced. Hard-tocontrol weeds generally require a higher rate and/or applic ation at a smaller growth stage in order to consistently achieve commercially acceptable control, and includes copperleaf, hemp sesbania, morningglory, pr ickly sida, velvetleaf, waterhemp, and a number of other broadleaf and grassy weeds. Refer to the Roundup WeatherMax label (U.S. EPA Reg. No. 524-537) fo r a listing of these weeds.

 Dicamba use on MON 87708 will also provide growers with the option to use an

herbicide with a different mode-of- action to manage weed species ( e.g. , Pigweed) where certain biotypes have demonstrated resistance to herbicide classes other than glyphosate, such as protoporphyrinogen oxida se (PPO) or acet olactate synthase (ALS) inhibitors (Sec tion L.5.3.3). Herbicide resistant weeds are those listed on the International Survey of Resistant Weeds website (www.weedscience.org).

 Dicamba use on MON 87708 will reduce the risk to soybean production that ensues when growers utilize alternate herbicides that have long ro tation restrictio n periods or pose potential for substantia l crop injury and loss of yield (Section L.5.3.2.1).

 Planting glyphosate tolerant soybean ther eby allowing the use of glyphosate to effectively control weeds in no-till fields has made no-till a viable production system for soybean (Pedersen, 2008). The use of dicamba on MON 87708 will reduce the risk of growers reverting back to conven tional tillage practices from conservation tillage practices due to concerns over weed control or resistance and thereby foster continued adoption of conservation tillage practices, which is an important goal for the agro-ecosystem and the long term sustainability of U.S. agriculture. The integration of MON 87708 into the Roundup Ready soybean system will allow the flexibility to incorporate a second herbic ide and mode-of-action in preemergent or postemergent applications a nd support the continued us e of conservation tillage production (Section L.5.3.3).

In conclusion, dicamba provides a similar and in some cases a more favorable comparative risk profile compared to other alternative herbicide products; the use of dicamba on MON 87708 will positively impact in tegrated pest management practices and sustainability of soybean production in the United States by providing a valuable weed management tool for this important agricultu ral crop that imparts greater flexibility and weed control options, and will help to slow the selection for herbicide-resistant weed biotypes for all herbicide modes-of-action (i.e. , ALS, HPPD, PPO, gl yphosate) currently registered for use in soybean; and will continue to support adoption of no-till and conservational tillage practices.Monsanto Company 10-SY-210U 609 of 721 

### L.2. Background

L.2.1. Chemical Name and Structure

Dicamba (3,6-dichloro-2-methoxybenzoic acid) is a carboxylic acid that can form salts in aqueous solution. The chemical structure is provided in Figure L.1. Dicamba products registered for agricultural uses are form ulated with various dicamba salts. The formulated products Clarity and M1691 contain the diglycolamine salt of dicamba at a nominal level of 56.8% by weight, which is eq uivalent to 38.5% by weight dicamba acid (also referred to acid eq uivalents or a.e.).



## O
CH 3 

## Cl

Cl O OH

Figure L-1. Structure of Dicamba

L.2.2. Herbicide Class and Herbicidal Mode-of-Action

Dicamba belongs to the “benzoic acid class” of herbicides. The Weed Science Society of America (WSSA) places dicamba in herbicide group number 4. 48 

### It is an auxin agonist; it is a plant hormone (indole acetic acid, I AA) mimic that causes rapid uncontrolled cell division and growth, leading to plant death. Dicamba is main ly used to control broadleaf weeds and woody plants.

L.2.3. Current U.S. Uses and Established Tolerances

Dicamba is a selective herbicide labeled fo r control of certain broadleaf weeds and woody plants. It was first registered for agri cultural use in the U.S. in 1967 and is widely used today in agricultural, industrial, and residential se ttings. Dicamba salts have approved uses on asparagus, corn, cotton, gr ass seed production, pasture and rangeland grasses, small cereals includi ng barley, oats, rye, and wheat, sorghum, soybean, and sugarcane. Dicamba is also used for industrial vegetation management ( e.g. , forestry and roadside right-of-ways), prof essional turf management ( e.g. , golf courses, sports complexes), and residential turf (U.S . EPA, 2009b; Durkin and Bosch, 2004).

48 There are several systems of herbicide mode-of-action classification. Among the most widely used are

those of the Herbicide Resistance Action Committee (HRAC) and the WSSA. The classifications are compared in a chart at: http://www.plantprotection.org/ HRAC/Bindex.cfm?doc=MOA.html . Accessed May 27, 2010Monsanto Company 10-SY-210U 610 of 721 

### Established food and feed tole rances are listed at 40 CFR 180.227, and in addition to the crop plants, they include residue limits fo r meat, milk, and meat by-products that may

arise when livestock consume dicamba-treated commodities.

L.2.4. Proposed use pattern for dicamba on MON 87708

Monsanto has submitted to the EPA an application to amend EPA Reg. No. 524-582 to register a new use pattern for dicamba. The new use pattern allows for sequential applications of dicamba, formulated as the diglycolamine salt, to MON 87708:

 Preplant / preemergent applications, totali ng up to 1 pound per acre of dicamba a.e.

 One or two postemergent in-crop applicati ons (up to 0.5 pounds a.e./acre each), timed between soybean emergence (cracking) and ea rly flowering (the R1/R2 growth stage) of the soybean.

The proposed use pattern for dicamba on M ON 87708 is within the maximum single and annual application rates that have previously been assessed by EPA. Once approved by EPA, this label will become a legally-bi nding constraint on the use of dicamba on

MON 87708.

Residue analyses conducted by Monsant o, in which MON 87708 was treated at the proposed maximum application rates, were found to produce to tal dicamba-derived residues in soybean seeds at a median level of <0.065 ppm (range of <0.041 to 0.471 ppm). These levels are substantially less than the current established food tolerance; the highest measured residue from the M ON 87708 residue study (0.471 ppm) was only 4.7% of the established 10 ppm dicamba soyb ean tolerance. Therefore, the proposed maximum dicamba treatments to MON 87708 (allowing up to 1 lb a.e. of dicamba per acre applied at a preemergent timing plus up to two postemergent in-crop applications of up to 0.5 lb a.e./acre each), will not increas e dietary exposure to dicamba residues in soybean seed beyond that which is already permitted within the existing legal food

tolerance.

Residues were also measured in soybean forage and hay of treated MON 87708. Monsanto is seeking the establishment of feed tolerances for soybean forage and hay to allow feeding of these dicamba-treated M ON 87708 commodities to livestock. However,

these dicamba residues will not increase the livestock dietary burden used to establish the current animal commodity food tolerances, so all dietary exposures from the proposed new dicamba use on MON 87708 have already been accounted for within the existing

dicamba food tolerances.

L.3. Properties of Dicamba Herbicide

L.3.1. Human Health

MON 87708, treated with preemergent and over-the-top applications of dicamba herbicide has the opportunity to redu ce risks to human health by:Monsanto Company 10-SY-210U 611 of 721 

###  Replacing, in part, currently used or fores eeable future alternative herbicide products with less favorable risk profiles, and

 Providing an effective tool to manage hard -to-control broadleaf weeds or broadleaf weeds that are resistant to glyphosate, PPO inhibitors, or ALS inhibitors, thereby preserving the ability of gr owers to manage broadleaf weed problems and maximize yields in a less risky manner, recognizing the human health benefits offered by the superior toxicity and reduced risk pr ofile of glyphosate and the risk profile demonstrated by dicamba.

Both dicamba, as the Clarity or the M1691 formulation, and glyphosate, as various Roundup-branded formulations, have “CAUTION !” signal words (CAUTION is the most favorable of three possible label signal words that can be required by EPA) and favorable chronic toxicity profiles. In a comparative analysis with other a lternative soybean weed control products, dicamba products offer bette r, or at least equi valent, human health safety profiles, as discussed below.

In 2006, EPA issued the Reregistration Eligib ility Decision (RED) document for dicamba

and its associated sa lts (U.S. EPA, 2009b). The RED doc ument, and the related Health Effects Division (HED) chapter (U.S. EP A, 2005b), presented an overview of the toxicological properties of dicam ba, which is summarized below.

The measurement of human health is the resu lt of conventional labor atory testing against standard indicator species (generally ra ts, mice, and dogs) and is required for the registration of a pesticide by the EPA. Resu lts are presented using standard toxicity indices, such as the concentration or dose required for 50% lethality (LC 50 

### or LD 50 

### ), the highest dosing level that pr oduced No Observable Advers e Effects (NOAEL), or the lowest dosing level that produced an Observ able Adverse Effect (LOAEL). The results of the acute (single exposure) toxicity studies for dicamba are presented in Table L-1. Results for developmental, reproduction, muta genic and neurotoxicol ogical studies are presented in Table L-2. Subchronic, ch ronic and carcinogenici ty study results are presented in Table L-3.

Table L-1. Dicamba Acid Acute Toxicity Study Findings

Study Endpoint EPA Category 1



### Acute oral (rat) LD 50 

### 2740 mg/kg III
Acute dermal (rat) LD 50 

### 2000 mg/kg III
Acute inhalation (rat) LC 50 

### 5.3 mg/L IV
Primary eye irritation (rabbit) Irritant II
Primary dermal irritation (rabbit) Irritant II
Dermal sensitization (guinea pig) 2 

### Negative NA

1 EPA acute toxicity categories range from I (worst) to IV (best).

2 Determination of the potential to cause or elicit skin sensitization reactions (allerg ic contact dermatitis) is an important element in evaluating a substance’s toxicity.Monsanto Company 10-SY-210U 612 of 721 

### Table L-2. Dicamba Acid Reproducti ve, Developmental, Mutagenic, and Neurotoxicologic Findings

Study Systemic Toxicity Endpoint (mg/kg body wt/day) Offspring Toxicity Endpoint (if any) (mg/kg body wt/day) Developmental (rat) Matern al NOAEL 160; LOAEL 400. Clinical signs: decreased food consumption and weight gain, increased mortality. Developmental NOAEL 400 (HDT 1 

### ).

Developmental (rabbit) Maternal NOAEL 62.5; LOAEL 150. Clinical signs: decreased motor activity, ataxia, increased abortion. Developmental NOAEL 62.5; LOAEL 150. Clinical signs:

Increased abortion. Developmental Neurotoxicity Not Required

Reproduction, multigeneration (rat) Parental NOAEL 122/136 (M/F 2 

### ); LOAEL 419/450 (M/F). Clinical signs: reduced righting reflex. Reproductive NOAEL 122; LOAEL 419. Delayed F1 male maturation. Offspring NOAEL 45;

LOAEL 136. Clinical signs: Decreased pup weights, all generations. Acute Neurotoxicity (rat) NOAEL not established; LOAEL 300. Clinical signs: Impaired gaits and righting reflex, impaired respiration, rigidity.

Subchronic Neurotoxicity (rat) NOAEL 401/472 (M/F); LOAEL 768/1029 (M/F). Clinical signs: Rigidity, slightly impaired gait and righting reflex.

Gene Mutation –

Salmonella Not mutagenic.

Chromosome aberration (CHO 3 

### ) Aberrations not indu ced at any tested

concentration with or without S9

activation.

Unscheduled DNA Synthesis (UDS) No Evidence of UDS up to 3000 μg/mL

1 

### HDT stands for the highest dose tested.

2 

### M/F stands for males/females.

3 

### Chinese hamster ovaries.Monsanto Company 10-SY-210U 613 of 721 

### Table L-3. Dicamba Acid Subchronic, Chronic and Cancer Findings

Study Toxicity Endpoints (mg/kg body weight/day) Subchronic Oral (rat) NOAEL 479/536 (M/F 1 

### ); LOAEL 1000/1065 (M/F). Clinical signs: decreased weight gains, liver effects. 28-day dermal (rat) NOAEL 1000 (HDT 2 

### ). Chronic / Carcinogenicity (rat) NOAEL 107/127 (M/F; HDT). Not carcinogenic. Chronic (dog) NOAEL 52 (HDT). Carcinogenicity (mouse) NOAEL 358/354 (M/F); (HDT). Not carcinogenic.

1 

### M/F stands for males/females.

2 

### HDT stands for the highest dose tested.

The EPA has classified dicamba as “Not Like ly to Cause Cancer in Humans” (the most favorable among EPA’s cancer categories), and concluded that dicamba is not mutagenic and is not a developmental toxin. There was no evidence of behavioral or neurological effects on offspring and, theref ore, a developmental neurot oxicity study was not required by the EPA.

In the human health risk assessments for dicamba, the EPA employed a chronic Population Adjusted Dose (cPAD) of 0.45 mg /kg/day, based on a 100-fold safety factor applied to the offspring NOAEL in the multig eneration rat study. For evaluating acute exposures to dicamba, the EPA employed an acu te Population Adjusted Dose (aPAD) of 1 mg/kg, based on a 300-fold safety factor (for use of the LOAE L rather than the NOAEL) applied to the LOAEL in the rat acute neurotoxicity study (U.S. EPA 2008a).

Using a dietary exposure model to estimate combined exposures from all presently approved uses, including both food and wate r exposure routes, and assuming that 100% of all labeled crops ar e treated with dicamba and that the resulting foods have tolerancelevel residues, total dietary exposure reache d only 4.4% of the aP AD level and 2.7% of the cPAD level for the general U.S. populati on; and 11% of the aPAD and 6.8% of the cPAD for the most highly exposed subpopulati ons of children 1-2 years old (U.S. EPA 2008a). The proposed dicamba use on MON 87708 does not require an increase in the soybean seed food tolerance. Monsanto has requested new tolerances for soybean forage and hay; however, these do not increase the li vestock dietary burden utilized in the RED assessment, so all dietary exposures from the proposed new dicamba use on MON 87708 are already accounted for in these assessments.Monsanto Company 10-SY-210U 614 of 721 

### L.3.2. Toxicology of Dicamba Pl ant and Animal Metabolites

MON 87708 has been genetically enhanced to express a dicamba metabolizing enzyme (dicamba mono-oxygenase). The enzyme catalyzes a mono-oxygenation reaction resulting in an oxidative demethylati on of dicamba, forming 3,6-dichloro-2- hydroxybenzoic acid, also known as 3,6-dichlorosa licylic acid (DCSA) . In the dicambatreated MON 87708, glucoside conjugates of DC SA were the major plant metabolites.



## OH Cl

Cl O OH

Figure L-2. Structure of DCSA

DCSA is a known metabolite of dicamba in soil, plants, and livestock. It is presently included in the residue expressi on specified in the food and f eed tolerance for dicamba in 40 CFR 180.227(a)(3). Therefore, the existing food tolerance for soybean seed includes

DCSA residues.

In the RED, EPA considered that DCSA ha s structural similarity to dicamba, and

concluded that it would have similar toxicity to the pare nt dicamba. Monsanto has conducted and submitted additional toxicity st udies involving direct dosing with DCSA (U.S. EPA OPP Decision Number D432752) to further substantiate the conclusion reached by the EPA in the RED. The results of these studies, summarized in Table L-4, can be compared to those of dicamba (Table s L-1 through L-3). Monsanto has submitted these studies to the EPA in support of our app lication to register th e use of dicamba on

MON 87708.Monsanto Company 10-SY-210U 615 of 721 

### Table L-4. Summary of Toxicologi cal Findings from Testing of DCSA

Study Systemic Toxicity Endpoint (mg/kg body wt/day) Offspring Toxicity Endpoint (if any) (mg/kg body wt/day) Acute oral (rat) LD 50 

### 2641 (Category III) NA Developmental (rat) Maternal NOAEL 100 Developmental NOAEL

100 Developmental (rabbit) Maternal NOAEL 25 Developmental NOAEL 65 (HDT) Reproduction, multigeneration (rat) Parental NOAEL 42 (M/F 1


### combined) Offspring NOAEL 42

Gene mutation – S. typhimurium & E. coli Not mutagenic NA

In vitro chromosome aberration (CHO) Aberrations not induced NA

Micronucleus (mouse) Negative NA Subchronic (90-day) oral (Rat) NOAEL 362/222 (M/F) NA

Chronic (12-month)(rat) NOAEL 171/206 (M/F). NA Subchronic (90-day) (dog) NOAEL 50 NA

In Vitro cytogentics (human lymphocytes) Weakly positive with S9

activation NA

In Vivo cytogenetics (rat) Negative NA Carcinogenicity Ongoing NA NA denotes Not Applicable.

1 

### M/F stands for males/females.

Smaller amounts of a glucoside of a nother known metabolite, 5-dichloro-3,6- dihydroxybenzoic acid (3,6-dichlo rogentisic acid, DCGA) were also identified in the soybean metabolism study. Levels of the DCGA glucoside were less than 10% of total MON 87708 soybean-contained radioactivit y. Monsanto conducted a limited set of toxicity studies on DCGA; and has provide d these studies to EPA to support our application to register dicamba on MON 87708.

The results of the DCSA and DC GA toxicity studies substan tiate the EPA conclusion that

dicamba metabolites will have simila r toxicity to parent dicamba.

L.3.3. Dicamba Ecological Eff ects – Non-target Species

The following tables summarize th e hazard potency of dicamb a to non-target species.

These data were taken from the Environm ental Fate and Effects Division (EFED) Chapter of the dicamba RED (U.S. EPA, 2005 a). These measures are the result of conventional laboratory testing against standard indi cator species of fish, birds, mammals, and plants as required for EPA re gistration. Results are presented usingMonsanto Company 10-SY-210U 616 of 721 

### standard toxicity indices, such as the concentration or dose required for 50% lethality (LC 50 

### or LD 50 

### ) or the concentration required for a 25 or 50% reduction in growth or biomass (EC 25 

### or EC 50 

### ). In some cases the “No Obse rvable Effect Concentration” (NOEC) or “No Observable A dverse Effect Level” (NOAEL) is also listed. A summary of the findings of the ecotoxic ity studies conducted on dicamba acid is provided in Tables L-5 through L-7. Ecotoxicity studies have also been conducted on the diglycolamine (DGA) salt of dicamba and are summarized in Table L-8.

Table L-5. Dicamba Acid Ec otoxicity Findings on Mammals, Birds and Fish (U.S. EPA. 2005a)

Study Toxicity Endpoint Comment

Mammals
Dicamba acid acute oral (rat) LD 50 

### 2740 mg/kg body wt
Multigeneration reproduction (rat)

NOAEL/LOAEL 45 /136mg/kg/day

Birds
Dicamba acid acute oral (quail) LD 50 

### 188 mg/kg body wt
Dicamba acid acute oral (duck) LD 50 

### 1373 mg/kg body wt
Dicamba acid sub-acute oral (quail) LC 50 

### >10,000 mg/kg diet

Dicamba acid sub-acute oral (duck) LC 50 

### 2009 mg/kg diet

Dicamba acid sub-acute oral (duck) LC 50 

### >10,000 mg/kg diet Replicate
Dicamba acid reproduction (quail)

NOEC/LOEC 1390 (HDT 1 

### ) mg/kg

diet
Dicamba acid reproduction (duck)

NOEC/LOEC 695/1390mg/kg diet

Fish
Dicamba acid (trout) LC 50 

### 28 mg/L
Dicamba acid (trout) LC 50 

### 135 mg/L Replicate
Dicamba acid (trout) LC 50 

### 153 mg/L Replicate
Dicamba acid (Bluegill) LC 50 

### 135 mg/L
Dicamba acid (Bluegill) LC 50 

### >50 mg/L Replicate
Dicamba acid (Sheepshead) LC 50 

### >180 mg/L
Dicamba acid fish early life stage NA
Dicamba acid fish life cycle NA
NA denotes Not Applicable.

NOEC stands for No Observab le Effect Concentration.

LOEC stands for Lowest Observ able Effect Concentration.

1 

### HDT stands for the Highest Dose Tested.Monsanto Company 10-SY-210U 617 of 721 

### Table L-6. Dicamba Acid Ecotoxicity Findings on Aquatic and Terrestrial Invertebrates (U.S. EPA. 2005a)

Study Toxicity Endpoint Comment

Aquatic Invertebrates
Dicamba acid (Daphnia) LC 50 

### 111 mg/L
Dicamba acid (Daphnia) LC 50 

### >100 mg/L Replicate
Dicamba acid (Sowbug) LC 50 

### >100 mg/L
Dicamba acid (Scud) LC 50 

### >100 mg/L
Dicamba acid (Grass Shrimp) LC 50 

### >132 mg/L
Dicamba acid (Fiddler Crab) LC 50 

### >173 mg/L
Dicamba acid (Oyster) LC 50 

### >1 mg/L
Dicamba acid (Glass Shrimp) LC 50 

### >56 mg/L
Invertebrate life cycle NA

Terrestrial Invertebrates
Honeybee LD 50 

### >90.6 μg/bee
NA denotes Not Applicable.

Table L-7. Dicamba Acid Ecotoxicity Re sults on Aquatic and Terrestrial Plants (U.S. EPA. 2005a)

Study Toxicity Endpoint Comment

Aquatic Plants

Dicamba acid duckweed EC 50 

### / NOEC >3.25 / 0.2 mg/L
Dicamba acid green alga EC 50 

### / NOEC >3.7 / 3.7 mg/L

Dicamba acid marine diatom EC 50 

### / NOEC 0.49 / 0.011 mg/L
Dicamba acid blue-green alga EC 50 

### / NOEC 0.061 / 0.005 mg/L

Dicamba acid freshwater diatom EC 50 

### / NOEC 2.3 / 0.5 mg/L

Terrestrial Plants
Dicamba acid seedling emergence monocot

EC 25 

### / NOEC 0.004 / <0.032 lb/a Onion

Dicamba acid seedling emergence dicot EC 25 

### /

NOEC 0.0027 / < 0.0022 lb/a Soybean

Dicamba acid vegetative vigor monocot EC 25 

### /

NOEC 0.15/ 0.13 lb/a Onion

Dicamba acid vegetative vigor dicot EC 25 

### /

NOEC 0.0068 / <0.004 lb/a SoybeanMonsanto Company 10-SY-210U 618 of 721 

### Table L-8. Dicamba, Diglycolamine (DGA) Salt Ecotoxicity Findings (U.S. EPA. 2005a)

Study Toxicity Endpoint Comment

Birds
Dicamba DGA salt acute oral (quail) LD 50 

### 262 mg a.e./kg body
weight
Dicamba DGA salt sub-acute oral (quail) LC 50 

### >1522 mg a.e./kg diet
Dicamba DGA salt sub-acute oral (duck) LC 50 

### >1522 mg a.e./kg diet

Fish
Dicamba DGA salt (trout) LC 50 

### >270mg a.e./L
Dicamba DGA salt (bluegill) LC 50 

### >270mg a.e./L

Aquatic Invertebrates
Dicamba DGA salt (Daphnia) LC 50 

### >270 mg a.e./L

 As a part of the reregistration evaluati on under FIFRA, EPA conducted an ecological screening assessment for dicamba. This assessment compared the results from toxicity tests with dicamb a conducted with various plant and animal species to conservative (high end) estimates of the concentration of dicamba to which an organism might be exposed in the envi ronment. These estimates, called the Estimated Environmental Concentrations (EECs), are point estimates for specific types of exposure ( e.g. , aquatic or dietary) that assu me constant high concentrations throughout the lifespan of the organism. Th ese estimates do not take into account

normal variation in environmen tal concentrations, the dilu tion or dissipation of those concentrations, or the frequency of exposur e of wildlife to the pesticide. Such assumptions provide a screening level of assessment where conclusions of no harm can be drawn with high confidence; however, it is also possible to reach a conclusion of no harm with more refined exposur e assumptions. EPA derived exposure estimates for a number of use patterns (U .S. EPA, 2005a), including two that are reasonably close to the pr oposed use pattern of dicam ba on MON 87708. These use patterns are: 1) for wheat, with two 1.0 lb a.e. /acre applications; and 2) for sorghum, with two 0.5 lb a.e./acre applications. For both crops, the two applications were considered to occur on May 1st and June 1st. Based on these hi ghly conservative screening level assessments, EPA’s Environm ental Fate and Effects Division (EFED) identified the concerns for non-target spec ies effects (terrestrial plants, Risks to terrestrial plants; acute risks to birds, a nd chronic risk to mammals in their risk assessment for the 2006 Dicamba RED (U.S. EPA, 2009b), however EPA concluded in the final reregistration decision doc ument that no specifi c additional drift mitigations were needed to support the con tinued registration of all dicamba uses.Monsanto Company 10-SY-210U 619 of 721 

### L.3.4. Environmental – Soil, Air, and Water Quality

The following tables summarize the physical and chemical properties of dicamba that can influence its mobility and persistence in the environment. These measures are the result of conventional laboratory testing, as re quired for EPA registration. Results are presented using standard environmental fate indices, such as the time required for 50% degradation (half-life) of th e initial concentration.

Table L-9. Environmental Fate and Ph ysical Properties of Dicamba Acid

Environmental Physical Properties Results
Dicamba acid vapor pressure 3.4 x 10 -5 

### torr (25° C)
Dicamba acid acidity (pKa) 1.87
Dicamba acid water solubility 6100 mg/L

Dicamba acid octanol / wate r partition coefficient 0.1

Dicamba acid K

oc 

### (Freundlich soil-bindi ng constant) 3.5 – 21.1 mL/g
Dicamba acid field dissipation half-life (conducted
with salt formulations) 3 – 19.8 days

Dicamba acid aerobic soil half-life, Laboratory 6 days

Dicamba acid anaerobic soil ha lf-life, Laboratory 141 days
Dicamba acid aerobic aquatic half -life, Laboratory 20.2 – 24.3 days
Dicamba acid aqueous photolysis half-life 38.1 days
Dicamba acid soil photolysis degrad ation rate ~20% after 30 days
Hydrolysis half-life Stable

L.3.4.1. Risks of Leaching and Runoff

The high water solubility, relatively low soil -binding coefficient, and a pKa that requires dicamba to exist primarily as an anion at normal soil pH suggest that dicamba may have risk of leaching or runoff. These indicators of low soil-binding are largely offset by the rapid soil microbial breakdown of both dicam ba and its DCSA metabolite under aerobic conditions, leading overall to only a low to mode rate risk of leaching or runoff. Using the Pesticide Root Zone Model/Exposure Analysis Modeling System (PRZM/EXAMS 51 

### ) and Screening Concentration in Ground Water (SCI-GROW 52 

### ) models, EPA estimated potential drinking water concen trations originating from gr ound and surface water for use in the dicamba dietary risk assessment. Mo del results indicated potential dicamba ground and surface water concentrations of 0.016 μg /L and 13.8 μg/L, respectively (U.S. EPA,

51 PRZM-EXAMS Model Description. Environmental Fate and Effects Division, Office of Pesticide Programs, U.S. Environmental Protection Agency, Washington, D.C. http://www.epa.gov/oppefed1/m odels/water/models4.htm#przm . Accessed May 28, 2010

52 SCI-GROW Model Description. Environmental Fate and Effects Division, Office of Pesticide Programs, U.S. Environmental Protection Agency, Washington, D.C. http://www.epa.gov/oppefed1/models /water/scigrow_description.htm . Accessed May 28, 2010.Monsanto Company 10-SY-210U 620 of 721 

### 2008). Surface water modeling using PRZM -EXAMS described in the EFED RED Chapter (U.S. EPA, 2005a) predic ted levels ranging from 4.9 to 18 μg/L for a one lb per acre dicamba a.e. application ra te to soybean (the highest si ngle proposed application rate for MON 87708). Dicamba has been in use for many years on a significant number of acres in the corn belt, with approximatel y 36 million treated-acres in 1994 alone (Table VIII-11), so that data from groundwater moni toring studies provide additional evidence pertaining to the potential for dicamba to leach. There has been a low incidence of dicamba detections (approximately 3% of s ites) in the U.S. Geological Survey National Water Quality Assessment (NAWQA) mon itoring program (U.S. EPA, 2005b). The highest detection levels were approxima tely 2 μg/L. Altogether, the modeling predictions and the NAWQA mon itoring results show that dicamba concentrations that might occur in drinking water are very low, and it confirms that the potential risk of dicamba leaching or runoff does not threaten human health or acceptable water quality. The lifetime Health Advisory Level (HAL) 53 

### for dicamba is 4,000 μg/L (U.S. EPA, 2011).

L.3.4.2. Other Hazards

Dicamba does not include any fluorine atoms, which are characteristic of ozone depletion risk. Based on the vapor pre ssure presented in Table L-9, dicamba is not sufficiently volatile to be present in the stratosphere at ozone-threatening leve ls. Dicamba acid, nor

its water soluble salts or derived formulation, does no t have the pr operties of explosiveness, flammabi lity, or corrosion.

L.3.4.3. Efficacy and Weed Management Practices for Dicamba on MON 87708

Since the introduction of the Roundup Ready ® 

### soybean crop product in 1996, glyphosate herbicides have become the predominant weed control tool in soybean production. That has been beneficial for gr owers, because glyphosate us e in the Roundup Ready soybean system offers unmatched flexible weed contro l at a relatively low pr ice; the high adoption rate of the Roundup Ready soybean system by growers is a testament to its ease of use and ability to provide full spectrum weed management. The adoption of the Roundup Ready soybean system has provided additional be nefits from a risk perspective, because glyphosate is widely-recognized as an herbicide that has a favorable profile in terms of human health, non-target animal species, a nd ground and surface water. A significant step that can be taken toward minimizing fu ture potential risk from herbicides is to strengthen and support the co ntinued sustainability of glyphosate use in soybean production by providing a complementary tool to manage hard-to-control weeds and, as a result, optimize soybean yield. As stated earlier, EPA ha s acknowledged the value of the glyphosate-based weed control sy stem and the need to have other herbicide modes of action available for IPM purposes, including weed resistance management, particularly in

53 The concentration of a chemical in drinking water that is not expected to cause any adverse noncarcinogenetic effects for a lifetime of exposure.

® Roundup Ready is a registered trademark of Monsanto Technology LLC. All other trademarks are the property of their respective owners.Monsanto Company 10-SY-210U 621 of 721 

### its decision to continue regist ration of herbicides in cotton (U.S. EPA, 2009a). In this revised RED decision on MSMA (an organic ar senic herbicide), EPA reversed it decision on continued registration of this active ingredient in cotton based in part on the

aforementioned IPM benefits.

According to 2006 market data 75.59 million ac res of soybeans were planted in 2006, and 74.6 million acres were harvested (USDA-NA SS, 2007a). Soybean-growing acres received approximately 2.0 herbicide tr eatments (AgroTrak, 2009), leading to approximately 149 million herbicide treated acres . Based on the most recently available USDA-NASS figures from 2006, 97% of the herbic ide total treated acre s were made with glyphosate herbicides, and the remaining 32% of treatments were made with more than 25 other active ingredients (USDA-NASS 2007b).

In 2008, dicamba-treated soybean acres only accounted for 0.53 million acres, or 0.7% of the soybean acres (Table VIII-1 2). This is primarily becau se current commercial soybean

varieties are not tolerant to dicamba, and therefore dicamba is only labeled for application at timings that avoid contact with the growin g plant or when some level of injury is acceptable: (1) preplant treatments 2 to 4 weeks prior to planting, depending on rate and rainfall, or (2) preharvest trea tments after the seeds have ma tured, at least 14 days before

harvest.

As a consequence of the intensive use of glyphosate for weed control in Roundup Ready soybean over the past 13 seasons, certain pr oblematic weeds (glyphosate hard-to-control and-resistant) have become more common, although glyphosate is s till considered to provide very broad spectrum weed contro l. Since the registration and market introduction of glyphosate, it has been known that certain weeds are more difficult to control than others at any gi ven herbicide rate and timing of application. Glyphosate, as with any herbicide, when used repeated ly season-after-season without other weed management practices, can select for those weed s that fall in this “difficult-to-control” category, including weeds such as morning gl ory, nightshade, and wild buckwheat, so that their populations can in crease if the correct rate and application timing are not practiced. For some weed sp ecies biotypes, selection and resistance have occurred in some geographical locations. In addition, resistance has de veloped for some populations of weeds that were formerly well-contro lled by glyphosate in soybean; these include glyphosate-resistant waterhemp, Palmer am aranth, horseweed, common ragweed, and giant ragweed. As a group, the glyphosate hard-to-contro l and glyphosate-resistant weeds are generally broadleaf dicotyledonous weeds. Accordingly, in Roundup Ready soybean fields where these problem weed s are widespread, affecting yield and complicating harvest, grower s have begun to use a variety of broadleaf herbicides sequentially or as a mi xture with glyphosate.

Monsanto, and other companies, have rec ognized this weed c ontrol need, and now recommend the incorporation of multiple mode s-of-action for weed control. Companies are also searching for a companion herbicid e for use with glyphosate to manage these populations of problematic broadleaf weeds. A complementary herbicide would provide new weed control options that strengthen the utility and sustainability of glyphosate as a

weed control tool in the Roundup Ready soybean system. In the absence of MON 87708Monsanto Company 10-SY-210U 622 of 721 

### integrated into the Roundup Ready soybean syst em, growers will need to rely on multiple herbicide applications at different timings to gain similar weed control. Dicamba is considered an optimum herb icidal companion to glyphosat e in control of broadleaf weeds, since soybean injury can be elim inated through the introduction of MON 87708, because it: 1) has a different mode-of-act ion from glyphosate; 2) dicamba has shown little propensity toward resistance developmen t; 3) is very effective in controlling the problematic broadleaf weed spectrum descri bed above; 4) has a sh ort soil half-life, without the potential for carry-over problems or plant-back restri ctions; 5) can offer superior crop safety relative to other pos temergent herbicides available for use in soybean; 6) is available at a cost appropriate to its benefit; and 7) is compatible in mixtures with glyphosate and other herbicides.

Monsanto has developed MON 87708 by introduc ing a gene that encodes an enzyme to catalyze the breakdown of dicamba. The dicamba mono-oxygenase enzyme cleaves a specific carbon-oxygen bond and converts the arom atic methoxyl moiety into a hydroxyl group, to form the non-herbicidal metabolite 3,6- dichlorosalicylic acid (DCSA). The rate

of dicamba breakdown in MON 87708 is suffi ciently rapid to provide complete protection from herbicidal symptoms, there by allowing dicamba app lications anytime up to crop emergence (cracking) as well as tw o postemergent in-crop applications between cracking and the R1/R2 growth stage. Monsan to seeks to offer growers soybean varieties that include both the glyphosate- and dicamba tolerance traits (MON 89788 × MON 87708), allowing use of either herbicide alone or together in sequence or as a mixture, to provide wide flexibility to meet diverse and specific weed control needs in

individual fields.

L.4. Properties of Alternative Herbicide Products

As discussed in L.3.4, ninety -seven percent (97 %) of soybean treated acres receive an application of glyphosate; the remaining acres are treated with more than 25 other active ingredients. In some of the soybean acres, these other active ingred ients are applied on acres that also receive a glyphosate application. The ten most widely used herbicides are shown in Table L-10. Integration of MON 87708 into the Roundup Ready soybean system and the subsequent use of dicamba will result in the displacement of some currently used, or foreseeable future use herb icides, and therefore th e properties of these alternative herbicides are summarized in this section to provide a baseline for comparison

to dicamba use on MON 87708.Monsanto Company 10-SY-210U 623 of 721 

### Table L-10. Ten Most Widely Used Alte rnative Herbicides in U.S. Soybean

Production

Herbicide Treated acres (millions) 1



### 2,4-D (acid, salts, and esters, combined) 6.32

flumioxazin 3.45 imazethapyr 3.35 cloransulam-methyl 2.90 chlorimuron-ethyl 2.66

fomesafen 2.26

clethodim 2.16 pendimethalin 2.15

tribenuron 2.05 flumiclorac-pentyl 1.32

1 AgroTrak, 2009



### Table L-11 also summarizes key information fr om alternate herbicid e product labels that are included in the comparative analysis in Section L.5. Table L-12 lists the eighteen active ingredients that make up the products in Table L-11. 2,4-D, being used primarily as a pre-plant application, is the most widely -used herbicide in this alternate herbicide list, representing about 10% of treated acres ; whereas acifluorfen, carfentrazone-ethyl, and flufenacet are the least used among these, representing <0.5% of treated acres. Mesotrione has not been used in soybean production previously; th e use on soybean was only recently registered by the EPA (2009d). Table L-12 also lists general regulatory

information about each herbicide. Note that only paraquat is classified as a Restricted Use pesticide among this group, on the ba sis of acute toxicological concern.Monsanto Company 10-SY-210U 624 of 721Table L-11. Alternative Registered Soybean HerbicidesBrand(U.S. EPA Reg.No.)ActiveIngredientSignalWord1ActiveIngredientContentRe-entryInterval(REI)2Max.Soybeanlb/a (singletreatment)3Max.Soybeanlb/a(season) Label Warnings or Special Directions4Aim®(279-3241) CarfentrazoneethylCaution 2 lb/gal 12 hr0.00850.023 "toxic to fish";"toxic to algae"; V3 - V10;do not feed foliage; some burn injuryAuthority®FirstDF (279-3246)Sulfentrazone Caution 0.62 lb/lb 12 hr 0.31 0.31 "known to leach"; "toxic to marine /estuarine invertebrates.";65-day PHI; croprotation restrictions, up to 30 mts; soilO.M. limits (sands <1% organic matter)Cloransulammethyl0.08 lb/lb 0.04 0.04Authority MTZ(279-3340)Sulfentrazone Caution 0.18 lb/lb 12 hr 0.028 0.046 "known to leach"; "toxic to marine /estuarine invertebrates. 120-day PHI (notOver The Top); sensitive varieties, injurypossibleMetribuzin 0.27 lb/lb 0.042 0.07Basagran®(7969-45)Bentazon Caution 4 lb/gal 48 hr 1 2 "known to leach";30-day PHI for feedingtreated forage and hay; minor injuryButoxone®7500(71368-49)2,4-DB Caution 0.75 lb/lb 48 hr 0.375 soil type limitsButyrac®200(42750-38)2,4-DB DMA salt Danger 2 lb/gal 48 hr 0.4 0.4 "toxic to fish";60-day PHI; injury mayoccur, especially with tank mixturesCadet®(279-3338)Fluthiacet-methyl Warning 0.91 lb/gal 12 hr 0.0065 0.009 do not feed foliage; minor injuryCallisto®(100-1131)Mesotrione Caution 4 lb/gal 12 hr 0.1875 0.1875 “high potential for runoff”; crop rotationrestrictions up to 18 mts; "transientbleaching" may occur; pre-emergence useonly, no in crop useMonsanto Company 10-SY-210U 625 of 721Table L-11 (continued). Alternative Registered Soybean HerbicidesBrand(U.S. EPA Reg.No.)ActiveIngredientSignalWord1ActiveIngredientContentRe-entryInterval(REI)2Max.Soybeanlb/a (singletreatment)3Max.Soybeanlb/a(season)Label Warnings or Special Directions4Classic®(352-436)ChlorimuronethylCaution 0.75 lb/lb 12 hr 0.14 0.14660-day PHI; crop rotation restrictions upto 30 mts and complicated description of3 different intervals specific to US regionsand soil pH; do not feed foliage; soil typelimits; "temporary leaf yellowing"Cobra®(59369-34)Lactofen Danger 2 lb/gal 12 hr 0.2 0.46"toxic to fish"; Do not apply past soybeangrowth stage R6 / 45-day PHI; minorinjuryExtreme®(241-405)Imazethapyr Warning 0.17lb/gal 48 hr 0.06460.0646"properties & characteristics associatedwith chemicals detected in ground water";crop rotation limitsGlyphosate-IPA 2 lb/gal 0.75 0.75FirstRate(62719-275)CloransulammethylCaution 0.84 lb/lb 12 hr 0.04 0.055 65-day PHI; crop rotation restrictions upto 30 mts; soil types; 14-day forage andhay feeding restrictionFlexstar®(100-1101)Fomesafen Warning 1.88 lb/gal 24 hr 0.35 0.3756"cause tumors"; "known to leach"; 45-dayPHI; do not feed foliage; crop rotationlimitsGangster®Copack (59639-131)Flumioxazin Caution 51% 12 hr 0.096 0.096 "toxic to aquatic invertebrates.";"Preemergent only. “properties &characteristics Associated with chemicalsdetected in ground water"; "toxic toinvertebrates."Cloransulammethyl84% 0.032 0.032GramaoxoneInteon®(100-1217)ParaquatdichlorideDanger 2 lb/gal(cationbasis)12 hr 1.5 2.9 "toxic to wildlife"; Restricted Use; noOver-the-Top useMonsanto Company 10-SY-210U 626 of 721Table L-11 (continued). Alternative Registered Soybean HerbicidesBrand(U.S. EPA Reg.No.)ActiveIngredientSignalWord1ActiveIngredientContentRe-entryInterval(REI)2Max.Soybeanlb/a (singletreatment)3Max.Soybeanlb/a(season)Label Warnings or Special Directions4Ignite®(264-829) GlufosinateammoniumWarning 2.34 lb/gal 12 hr 0.66 0.8 "runoff potential"; "toxic to vascularplants"; 70-day PHI; some crop rotationlimits up to 180 days; only Over-the-Top toLiberty Link soybeanLiberty®(264-660) GlufosinateammoniumWarning 1.67 lb/gal 12 hr 0.44 0.8 "runoff potential"; "toxic to vascularplants"; 70-day PHI; do not feed foliage;crop rotation limits up to 120 days;Phoenix®(59639-118)Lactofen Caution 2 lb/gal 12 hr 0.3 0.46"toxic to fish"; Do not apply past cropgrowth stage R6 / 45-day PHI; minor injuryPursuit®(241-310) Imazethapyr Caution 2 lb/gal 4 hr 0.063 0.063 "properties& characteristics associated withchemicals detected in ground water"; 85-day PHI; do not feed forage and hayPursuit®Plus(241-331)Pendimethalin Caution 2.7 lb/gal 24 hr 0.84 0.84 "properties & characteristics associated withchemicals detected inground water"; "toxicto fish"; 85-day PHI; crop rotation limits upto 40 monthsImazethapyr 0.2 lb/gal0.063 0.063Raptor®(241-379) ImazamoxammoniumCaution 1 lb/gal 4 hr 0.04 0.04 "phytotoxic to all plants"; plantback / croprotation limits up to 26 months, two regionswith complicated warningsMonsanto Company 10-SY-210U 627 of 721Table L-11 (continued). Alternative Registered Soybean HerbicidesBrand(U.S. EPA Reg.No.) Active IngredientSignalWord1ActiveIngredientContentReentryInterval(REI)2Max.Soybeanlb/a (singletreatment)3Max.Soybeanlb/a(season)Label Warnings or Special Directions4Reflex®(100-993) Fomesafen Danger 2 lb/gal 24 hr 0.375 0.3756"known to leach"; 45-day PHI; crop rotationlimits up to 18 mts; minor injury, significantgeographical restrictions (5 regions eachwith different rate structure)Resource®(59639-82)FlumicloracpentylWarning 0.86 lb/gal 12 hr 0.081 0.11 "toxic to shrimp"; 60-day PHI; do not feedforage or hay to livestock; temporaryspotting or burn to soybeanScepter®70 DG(241-306)Imazaquin Caution 0.7 lb/lb 12 hr 0.123 0.1236"properties & characteristics associated withchemicals detected inground water"; 90-dayPHI; do not feed forage or hay to livestock;crop rotation limits up to 40 mts; regionallimitations (3 regions)Sencor®(DF 75%)(264-738)Metribuzin Caution 0.75lb/lb 12 hr 0.6661.36"can seep or leach"; 70-day grain PHI; 40-day PHI on feeding forage to livestock; noOver-the-Top application, directed sprayOK; injury in high pH or low O.M. soils oron certain crop varieties, crop rotation limitsup to 18 mtsSynchrony®XP(352-648)Thifensulfuron Caution 0.069 lb/lb 12 hr 0.013 0.013 45-day planting restriction applied prior tosoybean planting / emergence; 60-day PHI;complicated crop rotation restrictions (3regions, 4 intervals) with limits up to 30mts; do not feed forage or hay to livestock;soil types; injury if adjuvants or tank mixedUltraBlazer(70506-60)AcifluorfensodiumDanger 2 lb/gal 48 hr 0.374 0.5 50-day PHI; minor injuryChlorimuron-ethyl 0.215 lb/lb 0.04 0.04Monsanto Company 10-SY-210U 628 of 721Table L-11 (continued). Alternative Registered Soybean HerbicidesBrand(U.S. EPA Reg.No.)ActiveIngredientSignalWord1ActiveIngredientContentReentryInterval(REI)2Max.Soybeanlb/a (singletreatment)3Max.Soybeanlb/a(season)Label Warnings or Special Directions4Valor®SX(59639-99)Flumioxazin Caution 0.51 lb/lb 12 hr 0.096 0.096 "runoff potential"; "toxic to aquaticinvertebrates."; preemergence use only, noin crop use; do not feed forage or hay tolivestock; crop rotation limit up to 18 mts. &soil type limits; injury under cool wetconditions or poorly drained soil; restrictionson use with flufenacet, alachlor,metolachlor, or dimenthenamidValor®XLT(59639-117)Flumioxazin Caution 0.3 lb/lb 12 hr 0.094 0.094 "toxic to aquatic invertebrates.";preemergence only, no in crop use; do notfeed forage or hay to livestock; crop rotationlimits up to 30 mts; injury under cool wetconditions or poorly drained soilChlorimuronethyl0.103 lb/lb 0.032 0.032Weedone®(650,638, LV4, LV6)and other 2,4-Dbrands (71368-3, -6, -10, -11, -14, -19)2,4-D; 2,4-D salts;2,4-D estersVaries Varies 0.93 0.93 Weedone 650 as an example: "toxic toaquatic invertebrates."; do not use on sandysoils (<1% O. M.); preplant to emergedweeds only , no in crop use; do not feedforage or hay to livestock.1The EPA-required statement to convey to applicators the overall acute toxicity hazard posed by the product. Caution is more favorable than Warning, which ismore favorable than Danger.2The period of time following application during which worker reentry into the treatedarea is restricted, according to EPA’s Worker Protection Standard (WPS).3The highest single-treatment and seasonal rates that can be applied to soybean according to the product Directions for Use label.4Lists specific statements extracted from the product label that represent specific hazards orlimitations that may reduce the utility of the product for soybean weedcontrol5Higher rates with directed / hooded sprayers.6Regional or soil type limitations may lower this rate.7Soybean label not yet publically available. Corn label comments are citedPHI – preharvest interval, O. M. – organic matter, mts - months.Monsanto Company 10-SY-210U 629 of 721Table L-12. Active Ingredients Contained in Alternative HerbicidesActive IngredientFirstRegistered12006 TreatedSoybeanAcreage(%)2RegistrationReviewStatus3RED Date4Max.Soybean lb/a(singletreatment)5Max.Soybeanlb/a(season)Tolerances(40 CFR180)6RestrictedUse7glyphosate salts3-May-7697open 2009Sep-931.56364Nodicamba-diglycolaminesalt2-Feb-56<0.5NAJun-09,corrected1929227No2,4-D acid, salts, andesters3-Jun-52 10 2013 Jun-05 0.93 0.93 142 Noflumioxazin 12-Apr-2001 3 unscheduled NA 0.096 0.096 568 Noimazethapyr 30-Jan-87 3 2014 Jun-06 0.06480.0648447 Nocloransulam-methyl 29-Oct-1997 1 2011 NA 0.04 0.055 514 Nochlorimuron-ethyl 4-Apr-86 4 2011 Sep-04 TRED 0.14 0.148429 Nofomesafen 10-Apr-87 2 open 2007 TRED Aug-07 0.375 0.3758433 Noflumiclorac-pentyl 23-Mar-94 1 open 2009 Aug-05 TRED 0.081 0.11 477 Nosulfentrazone 22-Nov-93 1 open 2009 NA 0.31 0.31 498 Nothifensulfuron 25-Apr-86 1 2011 NA 0.013 0.013 439 Noimazaquin 20-Mar-86 1 2014 TRED Dec-05 0.123 0.1238426 Noimazamox-ammonium 17-Apr-95 <0.5 2014 NA 0.04 0.04 1223 Noparaquat dichloride 8-Jan-80 1 2012 Aug-97 1.0 2.9 205Yeslactofen 1-Apr-87 <0.5 open 2007 TRED Sep-03 0.3 0.4 432 Noglufosinate-ammonium 29-May-91 <0.5 open 2008 NA 0.66 0.8 473 No2,4-DB 30-Jun-66 <0.5 2014 Jan-05 0.4 0.4 331 Nofluthiacet-methyl 14-Apr-99 <0.5unknown NA 0.0065 0.009 551 Noacifluorfen sodium 29-May-81 <0.5 unscheduled Sep-09 0.374 0.5 383 NoMesotrione 4-Jun-01 0.0 unscheduled NA 0.1875 0.1875 571 NoMonsanto Company 10-SY-210U 630 of 721TRED denotes Tolerance Reregistration Eligibility Decision1The date the herbicide was first approved for any use (e.g., industrial) by U.S. EPA.2The percentage of the herbicide-treated soybean acres that were treated with each herbicide in AR, IA, IL, IN, KS, KY, LA, MI,MN, MS, MO, NE, NC, ND, OH,SD, TN, VA, and WI in 2006 (USDA-NASS, 2007b) .3The herbicide’s progress in the ongoing EPA program named as Registration Review. Year indicates when the official docket wasor will be opened. EPA isrequired by law to re-evaluate pesticides periodically, generally every 10-15 years.4The date when EPA issued a Reregistration Eligibility Decision document. Reregistration was anearlier re-evaluation program designed to ensure that supportingdata are up-to-date for a.i.s first registered before 1984.TRED means Tolerance Reassessment Eligibility Decision, which refers to an alternative review path thatsome post-1984 a.i.s followed.5The maximum amount of the herbicide that can be applied to soybean in a single treatment or during the entire season, accordingto product labels.6The number of the paragraph in the Code of Federal Regulationswhere that herbicide’s food and feed tolerances are listed.7An EPA pesticide classification that restricts a product, or its uses, to use bya certificated pesticide applicator or under the direct supervision of a certifiedapplicator. See 40 CFR 152.160.8Regional or soil type limitations may lower this rate.9Maximum treatment rates for the proposed dicamba label.Monsanto Company 10-SY-210U 631 of 721 

### L.4.1. Human Health Effects of Alternative Herb icide Products

Table L-13 provides information concerni ng human health parameters for each alternative herbicide compared to dicam ba. The listed parameters include:

 Acute Toxicity Categori es for the herbicide.

 Cancer Classification of the herbicide.

 FQPA (Food Quality Protection Act) safe ty factor employed in EPA’s risk assessment process for the herbicide.

 Level of Exposure representing the accep table safe range for acute (acute population adjusted dose, aPAD) and chr onic (chronic population adjusted dose, cPAD) exposures.

 Extent to which all presently approved uses exhaust th e acceptable safe exposure range (% aPAD and % cPAD utilized for the most highly exposed population subgroups), according to recent Federal Re gister Rules or other public risk

assessment documents.

A variety of chemical-specific public data s ources were used to compile this comparison. Columns 9 to 11 in Table L-13 (to the right of the vertical gray bar) pertain specifically to the use of the herbicide in soybean:

 The established soybean seed food toleran ce in 40 CFR 180 that supports the uses in soybean.

 The Theoretical Maximum Residue Concentration (TMRC) arising from this soybean tolerance using the DEEM diet ary exposure software, assuming that

residues are at tole rance levels and that 100% of the crop has been treated.

 The percentage of the acceptable chronic exposure (cPAD) that is contributed by the soybean TMRC dietary exposure.Monsanto Company 10-SY-210U 632 of 721Table L-13. Human Health Risk Parameters for Alternative HerbicidesActive IngredientAcute (Oral, Dermal,Inhalation, Eye Irr.,Skin Irr., Sens.)1CancerClassification2cPADmg/kg/day3% cPADUtilized4,5aPADmg/kg/day6% aPADUtilized6,5FQPASF7Soy SeedTol.(ppm)8,9Soybean dietexposureμg/kg/day5,8,10%Soybeandietexposure /cPAD8,11glyphosate acid /potassium saltIV / IV / NA / II / IV / N;IV / IV / III / III / IV / NE1.757NANA1X2033.241.90%dicamba aciddiglycolamine salt12III / III / IV / II / II / N;III / III / IV / III / III / Nnot likely0.456.61111x1016.623.69%2,4-D acid /salts /estersIII / III / III/I/ III-IV /N;III / III / IV / III / IV / ND 0.005 38 0.067 58 1X0.02 0.03 0.66%flumioxazin IV/III/IV/III/IV/Nnot likely 0.02 18 0.03 8 1X0.02 0.03 0.17%imazethapyr IV / III / IV / IV / III / N not likely 2.5 < 1 NA NA 1X0.1 0.17 0.01%cloransulam-methyl IV / III / III / III/ IV / N not likely 0.1 <1 NA NA 1X0.02 0.03 0.03%chlorimuron-ethyl IV / III / IV / III / IV / N not likely 0.09 0 NA NA 1X0.05 0.08 0.09%fomesafen III / II /III /I/ II-III / Y not likely 0.0025 31 NA NA 1X0.05 0.08 3.32%flumiclorac-pentyl IV / III/ IV/ IV/II/ Y no evidence 1 < 0.01 NA NA 1X0.01 0.02 0.00%sulfentrazone III/ III / IV /I/ IV / Y not likely 0.14 <1 0.25 1 1X0.05 0.08 0.06%thifensulfuron IV / III / IV / IV / III / N not likely 0.07 <1 1.59 <0.1 1X0.1 0.17 0.24%imazaquin IV / III / III / IV / IV / N no evidence 0.25 <1 NA NA 1X0.05 0.08 0.03%imazamoxammoniumIV / III / IV / III / IV / N not likely NA NA NA NA 1Xex '03 NA NAparaquat dichloride II / III /I/II / IV / N E 0.00045 26 0.0042 66 1X0.7 1.16258.53%Monsanto Company 10-SY-210U 633 of 721Table L-13 (continued). Human Health RiskParameters for Alternative HerbicidesActive IngredientAcute (Oral, Dermal,Inhalation, Eye Irr.,Skin Irr., Sens.)1CancerClassification2cPADmg/kg/day3% cPADUtilized4,5aPADmg/kg/day6% aPADUtilized6,5FQPASF7Soy SeedTol.(ppm)8,9Soybean dietexposureμg/kg/day5,8,10%Soybeandietexposure /cPAD8,11lactofen IV / III / IV / III / IV / Nlikely/unlikely 0.008 <0.1 0.17 <0.13X (A)0.01 0.02 0.21%glufosinateammoniumIII / III / III / III / IV / N no evid. of 0.006 27 0.0063 48 1X2 3.3255.40%2,4-DB III / III / IV / III / IV / not likely 0.03 2 0.6 <1 1X0.5 0.83 2.77%fluthiacet-methyl IV / III / IV / IV / IV /Nlikely(7.5x10-7)0.001 <1 NA NA 1X0.01 0.02 1.66%acifluorfen sodium III / III / IV /I/ II / N likely/unlikely 0.013 <1 0.02 <11X/3X/10X0.1 0.17 1.28%mesotrione IV / III / IV / III / IV/ N not likely 0.007 5.8 NA NA3X0.01 0.02 0.23%Sources of the information summarized in this table are listed in the Alternative HerbicideSpecific References Section.N denotes negative for dermal sensitizationNA stands for not applicable.1EPA categories for the standard six acute toxicity tests of the active ingredient. Categories I and IV denote the least and most favorable findings, respectively.Category I findings are highlighted inboldfont.2The conclusion reached by the EPA Office ofPesticide Programs Cancer Assessment Review Committee. The system of classification has changed over the years,resulting in a combination of different terminology. Generally, Group E, “not likely” or “no evid.” are the most favorable conclusions, Group D indicates someuncertainty, and “likely/not likely” or “likely” indicate that a potential to inducecancer exists. More information can be found athttp://www.epa.gov/opp00001/health/cancerfs.htm. |Accessed March 26, 2010| “Likely” findings are highlighted in bold font.3The EPA-determined chronic Population Adjusted Dose, against which chronic exposure, primarily from combined food and water residues, are compared forhuman health risk assessment. This key risk assessment parameter is derived from consideration of all the chronic toxicity studies, and includes all necessary safetyfactors. Higher values indicate herbicideswith less severe chronic toxicity effects.4The percentage of the cPAD that is represented by all presentlyapproved uses of that herbicide for the most highly-exposed population subgroup.It is calculatedby summing estimated chronic exposures from dietary and water, and dividing by the level of exposure that is considered safe (i.e., cPAD). Lower percentagesindicate that current estimated exposure is a smaller proportion ofthe safe level, and thereforeimplies a greater margin of safety. EPA presents this calculationwhen new risk assessments are conducted, such as when a new food tolerance is petitioned.5Most highly exposed population subgroup.6The acute risk assessment parameters correspond to those described above for chronic exposure. In some cases, EPA’s review ofthe acute toxicity testing does notresult in an acute effect of concern, and noaPAD is needed, indicated in the table as NA.7The Safety Factor EPA has utilizedaccording to the requirements ofthe Food Quality Protection Act. FQPA thatrequires EPA utilize an additional 10-fold (10x)safety factor to protect infants and children, unless the scientific results indicate that a different level is protective. Ifthe database is complete, and the reproductiveMonsanto Company 10-SY-210U 634 of 721and developmental toxicity studies do not indicate that pre- and postnatal exposure results in increased sensitivity, EPA oftenreduces the FQPA SF to 1x. If there isindication of increased sensitivity, or the necessary data are lacking, SFs of 3X or10X are sometimes used. These higher safety factors are considered in thisanalysis to denote higher risk to infants and children, and such cases are highlighted in bold font.8The 3 columns to the right of the gray bar pertain to the use ofeach herbicide on soybean only,other current uses are excluded from this analysis.9The soybean seed food tolerance established in the relevant numbered paragraph in 40 CFR 180.10The Monsanto-calculated theoretical maximum dietary exposure to the most highly-exposed U.S. population subgroup if 100% of allsoybean were treated withthat herbicide, and residues were at tolerance levels. This theoretical exposure doesnot occur in real life, but allows a consistent comparison for all alternateherbicides. The calculation is made using the same DEEM dietary exposure model that EPA routinely useshttp://www.durango-software.com/software/deem.html.|Accessed March 26, 2010|.11The ratio of the prior column divided by the cPAD. It denotes the percentage of the safe exposure level that is attributed tosoybean residues, as a soybean riskindex. High numbers are less favorable. Any value above 100% requires further risk refinementto be deemed acceptable, such as that for paraquat. Values greaterthan 10-times that of dicambaare highlighted in bold font.12Dicamba diglycolamine data are for Clarity(U.S. EPA Reg. No. 7969-137) and M1691 formulations (U.S. EPA Reg. No. 524-582).Monsanto Company 10-SY-210U 635 of 721 

### L.4.2. Ecological Effects of Alternative H erbicide Products – Non-target Species

L.4.2.1 Fish and Aquatic Invertebrates.

Table L-14 provides information about the hazards, exposures, and risks of dicamba and each of the eighteen alternative herbicides to fish and aquatic invertebrates (considering 2,4-D acid, salts, and esters as one alternative herbicide). The listed parameters include:

 LC 50 

### endpoints from acute fish toxicity st udies, as reported in EPA’s one-liner

database 55 

### , published in a RED or Registratio n Review risk assessments. The highest and lowest available LC 50 

### values for any fish study are listed, regardless of species, including both fresh and marine species together. The purpose is to define a range of concentrations that spans the expected fish-toxic levels.

 EC 50 

### endpoints from acute aquatic invertebrate studies, as reported from the same sources cited above. The highest and lowest available EC 50 

### values for any invertebrate study are listed, regardless of species, including both fresh and marine species together. The purpose is to define a range of concentrations that spans the expected aquatic invertebrate-toxic levels.

 Estimated environmental exposure concen trations (EECs) in surface water for each of the eighteen alternative herbicides. The third column in Table L-14, identified as “Calculated EEC”, provides a simple standard estimate based on the maximum single application rate in soyb ean, using EPA’s standard field-farm pond scenario. This scenario examines a 1-acre pond in a 10-acre field in which (1) 5% of the application drifts into the 6-foot-deep pond and (2) 5% of the application onto the 10 acres runs off in to the same pond. The fourth column in

Table L-14 lists other model estimates of surface water concentrations as provided by one or more modeling pr ograms, as cited by EPA in public documents, such as Federal Register final tolerance rule drinking water assessments. The purpose is to define a range of concentrations that spans available estimates of potent ial aquatic exposure levels.

 Calculated Risk Quotients (RQs) for a quatic animals, comprised of fish and aquatic invertebrates combined together. Rather than calculate a single RQ for each species, Monsanto has calculate d a range of potential RQs for each herbicide, bracketed by the best- and worst- case values. The “best” RQ is derived from the ratio of the lowest reported EEC concentration divided by the highest

LC 50 

### or EC 50 

### for any aquatic animal. Conversely, the “worst” RQ is derived from the ratio of the highest EEC concentration divided by the lowest LC 50 

### or EC 50 

### for any aquatic animal. (Note: the RQ figur es are rounded to two decimal places, so that entries that appear as “0.00” mean that the specific RQ is less than 0.005.) The purpose is to define a range of RQs th at span and describe the risk posed by

55 National Information System – Regional IPM Ce nters. OPP Pesticide Ecotoxicity Database. http://www.ipmcenters.o rg/Ecotox/index.cfm [Accessed May 27, 2010].Monsanto Company 10-SY-210U 636 of 721 

### the alternative herbicide to aquatic anim als. The RQs that exceed the EPA’s Level of Concern (LOC) of 0.5 are marked in bold font.

Using the worst case risk quotient, ten of 18 alternative herbicides have risk quotients greater than or equal to 0.01, while the worst case risk quo tient for dicamba and seven other herbicide is <0.01. Only three of these 10 herbicides have risk quotients greater than 0.05 or 0.1, the levels of concern for threatened or endangered species and acute restricted use, respectively. Tw o of these 10 herbicides have RQ values greater than 0.5, the highest acceptable level of concern. Monsan to believes that based on risk quotients, dicamba offers a lower risk to aquatic anim als relative to three of the 18 alternative herbicides: 2,4-D esters, flumiox azin, and lactofen. This conc lusion is tabulated in Table

L-17.Monsanto Company 10-SY-210U 637 of 721Table L-14. Aquatic Toxicity Parameters for Fish and Aquatic Invertebrates for Alternative HerbicidesActive IngredientMaximumSoybeanlb/acre(singletreatment)1CalculatedEEC (ppm)2,3FIRST, GENEECor PRZX/EXAMSSurface Waterppm (RED orTolerance Rule)4Fish LC50Range(ppm)5AquaticInvertebrate LC50or EC50Range(ppm)fRisk Quotient forAquatic AnimalsRange6Label Warnings7low high low high best worstglyphosate salts1.50.0500.0008 - 0.02145>1000557800.000.00dicamba /DGA salt10.0340.01 - 0.03628> 270>100>2700.000.002,4-D acid + salts 0.93 0.031 0.064 - 0.118>80 2244 25 820 0.00 0.002,4-D esters 0.93 0.031 0.064 - 0.118>0.15 14.5 2.2 12 0.00< 0.79flumioxazin 0.096 0.003 0.018 - 0.0342.3 21 0.23 5.5 0.00 0.15"toxic to invertebrates”imazethapyr 0.064 0.002 0.006> 112 423 > 109 > 1000 0.00 0.00cloransulam-methyl 0.04 0.001 0.002> 86 > 154 > 111 > 121 0.00 0.00"toxic to invertebrates”chlorimuron-ethyl 0.14 0.005 0.003 - 0.005> 2 8.4 >10a> 10 0.00 0.00fomesafen 0.375 0.013 0.006 - 0.012126 > 163 25 376 0.00 0.00flumiclorac-pentyl 0.081 0.003 0.00024>0.189 > 24 >0.189 >38 0.00 0.02"toxic to shrimp"sulfentrazone 0.31 0.010 0.004 - 0.01694 > 120 1 60.4 0.00 0.02"toxic to invertebrates”thifensulfuron 0.013 0.000 0.0003 - 0.004>100> 100 >1000> 1000 0.00 0.00Monsanto Company 10-SY-210U 638 of 721Table L-14 (continued). Aquatic Toxicity Parameters for Fish and Aquatic Invertebrates for Alternative HerbicidesActive IngredientMaximumSoybeanlb/acre(singletreatment)1CalculatedEEC (ppm)2,3FIRST, GENEECor PRZX/EXAMSSurface Waterppm (RED orTolerance Rule)4Fish LC50Range(ppm)5AquaticInvertebrate LC50or EC50Range(ppm)5Risk Quotient forAquatic AnimalsRange6Label Warnings7low high low high best worstimazaquin 0.123 0.004 0.004 - 0.008280 420 280a280 0.00 0.00imazamox-ammonium 0.04 0.001 0.002> 94.2 > 122 >94.3 > 122 0.00 0.00paraquat dichloride 1.0 0.033 0.0015> 1 156 1.2 1.2 0.00 < 0.05lactofen 0.3 0.011 0.000008 - 0.40.46 0.46 0.02 4.85 0.0020"toxic to fish"glufosinate-ammonium 0.66 0.022 0.043 - 0.094> 320 > 1000 8 668 0.00 0.012,4-DB 0.4 0.013 0.013 - 0.0152 18 25a25 0.00 0.01"toxic to fish"fluthiacet-methyl 0.00650.000 0.0005 - 0.00080.043 0.16 0.3 >2.3 0.00 0.02acifluorfen sodium 0.374 0.013 0.0024 - 0.01031 204 28.1 > 1000 0.00 0.00mesotrione 0.1875 0.011 0.004 - 0.02> 114 520 3.3 840 0.00 0.011The highest single-treatment rate permitted by the herbicide’s product labels. This rate is used to calculate potential acuteexposure to aquatic non-target speciesvia spray drift or runoff.2Based on the maximum single treatment rate, with 5% spray drift and 5% runoff from 10 treated acres into a 1-acre 6-foot-deep pond.3The Estimated Environmental Concentration in surface water. Itwas calculated by Monsanto using the EPA’s “standard field-farmpond scenario”http://www.epa.gov/oppefed1/models/water/geneec2_description.htm|Accessed May 28, 2010|. In this scenario, itis assumed that the herbicide is applied to a10 acre farm field containing a 1 acre pond that is six feet deep. The pond experiences 5% of the application rate by spray drift and 5% of the application on thesoil enters the pond via runoff. This concentration estimationis a simple, conservative Tier 1 procedure that utilizes only the application rate to estimate aquaticMonsanto Company 10-SY-210U 639 of 721exposures, and allows quick comparison of many different herbicides. Other more increasingly-detailed computer models can be used to obtain more refinedEEC estimates, but these require the userto input various physical chemical parameters and weatherdata for each product to bemodeled. Examples of thesemethods are listed in the next column.4Modeling estimates of potential aquatic exposure levels. When EPA conducts risk assessments, it uses computer models to obtainestimates of potential surfacewater concentrations. These are published inthe RED for each herbicide, or in tolerance rules in the Federal Register. The range of estimates is listed, whichcan be compared to the single number in the prior column. Sources of these estimates are listed in the “ALTERNATIVE HERBICIDE-SPECIFICREFERENCES“ section.5“Fish LC50Range (ppm)” and “Aquatic Invertebrate LC50or EC50Range (ppm)”. These four columns describe the range of hazard data found in public datasources representing the toxicityof each herbicide versus freshwater and marine animals. The LC50or EC50means the water concentration needed to kill orimmobilize half of the test species, which is a standard potency descriptor. The highest and lowest values found for any fishspecies (trout, bluegill, sheepshead,etc.) were tabulated. Likewise, the highestand lowest values found for any aquatic invertebrate species (Daphnia, shrimp, crab, etc.) were included. Sources ofthese data are listed in the “ALTERNATIVE HERBICIDE-SPECIFIC REFERENCES“ section and in available public databases (National Information System –Regional IPM Centers. OPP Pesticide Ecotoxicity Database.http://www.ipmcenters.org/Ecotox/index.cfm) |Accessed May 28, 2010|.6EPA’s EFED uses a Risk Quotient (RQ) method for ecological risk assessment. The RQ equals the potential exposure level dividedby the hazard level. Higherexposures or more potent hazard findings lead to higher RQs. EFED has established Levels of Concern (LOCs) for various non-target species categories. Whenthe RQ exceeds the LOC, further refinement is needed to determine whether risk mitigation might be needed. For non-listed aquatic animals, the LOC for acuterisk is 0.5, for acute risk restricted useis 0.1, and for threatened or endangeredspecies it is 0.05. In this analysis, Monsanto calculated best-case RQs by dividingthe lowest EEC estimate by the highest hazard (LC50or EC50) value, and calculated the worst-case RQ fromthe highest EEC and lowest hazard value. Thepurpose was to bracket a range that typifiedthe aquatic animal risk presented by eachherbicide. When neither the worst-casenor the best-case RQs exceed aLOC of 0.05, Monsanto concluded that risk to aquatic animals is minimal. Instances where the LOC threshold of 0.5 is exceededare highlighted in bold font.7Lists instances where the product label includes warning statements about aquatic animal exposure.Monsanto Company 10-SY-210U 640 of 721 

### L.4.2.2. Aquatic Plants

Table L-15 provides information about the hazards, exposures, and risks of dicamba and each of the eighteen (18) alternative herbicid es to aquatic plant species, specifically duckweed and aquatic algae sp ecies (considering 2,4-D acid, salts, and esters as one alternative herbicide). The data form at, sources, and methods of Estimated Environmental Exposure Concentration (EEC) ca lculation are identica l to those described above for the aquatic animals (Table L-14). A Level of Concern (LOC) value of 1.0 has been used for judging RQ exceedances in the ca se of aquatic plants, consistent with EPA EFED’s normal practices.

The assessment and comparison summarized in Table L-15 establishes that dicamba poses little acute risk to aquatic plants at use rates of 0.05 – 1.0 lb dicamba a.e./acre, which is consistent with EFED’s assessment published in the RED EFED Chapter.

Monsanto was unable to identif y aquatic plant hazard data for three of the alternative herbicides (imazaquin, chlorimuron-ethyl, and fl umiclorac-pentyl). For nine (9) of the eighteen (18) alternative herbicides, the range of RQs is < 0.005 to 0.75; that is, none of these nine a.i.s present an aquatic plant risk, which even in the worst-case calculation, reach EFED’s Level of Concern (LOC) for aq uatic plants. However, for seven of the alternative herbicides, the worst-case RQs did exceed EFED’s LOC of 1.0. It is not surprising that some herbicides are quite toxic to aquatic plants, and the worst-case RQs for three of the alternate herbicides (flu mioxazin, lactofen, and paraquat dichloride) exceeded the LOC by a factor of more than 50-fold.

Monsanto believes that dicamba offers a lower risk to aquatic plants relative to seven of the 18 alternative herbicides (2,4-D, flumi oxazin, sulfentrazone, th ifenslufuron, paraquat dichloride, lactofen, and meso trione). This conclusion is tabulated in Table L-17.Monsanto Company 10-SY-210U 641 of 721Table L-15. Aquatic Toxicity Parameters for AquaticPlants for Alternate Herbicide Active IngredientsActive IngredientMaximumSoybeanlb/acre(singletreatment)1CalculatedEEC(ppm)2,3FIRST,GENEEC orPRZX/EXAMSSurface Waterppm (RED orTolerance Rule)4Duckweed andAlgae EC50Range(ppm)5Risk Quotient forAquatic PlantsRange6low high best worstglyphosate salts1.50.0500.0008 - 0.0210.7738.60.000.06dicamba /DGA salt10.0340.01 - 0.0360.06> 3.70.000.602,4-D acid + salts 0.93 0.031 0.064 - 0.1180.29 156 0.00 0.412,4-D esters 0.93 0.031 0.064 - 0.1180.066 >19.8 0.001.79flumioxazin 0.096 0.003 0.018 - 0.0340.0005 0.019 0.1668.00imazethapyr 0.064* 0.002 0.0060.008 59.2 0.000.75cloransulam-methyl 0.04 0.001 0.0020.003 135 0.00 0.67chlorimuron-ethyl 0.14 0.005 0.003 - 0.005NA NA NA NAfomesafen 0.375 0.013 0.006 - 0.0120.09 71 0.00 0.14flumiclorac-pentyl 0.081 0.003 0.00024NA NA NA NAsulfentrazone 0.31 0.010 0.004 - 0.0160.002 0.033 0.128.0thifensulfuron 0.013 0.000 0.0003 - 0.0040.0016 > 0.026 0.022.50imazaquin 0.123 0.004 0.004 - 0.008NA NA NA NAimazamox-ammonium 0.04 0.001 0.0020.011 > 0.038 0.03 0.18paraquat dichloride 1.0 0.033 0.00150.00055 2.84 0.0090.9lactofen 0.3 0.011 0.000008 - 0.40.001 0.001 0.00400glufosinate-ammonium 0.66 0.022 0.043 - 0.0941.5 7.8 0.00 0.062,4-DB 0.4 0.013 0.013 - 0.015>0.932 >0.932 <0.01 <0.02fluthiacet-methyl 0.00650.000 0.0005 - 0.00080.0022 >0.018 <0.01 0.36acifluorfen sodium 0.374 0.013 0.0024 - 0.010> 0.26 0.38 0.01 <0.05mesotrione 0.1875 0.011 0.004 - 0.020.018 132 0.001.11Monsanto Company 10-SY-210U 642 of 721The first three columns in this table are identical to- those in Table L-14.1The highest single-treatment rate permitted by the herbicide’s product labels. This rate is used to calculate potential acuteexposure to aquatic non-target species viaspray drift or runoff.2Based on the maximum single treatment rate, with 5% spray drift and 5% runoff from 10 treated acres into a 1-acre 6-foot-deep pond.3The Estimated Environmental Concentration insurface water. It was calculated by Monsanto using the EPA’s “standard field-farmpond scenario”http://www.epa.gov/oppefed1/models/water/geneec2_description.htm|Accessed May 28, 2010|. In this scenario, it isassumed that the herbicide is applied to a 10acre farm field containing a 1 acre pond thatis six feet deep. The pond experiences 5% of the application rate by spray driftand 5% of the application on the soilenters the pond via runoff. This concentration estimation is asimple, conservative Tier 1 procedure that utilizes only the application rate to estimate aquaticexposures, and allows quick comparison of many different herbicides. Other more increasingly-detailed computer models can be used to obtain more refined EECestimates, but these require the user to input various physical chemical parameters and weather data for each product to be modeled. Examples of these methods arelisted in the next column.4Modeling estimates of potential aquatic exposure levels. When EPA conducts risk assessments, it uses a computer models to obtain estimates of potential surfacewater concentrations. These are published in the RED for each herbicide, or in tolerance rules inthe Federal Register. The range of estimates is listed, which can becompared to the single numberin the prior column. Sources of these estimatesare listed in the “ALTERNATIVE HERBICIDE-SPECIFIC REFERENCES” section.5These two columns describe the range of hazard data found in public data sources representing the toxicity of each A.I. versusfreshwater and marine plants. TheEC50means the water concentration needed to kill or prevent growthof half of the test species, which is a standard potency descriptor. The highest and lowestvalues found for any aquatic plant species (diatom, duckweed, alga, etc.) were tabulated. Sources of these data are listed inthe “ALTERNATIVE HERBICIDESPECIFIC REFERENCES” section of this document and in available public databases cited in footnote 37 (National Information System – Regional IPM Centers.OPP Pesticide Ecotoxicity Database.http://www.ipmcenters.org/Ecotox/index.cfm|Accessed May 28, 2010|)6As described above for Table L-15, EPA’s EFED uses a Risk Quotient (RQ) method for ecological risk assessment. For non-listedaquatic plants or for threatenedor endangered species the LOC is 1.0. In this analysis, Monsanto calculated best-case RQs by dividing the lowest EEC estimateby the highest hazard (EC50) value,and calculated the worst-case RQ from the highest EEC and lowesthazard value. The purpose was tobracket a range that typified the aquatic plant risk presentedby each herbicide. When neither the worst-case nor the best-caseRQs exceed a LOC of 1.0, Monsantoconcluded that risk to aquatic plants is minimal. Instanceswhere the LOC threshold of 1.0 is exceeded are highlighted in bold font.Monsanto Company 10-SY-210U 643 of 721 

### L.4.3. Looking Toward the Foreseeable Future

As noted, weeds that are difficult to cont rol with glyphosate and weeds that are glyphosate-resistant, PPO-resistant, or AL S-resistant represent an opportunity for improved weed control in soybean. To addr ess this need, Monsan to is seeking to commercialize MON 87708, which will allow dicamba to be used as a weed control tool in this important U.S. crop. Monsanto is al so aware that other companies are developing biotechnology-derived soybean enhanced w ith other herbicide-tolerant traits.

Biotechnology-derived soybean developed to be tolerant to applications of 2,4-D has been submitted to USDA-APHIS for deregulation. 56 

### Monsanto proposes that 2,4-D presents more relative risk than dicamba in the area of acute human health and chronic exposure risk, and for 2,4-DB relatively more ecological risk for aquatic animals. Applications of 2,4-D will also require management of offsite movement.

In addition, biotechnology-derived soybean enhan ced to be tolerant to herbicides that inhibit 4-hydroxyphenylpyruvate dioxygenase (HPPD ) is one such trait that Monsanto believes is in development, based on public announcements from companies other than Monsanto; evidence of these developments is apparent in the r ecent EPA approval of

mesotrione for use on mesotrione-tolerant s oybean (U.S. EPA, 2009e). HPPD-tolerance

allows use of these broadleaf herbicides in soybean production, wh ich is a good technical fit as highlighted by Table L-16 which shows many of the troublesome soybean weeds are effectively controlled by HPPD herbic ide products such as Balance Pro (active ingredient: isoxaflutole), Laudis (active ingredient: tembotrione) or Callisto Herbicide (active ingredient: mesotrione). Since some of these products are not yet registered for soybean use, and the label for Callisto defini ng application parameters in soybean is not yet commercially available, it is not possible to undertake a rigorous application-ratebased risk comparison with dicamba, but it is possible to make a hazard comparison, using isoxaflutole and tembotri one as typical examples (mes otrione is incl uded above in

the discussion of alternate herbicides , since has been approved by EPA).

Inhibition of HPPD in plants results in a disruption of carotenoid biosynthesis, which leaves the plant’s chlorophyll pigments unprotected from rapid degradation via photooxidation, and results in a very charac teristic white bleached symptomology of plant parts that are normally gr een. HPPD is also an animal liver enzyme involved in the catabolic breakdown of tyrosine, and its inhi bition in laboratory animals results in elevated tyrosine plasma concentrations (tyrosinemia), which can cause adverse ocular, developmental, liver, and kidney effects. St ated simply, the mechanism of herbicidal efficacy based on HPPD inhibition is inheren tly linked to a potential for negative human

health effects.

Publicly available study results show th at isoxaflutole and tembotrione both caused

ocular and liver effects in test animals, and both caused developmental t oxicity at non-

56 Dow Agrosciences press release, December 15, 2009.

http://www.dowagro.com/newsroom/corporatenews/2009/20091215a.htm . [Accessed March, 26, 2010].Monsanto Company 10-SY-210U 644 of 721 

### maternally toxic levels. Because of the pot ent toxic effects, EPA has calculated cPADs

for isoxaflutole and tembotrione that are lowe r than that of dicamba by factors of 225 and 1125, respectively. EPA is also considering the need for a cumulative risk assessment approach for these two chemicals, along w ith mesotrione and other HPPD-inhibiting herbicides, because they share a common toxic mechanism (U.S. EPA, 2009d, e). In addition, both isoxaflutole and tembotrione had carcinogenic effects in long-term testing summarized by EPA. For tembotrione, carcin ogenicity was limited to rats only, but

isoxaflutole was found to have carcinogenic effects in two species, and was categorized by EPA as a B2 carcinogen in 1998 when first registered. Isoxaflut ole products, such as Balance Pro, are Restricted Use pesticides b ecause of a very high level of concern about damage to non-target plants caused by spray drift, and isoxaflutole labels also bear warning statements about the likelihood of pe rsistence and leaching. There have also

been concerns about isoxafluto le use leading to levels of herbicid ally-active isoxaflutole metabolites in surface water utilized for irriga tion purposes. Because of these properties of HPPD chemicals, Monsanto believes that di camba offers a lower risk relative to these potential future soybean herbicides.

L.4.4. Efficacy and Weed Management Prac tices of Alternative Herbicide Products

Table L-16 provides weed control effectiv eness of formulated products containing dicamba, glyphosate and alternate herbicides. Weed control less than 70% is considered insufficient (white), control between 70 and 85 % is considered as marginal effectiveness (black-white), and control of more than 85% is considered commercially acceptable (black). The data presented in Table L- 16 are derived by combining state and dealer 57


### herbicide guidance for soybean production across major s oybean-producing states (Ohio

and Indiana 58 

### , Iowa 59 

### , Tennessee 60 

### , North Dakota 61 

### ). Weed control herbicide recommendations provided by University Extens ion scientists to control specific weeds

were converted to a common scale and combin ed to reflect an av erage herbicide weed control rating across geographies . Weed control ratings speci fic to resistant weeds were based mainly on recommendations from Ohio and Indiana. Monsanto weed scientists applied their own expert scientific judgm ent during the conversion of University

Extension recommendations into a common scal e. The weeds chosen for inclusion in Table L-16 represent current problem weeds in soybean, either because they exhibit herbicide resistance or because they are generally hard-to-control species.

As Table L-16 shows, the altern ative herbicides can be used in preemergent applications, postemergent applications, or, like dicam ba and glyphosate, as both pre- and

57 2009 Crop Protection Guide - Information for Dealers http://www.agrisolutionsinfo.com/ Accessed

May 28, 2010

58 2010 Ohio and Indiana Weed Control Guide (Bulletin 789; Pub. WS16)

www.btny.purdue.edu/Pubs/WS/WS-16/ Accessed May 28, 2010

59 2009 Herbicide Guide for Corn and Soybean Production - http://www.weeds.iastate.edu [Accessed May

28, 2010]

60 2010 Weed Control Manual for Tennessee

http://www.utextension.utk.edu/publications/pbfiles/pb1580.pdf Accessed may 28, 2010

61 2010(a) NDSU Weed Control Guide - http://www.ndsu.edu/weeds Accessed May 28, 2010Monsanto Company 10-SY-210U 645 of 721 

### postemergent applications. Several herbicid es, such as 2,4-D, 2,4-DB, sulfentrazone and paraquat, do not have sufficient soybean safe ty for application in-crop, so their use is limited to control of existing weeds and pree mergent control of later emerging weeds via soil residual activity, if any. Others, such as the protoporphyrinogen oxidase (PPO) inhibitors acifluorofen, lact ofen, and fomesafen, are most effective as postemergent treatments, even though they may cause some soybean leaf injury. Acetolactate synthase (ALS) inhibitors like chlorimu ron-ethyl and cloransulam-met hyl can be used at either timing. Glufosinate, like glyphosate and di camba, does not have intrinsic soybean selectivity and can only be used as a poste mergent application ove r soybean that is genetically enhanced to provi de glufosinate tolerance ( i.e. , Liberty Link).

Table L-16 also highlights ex pected weed efficacy trends . Glyphosate does not provide commercial control of glyphosat e-resistant weed biotypes such as common ragweed orhorseweed, but does control the wild type plants of these species. Similarly, ALSinhibiting herbicides provide poor control of ALS-resistant weeds, and PPO inhibitors do not adequately control PPO-resistant weeds. Table L-16 focuses on problem weeds found in soybean and does not include any weeds with auxin re sistance, although 2,4-D provides good control of many he rbicide-resistant broadleaf w eed biotypes in this group. Extreme, which contains two modes-of-act ion (glyphosate and imazethapyr), provides better control of this target weed spectrum than most of the herbicide products having only a single mode-of-action.

Further discussion of the comparative eff ectiveness of dicamba versus alternative herbicide products against herb icide-resistant and hard-to-co ntrol weeds can be found in

L.5.3.Monsanto Company10-SY-210U646 of 721Table L-16. Herbicide Efficacy Comparison: Herbicide Resistant Weeds and Hard-to-Control Weeds in Soybean2>85% control170% - 85% control0< 70% controlHerbicideHerbicide BrandClass GR Waterhemp GR Palmer amaranth GR Horseweed GR C. ragweed GR G. ragweed GR Johnsongrass ALS C. ragweed ALS+PPO C. ragweed ALS+GR C. ragweed ALS G. ragweed ALS+GR G. ragweed ALS+GR Horseweed ALS Kochia TRI Lambsquarters A. Morningglory B. Nightshade Cockelbur C. Ragweed G. Ragweed Hemp sesbania Kochia Lambsquarters Horseweed Palmer Amaranth Prickly Sida Sicklepod Velvetleaf WaterhempGlyphosateRoundupEPSPSDicambaClarityauxin2,4-D2,4-Dauxin2,4 DBButyrac 200auxinChlorimuron + MetribuzinCanopy DFALS/PSIIChlorimuron + TribenuronCanopy EXALSImazaquinScepterALSSulfentrazone + ImazethapyrAuthority AssistPPO/ALSSulfentrazone + MetribuzinAuthority MTZPPO/PSIISulfentrazone + CloransulamAuthority First / SonicPPO/ALSFlumioxazinValorPPOFlumioxazin + ChlorimuronValor XLTPPO/ALSFlumioxazin + CloransulamGangsterPPO/ALSCloransulamFirstrateALSParaquatGramoxone InteonPSIIsoxaflutoleBalance PROHPPDMesotrioneCallistoHPPDMesotrioneCallistoHPPDTembotrioneLaudisHPPDTropramezoneImpactHPPDHard-to-Control Weeds2HPPD POSTEMERGENCE5Resistant Weeds1PREEMERGENCE/BURNDOWN3HPPD PREEMERGENCE4Monsanto Company10-SY-210U647 of 721Table L-16 (continued). Herbicide Efficacy Comparison: Herbicide Resistant Weeds and Hard-to-Control Weeds in Soybean2>85% control175% - 85% control0< 75% controlHerbicideHerbicide BrandClass GR Waterhemp GR Palmer amaranth GR Horseweed GR C. ragweed GR G. ragweed GR Johnsongrass ALS C. ragweed ALS+PPO C. ragweed ALS+GR C. ragweed ALS G. ragweed ALS+GR G. ragweed ALS+GR Horseweed ALS Kochia TRI Lambsquarters A. Morningglory B. Nightshade Cockelbur C. Ragweed G. Ragweed Hemp sesbania Kochia Lambsquarters Horseweed Palmer Amaranth Prickly Sida Sicklepod Velvetleaf WaterhempGlyphosateRoundupEPSPSDicambaClarityauxinImazethapyr + GlyphosateExtremeALS/EPSPSFomesafenFlexstarPPOImazethapyrPursuitALSCloransulamFirstrateALSFlumicloracResourcePPOChlorimuronClassicALSImazamoxRaptorALSLactofenCobra / PhoenixPPOFomesafenReflexPPOAcifluorfenUltra BlazerPPOGlufosinateIgniteGluChlorimuron + ThifensulfuronSynchrony XPALSFluthiacetCadetPPOHard-to-Control Weeds2Resistant Weeds1POSTEMERGENCE5Horseweed is synonymous to marestail.GR = Glyphosate resistantALS = Acetolactate synthaseTri = TriazinePPO = protoporphyrinogen oxidaseMonsanto Company 10-SY-210U 648 of 721Note:This table was based primarily on State University Extension weed control recommendations for soybean growing areas, Table L-16 indicates the degree towhich each product controls the targeted weeds in soybean. The legend describes the meaning of the symbols. See L.5.3 for additional detail.1The section of the table describes control of populations (biotypes) of weeds that are known to have genetic resistance to specific herbicidal modes-of-action2This section describes control of weeds that are difficult tocontrol in soybean with existing herbicide treatments. Generallythese are broadleaf weeds whoseremoval would require herbicide rates that would severely damage the crop.3This section describes weed control by herbicides that are applied prior to soybean emergence. The weeds may or may not be emerged.4This section describes foreseen use of herbicides that are notyet approved for use in soybean that have inhibition of 4-hydroxyphenylpyruvate dioxygenase.5The section describes weed control by herbicides that are applied after the soybean emergence, generally over-the-top of the crop.Monsanto Company 10-SY-210U 649 of 721 

### L.5. Comparison of Dicamba Use in the Dicamba-Tolerant Soybean System (MON 87708) to Altern ative Herbicides

The intent of the comparative analysis is to define current herbicide use in U.S. soybean production, and to compare dicamba’s human health and environm ental properties to herbicides currently used by growers for weed control. In order for a pesticide (herbicide) to be registered by EPA it must meet the FIFRA and FFDCA standards for safety to human health and the environment. The EPA must conclude that the herbicide when used according to the label does not pose an unreasonable risk to humans or the environment; and in order to establish a tolera nce for the use of an herbicide on a food or feed crop, find there is a reasonable certain ty of no harm to human health from nonoccupational (food, water and re sidential/recreational) expos ures to the herbicide. Consequently all alternative s oybean herbicides can be used safely, and do not pose a risk

to humans or the environment.

L.5.1. Comparisons for Human Health Risks

Table L-13 provides information concerning human health parameters for dicamba and the alternative herbicide products. These data allow a comparison of the relative human health safety among the optional weed control products on the basis of:

 Acute toxicity.

 Chronic toxicity.

 Cancer risks and classification.

 Risk to infants and children.

 Magnitude of potential exposure that is considered to be within the acceptable safe range.

 Extent to which all presently approved us es exhaust the acceptable safe acute and chronic dietary exposure ranges fo r the most highly exposed population subgroups.

 The proportion of present dietary exposu re that arises through use on soybean.

A compilation of Monsanto’s comparative determinations of the risks posed by the 18 alternate herbicides compared to dicamba in the four basic human health risk categories (acute, cancer, chronic and infants/children) is tabulated in Table L-17. A “Yes” entry indicates a benefit for dicamba compared to that alternative herbicide. Entries not

indicated with a “Yes” mean that dicamba is ei ther comparable or less favorable than the

alternative herbicide. 

### A “Neutral” entry means that dicamba and the alternative herbicide have similar risks, and a “No” entry denotes alternative herbicides that have a benefit compared to dicamba. Table L-17 is intende d as a 1-page scorecard summary of theseMonsanto Company 10-SY-210U 650 of 721 

### benefit comparisons. Monsanto has conc luded that dicamba use on MON 87708 will provide benefits compared to the alternate herbicides used for soybean weed control.

L.5.1.1. Acute Health Risks

Dicamba acid has a Category II classification for eye and skin irritation, but once the acid

is neutralized to form the DGA or othe r salt forms used in herbicide product formulations, all acute Categories are III or IV. These classifications are the most favorable acute toxicity categories in EPA’s acute hazard paradigm. Several alternative herbicides (acifluorfen, sulfen trazone, and some forms of 2,4-D) have high risk of eye irritation (Category I). Principal formulati ons of fomesafen, paraqua t, lactofen, 2,4-DB (DMA salt), and acifluorfen have a “DANGE R!” signal word (Table L-11). The 18

alternate herbicide actives, as a group, also have relati vely low acute (oral, dermal or inhalation) toxicity. For eight of the eightee n, EPA determined that an acute dietary risk

assessment is not needed because there are no relevant acute toxicological effects. One notable exception is paraquat, which has high ri sk (Category I) of acute toxicity by the inhalation route, and present uses occupy 66% of the aPAD (t he safe exposure level). 2,4-D and glufosinate have relatively low aPADs compared to their dietary residues, and present uses occupy 58% and 48% of thei r respective aPADs. Overall, Monsanto concludes that dicamba has a lower acute toxicity risk compared to six of the eighteen

alternate herbicides and thei r formulated products (para quat, 2,4-D, glufosinate, acifluorfen, sulfentrazone, and fomesafen). These six are indicated by a “Yes” in the “Acute Toxicity Risk” column of Table L-25. Flumioxazin and 2,4-DB were judged to have similar acute toxicity risk as dicamba, and are marked as “Neutral” in Table L-17. Ten herbicides, which either did not have re levant acute toxicological effects or which

utilized 1% or less of the allowable acute exposure (aPAD), were judged to have less acute toxicity risk than dicamba, and are marked as “No” in Table L-17.

L.5.1.2. Chronic Risk

Chronic dietary risk can be evaluated by consideration and comparison of the percentage of the safe exposure level (chr onic population adjusted dose: cPAD) that is used up by all currently registered uses of an active ingredient. Table L-17summarizes the % cPAD

utilized for each alternative herbicide, according to recently published Federal Register

Final Rule information. A number of the alte rnative herbicides have very low use rates, and result in very low residues in treated food or feed and, accordingly, utilize only a small percentage of the cPAD. For dicamba and twelve of the alternate herbicides, the percentage of the cPAD utilized for all approved uses is <10%, so that this group has at least an added 10-fold margin of safety beyond that which EPA has determined is protective of human health. For imazamox, EP A has determined that residues in food or feed are not likely to approach levels of c oncern, and exempted it from the requirement of food and feed tolerances. The remaining five herbicides (2,4-D, flumioxazin, fomesafen, paraquat, and glufosinate) utilize a somewhat higher portion of the cPAD, ranging from 18% to 38%. Although the 10% cutoff is an arb itrary one, and the increased risk of even

the worst-case alternative herbicide for chronic risk is only 6-fold greater than that of dicamba, Monsanto concludes that dicamba offers a lower chronic toxicity risk compared to 2,4-D, flumioxazin, fomesafe n, paraquat and glufosinate.Monsanto Company 10-SY-210U 651 of 721 

### Another way to compare chronic dietary risk would be to focus directly on the soybean

seed residues and their contribution to cPAD utilization. In Table L-13, columns 9 to 11 (those to the right of the gray bar) present th e established soybean seed tolerance for each herbicide. Using DEEM (a computer di etary exposure model used commonly by EPA,) and a theoretical worst-case approach (assume 100% of soybean is treated with a given

herbicide that results in residues at the tolerance level), Monsanto calculated the

theoretical maximum residue c ontribution (TMRC, a standard Tier 1 dietary risk method) to dietary exposure of each herbicide via soyb ean seed in μg/kg/day and as a percentage of the cPAD, as shown in Table L-13. This an alysis highlighted para quat and glufosinate as alternative herbicides with notably higher cPAD utilization by the soybean residue component, both herbicides were considered to presen t more relative risk than dicamba in the previous comparative method as well. The TMRC soybean calculation for paraquat yields a number that is 2.6-fold higher than the cPAD, because the EPA’s risk assessment methodology for paraquat assumes a market pene tration in soybean of <5%. If this market share were increased, the risk attributed to soybean residue could become a substantial portion of the cPAD.

Based on this reasoning, Monsanto concluded th at dicamba offers lower chronic toxicity risk than five alternative herbicides ( 2,4-D, flumioxazin, fomesafen, paraquat and glufosinate), and these a.i.s are marked with a “Yes” in the chronic toxicity column of

Table L-17. Imazamox-ammonium was considered by EPA to have such low toxicity that no food or feed tolerances were required by EPA, and therefore imazamox-ammonium was judged by Monsanto to have lower chronic toxicity risk than dicamba, indicated by a

“No” in Table L-17. The alternate herbicide other a.i.s under consid eration were judged

to have similar chronic toxici ty risk as dicamba, and are ma rked “Neutral” in Table L-17.

L.5.1.3. Cancer Risk

EPA classified dicamba as “not likely” fo r human carcinogenicity. Fourteen of the

alternative herbicides are clas sified similarly – “not likely” , “no evidence”, or “Group E (Evidence of Non-Carcinogenicity for Humans )”. 2,4-D is classified as “D”, meaning that although the studies are acceptable, the evidence is unclear and some uncertainty

remains. Two of the alternative herbicides (lactofen and acifluorfen) are classified as “not likely” at low doses but “l ikely” at high doses, due to liver and other effects. A peroxisome proliferation mechanism is estab lished for acifluorfen. Since acifluorfen is a metabolite of lactofen, the similarity in toxicology and cancer classification is appropriate. One alternative herbicide, flut hiacet-methyl, is categor ized as a “likely” human carcinogen, due the occurrence of dose-rela ted tumors in both rats and mice; a Q*

of 7.5 X 10 -7 

### has been calculated, which is a m easure of carcinogenic potency that EPA utilizes in risk assessment. Overall, Monsanto concludes that dicamba has a cancer risk benefit compared to four of the alternative herbicides (2,4-D, lact ofen, acifluorfen, and fluthiacet-methyl), and this judgment is indi cated by a “Yes” in the Cancer Risk column

of Table L-17. Four alternative herbicid e a.i.s (flumiclorac-pe ntyl, glufosinate, imazaquin, and paraquat) have been categor ized by EPA as Group E or “no evidence”, and Monsanto judges that these four have lower cancer risk th an dicamba, and are

marked as “No” in Table L-17. The other alte rnate herbicide a.i.s that are in the “notMonsanto Company 10-SY-210U 652 of 721 

### likely” category have similar cancer risk to di camba, and are marked as “Neutral” in table

L-17.

L.5.1.4. Risks to Infants and Children

The Food Quality Protection Act (FQPA) require s that EPA take special care in its risk

assessments to establish that infants and ch ildren do not have increased sensitivity to pesticides and thereby experi ence greater risks from residue s in food than the general U.S. population. EPA’s implementation of this requirement is embodied in the additional FQPA safety factor, which is established by the statute at 10-fo ld, unless there is

evidence that another level is protective. When making determinations about the magnitude of the FQPA safety factor, the EPA considers the completeness of the database and specifically the findings from the developmental and reproductive toxicity

studies to determine if there is evidence that infants and children are more sensitive than adults. Therefore, the magnitude of the FQPA safety factor (usually 1X, 3X, or 10X) is a comparative parameter that may be used as an indication of the poten tial risks to infants and children and for overall developmenta l and reproductive t oxicity findings.

Dicamba risk assessments for both acute and chronic effects utili ze an FQPA safety

factor of 1X. This is also the case for 15 of the 18 alternate herbicides, although in some cases, an acute dietary risk assessment was not necessary due to a lack of acute toxicity effects. For lactofen, acifluorfen, and mesotr ione a 3X or 10X FQPA safety factor was

used for the acute and/or chronic assessme nts, indicating a higher level of risk or uncertainty. Therefore, Monsanto concludes th at dicamba offers an improved risk profile for infants and children compared to these th ree alternative herbicid es, which are marked

with a “Yes” in the Infants & Children Risk column in Table L-17. All other alternate herbicide a.i.s which have an FQPA safety factor of 1X are judged to have similar potential risk as dicamba, and are mark ed with “Neutral” in Table L-17.Monsanto Company 10-SY-210U 653 of 721Table L-17. Summary of Comparative Analysisof Dicamba and Alternative HerbicidesActiveIngredientMode-ofAction(WSSAGroup1)Human Health Risk Measures2Aquatic Non-TargetSpecies RiskMeasures3KnownResistantWeedSpecies4HerbicidalEfficacy5(< 50% ofdicamba)LongRotationalCropRestriction6SeriousCropInjuryPotential7Numberof “Yes”Entries8AcuteToxicityRiskCancerRiskChronicRiskInfants &ChildrenRiskAquaticAnimalRiskAquaticPlantRiskNumberof “No”Entries92,4-D acid /estersAux (4)Yes Yes YesNeutralYes Yes28NeutralNeutralNeutral602,4-DB NeutralNeutralNeutralNeutralNeutralNeutralYesNeutralNeutral20imazethapyrALS (2)NoNeutralNeutralNeutralNeutralNeutral107 (Yes)Yes YesNeutral31cloransulammethylALS (2)NoNeutralNeutralNeutralNeutralNeutralYes YesNeutral41chlorimuronethylALS (2)NoNeutralNeutralNeutralNeutralNAYes YesNeutral31thifensulfuronALS (2)NoNeutralNeutralNeutralNeutralYes Yes YesNeutral41imazaquinALS (2)NoNoNeutralNeutralNeutralNAYes YesNeutral32imazamoxammoniumALS (2)NoNeutralNoNeutralNeutralNeutralYes YesNeutral42flumioxazinPPO (14)NeutralNeutralYesNeutralYes YesYes YesNeutral50fomesafenPPO (14)YesNeutralYesNeutralNeutralNeutralNeutralYes Yes 4 0flumicloracpentylPPO (14)NoNoNeutralNeutralNeutralNAYesNeutralNeutral12sulfentrazonePPO (14)YesNeutralNeutralNeutralNeutralYes5YesNeutralNeutral40lactofenPPO (14)NoYesNeutralYes Yes YesNeutralNeutralYes 51fluthiacetmethylPPO (14) NoYesNeutralNeutralNeutralNeutralYesNeutralYes 31acifluorfensodiumPPO (14)Yes YesNeutralYesNeutralNeutralYesNeutralYes 50paraquatdichlorideBiPyr (22)YesNoYesNeutralNeutralYes24YesNeutral Neutral40glufosinateammoniumGlu (10)YesNoYesNeutralNeutralNeutralNo reports NeutralNeutralNeutral30mesotrione HPPD (28)NoNeutralNeutralYes Neutral Yes No reports Yes Yes Neutral4 1Monsanto Company 10-SY-210U 654 of 721Table L-17 is intended to be a 1-page scorecard to track the benefits ofdicamba use on MON 87708 according to the different listed criteria. Each “Yes” entrysignifies that Monsanto has concluded that dicamba represents abenefit versus the relevant alternative herbicide on the basisof the risk factorin that column’sheading. The basis for entering a “Yes” under each risk factor is further explained in the relevant portions of this document.”Neutral” entriesindicate similar risksexist for dicamba and the alternative herbicide. “No” means the alternative herbicide offers a risk benefit compared to dicamba1The herbicidal biochemical mechanismof weed-killing activity, according to the Weed Science Society of America.www.plantprotection.org/HRAC/Bindex.cfm?doc=MOA.html|Accessed May 27, 2010|2A tally of the benefits dicamba use on MON 87708 offers over thealternative herbicide in the four categories of human health risk, according to the criteriadescribed in L.5.1.3A tally of the benefits dicamba use on MON 87708 offers over the alternative herbicides in the two categories of aquatic non-target species risk, according to thecriteria described in L.5.2.4A listing of the worldwide numbers of known resistant weeds for each herbicide based on its mode-of-action group.Dicamba has 5 known resistant spp worldwide.A“Yes” indicates that the number of resistant weeds in this herbicide class is many more than the known five dicamba resistant species biotypes. A comparison ofeach individual herbicide in the class is not provided. See L.5.3.3.www.weedscience.org/summary/MOASummary.asp|Accessed May 28, 2010|5Alternative herbicides that provide commercial control of fewer than 50% of targeted problem weeds in soybean compared to dicamba, according to the data inTable L-16, where commercial control is considered to be >85%, as indicated bya fully-black circlesymbol. See L.5.3.1.6Alternative herbicides that require long waiting periods betweenapplication and subsequent planting of a crop other than soybean. This constraint is a disadvantageto growers. See Table L-18 and L.5.3.2.7“Alternative herbicides that can substantially injure the soybeancrop when applied for weed control, potentially reducing soybean yield. See L.5.3.2.8Tabulation of the number of “Yes” entries in each row, indicating a total scorefor improved risk profile for dicamba use on MON 87708 offers versus an alternativeherbicide. This summation is not presented as a net value (i.e., subtracting where an alternative herbicide has a benefit over dicamba).9Tabulation of the number of “No” entries ineach row, indicating a total score for worse risk profile for dicamba use on MON 87708 offers versus an alternativeherbicide. This summation is not presented as a net value (i.e., subtracting where an alternative herbicide has a benefit over dicamba).NA – not available.Monsanto Company 10-SY-210U 655 of 721 

### L.5.2. Comparisons for Ecological Effects

MON 87708 can be treated with preemergence and postemergence in-crop applications

of dicamba. Such a weed control treatment re gime has the opportunity to reduce risks to aquatic fish and invertebrates by:

 Replacing currently-used or foreseeable futu re alternative herbicide products that have higher aquatic toxic ity risk profiles, and

 Addressing hard-to-control broadleaf weeds or broadleaf weeds that are resistant to glyphosate, PPO inhibitors, or ALS inhi bitors, thereby preserving the ability of growers to manage weed problems and ma ximize soybean yield in the least risky manner, recognizing the ecological benef its offered by the superior hazard and reduced risk profile of gl yphosate and the favorable profile of dicamba compared

to some of the alternative herbicides..

L.5.2.1. Aquatic Animals

As mentioned, Table L-14 provides informa tion concerning hazards, potential exposures, and risks to fish and aquatic invertebrates for each alternat e herbicide and for dicamba. These data allow a comparison of the relati ve aquatic animal safety among the optional weed control products on the basis of:

 Potency against the indicator species.

 Estimates of potential exposure to aquatic animals.

 Calculated RQs for aquatic animals, by combining the hazard and exposure parameters. The RQs that exceed the EPA’s LOC for non-listed aquatic animal species of 0.5 are marked in bold font.

The assessment and comparisons summarized in Table L-17 establish that dicamba poses little acute risk to aquatic animals, which is consistent with EFED ’s assessment published in the RED EFED Chapter and described in mo re detail in L.3.3. Furthermore, for 16 of the 18 alternate herbicides, aquatic anim al RQs range from < 0.005 to 0.05, and do not present a risk to aquatic animals ev en using a worst-cas e upper bound exposure estimation. Only 2,4-D (the esters form) and lactofen exceed the LOC of 0.5 using conservative “worst-case” exposure estimates. These are highlighted in bold font.

Monsanto notes that EPA considers the LOC for aquatic animals that are listed as endangered or threatened species to be 0.05 an d the LOC for acute restricted use to be

0.1. If the 10-fold to 5-fold lower level of concern were applied, flumioxazin would also exceed the LOC based on this “worst-case” exposure estimate.

In addition to the calculated RQs, product labels for five of the altern ative herbicides bear EPA-required warning statements for toxicity to fish or inve rtebrates, based on the hazard values (low LC 50 

### or EC 50 

### ) of those herbicides. These are flumioxazin, cloransulammethyl, flumiclorac-pentyl, lactofen, and 2,4-DB.Monsanto Company 10-SY-210U 656 of 721 

### Therefore, Monsanto concludes that dicamba presents a risk benefit for aquatic animals compared to three of the 18 alternate herbicid es: 2,4-D, flumioxazin, and lactofen. This

conclusion is tabulated in the Table L-17 scor ecard by a “Yes” in the relevant column.

The alternative herbicide a.i.s that have a “worst-case” RQ < 0.05 are similar to dicamba in regards to aquatic animal risk, and are marked “Neutral” in Table L-17

.



### L.5.2.2. Aquatic Plants

As mentioned, Table L-15 provides informa tion concerning hazards, potential exposures, and risks to aquatic plants fo r each alternate herbicide and for dicamba. These data allow a comparison of the relative aquatic plant sa fety among available weed control products

on the basis of:

 Potency against the indicator species.

 Estimates of potential exposure to aquatic plants.

 Calculated RQs for aquatic plants, by co mbining hazard and exposure parameters. The RQs that exceed the EPA’s LOC for non-listed aquatic plant species of 1.0

are marked in bold font.

The data format, sources, and methods are identical to those de scribed above for the aquatic animals (Table L-14). A Level of Co ncern (LOC) value of 1.0 has been used for judging RQ exceedances in the case of aquatic plants, consistent with EPA EFED’s normal practices.

The assessment and comparison summarized in Table L-17 establishes that dicamba poses little acute risk to aquatic plants, which is consistent with EFED’s assessment published in the RED EFED Chapter (U.S. EP A, 2005a) and discussed above. Monsanto was unable to locate public aquatic plant hazard data for three of the alternate herbicides (imazaquin, chlorimuron-ethyl, and flumiclorac- pentyl). For 9 of the 18 alternatives, the worst case RQs ranged between < 0.02 and 0.75; and do not pr esent a risk to aquatic plants even using a worst-case upper bound ex posure estimation. However, for seven alternative herbicides, the wo rst-case RQs did exceed EFED ’s LOC of 1.0. It is not surprising that some herbicides are quite toxi c to aquatic plants. The worst-case RQs for three of the alternate herbicides (flumioxazi n, paraquat dichloride, a nd lactofen) exceeded the LOC by a factor of more than 50-fold.

Therefore, Monsanto concludes that dicamb a presents less risk to aquatic plants compared to seven of the 18 alternative he rbicides: 2,4-D (ester form), flumioxazin, sulfentrazone, thifensulfuron, paraquat dich loride, lactofen, and mesotrione. This

conclusion is tabulated in the Table L-17 scor ecard by a “Yes” in the relevant column. The other alternative herbicide a.i.s, fo r which RQs are less than EPA’s LOC have similar non-target aquatic plant risk as di camba, and are marked in Table L-17 with

“Neutral”. The three herbicides for which no re levant data were available are marked as

“NA”

.Monsanto Company 10-SY-210U 657 of 721 

### L.5.3. Comparison for Efficacy and Weed Management, Including Weed Resistance

L.5.3.1. Comparison of Weed Management Efficacy

As mentioned, Table L-16 compares weed cont rol effectiveness of formulated products containing dicamba and the alternative he rbicides. The various products’ overall effectiveness can be compared using the simple method of c ounting the fully-black circle symbols, which identify instances of commerci al level weed contro l. Dicamba provides

commercial control for 23 of the listed weeds (primarily broadleaf species), which is the most of any of the herbicides in the table; dicamba offers commercial control for 13 of the herbicide-resistant biotypes and 10 of the hard-to-control species. After dicamba, preemergent treatments with 2,4-D, or pos temergent treatments with glufosinate, tembotrione, and imazethapyr plus glyphosate ar e the next most effective herbicides against this target group of weeds. The least effective herbicides in this analysis against these targeted weeds and resistant biotype s are 2,4-DB, paraquat, imazethapyr alone, flumiclorac-pentyl, chlorimuron-methyl and fluthiacet. To summarize the comparative herbicidal effectiveness in this analysis, the scorecard in Table L-16 contains a column in which those herbicides that provide commercia l control of 50% or fewer weeds compared to the number of weeds controlled by dicamba (i .e., eleven or fewer fully-black circles in Table L-16) are marked with a “Yes”, to de note a clear dicamba advantage. Alternative herbicide a.i.s that provide commercial cont rol of greater than 50% of the number of weeds controlled by dicamba (i.e., 12 or more fully-black circles in Table L-16) are marked in Table L-17 with “Neutral”, meaning are judged similar to dicamba. The 50%

threshold criterion is Monsanto ’s arbitrary choice that is intended to identify herbicide options that are expected to provide subs tantially poorer weed control compared to

dicamba.

L.5.3.2. Comparison for Weed Management Practices

Beyond direct efficacy against the key weed s, other advantages of MON 87708 treated

with dicamba exist.

L.5.3.2.1. Increased Weed Control Flexibility

MON 87708 will allow more flexibility for control of weeds just prior to or at planting of the crop, due to elimination of preplant intervals or plant back restrictions on present

dicamba labels. These restrictions were in place due to concern over potential soybean injury, which is not a concern for MON 87708. Current common practice is to use 2,4-D for preemergent burndown treatment. When ap plied too close to soybean planting, 2,4-D can potentially reduce crop stands and cause in jury to new seedlings (Thompson et al., 2007). Restrictions have been implemented fo r most products and are as follows: rates up to 0.5 lb a.i. per acre must be applied at least seven da ys prior to planting; rates between 0.5 and 1 lb a.i. per acre must be applied at least 30 days prior to planting. Additionally, several sulfonylur ea herbicides can also be utilized for preemergent burndown weed control in soybean. For exam ple, Canopy herbicide can be applied prior to planting with the following restrictions : rates of 2.2 oz per acre or less should beMonsanto Company 10-SY-210U 658 of 721 

### applied at least seven days pr ior to planting; ra tes of 2.2 to 3.3 oz per acre should be applied at least 14 days before planting.

Current recommendations to control glyphos ate-resistant biotypes of waterhemp and Palmer amaranth include the use of a residual herbicide treatment and postemergent applications of PPO-inhibiting herbicides such as Cobra (lactofen), Ultra Blazer (acifluorfen), or Cadet (flu thiacet). It is commonly known that these postemergent herbicides can cause excessive injury to soybean, especial ly under hot and sunny conditions. Soybean injury, cau sed by acifluorfen from early (V 2 to V3 growth stage) or late postemergence (V5 to V6) applications, wa s seen as increased chlorosis and stunting that translated into yield reduction (Young et al., 2003). Furthermore, Legleiter et al. (2009) illustrated the effectiveness of preemergent herbicides for the control of resistant waterhemp populations and demonstrated inconsistent control with PPO-inhibiting

herbicides or herbicide combinations for the control of waterh emp populations with multiple resistance to glyphosat e and PPO-inhibiting herbicides. The introduction of MON 87708 will provide an effective management option for herbicide-resistant biotypes of waterhemp and Palmer amaranth that is not expected to cause either crop injury or yield loss.

Residual herbicides containing herbicides including sulfentrazone, dimethenamid, pendimethalin, metribuzin, or metolachlor ha ve been shown to control waterhemp and Palmer amaranth; however, such products can present other challenges. Limitations include the need of adequate soil moisture for activation, potenti al crop injury, crop rotation restrictions, and use rest rictions based on soil type. As an example, metribuzin should not be used on sands, loamy sands, a nd sandy loams with less than 1% organic

matter 62 

### , and pendimethalin applied after crop- emergence can result in soybean injury 63



### .

For some residual herbicides there are extens ive rotational crop rest rictions, ranging from four to 40 months after applic ation, to avoid subsequent crop injury to the rotated crop caused by herbicide remaining in the soil. These limitations reduce the choice of crops that can be planted in case the soybean cr op is destroyed by weather or even the following growing season. Examples of planting limitations among the alternate

herbicides are shown in Table L-18.

62 Product label for Metribuzin 75 DF (http://www.c dms.net/LabelsMsds/LMDefault.aspx?pd=7614&t=). [Accessed 5/14/2010]

63 Product label for Pendimax 3.3 (http://aesop.rutgers.edu/~plantb iopath/links/bbcpestweb/GrapeLab els/pendimax33.pd f). [Accessed 5/14/2010]Monsanto Company 10-SY-210U 659 of 721 

### Table L-18. Planting Restrictions (month s) for Alternative Herbicide Products

CROP Herbicide Product (Active Ingredient) Authority MTZ (sulfentrazone + metribuzin) Pursuit (imazethapyr) CANOPY EX (chlorimuron +

tribenuron) Raptor (imazamox)

Field corn 4 8.5 8 8.5

Wheat 4 4 4 9

Cotton 12 18 8 9

Peanut 12 0 15 9 Sorghum 12 18 9 3

Onions 18 40 30 9

Dicamba does not have rotational restrict ions for such extended time periods, and provides a substantial advantage in flexibility. For the majo rity of crops, no rotational restrictions apply afte r 120 days following dicamba applic ations. Specifically, there are

no rotational restrictions for planting corn following a dicamba application. For cotton, a

rotational restriction of 21 days is recommended. To su mmarize this advantage, Table L17 includes a column in which a “Yes” is mark ed for those alternative herbicides that include substantial rotational or replanting restrictions. Entries for alternative herbicide a.i.s that do not require l ong rotation intervals are judged similar to dicamba, and are

marked in Table L-17 with a “N eutral” in the relevant column

. 

### Furthermore, in situations where sequentia l herbicide applications were employed to control common waterhemp populations, reduced soybean yields have been reported. Yield reductions up to 19% compared to a non-treated control were reported when

acifluorfen or fomesafen was th e postemergent herbicide (S oltani et al., 2009). These reduced yields were associated with crop injury (chlorosis and stunting) following postemergent applications of certain herbicid es, especially PPO inhibitors (Baumann et al., 2010; Loux et al., 2009).

Dicamba treatments in MON 87708 provide excellent crop safe ty. Table L-17 includes a column in which the crop injury potential versus dicamba is summarized; a “Yes” entry indicates a substantial potential for visible soybean injury. Entries for alternative herbicide a.i.s with good soybean crop safety ar e judged to be similar to dicamba, and are

marked with a “Neutral” in the relevant column of Table L-17. In conclusion, the introduction of MON 87708 will provide an additional mode-of-action for postemergent weed control with excellent crop safety.

L.5.3.2.2. Weed Spectrum Benefits

Dicamba provides control of over 95 annual an d biennial weed species, and control or suppression of over 100 perennial broadleaf and woody species. Dicamba provides more effective preemergent weed control than 2,4- D on cutleaf evening primrose, clover, and chickweed (Loux et al., 2009). Furthermor e, dicamba provides excellent control compared to 2,4-D on summer annuals including those with a prostrate growth habit suchMonsanto Company 10-SY-210U 660 of 721 

### as knotweed and purslane. With regard to pe rennial weeds, research conducted at North Dakota State University indicates that dicamb a is more effective in controlling Canada thistle compared to 2,4-D and effectivel y controls field bindw eed (Zollinger, 2000; NDSU, 2010b).

Dicamba also provides excellent control of wild buckwheat while 2,4-D has only limited activity and provides inadequate control (Zollinger et. al, 2006). Other preemergent or postemergent herbicides often provide incomplete control of wild buckwheat including

dinitroanilines or PPO inhibi tors. The most effective herbicides for buckwheat are dicamba, and some sulfonylurea products; however , some of the sulfo nylurea herbicides may persist and carry over for more than one growing season, especially in high pH soils.

Dicamba has been valued as more efficaci ous on lambsquarters than fomesafen or acifluorfen based on university weed cont rol guidelines (Moec hnig et al., 2010; University of Illinois, 2008; Legleiter et al., 2009; Loux et al., 2009). In addition, dicamba exhibits improved control of sick lepod (Loux et al., 2009), kochia and common ragweed (Legleiter et al., 2009) , and waterhemp (Soltani et al., 2009) compared to

fomesafen and acifluorfen.

L.5.3.3. Comparison for Herbicide-Resistant Weeds

The development of weed resistance reduces the effectiveness of all major herbicide classes used in soybean pr oduction today, including glyphos ate, thereby jeopardizing soybean yields and requiring the introducti on of new tools to control populations of resistant weeds. It is widely recognized thatutilizing herbicides with different modes-ofaction in conjunction with esta blished products is especially effective combating further weed resistance development and to provide control of existing populations (Beckie, 2006). Preplant / preemergent or early-posteme rgent in-crop applications of dicamba in MON 87708 will introduce a new mode-of-action; thus the introduction of dicamba use on MON 87708 holds great promise for addressi ng current and future weed management needs in soybean. The primarily basis for this promise is the wide spectrum of activity of dicamba on broadleaf weed sp ecies, which are the most common hard-to-control species and resistant weed biotypes in soybean production today.

Dicamba belongs to the auxin agonist class of herbicides, which is the oldest class of known synthetic herbicides. This clas s includes 2,4-D, 2,4-DB, mecoprop, MCPA, clopyralid, and several other active ingredie nts, and is WSSA Herbicide Group Number

4. 64 

### On the basis of their struct ural and chemical properties, auxinic herbicides have been classified into several sub-groups, i.e. , phenoxyalkanoic acids ( e.g. , 2,4-D, MCPA), benzoic acids ( e.g. , dicamba, chloramben), pyridines ( e.g. , picloram, clopyralid), and quinolinecarboxylic acids ( e.g. , quinclorac, quinmerac). Ge nerally, auxinic herbicides are effective against broadleaf (dicotyledonous) plant species, allowing them to be used in production of narrow leaf (monocotyledonou s) crops such as corn and wheat. The

64 There are several systems of herbicide mode-of action classification. Among the most widely used are

those of the Herbicide Resistance Action Committee (HRA C) and the Weed Science Society of America. The classifications are compared in a chart at http://www.plantprotection.org/HRAC/Bindex.cfm?doc=MOA.html [Access May 28, 2010]Monsanto Company 10-SY-210U 661 of 721 

### relative occurrence of herbicid e-resistant weeds differs between the different sub-groups of auxinic herbicides. The largest number of resistant weed biot ypes has been found for 2,4-D. Considering that auxinic herbicides have been widely used in agriculture for more than 60 years, the occurrence of weed resistan ce to this class is relatively low (28 species worldwide, to date) and its development has be en slow especially when compared to the speed of appearance of resistance to ALS inhibitors (107 species) or triazine-resistant populations (68 species). 65 

### The relatively low incidence of auxinic herbicide resistance is believed to be attributable to the fact that there are multiple mechanisms of action for these herbicides (Gressel et al ., 1982; Morrison and Devine, 1994).

Only five weed species have been reported to date to be resistant to dicamba worldwide: kochia ( Kochia scoparia ) 66 

### , lambsquarters ( Chenopodium album ) 67 

### , prickly lettuce ( Lactuca serriola ), common hempnettle (Galeopsis tetrahit ) and wild mustard ( Brassica

caber ) 68 

### . Of these five species, resistant popula tions of lambsquarters have only been reported in New Zealand. Rega rding the two species with re sistant populat ions in the U.S. (kochia, prickly lettuce) and Canada (common hempnettle and wild mustard), all were common to cereal production areas in th e Western U.S. and Canada. No dicambaresistant populations have been reported in the main soybean production areas, including the Midwest, the South and the East coast of the U.S. Table L-17 shows the number of U.S. weed species known to have resistance to each of the major herbicide groups (or sub-groups within groups, as appropriate) to which the alternative he rbicides belong. It

also contains a “Yes” marker for those herb icides that have many more known resistant weed biotypes than the five known for dicamba, indicating a potential lower risk for weed resistance development for dicamba compared to alternative herbicides. Entries not

indicated with a “Yes” mean that dicamba is ei ther comparable or less favorable than the

alternative herbicide. 

### A comparison of each individual he rbicide in the class is not provided.

Although dicamba resistance exis ts, weed populations that ar e resistant to dicamba in U.S. soybean cropping areas have not been overly problematic to date, possibly because selection pressure on summer annual weeds has been low. Dicamba has seen very limited use in soybean (0.7% of treated soybe an acres as a preplant and pre-harvest applications) and currently has relatively low usage in the crops that are commonly rotated with soybean (9.4% of corn acres as preplant and in-crop applications, 6.3% of cotton acres as a preplant application, and 8.4% of wheat acres, respectively) (Table VIII-12), although historically dicamba was used more extens ively in corn. In addition, there are over 20 commercially -available pre-mixed multiple-herbicide formulations that contain dicamba, so dicamba is often used in combination with other

65 Weed Science Society of America. http://www.weedscience.org/summary/MOASummary.asp

[Accessed May 28, 210]

66 “Dicamba Resistance in Kochia”. H. J. Cranston, A. J. Kern, J. L. Hackett, E. K. Miller, B. D. Maxwell

and W. E. Dyer, Weed Science 49:164-170, 2001.

67 “Chemical Control Options for the Di camba Resistant Biotype of Fathen ( Chenopodium album )”. A.

Rahman, T. K. James, and M. R. Trolove, New Zealand Plant Protection 61: 287-291, 2008.

68 “Inheritance of Dicamba Resistance in Wild Mustar d (Brassica Kaber)”. M. Jasieniuk, I. N. Morrison

and A. L. Brûlé-Babel, Weed Science 43:192-195, 1995.Monsanto Company 10-SY-210U 662 of 721 

### herbicide modes-of-action. It is expect ed that selection pressure favoring the development of dicamba-resistant weeds will continue to be low even after in-crop use of dicamba on MON 87708 is approved. Monsanto believes this is because dicamba will predominantly still be used in combination with other herbicides exhibiting different modes-of-action, principally glyphosate, but al so with other soil-act ive herbicides. The presence of multiple herbicides in the weed management system greatly diminishes the

likelihood of weed resistance to dicamba deve loping to a level of predominance in weed populations. Dicamba is an excellent comple ment to the weed control spectrum of glyphosate, and it has a relatively low cost and low potential for crop injury to

MON 87708 relative to other broa dleaf weed options. Taken t ogether, these factors will help to ensure that more fields receive w eed-control treatments using multiple herbicide modes-of-action, in a proactive way, aimed at slowing the evolution of resistance. In general, this will serve to further reduce th e development of weed resistance problems to all herbicides targeting broadleaf weeds in soybean production, and in crops rotated with soybean.

As part of the projected role of dicamba as a companion herb icide for glyphosate, dicamba will provide growers with a new m ode-of-action for use in-crop against summer annual broadleaf weeds. Dicamba will help prevent and/or combat existing weed resistance issues that can limit effectiveness of the PPO- and ALS-inhibiting herbicide

classes. Herbicides from these two herbic ide classes have historically dominated the non-glyphosate broadleaf weed control tools available in soybean (13 of the 18

alternative herbicides consid ered here are PPO- or ALS-i nhibiting herbicides (Table L17)), and were the predominant modes-of-a ction used for weed control in soybean production prior to the introduction of th e Roundup Ready soybean system; and remain the primary options recommended for manage ment of glyphosate-resistant or hard-tocontrol broadleaf weeds in soybean. For ex ample, fomesafen and flumioxazin, both PPO Inhibitors, are the primary he rbicides recommended for cont rol of glyphosate-resistant Palmer amaranth (pigweed) in soybean.

Dicamba will foster the adoption of IPM pr actices in soybean by allowing growers to continue to primarily focus on postemergent in-c rop weed control, as they have practiced with Roundup Ready soybean, and will make current practices more effective by providing an additional mode-of-action herbicide. This will allow growers to delay some herbicide treatments until field scouting indicates a need for additional weed control which is consistent with the principles of Integrated Pest Management. Dicamba, as a companion product to glyphosate, will also con tinue to foster adoption of and maintain

the use of conservation tilla ge practices, because of grower preference to usepostemergent products, such as dicamba on MON 87708, compared to reliance on soilactive preemergent products

In summary, the ability to use dicamba as pa rt of a weed management program in U.S. soybean production will provid e significant benefits for managing broadleaf weed resistance not only relative to glyphosate but also to othe r herbicides such as those

included in PPO- and ALS-inhibitor classes. Th ere is evidence in the scientific literature from data generated in field studies and from model simulations that the application of multiple herbicides, each effective in control ling a weed spectrum, with more than oneMonsanto Company 10-SY-210U 663 of 721 

### mode-of-action can significantly delay the ev olution of resistant populations within a field (Powles et al., 1996; Beckie, 2006). In addition, there is evidence that resistance would be delayed longer by use of herbicide mixtures than by using an herbicide rotation strategy (Diggle et al., 2003; Beckie and Reboud, 2009). Based on this general information on resistance, Monsanto believes that application of dicamba on MON 87708 integrated into the Roundup Ready soybean system will reduce the development of

herbicide-resistant br oadleaf populations to glyphosate and other herbicides. This conclusion is based on the following: 1) th e efficacy and broad spectrum of glyphosate and weed control spectrum of dicamba, 2) the low level of dicamba-resistant broadleaf weeds in the major soybean production areas, 3) the low number of species resistant to glyphosate, 4) the expected use of dicamba applied to MON 87708, and 5) the fact that dicamba will be used in combination with glyph osate, and other alternative herbicides as necessary to control problem atic weeds. Using available information, Monsanto conservatively estimates that dicamba us e on MON 87708 could reduce the growth of resistant weed populations on five to ten percent of the expe cted 75 million U.S. planted soybean acres (Table VIII-1), which is equa l to approximately 3.6 to 7.2 million acres.

Furthermore, Monsanto believes the opportunity for dicamba use on MON 87708 will foster continued adoption of conservation tillage practices, an important goal for the agro-ecosystem and the long term sustainabil ity of U.S. agriculture. Presently, 41.5% of an estimated 74 million acres of U.S. soybean acres, or 30.6 million acres, employ no-till or conservation tillage production syst ems (CTIC, 2007). The introduction of MON 87708 into the Roundup Ready soybean sy stem would allow th e flexibility to incorporate an additional herbicide mode -of-action in both pre- or postemergent applications, and support the continued use of conservation tillage production systems. The benefits of conservation tillage are we ll known and demonstrated including soil and water conservation, improved environmental ( e.g. , water) quality, an d a reduced carbon footprint (Arriaga and Balkcom, 2005; Reicos ky, 2008). These conservation tillage acres will represent a significant portion of the 30 million total acres projected for MON 87708 planting, in turn representing a significant number of acres where a delay in the development of glyphosate-resistant biot ypes could be expected to occur.

L.6. Statement of Benefits for Dicamba-Tolerant Soybean MON 87708

Monsanto intends to commercialize M ON 87708 in combination with the Roundup Ready 2 Yield soybean platform (glyphosate-to lerant soybean), so that both herbicide tolerance traits occur in the same soybean plant. The addition of MON 87708 to the Roundup Ready 2 Yield soybean platform enab les the use of dicamba withglyphosate or other herbicides as a mixture or sequential applications for pre-plant burndown and inseason weed control, resulting in a highl y effective weed management system for soybean growers. Combined use of dicamba plus glyphosate will provide excellent grass and broadleaf weed control, including those br oadleaf weeds that are hard–to-control or glyphosate-resistant. The use of dicamba and glyphosate should deter the spread of existing glyphosate-resistant broadleaf weed s, and possibly delay the development of new resistant populations through use of multip le modes-of-action. The decision by growers to adopt MON 87708 x Roundup Ready 2 Yield soybean system and to include a dicamba herbicide treatment, either preemerge nt or in-crop, will be dependent up on threeMonsanto Company 10-SY-210U 664 of 721 

### factors: weed spectrum in a grower’s field, efficacy of dicamba against broadleaf weeds, and costs and flexibility versus a lternative herbicide programs.

The use of dicamba in combination with glyphosate for burndown treatments prior to planting on no-till acres will provi de growers with a more flex ible weed management tool than is presently available. MON 87708 (in combination with appl ication of dicamba) will provide growers with greater flexibil ity in timing of application of burndown treatments, eliminating the need for any interval between dicamba application and planting, and improving overall efficacy on hard-t o-control and larger broadleaf weeds. In addition, dicamba is an effective comple ment to glyphosate in cropping systems where glyphosate-resistant weeds such horseweed ( Conyza canadensis) have developed. Approximately 40% of the total soybean acres in the United States are produced in a notill system (CTIC, 2007). In these cropping systems, it is an ticipated that dicamba will displace other herbicides, such as 2,4-D, becau se a preplant interval is not needed with MON 87708 to avoid potential crop injur y, and because dicamba has a better weed spectrum profile, as discussed previously. However, the potential future introduction of 2,4-D-tolerant soybean will likel y allow for a decreased prep lant interval for soybean containing this trait.

In addition to soybean production under no- till cropping systems, MON 87708 offers an effective solution for improving weed control in conventional tillage cropping systems. Herbicides with soil residual and posteme rgent efficacy are frequently used in combination with glyphosate to control weeds that are hard -to-control, resistant to glyphosate, or are capable of producing multiple flushes. Examples include, but are not limited to, the following species: Amaranthus tuberculatus (tall waterhemp), Palmer amaranth ( Amaranthus palmeri ), giant ragweed ( Ambrosia trifida ) and lambsquarters ( Chenopodium album ). In these cropping situations, it could be assumed that dicamba would replace some of the less effective PPO inhibitors (such as acifluorfen and lactofen) that have greater potential for crop injury. The opportunity to use dicamba as yet a third

mode-of-action in these situations would also reduce the potential fo r selection pressure for resistance to any single herbicidal m ode-of-action.

Assuming that the U.S. soybean crop is gr own on approximately 75 million acres (Table VIII-1), it is estimated that dicamba herbicid es could be used for weed control on a total of approximately 30 million acres. There are currently 11 active ingredients that account for 90% of the herbicide usage (based on expenditure) in s oybean production today (AgroTrak, 2009). The estimated percentage of treated soybean acreage by active ingredient is presented in Table L-12 base d on the latest USDA-NASS use data from 2008. The use of dicamba as a preplant burndow n or in-crop applica tion has the potential to reduce the rates, acreage, and/or the num ber of applications of these alternative herbicides in soybean production.

There are widely varying estimates of the current number of soybean acres that have populations of weeds that are resistant to one or more of the classes of herbicides currently registered for use in soybean. It is difficult to determine the validity and comparability of these estimates, as different regions and weed scientists have a different approach or method for deriving these estima tes. Regardless of the accuracy of theseMonsanto Company 10-SY-210U 665 of 721 

### estimates of the current state of herbic ide resistance in soybean production, the application of dicamba on MON 87708 will provide a benefit in broadleaf weed resistance management for all the herbicide modes-of-action currently used in soybean for the following reasons: 1) the broad spect rum of broadleaf weed control provided by dicamba, 2) the limited number of dicamba-resi stant biotypes present after decades of dicamba use, 3) the projected soybean acreage (40%) adopting MON 87708 into the Roundup Ready soybean system and the combin ation with other herbicide modes-ofaction on these acres, and 4) the compatib ility of dicamba e nd-use products with herbicides having othe r modes-of-action.Monsanto Company 10-SY-210U 666 of 721 

### References for Appendix L

AgroTrak. 2009. Unpublished 2008 agricultural survey datra. Gfk Kynetec Marketing Research, Inc. http://www.gfk.com/gfk-kynetec/.

Arriaga, F.J. and K.S. Balkcom. 2005. Bene fits of conservation tillage on rainfall and water management. 2005 Georgia Water Resour ces Conference, University of Georgia, Athens, Georgia.

Baumann, T.T., A.F. Dobbels, W.G. Johnson, M.M. Loux, G.R.M. Nice, and S. J.M.

2010. Weed Control Guide for Ohio and Indian a. Purdue University and Ohio State GUniversity Extension Bulletin 789.

Beckie, H.J. 2006. Herbicide-resistant weeds: Management tactics and practices. Weed Technology 20:793-814.

Beckie, H.J. and Xavier Reboud. 2009. Selecting for weed resistance: Herbicide rotation and mixture. Weed Technology 23:363-370.

CTIC. 2007. 2007 amendment to the national crop residue management survey summary. Conservation Technology Information Center, West Lafayette, Indiana. http://www.conservationinformation.org/ pdf/National_Summary_2007_Amendment.pdf
Accessed June 14, 2010

.

Crop Protection Guide - Information for Dealers. 2009. http://www.agrisolutionsinfo.com / [Accessesd March 26, 2010].

Diggle A.J., P. Neve, and F.P. Smith. 2003. Herbicides used in combination can reduce the probability of herbicide resist ance. Weed Research 4: 371-382.

Durkin, P. and S. Bosch. 2004. Dicamba - Huma n health and ecological risk assessment - Final report. SERA TR 04-43-17-06d. U.S. De partment of Agricultu re, Forest Service, Washington, D.C. http://www.fs.fed.us/fo resthealth/pesticid e/pdfs/112404_dicamba.pdf Accessed May 26, 2010

.

Gianessi, L.P. 2005. Economic and herbicide use impacts of glyphosate-resistant crops. Pest Management Science 61: 241-245.

Gressel, J., and Segel, L. A. 1982. Interrela ting factors controlling th e rate of appearance

of resistance: the outlook for the future . Pages 325–347in Herbicide Resistance in Plants. H.M. LeBaron and J. Gressel, eds. John Wiley & Sons, Inc., New York, New York.

Legleiter, T.R., Bradley, K.W., and Massey, R.E. 2009. Glyphosate-resistant waterhemp ( Amaranthus Rudis ) control and economic returns with herbicide programs in soybean. Weed Technology 23:54-61.Monsanto Company 10-SY-210U 667 of 721 

### Loux, M.M., D. Doohan, A.F. Dobbels, W.G. Johnson, G.R.W. Nice, T.N. Jordan and T.T. Bauman. 2009. Weed control guide for Ohio and Indiana. Bulletin 789. Purdue University and Ohio State University Extension, West Lafayette, Indiana.

Moechnig, M., D.L. Deneke and L.J. Wr age. 2010. Weed control in soybeans: 2010.

South Dakota State Universit y, Brookings, South Dakota. http://agbiopubs.sdstate .edu/articles/FS525B.pdf Accessed May 12, 2010

Morrison, I.N., and M.D. Devine. 1994. Herbic ide resistance in the Canadian prairie provinces - 5 years after the fact. Phytoprot ection 75:5-16.

NDSU. 2010a. Weed control guide. http://www.ndsu.edu/weeds Accessed March 26,

2010

.

NDSU. 2010b. Canada thistle: Cirsium arvense (L.) Scop. North Dakot a State University, Fargo, North Dakota. http://www.ag.ndsu.nodak.edu/invasi veweeds/canda%20thistle.htm

. Accessed March 30, 2010

Pedersen, P. 2008. Soybean variety selection. Iowa State University Extension, Ames, Iowa. http://extension.agron.iastate.edu/ soybean/production_varietyselect.html Accessed April 19, 2011

.

Powles, S.B., C. Preston, I.B. Bryan and A. R. Jutsum. 1996. Herbicid e resistance: Impact and management. Pages 57-93 in Advances in Agronomy. Volume 58. D.L. Sparks (ed.). Academic Press, San Diego, California.

Reicosky, D.C. 2008. Carbon sequestration and environmental benefits from no-till systems. Pages 43-58 in No-Till Farming System s. T. Goddard (ed.). World Association of Soil and Water Conservation, Bangkok, Thailand.

Soltani, N., Vyn, J. D., and Sikkema, P. H. 2009. Control of common waterhemp ( Amaranthus tuberculatus var. rudis ) in corn and soybean w ith sequential herbicide applications. Canadian Jour nal of Plant Sc ience 89:127-132.

Thompson, M.A., Steckel, L.E., Ellis, A.T., a nd Mueller, T.C. 2007. Soybean tolerance to early preplant applications of 2,4- D ester, 2,4-D Amine, and Dicamba. Weed Technology 21:882-885.

U.S. EPA. 2005a. EFED Reregistration Chap ter For Dicamba / Di camba Salts. U.S. Environmental Protection Agency, Office of Pesticides, Prevention, and Toxic Substances, DP Bar Code 317976, August 31, 2005.

U.S. EPA. 2005b. Dicamba: HED Chapter of the Reregistration Eligibility Decision Document, Phase 1. PC Code 029801; DP Barcode: D317720 (Memorandum, C. Olinger and Y. Yang, September 13). Health Effect s Division, Office of Prevention, Pesticides and Toxic Substances, U.S. Environmen tal Protection Agency, Washington, DC.Monsanto Company 10-SY-210U 668 of 721 

### EPA. 2008. Dicamba pesticide tolerance. U.S. Environmental Protection Agency, Washington, D.C. Vol. 73, No. 64.

U.S. EPA. 2009a. Amendment to organic arse nicals RED. U.S. EPA U.S. Environmental Protection Agency, Office of Pesticides, Pr evention, and Toxic S ubstances, April 22, 2009. http://www.epa.gov/oppsrrd1/REDs/organic-arsenicals-amended.pdf.

U.S. EPA. 2009b. Registration eligibility decision for dicamba and associated salts. U.S. Environmental Protection Agency, Office of Pesticides, Prevention, and Toxic Substances, June 8, 2006, as amended June 17, 2009.

U.S. EPA. 2009c. Reduced risk/organophosphate alternative decisions for conventional pesticides, updated 7/1/09”. Washington, D.C. http://www.epa.gov/opprd001/workpl an/completionsportrait.pdf.

U.S. EPA. 2009d. Mesotrione: human-health risk assessment for Section 3 new use on soybeans. U.S. Environmental Protection Ag ency, Office of Pesticides, Prevention, and Toxic Substances, Washington, D.C. DP Barcode: D357890, August 12, 2009.

U.S. EPA. 2009e. Mesotrione; Pesticide To lerances. U.S. Environmental Protection Agency, Washington, D.C. 74 FR 67119 - 67124, December 18, 2009.

U.S. EPA. 2011. 2011 Edition of the drinking wate r standards and health advisories. U.S. Environmental Protection Ag ency, Washington, D.C.

University of Illinois. 2008. Illinois Agricultural Pest Management Handbook.. www.ipm.illinois.edu/pubs/iapmh/00contents.pdf Accessed June 14, 2010

.

USDA-NASS. 2007a. Acreage - June 2006. U.S. Department of Agriculture, National Agricultural Statistics Service, Washington, D.C.

USDA-NASS. 2007b. Agricultural chemical usage 2006 field cr ops summary. U.S. Department of Agriculture, National Agricult ural Statistics Service, Washington, D.C.

USDA-NASS. 2009. Crop production 2008 summary. U.S. Department of Agriculture, National Agricultural Statistic s Service, Washington, D.C.

Young, B.G., Young, J.M., Matthews, J.L., Owe n, M.D.K., Zelaya, I.A., Hartzler, R.G., Wax, L.M., Rorem, K.W., and Bollero, G.A.. 2003. Soybean development and yield as affected by three postemergence herb icides. Agronomy Journal 95:1152-1156.

Weed Control Manual for Tennessee. 2010. http://www.utextension.utk.edu/ publications/pbfiles/pb1580.pdf.

Zollinger, R. and Rodney, L. 2000. Identificat ion and control of field bindweed. W-802 (Revised). http://www.ag.ndsu.e du/pubs/plantsci/weeds/w802w.htm Accessed March 26,

2010

.Monsanto Company 10-SY-210U 669 of 721 

### Zollinger, R., Bradley, K. W., and Massey, R. E. 2006. The glyphosate weeds and crop series: biology and management of wild buc kwheat. Purdue Extension – Knowledge to

Go: GWC-10.Monsanto Company 10-SY-210U 670 of 721 

### Alternative Herbicide-Specific References

The following active ingredient specifi references were used to generate the comparative summary information contained in Tables L-13, L-14 and L-15.

2,4-D

U.S. EPA. 2004. Environmental Fate and Eff ects Division’s Risk Assessment for the Reregistration Eligibility Document for 2,4-Dichlorophenoxyacetic Acid (2,4-D). OPP Public Docket (OPP-2004-0167-0003).

U.S. EPA. 2005. 2,4-D RED Facts. Registra tion Eligibility Decision for 2,4-D. U.S. Environmental Protection Agency, Office of Pesticides, Prevention, and Toxic Substances, Document EPA 738-R-05-002, June, 2005.

2,4-DB

U.S. EPA. 2005. Registration Eligibility Decision for 2,4-DB”. U.S. Environmental Protection Agency, Office of Pesticides, Pr evention, and Toxic Substances, Document EPA738-R-05-001, January, 2005.

Acifluorfen, Sodium

U.S. EPA. 2003. R.E.D. Facts Sodium Ac ifluorfen (Blazer®). U.S. Environmental Protection Agency, Document EPA-738-F-04-001, September 2003.

U.S. EPA. 2004. Registration Eligibility Decision for Sodium Acifluorfen, Case No. 2605. U.S. Environmental Protection Agency, O ffice of Pesticides, Prevention, and Toxic Substances, (undated). See also “Sodium Acif luorfen RED; Notice of Availability”. 69 FR 4139 - 4138, January 28, 2004.

U.S. EPA. 2002. Sodium Acifluorfen, HED Chapter for the Reregistration Eligibility Decision. U.S. Environmental Protection Agen cy Office of Pesticides, Prevention, and Toxic Substances, DP Barcode: D279497, January 15, 2002.

U.S. EPA. 2010. OPP Pesticide Ecotoxicity Da tabase. National Information System for the Regional IPM Centers. http:// www.ipmcenters.org/Ecotox/index.cfm http://www.ipmcenters.o rg/Ecotox/index.cfm Accessed May 28, 2010

.

Chlorimuron

U.S. EPA. 2009. Chlorimuron-ethyl: Revi sed Human Health Risk Assessment for Proposed Uses On Cranberry and Lo w-growing Berry Subgroup 13-07H, except Strawberry, PP# 6E7153. U.S. Environmental Protection Agency, Office of Pesticides, Prevention, and Toxic Substances, DP Barcode D361368, February 5, 2009.

U.S. EPA. 2004. Report of the Food Quality Protection Act (FQPA) Tolerance Reassessment Progress and Risk Management Decision (TRED) for Chlorimuron Ethyl.Monsanto Company 10-SY-210U 671 of 721 

### U.S. Environmental Protection Agency, Office of Pesticides, Prevention, and Toxic Substances, September 22, 2004.

U.S. EPA. 2010. OPP Pesticide Ecotoxicity Da tabase. National Information System for the Regional IPM Centers. http:// www.ipmcenters.org/Ecotox/index.cfm http://www.ipmcenters.o rg/Ecotox/index.cfm Accessed May 28, 2010

.

Cloransulam

U.S. EPA. 1997. Pesticide Fact Sheet; Name of Chemical: Cloransulam-methyl. U.S. Environmental Protection Agency Office of Pe sticides, Prevention, and Toxic Substances, October 29, 1997.

U.S. EPA. 1997. Cloransulam-methyl; Pes ticide Tolerances. U.S. Environmental Protection Agency, 62 FR 49158-49163, September 19, 1997.

U.S. EPA. 2010. OPP Pesticide Ecotoxicity Da tabase. National Information System for the Regional IPM Centers. http:// www.ipmcenters.org/Ecotox/index.cfm http://www.ipmcenters.o rg/Ecotox/index.cfm Accessed May 28, 2010

.

Dicamba

Hoberg, James R. “Dicamba technical – Dete rmination of Effects on Seed Germination, Seedling Emergence, and Vegetative Vigor of Ten Plant Species”. Springborn Laboratories, 1993.

U.S. EPA. 2009. Registration Eligibility D ecision for Dicamba and Associated Salts. U.S. Environmental Protection Agency, Office of Pesticides, Prevention, and Toxic Substances, June 8, 2006, as amended June 17, 2009.

U.S. EPA. 2005. Dicamba: HED Chapter of the Reregistration Eligibility decision Document (RED) - Phase I. U.S. Environmen tal Protection Agency, O ffice of Pesticides, Prevention, and Toxic Substances, DP Bar Code D317720, September 13, 2005.

U.S. EPA. 2005. EFED Reregistration Chap ter For Dicamba / Dicamba Salts. U.S. Environmental Protection Agency, Office of Pesticides, Prevention, and Toxic Substances, DP Bar Code 317976, August 31, 2005.

Flumiclorac-pentyl

U.S. EPA. 2009. Registration Review - Preliminary Problem Formulation for the Ecological Risk Assessment of Flumiclora c-pentyl. U.S. Envi ronmental Protection Agency, Office of Pesticides, Prevention, and Toxic Substances, DP Bar Code 362087, July 10, 2009. EPA-HG-OPP-2009-0084-0004.

U.S. EPA. 2009. Flumiclorac Pentyl: Re vised Human Health Assessment Scoping Document in Support of Registration Review . U.S. Environmental Protection Agency,Monsanto Company 10-SY-210U 672 of 721 

### Office of Pesticides, Prevention, and Toxi c Substances, DP Bar Code D368451, August 28, 2009.

U.S. EPA. 2005. Report of Food Qualit y Protection Act (FQPA) Tolerance reassessment Progress and Risk Management decision (TRED) for Flumiclorac pentyl. U.S. Environmental Protection Agency, Office of Pesticides, Prevention, and Toxic Substances, August 23, 2005.

U.S. EPA. 2006. Flumiclorac pentyl; Pes ticide Tolerance. U.S. Environmental Protection Agency, 71 FR 11526-11533, March 8, 2006.

Flumioxazin

U.S. EPA. 2007. Flumioxazin. Human Health Risk Assessment for the Proposed Food Use of the Herbicide Flumioxazin on Alfalfa, Asparagus, Dry Beans, Fruiting Vegetables (Group 8, Including Okra), Melons (Subgroup 9A), Bushberries (Subgroup 13B), and Tree Nuts (Group 14), and a Request for an Amended Use on Garlic. U.S. Environmental Protection Agency, Office of Pesticides, Prevention, and Toxic Substances, DP Bar Code 336194, November 28, 2007.

U.S. EPA. 2008. Flumioxazin; Pesticide To lerance. U.S. Environmental Protection Agency, 73 FR 11826-11831, March 5, 2008.

U.S. EPA. 2010. OPP Pesticid e Ecotoxicity Database. National Information System forthe Regional IPM Centers. http ://www.ipmcenters.org/Ecotox/index.cfm Accessed May 28, 2010

.

Fluthiacet-methyl

U.S. EPA. 1999. Pesticide Fact Sheet; Name of Chemical: Fluthiacet-methyl. U.S. Environmental Protection Agency Office of Pe sticides, Prevention, and Toxic Substances, April, 1999.

U.S. EPA. 2001. Fluthi acet-methyl; Pesticide To lerance. 66 FR 65839-65850, December 21, 2001.

U.S. EPA. 2006. Fluthi acet-methyl; Pesticide To lerance. 71 FR 77620-77625, December 27, 2006.

U.S. EPA. 2010. OPP Pesticide Ecotoxicity Da tabase. National Information System for the Regional IPM Centers. http://www.ipmcenters.org/Ecotox/index.cfm Accessed May
28, 2010

.

Fomesafen

U.S. EPA. 2006. Fomesafen; Pesticide To lerance. U.S. Environmental Protection Agency, 71 FR 25945 - 25951, May 3, 2006.Monsanto Company 10-SY-210U 673 of 721 

### U.S. EPA. 2007. Fomesafen Summary Document; Registrati on Review - Initial Docket

March 2007. U.S. Environmental Protection Agency, Office of Pest icides, Prevention, and Toxic Substances, Docket N o. EPA-HQ-OPP-2006-0239, March 22, 2007.

U.S. EPA. 2010. OPP Pesticide Ecotoxicity Da tabase. National Information System for the Regional IPM Centers. http:// www.ipmcenters.org/Ecotox/index.cfm http://www.ipmcenters.o rg/Ecotox/index.cfm Accessed May 28, 2010

.

Glufosinate

U.S. EPA. 2003. Glufosinate Ammonium; Pe sticide Tolerance. U.S. Environmental Protection Agency, 68 FR 55833 - 55849, September 29, 2003.

U.S. EPA. 2009. Glufosinate Summary Docume nt; Registration Review - Initial Docket March 2008, Case #7224. U.S. Environmental Protection Agency, Of fice of Pesticides, Prevention, and Toxic Substances, Do cket No. EPA-HQ-OPP-2008-0190, March 19,

2009.

Glyphosate

U.S. EPA 2009. Problem Formulation for the Ecological Risk an d Drinking Water Exposure Assessments in Support of the Regi stration Review of Glyphosate and Its Salts. EPA-HQ-OPP-2009-0361-0007.

Imazamox

U.S. EPA. 1997 Pesticide Fact Sheet. Name of Chemical: Imazamox (Raptor Herbicide) Reason for Issuance: Conditiona l Registration. Date Issued: May 22, 1997.

U.S. EPA. 2010. OPP Pesticide Ecotoxicity Da tabase. National Information System for the Regional IPM Centers. http://www.ipmcenters.org/Ecotox/index.cfm Accessed May
28, 2010

.

Imazaquin

U.S. EPA. 2005. Imazaquin and its Salts: HED Chapter of the Tolerance Reassessment Eligibility Decision (TRED) Document DP Barcode: D302939. U.S. Environmental Protection Agency, Office of Pesticides, Pr evention, and Toxic Substances, October 31,

2005.

U.S. EPA. 2005. Report of the Food Qu ality Protection Act (FQPA) Tolerance Reassessment Progress and Risk Management Decision (TRED) for Imazaquin. U.S. Environmental Protection Agency, Office of Pesticides, Prevention, and Toxic Substances, Document No. EPA 738-R-00-009, December, 2005.

U.S. EPA. 2005. Drinki ng Water Assessment for Imazaquin and its Salts. Environmental Protection Agency, Office of Pesticides, Prevention, and Toxic Substances, DP Barcode: D313182, June 21, 2005.Monsanto Company 10-SY-210U 674 of 721 

### U.S. EPA. 2010. OPP Pesticide Ecotoxicity Da tabase. National Information System for the Regional IPM Centers. http://www.ipmcenters.org/Ecotox/index.cfm Accessed May
28, 2010

.

Imazethapyr

U.S. EPA. 2002. Imazethapyr; Pesticide To lerance. U.S. Environmental Protection Agency 67 FR 55323 - 55331, August 29, 2002.

U.S. EPA. 2010. OPP Pesticide Ecotoxicity Da tabase. National Information System for the Regional IPM Centers. http://www.ipmcenters.org/Ecotox/index.cfm Accessed May
28, 2010

.

Isoxaflutole

U.S. EPA. 1998. Pesticide Fact Sheet; Na me of Chemical: Isoxaflutole. U.S. Environmental Protection Agency Office of Pe sticides, Prevention, and Toxic Substances, September 15, 1998.

U.S. EPA. 1998. Isoxaflutole; Pesticide To lerance. U.S. Environmental Protection Agency, 63 FR 50773 - 50784, September 23, 1998.

Lactofen

U.S. EPA. 2006. EFED Problem Formulati on for Lactofen Review. DP Barcode: 323196. December 13, 2006. EPA-HQ-OPP-2005-0287-0004.

U.S. EPA. 2004. Lactofen; Pesticide Toleranc e. U.S. Environmental Protection Agency, 69 FR 57207 - 57216, September 24, 2004.

U.S. EPA. 2003. Report of the Food Qu ality Protection Act (FQPA) Tolerance Reassessment Progress and Risk Management Decision (TRED) for Lactofen. U.S. Environmental Protection Agency, Office of Pesticides, Prevention, and Toxic Substances, Document No. EPA 738-R-04-002, September 24, 2003.

U.S. EPA. 2000. Lactofen: Preliminary Hu man Health Risk Assessment for Tolerance Reassessment, Incorporating Revised Cancer Unit Risks. U.S. Environmental Protection Agency Office of Pesticides, Prevention, a nd Toxic Substances, DP Barcode: D269621, October 12, 2000.

Mesotrione

U.S. EPA. 2001. Pesticide Fact Sheet; Name of Chemical: Mesotrione. Office of Pesticides, Prevention, and T oxic Substances, June 4, 2001.

U.S. EPA. 2009. Mesotrione; Pesticide To lerances. U.S. Environmental Protection Agency, 74 FR 67119 - 67124, December 18, 2009.Monsanto Company 10-SY-210U 675 of 721 

### U.S. EPA. 2009. Mesotrione: Human-Health Risk Assessment for Section 3 New Use on Soybeans. U.S. Environmental Protecti on Agency, Office of Pesticides, Prevention, and Toxic Substances, DP Barcode: D357890, August 12, 2009.

U.S. U.S. EPA. 2010. OPP Pesticide Ecotoxici ty Database. National Information System for the Regional IPM Centers. http ://www.ipmcenters.org/Ecotox/index.cfm Accessed May 28, 2010

.

Paraquat

U.S. EPA. 1997. Registration Eligibility Decision for Paraquat Dichloride. U.S. Environmental Protection Agency Office of Pe sticides, Prevention, and Toxic Substances.

Document No. EPA 73 8-F-96-018, August, 1997.

U.S. EPA. 2006. Paraquat Dichloride; Pest icide Tolerance. U.S. Environmental Protection Agency, 71 FR 52487 - 52494, September 6, 2006.

U.S. EPA. 2010. OPP Pesticide Ecotoxicity Da tabase. National Information System for the Regional IPM Centers. http://www.ipmcenters.org/Ecotox/index.cfm Accessed May
28, 2010

.

Sulfentrazone

U.S. EPA. 2009. Revised EFED Problem Fo rmulation for Sulfentrazone Registration Review. U.S. Environmental Protection Agen cy, Office of Pesticides, Prevention, and Toxic Substances, DP Bar Code 363522, EPA-HQ-OPP-2009- 0624-0006. September 9,

2009.

U.S. EPA. 2009. Sulfentrazone. RE VISED Human Health Assessment Scoping Document in Support of Registration Review . U.S. Environmental Protection Agency, Office of Pesticides, Prevention, and Toxic Substances, DP Bar Code 369064, September 9, 2009.

Tembotrione

U.S. EPA. 2007. Pesticide fact Sheet: Tem botrione. U.S. Environmental Protection Agency, Office of Pesticides, Prevention, and Toxic Substances, September, 2007.

Thifensulfuron

U.S. EPA. 2007. Thifensulfuron Methyl; Pe sticide Tolerance. U.S. Environmental Protection Agency, 72 FR 13179 - 13184, March 21, 2007.

U.S. EPA. 2008. Ingredient: Thifensulf uron Methyl; Title: Label Amendments and petition for Tolerances on Wheat Forage and Ha y, Oat Forage and Hay, and Barley Hay. U.S. Environmental Protection Agency, Office of Pesticides, Prevention, and Toxic Substances, DP Bar Code 350544, April 17, 2008.Monsanto Company 10-SY-210U 676 of 721 

### Appendix M: Potential Impact of Dicamb a on Human Health and the Environment

M.1. Overview

Dicamba use is regulated at the federal level by EPA, not APHIS. APHIS’s authority under the Plant Protection Act does not allow it to specify conditions for the use of herbicides. Instead, EPA specifically approves labelin g for any herbicide use includi ng uses on agricultural crops. Before any dicamba product could be used over MON 87708 it must first be approved by the EPA as required by the Federal Insecticide Fung icide and Rodenticide Act (FIFRA); EPA must approve the pesticide (herbicide ) product labeling for that specifi c use. Nevertheless, Appendix M examines the potential im pact of dicamba use on MON 87708 on human health and the environment. It first provides a background di scussion of U.S. EPA’s authority to regulate pesticides. Next, Appendix M addresses the regulatory background for dicamba, including the proposed use on MON87708. Appendix M then disc usses the potential impact of dicamba on human health, including impact s on pesticide applicators. Appendix M concludes with a discussion of drift and offsite movement aspe cts associated with dicamba use.

M.2. Regulation of Pesticides in U.S.

M.2.1. Pesticide Registration, Rere gistration and Tolerance Setting

FIFRA requires that before the sale or distribution of a new pesticid e or a new use of a registered pesticide, a company must obtain a registration, or license, from EPA. The EPA must ensure that the pesticide, when used according to its label directions, wi ll not cause unreasonable adverse effects on human health and the environment. In order to address this standard, EPA evaluates potential risks to hum ans and the environment, and may require applicants to submit

more than 100 different scientif ic studies conducted according to EPA guidelines. According to EPA, more than 1000 active ingredients are currently registered as pesticides in the U.S., which are, in turn, formulated into many thousands of pesticide products that are available in the marketplace (U.S. EPA, 2010).

Pesticide registration is a scie ntific, legal, and administrati ve procedure through which 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 ev aluating a pesticide registration application, EPA assesses a wide variety of data indicating the pot ential human health and environmental effects associated with use of the pesticide product. The data required by EPA are used to evaluate a wide array of potential impacts, including whet her a pesticide has the potentia l to cause adverse effects on humans, wildlife, fish, and plan ts (including endangered species and other non-target organisms,

i.e. , organisms that the pesticide is not intended to act against). The registration applicant must also supply data on the pesticide’s potential impact on surface water and ground water, shouldleaching or runoff occur. The potential human health and safety risks assessed range from shortterm toxicity to long-term effects such as cancer and reproductive system disorders.

Pesticide label directions are c onsidered as part of EPA’s ev aluation to determine whether a pesticide can be used safely, as required by FIFRA, and the language that appears on each pesticide label has been expres sly approved by EPA. A pesticide product can only be usedMonsanto Company 10-SY-210U 677 of 721 

### legally according to the direc tions for use on the labeling acco mpanying the product at the time

of sale.

The registration of a new pesticid e is not EPA’s only opportunity to evaluate the product’s safety. EPA has recently completed a program to review older pesticides (those initially registered before November 1984) under FIFRA to ensure that they meet current scientific and regulatory standards. Reregistrati on, like the initial registration process, consid ers the human health and ecological effects of pesticides and results in actions to reduce risks that are of concern.

Where pesticides may be used on food or feed cr ops, EPA also sets pesticide tolerances, i.e., maximum pesticide residue levels that can legally remain in or on foods. EPA undertakes this analysis under the auth ority of the Federal Food, Drug, and Cosmetic Act (FFDCA). Under the FFDCA, EPA must find that such tolerances w ill be safe, meaning that there is a reasonable certainty that no harm will result from aggregat e exposure to the pesticide residue. This finding must be made and the appropriate tolerance estab lished before a pesticide can be registered for use on the particular food or feed crop in questi on. EPA must consider several factors before a

tolerance can be esta blished, including:

• the aggregate, non-occupational exposure fr om the pesticide (exposure through diet, from using pesticides in and around the home, and from drinking water);

• the cumulative effects from exposure to differe nt pesticides that produce similar effects in the human body;

• evidence of increased susceptibility to infants and children, or other sensitive subpopulations, from exposure to the pesticide; and

• evidence that the pesticide produces an effect in humans si milar to an effect produced by a naturally occurring estroge n or produces other endoc rine-disruption effects.

M.2.2. Pesticide Huma n Risk Assessment

The process EPA uses for evaluating the health impacts of a pesticide, under either FIFRA or FFDCA, is called risk assessment. EPA uses th e National Research Council’s four-step process to assess potential human health risks. This process involves hazardidentification, doseresponse assessment, exposure assessment and risk characterization. Each of these steps is

discussed below.

The first step in the risk assessment process is hazard identification to identify potential health effects, or hazards, which may occur from differe nt types of pesticide exposure. EPA considers the full spectrum of a pesticide’s potential he alth effects. Hazards are identified through a battery of studies that examine th e potential toxicity of the pest icide in various tests including, where appropriate, tests with laboratory animals.

To assess human health risk of the pesticide, pesticide companies conduc t many toxicity studies based on EPA guidelines and the Good Laborator y Practice (GLP) standards. Results are evaluated for acceptability by EPA scientists. EP A evaluates pesticides for a wide range ofMonsanto Company 10-SY-210U 678 of 721 

### effects, from eye and skin irritation to cancer a nd birth defects. EPA may also consult the public

literature or other sources of informa tion on any aspect of the chemical.

The next step of the risk assessment is dose-re sponse assessment which considers the levels at which the pesticide produces adve rse effects. Dose levels at which adverse effects were

observed in test animals are then transl ated into equivalent doses for humans.

Step three of the process involve s 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 food (following agricultu ral uses); home and personal use pesticides; pesticides applied to lands that make their way into the drinking water; or occupational exposure for agricultural workers or pe sticide applicators.

Risk characterization is the final step in assessing human health risks from pesticides. It is the process of combining the hazar d, dose-response and exposure assessm ents 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 dos e-response assessment. Th e strength of the overall database is considered, and broad conclusions are made. EPA’s role is to evaluate both toxicity and exposure and to determine the risk a ssociated with use of the pesticide.

The risk to human health from pesticide exposur e depends on both the toxi city of the pesticide

and the likelihood of people coming into contact with it, i.e. , the probability of exposure. At least some exposure and some toxicity are requir ed to result in a risk. For example, if the pesticide is found to have a high level of toxicity, but people ar e not exposed to the pesticide, there is no risk. Likewise, if there is ample exposure but the pesticide is nontoxic, there is no risk. Typically, however, there is some toxicity and exposure, wh ich results in a potential risk.

EPA recognizes that effects vary between animals of different species and from person to person. To account for this variability, a 100 -fold uncertainty factor is built into the risk assessment with

a 10X factor to account for diff erences between test species and humans, and a 10X factor to account for differences between people. This uncer tainty factor creates an additional margin of safety for protecting people who may be exposed to the pesticides. The Food Quality Protection Act (FQPA) requires EPA to use an additional, up to a 10-fold safety factor, if necessary, to protect special subpopulations if th ey show potential increased susc eptibility to effects of the pesticide, typically infants, childr en or women of child-bearing age.

Once EPA completes the risk assessment proce ss for a pesticide, the Agency uses this information to determine if there is a reasonable certainty of no harm to human health as a result of the use of the pesticide according to label directions as required by FIFRA. Using the conclusions of a risk assessment, EPA can then make an informed decision regarding whether toMonsanto Company 10-SY-210U 679 of 721 

### approve a pesticide chemical or use, as propos ed by the product label, or whether additional protective measures are necessary to limit expos ure to a pesticide. For example, EPA may prohibit pesticide use on certain crops because consuming the treated commodity may result in an unacceptable risk to consumers. Another exam ple of protective measures is requiring workers to wear personal protective equipment (PPE) such as a respirator or chemical resistant gloves, or not allowing workers to enter treated crop fiel ds until a specific period of time has elapsed (Restricted Reentry Interval or RE I). If, after considering all a ppropriate risk re duction measures, the pesticide still does not meet EPA’s safety standard, the Agency will not allow the proposed chemical or use. Regardless of the specific meas ures enforced, EPA’s primary goal is to ensure that legal uses of the pesticide are protective of human health, especially the health of children,

and the environment.

M.3. Dicamba Regulatory Status in U.S.

M.3.1. Dicamba – Registration History Dicamba is a selective broadleaf herbicide belong ing to the auxin agonist class, the oldest known class of synthetic herbicides, and is a member of the benzoic acids sub-group. Dicamba mimics the action of the plant hormone indole acetic acid, and causes rapid un controlled cel l division and growth, leading to plant deat h. Dicamba has been registered in the U.S. for use on food crops since 1967 (U.S. EPA, 2009) a nd has been widely used in ag ricultural production for over forty years. Dicamba is pres ently approved for use on aspara gus, corn, cotton, grass seed production, pastures and rangelands, small cer eals (including wheat), sorghum, soybean, and sugarcane. Dicamba is also used for industrial vegetation management ( e.g. , forestry and roadsides), professional turf management ( e.g. , golf courses, sports co mplexes), and residential turf (U.S. EPA, 2009; Durkin and Bosch, 2004).

Dicamba has a complete and comprehensive regul atory database (toxicity, environmental fate, and ecological toxicity) that has been evaluate d by the United States Environmental Protection Agency (U.S. EPA). A Registration Eligibility Decision (RED) for dicamba was completed by EPA in 2006 and subsequently amended in 200 8 and 2009 (U.S. EPA, 2009), as required for continued registration of all pest icides originally registered pr ior to 1984. EPA concluded the available data submitted for dicamba are comp lete and adequate to support the continued registration of dicamba products. EPA has evalua ted the available toxicity data and concluded that a high level of confidence exis ts in the quality of the dicamba data base and in the reliability of the dicamba toxicity endpoints for risk assessm ent. EPA also consider ed toxicity data and available information concerning the variability of sensitive subpopulations, including infants and children. The EPA concluded there is reasonable certainty that no harm will result to the general population, or to infants and children, as a resu lt of aggregate (all) exposure to dicamba residues. Thus, all current dicamba uses were eligible for reregistrati on (U.S. EPA, 2008a; U.S. EPA, 2008b; U.S. EPA, 2008c). In 2008, dicamba also successfully completed reevaluation by the Pest Management Regulatory Agency of H ealth Canada (PMRA, 2008) and the European

Commission Health and Consumer Protectio n Directorate-Genera l (European Commission, 2008). Dicamba has been approved by the EPA for a number of food and feed uses, including the major agricultural crops of corn, soybean and small grains ( e.g. , wheat or barley). Dicamba presently has 68 food and feed pesticide tolerances (40 CFR §180.227) established in support of

these uses.Monsanto Company 10-SY-210U 680 of 721 

### Dicamba is presently registered for preemergen ce (early pre-plant) a pplications up to 0.5 pound acid equivalence per acre (lb a.e./A) and late postemergence (pre-harve st) applications to soybean at rates up to 1.0 lb a.e./A, and a pest icide tolerance is established for residues of dicamba on soybean seed (10 ppm) in support of these uses.

M.3.2. Dicamba – Proposed New Use on MON 87708

An application has been submitted to the EPA to amend Registration Number 524-582 (a diglycolamine (DGA) salt formulat ion) to register a new use pa ttern for dicamba on MON 87708. In support of the new use of dicamba on MON 87708, mammalian toxicity data on the dicamba metabolites DCSA (3,6-dichlorosalicylic acid) and DCGA (3,6-dichlorogentisic acid), and crop

metabolism and residue data on dicamba-treated MON 87708 have been generated. These data confirm that existing dicamba food tolerances ar e sufficient to address any incremental exposure to dicamba resulting from its use on MON 87708. Furthermore, the proposed use pattern for dicamba on MON 87708 maintains the presen tly established maximum single and annual application rates. Therefore th e existing ecological risk assessment conducted by EPA as part of the dicamba RED is sufficient to address any incr emental ecological effects resulting from this

new use.

M.4. Potential Impact of Dicamba on Human Health

M.4.1. Dicamba Safety Evaluations for Consumers

On the basis of the risk assessments summari zed below, EPA has conc luded that there is a reasonable certainty that the existing uses of dicamba will not pose a risk to consumers, including infants and children.

Dicamba presently has 68 establis hed food and feed pesticide tole rances in the U.S. (40 CFR § 180.227). Each time EPA reviews an application to add a new food or feed use to the dicamba label, the EPA is required by FFDCA to conduct an aggregate risk assessment. This assessment considers potential exposure from the propose d new use with all ot her existing exposures, including non-occupationa l sources of exposure to the pest icide, and must conclude that aggregate exposure to the pesticide will be safe as defined by the statute and regulations. Risks associated with potential occupational expos ure for each new use are considered under the

FIFRA standard of no unreasonabl e adverse effects to the environment, which includes humans and workers (hereafter referred to as FIFRA unreas onable risk standard). Over the course of numerous reviews, the toxicol ogy of dicamba has been extensiv ely studied. Dicamba does not pose any unusual toxicological concerns (U.S. EPA, 2009; Durk in and Bosch, 2004; European Commission, 2007a) and is classifi ed by EPA as “Not likely to be Carcinogenic to Humans” (U.S. EPA, 2009). PMRA and the European Comm ission have also cla ssified dicamba as noncarcinogenic (PMRA, 2007, 2008; European Commission, 2007a).

Dietary exposure, as previously stated, is incl uded in the aggregate exposure assessment, and considers pesticide residues that may remain on food from crops on which the pesticide is applied (pre- or postemergence), as well as any residue in drinking water as a result of pesticide use. Non-dietary exposure is also included in this assessment, and includes exposure to the pesticide through residential use, such as on lawns, as well as e xposure in a recreational context,Monsanto Company 10-SY-210U 681 of 721 

### such as from a golf course or s ports field. Based on these data , EPA must be able to make a determination of reasonable certainty of no harm to human health as required by the FFDCA. At the time that dicamba was undergoing reregist ration review, occupati onal exposure was not considered as part of the aggregate exposur e and is evaluated separately under the FIFRA

unreasonable risk standard.

In making a determination of whether a reasonable certainty of no harm to human health exists, dietary risk assessments are performed that consid er both the potential expos ure and toxicity of a given pesticide. The dietary risk is then described as a percentage of a le vel of concern. The level of concern, which is also referred to as the population adjusted dose (PAD), is the dose (level of exposure) predicted to result in no adverse health effects to any human population subgroup, including sensitive subgroups, such as infants and children. The PAD may be expressed based on acute (aP AD, one day or less) or chr onic exposures (cPAD, lifetime exposure). The PAD is the reference dose (R fD) for the compound but with any additional safety/uncertainty factors to protect sensitiv e subpopulations, or to ad dress the completeness, quality or reliability of the toxi city data. The PAD is an estimat e of the amount of daily pesticide exposure to the human population that can occur acu tely (less than one da y) or over a lifetime with a reasonable certainty of no harm to human health. 69 

### An estimated exposure less than 100%

of the PAD is below the level of concern for the EPA.

The EPA evaluated the potential ri sks to human health associat ed with all then-registered dicamba uses as part of the reregistration of di camba, and published the results and conclusion in the dicamba RED (U.S. EPA, 2009). Since rere gistration, the EPA has approved an additional use on sweet corn, and as part of the approval included this new use in an updated dietary risk assessment. Since the sweet corn use pattern was within that review ed during reregistration, EPA determined that the ecological and environm ental fate assessments did not need to be updated and utilized the drinking water concentration from the RED in the updated sweet corn dietary risk assessment. The sweet corn use di d not result in any noticeable increase in dietary exposure compared to the assessment from the RE D; therefore, the risk assessment conduced for the dicamba RED is representative of all current registered uses of dicamba. EPA has conducted acute and chronic dietary (food and water) risk assessments for dicamba based on a theoretical worst case exposure estimate. For food, this es timate assumes that dicamba 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 resi dues found on all harvested food crops and derived animal food commodities ( e.g. , meat and milk) are at the level of the legally established tolerance ( i.e. , the maximum allowable pesticide residue level). Re sidues of dicamba are defined as dicamba and its metabolites 5-hydroxy dicamba and 3,6-dich lorosalicylic acid (D CSA) in soybean commodities, and as dicamba and DCSA in animal food commodities, as currently regulated in 40 CFR § 180.227 (U.S. EPA, 2005a; U.S. EPA, 2005b). For water, EPA assumed that dicamba could potentially move offsite to adjacent surface water bodi es as a result of drift or runoff. EPA also assumed dicamba could move throug h soil to groundwater; however, estimated concentrations in groundwater were significant ly lower and therefore surface water estimates were used in the worse case dietary assessments. Surface water estimates were generated with the conservative screening level models SC IGROW and PRZM/EXAM using an exaggerated

69 RfD is the current terminology used by EPA; however, earlier EPA risk assessment terminology used the term Acceptable Daily Intake (ADI). RfD and ADI are synonymous.Monsanto Company 10-SY-210U 682 of 721 

### application rate that is 2.8 tim es higher than the current 1.0 lb a.e./A maximum single application rate established in the dicamba RED (U.S. EPA, 2009, U.S. EPA, 2005b), and the maximum single application rate proposed for MON 877 08. EPA mandated reductions in dicamba use rates as part of the dicamba RE D (1 lb a.e./A and 2 lb a.e./A for a single application and for annual application, respectively).

The acute PAD for dicamba is 1 mg per kg body weight per day (mg/kg/day), based on an acute neurotoxicity study in rats (U.S. EPA, 2009). Based on the worst-case assumptions outlined above, the result of the dietary assessment ga ve a conservative, high -end (95th percentile) estimate of risk, which was well below the Agency’s level of concern for both the U.S. population in general and for all population subgroups. As shown in Table M-1, when both food and water are combined, infants were the most highly exposed subgroup w ith 11% of the aPAD, or acute level of concern, consumed. Beca use even this most highly exposed subgroup consumes a small percentage of the acute level of concern for dicamba, EPA concluded there is a reasonable certainty that acute di etary exposure to dicamba will not pose a risk to human health, including that of infants and children (U.S. EPA, 2009).

The chronic PAD for dicamba is 0.45 mg/kg/da y, based on a two-genera tion reproduction study in rats (U.S. EPA, 2009). Based on worst-ca se assumptions outlined above, EPA developed exposure estimates for the general U.S. populati on and the 8 other subpopulations of consumers evaluated by EPA, the major subpopulations are summarized in Table M-1. EPA determined that the most highly exposed subgroup for chr onic dietary exposure (i ncluding both food and water) was children aged 1-2 years old, which cons umed 6.6% of the cPAD, or chronic level of concern. Since even the most highly exposed subgroup consumes a small percentage of the chronic level of concern, EPA co ncluded there is a reasonable certainty that chronic dietary exposure to dicamba will not pose a risk to human health, including that of infants and children (U.S. EPA, 2009).

Table M-1. Summary of Dietary Exposure a nd Risk for Dicamba: Food and Water

Population subgroup Acute dietary (95th percentile) Chronic dietary Dietary exposure (mg/kg/day) % aPAD Dietary exposure (mg/kg/day) % cPAD

General U.S. population 0.0435 4.4 0.0118 2.6

All infants (<1 year old) 0.108 11 0.0199 4.4

Children 1-2 years old 0.0756 7.6 0.0297 6.6

Source: EPA 2005b.



### The EPA also conducted an aggregate risk as sessment which included dietary exposure (food and water) as well as othe r non-occupational exposures ( e.g. , from residential uses such as lawns and recreational uses such as golf courses or sports fields). For acute and chronic aggregate risk assessment, the aggregate exposure is the same as the dietary exposure. EPA does not typically aggregate acute dietary exposures with acute non-occ upational exposures beca use it is unlikely that high end dietary exposure will occur on the same day as maximum non-occupationalMonsanto Company 10-SY-210U 683 of 721 

### exposures (U.S. EPA, 2005b). Current residential uses of dicamba do not result in long term residential exposure scenarios, so chronic aggregate exposure/risk is equal to chronic dietary exposure/risk. Therefore, the acute and chronic dietary exposure and risk summarized in Table M-1 also represents the acute and ch ronic aggregate exposure and risk.

For short-term aggregate risk assessment of dicamba, EPA considered exposures from food, water, and residential handling and post-application. As show n in Table M-2, the highest exposed individuals in this assessment were adult males mixing, loadin g and applying dicamba using a hose-end sprayer, result ing in an aggregate exposure of 5.1% of the cPAD, and toddlers playing on treated turf which results in an aggreg ate exposure of 9.7% of cPAD. This short-term risk aggregate risk assessment was also considered to be protective of in termediate and long term exposures to dicamba. A large marg in of safety exists for exposure to dicamba, even considering all the approved food, feed and non-crop uses of dicamba.

Table M-2. Aggregate (Short-term) Exposure Assessment for Dicamba

Population Food + Water Exposure mg/kg/day Incidental Oral Exposure,

mg/day Dermal
Dose, mg/kg/day Combined Exposure, mg/kg/day %PAD

Adult Male -

Handler 0.0128 0 0.0102 0.023 5.1

Adult Male –

Post Application 0.0128 0 0.0037 0.0165 3.7

Child – 1-2

years 0.0297 0.0078 0.0062 0.0437 9.7

Source: EPA, 2005b.



### In summary, based on the EPA risk assessments discussed above, there is a reasonable certainty that the existing uses of dicamba will not pose a risk to consumers, including infants and children.

M.4.1.1. Dicamba Safety Evaluation for Use on MON 87708

Monsanto has submitted to EPA an application to register the use of dicamba on MON 87708 (U.S. EPA OPP Decision Numb er D-432752). The proposed us e of dicamba on MON 87708 will not result in measurable increases in the exposure to dicamba or significant changes in the

human health risk assessment. Plant metabolism studies have shown that the majority of the dicamba residue found in MON 87708 is DCSA, with lesser amounts of 3,6-dichlorogentisic acid (DCGA), 5-hydroxydicamba (5-OH dicamba) and parent dicamba (Table M-3). Toxicology

studies submitted to EPA have demonstrated th at the toxicology profiles for both DCSA and DCGA are comparable to that of parent dicam ba, and that the existing hazard endpoints (RfDs and PADs) for dicamba are adequate to assess the potential human health risks from the

metabolites as well. Studies have shown that the total dicamba residues in soybean seed following application of dicamba to MON 87708 will be well below the current 10 ppm soybean seed tolerance, an expos ure level which has been determined as acceptable by EPA. In addition, Monsanto is proposing to establish new feed to lerances for soybean forage (45 ppm) and hay (70 ppm), which will allow the feeding of forage and hay to livestock. This practice is notMonsanto Company 10-SY-210U 684 of 721 

### presently allowed because the preharvest applica tion occurs after the stage where the crop would be useful as forage and hay. Note that the ma ximum residue for forage at 51.2 ppm is above the proposed MRL of 45 ppm. This is due to the way the data are distributed and the use of the NAFTA MRL calculator, which is the standard method used by EPA to calculate pesticide tolerance levels (U.S. EPA, 2011a). The EPA will perform its own calculations based on the data summarized in the table below to establish an appropriate feed tolerance for dicamba on soybean forage and hay.

Table M-3. Residues of Dicamba, DCSA, 5-Hydroxydicamba and DCGA in DicambaTolerant Soybean Forage, Hay and Seed

Commodity PPM (Expressed as Each Analyte per se) PPM (Dicamba Acid Equivalents)

Dicamba DCSA 5-OH

dicamba DCGA Total as current definition of residue (dicamba + DCSA + 5-OH dicamba)
Forage

Mean 0.342 15.8 <0.006 2.04 17.3

Median 0.068 14.0 <0.005 1.95 15.2

Minimum <0.021 8.34 <0.005 0.359 10.0

Maximum 2.62 47.9 0.010 5.95 51.2

Hay

Mean 0.130 30.1 <0.014 2.68 32.3

Median 0.051 29.8 <0.014 2.02 31.9

Minimum <0.014 11.4 <0.014 0.169 12.2

Maximum 1.16 57.1 <0.014 7.33 61.1

Seed

Mean <0.013 0.055 <0.021 0.032 <0.091

Median <0.013 0.031 <0.021 0.017 <0.065

Minimum <0.013 0.009 <0.021 <0.011 <0.041

Maximum <0.013 0.411 <0.021 0.136 0.471



### Human dietary exposure will not increase beyond what has already been evaluated and determined acceptable by the EPA because estab lished tolerances for animal food commodities ( e.g. , meat or milk) are sufficient to address livestock consumption of soybean forage or hay. This is because the proposed soybean forage and hay tolerances (residues) are lower than existing livestock dietary constituents that could potentially be replaced with soybean forage or hay in the livestock diet ( e.g. , grass forage at 125 ppm could be replaced with soybean forage at 45 ppm). Soybean forage and hay are also not common livestock dietary constituents, as concluded by the EPA in its policy to permit labe l restrictions for livestock feeding of soybean forage and hay (U.S. EPA, 1996).

Furthermore, since existing dicamba food tolera nces (soybean seed and animal by-products) are inclusive of dicamba exposures arising from its use on MON 87708, the most recent EPA dietary and aggregate risk assessments for dicamba also address exposure from the proposed use in MON 87708. Therefore, these risk assessments de monstrate that there is a reasonable certainty that the use of dicamba on MON 87708, together w ith all other approved uses of dicamba, willMonsanto Company 10-SY-210U 685 of 721 

### not pose a risk to human health, including that of infants and chil dren. While the use of dicamba is expected to increase as a re sult of the availabil ity of MON 87708 integrat ed into the Roundup Ready ® 

### soybean system, the risk associated with the new use has been adequately assessed by EPA through the risk assessment conducted as pa rt of the dicamba RED. Lastly, EPA will review and confirm the acceptability of dietar y exposure of dicamba residues on MON 87708 as part of the review of our pending application.

M.4.2. Dicamba Safety Evaluation for Applicator

Other potential impacts considered by EPA in its human health assessment are occupational exposure of the pesticide handler/applicator, and post-application exposure resulting from reentry to treated fields or areas . The occupational exposure scen arios evaluated by the EPA as a part of the dicamba RED are also applicable to the propo sed use of dicamba on MON 87708 (U.S. EPA, 2005b; U.S. EPA, 2005d).

Using exposure data from the Pesticide Ha ndler Exposure Database (PHED), Outdoor Residential Exposure Task Force (ORETF), and Ca lifornia Department of Pesticide Regulations,

the EPA assessed short-term and intermediate-t erm occupational handler and post-application exposures. Handler exposure scenarios included mi xer-loader, applicator and flagger activities. When exposure assumptions included the wear ing of chemical re sistant gloves during mixer/loader operations involving liquids requi red by dicamba product labeling, all occupational handler and post-application re-entry scenarios exhibited margins of exposure greater than 100

and did not exceed the EPA level of concern (U.S. EPA, 2009). The use of dicamba on MON 87708 does not pose any ne w exposure considerations fo r workers beyond those which have been previously evaluated by EPA as part of the dicamba RED. Therefore the use of dicamba on MON 87708 will not pose a risk to agricultural workers.

M.5. Potential Impact of Dicamba on the Environment

As discussed above, environmental effects are car efully considered as a part of the FIFRA pesticide registration process. Pr ior to the approval of a new pest icide or a new us e (including a change in pesticide application rates and/or timing) and before reregistering an existing pesticide, EPA must consider the potential for environmental effects and conclude no unreasonable adverse

effects to the environment will result from the new pesticide, new use or continued use.

To make this determination, EPA requires a co mprehensive set of environmental fate and ecotoxicology data on the pesticide active ingredient (40 CFR Part 158). EPA uses these data to assess the pesticide’s potential environmental risk (risk = hazard × exposure). The required data include both short and long-term hazard data on repr esentative organisms that are used to predict hazards to terrestrial animals (birds, non-targ et insects, and mammals), aquatic animals (freshwater fish and invertebra tes, estuarine and marine orga nisms), and non-target plants (terrestrial and aquatic).

EPA reevaluated the environmenta l safety of dicamba in 2006 as part of the FIFRA-required reregistration of all pesticides. At the end of this evaluation, EPA c oncluded that all then-

® Roundup Ready is a trademark of Monsanto Technology LLC. All other trademarks are the property of their respective owners.Monsanto Company 10-SY-210U 686 of 721 

### registered uses of dicamba were eligible fo r reregistration, including single terrestrial ( i.e. , landbased) applications up to 1.0 lb a.e./A dicamba, and annual terrest rial applications up to 2.0 lb a.e./A. (U.S. EPA, 2009). This EPA assess ment fully supports the use of dicamba on MON 87708 as the proposed use pattern is with in that assessed in the RED assessment.

M.5.1. Persistence of Dicamba in the Soil

Dicamba’s fate in soil has been evaluated to determ ine the extent of its persistence in soil. While dicamba is stable to hydrolysis and photodegrades slowly on soil, dicamba is readily degraded in soil by aerobic soil microorganisms (U.S. EPA, 2009). Both laboratory and field studies demonstrate that dicamba dissipate s quite rapidly in soil and has a low potential for accumulation.

In a laboratory aerobic soil metabolism study, dicamba was rapidly metabolized to the nonpersistent degradate DCSA with a half-life of si x days. DCSA was rapidl y degraded at a rate comparable to that of dicamba producing carbon dioxide and being incorporated into biological natural products (U.S. EPA, 2005c). Other laboratory aerobic soil studies (Krueger et al., 1991)

have been conducted for dicamba. In these stud ies, conducted in a numb er of different soils under various moisture and temperature conditions , dicamba degraded with half-lives ranging from 17 to 45 days. The European Union did not consider these half-lives indicative of the degradation rate of dicamba after evaluating the Krueger and other aerobic soil degradation studies (European Commission, 2007b).

Field dissipation studies conducte d with dicamba formulations al so demonstrate rapid dissipation of dicamba in soil. Application of dicamba formulated as various salts (diglycolamine, dimethylamine and sodium salts corresponding to commercial products) to bare-ground plots at labeled rates (1 or 2 lb a.e./A) resulted in half -lives for dicamba of 3-20 days. Results also confirmed low vertical mobility under the conditions of the studies as demonstrated by only low concentrations (<20 ppb) of di camba and its major degradate DC SA present in soil segments deeper than 10 cm (U.S. EPA, 2005c). Th e proposed maximum use rates of dicamba on MON 87708 (single application rate of 1 lb a.e./A dicamba, total annual application rate of 2 lbs a.e./A) are within the rates investigated in these dissipation studies. In addition, dicamba applications, which can be applied prior to planting up through the R1/R2 reproductive stage to MON 87708, should occur when soil temperatur es are favorable for aerobic microbial degradation of dicamba.

As with many chemicals, dicamba is metabolized somewhat slowly under anaerobic conditions, degrading with a half-life of 141 days (U.S. EPA, 2005c). However, under conditions amenable to aerobic degradation of dicamba and where re tention of dicamba in aerobic soil layers is adequate for microbial degradation to occur, movement of dicamba to anaerobic soil layers would be minimal. Under normal soybean growing conditions of adequate soil temperatures and soil moisture, dissipation of dicamba through aer obic degradation would be rapid and it is unlikely that significant amounts of dicamba would reach anaerobi c soil layers.

Based on these data, EPA concluded that dicam ba and DCSA (the primary environmental degradate) are “somewhat persistent” under aerobic and anaerobic c onditions (U.S. EPA, 2009).

This statement reflects the difference in degradation rates under aerobic conditions (nonpersistent) and anaerobic conditions (persistent). As discussed above, applications of dicambaMonsanto Company 10-SY-210U 687 of 721 

### on MON 87708 are likely to occur when envir onmental conditions are conducive to rapid aerobic microbial degradation; therefore, dicamba is not expected to persist in soil as a result of the use on MON 87708. Furthermore, EPA concl uded, based on the registered use patterns of dicamba and product label mitigation statemen ts, no unreasonable adverse effects to the environment would result from the (a ll) registered uses of dicamba.

M.5.2. Surface Water and Groundwater

The EPA evaluated potential risks to surface and groundwater from the approved uses of dicamba as part of the 2006 Re registration Eligibility Decisi on (RED). The EPA estimated levels of dicamba in surface and ground water us ing screening level (high-end exposure) models PRZM/EXAMS and SCIGROW, respectively, wh ich estimate concentrations based on the physical, chemical and environmental fate proper ties of dicamba, and high-end use patterns.

These conservative estimates do not take in to account normal variation in environmental

concentrations that can occur from dilution and dissipation. Based on si mulated sugarcane crop scenarios for both ground and aeri al applications of 2.8 lbs dicam ba a.e./A (2.5 to 5 times the proposed maximum single application ra te for MON 87708),estimated groundwater concentrations were 0.016 μg/L dicamba and 0.008 μg/L for DCSA, the major environmental degradate (U.S. EPA, 2005b). In addition, EPA measured dicam ba as part of its 1971-1991 pesticides ground water survey. Dicamba was detected at a low level of incidence, less than 3% of the 3172 samples, at levels rang ing from trace to 44 μg/L(U.S. EPA

, 

### 1992). Dicamba also had a low incidence of detections in groundwater (le ss than 3% of the 6571 samples) in the U.S. Geological Survey National Water Quality As sessment (NAWQA) monitoring program during 1993-2003, where dicamba was detected at levels ranging from 0.008 to 2.50 μg/L (U.S. EPA, 2005b). Only parent dicamba and not the DCSA metabolite was analyz ed in these water monitoring surveys.

For drinking water derived from surface water, es timated concentrations (1 in 10 year annual mean) using the simulated sugarcane crop scenario for both ground and aeri al applications of 2.8 lbs dicamba a.e./A, ranged from 9.7 to 13 μg/L dicamba a.e. and 0.66 to 0.81 μg/L for DCSA (U.S. EPA, 2005b) Estimated surfa ce water concentrations consider ed for aquatic organism risk assessment using simulated soybean crop scenario for ground and aerial applications of 2.0lb dicamba a.e./A ranged from 33.3-36.1 μg/L dica mba a.e. (U.S. EPA, 2005c). The NAWQA program also analyzed for dicamba in surface water samples in the 1993-2003 survey, where dicamba was detected at a low incidence (3% of the 6614 samples) at levels ranging from 0.009 to 1.76 μg/L (U.S. EPA, 2005b).

Measured dicamba concentrations in ground a nd surface water were also evaluated from a collection of publicly-available water monitoring data assembled by Monsanto Company from across the U.S. during 1990-2010. Data sources incl ude the U.S. EPA (Pesticide Data Program, Safe Drinking Water Information System, Storage and Retrieval Data Warehouse and Unregulated Contaminant Monito ring Regulation data), the U.S. Geological Survey (NAWQA, National Water Information System), and various state water quality databases. From this collection of water monitoring data, relevant dicamba samples for ground and surface water were identified. First, data were restricted to th e major soybean production states in the Midwest (IA, IL, IN, KS, KY, MI, MN, MO, ND, NE, NM, OH, SD and WI) and Arkansas. Second, the following samples types were excluded from the ev aluation: finished dri nking water, qualityMonsanto Company 10-SY-210U 688 of 721 

### control samples, urban stream monitoring samp les, and alternative purpose sampling (e.g., contaminated site investigation). Records for which a sampling date or location was not reported were also excluded from the evaluation. Finally, fo r surface water, the data were limited to those samples collected during the months of April – Ju ly, which is considered the typical period when dicamba would be applied for agricultural uses , and for which maximum concentrations of dicamba in surface water would be expected.

Dicamba concentrations in ground water are presen ted in Table M-4 for several year ranges and for the entire dataset evaluated. Overall, traces of dicamba were detected in less than 6% of the samples. For samples with detected dicamba levels, the minimum, median, 90 th 

### percentile and maximum concentrations are provided. The maxi mum level of dicamba detected in groundwater between 1990 and 2010 was 2.2 μg/L. However the 90 th 

### percentile of dicamba detections during 1994-1998, corresponding to the peak dicamba usag e period (see Table VIII-11), and across the entire monitoring period was 0.25 μg/L.

Table M-4. Dicamba Concentrations (μg/L) in Groundwater in Major Soybean Growing

States

1990-

1993 1994-

1998 1999-

2004 2005-

2010 1990-

2010

Number of

Detects & Nondetects n 1796 2781 2558 2568 9703

Number of

Detects n 121 413 1 3 538

Dicamba

Concentration (μg/L) in Ground

Water Based on Detects Only Min 0.01 0.01 0.73 0.1 0.01

Median 0.25 0.25 n/a 0.1 0.25

90th

Percentile 0.25 0.25 n/a 0.18 0.25

Max 1 2.2 0.73 0.2 2.2

n/a: not applicable due to single data point



### The geographical coverage of th e groundwater sampling sites is provided in Figure M-1, which shows the locations of the sampli ng sites associated w ith all samples (detects and non-detects) and for detects only. The location of sampling sites during the 1994-1998 timeframe, which corresponds to dicamba’s peak hi storical use period, provide a ge ographical representation of the areas of dicamba historical agricultural use inte nsity (see Figure VIII-1) and the major areas of soybean cultivation (see Figure VIII-2).Monsanto Company 10-SY-210U 689 of 721 

### Figure M-1. Ground Water Sampling Sites for Dicamba (Top: Detects only; Bottom: detects and non-detects)Monsanto Company 10-SY-210U 690 of 721 

### Dicamba concentrations in surf ace water are presented in Table M-5 for several year ranges and

for the entire dataset evaluated. Overall, traces of dicamba were de tected in less than 11% of the samples. For samples with detected dicamba levels, the minimum, median, 90 th 

### percentile and maximum concentrations are provided. The ma ximum level of dicamba detected in surface water between 1990 and 2010 was 17 μg/L. However the 90 th 

### percentile of dicamba detections during 1994-1998, corresponding to the peak dicam ba usage period (see Table VIII-11), and across the entire monitoring period wa s 0.74 and 0.883 μg/L, respectively.

Table M-5. Dicamba Concentrations (μg/L) in Surface Water in Major Soybean Growing

States

1990-

1993 1994-

1998 1999-

2004 2005-

2010 1990-

2010

Number of

Detects & Nondetects n 588 1,362 2,443 1,033 5,426

Number of

Detects n 146 206 222 14 588

Dicamba

Concentration (μg/L) in Surface

Water Based on Detects Only Min 0.01 0.008 0.01 0.03 0.008

Median 0.2 0.25 0.29 0.2935 0.25

90th

Percentil

e 1.39 0.74 0.944 0.497 0.883

Max 17 9.4 3.3 0.94 17

The geographical coverage of surface water sampling sites is provided in Figure M-2, which shows the locations of the sampli ng sites associated w ith all samples (detects and non-detects) and for detects only. The location of sampling sites during the 1994-1998 timeframe, which corresponds to dicamba’s peak hist orical use period, represent a se gment of the areas of dicamba historical agricultural use intensity (see Figure VIII-1) and the major areas of soybean cultivation (see Figure VIII-2).Monsanto Company 10-SY-210U 691 of 721 

### Figure M-2. Surface Water Sampling Sites for Dicamba (Top: Detects only; Bottom: detects and non-detects)Monsanto Company 10-SY-210U 692 of 721 

### Altogether, the modeling estimates, based on high-end exposure assumptions, and monitoring

results show that concentrations of dicamba in potential drinking wa ter resources as a result of its use are very low, and are well below the lifetime Health Advisory Level (HAL) for dicamba of 4 mg/L (U.S. EPA, 2011b). Similarly, the estimated and monitoring levels of dicamba in surface

water are low and do not exceed the EPA level of concern for aquatic plants 70 

### and animals (U.S. EPA, 2005c). Therefore, the potential risk of di camba from leaching or runoff as a result of incremental exposure from the use of dicamba associated with the planting of MON 87708 is very low and is unlikely to adversely affect human health or aquatic plants or animals.

M.5.3. Wildlife

M.5.3.1. Animals

As a part of the reregistration evaluation unde r FIFRA, EPA conducted an ecological screening assessment for dicamba. This assessment compared the results from toxicity tests with dicamba conducted with various plant and animal species to conservative (high end) estimates of the concentration of dicamba to which an organism might be exposed in the environment. These estimates, called the Estimated Environmental Exposure Concentrations (EECs), are point estimates for specific types of exposure ( e.g. , aquatic or dietary) that assume constant high concentrations throughout the lifespan of the or ganism. These estimates do not take into account

normal variation in environmen tal concentrations, the diluti on or dissipation of those concentrations, or the frequency of exposure of wildlife to the pesticide. Such assumptions provide a screening level of a ssessment where conclusions of no harm can be drawn with high confidence; however, it is also possible to reach a conclusion of no harm with more refined exposure assumptions. During the reregistration process for dicamba and associated salts, the EPA derived exposure estimates for a number of use patterns (U.S. EPA, 2005c), including two scenarios that are reasonably close to the proposed use pattern for dicamba on MON 87708. These use patterns are: 1) wheat, with two 1.0 lb a.e./A applicati ons; and 2) sorghum, with two 0.5 lb a.e./A applications. For both cropping scenarios, the two appl ications were considered to occur on May 1st and June 1st.

Based on the EPA screening-level analysis for relevant use patterns (wheat and sorghum) described above, no acute or chronic risk to aquati c animals was identified. Also, no chronic risk

to birds or acute risk to mammals was iden tified. Considering maximum and mean residue levels assumed from the Kenaga nomogram (Fletcher et al., 1994) 71, 

### and application at rates of 1.0 lb a.e./A and 0.5 lb a.e./A, the screening assessment did determine that the risk quotient (RQ) exceeded the Level of Concern (LOC= 0.5) for acu te risk to some non-endangered bird species based on size and diet (U.S. EPA, 2005c). Usi ng the toxicity endpoints from the avian oral

gavage 72 

### study, i.e., a study which forces a single high dose of dicamba directly into the birds’s crop and is not representative of realistic exposure scenarios for birds, EPA concluded acute exposure risks exceeded their level of concern for small (20 g) and medium (100 g) size birds.

70 Currently there are no federally list ed threatened or endangered nonvascular aquatic plants, so only risk to nonlisted aquatic nonvascular plants is considered.

71 The Kenaga nomogram was developed by the U.S. Environmental Protection Agency (U.S. EPA) to predict the maximum potential pesticide residue levels in the food chain of wildlife for use in risk assessment.

72 Forcing a single high concentration of dicamba directly in to the bird’s crop, and is not representative of realistic exposure scenarios for birds.Monsanto Company 10-SY-210U 693 of 721 

### Despite these predictions of potential effects, no incident reports have been filed with EPA for adverse effects on birds from dicamba use (U.S. EPA, 2005c). Furthermor e, when exposure via the avian diet (the most rele vant and realistic route of e xposure of birds to dicamba) is considered, the EPA classified di camba acid and its salts as pract ically non-toxic to birds (U.S. EPA, 2005c). Avian toxicity to dicamba (as th e DGA salt) via dietary e xposure is significantly less (> 5-fold) than toxicity from exposure via oral gavage. Theref ore, it is unlikely that dicamba will pose an acute risk to birds when a more realistic dietary exposure is considered where

dicamba residues are mixed with other food com ponents and eaten over the entire day as would

occur in the natural environment.

The screening level assessment predicts that ther e will be no chronic risk (RQ < 1) to mammals regardless of size or dietary habits when mean residues resulting from ap plication at a maximum

rate of 1.0 lb a.e./A are utilized in the assessmen t (U.S. EPA, 2009). Mean initial residues, rather than maximum initial residues, provide a more real istic, but still conservative, dietary exposure estimate for a chronic exposure which would occur over days to weeks of duration.

The EPA has classified dicamba as practically n ontoxic to honey bees based on an acute contact LD50 value of >90.65 μg/bee (the honeybee is used to assess effects on non-target insects in general), and practically nontoxic to birds from dietary exposure. Dicamba presents a low hazard to aquatic organisms. Co nsequently, aquatic animals were predicted not to be at risk from rates of dicamba up to 2.8 lbs a.e./A, the highest rate assessed (U.S. EPA, 2005c). Dicamba has a low octanol-water coefficient (Log P < 3), indicating that it has a tendency to remain in the water phase rather than move from the water phase into fatty substances; therefore, dicamba is not expected to bioaccumulate in fish or other animal tissues (U.S. EPA, 2005c).

In conclusion, no acute or chroni c risk to aquatic or terrestria l animals, as defined above, is anticipated to result from potential incremental exposure of dicamba associ ated with the use of

MON 87708.

M.5.3.2. Plants

Dicamba is a selective herbicide with more ac tivity on dicotyledonous (dicot) plants than monocotyledonous plants (Asht on and Crafts, 1981). The EFED Reregistration Chapter for

Dicamba / Dicamba Salts concluded that listed and non-listed terrestrial plants are potentially at

risk from runoff and drift associated with all us es, but no risk to liste d and non-listed aquatic plants (vascular or non-vascular) is expected as result of dicamba use at single application rates up to 1.0 lb a.e./A (U.S. EPA, 2005c).

In the Reregistration Eligibility Decision (U.S . EPA, 2009), EPA concluded that a reduction in the maximum rate for a single application to 1.0 lb a.e./A and reduction in the total annual application rate to 2.0 lb a.e./A reduced the risk to te rrestrial plants, and further determined that all currently registered uses of dicamba were eligible for reregi stration provided th at these rate mitigation measures were adopted on product labels.

In conclusion, since the application rates for use of dicamba on MON 87708 fall within the reduced application rates mandated by the RED, the use of dicamba on MON 87708 will not pose a risk to non-target aquatic plants. Further, risks to non-target terrestrial plants areMonsanto Company 10-SY-210U 694 of 721 

### adequately managed through approp riate application of dicamba as directed on the pesticide label and any effects will be mostly limite d to non-lethal, temporary visible effects.

M.5.4. Endangered and Threatened Species

In the dicamba RED EPA discussed the changes in the registered use pattern of dicamba that were required to assure that dicamba meets the regulatory standards for pesticides in the United States. The primary use restriction that EPA determined was necessary to provide adequate protection of the environment, including non-targ et species, was the reduction of the maximum single dicamba application rate to 1.0 lb a.e./acre and the maximum annual rate of 2.0 lb a.e./A.

EPA went further in the RED to state that no specific additional mitigations of off-target movement due to spray drift were needed (U.S. EPA, 2009). Because these limits were mandated by EPA to provide protection to non-tar get organisms, and because the proposed use of dicamba on MON 87708 falls within these limits, the risks to these non-target organisms from

the use of dicamba on MON 87708 has also been addressed.

As stated earlier, because the maximum single and annual applica tion rates for the proposed use pattern for dicamba on MON 87708 are within the range of maximum application rates implemented as the primary use restriction ma ndated by the dicamba RED, the same conclusion

can be reached for this use. Monsanto ha s requested approval of the use of dicamba on MON 87708; in its review of this registration request, EPA will address this use of dicamba under applicable statutory and regulat ory requirements for pesticides.

In addition, Monsanto has gone beyond the existing restrictions in place for dicamba use under

FIFRA in order to assure ade quate protection of endangered and threatened species under the proposed use of dicamba on MON 87708. Specifical ly, Monsanto’s registration submission for dicamba use on MON 87708 requested use only for a low volatile salt form of dicamba (diglycolamine or DGA) and requested only ground applications be allowed ( i.e. , no aerial application).

M.5.5. Potential Effects on Endangered An imal Species Identi fied in Litigation

In response to litigation brought by the Wash ington Toxics Coalition, the EPA conducted an assessment for dicamba and its impact on the en dangered Pacific anadromas salmonids and their critical habitat. In this analysis, the EP A concluded that dicamba does not impact the endangered salmonids or their crit ical habitat (U.S. EPA, 2003). EPA also considered risks to endangered species as part of the reregistra tion of dicamba in 2006. Using an extremely conservative deterministic screening level ecolog ical risk assessment (U.S. EPA, 2004), the EPA

concluded that use of dicamba would have no eff ect on threatened or endangered freshwater fish, estuarine fish, and aquatic inve rtebrates (U.S. EPA, 2009).

The EPA has also evaluated the potential eff ect of dicamba on salmon in eleven areas of California and Southern Oregon 73 

### in response to the consent agreement reached in the Washington Toxics lawsuit. 74 

### The conclusion of EPA’s risk assessment is as follows:

73 These areas are called Evolutionarily Significant Units ba sed on the salmonid populations present in these areas.

74 Washington Toxics Coalition v. Environmental Protection Agency, 413 F.3d 1024 (9th Cir. 2005).Monsanto Company 10-SY-210U 695 of 721 

### “Regardless of the specific dicamba compou nds, I conclude that dicamba compounds with currently registered uses will have “no effect” on listed Pacific salmon and steelhead and their critical habitat...” (U.S. EPA, 2003).

Since the use rates of dicamba proposed for M ON 87708 are within the curre ntly registered and evaluated use rates, no further cons ideration of the risk to Pacific salmon or steelhead is needed.

This conclusion is consistent with EPA’s determina tion of no risk to fish or other aquatic animals in the dicamba screening assessment in the di camba RED (U.S. EPA, 2005c). Furthermore, soybean production is not commonly practiced in the Pacific Northwest. However, based on the analysis above, risk to endangered Pacific salmon or steelhead is not expected from the use of

dicamba on MON 87708.

M.5.6. Other Potential Environmental Impa cts Associated with Dicamba Use on

MON 87708

M.5.6.1. Potential Offsite Movement of Dicamba

Herbicide drift and offsite movement are activel y managed by farmers and applicators specially trained to use such products cons istent with product labels and othe r state or local restrictions. Depending upon the herbicide being used, factors for managing th e potential for spray drift include the selectivity and sensitivity of the he rbicide, local weather conditions at the time of application (wind, temperature, humidity, inversion potentia l), droplet size distribution, application volume, boom height (height of the application equipment above the crop canopy), sprayer speed, and distance from the edge of the application area (SDTF, 1997; Felsot et al., 2010). A variety of measures can be employed to control the potential for spray drift and offsite movement, including nozzle selection and appl ication techniques and restrictions.

The potential for offsite movement is regulated at the federal level by EPA. As indicated, EPA specifically approves product labeli ng for all crop uses of the herb icides. Before any dicamba formulation could be used on MON 87708, EPA is required to approve the pesticide label for that respective use, including specific directions for the use and application of the herbicide on the proposed crop. Label use directions would a ddress not only applicati on rates and timing, but as appropriate, could also include other measures to address the potential for offsite movement. EPA considers possible effects from offsite move ment as part of the pesticide registration process required under FIFRA. Specifically, in order to a pprove the use of a pesticide (herbicide), EPA must conclude that no unreasonable adverse e ffects on non-target vegetation will result from potential offsite movement when th e pesticide is used according to the product

label.

EPA reassessed the potential risks to non-target plants in its analysis in the dicamba RED, concluding that no specific additional drift m itigations were needed to support the continued registration of all dicamba uses (U.S. EPA, 2009). Since the proposed use pattern for dicamba

on MON 87708 is consistent with us e patterns evaluated and deemed eligible for reregistration in the dicamba RED, it is reasonable to conc lude that dicamba use on MON 87708 meets the

FIFRA standards related to drift and offsite move ment. Thus, when herbicides are applied in accordance with the application in structions on the FIFRA label, offsite impacts can be avoided.Monsanto Company 10-SY-210U 696 of 721 

### The use of dicamba on MON 87708 does not pose any greater risk to non-ta rget vegetation over existing dicamba agricultural uses approved by EP A. Nevertheless, Monsanto has already taken additional steps to manage dicamba offsite move ment even though EPA stated in the RED that no specific drift mitigation measures were needed. In the pending application to EPA, Monsanto requested the use of dicamba on MON 87708 on the low volatility DGA salt formulation (U.S. EPA Reg. No. 524-582) and limited dicamba appli cations to ground application equipment. Monsanto also plans to further address the sp ecific use of dicamba on MON 87708 with US EPA, to evaluate whether additional measures ma y be appropriate to addr ess potential drift and

offsite movement.

In addition, Monsanto will implement a robust st ewardship program that will include a strong emphasis on grower and applicat or training by working with Amer ican Association of Pesticide Safety Educators (AAPSE) and othe r stakeholders in applicator tr aining to further facilitate ontarget applications of dicamba. There is an extensive pesticide education system within the United States as evidenced by the existence of the AAPSE. Members of this organization train and certify 500,000 applicators in ag riculture and other pesticide us e areas, working in land-grant university cooperative extension services and tribal , state, trust, territory, and federal agencies across the nation. Furthermore, EPA actively suppor ts pesticide applicator training by funding the pesticide safety education program (PSEP). Sin ce 1975, EPA has had an interagency agreement with USDA to distribute funds to th e state cooperative extension service for the purpose of training pest icide applicators.

M.5.6.2. No-Till Practices

Conservation (no-till and mini mum-till) production practices are used on approximately 40% of the U.S. soybean acres (CTIC, 2007). The increase in the number of resistant weed biotypes to soybean herbicides, including gl yphosate, could adversely affe ct the sustainability of conservation production acres. The availabili ty of MON 87708 and the flexibility to use dicamba for both preemergent and postemergent control of resistant and hard-to-control broadleaf weeds will provide a tool to help maintain this important agricultural practice. Furthermore, the use of dicamba on MON 877 08 integrated into the Roundup Ready soybean system will help to delay the development of new re sistant weed biotypes as a result of a second mode-of-action in the weed management system.

The benefits of conservation tillage are well kno wn and demonstrated, and include soil and water conservation, improved water quality and a re duced carbon footprint (CTIC, 2011). No-till production is the practice of establishing an agri cultural seed bed and controlling weeds without mechanically tilling the so il. Instead, the only tillag e 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 pr oduction reduces soil erosion a nd the use of petroleum-based fuels for tractors. The practice has been show n to minimize surface water runoff and to improve soil quality by increasing the soil organic matter th at helps bind soil nutr ients and prevent their loss to runoff, erosion and leaching (Arriaga an d Balkcom, 2005; Leep et al., 2003). Less soil erosion into surface waters would positively impact stream dynamics (McVay et al., 2005; Reicosky, 2008).Monsanto Company 10-SY-210U 697 of 721 

### No-till agriculture can provide be nefits to water bodies as well. No-till practices reduce soil erosion to surface water bodies, decreasing the am ount of sediment in rivers and streams. Sedimentation increases the turbidity (cloudi ness) of surface water bodies, reducing light penetration, impairing photosynthesis and alte ring oxygen levels, which cause a reduction of food sources for some aquatic organisms. Sedi ment 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).

No-till practices have also been shown to reduce the carbon footprint from agricultural production by decreasing carbon emi ssions due to fewer cultivatio n activities. In addition, deposits of crop residue on the soil surface are converted to or ganic matter and humus through carbon cycling, preserving soil carbon reserves and increasing fertilit y and nutrient cycling (Reicosky, 2008).

M.5.6.3. Soil Microorganisms

Results of standardized tests with dicamba a nd dicamba formulations performed for submission to regulatory agencies indi cate no long-term effects on functional processes of soil microorganisms (carbon respiration and nitrogen tr ansformation) when exposed to dicamba at rates up to 3.8 pound a.e./A. These levels ex ceed the current maximum single and annual

dicamba use rates of one lb a.e./A and two lbs a.e./A, respectivel y (European Commission, 2007b).

M.5.6.4. Impact on Beneficial Arthropods

Dicamba is a broadleaf herbicide that controls a range of dicot plants in the presence of monocots and other crops. Its effects primarily ar ise from direct applications in the field to reduce the plant mass and reproductive potential of su sceptible weeds. Indirect effects outside of

the treated areas as a result of spray drift would be significantly less since the amount of dicamba that can drift offsite at the field margins is typi cally less than 1% of th e amount directly applied to the field (U.S. EPA,2005c). Dicamba has lit tle or no effect on monocot plants and is considered by EPA to be pract ically non-toxic to insects, including honeybees and other arthropods (U.S. EPA, 2005c). Most available studies on the effects of herbicide treatment on arthropod communities are done with direct applications, and poten tial indirect effects from

dicamba outside of the treated ar ea are extrapolations from the direct effects at high exposure levels. Within the application area, the expect ed effects would be re duction in food sources (foliage, nectar, seeds) and cover for insects due to the intended weed control in the crop provided by dicamba application.

Indirect effects of dicamba and related herbicides have been studied as potential effects on community structure, impacts to butterfly comm unities and other insect communities in areas adjacent to agricultural fields, and in the treate d fields themselves. Fuhlendorf et al. (2002) studied the impact of direct application of selective broadleaf herbicides on forb 78 

### community

78 Forbs are herbaceous flowering plants that are not graminoids (grasses, sedge s and rushes). Examples of forbs are clover, sunflower and milkweed.Monsanto Company 10-SY-210U 698 of 721 

### richness and arthropod composition in a tall gra ss prairie. The authors reached the following conclusions: “This study demonstrates that the application of a select ive herbicide can reduce the abundance and species ri chness of forbs that may serve as critical hosts to some arthropod species. However, analysis of these tallgrass communities did not yield significant differences in arthropod abundance or richness betw een grasslands treated with an herbicide and grasslands not treated with an herbicide... These results suggest that a single application of herbicide in tallgrass prairies of North America, although reducing the abundance and richness of forbs, neither eradicates the forb growth form nor substantially changes fo rb composition in the grassland community. Hence, neither does the arthropod community change. Relationships between forb cover and the arthropod community suggest that single app lications of broadleaf herbicides may only have an eff ect on arthropods when forb a bundance is dramatically reduced (below 5% cover). This study is incapable of concluding that herbicides targeted at forbs do not always influence the forb and ar thropod communities, becau se herbicides are applied typically to native grasslands on southern Gr eat Plains at 3–5 y ear intervals (Vallen tine, 1989; New, 1997; Hanselka et al., 1990).” (F uhlendorf et al., 2002).

Longley and Sotherton (1997) also reviewed the li terature regarding eff ects of pesticides on butterfly populations in ag ricultural areas. Ex cerpts from this review follow: “The main impact on butterfly populations is acciden tal drift or intenti onal direct spraying of broad-spectrum

herbicides onto field boundaries to reduce certain pe rnicious perennial weeds (Marshal and Smith, 1987). However, the production of annual gr ass species is encouraged by these herbicide applications leading to a species-poor commun ity (Smith and Macdonald, 1992). Consequently, herbicides may affect bu tterfly populations through the direct mo rtality of their larval food plants and flowering-nectar sources such as bramble ( Rubus fruticosus ), mayweed ( Matricaria perforata ), marjoram ( Origanum vulgare) and thistle-like Compositae . The removal of host plants or premature senescence of plant parts can lead to larval starvation or the exclusion of less-fit adults (Courtney, 1981)”. Later in the review, Longley and Sotherton indicate that species vulnerability depends on larval food plants ut ilized by the species. “With the various factors influencing the extent of spray deposition on plants, the de gree of pesticide (herbicide) exposure encountered by different butterfly fam ilies, and hence their vulnerability, will depend upon their choice of larval f ood plants. Members of the Hesperiidae and Satyridae families feed mainly on a range of coarse, densely tufted perennial grass species which are commonly found growing within many field boundaries”. Applicati on of broadleaf selectiv e herbicides such as dicamba that allow for selective growth of gr asses would increase the food source for these species. Indirect exposures from spray drift at greatly reduced rates would resu lt in substantially less impact to plant and arthropod communitie s surrounding the agricultural fields since exposures at drift rates are usually insufficien t to reduce the survivability and reproductive potential of plants (Al-Khatib and Tamh ane, 1999; Auch and Arnold, 1978; Behrens and Lueschen, 1979; Andersen et al., 2004). Since cont rol of vegetation other than soybeans within the field is a component of soybean production, a nd since dicamba is antic ipated to have little effect on forbs outside the field, the use of di camba on MON 87708 is anticipated to have little or no effects on arthropod communities that are dependent on plants for their survival.Monsanto Company 10-SY-210U 699 of 721 

### References for Appendix M

Al-Khatib, K. and Tamhane, A. 1999. Dry pea ( pisum sativum L.) response to low rates of foliar- and soil- applied sulfonylurea and growth regul ator herbicides. Weed Technology, 13:753-758.

Andersen, S.M., Clay, S.A., Matthees, D., a nd Wrage, L.J. 2004. Soybean foliage residues of dicamba and 2,4-D and correlation to application rates a nd yield. Agronomy Journal, 96:750-760.

Arriaga, F.J. and K.S. Balkcom. 2005. Bene fits of conservation tillage on rainfall and water management. 2005 Georgia Water Resour ces Conference, University of Georgia, Athens, Georgia.

Auch D.E. and Arnold W. E. 1978. Dicamba use and injury on soybeans ( Glycine max )

in South Dakota. Weed Science 26:471-475.

Ashton F.M. and Crafts A.S. 1981. Mode of action of herbicides. Second Edition. John Wiley & Sons, New York, New York.

Behrens, M.R., and W.E. Lueschen. 1979. Dicamba Volatility. Weed Science 27:5 486-

493.

Brown, L. R., Robinson, D. E., Nurse, R. E ., Swanton, C. J., and Sikkema, P. H. 2009. Soybean response to simulated dicamba/diflufenzopyr drift followed by postemergence herbicides. Crop Protection 28:539-542.

CTIC. 2007. Conservaton Tillage Information Ce nter. http://www.ctic.purdue.edu/
Accessed June 1, 2010

.

CTIC. 2011. Top 10 conservation tillage benefits : Conservation for agriculture's future. Conservation Technology Information Center, West Lafayette, Indiana. http://www.ctic.purdue.edu/resourcedisplay/293/ Accessed September 21, 2011

.

Durkin, P. and S. Bosch. 2004. Dicamba - Huma n health and ecological risk assessment - Final report. SERA TR 04-43-17- 06d. U.S. Department of Agri culture, Forest Service, Washington, D.C. http://www.fs.fed.us/f oresthealth/pesticide/ pdfs/112404_dicamba.pdf Accessed May 26, 2010

.

European Commission. 2007a. Draft Asse ssment Report, Volume 3, Annex B6: Toxicology and metabolism. Council Dire ctive 91/414/EEC, EU Review Programme.

European Commission. 2007b. Draft Asse ssment Report, Volume 3, Annex B9: Ecotoxicology. Council Directive 91/414/EEC, EU Review Programme.

European Commission. 2008. Review report for the active substance dicamba, Finalised in the Standing Committee on the Food Chain and Animal Health at its meeting on 14

March 2008 in view of the inclusion of di camba in Annex I of Directive 91/414/EEC.Monsanto Company 10-SY-210U 700 of 721 

### European Commission, Health and Consumer Protection Directorate-General, Paris,

France.

Felsot A.S., Unsworth J.B., Lindrs J.B.H.J ., Roberts G., Rautman D., Harris C., Carazo

E. 2010. J Environ Sci Hea. B

. 45 : 889-911.

Fletcher, J.S., J.E. Nellessen, and T.G. Pfleeg er. 1994. Literature review and evaluation of the EPA food-chain ( Kenaga ) nomogram, an instrument for estimating pesticide residues on plants. Environmental Toxi cology and Chemistry 13(9): 1383-1391.

Fuhlendorf, S.D., D.M. Engle, D.C. Ar nold and T.G. Bidwell. 2002. Influence of herbicide application on forb and arthropod communities of North American tallgrass prairies. Agriculture Ecosystems & Environment 92:251-259.

Hill, P.R. and J.V. Mannering. 1995. Conserva tion tillage and water quality. Purdue University Cooperative Extension Se rvice, West Lafayette, Indiana. http://www.ces.purdue.edu/extmedia/WQ/WQ-20.html Accessed July 2, 2009

.

Krueger, J.P., Butz, R.G. and Cork, D.J. 1991. Aerobic and anaer obic soil metabolism of dicamba. Journal of Agricultu ral and Food Chemistry 39:995-999.

Leep, R., D. Undersander, P. Peterson, D. -H. Min, T. Harrigan and J. Grigar. 2003. Steps to successful no-till establishment of forage s. Extension Bulletin E-2880. Michigan State University, University of Wisconsin, Univer sity of Minnesota, and Natural Resources Conservation Service, East Lansing, Michigan.

Longley, M. and N.W. Sotherton. 1997. Factor s determining the effects of pesticides upon butterflies inhabiting arable farmland. Agriculture Ecosystems & Environment

61:1-12.

McVay, K.A., D.L. Levlin and J. Neel. 2005. Effects of conservation practices on water quality: Sediment. MF2682. Kansas State Univer sity Agricultural Experiment Station and Cooperative Extension Service, Manhattan, Kansas.

PMRA. 2007. Re-evaluation – dicamba. The Use of Dicamba in Agricultural and Industrial Sites. Pest Management Regul atory Authority, Health Canada. Ottawa,

Canada.

PMRA. 2008. Re-evaluation – dicamba. Pest Management Regulatory Authority, Health Canada. Ottawa, Canada.

Reicosky, D.C. 2008. Carbon sequestration and environmental benefits from no-till systems. Pages 43-58 in No-Till Farming System s. T. Goddard (ed.). World Association of Soil and Water Conservation, Bangkok, Thailand.

SDTF. 1997. A summary of ground a pplication studies. Spray Drif t Task Force, St. Louis, Missouri. http://www.agdrift.com/PDF_FILES/Ground.pdf Accessed August 10, 2010
and November 26, 2011

.Monsanto Company 10-SY-210U 701 of 721 

### U.S. EPA. 1992. Pesticides in Groundwat er Database: A Comp ilation of Monitoring Studies: 1971-1991. National Summary (EPA 734-12-92-001). Office of Prevention

Pesticides and Toxic Substances. U.S. Environmental Protection Agency, Washington,

D.C.

U.S. EPA. 1996. Residue Chemistry Te st Guidelines, OPPTS 860.1000 Background. Office of Prevention, Pesticides and Toxic S ubstances, U.S. Envir onmental Protection Agency, Washington, DC.

U.S. EPA. 2003. No Effect Determination for Pa cific Anadromas Salmonoids (Memorandum, L. Turner, Environmental Fi eld Branch, Field a nd External Affairs Division). Field and Ex ternal Affairs Branch. Division, Office of Prevention, Pesticides and Toxic Substances, U.S. Environmenta l Protection Agency, Washington, DC. http://www.epa.gov/espp/litstatus/effects/ne-dicamba.pdf.

U.S. EPA. 2004. Overview of the ecological risk assessment process in the Office of Pesticide Programs. U.S. Environmental Pr otection Agency, Endangered and Threatened Species Effects Determinations, Washington, D.C.

U.S. EPA. 2005a. Dicamba. Residue Chemis try Considerations for the Reregistration Eligibility Decision (RED) Do cument. Summary of Analytic al Chemistry and Residue Data. DP Number D317703 (Memorandum, C. Olinger and W. Phang, September 13). Health Effects Division, Offi ce of Prevention, Pesticides and Toxic Substances, U.S. Environmental Protection Agency, Washington, DC.

U.S. EPA. 2005b. Dicamba: HED Chapter of the Reregistration Eligibility Decision Document, Phase 1. PC Code 029801; DP Barcode: D317720 (Memorandum, C. Olinger and Y. Yang, September 13). Health Effect s Division, Office of Prevention, Pesticides and Toxic Substances, U.S. Environmen tal Protection Agency, Washington, DC.

U.S. EPA. 2005c. Environmental Fate and Ecological Risk Assessment for the Reregistration of Dicamba and Dicamba Sodium, Potassium, Diglycoamine, Dimethylamine and Isopropylamine Salts (M emorandum, W. Erickson, I. Abdel-Saheb and S. Borges, August 31). Environmental Fa te Effects Division, Office of Prevention, Pesticides and Toxic Substances, U.S. E nvironmental Protection Agency, Washington,

DC.

U.S. EPA. 2005d. Dicamba: Occupationa l and Residential Exposure and Risk Assessment for the Reregistration Eligibili ty Decision (RED) Document. PC Code 029801, DP Barcode D317701 (Memorandum, T.C. Do le, September 6). Health Effects Division, Office of Prevention, Pesticides a nd Toxic Substances, U.S. Environmental Protection Agency, Washington, DC.

U.S. EPA. 2008a. Dicamba Pesticide Tole rance. U.S. Environmental Protection Agency, Washington, D.C. Vol. 73, No. 64.Monsanto Company 10-SY-210U 702 of 721 

### U.S. EPA. 2008b. Aldicarb, Ametryn, 2,4-DB, Dicamba, Dimethipin, Disulfoton, Diuron, et al.; Proposed Tolerance Actions. U.S. Environmental Protection Agency, Washington, D.C. Federal Register Vol. 73, No. 108.

U.S. EPA. 2008c. Aldicarb, Ametryn, 2,4-DB, Dicamba, Dimethipin, Disulfoton, Diuron, et al.; Tolerance Actions. U.S. Environmental Protection Agency, Washington, D.C. Vol. 73, No. 186.

U.S. EPA. 2009. Reregistration Eligibility Decision (RED) for Dicamba and Associated

Salts. Case No. 0065. U.S. Environmental Protection Agency Office of Prevention, Pesticides and Toxic Substances, Speci al Review and Reregistration Division, Washington, D.C.

U.S. EPA. 2010. Registration Review: C onventional Cases Schedule: 2010-2013. U.S. Environmental Protection Agenc y, Office of Prevention, Pesticides and Toxic Substances. June 7, 2010. http://www.epa.go v/oppsrrd1/registration_review/2010-13- conventional.pdf Accessed June 12, 2010

.

U.S. EPA. 2011a. NAFTA ma ximum residue levels (MRL) calculator. U.S. Environmental Protection Ag ency, Washington, D.C. http://www.epa.gov/oppfea d1/international/n aftatwg/mrl-calculator.html Accessed February 22 , 2012.

U.S. EPA. 2011b. 2011 Edition of the Drinking Water Standards and Health Advisories. U.S. Environmental Protection Agenc y, Office of Water. EPA 820-R-11-002. http://water.epa.gov/action/advisories /drinking/upload/dwstandards2011.pdfMonsanto Company 10-SY-210U 703 of 721 

### Appendix N: Overview of Evaluation of Potential Exposure and Biological Effects on Endangered Species for Dicamba Use in MON 87708

N.1. Introduction

This overview report provides a summary of a multi step approach that has been utilized to evaluate the potential effects on federally listed threatened and endangered (“listed”) species from the use of the herbicide dicamba on dicamba-tolerant (DT) soybean. This analysis follows the procedures described in the Overview of the Ecological Risk

Assessment Process in the Office of Pesticid e Programs, U.S. Environmental Protection Agency (U.S. EPA), Endangered and Threat ened Species Effects Determinations, published in 2004 (U.S. EPA, 2004), as well as methods utilized in more recent threatened and endangered species effect s determinations conducted by the U.S. Environmental Protection Agency (USEPA) fo r atrazine (U.S. EPA, 2007), and for a number of active ingredients in assessing their potential effects on the California red legged frog (U.S. EPA, 2008a). A four step pro cess is described in more detail below.

N.2. Endangered Species Exposu re and Effects Analysis

N.2.1. Screening Level Analysis

An initial analysis of the potential for expos ure and effects to all taxa from dicamba use in dicamba-tolerant soybean based on the us e pattern pending at EPA was conducted (Wright et al., 2011) using the EPA determin istic risk quotient approach (U.S. EPA,
2004). 79 

### The use pattern for dicamba utilized in this analysis was a pre-emergence application at a rate of 1.0 lb dicamba acid equivalents pe r acre (a.e/A) followed by two post-emergence applications each at a rate of 0.5 lb a.e./A with a 6- day interval between the pre-emergence application and the firs t post-emergence application, and a 6-day interval between the two post- emergence applications, with all applications being made using ground application equipment. 80 

### The 6-day application intervals utilized in this analysis are expected to be shorter than the intervals actually used in practice, since a grower is expected to wait at least 7 da ys before deciding to make a subsequent application in order to allow evidence of dicamba efficacy to develop.

Initial exposure estimates usi ng the above described use patte rn were based on standard EPA exposure models and default assumptions ; toxicity effects endpoints were taken

from the EPA Environmental Fate and Effect s Division (EFED) Science Chapter for the Reregistration Eligibility Decision for dicam ba (U.S. EPA, 2005) or from more recent EPA guideline studies conducted by BASF unde r an EPA data call-in (Porch et al.,

79 This approach calculates a risk quotient (RQ) by dividing the Estimated Environmental Concentration

(EEC) by the appropriate toxicity endpoint, and then compares that value with the appropriate Level of Concern (LOC). The LOC is established by EPA policy as the criteria used by EPA in comparison to thecalculated risk quotient (RQ) to assess the potential for a pesticide use to cause adverse effects to nontarget organisms.

80 The dicamba label for DT soybeans will not permit aerial application.Monsanto Company 10-SY-210U 704 of 721 

### 2009 81 

### ), when these endpoints were more consis tent with current EPA guidelines. The conclusion from this initial analysis, based on risk quotients (RQs) being less than the EPA Levels of Concern (LOCs), is that di camba use on dicamba-tolerant soybean would not affect threatened and endange red species in the following taxa : 82



###  Fish, aquatic phase amphibians, and aqua tic invertebrates (acute or chronic exposure)

 Birds, terrestrial-phase amphibian s, and reptiles (chronic exposure)

 Mammals (acute exposure)

 Insects, aquatic vascular plants and monocotyledonous terrestrial plant species 83,84



### Although the RQ for aquatic nonvascular plants exceeded the LOC additional analysis

was not conducted because there are no fede rally listed nonvascular aquatic plants.

These conclusions for aquatic plants and animals, birds, mammals and insects are consistent with the risk conclusions presen ted in the EFED science chapter for dicamba reregistration (U.S. EPA, 2005; 2006).

The conclusion for monocots is based on more recent nontarget plant studies with a typical dicamba end use product that were required by EPA during the reregistration process (Porch et al., 2009). 85 

### Using the no-observed-eff ect endpoints from these 2009

studies and the EPA model TerrP lant to calculate exposure a nd risk quotients, the RQs for monocots were below the LOC, and thus result in a conclusion of no effect. The levels of concern considered by EPA for thr eatened and endangered (listed) species risk assessments are given in Table N-1.

81 New studies were required by EPA because previous studies did not meet current regulations requiring

the use of formulated product for these studies. This new testing resulted in a more conservative endpoint for the vegetative vigor study and confirmed field observations of relative sensitivity for the seedling emergence study.

82 “If assumptions associated with the screening leve l action area result in RQs that are below the listed

species LOCs, a “no effect” determination conclusion is made with respect to listed species in that taxa, and no further refinement of the action area is necessary.” EFED Reregistration Chapter for Dicamba/Dicamba Salts,.

83 According to EPA methodology (U.S. EPA, 2004), risk to non-target plants is assessed only outside the

application area.

84 Exposure for insects and plants is not divided into acute and chronic durations.

85 The new study was conducted under the OPPTS 850.4225 draft guideline which is very similar to the

current OECD nontarget plant guidelin e, and the results are considered to be more representative of effects that would be expected to occur in the field because of an improved study design.Monsanto Company 10-SY-210U 705 of 721 

### Table N-1. EPA Levels of Concern for Threatened and Endangered Species

Risk Presumption Calculation for Risk Quotient Level of Concern

Birds:

Acute Risk EEC/LC 50 

### (application of a liquid)

or LD 50 

### /ft 2 

### or LD 50 

### /day (application as a granule, bait, or treated seed) 0.1

Chronic Risk EEC/NOAEC 1.0

Wild Mammals

Acute Risk EEC/LC 50 

### (application of a liquid)

or LD 50 

### /ft 2 

### or LD 50 

### /day (application of a granule, bait, or treated seed) 0.1

Chronic Risk EEC/NOAEC 1.0

Aquatic Animals

Acute Risk EEC/LC 50 

### or EC 50 

### 0.05

Chronic Risk EEC/MATC or NOAEC 1.0

Terrestrial and Semi-Aquatic Plants

Acute Risk EEC/EC 05 

### or NOAEC 1.0

Aquatic Plants

Acute Risk EEC/EC 05 

### or NOAEC 1.0

Source: U.S. EPA, 2004



### N.2.2. Refinement to the Screening Level An alysis - Use of Dicamba-specific Foliar

Residue Values

Consistent with EPA guidance found in the Overview Document (U.S. EPA, 2004), if screening-level assessments do not result in a “no effect” determination, EPA does not then conclude that an herbicide “may aff ect” threatened and endangered species; rather,

more refined assessments must be conducted to ascertain if any eff ects are expected to occur. Accordingly, for threatened and e ndangered animal species and the exposure durations for which the risk quotient exceeded the LOC in the screening level analysis (acute exposure for birds, amphibians and re ptiles, and chronic exposure for mammals), refined exposure estimates were develope d utilizing measured dicamba residues on pasture grasses and soybean forage 86 (as representative residues for short grass and broadleaf foliage 87 components 

### of animal diets) (Wright et al., 2011). These data were from residue

86 Residue values from these studies were converted to values expressed as parts per million per pound

dicamba acid from residues values for application rates of 0.5, 1.0, and 2.0 lb a.e./A for grasses and 0.5 and 1.0 lb a.e./A for soybeans.

87 EPA assumes that broadleaf foliage and small insects have the same residue values, so these dicambaspecific values were used both for broadleaf foliage and small insects.Monsanto Company 10-SY-210U 706 of 721 

### studies conducted under Good Laboratory Pr actices by dicamba registrants and by Monsanto, respectively, and are used to provide dicamba-specific residues for components of animal diets, instead of usi ng the EPA default residue values based on the Kenega nomogram (Hoerger and Kenega, 1972) as revised by Fletcher (1994). Both the soybean residue study and one of the grass re sidue studies, as well as a wheat residue study, included sites with several sampling poi nts for forage which occurred soon after application so that the rate of decline of dicamba residues on soybean, grass, and wheat foliage could be determined, and the time requi red for dissipation of fifty percent of the residues (DT50) calculated. 88 

### The Overview Document (U.S. EPA, , 2004) 89 

### ) indicates that chemical-specific foliar dissipation values can be used for multiple application exposure modeling for wildlife; accordingly, as a c onservative assumption, the longest of the representative dicamba-specific foliar DT50 values for these three crops (5.63 days, for pasture grass) was used in the calculation of the dicamba-specific residues for chronic exposure. For grass and broadleaf diet ary items, both mean and upper bound residue

values 90 

### were considered in the evaluation; these levels are suitable to estimate realistic (mean) and worst case (upper bound) levels of exposure. A compar ison of the default upper bound residue values and dicamba-specifi c upper bound residue values is given in

Table N-2.

88 For the pasture grass and wheat residue studies, there were over 50 treatments per study for which a DT 50

value could be calculated. With such a large number of data points, the 90 th percentile upper confidence

limit of the mean DT 50 value was selected as an appropriate DT 50 value to use for calculation of residue decline for these crops. For the soybean residue study with only six treatments available to calculate DT 50 values, the maximum value was considered repres entative of residue decline in soybean forage.

89 Overview Document (U.S. EPA, 2004),

90 A 90 th centile value was used for pasture grass and th e maximum residue value was used for soybean forage due to the difference in numbers of residue va lues available to calculate the upper bound residue.Monsanto Company 10-SY-210U 707 of 721 

### Table N-2. Comparison of Default Ken aga Residues and Dicamba-specific Residues

in Food-Items

Maximum dicamba residues in fo od items (ppm a.e.) based on a 1 lb a.e./A pre-emergence application followed by two sequential post-emergence 0.5 lb a.e./A applications

Short

Grass Tall

Grass Broadleaf

Plants/

Small Insects Fruits/Pods/Seeds/ Large Insects

Using upper bound Kenaga residues & default foliar half-life (35 days)

Day 0 (after 1 st


### application) 240.0 110.0 135.0 15.0

Maximum residue a 

### (after

3 rd 

### application) 415.8 190.6 233.9 26.0

Using Dicamba-specific residues & foliar half-life (5.63 days)

Day 0 (after 1 st


### application) 131.0 60.0 103.9 15.0

Maximum residue a 

### (after 1st application) 131.0 60.0 103.9 15.0

a Using the default foliar half-life, the highest residue occurs immediately after the third application. Using the dicamba-specific half-life, the highest residue occurs immediately after the first application



### These refined residue values for dietary items were cons idered when using the EPA model T-REX (v1.4.1) for the calculation of ri sk quotients and subse quent comparison to LOCs for acute exposures to birds, terrestr ial amphibians, and reptiles, and for chronic

exposures to mammals. 91 

### Figure N-1 describes the process for identifying the taxa that had risk quotients exceeding the EPA’s LO C after the screeni ng level analysis.

91 The T-REX model is not designed to allow modification of the dietary food item residues. Thus, the
dicamba-specific residues reported in Table N-2 were considered in the risk quotient calculations by
applying a correction factor to th e estimated environmental concentrations (EEC) values computed using
the standard T-REX model for a single 1-lb/A application.Monsanto Company 10-SY-210U 708 of 721 

### Figure N-1. Identification of Taxa Ex ceeding Endangered Species Levels of

Concern

a The basis for this conclusion is described in U.S. EPA, 2004. Overview of the Ecological Risk

Assessment Process in the Office of Pesticide Pr ograms, U.S. Environmental Protection Agency, Endangered and Threatened Species Effects Determinations



### Risk quotients for chronic exposure to ma mmals calculated using upper bound residues slightly exceed the LOC for small and medi um-sized mammals consuming short grass; however, utilizing mean residue values as a further refinement, risk quotients are all below the LOC (Table N-3) leading to a conc lusion of no effect on the species. This is consistent with the finding in the EFED Sc ience Chapter for dicamba (U.S. EPA, 2005) and the revised RED (U.S. EPA, 2006) for a si ngle application rate of 1.0 lb a.e./A. Because there will be at most only three ap plications per year and dicamba dissipates rapidly, an individual small or medium-s ized mammal will not chronically consume foliage containing maximum residue levels from dicamba-treated fields. Therefore, Exposure Estimate:

Worst Case

Estimated Environmental Concentration

(EEC) Using Standard EPA Models Or

Anal y sis Methods Toxicity Endpoint (TE):

Lowest Acute Or Chronic Value Meeting EPA Quality Standards

RQ > Level Of Concern (LOC)?

Refine EEC based on

experimental data Determine Risk Quotient (RQ = EEC/TE)

Yes For Each

Taxon

No Direct Effect Expected on the Taxa a No

RQ > Level Of Concern (LOC)?

No Direct Effect

Ex p ected on the Taxa a

No

Further Analysis YesMonsanto Company 10-SY-210U 709 of 721 

### reliance on dicamba-specific Day 0 mean residue values is also a conservative dietary exposure estimate for chronic exposure that is expressly countenanced by EPA. 92



### Table N-3. Risk Quotients for Chronic Exposure a for Mammalian Species Based on Dicamba-Specific Residue Values and DT 50 

### .

Diet Mammal Size (g) Risk Quotient using Upper Bound

Residues a,b 

### Risk Quotient using

Mean Residues

Short Grass 15 1.26 0.75

35 1.08 0.64

1000 0.58 0.34

Tall Grass 15 0.58 0.32

35 0.49 0.27

1000 0.27 0.15

Broadleaf Foliage /

Small Insects 15 1.00 0.75

35 0.86 0.64

1000 0.46 0.34

Fruits/Pods/Seeds/Large

Insects 15 0.14 0.07

35 0.12 0.06

1000 0.07 0.03

Grain 15 0.032 0.015

35 0.027 0.013

1000 0.015 0.007

a Dicamba-specific refined residue values based on a 1 lb a.e./A application rate were 131, 60.0, 103.9, and

15.0 mg a.e./kg diet, respectively, for upper bound values for short grass; tall grass; broadleaf foliage / small insects; and fruits/ pods/ seeds/ large insects, and 77.9, 33.0, 77.7, and 7.0 mg a.e./kg, respectively, for mean residues. The dicamba-sp ecific residue decline half-life (DT 50 ) of 5.63 days instead of the EPA default value of 35 days was also used in determining the peak residue value resulting from the three sequential applications.

b Numbers with Bold font indicat e the RQ exceeds the LOC (1.0).



### With respect to birds, analyses were performed using measured dicamba residue values (Table N-2) for RQ calculations for acute exposure. The following could be excluded from concern (and, therefore, no effects would be expected to occur): birds of all sizes consuming only grain or fru its/pods/seeds/large insects, and large birds consuming broadleaf foliage/small insects or tall grass. Small and medium-sized birds consuming broadleaf foliage/small insects or grasses and large birds consuming short grass required further analysis. RQ values are presented in Table N-4. Because birds are the surrogate

92 Overview Document (USEPA, 2004)Monsanto Company 10-SY-210U 710 of 721 

### species for terrestrial phase amphibians and re ptiles, further analys is was also conducted to ensure that amphibians and reptiles could be excluded from concern for adverse effect.

Table N-4. Risk Quotients for Acute Ex posure to Birds Using Measured Dicamba

Residue and Residue Decline Values

Diet Acute RQs: (Dose-based EEC/adjusted LD50) a



### Upper Bound Residues

20 g

Small 100 g

Medium 1000 g Large

Short grass 0.79 0.35 0.11

Tall grass 0.36 0.16 0.05

Broadleaf plants/small

insects 0.63 0.28 0.09

Fruits/pods/seeds/large

insects 0.09 0.04 0.01

Granivore 0.02 0.01 0.003

a Bold numbers indicate the RQ exceeds the LOC (0.1).



### N.2.3. Refined Analysis Considering Species County-Level Locations

Certain sizes of threatened and endangered bi rds, amphibians, reptiles, and mammals had risk quotients exceeding the LOC as a result of the use of dicamba on dicamba-tolerant soybean based on a default exposure analysis or an analysis using refined residues. Consistent with guidance set forth in th e EPA Overview Document, a more detailed

evaluation of the locations of these threatened and endangered species relative to potential areas of soybean production was undertaken.

First, the co occurrence of threatened and endangered species of these remaining

taxa 93 

### and the production of soybeans was determ ined at the county level. Listing status 94

, 

### , species habitat and proximitydata to soybean production at the county level were evaluated for these identified species to de termine which species in which counties can

be excluded from further evaluation and which re quire further evaluation. This process is referred to as the “county-leve l analysis” (Frank and Kemma n, 2012) and is discussed in

more detail in Section N.3 below.

Next, the Overview Document provides that – for those threatened and endangered species (avian, reptilian, amphibian (terre strial-phase), and mammalian) that require further evaluation – each species be consider ed individually to de termine whether it can

93 For completeness, all threatened and endangered avian, amphibian, reptilian, and mammalian species

were included in the county-level analysis.

94 Listing status refers to whether the species is classi fied as threatened, endanger ed, no longer considered

threatened or endanger ed (delisted), etc.Monsanto Company 10-SY-210U 711 of 721 

### be excluded from concern for potential effects based on body we ight or dietary considerations. Accordingly, for these animal species, refined risk quotient calculations were performed considering individual speci es body weight, food type and food intake rate, as described in Section N.4 and in Sc huler and Leopold (2012), and consistent with EPA guidance found in the Overview Document (U.S. EPA, 2004). Based on these refined county-level analyses, as discussed below, animal species were excluded from concern, and, therefore, would not be affected by dicamba use on DT-soybean.

N.3. County-level Analysis: Co-occurrence of Listed Species in Crop Production

Areas

The procedures used in the county-level analysis to identify counties containing threatened or endangered species that requir e further evaluation are depicted as a flow chart in Figure N-2 and are describe d in detail in Frank and Kemman ( 2012). 95 

### . The U.S. counties where soybean production was reporte d were identified using available data from the U.S. Census of Agriculture. Census data from 1997, 2002 and 2007 were utilized to identify these c ounties. This information was supplemented with soybean production data available from the Californi a Department of Pesticide Regulation. Counties without soybean production, but adj acent to counties with soybean production were also identified. In total, there were 2,728 counties consider ed in this an alysis (2,198 soybean counties, 530 adjacent counties). The counties considered at the beginning of county-level analysis are show n graphically in Figure N-3.

95 Overview Document (US EPA, 2004)Monsanto Company



### Figure N-2. County- Level Analysis Process

a Information from the California Pesticide Use Repor ting databas
herbicide applications to soybean fields in Califor nia.

10-SY-210U 

### Level Analysis Process

Information from the California Pesticide Use Repor ting databas e was also used to identify counties with
herbicide applications to soybean fields in Califor nia.

712 of 721 e was also used to identify counties withMonsanto Company 10-SY-210U 713 of 721 

### Figure N-3. Counties with Soybean Farms and Adjacent Counties

In the identified counties, av ailable county-level presence in formation for threatened or endangered avian, reptilian, amphibian, a nd mammalian species was evaluated, using county-level location information compile d by the FIFRA Endangered Species Task Force (FESTF) in the FESTF Information Management System (IMS). 96 

### Of the 2,728 counties initially considered, there were 2505 c ounties with listed speci es in the taxa of concern and where soybeans are produced (inc luding counties adjacent to counties where soybeans are produced). These counties are depicted graphically in Figure N-4 and contain 140 distinct species in 49 states (all states except Alaska) and the District of Columbia. There were 7,048 species/county co-o ccurrence records cons idered across all animal taxa evaluated. In these counties, e ach species was evaluated with respect to the current listing status, county-level locat ions, species biology, and species habitat requirements, in order to determine whet her exposure to dicamba from use on DTsoybean could potentially result in adverse effects to the species. Some listed species

could be removed from concern for adverse effects based on exclusions that currently

96 The FIFRA (Federal Insecticide, Fungicide, and Rodenticide Act) Endangered Species Task Force

(FESTF) Information Management System 2.7 (IMS) (referred to as the “FESTF IMS”) was developed in order to meet the legal obligations of its member companies to submit data required by EPA/OPP under FIFRA (as described in Pesticide Registration Notice 2000-2) in support of the members’ registration and re-registration actions. The purpose of the IMS is to meet the data requirements in a manner that significantly improves the consistency, quality, availability and use of existing information on threatened and endangered species and pestic ide use. http://www.festf .org/visitors/default.aspMonsanto Company 10-SY-210U 714 of 721 

### exist and are documented. Exclusions that have been employed include change in species listing status, not present due to extirpation 97 

### , habitat not in proximity to agriculture, product properties 98 

### , and species not in proximity to agriculture for other reasons.

For animal species that have been identified in the county-level analysis as requiring further analysis, species-spec ific refinements as describe d in Section N.4 have been

considered. T-HERPS was utilized to calculate risk quotients for amphibians of similar body weight as the California red-legged fr og. Diet informati on was utilized for individual species of birds, reptiles, and mammals to determine if actual diet considerations can justify rem oving a species from concern.

97 An extirpated species is defined as “a species no longer surviving in regions that were once part of its

range” ( http://www.fws.gov/midwest/endangered/glossary/index.html ).

98 A product properties exclusion is based on a quan titative analysis of dicamba properties beyond the

screening level analysis in the EFED Science Chapter. Specifically, this analysis involves assessing use

of measured foliar residues and foliar decline rates follo wing dicamba applications, adjusted to reflect the use pattern for DT-soybeans. The size and diet of the animal species are also considered. (See Sections 2 and 4)Monsanto Company 10-SY-210U 715 of 721 

### Figure N-4. Counties with Soybean Farm s (or Adjacent Counties) with Listed Species in the Taxa of Interest

N.4. Species-specific Risk Quotient Calculations

Consistent with EPA’s Overview Docume nt, species-specific dietary exposure was assessed considering animal body weight and diet (Schuler and Leopold, 2012) for birds and mammals of certain sizes because risk quotients exceeded the level of concern after

the initial refinements described above. A similar assessment was conducted for amphibians and reptiles because birds serve as the surrogate for terrestrial phase amphibians and reptiles. Biol ogical information was gathered from a number of sources, including U.S. Fish and Wildlife Service (USFWS) species recovery plans, primary literature, and other technical sources (e.g., NatureServe 99 )



### Mammals

For mammals, 38 species were examined for the potential for chronic adverse effects based on biological characteristi cs (i.e., dietary preferences and body size). The analysis progressed in a step-wise fashion:

99 NatureServe is a non-profit conservation organization that collects detailed local information on plants,

animals, and ecosystems. NatureServe has a public website available at http://www.natureserve.org.Monsanto Company 10-SY-210U 716 of 721 

### 1) From the baseline analysis, assuming the maximum-use pattern (as described Section N.2.1.) and using the upper bound dicamba-specific foliar residue and foliar half-life values, the LOC for wild mammals was exceeded only for chronic exposures to small and medium sized ma mmals consuming short grasses (Section N.2, Table N-3).

2) Using species-specific information, 27 species were given product property exclusions based on size (e.g. >1kg, larg e-sized mammals) and/or diet (e.g. carnivorous, frugivorous, etc.).

3) The remaining 11 mammals, small and me dium-sized species that consume grasses, were examined using the U.S. EPA T-REX model with species-specific weights used for the model input pa rameters. The predicted EECs were determined based on the upper bound di camba- specific foliar residue and half-life values, as described in Section N.2.2.

4) For 4 of these 11 species, RQs were below the LOC considering upper bound dicamba-specific residues and species-specifi c body weights, For another 3 of the 11 species, examination of the diet indicated that seeds were the primary constituent of the diet with grasses or other items being a very small component, and with this consideration RQs were below the LOC. For the remaining 4 species, considering the rapid rate of dicamba foliar residu e dissipation, chronic RQ’s calculated with upper bound residues exceed the RQ for such a short exposure duration ( i.e. , < 1 day to < 4 days) that it should not be considered a chronic exposure.

Furthermore, all mammalian species can be ex cluded from further consideration based on chronic RQ’s being below the LOC when RQs ar e calculated with initial mean residues. As indicated earlier, initial mean residues are considered more appropriate for assessing chronic risk. Thus, for these 38 species of mammals, chronic exposure to dicamba from application to DT-soybean can be excluded from concern and, therefor e, would not affect these species.

Birds

For birds, of the 43 species evaluated in this refined analysis:

1) Using species-specific information, 24 speci es were excluded from the need for further consideration because their refined T-REX acute RQs were less than the

LOC based on characteristics of diet and body weight considering dicambaspecific residues.

2) The 19 remaining species were excluded fr om the need for further consideration because the upper bound EECs calculated for these species were lower than dosebased No Observed Effect Levels ( NOELs) calculated from acute dietary exposure studies.Monsanto Company 10-SY-210U 717 of 721 

### The potential for acute adverse effects as determ ined in the analysis in Table N-4 is based on an oral gavage, a route of administration that can overestimate the expected exposure compared to more environmentally relevant methods with a slower rate of delivery (e.g., dietary exposure). Therefore, the use of the acu te oral gavage toxicity test to predict the potential for adverse effects to birds from dicamba use on DT-soybean should be considered a screening-level approach that is expected to overestimate the actual potential for effects. A more appropriate method to determine the acute risk to birds from consuming prey or food items containing dica mba residues is to estimate the dose-based effect levels from dietary t oxicity studies (Durda and Preziosi, 2000). This approach is

consistent with the EPA Overview Document (U.S .EPA, 2004) whic h indicates that for avian risk assessments, dietary residues are compared with dietary toxicity endpoints based on dietary concentrations (e.g. LC 50 

### s for acute effects) 100



### . This method was used for the 19 remaining species described above, with the conservative assumption that the avian species under evaluation are passerine sp ecies (highest metabolic/ingestion rates; U.S. EPA, 1993), and the toxic ity endpoint selected from the dietary studies was the NOEL value rather than a LD50 value. Even with the high ingestion rate relative to body weight, these birds cannot ingest enough food to achieve doses that would exceed the NOEL (highest dose tested). Also, it is likely that the birds will not be in soybean fields actively foraging, but rather fora ging in the adjacent habitats (e.g., tree canopy) outside of the fields. Threatened and endangered spec ies are “strongly associated” with their specific habitat type and, therefore, it is im probable that 100% of the bird’s diet will consist of dietary items exposed in the field. Thus, for these 43 species of birds, acute exposure to dicamba from application to DT -soybean can be excluded from concern and, therefore, would not affect these species.

Amphibians and Reptiles

For amphibians and reptiles, a total of 25 sp ecies (10 amphibians and 15 reptiles) were evaluated. The allometric equa tions used in the T-REX mode l assume that the ingestion rates for amphibians and reptiles are equal to thos e of birds, when in fact they are lower. Thus, the dose-based estimates of exposure calculated by T-REX are an over-estimation of exposure for amphibians and reptiles. Ther efore, the risk evaluation for amphibians and reptiles for dicamba from dietary e xposure was refined using the U.S. EPA THERPS model Version 1.0. Follo wing U.S. EPA guidance, the T-HERPS model is to be used only after the RQs exceed the LOC for endangered species using the standard TREX model (U.S. EPA, 2008b). T-HERPS is cu rrently approved for use for terrestrialphase amphibian and reptile species with diets similar to the federally threatened California red-legged frog, sin ce the exposure values are cons idered more representative of potential exposure to he rpetofaunal species.

The T-HERPS model was used to gene rate upper bound EECs resulting from the maximum application scenario using refined re sidue and half-life da ta (Section N.2). The avian acute and chronic toxicity data was used as a surrogate for the amphibian and reptilian species. A default scaling factor of 1.0 was used in the model calculations, as the relationship between body weight and toxici ty has not been examined for amphibian

100 Overview Document (U.S. EPA, 2004) p. 40.Monsanto Company 10-SY-210U 718 of 721 

### and reptile species (U.S. EPA, 2008b). Given the limited ava ilability of morphometric data for many species, similar species were analyzed collectively as a group (e.g., frogs and toads) using the weight data from a si ngle representative spec ies as a proxy for all species within the group. The exposure analys is only considered dicamba residues within prey items typical for each species group and an imal size. With these considerations, the T-HERPS model was used to calculate RQs for frogs, toads, salamanders, lizards and skinks. For these species, the acute, subacu te, and chronic RQ va lues based on refined

residue and half-life values do not exceed the LOC for endangered species. Thus, for frogs, toads, salamanders, lizards, and ski nks, exposure resulting from the dicamba maximum use pattern can be excluded from concern and, therefore, would not affect these species.

For snakes, turtles and tortoises, a speci es-specific exposure analysis was conducted using allometric equations for each speci fic dietary component (U.S. EPA, 1993). Exposure estimates based on refined dicamba-s pecific residue data and residue decline data were compared to acute and chronic av ian toxicity values, and the calculated risk quotients were all determined to be below the level of concern, indicating that these species can be excluded from concern and, therefore, would not be affected.

After considering species-spec ific body sizes and dietary co mponents, all the amphibian and reptilian species evaluated were determined to have risk quotients below the level of concern resulting in the conclusion that dicamba use on DT-soybean will not affect these species.

N.5. Indirect effects on Threatened a nd Endangered Species of Other Taxa resulting from Direct E ffects on Plant Species

To assess the potential for indi rect effects on threatened a nd endangered species, the U.S.

EPA evaluates the risk of direct effect s on non-endangered species from relevant taxonomic groups to make inferences concerni ng the potential for indirect effects upon threatened or endangered species that rely on non-endangered species in that category for critical resources (U.S. EPA, 2004). The RQ values used to assess the potential for indirect effects from dicamba applications to DT-soybean indicate that indirect effects to other taxa resulting from effects on monocot pl ant species from application of dicamba at rates up to 1.0 lb a.e./A would not occur. Furthe r, indirect effects to other taxa as a result of effects on dicot plant species from a combin ation of sheet runoff and spray drift to soil would also not occur (Wright et al., 2011). In addition, utilizing a refined exposure assessment, indirect effects to other taxa resulting from effects on dicot plant species exposed via a combination of channelized runof f and spray drift to soil would not occur (Wright et al., 2011). When RQ values are calculated for all six of the dicot species tested in the vegetative vigor study, RQs exceed the LOC for only two of the six species indicating that habitat and food sources from di cot plants will be present adjacent to areas treated with dicamba (Wright et al., 2011). Furthermore, fi eld studies demonstrate that even very sensitive plants at the edge of a field sprayed with dicamba are not affected to the extent that impacts on habitat would resu lt. Additionally, effects from drift can be further minimized through the use of best ma nagement practices such as drift reducing technologies (e.g., low drift spray nozzles, boom height, deposition aides). ConsideringMonsanto Company 10-SY-210U 719 of 721 

### these factors, indirect effects on threatened and endangered animals resulting from spray drift onto nontarget plants can be excluded from concern.

N.6. Conclusions

In the EFED ecological risk assessment condu cted for dicamba reregistration (U.S. EPA, 2005), using a screening level a ssessment, EPA scientists c oncluded that a “no effect”

determination can be made for all dicamba uses for listed fish, aquatic invertebrates, and vascular aquatic plant species. The potential for adverse effects to listed birds and mammals, non-vascular aquatic plants and terre strial and semi-aquatic plants could not be excluded based on the EPA screening level asse ssment, the initial phase of analysis. However, since there are currently no listed nonvascular aquatic pl ants, this taxon does not require further investigation at this time. Recognizing that screening level analysis is only the first step in evaluating impacts on threatened and endangered species, the EFED Science Chapter (U.S. EPA, 2005) indicates that “additional information on the biology of listed species, the locations of these speci es, and the locations of the use sites ... could be considered along with availa ble information on the fate an d transport properties of the pesticide to determine the extent to which sc reening assumptions regarding an action area apply to a particular listed organism.”

This report summarizes the conclusions of th ree other reports (Wright et al., 2011; Frank and Kemman, 2012, and Schuler and Leopold, 2012) that utilize some of the refinements described by EPA in the previous paragraph to evaluate the potential for adverse effects to listed birds and mammals as well as amphibi ans, reptiles, and terrestrial invertebrates. When the properties of dicamba and species-s pecific information are taken into account, these reports demonstrate that listed birds, mammals, amphibians, reptiles and terrestrial

invertebrates would not be a ffected by dicamba use in DT-s oybean. In addition, based on non-target plant studies c onducted as a requirement of dicamba reregistration, monocotyledonous terrestrial and semi-aquatic pl ants outside the treated area also would not be affected (Wright et al., 2011).Monsanto Company 10-SY-210U 720 of 721 

### References for Appendix N

Durda JL, Preziosi DV. 2000. Data Quality Evaluation of Toxicological Studies Used to Derive Ecotoxicological Benchmarks. Human and Ecological Risk Assessment 6(5):

747-765.

Fletcher JS, Nellessen JE, and Pfleeger TG . 1994. Literature Revi ew and Evaluation of the EPA Food-Chain (Kenaga) Nomogram, an Instrument for Estimating Pesticide

Residues on Plants. Environ. Tox. Chem. 13(9): 1383-1391. DOI:

10.1002/etc.5620130902.

Frank AR, Kemman R. 2011. Informa tion to Support an Endangered Species

Assessment for Dicamba Use in Dicamba-Tole rant Soybeans: A County-Level Analysis

for Animal Taxa. Monsanto T echnical Report MSL-22547. MRID 48644204.

Hoerger F, Kenaga EE. (1972) Pes ticide residues on plants: Correlation of representative data as a basis for estimation of their magnitude in the environment. In: Environmental Quality and Safety: Ch emistry, Toxicology, and Technology , F. Coulston and F. Korte, eds. Georg Thieme Pub lishers, Stuttgart, West Germany, pp. 9-28.

Schuler LJ, Leopold VA, Mortensen SR. 2011. Information to Support an Endangered Species Assessment for Dicamba Use in Dicam ba-Tolerant Soybeans: Refined Analysis

for Terrestrial Animals. Monsanto Technical Report MSL-22546. MRID 48644203.

U.S. EPA. 1993. Wildlife Exposures Handboo k. Office of Health and Environmental

Assessment. Office of Research and Development. EPA/600/R-93/187a.

U.S. EPA. 2004. Overview of the Ecological Risk Assessment Process in the Office of Pesticide Programs, U.S. Environmental Pr otection Agency, Endangered and Threatened Species Effects Determinations. Washington, D.C.

U.S. EPA. 2005. Environmental Fate a nd Ecological Risk Assessment for the Reregistration of Dicamba and Dicamba Sodium, Potassium, Diglycoamine, Dimethylamine and Isopropylamine Salts.

U.S. EPA. 2006. Reregistration Eligibilit y Decision for Dicamba and Associated

Salts.

U.S. EPA. 2007. Risks of atrazine use to federally listed endange red Pallid Sturgeon Scaphirhyncus albus Retrieved January 1, 2008, from http://www.epa.gov/oppfead1/endanger/litstat us/effects/pallid_sturgeon_eff_deter_08-31- 07.pdf

U.S. EPA. 2008a. Risks of Glyphosate Use to the Federally Threatened California Red legged Frog. Retrieved October 8, 2010, from http://www.epa.gov/espp/litstatus/effect s/redleg-frog/glyphosate/determination.pdfMonsanto Company 10-SY-210U 721 of 721 

### U.S. EPA. 2008b. T-HERPS (Terrestrial Herpetofaunal Exposure Residue Program Simulation). Version 1.0.

Wright JP, Schuler LJ, Carr KH, Honegge r JL. 2011. Information to Support an Endangered Species Assessment Dicamba Use in Dicamba-Tolerant Soybeans: An Evaluation of Potential Exposure and Biologi cal Effects. Monsanto Technical Report

MSL-22545. MRID 48644202.