Section 3.0 3  1 3  Environmental Assessment 3  Comment Excerpts Commenter Name: Lincoln Brower Commenter Organization Name: Sweet Briar College Comment Number: 30509B 084000 Excerpt Number: 2 Excerpt Text: In many respects, the potential for biodiversity damage that may ensue from a corn varietal that secretes Bt toxins in its roots seems even greater to me ....... affecting the biological integrity of the soil ecosystem in some of the richest and most biodiverse soils on this planet. Gene stacking seems particularly troublesome and fraught with long term toxicological uncertainty.

EPA Response: The fate of Cry3Bb1 protein in soils and indirect effects on soil biota have been evaluated. Test data show that most of the Cry protein deposited into soil is quickly degraded, although a residual amount may persist in biologically active form for a much longer period of time ( USEPA. 2002. MON 863 BRAD Section C. I. B. 4). It is also reported that the same degree of Bt Cry protein persistence takes place in soils that have been exposed to repeat Bt spray applications when compared to soil exposed to growing Bt crop ( USEPA. 2002. MON 863 BRAD Section C. I. B. 3). As expected, longer degradation times occur in corn tissues due to the time required for the tissue to decay and for the Cry3Bb1 protein to move from tissue to soil. Limited data do not indicate that Cry proteins have any measurable effect on microbial populations in the soil ( USEPA. 2002. MON 863 BRAD Section C. I. B. 5). Horizontal transfer from transgenic plants to soil bacteria has not been demonstrated. Published studies of Bt Cry protein in soil show no effect on bacteria, actinomyces, fungi, protozoa, algae, nematodes, springtails or earthworms ( USEPA. 2002. MON 863 BRAD Section C. I. B. 3). In addition, new plants planted in Bt Cry protein containing soil do not take up the Bt protein ( USEPA. 2002. MON 863 BRAD Section C. I. B. 6). However, because the Agency believes that additional studies would be useful in completing the database for long term effects assessment, it is requiring additional supplementary multi year studies regarding Cry3Bb1 protein degradation and persistence in various agricultural soils after tree year commercial planting .

Commenter Name: Berg, Mark Commenter Organization Name: Foundation E. A. R. T. H. Comment Number: 30509 078000 Excerpt Number: 6 Excerpt Text:

 It is apparent that these producers have concerns with usage of insecticides. While 12% have personally become ill due to exposure to insecticides, 20% claim that a family member or other associate has such an experience.

 By indicating either a 8, 9, or 10, exposure to the insecticide when filling insecticide boxes ( 76%) and exposure of other family members to insecticides ( 72%) surfaced as the primary concerns of growers with current soil applied insecticides  as they are well aware of the potential health hazards associated with Section 3.0 3  2 these chemicals. About half are extremely concerned about the impact of insecticide on the environment ( 53%) and exposure to livestock ( 47%).

EPA Response: The MON 863 corn will alleviate the concerns expressed by this comment. EPA has considered the potential for reductions in use of conventional pesticides in its Benefits Assessment of the Cry 3Bb1 registration application.

Commenter Name: Scott Black Commenter Organization Name: The Xerces Society Comment Number: 30509 L03 Excerpt Number: 9 Excerpt Text: Introduction of Cry 3Bb corn hybrids may lead to impacts on soil health and productivity. If approved, Monsanto's Cry3Bb corn could be adopted widely within a few years on millions of acres ( Gray, 2000). It will deliver into the root zone concentrations of Cry3Bb toxins that are orders of magnitude above what occurs naturally, possibly triggering profound effects on soils, soil food webs, and soil borne pest dynamics.

EPA Response: The fate of Cry3Bb1 protein in soils and indirect effects on soil biota have also been evaluated. Test data show that most of the Cry protein deposited into soil is quickly degraded, although a residual amount may persist in biologically active form for a much longer period of time ( USEPA. 2002. MON 863 BRAD Section C. I. B. 4). It is also reported that the same degree of Bt Cry protein persistence takes place in soils that have been exposed to repeat Bt spray applications when compared to soil exposed to growing Bt crop ( USEPA. 2002. MON 863 BRAD Section C. I. B. 3). Limited data do not indicate that Cry proteins have any measurable effect on microbial populations in the soil ( USEPA. 2002. MON 863 BRAD Section C. I. B. 5). Horizontal transfer from transgenic plants to soil bacteria has not been demonstrated. Published studies of Bt Cry protein in soil show no effect on bacteria, actinomyces, fungi, protozoa, algae, nematodes, springtails or earthworms ( USEPA. 2002. MON 863 BRAD Section C. I. B. 3). In addition, new plants planted in Bt Cry protein containing soil do not take up the Bt protein ( USEPA. 2002. MON 863 BRAD Section C. I. B. 6).

Commenter Name: Dennis Ward Commenter Organization Name: Monsanto Comment Number: 30509B 086000 Excerpt Number: 16 Excerpt Text: Environmental Fate and Ecosystem Impacts Cry3Bbl protein dissipates rapidly in soil with 90% degraded in 7.9 to 9.2 days.

 Cry3Bb1 protein is not likely to be secreted into the corn root zone and does not accumulate in the soil environment.  No adverse effects were observed with Cry3Bbl protein under maximum expected environmental exposures for nontarget organisms. Section 3.0 3  3  The levels of Cry3Bbl protein in various plant tissues of MON 863 were not found to cause any adverse effects in animal and nontarget organism feeding studies.  The maximum worst case exposure to earthworms is at least four fold lower than a level that is known not to cause adverse effects.

 Potential tritrophic impacts on predatory species have been assessed and there is minimal risk to insect predators, parasitoids or other animals.

 Beneficial insect populations have been shown to be higher in Bt crop fields ( i. e. corn and cotton) compared to fields treated with conventional broad spectrum insecticides.

EPA Response: The data received by EPA to date show that the above comments appear to be accurate. Additional and long range confirmatory studies are in progress and are expected to be completed prior to expiration of the current registration.

Commenter Name: Dennis Ward Commenter Organization Name: Monsanto Comment Number: 30509B 106000 Excerpt Number: 12 Excerpt Text: Environmental Fate and Ecosystem Impacts  Laboratory and field studies consistently show no adverse effects on ecologically and economically important nontarget species at or above the maximum expected environmental concentration to which the organisms would be exposed.

 Bt products have a 40 year history of safe use in traditional and organic agricultural systems and the Cry3 class of Bt proteins has been used during the past decade.

 The Cry3Bbl protein is less toxic to nontarget organisms than all organophosphate and pyrethroid currently insecticides registered and commonly used to control corn pests, including CRW.

 Studies submitted by Monsanto followed EPA protocols and provided sufficient information to establish the safety of MON 863 for nontarget organisms, including the adult honey bee, wasp, earthworm and C maculata.

 Field data validate the results of the maximum hazard dose laboratory studies and demonstrate that MON 863 poses a minimal risk to nontarget organisms, including beetle families commonly found in agricultural settings.

 Extensive laboratory and field tests demonstrate that only beetles in the family Chrysomelidae are susceptible to the Cry3Bb1 protein.

 A preliminary observation that MON 863 may reduce pest nematode populations, if confirmed, would Section 3.0 3  4 represent an unexpected benefit rather than an unintended ecological risk.

 Cry3Bb1 protein dissipates rapidly in soil with 90% degraded in 7.9 to 9.2 days.

 The maximum worst case exposure to earthworms is at least four times lower than a level that is known not to cause adverse effects.

 Some variation among individual plants in a composite sample would be expected, and in extreme cases where actual exposure exceeds the maximum expected environmental exposure concentration, then the uncertainty factor used in the risk assessment ( i. e.. 10 100X) will accommodate these excursions.

EPA Response: The data received by EPA to date show that the above comments appear to be accurate. EPA has considered the potential for reductions in use of conventional pesticides in its Benefits Assessment of the Cry 3Bb1 registration application.

Commenter Name: Lance Meinke Commenter Organization Name: NCR 46 Memebers Comment Number: OPP02 0016 Excerpt Number: 10 Excerpt Text:  The ECB has a broader host range than the predominant corn rootworm pest species. Other than corn, only a few weedy grasses serve as hosts for the western, Mexican, and northern corn rootworms. The contribution of rootworm adults from these alternate hosts that provide a refuge from toxins is unknown.

EPA Response: This comment on the comparative plant host range of the ECB and the CRW is duly noted and addressed by the EPA in its Insect Resistance Management section of the Cry 3Bb1 registration application. Section 3.1 3  5 3.1  Non target Organism Effects 3.1  Comment Excerpts Commenter Name: Gerald Wilde Commenter Organization Name: Kansas State University  Department of Entomology Comment Number: 30509 055000 Excerpt Number: 1 Excerpt Text: Having been involved in the testing of their product. I have seen first hand the remarkable level of western corn rootworm control this product can deliver. At the same time I am aware of numerous studies which have been conducted to deal with the insect resistance management aspect of this product and the studies to determine any possible adverse effects on non target organisms. These studies and others lead me to believe that this transgenic rootworm protected corn product should be registered as it will be a valuable pest management tool for the corn producers of the U. S. and other parts of the world.

EPA Response: This comment is consistent with EPA's conclusions.

Commenter Name: Caroline Cox Commenter Organization Name: The Northwest Coalition for Alternatives to Pesticides Comment Number: 30509 103000 Excerpt Number: 2 Excerpt Text: NCAP is particularly concerned about the levels of the Bt Cry3Bb proteins that will be expressed in Cry3Bb corn. The application refers to the expression levels of the Cry 3Bb. Protein as moderate to low." ( MRID # 451845 01, page 5). This characterization of " moderate to low" is made relative to the ability of Cry3Bb endotoxins at the levels expressed in plant roots, to control corn rootworm larvae, the principal target pest at its most vulnerable stage. But from the perspective of likely environmental impacts, the expression levels are remarkably high. Relatively high levels exist in plant leaf tissue, pollen and grain, where they are not needed and can do much harm. Potential impacts on nontarget organisms, already identified when monarch butterflies and green lacewings are exposed to Bt proteins, thus are serious concerns that need to be evaluated carefully.

EPA Response: The fate of Cry3Bb1 protein in soils and indirect effects on soil biota have also been evaluated. Test data show that most of the Cry protein deposited into soil is quickly degraded, although a residual amount may persist in biologically active form for a much longer period of time ( USEPA. 2002. MON 863 BRAD Section C. I. B. 4). It is also reported that the same degree of Bt Cry protein persistence takes place in soils that have been exposed to repeat Bt spray applications when compared to soil exposed to growing Bt crop ( USEPA. 2002. MON 863 BRAD Section C. I. B. 3). Limited data do not indicate that Cry proteins have any measurable effect on microbial populations in the soil ( USEPA. 2002. MON 863 BRAD Section C. I. B. 5). Horizontal transfer from transgenic plants to soil bacteria has not been demonstrated. Published studies of Bt Cry protein in soil show no effect on bacteria, actinomyces, fungi, protozoa, algae, nematodes, springtails or earthworms ( USEPA. 2002. MON 863 BRAD Section C. I. B. 3). In addition, Section 3.1 3  6 new plants planted in Bt Cry protein containing soil do not take up the Bt protein ( USEPA. 2002. MON 863 BRAD Section C. I. B. 6). Two season field studies provide further evidence that the Cry3Bb1 protein in corn event MON 863 produces no short term risk of unreasonable adverse effects for the environment ( USEPA. 2002. MON 863 BRAD Section C. I. B. 3. b). Because the Agency believes that additional studies would be useful in completing the database for long term effects assessment, it is requiring additional supplementary studies regarding Cry3Bb1 protein degradation and persistence in various agricultural soils after three year commercial planting. The EPA green lacewing and monarch butterfly hazard assessment shows that the effect on these species in the field is negligible ( USEPA. 2002. MON 863 BRAD Sections C. I. B. 3. a. iv and C. I. B. 3. a. viii).

Commenter Name: Thomas O'Connor Commenter Organization Name: National Grain and Feed Association Comment Number: 30509 112000 Excerpt Number: 2 Excerpt Text: It is our understanding that Monsanto intends to plant the new product on a limited number of acres for pre commercial inbred seed propagation and hybrid seed production registration. While the amount of total acreage planted to the corn under the requested registration is relatively minor, the acreage is being planted in a wide range of states. For this reason, we would raise the following issues with which we believe regulators should have some concern:

 Is the planned buffer strip between these crops and commercial corn crops adequate to prevent pollen drift from the fields planted with the new event from inadvertently mixing with commercial crops? The SAP for StarLink corn raised concerns that the 660 foot buffer foot zone may not be adequate.

 Is there any plan to notify neighboring farms adjacent to these fields, so that, if necessary, proper precautions can be taken? Is there any plan to notify first buyers in the marketing chain that crops, no yet intended for commercial use in food and feed, are being planted in the area? Should there be? Notification may be of particular interest in areas that export bulk corn to foreign markets that may not have approved the event for commercial use.

EPA Response: EPA's response to gene flow to conventional crops is addressed in the gene flow issues section of this document.

Commenter Name: Angelika Hilbeck Commenter Organization Name: Greenpeace Comment Number: 30509 113000 Excerpt Number: 4 Excerpt Text: The expression levels in the current commercial Bt crop varieties are considered ' high dose', i. e. a multifold concentration necessary to kill 99% of the susceptible target pest species is expressed. Thus, ' high dose' is very narrowly defined in terms of lethal effects exerted on the target pest species. The target pests of the transgenic Cry3Bb corn are CRW larvae feeding on the roots of corn. Although mean concentration levels of 41 mg/ g fresh weight of root tissue translate into a large amount of toxin compared to other Bt Section 3.1 3  7 producing plants, it is below the reported LC50 of 75 ppm according to the Monsanto documentation i. e. it is not expected to kill 50% of the CRW. Therefore, Monsanto Company describes it as a low to moderate expressing plant. Cry3Bb concentration levels in leaves and pollen are substantially higher, 2 and 1.5 times of the concentration achieved in roots, respectively. In an environmental risk assessment context, such large amounts of toxin call for rigorous testing of nontarget effects, including also pollen feeding nontarget insects, such as honey bees, certain predator species, in particular coleopteran species like Coleomegilla maculata, known to feed extensively on corn pollen, but also non coleopteran species like syrphids and butterflies, e. g. monarchs and swallowtails. It is not advisable to simply rely upon the described specificity of Bt proteins published in the literature. The employed protocols and narrow definitions of ' susceptibility' are typically designed to screen for efficacy of Bt toxins in herbivorous pest species, i. e. short term bioassays aimed at detecting and quantifying lethal effects often with an underlying interest to identify their potential economic utility for pest management ( MacIntosh et al. 1990, Moar et al. 1995). After all, whether or not Bt induced effects on nontarget organisms will occur depends aside of presence and density of receptors and gut enzyme composition also on dose, i. e. the plain amount of toxin an organism ingests.

EPA Response: The above concerns were addressed by the maximum hazard dose ( limit dose) toxicity testing performed on representative beneficials from several taxa ( USEPA. 2002. MON 863 BRAD Sections C. I. B. 3. a. iv and C. I. B). Testing at the maximum hazard dose level takes into account the variability in Cry Bb1 protein levels in Bt corn fields. The toxicity of the Cry3Bb1 protein has been evaluated following challenge of several species of invertebrates including: adult and larval honey bees, a parasitic hymenopteran ( Nasonia), green lacewings, lady beetles, collembola, monarch butterfly, and earthworms ( USEPA. 2002. MON 863 BRAD Sections C. I. B. 3. a. iv and C. I. B). Reproductive and developmental observations were also made on collembola, honeybee and lady beetle larva maturation studies ( USEPA. 2002. MON 863 BRAD Sections C. I. B. 3. a. iv and C. I. B). Insect host range bioassay and field survey data were used to assess the hazard to additional species not directly tested in the laboratory.

Commenter Name: Angelika Hilbeck Commenter Organization Name: Greenpeace Comment Number: 30509 113000 Excerpt Number: 9 Excerpt Text: Nontarget safety assessment Much of the ecotoxicology data with nontarget organsims followed, like previous such regulatory trials, an approach that has been developed for the side effect testing of synthetic pesticides. In our previous EcoStrat Report on ' Nontarget Organisms and Bt Plants' ( 2000) we discuss the short comings of this approach in detail and would like to refer to that part in that report and present only a brief summary here:

" These testing procedures are designed for pesticides and their mode of release  e. g. foliar application potentially causing drift into neighboring habitats, presence for a limited time period due to degradation, etc.  but these tests alone are insufficient for assessing ecological effects of transgenic, insecticidal plants on nontarget organisms. By making a plant produce an extra and novel protein ( e. g. Bt) important characteristics of Bt's mode of release are changed: the temporal and spatial presence of the insecticidal toxin is extended due to constitutive expression and, thereby, the mode of exposure to herbivorous insects and, consequently, to predators and parasitoids is altered. Furthermore, the altered plant's metabolism ( by Section 3.1 3  8 virtue of producing a novel protein) may in itself affect interactions between the plants, their herbivores or pathogens and organisms of higher trophic levels. Therefore, we consider it necessary to adapt and modify the testing procedures for transgenic plants by including more parameters than only for acute toxicity testing, such as testing protocols for chronic lethal and sublethal toxicity." ( EcoStrat Report 2000, ' Summary' and ' 2.1. General comments on ecotoxicological studies')

EPA Response: EPA has taken the approach outlined in this comment. The non target insect testing has been expanded to include species more likely to be affected by the particular active ingredient and testing of species more likely to be encountered in the particular agroecosystem in question. Protocols for laboratory testing of several coleopteran species are being developed. In addition, extensive field testing is being performed to assess the abundance of the fauna and flora. Also, monitoring of general ecosystem effects is being required during the duration of the registration.

Commenter Name: Angelika Hilbeck Commenter Organization Name: Greenpeace Comment Number: 30509 113000 Excerpt Number: 14 Excerpt Text: Resistance management The target pest CRW has an outstanding track record of adaptation to various management practises applied for its control including one that was or still is considered to be difficult to break, i. e. crop rotation. By current definition with respect to the target pest ( see above for discussion of that issue), the Cry3Bb corn expresses a low to moderate dose of Bt toxin and therefore does not comply with the requirements for current resistance management strategies designed for the use of ' high dose' plants. To our knowledge, no effective low dose expression resistance management plans have been developed or tested with transgenic Bt plants yet. However, it might be conceivable that the lower impact on the target pest CRW may translate into lower selection pressure. Therefore, these plants may in fact present a good chance to test whether a low dose approach for pest management via transgenic plant technology may not in fact be superior approach compared to high dose. The ' high dose' strategy accepts an ' overkill' from a pest management standpoint and resembles more a prophylactic blanket coverage of a pesticide. However, the question arising with these plants is to what degree the ' low dose' for CRW also is a low dose other nontarget organisms since in terms of quantity much toxin is produced than in any previous Bt crop plants. In any regard, these approaches need first to be explored in carefully designed, smaller scale field trials and laboratory trials before large scale commercialization is advisable.

EPA Response: The above mentioned low dose issue is addressed in detail in EPA's Insect Resistance Management section of this document. The low dose effects on non target organisms is also indirectly assessed during the field surveys being performed to observe the abundance of invertebrate fauna. Also, monitoring of any effects on the ecosystem in general is being required during the duration of the registration.

Commenter Name: Angelika Hilbeck Commenter Organization Name: Greenpeace Comment Number: 30509 113000Part2 Section 3.1 3  9 Excerpt Number: 20 Excerpt Text: The massive input of highly bioactive toxic proteins like those of Bacillus thuringiensis ( Bt) into the soil ecosystem due to commercial production of fransgenic Bt crop plants can potentially exert effects on all levels of the soil ecosystem via direct or indirect exposure of soil macro and micro organisms to the Bt toxins. The fact that Bt is a soil bacteria does not preclude effects on ecosystem components and food web organisms when the Bt proteins are released in such enormous quantities and in a more activated form that were/ was not experienced before. Specificity of Bt proteins has largely been determined for plant herbivores and in terms of lethal effects. With the production of vast acreages of Bt crop plants expressing the Bt toxins constitutively ( i. e., throughout the season) and in high concentration, sublethal longterm effects on soil inhabiting macro and micro organisms become important, in particular, with respect to their function in recycling of nutrients and soil ecosystem health, i. e., the intricate coupling of these processes and the balance between beneficial and detrimental organisms. From the overuse of insecticides, the detrimental consequences of uncoupling these processes on soil health and soil fertility are well known and include a decline in soil fertility, disease suppression capacity and the subsequent buildup of soil borne plant diseases and plant feeding pest organisms.

EPA Response: Unlike the effects on soil fertility by conventional pesticides, Bt Cry proteins which are biodegradable act as additional nutrients for soil microbes ( USEPA. 2002. MON 863 BRAD Sections C. I. B. 4). The fate of Cry3Bb1 protein in soils and indirect effects on soil biota have also been evaluated. Test data show that most of the Cry protein deposited into soil is quickly degraded, although a residual amount may persist in biologically active form for a much longer period of time ( USEPA. 2002. MON 863 BRAD Sections C. I. B. 4). It is also reported that the same degree of Bt Cry protein persistence takes place in soils that have been exposed to repeat Bt spray applications when compared to soil exposed to growing Bt crop ( USEPA. 2002. MON 863 BRAD Section C. I. B. 3). Limited data do not indicate that Cry proteins have any measurable effect on microbial populations in the soil ( USEPA. 2002. MON 863 BRAD Sections C. I. B. 5). Horizontal transfer from transgenic plants to soil bacteria has not been demonstrated. Published studies of Bt Cry protein in soil show no effect on bacteria, actinomyces, fungi, protozoa, algae, nematodes, springtails or earthworms ( USEPA. 2002. MON 863 BRAD Section C. I. B. 3). In addition, new plants planted in Bt Cry protein containing soil do not take up the Bt protein ( USEPA. 2002. MON 863 BRAD Sections C. I. B. 6). However, additional long term soil degradation and persistence studies in various agricultural soils after a 3 year commercial planting are being required by EPA.

Commenter Name: Angelika Hilbeck Commenter Organization Name: Greenpeace Comment Number: 30509 113000Part2 Excerpt Number: 25 Excerpt Text: 3) Effects on soil organisms The three studies analyzed ( studies 12, 14, and 15) investigated the effect of different plant expressed Bt toxins on microorganisms, protozoa and arthropods ( Appendix 2, Table 2). In study 12, the impact of three Bt cotton lines expressing either of two different Bt toxins ( Cry lAb or CrylAc) on soil microorganisms, and protozoa was in by mixing leaf tissues of the different plant lines or Section 3.1 3  10 purified toxins in various combinations in two soil types. Over a period of 28 and 56 days  depending on the experiment  persistence and activity of the toxin and effects on populations of protozoa and microorganisms was measured. No differences between the numbers of protozoa were found. However, changes in bacterial diversity, and bacterial and fungal population levels were observed ( measured by species identification, substrate utilization, and DNA fingerprints). In a number of experiments, incorporation of transgenic Bt cotton plant material into soil led to significantly increased bacterial and fungal population levels compared with the parental lines. These changes were described as ' transient' because they were observed only during a limited period of the entire experimental time. They disappeared until the end of the experimental period, except in one experiment ( examining the transgenic Bt cotton line 249) where fungal population levels were always higher in the transgenic treatment. But, except for one treatment in one experiment, this phenomena ( temporary increase of bacterial and fungal activity in the transgenic treatment) occurred consistently in all experiments. Since none of the purified toxins or control plants showed similar effects on microbial populations, it is suggested that other, unintended characteristics of the transgenic plants may be involved in this. These ' transient' effects were observed after one soilincorporation event of Bt proteins only. Further investigations need to reveal whether after repeated soilincorporation of Bt proteins, constant Bt protein exposure or accumulating Bt protein concentrations these ' transient', significant effects may turn into permanent, significant effects.

EPA Response: The issues raised by this comment will be addressed by studies of MON 863 Cry protein degradation rates over a three year period in a variety of MON 863 corn field soils with and without decaying Bt corn vegetation. These studies and are in developmental stages.

Commenter Name: Angelika Hilbeck Commenter Organization Name: Greenpeace Comment Number: 30509 113000Part2 Excerpt Number: 26 Excerpt Text: In a field study using Cry3A potatoes ( study 14), the leaf microflora and soil borne pathogens were investigated. Three treatments were applied: transgenic Bt potatoes, a non Bt potato variety ( Russet Burbank; probably not isogenic) treated with microbial Btt and a non Bt potato variety ( Russet Burbank; probably not isogenic) treated with a systemic insecticide. No untreated control for comparison was included, in the experiment but only ' treatments' were compared. For a number of reasons, we considered the omission of an untreated control in favor of another treatment in an attempt to simulate ' common agronomic practice' ( i. e. insecticide treatment) as scientifically problematic because it introduces a ' normative control'. The particular choice of treatments, in this case the choice of a systemic insecticide instead of an untreated control, will bias the outcome and none of the observed effects can be clearly attributed to an individual treatment. The authors concluded that Cry3A producing potato plants did not consistently affect the species composition and density levels of microorganisms associated with plant leaves or the incidence of plant diseases. However, the data provided do not fully support this conclusion. The authors did report significantly higher disease infections of Verticillium dahliae and viruses ( PLRV and PVY) in the transgenic treatments. The reasons why the authors believed this should be interpreted cautiously ( i. e. effects possibly confounded with the surprisingly early die back of the insecticide treated plants and the insecticide treatment itself) may well be valid but do not allow for a blanket dismissal . Further, this provided a case in point why the omission of an untreated control is scientifically problematic; Section 3.1 3  11 the interactions between insecticide treatment, insect virus vector control and the surprising die back of the insecticide treated plants could not be analyzed property because there was no untreated ( isogenic?) control for comparison. Important information was lost and the reasons for this phenomenon remained speculative. Therefore, the repetition of the whole experiment in a second and third growing season is recommended, including an untreated control. It was suggested that surveying leaf microflora should be considered as indicator for ecological effects in monitoring programs.

EPA Response: The inclusion of Cry3A producing potato plant data on comments regarding Cry3Bb corn is unusual. However, the recommendation that field survey data in general should include an untreated isogenic control has already been implemented by the EPA. The field studies submitted for MON863 have isogenic controls.

Commenter Name: Angelika Hilbeck Commenter Organization Name: Greenpeace Comment Number: 30509 113000Part2 Excerpt Number: 27 Excerpt Text: The last study ( study 15) investigated the effect of Bt cotton expressing CrylAb and CrylAc proteins and potatoes expressing Cry3A on two arthropod species, the springtail Folsomia candida and the mite Oppia nitens. The life history parameters oviposition, number of eggs produced, body length, etc., were measured over a period of 7 to 8 weeks. No effects on any of these parameters were found. However, it could not be demonstrated whether this was due to insensitivity of the test organisms to the Bt or whether the Bt toxins were not ingested or only in low concentrations. No gut analyses of the two test species were conducted to confirm the uptake of the Bt toxin. It was assumed that the Bt toxin was ingested because, at least for 0. nitens, ' green color was seen in the whitish insect gut...'. Both test species primarily feed on the fungi growing on the decaying plant material. However, it has not yet been demonstrated whether or not fungi growing on decaying Bt plant material also contain Bt toxins or not. Further, through the addition of brewers yeast as food, the organisms probably consumed a mixture of the different food types. Based on this study, no evidence was found that the transgenic cotton lines and potato lines investigated had adverse effects on F. candida and 0. nitens after the 2 month testing period. Since slightly lower ( but not statistically significant) reproduction rates for 0. nitens were observed in the transgenic treatment than in the control after 2 months, long term effects under persistent exposure to Bt proteins cannot be excluded and still need to be investigated.

EPA Response: The inclusion of comments investigating the effect of Bt cotton expressing CrylAb and CrylAc proteins and potatoes expressing Cry3A in comments on Cry3Bb corn is not strictly appropriate. However, the recommendation that two arthropod species, the springtail Folsomia candida and the mite Oppia nitens be looked at for long term effects is included in the USDA and EPA sponsored research , as well as the requirement that Monsanto conduct long term non target effects in the field during the MON 863 registration period.

Commenter Name: Lincoln Brower Commenter Organization Name: Sweet Briar College Section 3.1 3  12 Comment Number: 30509 114000 Excerpt Number: 1 Excerpt Text: In my article, I criticize EPA and the GMO Companies for totally inadequate toxicology testing on nontarget organisms. This is an even greater problem here. EPA has no business in approving these company applications without having bonafide and comprehensive toxicology testing.

EPA Response: The toxicology studies for protein Plant Incorporated Protectants ( PIP) done by the EPA on non target organisms have been submitted to a Scientific Advisory Panel ( SAP) review in by the December 9, 1999 SAP and found to be acceptable ( SAP Report No. 99 06, February 4, 2000)..

Commenter Name: Lincoln Brower Commenter Organization Name: Sweet Briar College Comment Number: 30509 114000 Excerpt Number: 5 Excerpt Text: In their article, " Transgenic Pollen Harms Monarch Larvae," the Cornell authors asked: Could windblown corn pollen accumulate on plants that grow extensively in and adjacent to cornfields and, like conventional insecticides, inadvertently kill native insects that are not pests? To test this question, they chose the monarch as their nontarget species. Female monarchs lay eggs on wild milkweed plants, the only plants that their caterpillars can eat. In their experiment, conducted in the laboratory, the authors dusted pollen gathered from one of the Bt corn strains onto the leaves of the common milkweed. They established that caterpillars that fed on the dusted leaves ate less, grew more slowly, and suffered higher mortality than caterpillars reared on milkweed leaves dusted with pollen from a non Bt corn strain. The scientists were circumspect about their results and stated clearly that more research was needed to determine the impact of the toxic pollen on monarchs in the natural environment.

EPA Response: Mon 863 Cry3Bb1 is a coleopteran active protein that is not expected to affect lepidopterans such as the monarch butterfly. In addition, extensive research conducted on the potential affects of monarch feeding on lepidopteran active Bt corn pollen has shown a lack of concern for subchronic toxicity. Nevertheless, Monsanto sponsored a monarch butterfly feeding study which demonstrated that corn pollen expressing the Cry3Bb1 protein will not result in toxic or developmental effects to monarch larvae. In addition, the August 27, 2002 SAP concluded that the monarch butterfly was not an appropriate indicator organism to be tested with MON 863 pollen ( SAP Meeting Minutes No. 2002 05, November 6, 2002) .

Commenter Name: Lincoln Brower Commenter Organization Name: Sweet Briar College Comment Number: 30509 114000 Excerpt Number: 9 Excerpt Text: Other studies warned of new threats. One determined Bt pollen to be toxic to later stage monarch larvaesignificant because older caterpillars had been assumed to be less sensitive than the young ones. Clarifying, a contentious point of the 1999 symposium, new data fed into revised computer models now led to Section 3.1 3  13 predictions that pollen shedding and monarch breeding happen simultaneously over wide geographic areas. This finding was made all the more important by new data showing that extensive monarch breeding occurs on milkweed growing inside cornfields. This, in turn, underscored the devastating effects that the long term use of herbicides, and genetically manipulated organisms such as Round up Ready crops, will have as their use totally eliminates milkweeds from the fields.

EPA Response: Extensive studies performed under the auspices of the USDA in the US and Canada, and published in the proceedings of the NAS show that the amount of pollen deposited from the currently approved corn producing anti lepidopteran Cry protein has no detrimental effect on the development of monarch larvae ( Proc. Natl. Acad. Sci. USA, 10.1073) MON 863 Cry3Bb1 it is a coleopteran active protein that is not expected to affect lepidopterans such as the monarch butterfly. Nevertheless, Monsanto sponsored a monarch butterfly feeding study which has demonstrated that corn pollen expressing the Cry3Bb1 protein will not result in toxic or developmental effects to monarch larvae.

Commenter Name: Charles Benbrook Commenter Organization Name: Union of Concerned Scientists Comment Number: 30509 115000 Excerpt Number: 5 Excerpt Text: These data show clearly that Cry 3Bb expression levels are extraordinarily high relative to previously approved Bt corn events. In addition, relatively high levels exist in plant leaf tissue, pollen and grain, where they are not needed and can do harm to nontarget organisms. High levels in these tissues are useless because the corn rootworm is only susceptible to the Cry 3Bb endotoxin in its larval stage, as it feeds in the soil on plant roots. Adults are essentially not susceptible and hence any feeding later in the season will have no appreciable impact on pest levels.

EPA Response: The above mentioned high dose issue is addressed in detail in EPA's Insect Resistance Management section of this document. However, the August 2002 SAP commented that the Cry3Bb expression levels in MON 863 are " not high dose". In addition, monitoring of the ecosystem non target effects in general is being required during the duration of the registration which will indirectly address any detrimental effects of the Cry3Bb expression levels in plants.

Commenter Name: Charles Benbrook Commenter Organization Name: Union of Concerned Scientists Comment Number: 30509 115000 Excerpt Number: 11 Excerpt Text: 5. High Toxin Levels May Jeopardize Margins of Safety for Soil Organisms Monsanto reports that the maximum environmental concentration expected in the soil is 13.3 mg/ kg, based on the assumption that corn plants are tilled into the top six inches of soil when leaf tissue expression levels are peaking. Yet exposure levels are likely to be much higher for organisms that move through the soil and seek out the nutrient enriched zone within a few centimeters of roots. Moreover, plant residues and stalks Section 3.1 3  14 worked into the soil will create an uneven distribution of Cry 3Bb in the soil profile; organisms that happen to be feeding in the vicinity of decomposing plant tissues will be exposed to relatively higher levels.

Cry 3Bb endotoxins are lethal to earthworms at 57 mg/ kg in the soil, resulting in just a 4 fold difference between the known high level concentrations in soil. Such a narrow margin of safety is not acceptable in any other regulatory context. While under " normal" conditions, a 4 fold margin of safety may spare most earthworms in fields planted to corn varieties expressing the Cry 3Bb endotoxin, many factors can enhance the vulnerability of earthworms  or other nontarget organisms  to the effects of any toxin.

Earthworms moving through " hot spots" could be exposed to lethal levels. Exposure to other insecticides or herbicides or drought stress also can make earthworms more vulnerable than normal. Tillage systems, irrigation, or soil conditions might alter their movement and feeding habits, increasing or decreasing their exposures levels.

EPA Response: Contrary to the submitted comment, a maximum hazard dose LC50 for earthworms exposed to Cry3Bb1 protein in soil was determined to be greater than 570 mg Cry3Bb1 protein/ kg dry soil, the only concentration tested. (( USEPA. 2002. MON 863 BRAD Section C. I. B. 3. a. vii, MRID No. 449043 16 ). This dose is greater than 10 times the maximum soil concentration of the cry protein. The data show that no adverse effects to earthworms are expected from exposure to Cry3Bb1 protein producing corn plants. The comments on the theoretically envisioned adverse effects of Cry3Bb protein on earthworms are also not supported by field survey data. The number of earthworms collected did not differ between Bt and non Bt plots, but there were significantly less earthworms in the plots treated with foliar insecticides than the other insecticide regimes. ( USEPA. 2002. MON 863 BRAD Section C. I. B. 3. b), Nevertheless, the EPA has required Monsanto to conduct additional long term field surveys during the MON 863 registration period.

Commenter Name: Charles Benbrook Commenter Organization Name: Union of Concerned Scientists Comment Number: 30509 115000 Excerpt Number: 28 Excerpt Text: 1. Data on Impacts on Nontarget Organisms Are Inadequate The need for a much more thorough assessment of nontarget impacts is among the lessons learned from the EPA's review and approval of Bt corn varieties engineered to control the European corn borer.

Most pesticides, including traditional Bt insecticides, have been tested in standard laboratory assays involving a half dozen to as many as ten indicator species. Such assays are reasonably useful as screening tools to determine whether there is much risk of adverse impacts on populations of beneficial or nontarget organisms. But there are profound differences in Cry3Bb exposure pathways and levels and qualitatively different studies will be needed to more fully assess impacts on nontargets. A British research team led by T. H. Schuler has published an excellent review of the impacts on Bttransgenic crops on arthropod natural enemies, which points out that  Section 3.1 3  15 " Feeding by herbivores induces changes in the emission of plant volatiles ( termed herbivore induced synomones) making the plant more attractive to parasitoids. Most current transgenes target the digestive system of insects and could therefore affect this induction process" ( Schuler et al., 1999).

Monsanto's registration application states that the Cry3Bb proteins are active against species in " several families of Coleoptera," some of which are likely to play beneficial roles as predators or part of food webs. Yet, Monsanto has submitted a bioassay on only one Coleoptera species ( ladybird beetles) in a family that is generally known not to be susceptible to Cry3Bb toxins. Much more comprehensive nontarget organisms testing is needed and must include both acute and sub acute effects, as well tritrophic level studies.

EPA Response: Contrary to the claim in this comment, EPA has 16 studies on a wide range of non target species. In addition insecticidal activity spectrum bioassays of six Families of the Order Coleoptera and two Lepidoptera species detected activity only against beetle species of the family Chrysomelidae ( corn root worm and Colorado potato beetle) ( USEPA. 2002. MON 863 BRAD Section C. I. B. 3). The October 2000 and August 2002 SAP reports commented that wide spectrum non target testing is not necessary for the Cry proteins and should be focused on groups of non targets exposed and susceptible to the crop being registered ( SAP Report No. 2000 07. March 12, 2001); SAP Meeting Minutes No. 2002 05, November 6, 2002). The Agency has determined that the non target organisms most likely to be exposed to the protein in transgenic corn fields were beneficial insects feeding on corn pollen and nectar, and soil invertebrates, particularly Coleoptera spp. ( USEPA. 2002. MON 863 BRAD Section C. I. B. 3). The field studies performed to date do not show an effect on non target coleopteran species ( USEPA. 2002. MON 863 BRAD Section C. I. B. 3. b). Protocols for laboratory testing of carabid beetles and other significant coleopteran soil species are not available and are under development by the Agricultural Biotechnology Stewardship Technical Committee ( ABSTC). Additional multi year field surveys to address the long range effects on significant soil beetles are in progress. A decreased abundance of parasitoids in fields where the pest larvae have been eliminated is a well known phenomenon and not unique to the cultivation of Cry protein containing crops. Comments from the August 2002 SAP recommended against tritrophic studies on green lacewings in corn because the lacewing prey in corn fields does not have Bt cry toxin because they feed on corn phloem which does not contain Cry proteins ( SAP Meeting Minutes No. 2002 05, November 6, 2002) The EPA will, however, require tritrophic studies on a case by case basis where they would appear to be warranted by the ecology of the crop in question.

Commenter Name: Charles Benbrook Commenter Organization Name: Union of Concerned Scientists Comment Number: 30509 115000 Excerpt Number: 29 Excerpt Text: The adverse impacts of Cry3Bb on nontarget organism will be largely indirect, the result of changes in species composition and food webs. Work by Angelika Hilbeck and colleagues at the Swiss Research Station for Agroecology and Agriculture has shown that Bt transgenic corn can have direct and indirect impacts on both nontarget organisms and beneficial insects.

Two studies published in 1998 showed that the Cry1 toxins expressed in Bt corn plant tissue can have an adverse impact on the development and populations of Chrysoperla carnea ( green lacewings), a common Section 3.1 3  16 generalist predator ( Hilbeck et al., 1998a; Hilbeck et al., 1998b). Mortality as high as two thirds was observed among green lacewing larvae reared on insects that had fed on corn plants expressing Bt toxins. Similar morality was found when activated Bt toxins ( the truncated form of Bt expressed in corn plant tissues) were fed to green lacewing larvae in an artificial diet ( Hilbeck et al., 1998a).

In both studies, Hilbeck and colleagues used Egyptian cotton leafworms. These insects fed on the Bt corn, or diets containing the activated Bt toxins, and suffered only minor, non lethal effects. But when the lacewings fed on the cotton leafworms, they suffered both impaired development and mortality. In addition, green lacewings feeding on Bt fed prey progressed through developmental stages more slowly than the controls fed on prey not exposed to Bt toxins.

One of the study's intriguing findings is that passage of Bt toxins and protoxins through the gut of certain insects, in this case Egyptian cotton leafworms, appears to potentiate the effects of the Bt toxins when another insect ( i. e., green lacewings) preys upon the cotton leafworms ( Hilbeck et al., 1999). This and other findings led the team to conclude " tritrophic level studies are necessary to assess the long term compatibility of insecticidal plants with common natural enemies" ( Hilbeck et al, 1999) and to make a critical point:

" The ubiquitous and temporally extended availability of B. thuringiensis proteins in the field in addition to its modified form of release, makes it necessary to verify and monitor the compatibility of this new pest management strategy with natural enemies. The long term agroecological safety of the combined use of transgenic crop plants and B. thuringiensis insecticides cannot simply be deduced from the past record of safe B. thuringiensis insecticide use when B. thuringiensis compounds were available in the field only during short periods." ( Hilbeck et al., 1999).

EPA Response: Comments from the the August 2002 SAP which addressed the tritrophic feeding issue indicate that the tritrophic scenario described above does not exist in corn fields ( SAP Meeting Minutes No. 2002 05, November 6, 2002). Comments from the the August 2002 SAP recommended against tritrophic studies on green lacewings in corn because the lacewing prey in corn fields does not have any Bt cry toxin because they feed on corn phloem which does not contain Cry proteins ( SAP Meeting Minutes No. 2002 05, November 6, 2002). The EPA will, however, require tritrophic studies on a case by case basis where they are warranted by the ecology of the crop in question. The USDA and EPA are also funding long term effects studies to address the effects of season long exposures to Bt Cry proteins on invertebrate communities. In addition EPA has required Monsanto to conduct long term non target effects studies in the field during the MON 863 registration period Commenter Name: Douglas Tallamy Commenter Organization Name: University of Delaware  Deparment of Entomology and Applied Ecology Comment Number: 30509 117000 Excerpt Number: 2 Excerpt Text: Effects on Nontarget Insects Section 3.1 3  17 In 1999 John Losey, Linda Rayor and Maureen Carter of Cornell University painted milkweed leaves with pollen from BT corn and fed those leaves to the larvae of monarch butterflies. Some of these larvae died. Some grew more slowly, and some developed with no ill effects. This study sparked a national concern for the monarch's well being that a century of mowing or paving over milkweed habitat, the annual slaughter of millions of migrating monarchs on our nation's highways, the continued logging of the monarch's over wintering habitats in Mexico, and monarch death from insecticide drift was never able to muster. Although the Losey study has been criticized as not depicting actual field conditions to which monarch larvae near BT corn are exposed, we need not discredit the study to find a flaw in the argument that BT corn will threaten the monarch. The question is not whether some monarchs might die if millions of acres of BT are planted. Instead we should ask whether BT plants will kill more monarchs than conventional corn plantings that rely on repeated insecticide applications for their success. How many monarchs and other nontarget organisms that are not quite as flashy die every time insecticides are sprayed on corn from airplanes? Most opponents to BT corn do not realize that more insecticide is deployed against the western corn rootworm than any other single pest in the world. Ironically, the people opposed to BT corn are often the same people concerned about the use of pesticides.

EPA Response: EPA recognizes that conventional pesticides potentially pose a greater hazard than PIPs to monarch butterflies, other invertebrates, and their interdependent interactions in the agroecosystem, as well as to animal and plant terrestrial and aquatic wildlife.

Commenter Name: Douglas Tallamy Commenter Organization Name: University of Delaware  Department of Entomology and Applied Ecology Comment Number: 30509 117000 Excerpt Number: 3 Excerpt Text: Wise use of BT corn against corn rootworms would considerably reduce the pesticides applied to US corn overnight, and, as a direct consequence, reduce monarch mortality associated with conventional corn plantings. The reduction of pesticide has been a national goal for decades. Now that we have the technology to attain this goal, registration of this technology is threatened because of misguided national hysteria. I urge that we return to logic and reason in this matter.

EPA Response: EPA recognizes the fact that PIPs have greatly reduced the use of conventional chemical pesticides with resultant reduction in exposure of wildlife to the more toxic chemicals.

Commenter Name: Scott Black Commenter Organization Name: The Xerces Society Comment Number: 30509 L03 Excerpt Number: 6 Excerpt Text: Cry 3Bb endotoxins are lethal to. earthworms at 57 mg/ kg in the soil, resulting in just a 4 fold.. difference between the known high level concentrations in soil. While under " normal" conditions, a 4 fold margin of safety may spare most earthworms in fields planted to corn varieties expressing the Cry 3Bb endotoxin, Section 3.1 3  18 many factors can enhance the vulnerability of earthworms  or other non target organisms  to the effects of any toxin.

For example, if the average level in the top six inches of soil is 13.3 mg/ kg, there surely will be places within the rhizosphere where levels are as much as two orders of magnitude higher. Earthworms moving through such " hot spots" could be exposed to lethal levels. Exposure to other insecticides or herbicides or drought stress also can make earthworms more vulnerable than normal. Tillage systems, irrigation, or soil conditions might alter their movement and feeding habits, increasing or decreasing their exposures levels.

EPA Response: Contrary to the submitted comment, a maximum hazard dose LC50 for earthworms exposed to Cry3Bb1 protein in soil was determined to be greater than 570 mg Cry3Bb1 protein/ kg dry soil, the only concentration tested. (( USEPA. 2002. MON 863 BRAD Section C. I. B. 3. a. vii, MRID No. 449043 16 ). This dose is greater than 10 times the maximum soil concentration of the cry protein. The data show that no adverse effects to earthworms are expected from exposure to Cry3Bb1 protein producing corn plants. The comments on the theoretically envisioned adverse effects of Cry3Bb protein on earthworms are also not supported by field survey data. The number of earthworms collected did not differ between Bt and non Bt plots, but there were significantly less earthworms in the plots treated with foliar insecticides than the other insecticide regimes. ( USEPA. 2002. MON 863 BRAD Section C. I. B. 3. b), Nevertheless, the EPA has required Monsanto to conduct additional long term field surveys during the MON 863 registration period.

Commenter Name: Scott Black Commenter Organization Name: The Xerces Society Comment Number: 30509 L03 Excerpt Number: 7 Excerpt Text: Indirect Impacts Must Be Assessed The adverse impacts of Cry3Bb on non target organism will be largely indirect, the result of changes in species composition and food webs. Work by Angelika Hilbeck and colleagues at the Swiss Research Station for Agroecology and Agriculture has shown that Bt transgenic corn can have direct and indirect impacts on both non target organisms and beneficial insects.

Two studies published in 1998 showed that the Cry1 toxins expressed in Bt corn plant tissue can have an adverse impact on the development and populations of Chrysoperla carnea ( green lacewings), a common generalist predator ( Hilbeck et al., 1998a; Hilbeck et al., 1998b). Mortality as high as two thirds was observed among green lacewing larvae reared on corn plants expressing B. thuringiensis. Similar morality was found when activated Bt toxins ( the truncated form of Bt expressed in corn plant tissues) were fed to green lacewing larvae in an artificial diet ( Hilbeck et al., 1998a).

More recent research by the Hilbeck team explores prey mediated impacts. Such impacts can arise when predatory insects like green lacewings feed on a herbivore that has, in turn, been feeding on B1 corn plants. In both the earlier and new work, Hilbeck and colleagues used Egyptian cotton leafworms. These insects fed on the Bt corn, or diets containing the activated B1 toxins, and suffered only minor, non lethal effects. Section 3.1 3  19 But when the lacewings fed on the cotton leafworms, they suffered both impaired development and mortality. In addition, green lacewings feeding on Bt fed prey progressed through developmental stages more slowly than the controls fed on prey not exposed to Bt toxins.

These and other findings led the team to conclude " tritrophic level studies are necessary to assess the long term compatibility of insecticidal plants with common natural enemies" ( Hilbeck et al, 1999) and to make a critical point:

" The ubiquitous and temporally extended availability of B. thuringiensis proteins in the field in addition to its modified form of release, makes it necessary to verify and monitor the compatibility of this new pest management strategy with natural enemies, The long term agroecological safety of the combined use of transgenic crop plants and B. thuringiensis insecticides cannot simply be deduced from the past record of safe B. thuringiensis insecticide use when B. thuringiensis compounds were available in the field only during short periods." ( Hilbeck et al., 1999).

EPA Response: Comments from the the August 2002 SAP which addressed the tritrophic feeding issue indicate that the tritrophic scenario described above does not exist in corn fields ( SAP Meeting Minutes No. 2002 05, November 6, 2002). Comments from the the August 2002 SAP recommended against tritrophic studies on green lacewings in corn because the lacewing prey in corn fields does not have any Bt cry toxin because they feed on corn phloem which does not contain Cry proteins ( SAP Meeting Minutes No. 2002 05, November 6, 2002). The EPA will, however, require tritrophic studies on a case by case basis where they are warranted by the ecology of the crop in question. The USDA and EPA are also funding long term effects studies to address the effects of season long exposures to Bt Cry proteins on invertebrate communities. In addition EPA has required Monsanto to conduct long term non target effects studies in the field during the MON 863 registration period Commenter Name: Scott Black Commenter Organization Name: The Xerces Society Comment Number: 30509 L03 Excerpt Number: 8 Excerpt Text: Consulting with the United States Fish and Wildlife Service There are many Coleoptera that are listed as threatened or endangered under the Endangered Species Act, The may be impacted in  some way by the EPA's new regulation due to the non target impacts of Cry 3Bb corn. Any federal agency that initiates new policies that may impact endangered or threatened species must consult with the United States Fish and Wildlife Service. The consultation is required under the Act. Some of these species may need special consideration because of their small population size or because their populations are declining. A thorough review of the endangered species that may be impacted by Cry 3Bb corn should be instituted to determine the impact on them. Impacts on Soil Health and Productivity Must Be Assessed EPA Response: EPA conducted a hazard assessment, exposure assessment and risk characterization to demonstrate that Section 3.1 3  20 Cry3Bb1 does not pose a risk to endangered Coleoptera ( USEPA. 2002. MON 863 BRAD Section C. I. C). It was determined that no adverse affects from Cry3Bb1event MON 863 are expected to endangered/ threatened Coleoptera species listed by the USFWS ( USEPA. 2002. MON 863 BRAD Section C. I. C). Non coleopteran endangered species are not expected to be susceptible to MON 863. Therefore since no " may effect" finding was made, according to the Endangered Species Act, a consultation with the USFWS is not required.

Additional studies on soil health will be conducted for a three year period.

Commenter Name: Janet Cutrona Commenter Organization Name: Janet Cutrona Comment Number: 30509B 030004 Excerpt Number: 1 Excerpt Text: The studies in this project showed that monarch caterpillars have to be exposed to pollen leaves greater than 1,000 grains/ cm. 2 to show toxic effects.

Caterpillars were found to be present on milkweed during the one to two weeks that pollen is shed by corn, but corn pollen levels on milkweed leaves were found to average only about 170 pollen grains/ cm2 in corn fields.

Reports from several field studies show concentrations much lower than that even within the cornfield. In Maryland, the highest level of pollen deposition was inside and at the edge of the corn field, where pollen was found at about 50 grains/ cm2. In the Nebraska study, pollen deposition ranged from 6 grains/ cm2 at the field edge to less than 1 grain/ cm. 2 beyond 10 meters. Samples collected from fields in Ontario immediately following the period of peak pollen shed showed pollen concentrations averaged 78 grains at the field edge.

EPA Response: Extensive studies performed under the auspices of the USDA in the US and Canada, and published in the proceedings of the NAS show that the amount of pollen deposited from the currently approved corn producing anti lepidopteran Cry protein has no detrimental effect on the development of monarch larvae ( Proc. Natl. Acad. Sci. USA, 10.1073). MON 863 Cry3Bb1 is a coleopteran active protein that is not expected to affect lepidopterans such as the monarch butterfly. Nevertheless, Monsanto sponsored a monarch butterfly feeding study which has shown that exposure to corn pollen expressing the Cry3Bb1 protein will not result in toxic or developmental effects in monarch larvae..

Commenter Name: Richard Hellmich Commenter Organization Name: USDA ARS Comment Number: 30509B 034000 Excerpt Number: 3 Excerpt Text: As with the lepidopteran Bt corn, there will be questions about non target effects and insect resistance management. Science cannot prove a negative  that there will never be any non target effects. But I believe most of the significant questions have already been addressed through the registration process. Section 3.1 3  21 Most important, positive and negative impacts of new technologies must be compared with those of existing technologies, not simply judged in a vacuum. AR possible impacts of any technology or farming practice are impossible to foresee, but we can focus on known and probable risks. When risks of a technology are characterized as low, based on actual data, then the potential impact should be evaluated proportional to that level of concern. This reasonable approach should reduce the chances of rejecting safe technologies simply because they are new and unfamiliar.

EPA Response: The comment does not require a response.

Commenter Name: Paul Mitchell Commenter Organization Name: Texas A& M University  Department of Agriculture Economics Comment Number: 30509B 082000 Excerpt Number: 6 Excerpt Text: The risk of significant monarch and other non target insect problems has proven minor ( see a nice summary of the issue at http: www. nbiap. vt. edu/ news/ 200j/ newsO2. apr. html# aprO2O4). The StarLink food contamination is not an issue, since the new event has or will be approved for human consumption, as have most of the other Bt events. The recent report of the spread of other Bt event genes into wild populations in Mexico seems to have serious scientific flaws as did the original claim that Bt corn was killing a substantial number of monarch larvae ( see http:// www. isb. vt. edu/ news/ 2002/ news02. mar. html# mar0202). I am glad that people are looking for possible problems with these transgenic crops. However, I believe that proper science should be used to make policy relevant claims.

EPA Response: The comment does not require a response.

Commenter Name: Dennis Ward Commenter Organization Name: Monsanto Comment Number: 30509B 086000 Excerpt Number: 3 Excerpt Text: Comment or Question: Cry3Bbl endotoxins are lethal to earthworms at 57 mg/ kg soil, resulting in just a four fold difference between the known high level concentration in soil Such a narrow margin of safety is not acceptable in any other regulatory context.

Scientific Background: Contrary to what has been alleged, no mortality of earthworms ( or any other effects) was observed at the 57 mg/ kg soil treatment level. Mortality was only observed at a ten fold higher treatment level. The 57 mg/ kg level was established as a no observable effect concentration. Therefore, the maximum worst case exposure to earthworms is at least four fold lower than a level that is known not to have an effect on earthworms. Under actual use conditions, the crop will be harvested and not tilled into the top 6 inches of soil. Moreover, the protein is known to degrade rapidly in soil. Therefore, MON 863 corn is expected to pose no more than minimal risk to earthworms.

EPA Response: Section 3.1 3  22 An independent review of the available data by the EPA has reached the same conclusion. The laboratory data and field surveys show that no adverse effects to earthworms are expected from exposure to Cry3Bb1 protein producing corn plants.

Commenter Name: Dennis Ward Commenter Organization Name: Monsanto Comment Number: 30509B 086000 Excerpt Number: 5 Excerpt Text: Comment or Question: There is a lack of data on nontarget species that may play beneficial roles as predators or part of food webs. The data submitted were limited to a bioassay on only one Coleopteran species ( ladybird beetles) in a family generally known not to be susceptible to Cry3Bb1 toxins.

Scientific Background: A standard battery of nontarget organism tests was conducted for the Cry3Bb1 protein as required by EPA. This battery of tests included the ladybird beetle, which is generally recognized as an important biological control agent. Therefore, the test was performed to document the absence of effects on this important beetle. Monsanto has tested other coleopteran families in addition to the Chrysomelidae ( target species) and Coccinellidae ( nontarget) families. These families include Bruchidae, Tenebrionidae, and Curculionidae. [ Footnote 6: Monsanto report MSL 17614; MRID 455770 03] No effects are predicted in these non target coleopteran families. Moreover, no apparent affects on beetles or other species were observed in the first year of a two year field study with MON 863 corn, [ Footnote 7: Monsanto report MSL 17179; MRID 455382 06] nor in university studies conducted at various locations. [ Footnote 8: Monsanto reports 99 894E and 00 CR 032E 7; MRIDs 453485 02 and 456530 03]

Comment or Question: More comprehensive testing of non target organisms is needed and must include both acute and subacute effects, as well as tritrophic studies. Tritrophic studies are necessary to assess the long term compatibility of insecticidal plants with common natural enemies.

Scientific Background: A comprehensive ecological risk assessment was conducted for the Cry3Bb1 protein as outlined in Subdivision M of the U. S. EPA FIFRA regulations. Crops with pesticidal traits are required to undergo acute and subacute evaluation for a series of surrogate species. Potential adverse effects to nontarget organisms from exposure to Cry3Bb1 were evaluated in a series of studies with representative avian, aquatic and terrestrial beneficial invertebrate species. These studies establish that the potential risk to nontarget organisms is negligible.

Tritrophic studies are mainly designed to examine the effects of food chain transfer of the toxin and to examine possible secondary effects on predator populations due to changes in the prey ( pest) populations. Since the  toxicity of Bt proteins has been assessed by directly feeding predators such as lacewings and ladybird beetles ( as per EPA's Subdivision M), this information is sufficient to assess risk from food chain transfer. Comparison of no observed effect levels to maximum exposure concentrations indicates minimal risk to insect predators, parasitoids or other animals. Recent FIFRA Scientific Advisory Panels concluded that tritrophic studies were not needed as part of the ecological risk assessment.

Laboratory studies conducted by Hillbeck et al [ Footnote 9: Hillbeck et al. ( 1998). Effects of transgenic Section 3.1 3  23 Bacillus thuringiensis corn fed prey on mortality and development time of immature Chrysoperla carnea ( Neuroptera: Chrysopidea). Environ. Entomol. 27: 480 487] showed that green lacewing larvae feeding on Bt toxin" intoxicated" preys ( Spodoptera littoralis larvae) had about 25% more mortality than those feeding on healthy (" nonintoxicated") preys. However, recent studies by Dutton et al. [ Footnote 10: Dutton et al. ( 2002). Update of Bt toxin by herbivores feeding on transgenic maize and consequences for the predator Chrysoperla carnea. Ecological Entolmol. ( in press)] indicated that this adverse effect on the lacewing larvae is largely due to the " sickness" of the Bt toxin intoxicated preys, rather than ingested Bt toxins. They showed that an alternative prey ( Tetranychus uticae Koch) feeding on Bt corn had a 2 to 3 fold higher body burden of Bt proteins than S. littoralis, yet produced no adverse effect on the development and survival of the green lacewing larvae tested.

EPA Response: An independent review of the available data by the EPA has reached the same conclusions. In addition, EPA intends to ask for tritrophic studies where they would appear to be warranted by the ecology of the crop in question.

Commenter Name: Rissler Commenter Organization Name: UCS Comment Number: 30509B 089000 Excerpt Number: 6 Excerpt Text: An ideal, precision engineered transgenic corn for rootworm control would express in and through roots a " high dose" for control of corn rootworms for the first two months of the corn growth cycle, with little or no expression in other tissues or later in the season. The degree to which Cry 3Bb proteins are expressed in the second half of the growth cycle, in any tissue, adds nothing to efficacy technology and increases the potential for risks, including impacts on nontarget organisms and the emergence of resistance. It will also expose adult rootworm beetles to sublethal levels of Cry 3Bb toxins via leaf tissues during a second stage of life, adding new complexity to the assessment and management of resistance.

EPA Response: The above mentioned high dose issue is addressed in detail in EPA's Insect Resistance Management section of this document. Monitoring of the agro ecosystem effects in general is being required during the duration of the registration which will indirectly show any detrimental effects of the Cry3Bb expression levels.

Commenter Name: Rissler Commenter Organization Name: UCS Comment Number: 30509B 089000 Excerpt Number: 9 Excerpt Text: Limited Field Study Data Indicate Possible Adverse Impacts.

A report of the one field study on ecological impacts of MON 863 corn on nontarget organisms ( Bhatti et al., 2001) states that " Preliminary field data were collected from large scale corn plots in 1999" whereas the Dudin et al. report on those 1999 field trials explains that in each location, a limited number of corn plants could be sampled for Cry 3Bb1 expression levels because of the scarcity of seeds and viable plants. Section 3.1 3  24 The 1999 trials apparently were not " large scale" as the term is usually used to describe corn experiments.

EPA Response: The reported discrepancy between reported test plot sizes in is duly noted by the EPA. Research to develop protocols for statistically valid large scale field studies is currently in progress.

Commenter Name: Rissler Commenter Organization Name: UCS Comment Number: 30509B 089000 Excerpt Number: 11 Excerpt Text: A Flawed Analysis of Bt Toxin Concentration in Soil Underestimates Risks to Earthworms and Other Soil Organisms.

In the ecological impacts section of the January 8, 2002, summary document ( Ward, 2002; Monsanto 00 CR 032E 6), the author states that no adverse impacts on nontarget organisms should be expected at " Maximum Expected Environmental Concentrations" ( MEECs). In the case of pollen, the MEEC was determined to be 93 ug/ g, a level probably no more than one half the true MEEC, since the 93 ug/ g value was based on a composite sample containing pollen from many plants collected over seven days ( see discussion above).

The MEEC for soil dwelling organisms is estimated to be 13.3 mg/ kg ( or 13.3 ug/ g) " based on the assumption that corn plants are tilled into the top six inches of soil at the time of maximum Cry 3Bb1 concentration ( i. e., 93 ug/ g)."

This method for calculating the soil MEEC is flawed and results in a far lower soil MEEC than the actual level of Cry 3Bb1 protein that many soil dwelling organisms will be exposed to, at least during the first 60 to 90 days of the corn plant growth cycle. Impacts on organisms that spend part of their life cycle near or in the rhizosphere ( zone of soil immediately surrounding roots and modified by root exudates) will, in particular, be exposed to much higher concentrations than other soil organisms. According to Monsanto scientists, Cry 3Bb1 protein levels are highest in the parts of roots that are growing rapidly ( Vaughn et al., 2001). The areas around root tips  clearly a fastgrowing area  also likely emit the highest soil concentrations of Cry 3Bb1 protein exudates, again especially when corn plants are growing rapidly. For these reasons, and drawing on the work of Saxena et al. ( 1999), the levels of Cry 3Bb1 proteins in the few centimeters of soil around the end of rapidly growing roots will be far higher than at any other place or at any other time. Mixing the Cry 3Bb1 protein in a corn plant in the top six inches of soil obviously results in significant dilution of the protein, and hence an MEEC low by perhaps orders of magnitude compared to levels in the rhizosphere for the first two months of plant growth.

It is worth emphasizing, as well, that data submitted to date by Monsanto report the concentration of Cry 3Bb1 protein in fresh root tissue, but not in the soil immediately surrounding roots. Root exudates, not levels in the roots, will drive adverse impacts on a wide range of soil organisms. But even with the far toolow soil MEEC, Monsanto's ecological effects studies have documented potentially serious impacts on earthworms, an organism with a MOE of only 4.3 ( the " No Observable Effect Concentration" is 4.3 times higher than the MEEC in soil). In most ecological impact studies, the EPA strives to assure a minimum Section 3.1 3  25 MOE of 10.

Two other nontarget organisms have MOEs under 10, based on relevant MEECs. The adult honey bee, a key pollinator, has an MOE of just 3.8; the common parasitic wasp Nasonia vitripennis has an MOE of only 4.3, again based on the pollen MEEC of 93 ug/ g. While not an important beneficial insect in corn production systems, Nasonia vitripennis is a common test organism that is easily reared in the laboratory. Impacts on this organism may be indicative of impacts on other parasitic wasps that are important in corn production fields.

EPA Response: Contrary to the claims by this commenter of possible adverse effects because of localized high soil Cry3Bb protein concentration, submitted laboratory toxicity data performed at a maximum hazard dose ( which takes into account localized high concentrations), and field survey results, as well as published reports of effects on soil microbial and invertebrate flora do not show significant adverse effects. However, additional multi year field surveys are being conducted to confirm this conclusion. Examination of the submitted nontarget studies shows that these were performed with 20x or higher safety margins, except where pure pollen or plant tissue were used where the concentration of the Cry protein cannot be increased. The parasitic hymenoptera were in fact tested at only 1X field concentration in plants rather than 10X. However, parasitic hymenoptera are not expected to feed directly on corn plant tissue. For this reason the SAP commented that parasitic hymenoptera are not an appropriate test species for MON863 corn. The LC50 for adults honey bees was determined to be greater than the highest concentration tested, 360 ug Cry3Bbl protein/ g diet, or 4.3 times the MEEC ( i. e., 93 ug Cry3Bbl protein/ g pollen). Since no adverse effects were noted at 360 ug/ g ( and this exposure will not be realized under field conditions), minimal risk to adult honey bees is expected.

Commenter Name: Rissler Commenter Organization Name: UCS Comment Number: 30509B 089000 Excerpt Number: 42 Excerpt Text: B. An Inappropriate Testing Paradigm, Poorly Designed Experiments, and Insufficient Data Render Monsanto's Assessment of the Environmental Risks of MON 863 Inadequate. [ Footnote 13: See Appendix A for additional comments on environmental risk assessment.]

A proper risk benefit assessment of MON 863 corn must analyze this technology's risks and benefits both in isolation and relative to other accessible, proven control measures. Data submitted by Monsanto on the nontarget organism impacts of MON 863 corn, while limited, raise serious concerns about this product's risks to the environment.

Many organizations and experts, including scientific advisory panels convened by EPA, have stressed that a conceptual framework different from the one traditionally used to evaluate impacts of chemical pesticides, and a different set of questions, should serve as the foundation for the ecological risk assessment of crops engineered to express toxins like Cry 3Bb. [ Footnote 14: For an overview of why a new conceptual framework is needed, see comments by Dr. Angelika Hilbeck and Matthias Meier on the ecological risks of Bt transgenic plants at www. ucsusa. org/ food/ by_ ren_ app1. pdf.] Section 3.1 3  26 The ecological risk assessment of chemical pesticides is generally limited to acute lethality in test animals. Simple toxicological experiments are designed to establish the dose at which 50 percent or 90 percent of a test population is killed. Little or no effort is made to explore other mechanisms of toxicity or adverse impacts other than death. There are virtually no studies carried out on:

 Reproduction and development, impacts that are likely to occur at much lower doses that acute poisoning; [ Footnote 15: Recent evidence of the adverse impact of atrazine on frog development documents developmental abnormalities at levels two to three orders of magnitude lower than required to kill 50 percent of adult frogs ( Hayes et al., 2002).]

 Impacts on classes of nontarget organisms that play key roles in food chains and/ or plant pest soil microbial community interactions;

 Unique impacts during a portion of the year or under conditions of environmental stress like drought, excessive heat, or in the presence of soil mineral or chemical imbalances; and  The synergistic impacts of exposures to multiple toxins, chemicals, and stresses.

EPA Response: Contrary to the commenter's claims, the data reviewed on the non target organism impacts of MON 863 corn, do not raise "... serious concerns about this product's risks to the environment". The` new concept' proposed by the commenter in risk assessment for genetically engineered plants is in fact being implemented to supplement single species laboratory testing. New protocols for more appropriate nontarget species are being developed and a heavy reliance of ecosystem effects as determined by field surveys have been implemented, and additional protocols for large scale multi year field studies are being developed concurrently with on going field surveys on MON863 corn. These field studies would also detect reproductive and interactive non target effects.

The comment that "... a conceptual framework different from the one traditionally used to evaluate impacts of chemical pesticides, and a different set of questions, should serve as the foundation for the ecological risk assessment of crops engineered to express toxins like Cry 3Bb" is inconsistent with the 1999, 2000 and 2002 SAP comments which agreed with he EPA approach of single species tier I testing followed by field testing when adverse effects were seen. While recognizing that different specific studies are appropriate for different B. t. crop risk assessment, the SAPs do not conclude or imply that EPA's ecological risk assessment methods for these plants are " fatally flawed" ( SAP Report No. 99 06, February 4, 2000; SAP Report No. 2000 07. March 12, 2001; SAP Meeting Minutes No. 2002 05, November 6, 2002).

Commenter Name: Rissler Commenter Organization Name: UCS Comment Number: 30509B 089000 Excerpt Number: 43 Excerpt Text: Genetically engineered crops expressing toxins in all or most plant tissues expose nontarget organisms within a given agroecosystem to qualitatively and quantitatively different risks over a more extended timeframe, in contrast to chemicals ( Hilbeck and Meier, 2001; Hilbeck et al., 1999). An applied chemical Section 3.1 3  27 typically has a predictable environmental fate and the pest manager has control over when it is applied; it is generally known where the chemical is likely to wind up, whether and how it breaks down, how long it lasts, and therefore, which organisms are exposed to what levels and for how long. With transgenic organisms however, the environmental fate of toxins is much more complicated, since toxin levels are determined by the development and growth patterns of living organisms. A myriad of feedback mechanisms within the environment govern plant development and growth, as well as reproductive success. The farmer has little control over these interactions once a transgenic crop is planted. For these reasons, the simple, one dimensional toxicological models EPA has adapted from its chemical testing and applied to GMO crops are likely to fail to detect most of the possibly important ecological impacts of transgenic crops.

Monsanto studies on the impacts of Cry 3Bb endotoxins on nontarget organisms suggest potential impacts but are far too limited in sample size and statistical design to support any firm conclusions.

For example, the November 5, 2001 submission included a study by Head et al. entitled " Insecticidal Spectrum of Activity for Cry 3Bb Protein in vitro" ( Monsanto Number 455382 07). This study assesses impacts on a range of nontarget organisms including Coleoptera from six families; two species each of Lepidoptera, Hymenoptera, and Neuroptera; and one species of Collembola. Six to eight concentrations of Cry 3Bb toxins from 1 ppm to 200 ppm were used in assays performed in Monsanto's laboratory covering the Colorado potato beetle, corn rootworm, boll weevil, corn earworm, ECB and monarch butterfly.

It is not possible to determine sample sizes and statistical reliability with the limited details offered on the Standard Operating Procedure ( SOP, i. e., experimental protocols), which is covered in Appendix 1 of the Head et al. document. The document references the SOP to Stone et al., 1989, which is incorrect, since there is no Stone et al., 1989 in the references. There is a similar reference Stone et al., 1991. In any event, the SOP describes a method for mixing purified Cry 3Bb toxins into artificial media, which are then fed to insects at varying Cry 3Bb concentrations. Exposing insect larvae in a laboratory bioassay to toxins via an artificial media is very different from larval exposure in crop fields as they feed on plant tissues ( Hilbeck and Meier, 2001). It is not possible to predict, at this time, how relevant or accurate such bioassays are in determining the toxicity of MON 863 corn under field conditions.

Monsanto contracted with various laboratories to do all other nontarget insect testing on key species including honey bees, parasitoid wasps, ladybird beetles and Collembola. According to the Head et al. report ( page 5):

These assays were carried out according to GLP at contract laboratories as part of the nontarget organism bioassays required for an EPA regulatory package . Briefly, one or two test concentrations of Cry 3Bb were used in these assays. The maximum concentration used in each case was based upon estimates of the highest level of Cry 3Bb to which that species might be exposed if Cry 3Bbexpressing crops were deployed.

EPA Response: The concept of evaluating multi year effects of non target invertebrates in the field and their interactions is in fact being implemented to supplement single species laboratory testing. New protocols for more appropriate non target species are being developed and a heavy reliance of ecosystem effects as determined by field surveys have been implemented, and additional protocols for large scale multi year field studies are Section 3.1 3  28 being developed concurrently with on going field surveys on MON863 corn. These field studies could also detect reproductive and interactive non target effects.

Commenter Name: Rissler Commenter Organization Name: UCS Comment Number: 30509B 089000 Excerpt Number: 44 Excerpt Text: The upper end feeding rate  200 ppm  is just over twice the maximum concentration reported in various corn tissues. Ecological impact studies are typically carried out at 2X, 5X, and even 10X or more of documented field concentrations to compensate for the generally low sample sizes used in such experiments and in order to establish a margin of exposure ( MOE) at a known effect level. The dose levels in these contract studies are inadequate to establish MOEs.

Head et al. goes on to explain that " concentration mortality regressions were estimated using a probit model and weight values" to estimate LC50 values. The appropriateness of such a methodology in a laboratory bioassay including just two concentration levels is subject to question. Indeed, in the Head et al. study, the single table reporting maximum concentrations tested and estimated LC50 values contains only two values  one for the Colorado potato beetle and the other for western corn rootworm.

A March 1, 2001, study carried out by Monsanto scientists ( Duan et al., 2001) assessed the impact of the variant Cry 3Bb1 protein on larvae of the ladybird beetle. The summary states:

Test pollen, containing a variant of the Cry 3Bb1 protein at a level of 93 ug/ g fresh weight pollen, was collected from corn rootworm protected corn event MON 863.

In the Dudin et al. study on MON 863 expression levels, the highest level reported of Cry 3Bb1 protein in pollen was 93 ug/ g. This level was found in the single sample collected from U. S. grown corn ( collected at the Monmouth, Illinois test site). This sample is a composite of samples from many plants collected over seven days. The Dudin et al. report does not make clear how many plants were sampled each day and hence there is no way of knowing the number of plants in the composite nor the variation in levels above and below the average composite level, 93 ug/ g. But since the 93 ug/ g is an average, it is clearly not the maximum concentration in samples collected. The Argentina pollen testing suggests that the range of Cry 3Bb1 levels in pollen could easily be twice the mean level, i. e., some values in a composite will be one half or less the composite mean and some will be twice or more higher. Accordingly, the actual maximum environmental concentration documented in the one U. S. test probably is above 150 ug/ g and could approach 200 ug/ g.

In addition to inadequate dose levels, the sample sizes ( 10 larvae per replicate) in the Duan et al. study were also too small to detect subtle impacts. Appropriate dose levels would range up to at least 3 times the maximum expected level in a single plant. In addition, the study protocol was too narrow in its exclusive focus on mortality. Data are also needed, for example, on the developmental and reproductive success of treated insects through at least one full generation.

EPA Response: Section 3.1 3  29 Examination of the submitted non target studies shows that these were performed with 20x or higher safety margins, except where pure pollen or plant tissue were used where the concentration of the Cry protein cannot be increased. This high safety margin showing no adverse effects is indicative of the absence of subtle effects at lower concentrations encountered in the field. However, additional multi year field surveys are being conducted to confirm the no effect conclusions drawn from high dose laboratory studies. The lady beetle study cited by the commenter was followed up by three other lady bird studies which showed no detrimental effects.

Commenter Name: Jeff Boeger Commenter Organization Name: Jeff Boeger Comment Number: 30509B 092031 Excerpt Number: 2 Excerpt Text: As for environmental effects, since corn in the USA is grown exclusively as monocultures; in environments with dissimilar plants around them, the chance of any interbreeding with native plant species is very low. This can't be said of all crops, but it is true of corn. The risk is extremely minimal compared to the great good this modification can do for farmers.

EPA Response: The information available to date indicates that the above comments appear to be accurate.

Commenter Name: Dennis Ward Commenter Organization Name: Monsanto Comment Number: 30509B 106000 Excerpt Number: 1 Excerpt Text: Many of the public comments fail to fully consider the collective evidence of laboratory and field studies submitted by Monsanto that consistently demonstrate no adverse effects of Cry3Bb1 on ecologically and economically important nontarget species at or above the maximum expected environmental concentration ( MEEC) to which such organisms would be exposed. Another important consideration, sometimes overlooked, is the 40 year history of safe agricultural use of Bt products that collectively express a diverse range of insecticidal crystal proteins. For example, Cry3 Bt products ( e. g., Raven Oil Flowable Bioinsecticide) have been used during the past decade in traditional and organic agricultural systems. Finally, any analysis of the potential risks and benefits for a new technology must be considered in the context of existing pest management practices and systems. The Cry3Bb1 protein produced in MON 863 corn is less toxic to nontarget organisms than all organophosphate and pyrethrold insecticides currently registered and commonly used to control corn pests, including CRW. In addition to the low toxicity of the Cry3Bbl protein, the in planta delivery system reduces exposure to nontarget organisms since they must ingest the plant tissue to be exposed.

EPA Response: The data received by EPA to date show that the above comments appear to be accurate. Additional and long range confirmatory studies are in progress.

Commenter Name: Dennis Ward Section 3.1 3  30 Commenter Organization Name: Monsanto Comment Number: 30509B 106000 Excerpt Number: 3 Excerpt Text: Comment or Question: Nontarget organism studies were not conducted for important beetle species closely related to the target pest ( also a beetle).

Scientific Background: Laboratory studies with nontarget beetle species closely related to CRW ( family Chrysomelidae), an analysis of potential impacts to endangered beetles from three additional families, as well as multi location field investigations by Monsanto and university researchers, demonstrate that beetles in only the family Chrysornelidae are susceptible to the Cry3Bb1 protein. Monsanto's initial data package included the nontarget test species as required in the EPA Subdivision M ( 1989) and OPPTS ( 1996) guidelines. Pursuant to EPA's Science Advisory Panel recommendation [ Footnote 3: SAP report No. 2000 7, March 12, 2001], Monsanto generated additional toxicity information for species closely related to the target species ( corn rootworm beetles in this case). Initially, one species of beetle was tested ( adult Hippodamia convergens) and no effects were observed at 8,000 ppin Cry3Bb1 protein in the diet. Subsequent studies were conducted with adult and larvae ladybird beetles of another species, Coleomegilla maculata. No adverse effects were observed in this specie when fed MON 863 pollen at up to 50% of the diet ( the maximum exposure level in the field).

In addition to data on Coccinellidae ( nontarget) and Chrysomelidae ( target species), Monsanto has conducted studies on representative species within the Bruchidae, Tenebrionidae, and Curculionidae families. [ Footnote 4: Monsanto report C3NTO; MRID 455382 07] These data demonstrate minimal potential risk for endangered beetle species.

Finally, field investigations were initiated in year 2000 by Monsanto. as well as by several university researchers. across multiple locations to validate the prediction of minimal risk to nontarget beetles in the field based on the laboratory data. An interim report summarizing the data collected for key nontarget and target species was submitted to EPA in 2001. [ Footnote 5: Monsanto report 99 894E; MRID 453484 02] These data indicate no significant adverse effects of MON 863 on carabid beetles or other nontarget beetles, while adverse effects of currently used conventional insecticides were identified. Thus, the field data validate the results of the maximum hazard dose laboratory studies and demonstrate that MON 863 poses minimal risk to nontarget organisms, including beetle families common1y found in agricultural settings.

EPA Response: The data received by EPA to date show that the above comments appear to be accurate. Additional and long range confirmatory studies are in progress.

Commenter Name: Dennis Ward Commenter Organization Name: Monsanto Comment Number: 30509B 106000 Excerpt Number: 4 Excerpt Text: Comment or Question: Additional nematode studies are needed since preliminary delta showed significant Section 3.1 3  31 nematode population reductions in a field study.

Scientific Background: Any reduction in the pest nematode population related to use of MON 863 would represent an unexpected benefit rather than an unintended ecological risk. Consequently, additional maximum hazard dose studies with pest and other nontarget nematodes would not be necessary. A preliminary report indicated the potential for corn expressing Cry3Bb1 to impact certain nematode species. [ Footnote 6: Monsanto report 00 CR 032E 7, Appendix F, MRID 456530 03] However, to put the observation in proper context, the author of the report stated that the results of this preliminary study "... make it seem very unlikely that Bt corn would have an impact on nematode fauna not associated with the corn plant itself, in other words a nematode that feeds on the plant in some way."  This qualification makes it clear that the potential for impacts on nematodes is focused on nematode corn pest species; consequently, any effects would represent a potential benefit rather than an unintended risk. Based on this preliminary research and the researcher's statement, there is no basis for conducting additional nontarget organism studies with nematodes.

EPA Response: EPA does not believe that impacts on nematodes are sufficient to constitute an unreasonable adverse effect. Nonetheless, to address this issue conclusively, EPA has requested additional data in order to make a risk assessment.

Commenter Name: Lance Meinke Commenter Organization Name: NCR 46 Memebers Comment Number: OPP02 0016 Excerpt Number: 9 Excerpt Text:  Both corn rootworm adults and larvae feed on the corn plant, which potentially exposes both life stages to any toxin that is expressed throughout the plant. This could lead to extended selection for resistance. In contrast, only ECB larvae are exposed to CrylAb as ECB moths do not feed on corn tissue.

EPA Response: The above mentioned resistance issue is addressed in detail in EPA's Insect Resistance Management section of this document.

Commenter Name: Clifford Habig Commenter Organization Name: Exponent, Inc. Comment Number: OPP02 0017 Excerpt Number: 3 Excerpt Text: EPA currently requires a suite of laboratory studies to support the registration of PIPs. Several of these studies, such as those conducted on avian and aquatic invertebrate species, flow directly from standard chemical and microbial testing on non target organisms. Interestingly, EPA's evaluation of conventional chemical pesticides includes very few data requirements concerning non target insects and soil invertebrates, whereas the microbial testing guidelines require more extensive testing of beneficial insects and soil  invertebrates. Similar to microbial pesticides, most non target testing with PIPs has focused on non target insects and soil invertebrate species. These data requirements represent a refinement of the Section 3.1 3  32 Series 885 Microbial Testing Guidelines, which require testing on several non target insect species, including honeybees, and a representative series of beneficial insects, such as lady bird beetles and lacewings.

EPA guidance and representative testing protocols for non target insects are not very detailed. Guidance on duration of the tests, type of testing material, appropriate test endpoints, and standard acceptability criteria are limited and registrants must interpret the available guidance when designing studies. The current studies are different from many of the traditional chemical beneficial insect tests, such as those used in Europe, in that the PIP tests focus on dietary exposure rather than contact and spray exposure, and many of the PIP non target insect tests tend to be conducted for longer exposure. times. For example, beneficial insects in PIP testing are typically exposed to high concentrations of dietary Bt proteins as the sole dietary source, for 10 to 30 days, depending on the test species. There are also questions concerning the appropriate selection of laboratory test organisms. Given the small number of validated protocols for nontarget insect testing, one possible approach involves having a core set of test species for all PIPs that is supplemented by additional species representative of those groups most likely to be impacted by the specific plant pesticide product.

EPA Response: The registrants do not need to be in the dark about interpretation of the available guidance for protocol development. They are not left without guidance in that the case by case protocol developed includes consultation and guidance from EPA scientists. The selection of a core of appropriate non target species to be tested has been addressed in several workshops and research proposals by the EPA and USDA. In addition, in 2003 EPA has commenced the development of testing guidelines specifically for PIPs.

Commenter Name: Clifford Habig Commenter Organization Name: Exponent, Inc. Comment Number: OPP02 0017 Excerpt Number: 4 Excerpt Text: EPA has traditionally employed a risk quotient approach to risk assessment, in which toxicity endpoints are compared to estimated exposure to determine whether an adequate margin of safety exists for labeled or proposed uses of a product. Toxicity end points used for acute risk assessments are typically LD50, LC50, or EC50 values, while toxicity endpoints used for subchronic or chronic ( longer term) risk assessments are typically no observed effect concentrations ( NOECs) or no observed adverse effect concentrations ( NOAECs). In rare cases, sufficient data are available to calculate ECX values ( e. g., EC05, EC 10, EC20) to use as toxicity endpoints for risk assessment. Exposures of non target organisms are typically estimated by environmental modeling techniques or calculations based on generic databases. Risk assessments are conducted based on the results of laboratory testing and the estimated exposure; field testing data, when available is primarily used to collaborate or confirm risk conclusions based on laboratory derived data.

Non target organism risk assessments for conventional chemical pesticides are conducted for birds, mammals, and aquatic organisms ( fish and aquatic invertebrates). For conventional chemical pesticides, risk assessments for terrestrial arthropods and soil invertebrates are limited to a hazard assessment for honeybees. Toxicity endpoints are typically selected based on data for the most sensitive species tested Section 3.1 3  33 among representative species for each taxonomic group evaluated. Assessments of multiple application scenarios are generally based on a longer term exposure estimate that incorporates a dissipation component following each application.

Risk assessments for conventional microbial pesticides are conducted for birds, mammals, aquatic organisms, and non target terrestrial arthropods and soil invertebrates. However, these risk assessments are generally simplified by using the maximum hazard dose approach to testing, so that if no adverse effects occur at the maximum hazard dose ( concentration), the product is determined to present low risk to non target organisms. Thus, for microbial products, the exposure component of the risk assessment is built into the dosing regime used for testing.

The nature of PlPs results in unique exposure scenarios compared to conventional chemical or microbial pesticides. Exposures to conventional products focus on off site movement of pesticide residues to water bodies and estimated residues in/ on feed items and in soil. For PIPs, the specificity of the plantincorporated proteins and incorporation into specific plant tissues focuses exposure of non target organisms on dietary ingestion of plant products and exposure in soil. Patterns of expression of PIPs in plant tissues over the course of the growing season represent a key component of the exposure assessment; depending on the pattern of expression some off site exposure ( eg., pollen expression) is also possible. However, off site movement through processes such as drift or runoff is greatly reduced compared to conventional products, resulting in reduced risk to several key groups of non target organisms.

EPA Response: This comment does not require a response.

Commenter Name: Clifford Habig Commenter Organization Name: Exponent, Inc. Comment Number: OPP02 0017 Excerpt Number: 9 Excerpt Text: Conclusions  Non target organism testing requirements for PIPs should include both a core set of studies required for all PIPs and a supplemental set of studies that reflect the specific properties of any given PIP. The testing strategy for MON 863 followed this type of approach.  Current testing and risk assessment guidelines for PIPs, with the exception of the field data requirements, are similar to well tested processes for conventional chemical and microbial pesticides. Risk assessment procedures used for MON 863 follow this guidance.  Risk assessment decisions for PIPs should be consider not only the overall safety of the individual PIP product, but should also be compared to risks associated with existing conventional pest control technologies.

EPA Response: This comment does not require a response.

Commenter Name: Monsanto Section 3.1 3  34 Commenter Organization Name: Monsanto Comment Number: OPP02 0021 Excerpt Number: 2 Excerpt Text: In summary, Monsanto supports a scientific approach to risk assessment where well designed laboratory studies with surrogate species that represent the spectrum of potentially exposed species are used in an early tier assessment of ecological risk for Cry proteins. If suitable laboratory surrogate species tests are not available then semi field or field studies focused on particular indicator organisms may be appropriate. If field studies are undertaken, the specific questions to be answered should be clearly stated prior to initiation.

EPA Response: This commenter mirrors the approach that EPA is taking in non target risk assessment for PIPs. The comments from the October 2000 and August 2002 SAP also endorse this approach.

Commenter Name: Monsanto Commenter Organization Name: Monsanto Comment Number: OPP02 0021 Excerpt Number: 4 Excerpt Text: Question 2: Duration of Field Abundance Studies Please comment on the adequacy of the 2 year field abundance study for making determination of the potential risks from commercial use of event MON 863.

Response: Monsanto believes that a two year field abundance study is adequate for assessing the risk of corn event MON 863 to nontarget organisms. Results from the first and second year field study conducted by Monsanto indicated no adverse effects in key beneficial insects including families of nontarget beetles. Collaborative nontarget organism studies have been conducted with university researchers in Illinois, Iowa, Kansas, Nebraska, New York, and South Dakota. The results of these studies were consistent across sites and across years, and confirmed the safety of the MON 863 event to nontarget organisms. [ Footnote 5: Head, G. ( 2002). Research on the Effects of Corn Rootworm Protected Transgenic Corn Events on Nontarget Organisms: Preliminary Results. An unpublished report prepared by Monsanto Company. MRID 456530 03]

A thorough laboratory based risk assessment using quail, earthworms, Collembola, ladybird beetles, parasitoid wasps, lacewing, honeybees, monarch butterfly, daphnia and catfish, demonstrated minimal risk of MON 863 to nontarget organisms. The field study undertaken by Monsanto to provided additional information to support the laboratory based risk assessment, especially for nontarget beetles. The field study was conducted in Monmouth, Illinois and utilized 60x60 ft plots replicated four times per treatment. Conventional corn rootworm control programs using seed insecticide, soil incorporated insecticide and foliar insecticide treatments were included. The first year of the Monsanto data for key beneficial insects was collated and summarized in an interim report that was submitted to EPA. These data showed no adverse effects on important beneficial insects and closely related nontarget insects. [ Footnote 8: Bhatti, M. A., C. L. Pilcher, M. J. McKee, T. E. Nickson, G. P. Head and C. D. Pilcher ( 2001). Field Evaluation for Section 3.1 3  35 the Ecological Impact of Corn Rootworm Insect Protected Corn on Nontarget Organisms. Report MSL 17179, an unpublished study conducted by Monsanto Company. MRID 455382 06.] The second year of field data have been compiled and it is our conclusion that the findings confirm the first year data in that there were no adverse effects observed in the second year. [ Footnote 7: Preliminary results for two year field study, Monsanto unpublished data ( 2002)]

EPA Response: EPA has reviewed the two year field data summarized by this commenter for the registration of MON 863. However, with the advice of the SAP and other public commenters, EPA is requiring studies that extend beyond a two year period.

Commenter Name: Monsanto Commenter Organization Name: Monsanto Comment Number: OPP02 0021 Excerpt Number: 31 Excerpt Text: A) Please comment on the relative strengths and weaknesses of such field data vs. laboratory feeding studies performed on a limited number of indicator organisms, for purposes of hazard assessment.

Response: Monsanto supports a scientific approach to risk assessment where a well designed laboratory assessment with surrogate species that represent the spectrum of potentially exposed species is used in a tiered assessment of ecological risk for Cry proteins. If suitable laboratory surrogate species tests are not available, or if adverse effects are observed in these laboratory studies, then semi field or field studies focused on particular indicator organisms may be appropriate. In the case of the Cry3Bb1 protein analysis, a field assessment was conducted to provide supplemental information and reinforce the minimal risk conclusion reached based on the laboratory studies. Since this is a beetle active Cry protein, additional information was desired for nontarget Coleoptera species closely associated with the soil environment. Field studies were conducted to address potential effects on nontarget beetles, such as staphylinid or carabid beetles. Results of the field study indicated no adverse effects on nontarget beetle or other nontarget insect populations. [ Footnote 2: Bhatti, M. A., C. L. Pilcher, M. J. McKee, T. E. Nickson, G. P. Head and C. D. Pilcher ( 2001). Field Evaluation for the Ecological Impact of Corn Rootworm Insect Protectant Corn on Nontarget Organisms. Report MSL 17179, an unpublished study conducted by Monsanto Company. MRID 455382 06.]

Laboratory and field approaches to nontarget organism risk assessment are valid and complementary. Traditionally, data collection and risk assessment are approached in a tiered analysis, with higher tier studies being conducted where risk is indicated at the lower tiers. Monsanto believes that well designed laboratory assessments with surrogate species, representing the spectrum of potentially exposed species, are a valid method to assess ecological risk of Cry proteins. If laboratory testing guidelines/ study protocols are not available for certain surrogate species, then small scale semi field or field studies could be used to supplement the laboratory data. Though not necessary or required for the Cry3Bb1 protein analysis, a field assessment was conducted to provide supplemental information and reinforce the minimal risk conclusion from the laboratory assessment.

EPA Response: Section 3.1 3  36 EPA supports the proposal to perform laboratory non target effects testing supplemented by field survey data on those invertebrates that cannot be propagated in a laboratory setting. The August 2002 SAP has also recommended this approach.

Commenter Name: Monsanto Commenter Organization Name: Monsanto Comment Number: OPP02 0021 Excerpt Number: 32 Excerpt Text: Laboratory studies, compared to field studies, have the advantage of rigorous control of the amount of protein to which the organisms are exposed. In the nontarget organism laboratory studies, the diet containing the Bt protein is fed directly to the nontarget organism. The bioactivity of the protein is confirmed in the diet using a known susceptible species. A laboratory test can more adequately characterize the hazard potential ( intrinsic toxicity) of the protein than in a field study since the ingestion of the material in the diet can be readily observed/ monitored. Using results from these studies and ' comparing them to conservative estimates of field exposure provides a highly conservative approach to assessing ecological risk. [ Footnote 3: Suter, G. W. ( 1993). Ecological Risk Assessment. Lewis Publishers, Chelsea, Michigan] [ Footnote 4: EPA( 1998). Guidelines for Ecological Risk Assessment. EPA/ 630/ R 95/ 002F, April, 1998. Risk Assessment Forum, U. S. Environmental Protection Agency, Washington, DC] A potential disadvantage of laboratory testing is that validated testing protocols may not be available for all taxonomic groups that need to be included to meet the objectives of the ecological risk assessment.

For the Cry3Bb1 protein, the focus of the laboratory analysis was on beneficial arthropods, particularly pollen feeders, and soil invertebrates. For pollen feeders, standard laboratory tests available to assess potential effects of Bt proteins, including honeybee larva and adults, are ladybird beetle larvae and adults, and adult parasitoid wasp. These nontarget organisms were not affected at the maximum estimated exposure level Cry3Bb1 protein in the field. Where no effects are observed in organisms ingesting proteins at levels above what they will see in the field, minimal risk is indicated.

EPA Response: The above commenter is summarizing the EPA approach to non target risk assessment; no response is necessary.

Commenter Name: Monsanto Commenter Organization Name: Monsanto Comment Number: OPP02 0021 Excerpt Number: 33 Excerpt Text: For soil invertebrates, standard tests are available for Collembola ( soil detritivore) and earthworms, as these species are routinely used to assess the toxicity of chemical insecticides. Results of the studies where Collembola were fed tissues containing the Cry3Bbl protein and earthworm were exposed to the protein in the soil, clearly indicated that no toxicity would be expected at maximum estimated exposure levels in the field.

Typically, field studies are hi her tier and would only be conducted where risk is not excluded at lower Section 3.1 3  37 tiers. Minimal risk of Cry3Bbl protein to nontarget organisms is concluded from the standard laboratory assessment. However, since this is a beetle active Cry protein, Monsanto desired additional information for nontarget Coleoptera species closely associated with the soil environment. Since reliable and validated laboratory feeding studies for nontarget Coleoptera such as beetles in the families Carabidae and Staphyllinidae were not available, field studies were conducted to address potential effects on these nontarget beetles, such as staphylinid or carabid beetles. Results of the field study indicated no unexpected effects on nontarget beetles or any other nontarget organism. [ Footnote 2: Bhatti, M. A., C. L. Pilcher, M. J. McKee, T. E. Nickson, G. P. Head and C. D. Pilcher ( 2001). Field Evaluation for the Ecological Impact of Corn Rootworm Insect Protectant Corn on Nontarget Organisms. Report MSL 17179, an unpublished study conducted by Monsanto Company. MRID 455382 06.]

In addition to Monsanto conducted studies, a number of collaborative nontarget studies also have been conducted with university researchers in Illinois, Iowa, Kansas, Nebraska, New York, and South Dakota. All studies compared the impacts of MON 863 corn expressing the Cry3Bbl protein, with a conventional corn isoline and the same isoline treated with the soil insecticide used locally to control corn rootworm. These studies included broad surveys of the arthropods present, as well as studies that concentrated on particular taxonomic groups like carabids. Plot sizes were greater than an acre in several cases. In all of these studies, no consistent significant differences have been seen between the MON 863 plots and those with the untreated isoline with respect to any nontarget species. [ Footnote 5: Head, G. ( 2002). Research on the Effects of Corn Rootworm Protected Transgenic Corn Events on Nontarget Organisms: Preliminary Results. An unpublished report prepared by Monsanto Company. MRID 456530 03] However, the soil insecticides adversely impacted a number of nontarget species. Thus, the results of these studies were as predicted from the Tier 1 tests.

EPA Response: EPA has reviewed the studies described in this comment and incorporated the findings into the MON 863 environmental risk assessment.

Commenter Name: Monsanto Commenter Organization Name: Monsanto Comment Number: OPP02 0021 Excerpt Number: 34 Excerpt Text: B) The panel is requested to comment on the logistics, validity, cost and expected scientific gain, if any, of conducting a census of the invertebrate community vs. concentrating the studies on specific indicator organisms. In addition, please comment on the suggested indicator groups such as Carabids and Staphyllinids in the case of Cry3Bb1, that would be most likely to provide the Agency with meaningful data for assessing potential hazards to nontarget invertebrates from corn rootworm PIPS.

Response: Monsanto believes the value of conducting an in field census analysis of nontarget arthropods is limited because much of the sample collection, handling and identification resources are expended on species already well documented to be at minimal risk and the variability in field populations is high, thereby making it difficult to draw conclusions from field insect population studies. Therefore, there would be limited scientific benefit for studies that are very time and resource intensive to conduct. Monsanto believes that of appropriate surrogates for potentially exposed species is an acceptable way to approach the Section 3.1 3  38 ecological risk assessment. If potentially exposed and susceptible species are identified and cannot be tested in laboratory assays, then focused semi field or field studies would be a reasonable alternative. The interpretation of any semi field or field studies should be made relative to the conventional pest management program. As more knowledge is gained from current laboratory and field assessment programs, the need for field studies to assess the ecological risk of plant incorporated protectants should decrease.

Census of the invertebrate community requires collection of invertebrate samples, analysis of specimens and interpretation of data. Arthropod communities are assemblages resulting from complex interactions with the physical environment as well as with other species. For a community analysis, interpretation of census data must consider these interactions as well as biases created by the type of collection method used ( i. e., some species are more likely to be trapped by some methods than other species) and by taxonomic resolution. Ideally all specimens should be identified to the species level, however, the taxonomy is sometimes complex or expertise may be limited. Exposure of organisms to the protein in the field is also complex which makes it difficult to relate changes in the trapping success for individual species to exposure. Interpretation of in field census data is often complicated by a lack of understanding for the impacts of agricultural processes such as present and past tillage practices, fertilizer inputs, and crop varieties on arthropod populations. Many of these factors have caused difficulties in the interpretation of field data in EPA's Office of Pesticide Program initiative to use field tests to assess impacts of chemical pesticides ( e. g., mesocosm assessments). In balance, minimal scientific gain relative to risk assessment will be obtained by conducting a census of in field arthropod communities for Cry proteins.

EPA Response: The above comments are reasonably consistent with the conclusions of the August 2002 SAP. As a result efforts are under way to identify indicator invertebrate species or physiological groups that can be identified in semi field studies. Protocols for these and for full field studies with appropriate statistical power are in various stages of development.

Commenter Name: Monsanto Commenter Organization Name: Monsanto Comment Number: OPP02 0021 Excerpt Number: 35 Excerpt Text: Where additional information is considered necessary to supplement the standard laboratory assessment, the information can be obtained through additional laboratory testing or through focused semi field or field studies. For the Cry3Bb1 protein, prior laboratory testing indicated that only beetles are highly susceptible to the Cry3Bb1 protein, specifically beetles in the family Chrysomellidae. [ Footnote 6: Head, G., M. Pleau, S. Sivausupramanian and T. Vaughn ( 2001). Insecticidal Spectrum of Activity for CryBb1 Protein in vitro. Report C3NTO, an unpublished study conducted for Monsanto Company. MRID 455382 07] Therefore, potential risk to nontarget species closely related to the target organism was included by Monsanto as part of the assessment.

One taxonomic group specifically focused on in the Cry3Bb1 protein assessment was ladybird beetles, since these species are important biological control agents in agricultural ecosystems. To evaluate potential effects of Cry3Bb1 protein on ladybird beetles Monsanto conducted four laboratory studies with both Section 3.1 3  39 purified Cry3Bb1 protein and MON 863 pollen using two species, Hippodamia convergens and Coleomegilla maculata. Additionally, Monsanto conducted a two year field study that estimated the abundance of nontarget organisms in the soil, on the soil surface and on foliage. Five species of ladybird beetle, including Harmonia axyridis, Coccinella septenipunctata, H. convergens, Cycloneda munda and C. maculata were assessed in this field study. These data clearly demonstrate that larval and adult ladybird beetles will not be at risk from the corn rootworm product. The adoption of MON 863 will be a clear benefit to in field ladybird beetle populations where adult corn rootworm foliar insecticide control programs significantly can reduce ladybird beetle populations. [ Footnote 7: Preliminary results for twoyear field study, Monsanto unpublished data ( 2002)]

Other nontarget Coleoptera taxa that could be potentially exposed include carabid and staphyllinid beetles. Since dietary feeding test guidelines for representatives of these families were not available, Monsanto collected information on their general abundance in cornfields. Results of these studies indicate that abundance of nontarget Coleoptera families was no different on MON 863 compared with the untreated nontransgenic corn isoline. Conventional insecticide chemical the control programs did tend to decrease the abundance of some of these nontarget Coleoptera families.

EPA Response: EPA has reviewed the studies described in this comment and incorporated the findings into the MON 863 environmental risk assessment.

Commenter Name: Monsanto Commenter Organization Name: Monsanto Comment Number: OPP02 0021 Excerpt Number: 36 Excerpt Text: Question 3: Green Lacewing Larva Test The Agency solicits the Panel's comments on an appropriate design for evaluating the toxicity of Cry3Bb1 proteins to lacewing larvae.

Response: Monsanto believes that the green lacewing study conducted with Cry3Bb1 protein treated moth egg diet is both practically and scientifically justified. The no observable effect concentration ( NOEC) from the lacewing study greatly exceeds the maximum expected environmental concentration ( MEEC), indicating minimal risk from exposure to MON 863. Results of field scale assessment of nontarget organism abundance ( see question 2) confirm minimal risk to green lacewing larvae under field conditions.

The green lacewing study conducted by Monsanto involved exposing newly hatched larvae to moth egg diet treated with purified Cry3Bbl protein at the maximum diet concentration of 8,000 ppm for a period of ten days. To determine if this exposure regime was capable of detecting an adverse effect of the test substance, moth ego, diet treated with arsenate ( a known stomach poison) was concurrently tested as a positive control. No significant differences in mortality were detected between Cry3Bb1 protein and water control diet treatments ( 23% vs 27%), while the arsenate diet treatments resulted in significant mortality of the test larvae ( 43% at 1,000 ppm and 100% at 10,000 ppm). The significant mortality of the lacewing larvae exposed to the arsenate treated diets indicated that the test system used in the study was capable of Section 3.1 3  40 detecting adverse effects of a toxicant applied to the moth egg diet.

Arsenical compounds have historically been designated as insect stomach poisons, as opposed to contact poisons, because contact with arsenicals does not provided control of insect pests; the insect pests must ingest these toxins to achieve adequate control. [ Footnote 9: Metcalf, C., W. Flint and R. Metcalf ( 1962). Pp. 1087. In Destructive and Useful Insects: Their Habits and control. McGraw Hill Book Company, New York, New York.] Although this observation does not preclude the possibility of dermal penetration, it does indicate that dermal penetration does not take place at a rate capable of producing substantial lethality. Furthermore, the arsenate treated moth egg diet used in the study was supplied to the lacewing larvae on a very limited area of the test arena ( only on the bottom of the test arena), and direct tarsal contact with arsenate treated moth egg diet by test larvae, if any, would have been limited. Thus, the significant lethal effect of the arsenate treatment detected in this study was likely to have resulted from ingestion of lethal doses of the toxicant by test larvae via feeding the treated moth egg diet, rather than from tarsal contact to the treated diet.

EPA Response: Comments at the August 2002 SAP cast doubt on the validity of the use of moth eggs for lacewing larval studies. It was noted that the Cry protein coats the outside of the eggs, while the lacewing larvae pierce the eggs and feed on the egg contents, thus it is not clear whether the lacewing larvae actually ingest Cry protein coating the outside of the egg. Likewise, arsenate was said to very likely diffuse into the egg contents and give a false sense of a positive control, whereas the Cry protein would appear to give negative results simply because of the inability to get into the eggs. As a result efforts are in progress to develop an artificial lacewing larva diet which can be mixed with Cry protein.