Commenter Name: Monsanto Commenter Organization Name: Monsanto Comment Number: OPP02 0021 Excerpt Number: 37 Excerpt Text: Some published studies have evaluated the toxicity of other Bt Cry proteins by using either encapsulated Bt Cry protein treated liquid artificial diet [ Footnote 10: Hilbeck, A., W. J. Moar, M. Pusztai Carey, A. Filippini and F. Bigler ( 1998). Toxicity of Bacillus thuringiensis Cry1Ab Toxin to the Predator Chrysoperla carnea ( Neuroptera: Chrysopidae). Environ. Entomol. 27: 1255 1263.] or natural prey pre fed with Bt Cry protein treated diet. [ Footnote 11: Hilbeck, A., M. Baumgartner, P. M. Fried and F. Bigler ( 1998). Effects of Transgenic Bacillus thuringiensis Corn fed Prey on Mortality and Development Time of Immature Chrysoperla carnea ( Neuroptera: Chrysopidae). Envrion. Entomol. 27: 480 487.] [ Footnote 12: Hilbeck, A., W. J. Moar, M. Pusztai Carey, A. Filippini and F. Bigler ( 1999). Prey mediated Effects of Cry1Ab Toxin and Protoxin and Cry2A Protoxin on the Predator Chrysoperla carnea. Entomologia Experimentalis et Applcata 91: 305 316.] [ Footnote 13: Meier, S. M. and A. Hilback ( 2001). Influence of Transgenic Bacillus thuringiensis Corn fed Prey on Performance of Immature Chrysoperla carnea ( Neuroptera: Chyrsopidae). Basic Appl. Ecol. 2: 35 44.] Hilbeck et al. fed the green lacewing larvae with natural prey ( larvae of Spodoptera littoralis) reared on the diet containing a lepidopteran active Cry protein, and observed adverse effect of the " Bt Cry protein intoxicated" prey on the mortality of test lacewing larvae. However, recent studies indicate that this adverse effect from secondary exposure to Bt toxins on the lacewing larvae is largely due to the " sickness" of the " Bt toxin" Intoxicated prey, rather than Section 3.1 3  41 ingested Bt toxins per se. [ Footnote 14: Dutton, A., H. Klein, J. Romeis and F. Bigler ( 2002). Uptake of Bt toxin by Herbivores on Transgenic Maize and Consequences for the Predator Chrysoperla carnea. Ecological Entomol. ( in press)] Therefore, tri trophic studies with natural prey exposed to Cry proteins may not be appropriate in determining the hazard of the Bt Cry protein toxin to green lacewings in the firsttier toxicity studies. In addition, recent studies indicated that some of the natural prey preferred by the green lacewing, such as aphids, feed from the phloem sap of transgenic Bt plants, and do not have detectable levels of the Cry protein in their body. [ Footnote 15: Raps, A., J. Kehr, P. Gugerli, E. J. Moar, F. Bigler, and A. Hilbeck. 2001. Immunological analysis of phloem sap of Bacillus thiringiensis corn and of the nontarget herbivore Rhopalosiphum padi ( Homoptera: Aphididae) for the presence of Cry1Ab. Molecular Ecology 10: 525 533.] [ Footnote 16: Head, G. P., C. R. Brown, M. E. Groth and J. J. Duan. ( 2001). Cry1Ac Protein Levels in Phytophagous Insects Feeding on Transgenic Corn: Implications for Secondary Exposure Risk Assessment. Entomologia Experimentalis et Applicata: 99: 37 45.] Thus in nature, exposure to plant produced Cry proteins for lacewing via tri trophic interactions ( i. e., secondary exposure) would be extremely limited.

In summary, we believe that the green lacewing study conducted by Monsanto with Cry3Bbl proteintreated moth egg diet is both practically and scientifically Justifiable and was deemed acceptable by EPA. Based on our laboratory study as well as field studies, minimal risk of event MON 863 is expected for the green lacewing.

EPA Response: An independent review of the cited published studies by the EPA has reached the same conclusions which is that the tritrophic studies on green lacewing do not apply to the corn field environment where the prey, such as aphids, do not ingest BT toxin while feeding on the phloem of the corn plant. In addition, EPA has determined that in the reported tritrophic studies the prey were fed excessive doses of Cry protein in an artificial diet, which far exceeded the amounts present in corn plants. Nevertheless, EPA intends to ask for tritrophic studies where they would appear to be warranted by the ecology of the crop in question. EPA disagrees with the commenter on the usefulness of the lacewing testing as performed by Monsanto. 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 which cannot get into the eggs. 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 penetrate the outside of the eggs. As a result efforts are in progress to develop an artificial lacewing larva diet which can be mixed with Cry protein.

Commenter Name: Monsanto Commenter Organization Name: Monsanto Comment Number: OPP02 0021 Excerpt Number: 38 Excerpt Text: Question 5: Preliminary Risk Assessment for Nontarget Invertebrates and Soil Fate Please comment on the Agency's nontarget invertebrate and soilfate assessment. Section 3.1 3  42 Response: The collective evidence of laboratory and field studies submitted by Monsanto consistently showed no adverse effects on nontarget species, including nontarget beetle families, at maximum estimated environmental concentrations of the CRW protein. The process by which Monsanto, in communication with EPA, approached the ecological risk assessment for MON 863 was logical, utilized state of the art methods for assessing ecological risk, and incorporated input from recent EPA Science Advisory Panel reports. The initial laboratory based risk assessment was conducted similar to that for past plant incorporated pesticide products. Since Cry3Bb1 is a known beetle active protein, the need was identified to supplement the standard laboratory assessment with more data on nontarget beetles. Since standard laboratory dietary tests did not exist for nontarget beetles, Monsanto conducted a field study to provide the additional information, as well as encouraged independent academic institutions to engage in field research. The overwhelming conclusion from all of these data is that use of event MON 863 will result in minimal risk to nontarget organisms.

The laboratory assessment for MON 863 was based on EPA guidance and previous testing with pesticide incorporated pesticides. Risk characterization was based on Office of Pollution Prevention and Toxic Substances ( 1996) 885.4000 Guidelines which state ( p. 3), "... data that establishes an LC50, ED50, or LD50 that is greater than the maximum hazard dosage level ( e. g., LD50> 1,000 mg/ kg) would often be adequate for the purposes of hazard assessment". For all of the nontarget organism studies conducted with the Cry3Bbl protein, the LC50 was found to be greater than the highest concentration tested ( i. e., maximum hazard dose), indicating minimal risk. The ratio of the LC50: MEEC was greater than 10 for all species tested ( including wasp, earthworm and ladybird beetle.), except the adult honey bee. The LC50 for adults honey bees was determined to be greater than the highest concentration tested, 360 ug Cry3Bbl protein/ g diet, was 4.3 times the MEEC ( i. e., 93 ug Cry3Bbl protein/ g pollen). Since the NOEC is 360 ug/ g and this exposure will not be realized under field conditions, minimal risk is indicated for adult honey bees as well. In addition to standard GLP data on Coccinellidae ( nontarget), Monsanto has conducted laboratory bioassays with representative species of the Bruchidae, Tenebrionidae, and Curculionidae families and found no evidence of toxicity in these nontarget Coleoptera families. [ Footnote 19: Head, G., M. Pleau, S. Sivausupramanian and T. Vaughn ( 2001). Insecticidal Spectrum of Activity for Cry3Bb1 Protein in vitro. Report C3NTO, an unpublished study conducted for Monsanto Company. MRID 455382 07.] Consequently, the laboratory studies conducted by Monsanto indicate minimal risk of MON 863 to nontarget organisms.

EPA Response: The above commenter is summarizing the EPA approach to non target risk assessment. EPA is in agreement with these comments.

Commenter Name: Monsanto Commenter Organization Name: Monsanto Comment Number: OPP02 0021 Excerpt Number: 39 Excerpt Text: Monsanto has submitted a soil degradation study for Cry3Bbl protein using field collected soil that indicates the protein will dissipate rapidly under field conditions. [ Footnote 17: Martin, J. W., M. J. McKee, S. Dubelman and Y. A. Dudin ( 2000). Aerobic Soil Degradation of the B. t. Protein 11098 as a Component of Insect Protected Corn. Report MSL 16440, an unpublished study conducted by Monsanto Company. Section 3.1 3  43 MRID 451568 04.] The design of this study employed exaggerated doses to simulate worst case soil deposition from the variety of potential mechanisms, including potential secretion, shedding of root hairs, degradation of biomass and pollen deposition. The calculated DT50 ( time to 50% degradation) was 2.4 to 2.8 days, and the calculated DT90 ( time to 90% degradation) was 7.9 to 9.2 days. There was no detection of Cry3Bbl, by either ELISA or insect bioassay, in any sample incubated for over 21 days.

Field investigations were initiated in 2000 by Monsanto and separately by a number of university researchers across multiple locations to validate the prediction of minimal risk to nontarget arthropods in the field. Interim reports summarizing the data collected for key nontarget and target species were submitted to EPA in 2001 and 2002. These data indicate no significant 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 a minimal risk to nontarget organisms, including beetle families commonly found in agricultural settings.

The collective evidence of laboratory and field studies submitted by Monsanto consistently showed no adverse effects on nontarget species at maximum estimated environmental concentrations of the Cry3Bb1 protein. Another important consideration, sometimes overlooked, is the long history of safe agricultural use of Bt products in general, and specifically, the Cry3 class of Bt products presently used in traditional and organic agricultural systems ( e. g., Raven Oil Flowable Bioinsecticide). Finally, any analysis of the potential risks and benefits of a new technology must be considered in the context of existing pest management practices and systems. The Cry3Bbl protein is less toxic to nontarget organisms than existing organophosphate and pyrethroid insecticides currently approved and commonly used to control corn pests, including CRW. Furthermore, in addition to the lower toxicity of the Cry3Bb1 protein, the in plant delivery system further reduces exposure to nontarget organisms since target organisms must be closely associated with the plant tissue to be exposed.

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

Commenter Name: John Foster Commenter Organization Name: John Foster Comment Number: OPP02 0027 Excerpt Number: 1 Excerpt Text: Having been involved with the Bt corn and issues focused on the monarch butterfly also made several things clear to me. First, it is easy to focus on potential hazards of new technologies and lose sight of its potential benefits ( and the risks associated with existing technologies). Secondly, with a new technology, appropriate field studies are the only way to really understand the performance of a product. Over the. past 3 years, we have been conducting field research on the impact of MON 863 on non target arthropods at multiple sites in Nebraska. We have compared MON 863 with standard used soil insecticides, we have used increasing plot sizes as seed became available, and variety of standard sampling techniques, and we have focused on a number of groups of arthropods that are of ecological and economic importance, and that might be impacted by a transgenic coleopteran active protein that is expressed in the roots and above Section 3.1 3  44 ground tissues of a corn plant.

To date, when comparing the arthropods found in plots of MON 863 with those in plots containing untreated isolines, we have seen no significant adverse effects of MON 863 on any non target group. We have seen some differences in the communities among sites but nosignificant impacts of the variety ( MON 863 compared to its isoline). Our analyses have included ground dwelling beetles, Collembola, and spiders. Furthermore, I also have communicated with other academics performing similar studies, with comparable results, over the past 3 years. With locations ranging from New York to South Dakota to Kansas, and a variety of techniques and points of focus, and with very large plot sizes in some cases, these studies represent a comprehensive investigation of the potential impact of MON 863 on agro ecosystems. In all of the studies that I am aware of, no significant adverse impacts have been observed. Together with the results generated by my students in my laboratory, these studies give me confidence that MON 863 is a safe product with respect to non targets, and hence, the EPA's ecological assessment is an accurate one. Finally, I know what the currently used soil insecticides can do to agro ecosystems. In Nebraska, current technologies used to control corn rootworm have obvious adverse effects on many non target species.

EPA Response: The above commenter confirms EPA's ecological risk assessment on MON 863 as accurate from his personal experience and asserts that MON 863 is much friendlier to the ecosystem than conventional pesticide treatments. EPA concurs with these comments.

Commenter Name: John Foster Commenter Organization Name: John Foster Comment Number: OPP02 0027 Excerpt Number: 2 Excerpt Text: I believe that the introduction of MON 863 has the potential to bring clear and measurable ecological benefits to corn production systems in terms of reduced insecticide usage and hence reduced worker exposure EPA Response: EPA concurs with this comment.

Commenter Name: N/ A Commenter Organization Name: Center for Science in the Public Interest Comment Number: OPP02 0028 Excerpt Number: 5 Excerpt Text: 3. Monsanto did not conduct non target organism toxicity testing according to EPA guidelines. Several of the non target organism tests used to predict environmental risks were not conducted with sufficiently high doses of Cry3Bb1. Monsanto should repeat non target testing at maximum hazard doses ( at least 10 fold higher than the maximum expected environmental exposure) where those doses were not used.

EPA Response: Section 3.1 3  45 Contrary to the claims by this commenter, examination of the submitted non target studies shows that these were performed with 20x or higher safety margins, except where pure pollen of plant tissue were used and where concentration of the Cry protein cannot be increased. The parasitic hymenoptera were in fact tested at only 1X field concentration in plants rather than at 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 level will not be realized under field conditions), minimal risk to adult honey bees is expected.

Commenter Name: N/ A Commenter Organization Name: Center for Science in the Public Interest Comment Number: OPP02 0028 Excerpt Number: 6 Excerpt Text: 4. Monsanto did not conduct maximum hazard dose tests of important non target beetle and nematode species. Additional beetles species, such as ground beetles, should be tested because beetles are most closely related to CRW and therefore most likely to be harmed by Cry3Bb1. Monsanto only tested two ladybird beetle species, and they were not tested at a maximum hazard dose. In addition, further testing of free living nematodes should be conducted since preliminary tests showed significant population reduction of two nematode species when exposed to MON 863 corn.

EPA Response: Contrary to the claims by this commenter, one lady beetle study was conducted at the maximum hazard dose ( 20xEEC), and three other lady beetle studies were conducted with pollen where the Cry protein content cannot be manipulated in the laboratory. Protocols for testing of additional ground beetles are being developed . In the meantime EPA made a risk assessment on these species from field survey results. EPA has requested additional data in order to make a risk assessment. An initial observation is that an adverse effect on parasitic nematodes in the corn root zone may have beneficial effects.

Commenter Name: N/ A Commenter Organization Name: Center for Science in the Public Interest Comment Number: OPP02 0028 Excerpt Number: 18 Excerpt Text: According to EPA's Microbial Test Guidelines ( 8), EPA can require the testing of different non target organisms than are specified if the agency believes that those other organisms would be more appropriate. EPA should require Monsanto to perform maximum hazard dose tests with several additional Coleoptera, such as ground beetles and other species, to gain confidence that MON 863 will not harm beneficial insects found in corn fields.

Only two species of Coleoptera, both ladybird beetles, have been tested for sensitivity to MON 863, and in at least one case, the concentration of Cry3Bb1 was too low. Since Coleoptera are more closely related to the target organisms than most of the indicator species currently used, and therefore have a higher Section 3.1 3  46 likelihood of being susceptible to Cry3Bbl, EPA should require appropriate maximum hazard dose testing of additional beetle species.

In addition, preliminary results strongly suggest that free living nematodes may be harmed by CryBb1 ( 10). The highest concentration used in those tests was about 10 times less than EEC ( based on the root concentration of Cry3Bb1 in MON 863), and was therefore 100 1000 fold below the appropriate maximum hazard dose. Several species of free living nematodes should be included in additional maximum hazard dose tests ( see section III. C.).

EPA Response: EPA has required additional non target beetle testing. Protocols for these are being developed under the auspices of the ABSTC. In the meantime EPA made a risk assessment on these species from observations of their abundance in corn fields. Multi year field surveys are also addressing this issue. Regarding the lady beetle comment, one lady beetle study was conducted at the maximum hazard dose ( 20xEEC), and three other lady beetle studies were conducted with pollen where Cry protein content cannot be manipulated in the laboratory. EPA requires maximum hazard dose testing whenever the testing conditions permit it. However, the commenter should note that in the final risk assessment when the LD50 and the EEC are factored together, an LD50 greater than 5xEEC is considered as acceptable. EPA has requested additional data in order to make a risk assessment. The initial observations also indicate that an adverse effect on parasitic nematodes in the corn root zone may have beneficial effects.

Commenter Name: N/ A Commenter Organization Name: Center for Science in the Public Interest Comment Number: OPP02 0028 Excerpt Number: 30 Excerpt Text: A. High Dose Non Target Organism Acute Toxicity Tests These tests were not performed in accordance with EPA's microbial test guidelines that are also used to evaluate genetically engineered plant incorporated protectants ( 8), or in accordance with the recommendations of an EPA convened SAP that evaluated thhose guidelines ( 9). Monsanto did not use maximum hazard doses in several of its non target organism tests.

Acute maximum hazard dose toxicity tests should be performed at a high multiplicity of expected exposure to increase the possibility that any adverse effects would be detected in those short term tests. Also, use of a high dose increases the chance that adverse effects on a less sensitive test species may be detected. Different levels of sensitivity between species have commonly been recognized with Cry proteins, such as between Colorado potato beetle and CRW in response to Cry3Bb1.

Instead of using the accepted definition of maximum hazard dose, which is typically at least 10 100 times the estimated environmental concentration ( EEC), Monsanto's tests are based on the absolute concentration of Cry3BbI in the plant for non soil organisms ( 11). Monsanto characterizes this as "... very high dietary concentrations ( Table 7)" ( 12). While Table 7 of Monsanto's safety assessment document ( 12) confirms that high concentrations of Cry3Bb1 were used compared to some tests for previous Bt toxins, the table fails to disclose that those other Bt toxins were expressed at many times lower concentrations in crop plants Section 3.1 3  47 than Cry3Bb1 in MON 863. The absence of comparative expression data in Table 7 gives a false impression of greater margins of exposure for MON 863 in toxicity tests than actually occur.

EPA's SAP stated the importance of maximum hazard dose testing, saying "... the higher the maximum dose the better." The Panel recognized "... that it is not always practical to use the 100X [ EEC] level" but that the maximum hazard dose should remain at least 10 times the EEC ( 9).

EPA Response: The EPA testing guidelines have been reviewed by two previous SAPs ( 1996, 1999). The hazard assessment process is explained in Section C. I. A of the MON 863 BRAD Document. Briefly, the process is as follows: the testing is performed at the prescribed maximum hazard dose ( MHD), mortality is the end point, and if greater tan 50 % mortality is seen at the MHD, then a LD50 has to be determined. If the LD50 is at least 5x the EEC ( RQ 0.2) then a no risk finding is made. It is notable that the guidelines state that the reason for requiring MHD testing is to reduce the cost of LD50 determinations, not to detect low level effects as claimed by the commenter. This process also makes it apparent that testing at only 5x without apparent mortality can also be used for risk assessment under extenuating circumstances. Examination of the submitted non target studies for MON863 shows that these were performed with 20x or higher safety margins, except where pure pollen of plant tissue were used where concentration of the Cry protein cannot be increased. The parasitic hymenoptera were in fact tested at only 1X field concentration in plants rather than at 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, which was 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. The goal of risk assessment is to determine what the effects are likely to be at the field concentration. Toxicity at levels higher than the field concentration are not indicative of hazard in the field.

Commenter Name: N/ A Commenter Organization Name: Center for Science in the Public Interest Comment Number: OPP02 0028 Excerpt Number: 31 Excerpt Text: While Monsanto's use of MON 863 corn tissue in several non target tests is useful for detecting possible unintended deleterious changes in the plant, the dose of Cry3Bb1 delivered in those tests is not high enough for acute maximum hazard testing. In tests with some organisms, Monsanto used purified Cry3BbI protein at higher concentrations than occur in MON 863, but those concentrations remain below the recommendations of both EPA testing guidelines and the SAP. EPA should require Monsanto to repeat maximum hazard toxicity tests at 100 times higher than the EEC for the following invertebrates where the test dose was less that 10 times higher than the EEC: adult honeybee, parasitic hyrnenoptera, earthworm, and the ladybird beetle ( Coleoineggilla maculam). The SAP recognized that a 100X dose may sometimes not be achievable. If that is the case with some of the above organisms, a dose between 10X and I00X should be used and justified.

In addition, Monsanto should be required to repeat those tests for other non target insects that were Section 3.1 3  48 conducted at less than I 00 times EEC, unless the dose used by Monsanto can be justified. Those organisms include:. collembola, larval honeybee, ladybird beetle ( Hippodainia covergens), and green lacewing.

The method used by Monsanto to determine the soil EEC of 13.3 ug Cry3Bbl/ g soil underestimates the exposure of soil organisms ( see section II. D. " Persistence of MON 863 in the Soil"). The EEC should be based, as a minimum, on the concentration of Cry3Bb1 in the roots of MON 863 corn, or 58 ug Cry3Bbl/ g ( 13), since root herbivores, detritus feeders, or their predators and parasites could be exposed to those levels of Cry3Bbl. On this basis, the tests performed by Monsanto on collembola were only about 15 times the more realistic EEC, while the earthworm test was done at EEC levels.

In summary, the only tested non target invertebrate where the dose met  or exceeded the desired 100 times EEC was daphnia. Even if doses between 10 times and 100 times EEC are accepted, only four terrestrial insect species were adequately tested, including only a single beetle species. While EPA microbial pesticide test guidelines only recommend testing of three non target insect species, those guidelines are not always appropriate for plant pesticides. The SAP ( 9) recognized that plant pesticides differ significantly from microbial pesticides and may require testing of more than three non target insect species. It is important to test additional Coleoptera species since important non target species related to the target corn rootworm. ( Coleoptera) are often common in corn fields ( see section III. B.).

EPA Response: Testing of aquatic invertebrates at 100x the terrestrial EEC is not a requirement when aquatic exposure is low to nonexistent. The EPA testing guidelines have been reviewed by two previous SAP's ( 1996, 1999). The hazard assessment process is explained in Section C. I. A of the MON 863 BRAD Document. Briefly, the process is as follows: the testing is performed at the prescribed maximum hazard dose ( MHD), mortality is the end point, and if greater tan 50 % mortality is seen at the MHD, then a LD50 has to be determined. If the LD50 is at least 5x ( 10x for aquatics) the EEC ( RQ of 0.2 and 0.1 respectively) then a no risk finding is made. It is notable that the guidelines state that the reason for requiring MHD testing is to reduce the cost of LD50 determinations, not to detect low level effects at unrealistically high doses as claimed by the commenter. This process also makes it apparent that testing of terrestrial animals at only 5x without apparent mortality can also be used for risk assessment under extenuating circumstances. Examination of the submitted non target studies for MON863 shows that these were performed with 20x or higher safety margins, except where pure pollen of 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 at 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, which was 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. The goal of risk assessment is to determine what the effects are likely to be at the field concentration. Toxicity at levels higher than the field concentration are not indicative of hazard in the field. The EPA has the option to agree to testing conditions that deviate from the testing guideline requirements, and EPA agreed that Monsanto could test the collembolla and some lady beetles in the presence of 50% MON863 plant tissue. Monsanto did perform an earthworm study at 10x Section 3.1 3  49 EEC concentration ( 570 mg Cry3Bb1 protein/ kg dry soil) ( MRID# 449043 16). Protocols for additional soil insects are being developed, and multi year field surveys are being commenced to evaluate the effects on the soil invertebrates and their interactions.

Commenter Name: N/ A Commenter Organization Name: Center for Science in the Public Interest Comment Number: OPP02 0028 Excerpt Number: 32 Excerpt Text: Greater reliance on non target field data was recommended by a recent SAP, and is a welcome part of the environmental assessment of MON 863. However, only preliminary results of those field tests are currently available, and those results are insufficient to draw conclusions about environmental impacts of MON 863. Field testing typically needs to be conducted for several years before enough data can be altered, because it involves more uncontrollable variables than laboratory tests. In addition, environmental conditions that may influence results are variable and often sporadic, and may only be encountered after several years. Therefore it is not possible to predict whether Monsanto's proposed two years of field testing will be sufficient. EPA should require Monsanto to continue its field tests for as long as necessary to acquire acceptable data rather than for a period fixed two years as proposed by Monsanto.

In addition to concern about the duration of the field tests, Monsanto's experimental design does not adequately address certain limitations of small field tests. For example, edge effects, such as the immigration of certain beneficial organisms, can mask adverse effects occurring in small field tests. Several of the current field tests do not address the issue of possible edge effects. Monsanto should explicitly address such effects to the extent feasible in all of the current field tests, or at least attempt to address how uncontrollable edge effects may influence those tests.

In preliminary experiments, two of three species of nematodes, including a plant pathogen and a soil inhabiting ( free living) species, suffered significantly reduced populations when exposed to MON 863. The free living species was exposed to a root homogenate ( root extract) that was diluted 10 fold compared to the Cry3Bbl concentration in MON 863 roots ( 10). The investigator concluded that because the free living nematode species were not harmed by a MON 863 soil leachate used in another test, free living species probably were not at risk. This conclusion was based on the investigators' unsupported assessment that the leachate more accurately represents the likely means of exposure of free living nematodes in MON 863 fields.

That conclusion does not account for the many untested species of nematodes that occur in soil and occupy many different important ecological niches. For example, species that feed on detritus containing MON 863 roots may be exposed to higher levels of Cry3Bb I than occur in the leachate or root extracts. Some untested species of nematodes may be more sensitive than the tested species, and therefore susceptible to the concentrations lower than found in root extracts. It should also be noted that some Cry5 and CryI4 proteins are toxic to nematodes. Cryl4AI is toxic to nematodes and CRW, so it may not be surprising that Cry3Bbl is toxic to both CRW and nematodes ( 14).

EPA Response: EPA did require Monsanto to continue its field surveys to evaluate the effects on the soil invertebrates and Section 3.1 3  50 their interactions. USDA and also EPA are sponsoring research to develop statistically appropriate large scale field testing protocols to overcome effects like the " edge effects " mentioned by this commenter. EPA has requested additional data in order to make a nematode risk assessment. However, the initial observations also indicate that an adverse effect on parasitic nematodes in the corn root zone may have beneficial effects.

Commenter Name: N/ A Commenter Organization Name: Center for Science in the Public Interest Comment Number: OPP02 0028 Excerpt Number: 33 Excerpt Text: Furthermore, the leachate method may greatly underestimate the concentration of Cry3Bb1 in MON 863 corn field soils. That is because the method used to make the leachate  suspension of soil that contained MON 863 corn plants in an aqueous so solution followed by centrifugation, where the supernatant is the leachate  would probably leave most of the Cry3Bb1 in the soil based on the tight binding of active Cry protein to soil clay particles ( 15,16). Previous studies have shown that aqueous solutions similar to that used in the Monsanto submission are leave much of the active Cry protein in the soil ( 15,16). Notably, the researchers did not indicate that they tested the leachate for the presence or concentration of Cry3Bbl.

In addition, the a second free living nematode species unaffected by the MON 863 root extract was reported not to be feeding. Since ingestion is required for Cry protein toxicity, that test was not valid. Therefore, both of the species that were actually exposed to Cry3Bb1 in a realistic manner were harmed. Those results suggest that many other nematode species might also be harmed by Cry3Bb1.

Since nematodes are important and abundant soil organisms, EPA should require Monsanto to perform additional testing of nematodes to determine the impact of Cry3Bb1 prior to registration. Such testing must include the nematode species already examined, field studies on nematode populations, as well as maximum hazard tests and field tests on additional free living nematode species.

Finally, even after several years of testing, the data from field experiments often result in a substantial amount of statistical variation that can mask adverse effects. In addition, some environmental impacts may take many years to develop. A recently published report by the National Research Council ( 17) recognized those limitations of small scale field trails and recommended follow up monitoring programs. EPA should work with USDA/ APHIS to develop such programs.

EPA Response: USDA and also EPA are sponsoring research to develop statistically appropriate large scale field testing protocols to overcome new insect incursion effects. EPA has requested additional data in order to make a nematode risk assessment. However, the initial observations also indicate that an adverse effect on parasitic nematodes in the corn root zone may have beneficial effects. Complete nematode effects assessment was not required prior to registration. Current agricultural practices such as use of fertilizers, soil pH manipulation, chemical pesticide and herbicide uses, as well as soil fumigation and sterilization practices all have a detrimental impact on nematode populations. Therefore immediate resolution of the possible effects on nematodes by MON863 corn does not appear critical. Section 3.1 3  51 Commenter Name: Teresa Gruber Commenter Organization Name: Council for Agricultural Science and Technology Comment Number: OPP02 0037 Excerpt Number: 4 Excerpt Text: In general, the EPA has identified and considered a reasonable/ rational set of taxa and species for pestincorporated protectants. Some additional attention to specific species related to the target pest maybe appropriate. In the case of Cry3Bb1, the protein is an effective toxin to a few species of Coleoptera, therefore some emphasis should be placed on other Coleoptera typically present in cornfields or in the soil with some history of corn production. Thus, the Coccinelids ( ladybird beetles) were logical choices for study because of their relatedness to the target pest and their utility as natural enemies in cornfields. Tests and resulting, decisions should emphasize concentrations of the toxin likely to be encountered by natural enemies and other nontarget organisms under natural/ field conditions.

EPA Response: EPA has required additional non target beetle testing. Protocols for these are being developed under the auspices of the ABSTC. In the meantime EPA made a risk assessment on these species from observations of their abundance in corn fields. Additional multi year field surveys are also addressing this issue.

Commenter Name: Angelika Hilbeck Commenter Organization Name: Greenpeace Comment Number: 30509 113000Part2 Excerpt Number: 18 Excerpt Text: Current knowledge of Bacillus thuringiensis spores and crystal proteins, and their inactivation in soil Bacillus thuringiensis seems to be indigenous to many different environments since it has been isolated worldwide from many habitats such as soils, insects, stored product dusts. and deciduous and coniferous leaves ( Schriepf et al. 1998). It produces large amounts of insecticidal delta endotoxins during sporulation. In these bacteria, the endotoxins are present in a crystalline, biologically inactive form. Microbial Btinsecticides targeting lepidopteran pests typically contain Bt proteins of the Cryl class. In addition to Cryl 6 endotoxin crystals, spores produced by Bacillus during the fermentation process also are present in these insecticides ( Feitelson et al. 1992). Other potential bioactive substances occur in many Btinsecticides, such as VIPs ( vegetative insecticidal proteins), phospholipases, zwittermycin, carrier and formulation ingredients, all of which exert an insecticidal effect in concert. This potpourri of biochemicals is ingested by the insects. The Bt crystals must undergo a complex cascade of biochemical reactions before they become active against susceptible insects. Firstly, the crystals must be dissolved in an alkaline midgut milieu and the presence of certain enzymes to yield a biologically inactive protoxin of 130 kDa in molecular weight for the type of Bt toxin produced in currently commercially available lepidopteran toxic Bt com ( except Cry9C corn) and Bt cotton. Secondly, again in a stepwise chain of reactions, a 65 kDa toxic fragment is proteolytically cleaved from the protoxin. This fragment, commonly referred to as the activated toxin, binds to receptors located on the epithelium of the insect midgut. The receptor bound toxin then induces pore formation leading to lysis of the midgut which results in the death of the insect. For induction of death of a susceptible insect, binding and pore formation are essential. However, all steps proceeding pore formation are equally crucial for determining the specificity of the Bt proteins in killing insects Section 3.1 3  52 ( Goldburg and Tjaden 1990) ( Figure 2). Lu transgenic plants, the truncated semi activated protein is expressed and will be ingested by the herbivorous insects feeding on it. Consequently, no crystal solubilization and relatively little protoxin toxin conversion is necessary within the insect gut. It is conceivable that insects lacking the appropriate midgut enzyme composition or pH milieu may well have appropriate receptors. [ see PDF file for " Figure 2. Differences Bt insecticides and Bt plants"]

EPA Response: This comment does not require a response. The above commenter is summarizing the mode of action of Bt Cry proteins and soluble toxins.

Commenter Name: Lincoln Brower Commenter Organization Name: Sweet Briar College Comment Number: 30509 114000 Excerpt Number: 2 Excerpt Text: Another biological approach was to manipulate the soil bacterium Bacillus thuringlensis. The Bt bacterium secretes a protein that, when ingested by a sensitive insect, cases the larval gut to break down and a gooey, black death ensues. Industrial and academic scientists have selected numerous Bt strains that are toxic to the larvae of different groups of insects. The Bt kurstaki strain is lethal to the caterpillars of virtually all moths and butterflies and is produced in mass cultures that are harvested and sold as Dipel. Used in home gardens as a " natural" toxic powder to kill tomato hornworm is and cabbage caterpillars. Dipel is also sprayed to kill gypsy moth caterpillars in the eastern deciduous forests, spruce budworms HI the northern boreal forests. and tussock moth caterpillars in the western Douglas fir forests. Extensive sprayings of Dipel and its derivatives, along with repeated releases of exotic parasitic insects. Have severely reduced the populations of many benign and beneficial native insects, including several of the New England silk moths renowned for their elegance and bizarre caterpillars The danger to nonpest species was raised to a far more sophisticated level by the new science of genetic engineering, which makes it possible to transfer genes between any species on earth. When successful, the transferred genes give the recipient species the ability to synthesize proteins that were specific to the donor species. An obvious strategy would be to insert various Bt genes into crop plants. Then as the seeds of the genetically modified strain sprout and grow, the inserted DNA would express itself in every single cell of the growing seedling. Wonder of wonders, the roots, stems, leaves, and seeds of the plant contain the Bt toxin and are toxic to virtually all caterpillars. Agricultural companies introduced the Bt genes into several crops, including potatoes, soybeans, cotton, and corn. One major target was the European corn borer moth, an economically damaging species that is found throughout the eastern United States and southern Canada.

EPA Response: This comment does not require a response. The above commenter is summarizing the theoretical consequences of use of spray and plant incorporated Bt toxins on insect wildlife.

Commenter Name: Lincoln Brower Section 3.1 3  53 Commenter Organization Name: Sweet Briar College Comment Number: 30509 114000 Excerpt Number: 4 Excerpt Text: Many biologists heralded the new Bt corn technology because they believed it would mitigate the need to spray insecticidal chemicals. The corporations involved in marketing the seeds ( for the most part the same ones that had developed the synthetic insecticides several decades earlier) sponsored a multimillion dollar campaign touting them as an environmental panacea. The response was stunning: by the 1998 season, twenty five percent of the total U. S. corn crop ( of eighty million acres) was planted with Bt corn.

EPA Response: This comment does not require a response.

Commenter Name: Lincoln Brower Commenter Organization Name: Sweet Briar College Comment Number: 30509 114000 Excerpt Number: 8 Excerpt Text: Aware that new data and more sophisticated analyses would be forthcoming, the Union of Concerned Scientists and eleven other public interest organizations made a request to the EPA: to postpone the next Scientific Advisory Panel ( SAP) meeting until more data were collected and made available to the public, including, the scientists' findings gathered over the summer of 2000. The EPA, however, held the SAP meeting on October 19, a month before the scientific symposium was scheduled to take place. Prior to the SAP meeting, the EPA had allowed several corporations to review the agency's preliminary assessment and suggest modifications. In addition, the EPA allowed the companies to withhold important data as confidential business information. One of the principal documents, contained approximately forty deletions of so called " proprietary" data. It was therefore impossible for the EPA panel or independent scientists to evaluate the data. Both industry and the EPA documents also ignored relevant data readily available in the scientific literature. Thus, without considering the new information that would be presented the following month, and drawing passages almost verbatim from documents prepared by industry, the EPA's interim assessment of the risks and benefits presented to the SAP stated that " the published preliminary monarch toxicity information is not sufficient to cause undue concern of harmful widespread effects to monarch butterflies at this time."

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. Section 3.2 3  54 3.2  Environmental Fate/ Persistence/ Indirect Effects on Soil Biota 3.2  Comment Excerpts Commenter Name: Dan Bolling Commenter Organization Name: Dan Bolling Comment Number: 30509 045000 Excerpt Number: 1 Excerpt Text: Although we all agree on the goal of reducing the use of pesticides  for environmental and economic reasons  I believe we should not assume that the proposed adoption of Bt protected corn is necessarily the best approach. Undoubtedly it is the approach with the greatest potential economic return to its developers. And it may yet prove to be the environmentally preferred approach, as well.

But as yet the assessment of risk from Bt resistant pest development, Bt soil residuals, and unintended impacts on beneficial species have not been adequately communicated to the public. Many thoughtful voices have suggested that this reflects the fact that inadequate risk assessment has been performed.

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 December, 1999 and August 2002. The SAPs found to studies adequate with recommendations that EPA test more species appropriate to the agroecosystems in question. ( SAP Report No. 99 06, February 4, 2000; SAP Meeting Minutes No. 2002 05, November 6, 2002). These recommendations, as well as the recommendation to do long range field monitoring, are being implemented by the EPA.

Commenter Name: Angelika Hilbeck Commenter Organization Name: Greenpeace Comment Number: 30509 113000 Excerpt Number: 8 Excerpt Text: III. Degradation/ dissipation of material From the little information on the employed protocols provided in the Monsanto Document, the presented degradation studies appear to have been conducted according to the protocols described in studies 1 and 2 of the EcoStrat report on ' Bt proteins in soils' ( 2001). Also here, unrealistic short half life dissipation times ( 2.37 and 2.76 days) were reported based on fitted data calculated from insect bioassay results with the Colorado potato beetle. Additionally, ELISA analyses were conducted. From that we speculate that the trials were not carried out with unprocessed plant material but probably with microbial derived Cry3Bb protein or processed plant powder similar to the reviewed studies 1 and 2 mentioned above. However, because also no information is provided in the Monsanto Document on the experimental conditions like temperature, soil moisture and ambient air humidity levels, no independent scientific evaluation is possible. If similar settings, materials and methods were employed as described in the studies 1 and 2 mentioned above, the same short comings apply of course that were outlined there ( EcoStrat Report 2001). In an agricultural ecosystem, in particular in temperate regions, unprocessed plant material will enter the soil Section 3.2 3  55 upon harvest at the end of the vegetation period during fall when temperatures drop and the metabolism of all organisms slows down to an absolute minimum in order to survive the cold winter season. Therefore, it must be expected that also dissipation and degradation of plant material and the respective Bt proteins are reduced and will remain much longer in the soil ecosystem than observed under optimal laboratory conditions.

Commenter Name: Rissler Commenter Organization Name: UCS Comment Number: 30509B 089000 Excerpt Number: 29 Excerpt Text: 1. Require Monsanto to submit data on Cry 3Bb expression levels in MON 863 tissues under a range of environmental conditions and on the quantity and fate of Cry 3Bb proteins exuded into the soil.

Commenter Name: Rissler Commenter Organization Name: UCS Comment Number: 30509B 089000 Excerpt Number: 31 Excerpt Text: 4. Direct its Scientific Advisory Panel ( SAP) to determine whether the technology currently exists, or is likely to exist within five years, to direct the expression of Bt endotoxins toward plant roots such that levels in roots exceed levels in grain, pollen and leaf tissue by 10 fold or more.

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. 4) 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. ( USEPA. 2002. MON 863 BRAD Section C. I. B. 4). However, because the Agency believes that additional studies would be useful in completing the database for long term effects assessment, it is requesting supplementary multi year studies regarding Cry3Bb1 protein degradation and persistence in various agricultural soils.

The Agency has also required the submission of additional expression data in terms of dry weight, as the amount of protein present in the given tissue. Tissues for which expression data must be provided include: leaf, root, pollen, seed, and whole plant. In addition, data for each of these tissues should be provided for young plants in rapid growth, during flowering, and mature plants before harvest when that part of the plant is present. Data obtained for roots should also include Section 3.2 3  56 typical times when corn rootworm would be feeding.

Commenter Name: Angelika Hilbeck Commenter Organization Name: Greenpeace Comment Number: 30509 113000 Excerpt Number: 17 Excerpt Text: II. Routes of input and amount of transgenic plant material and plant expressed novel protein into the soil ecosystem In our Ecostrat report on ' Bt in soils' ( 2001), we describe the various routes transgenic Bt plant material enters the soil ecosystem. In order to identify possibly exposed nontarget organisms and, subsequently study whether they will be affected or not by the released Bt toxins, tissue specific information on Bt concentration in plant residues and plant excretions as well as their dissipation/ degradation in soils is important. Tissue specific Bt concentration levels have been discussed above already and were provided in the Monsanto Document. However, no data was provided regarded Bt concentrations in plant excretions like root exudates or mucilage produced by the roots ( see Introduction in EcoStrat report 2001 on ' Bt in soils'). These excretions are produced throughout the life time of the plant in addition to small amounts of broken off plant debris and continuously enter the soil ecosystem. Many micro organisms but also organisms like nematodes or macrofauna like earthworms etc. are thereby continuously exposed to these toxins. In particular when considering the large amounts of Cry3Bb toxin, it must be of primary interest to assure that such organisms providing very important services for soil health and soil ecosystem function will not be adversely affected by the high input of Bt toxin. However, no data on this is provided in the Monsanto Document. Further, also no data is provided on binding of Cry3Bb proteins to surface active particles. In particular with regard to the on going production of transgenic Cry1Ab corn on large areas lasting several years already, this becomes an interesting and probably scientifically challenging task to study, since both types of toxins may interact, for example by competing for binding sites on surface active particles. From the results of previous such studies with Cry1Ab corn, it cannot be excluded that Bt Cry1Ab levels in soils are already elevated. Therefore, and additionally because Monsanto Company recommends to grow both types of transgenic Bt corn varieties, Cry3Bb and Cry1Ab corn, simultaneously for resistance management purposes, soil nontarget testing must account for interaction effects of Cry3Bb and Cry1Ab. To date, this has not at all been addressed.

EPA Response: The fate of Cry3Bb1 protein in soils and indirect effects on soil biota have been evaluated by the EPA. 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. 4). Soil heath evaluation from limited data do not indicate that Cry proteins have any detrimental 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 except under artificial laboratory conditions created to favor DNA transfer. Published studies of Bt Cry protein in soil show no effect on bacteria, actinomyces, fungi, protozoa, algae, Section 3.2 3  57 nematodes, springtails or earthworms.( USEPA. 2002. MON 863 BRAD Section C. I. B. 5). 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). 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. iii). Because the Agency believes that additional studies would be useful in completing the database for long term effects assessment, it is requesting additional supplementary studies regarding Cry3Bb1 protein degradation and persistence in various agricultural soils. Additive effects of several Cry proteins have been looked at by the EPA in laboratory studies on non target organism effects. No additional effects by the combination were seen on susceptible insect species. The root exudate issue has been indirectly addressed by testing the springtails and earthworms at Cry protein doses exceeding any possible soil concentrations from plant decay and root exudation combined.

Some evidence suggests that Cry proteins while bound to some soil components, e. g. clays and humic acids, are recalcitrant to degradation by soil microorganisms, but without eliminating their insect toxicity. Several factors influence either the affinity of binding or the rate of degradation. Low pH favors mineral binding, while pH near neutrality generally substantially increases degradation. Corn does not grow well below ~ pH 5.6 and therefore most corn growing soils are expected to be at a higher pH. Therefore, under most production conditions, corn would not be grown on soils that would favor Cry protein binding to clay particles and accumulation in soil. Therefore, corn production would not inhibit the rate of degradation compared to what is seen in soil of lower pH. Nevertheless, these issues are being evaluated on a case bycase basis by environmental fate studies designed to determine the rate of Cry protein degradation over sufficiently long periods to assure an accurate assessment of degradation in agricultural soils of various compositions and pH.

Commenter Name: Angelika Hilbeck Commenter Organization Name: Greenpeace Comment Number: 30509 113000 Excerpt Number: 18 Excerpt Text: Soil nontarget studies presented in Monsanto Document Only two soil inhabiting organisms were studied, the collembola Folsomia candida and the earthworm ( probalby Eisenia foetida). No details on the protocols applied were presented but from the little information given, we speculate that largely the same protocols were applied as in other previous regulatory studies. These have been reviewed by us and severe short comings were identified. If our assumption of similar protocols is correct, the same short comings will apply for the studies presented here ( EcoStrat Report 2000).

" In the experiments, no plant foliage was tested but ... lyophilized corn leaf protein ( a powder) was used as dietary test material. E. foetida was added to a mixture of soil and lyophilized leaf protein powder, although, this species hardly eats soil and prefers organic material. F. candida feeds on saphrophytic fungi growing on decaying plant material but was also offered lyophilized leaf protein powder mixed into soil. In acknowledgment of the feeding preference of this species yeast was also added. But it is unclear whether F. candida actually ingests any of the leaf protein powder when ingesting the yeast cells." Section 3.2 3  58 " None of the ecotoxicological tests considered multitrophic interactions between plants, herbivores or pathogens and natural enemies."

The regulatory trials with E. foetida reviewed in the EcoStrat Report ( 2000) lasted only 14 days although these organisms can live for more than a year and the Bt plants grow in the field for several months and there is strong evidence that the Bt proteins and the transgenic plant material remain even longer in the soil.

EPA Response: There are reports in the open literature that earthworms tested for ingestion of Cry protein from soils do ingest it, and Cry protein is also detected in earthworm casts. ( USEPA. 2002. MON 863 BRAD Section C. I. B. 3. a. vii). Lyophilized leaf tissue is actually dehydrated plant tissue, therefore, tissue was tested. The collembola in the tests performed exhibited a greenish color indicative of leaf tissue ingestion. However, even if collembola do not ingest plant tissue, no hazard to collembola would exist. Testing of collembola for more than 14 days ( chronic testing) is not deemed appropriate at this time because the collembola populations are evaluated during field testing. In addition, the collembola studies did go through a reproductive cycle. This amounts to a chronic and a reproductive test.

The August, 2002 SAP cast doubt on the usefulness of tritrophic studies, nevertheless, EPA intends to request tritrophic studies where they appear to be warranted by the ecology of the crop in question.

Commenter Name: Angelika Hilbeck Commenter Organization Name: Greenpeace Comment Number: 30509 113000 Excerpt Number: 19 Excerpt Text: It appears that in the E. foetida study presented in the Monsanto Document microbial derived Cry3Bb protein was added to the soil while F. candida was actually provided transgenic Cry3Bb plant material. But a 10 fold safety margin is not outstanding for a laboratory ecotoxicity trial. In such studies larger safety margins are commonly applied, certainly for human exposure. Additionally, the calculations relating to expected exposure of soil inhabiting organisms presented in the Monsanto Document are unconvincing and speculative. On page 25 of 56 it is stated:

' The maximum environmental concentration for soil dwelling organisms is predicted to be 13.3 mg/ kg based on the assumption that corn plants are tilled into the tip 6" of soil at the time of maximum leaf expression for Cry3Bb1.11098 ( i. e., 93 ? g/ g). The measured NOECs form these tests exceed the maximum predicted environmental concentration by 3 to 140 fold, demonstrating an adequate margin of safety for these organisms.'

However, in particular E. foetida, commonly called the compost worm and sold commercially for improvement of garden composts consisting almost exclusively of organic material, and other earthworms in the field prefer to feed on organic, i. e. plant, material and ingest little soil. So, the expected exposure is higher and closer to the actual expression levels in the transgenic plant residues. At the time of harvest expression levels recorded in the Monsanto Document on page 24 of 29 ( Table 2) ranged between 13  35 ? g/ g ( mean 25 ? g/ g) for whole plants and 3.2  36 ? g/ g ( mean 24 ? g/ g) for roots ( both at day 126 after planting), so in fact up to 3 times higher than suspected in the Monsanto Document. These concentrations Section 3.2 3  59 add to the Cry3Bb and Cry1Ab toxin concentrations potentially present already in the soil due to root exudation and previous Bt corn production. In the Monsanto Document a NOEC of 57 mg/ g which is the equivalent of 57 ? g/ g is reported above which effects must be expected. Therefore, the safety margin has decreased to 0  1.5 fold and adverse effects on this organism seem likely and merit further laboratory and field experimentation with relevant organisms, more realistic exposure levels and preferred diet, i. e. unprocessed plant material.

However, no details on rearing and experimental conditions nor experimental design were given. Therefore, no final evaluation of the data or conclusions can be drawn from these studies.

We would also like to point out that soil organisms like earthworms may accumulate fairly high concentrations of Bt and serve as mediators of high Bt doses to their natural enemies like birds, rodents and other small mammals even if the toxin does not affect the earthworms themselves. To our knowledge, this issue has not been addressed yet neither in private nor public sector research.

EPA Response: The available data do not support these comments. There are reports in the open literature that earthworms tested for ingestion of Cry protein in the system used by Monsanto do ingest Cry protein, and Cry protein is also detected in earthworm casts. ( USEPA. 2002. MON 863 BRAD Section C. I. 3. a. vii) The earthworms were not affected by the Cry proteins. Monsanto testing was done with purified cry protein in order to expose the earthworms to large doses exceeding the possible soil concentration. Birds and mammals fed Cry proteins do not show any adverse effects, therefore ingestion of earthworms with cry protein would not pose a hazard. The calculations presented by this commenter are not based on the same figures supplied with the earthworm studies.

The study referred to by the above commenter ( MRID No. 449043 16) shows that the 570mg/ kg soil was the highest concetration tested, so that the LC50 is above that concentration, not at that concentration. This study has a 10 x safety factor. The data show that no adverse effects to earthworms are expected from exposure to Cry3Bb1 protein producing corn plants. The calculations for soil concentration in the top 6" were done by standard EPA procedures that have been approved by Scientific Advisory Panels. As for the suggestion that a test concentration greater than 10x is required to assess toxicity, the EPA hazard evaluation procedure considers an LD50 ( not a NOEC) at 5x the environmental concentration ( EEC) as indicative of no hazard to terrestrial organism populations, while in the submitted studies there was no LD50, or mortality seen at 10 x, the highest concentration tested.

Commenter Name: Angelika Hilbeck Commenter Organization Name: Greenpeace Comment Number: 30509 113000Part2 Excerpt Number: 1 Excerpt Text: 1) Only a limited number of studies is currently available that explored input. persistence/ degradation of Bt proteins from transgenic plants and/ or their effects on soil organisms. These studies were conducted in four different laboratories and do not yet allow a meaningful assessment of the ecological impact of transgenic Bt plants in soil ecosystems for the following reasons: Section 3.2 3  60 a) The studies analyzed are difficult to compare, because of the different experimental conditions, methods and materials used. For example, in some studies, Bt toxins and protoxins derived from commercial formulations were used, in others intact and/ or frozen plant material was used, and in others, processed, sieved plant powder was used to explore persistence/ degradation and impact on soil organisms. Furthermore, different and often rather unspecified soil types were used.

b) Temperatures for incubation were in some studies high, which provides for maximal microbial activity and are not representative for soils in temperate regions. Therefore, it must be assumed that microbial degradation under field conditions is reduced compared with what was observed under high humidity and high temperature conditions in the laboratory. Consequently, toxin persistence can be expected to be greater at least in temperate regions with distinct winter seasons and Bt crops harvested at the end of the growing season when temperatures begin to drop.

For these reasons, it is difficult to combine the information provided in these studies for a final synthesis and conclusions.

EPA Response: EPA agrees that the long range effects data base needs to be enlarged over a longer time period to supplement the existing information. To this end, the EPA is granting time limited registrations and is requiring multi year Cry protein soil accumulation studies in a variety of soil and temperature conditions. ( USEPA. 2002. MON 863 BRAD Section C. I. F)

Commenter Name: Angelika Hilbeck Commenter Organization Name: Greenpeace Comment Number: 30509 113000Part2 Excerpt Number: 2 Excerpt Text: 2) From the few studies, it cannot be excluded that Bt proteins may accumulate in the soil under continuous large scale production of transgenic Bt plants. After analyzing the relevant studies, there is reason to believe that Bt toxins can persist in soil for a long time; according to the data collected in the laboratory trials possibly for more  than eight months. This will depend on the soil type, predominantly on the content of surface active particles like clay minerals or humic acids. There is further evidence that coupled with persistence, the insecticidal activity of the Bt toxins is retained at ecologically relevant levels. In temperate regions, the toxin may well persist over the winter months. In this context, root exudation of Bt from transgenic corn becomes an important aspect to consider because Bt toxins are excreted already in the seedling stage, i. e., early in the season. In practice, this may mean that before the incorporated Bt toxin from the previous growing season is fully degraded, more Bt toxin from the following vegetation period is introduced again. Over many years, this could potentially lead to an accumulation of Bt toxins in some soils, i. e. with high content of surface active particles. No coordinated efforts are under way to monitor the Bt contents in the soils of those areas where Bt crops are produced since several years, nor has any research been conducted and published to date studying fate and movement, of free and bound Bt proteins in such soils with high content of surface active particles.

3) The evidence for persistence of Bt proteins in soils and their retained insecticidal activity poses potential hazards for soil micro and macro organisms. Firstly, nontarget effects could arise that may impact soil Section 3.2 3  61 food web structure and biological regulation mechanisms. Further, it could also select for reduced susceptibility of exposed nontarget soil anthropods, e. g. soil inhabiting lepidopteran and/ or coleopteran larvae. This may have repercussions for future transgenic Bt plants designed to control such soil inhabiting lepidopteran or coleopteran larvae that are serious pests of crop plants, e. g. Diabrotica species. However, enhanced unintended control of soil inhabiting pest species may occur as well, as a ' positive' side effect, at least until resistance develops, in some of the reviewed studies, temporary, statistically significant or slight, statistically insignificant effects on soil micro or macroorganisms were observed. However, the research conducted to date is far too limited to allow for ' safe' conclusions yet, in particular, with respect to long term effects under constant and/ or repeated exposure to Bt proteins.

4) Soil ecology is so poorly understood that interpretation of the limited number of studies is almost impossible due to the fundamental lack of knowledge about functioning and ecological interactions in soils.

EPA Response: The information available to date indicates that soil accumulation of significant amounts of Bt Cry protein and ecosystem perturbation does not take place. However, EPA recognizes the value of additional multiyear studies to confirm these findings. To this end the EPA is requiring multi year non target effects and Cry protein soil accumulation studies in a variety of soil and temperature conditions. ( USEPA. 2002. MON 863 BRAD Section C. I. F). These issues are also one of the reasons why the Agency is granting time limited registrations of Cry protein containing crops.

Commenter Name: Angelika Hilbeck Commenter Organization Name: Greenpeace Comment Number: 30509 113000Part2 Excerpt Number: 3 Excerpt Text: Pre commercial release research Research activities in basic soil ecology and soil ( bio) chemistry and, specifically, on all aspects necessary for hazard identification, including input, fate and impact of Bt proteins should be increased significantly. It would further be desirable if standard protocols could be developed and agreed upon to facilitate the comparison of the various studies in the future. Pre commercial release research should also investigate the movement of the Bt proteins released from transgenic plants in soils, including soils that are already in Bt plant production since many years. Further, the impact of Bt proteins from transgenic plants on the soil macro and microbiota diversity and population dynamics should be assessed, and, vice versa, also the degree to which varying communities of microorganisms affect persistence/ degradation of Bt proteins in different soil environments. Because rhizosphere microorganisms are very important for plant health and development, it is essential that proper testing of these microorganisms with Bt, is undertaken prior to the large scale release of the transgenic Bt plants. In particular with regard to modem, sustainable agricultural production methods aiming at reducing external input like fertilizers, rhizosphere microorganisms will become increasingly important again.

Post commercial release continued oversight The most obvious and straightforward approach to study the fate of Bt toxins in soil ecosystems is to monitor the content of Bt proteins in soils that have been under continuous Bt plant production for several Section 3.2 3  62 years. Mathematical modeling and computer simulations might be useful in the design of monitoring schemes. Many of the questions posed in this document could be answered by now if monitoring had been in place from the beginning of commercial field production of transgenic Bt plants. Today, in the 6~ h year of Bt crop production, it may be difficult to find appropriate control fields for comparison. However, this is only true for the United States and, perhaps, Canada, where most of the transgenic Bt crops are grown. Therefore, determination of natural background levels of Bt protein in soils not yet in production of transgenic Bt plants in Europe and most other countries of the world should be initiated before their largescale commercialization.

EPA Response: The information available to date indicates that soil accumulation of significant amounts of Bt Cry protein and ecosystem perturbation does not take place. However, EPA recognizes the value of additional multiyear studies to confirm these findings. To this end the EPA is requiring multi year non target effects and Cry protein soil accumulation studies in a variety of soil and temperature conditions. ( USEPA. 2002. MON 863 BRAD Section C. I. F). These issues are also one of the reasons why the Agency is granting time limited registrations of Cry protein containing crops.

Commenter Name: Angelika Hilbeck Commenter Organization Name: Greenpeace Comment Number: 30509 113000Part2 Excerpt Number: 5 Excerpt Text: 1) Input of Bt proteins from transgenic plants. Bt proteins from transgenic plants entering the soil ecosystem stem from both living plants and dead plant debris/ residues. The temporal distribution of the amounts of Bt toxins entering the soil ecosystem varies considerably:  input through root exudation, root leakage and/ or root debris left behind during growth is a permanent process of probably small amounts ( not quantified yet).  input via plant parts ( pollen, leaves, etc.) falling to the ground throughout the season prior to harvest is also a permanent process of smaller amounts.  input via plant residues ( dead or alive, e. g. as volunteer seeds) remaining in the field after harvest, and, possibly, incorporated into soil is a cyclic process of large amounts.

2) Fate of Bt proteins from transgenic plants. Depending on the environmental conditions and soil type, Bt proteins can either persist or degrade more or less quickly. Points to consider here are:  Toxin inactivation.  Mechanisms of persistence.  Chemical degradation.  Microbiological degradation.  Removal from soil ( e. g. through leaching).  Uptake by higher soil organisms or plants.

3) Effects on soil organisms. Bt proteins released from transgenic plants may have various impacts on the soil biota. Section 3.2 3  63  Impact on biodiversity or biomass of soil organisms ( e. g. abundance, population dynamics, community composition).  Impact on soil and plant health if check balance systems of pathogenic and beneficial microorganisms are disrupted.

EPA Response: The October 2000 SAP concluded that invertebrates such as earthworms and springtails ( collembola) are appropriate indicator species for Cry protein testing because of the specific nature of the Cry protein toxicity to select target species. ( SAP Report No. 2000 07. March 12,2001) When it initially reviewed the applications for PIP products that were registered in 1995, EPA considered requiring studies evaluating effects upon the representative beneficial soil invertebrates Collembola and earthworms. The Agency was concerned ( 1) that such soil organisms may be subject to long term exposure as a result of soil incorporation of crop residues or when crop residues are left on the soil surface and ( 2) that adverse effects on such soil organisms could result in an accumulation of plant detritus in fields. Recent reports of exudation of Cry proteins by corn roots throughout the growing season add to this concern. However, the Agency understands that routine agronomic practices have included the long term use of chemical insecticides, which have adverse effects on soil organisms, but there has not been an accumulation of significant amounts of plant detritus in soils. Thus, Cry3Bb1 corn, which is expected to have less impact on these species than chemical pesticides, should not result in any increased build up of plant detritus or Cry proteins at toxic levels. Supporting this conclusion are data received by EPA that indicate that such proteins are known to degrade rapidly in field soils. ( USEPA. 2002. MON 863 BRAD Section C. I. B. 4) Cry proteins that are bound to soil particles have been shown to be rapidly degraded by soil microbes upon elution from the soil particles. ( USEPA. 2002. MON 863 BRAD Section C. I. B. 4) Therefore, the potential for significant soil buildup and adverse effects to non target soil organisms are not anticipated. It has been confirmed in published literature that Bt Cry protein released from root exudates and biomass of Bt corn plants has no apparent effect on earthworms, nematodes, protozoa, algae, bacteria, actinomyces and fungi in soil in spite of the fact that enough detectable Cry protein is bound to soil particles to show toxicity to the target pest. ( USEPA. 2002. MON 863 BRAD Section C. I. B. 5) These results suggest that despite its presence in soil, the Cry protein released in root exudates of Bt corn, or from the degradation of the biomass of Bt corn, is not toxic to a variety of organisms in the soil environment. It has also been reported that the same degree of Bt Cry protein persistence takes place in soils that have been exposed to repeated Bt microbial spray applications. ( USEPA. 2002. MON 863 BRAD Section C. I. B. 4) In addition, new plants grown in Bt containing soil do not take up the Bt protein. ( USEPA. 2002. MON 863 BRAD Section C. I. B. 6) Nevertheless, data on insects closely related to the target pest, as well as other studies to address the published data requirements for registration of microbial toxins ( 40 CFR § 158) have been received and reviewed. However, EPA is a party to ongoing research to address the long range concerns expressed. In addition, the resolution of these questions are also one of the reasons why the Agency is granting time limited registrations of Cry protein containing crops.

Commenter Name: Angelika Hilbeck Commenter Organization Name: Greenpeace Comment Number: 30509 113000Part2 Excerpt Number: 9 Excerpt Text:  Degradation Section 3.2 3  64 Purified microbial and plant produced Bt toxin: Seven studies provided information about the retained insecticidal activity of purified toxin, protoxin derived from microbial Bt preparations and of plantproduced toxin in soil ( studies 6 9, 11 and 12). The experimental period in these studies varied from 50 hours to 234 days, and in most of the studies, the toxin could be detected at every time point.

Studies 6 8 investigated binding and insecticidal activity of purified Bt toxins obtained from microbial Bt preparations when bound on surface active particles such as clay minerals or humic acids in the presence of microorganisms. In all studies, it was demonstrated that Bt toxins adsorbed rapidly on the clay minerals, thus, confirming the results of earlier studies ( see above, studies 3 to 5 and 7). In study 6, it was shown that Bt toxins were resistant to microbial degradation when bound on the clay minerals montmorillonite and kaolinite or on the clay size fraction of different soils. Insecticidal activity was retained after 40 days ( longest time period measured) when toxin containing soils were incubated with microbial cultures. Similar results were obtained with humic acids, i. e., protection from biodegradation and retained insecticidal activity ( study 7). In study 8, soils containing kaolinite and/ or montmorillonite were incorporated with Btktoxins and supematants from fresh garden soil to enhance microbial activity. Again, insecticidal activity was retained after 234 days of incubation ( 240C). During this time, for example, the LC50 of Manduca sexta to Btk bound on kaoliite increased fourfold over the LC50 measured before incubation. Hence, activity had declined to 25% of the initial activity of the toxin. The amount of retention of insecticidal activity varied with soil type, clay mineral content, clay mineral composition and pH.

Study 11 describes a method to optimize extraction and immunological detection of Bt toxin from soils. Highest recovery of Bt was found in soils with Low clay content and low organic matter. Buffer characteristics, such as ionic interactions and pH, were important, e. g. neutral or alkaline conditions promoted recovery, and there was also an interaction between buffer characteristics and soil type.

In study 13, Bt toxins rapidly degraded to a low concentration at the beginning of the experiment and then remained unchanged for several weeks. The authors speculated that this may have been the result of binding of the proteins on soil particles, which protected them from biodegradation ( see above ' mechanisms'). A similar observation was made in study 7 when free toxin was mixed with free humic acids. After 4 to 6 hours, microbial growth was lower in a medium ( DCMM) containing both humic acids and free toxin than in a medium containing free toxin only; i. e., without humic acids. The authors concluded that this effect was probably the result of adsorption of the toxin to the humic acids which resulted in their protection from biodegradation.

EPA Response: EPA recognizes the fact that Cry proteins do adsorb to clay particles, but are quickly inactivated by soil microbes upon elution. EPA also recognizes that Cry proteins within plant tissues may remain active for the same long period of time that it takes for the plant tissue to decay. The long term implications of the fate of Cry proteins is soil after several seasons of Cry protein containing crop cultivation are being addressed by environmental fate studies designed to determine the rate of Cry protein degradation over sufficiently long periods to assure an accurate assessment of degradation in agricultural soils of various compositions and pH.

Commenter Name: Angelika Hilbeck Commenter Organization Name: Greenpeace Section 3.2 3  65 Comment Number: 30509 113000Part2 Excerpt Number: 10 Excerpt Text: Differences between transgenic plant toxins and purified microbial toxins. In three studies ( 11, 12, and 13) purified and transgenic plant toxin were compared. In studies 11 and 13, transgenic plant toxin degraded faster than purified toxin suggesting that the purified toxin was more stable. The authors speculated that this could have been due to degrading enzymes and compounds released when plants decay, or due to the toxin being more accessible to degrading soil microorganisms than the purified toxin, and/ or due to the increase in microbial populations observed when plant material was added ( typically, a 100 1000 fold increase). However, when purified toxin was added to non Bt plant material, the purified toxin had the slowest degradation rate of all treatments measured. This is an indication that other factors may have a role and that it is important whether the toxin is within or outside of the plant material.

In contrast, in study 12, purified CrylAb and CrylAc degraded a little faster than plant expressed toxin: purified toxin was detected after 28 days~ but not after 56 days, whereas plant expressed toxins and purified toxins together with plant material from parental plant lines were detected after 56 days.

In summary, a general pattern of Bt toxin degradation emerged: initially, a rapid decline of the extractable toxin concentration is observed, followed by a more gradual decline to low concentrations of the toxin, which then remained unchanged for several weeks. This is somewhat similar to the observations made in early studies on crystalline Bt protein degradation ( see ' Introduction'). However, the results are inconsistent, and it is not clear what influences the somewhat different degradation of purified microbial toxins versus transgenic plant expressed toxins. According to Stotzky ( 2000), adsorption of the toxins increased with their concentration and then leveled off, suggesting that the clays became saturated with the toxins, as observed with other proteins and organic compounds. However, it still remains to be investigated to what extent this occurs in the field, whether or not satiation of surface active particles in the field can be reached and how free Bt toxin in soils move; especially, in soils that are under continuous Bt plant production since several years where surface active particles are perhaps exhibiting increasing Bt satiation levels?

EPA Response: This comment is a summary of soil degradation findings which were considered by the EPA during the risk assessment process. Additional research on the long term accumulation and effects of Bt proteins in soils are in progress.

Commenter Name: Angelika Hilbeck Commenter Organization Name: Greenpeace Comment Number: 30509 113000Part2 Excerpt Number: 11 Excerpt Text: Toxin in plant powder. The reported half time for degradation of 1.6 days in study 1 is short. Most likely, the optimal experimental conditions led to such a fast degradation. Leaf tissue of transgenic Bt corn and isogenic non transgenic corn was lyophilized, ground and sieved. Thereby, also ' hard' leaf material was well prepared for microbial degradation, i. e., offering a large surface area for attack by microorganisms. The plant powder used and the favorable laboratory settings ( high temperature) make it difficult, if not impossible, to draw conclusions about degradation under field conditions. Section 3.2 3  66 However, despite the optimal conditions, 20% of the initial toxin activity was still detected after 15 days in study 1 using transgenic Bt corn, and after 120 days with transgenic Pt cotton in study 2. In study 2, the authors gave no information whether the 3ctivity could be measured after 43 days, which represented the end of the experimental period. Again, this supports the observations made by others that the toxin may persist in low concentrations for a long time in the soil.

Further, the degradation data provided were not experimentally obtained but estimated indirectly based on a number of assumptions whose validity are unproven. Only larval growth of H virescens larvae was experimentally measured, i. e., bioactivity. H virescens larvae were sublethally affected when feeding on an artificial diet that contained a soil solution that had been incubated with 1.46 j. ig Bt plant powder/ g soil for up to 43 days in a time series experiment. The authors used these data to carry out a nonlinear regression analysis by fitting a 3 parameter function and, thereby, obtained an EC50 value, i. e.,, ' effective concentration of test suspension' at which larval growth of H virescens larvae was only half of that in the control. The authors assumed that the EC50 value is inversely proportional ( i. e.,, linear relationship) to the concentration of biologically active Bt protein in the soil and that dissipation follows first order kinetics. No data or references are given in support of these assumptions. First order kinetics in dissipation processes is commonly assumed for pesticides and other chemicals yielding half life values. These half life values are then often used for extrapolation of long term dissipation processes. Even for pesticides and chemicals, this assumption often does not hold. For proteins, this certainly needs to be verified experimentally, because they are far more complex in their molecular structure and behavior, and protein degradation in soil, in general, is far less explored. A first order kinetics rate law would imply a linear relationship between ln( 1/ EC5o) and incubation time. However, the figures shown in both papers suggest a non linear relationship indicating that a simple first order kinetics rate law may be inadequate to describe fully the dissipation of Bt toxin. Additionally, the data were associated with large variances, which also urge for cautious interpretation. The authors used the relationship between In( 1/ EC5o) and incubation time to estimate the DT50/ 90 values. However, because the basic assumptions are questionable, the DT50/ 90 values are also questionable and should be interpreted cautiously.

The conclusions drawn by the authors: ' CrylAb protein is likely to degrade rapidly under the reduced cultivation practices associated with no till corn production as well as under conventional cultivation in which corn plant residue is plowed into the soil.' are not supported by these experiments that did not test degradation under such field conditions nor measured it directly under the optimal conditions in the laboratory.

EPA Response: EPA is aware of the non linear degradation of Cry proteins in soils with the resultant ` tailing' effect. In addition, it is general knowledge that microbial degradation of proteins in soil is ubiquitous. This observation was reaffirmed by the SAP ( SAP Report No. 99 06, February 4, 2000). However, studies to confirm the long term ecological effects experimentally, and the time for complete inactivation of Cry proteins in agricultural fields over several planting seasons are in progress.

Commenter Name: Angelika Hilbeck Commenter Organization Name: Greenpeace Comment Number: 30509 113000Part2 Excerpt Number: 12 Section 3.2 3  67 Excerpt Text:  Uptake by plants One study ( study 10) investigated the uptake of Bt toxins from soil by different plants. Soils were used in which previously either Bt or isogenic corn had been grown, or to which Bt or isogenic biomass or purified Bt toxin had been added. The test plants were corn, carrot, radish and turnip plants grown for 120 to 180 days in the differently treated soils. Additionally, isogenic corn was grown in a soil free, hydroponic medium ( Hoagland's solution) in which previously Bt corn had been grown for 15 days.

All experiments indicated that plants do not take up Bt toxin released into the soil or hydroponic solution from root exudation, degradation of Bt corn biomass or purified toxin. However, the study reconfirmed results of previous studies conducted by the same group investigating the persistence of Bt toxins in soils ( studies 3 7) when bound to surface active particles and corn root exudation of Bt toxin into soir ( study Q). In the experiments Bt toxin was still detectable after 180 days after its release ( i. e. the longest time evaluated).

To our knowledge, this is the first report about experiments conducted to test for uptake of Bt toxins by plants. Because of the large acreages grown to Bt plants in the United States, this topic becomes increasingly relevant. Such studies could serve as a model testing system for future transgenic plants releasing novel pesticidal compounds into the soils either via exudation or biomass incorporation.

EPA Response: The above comments confirm the EPA determination that plants do not take up Bt protein released into the soil. ( USEPA. 2002. MON 863 BRAD Section C. I. B. 6). The testing approach recommended by the commenter extends beyond a finding of an ` unreasonable adverse environmental effects determination' and into the areas of academic research and protocol development. Currently, EPA is limiting the testing process to that recommended by the FIFRA Scientific Advisory Panels.

Commenter Name: Angelika Hilbeck Commenter Organization Name: Greenpeace Comment Number: 30509 113000Part2 Excerpt Number: 14 Excerpt Text: Soil ecosystems are complex and little explored to date. Changes taking place in soil ecosystems can often not be observed directly, and it may take years before they become evident. Early indications are subtle, and a good farmer for example must be an alert observer to detect such subtle changes which, later, may result in drastic effects when they go unobserved.

Soil organisms contribute to the maintenance of soil quality in that they promote plant growth, influence decomposition of plant and animal materials, influence the biogeochemical cychng, control the formation of soil structure and the fate of organic material ( Turco et al. 1994; Linden et al. 1994). Soil contains unknown numbers of different species of microorganisms ( Angle 1994). Of all soil bacteria, it is claimed that approximately 90% cannot be isolated or cultured on laboratory media ( Turco et al. 1994). Therefore, determination of microbial biomass is often restricted to indirect measures like metabolic activity ( respiration by measuring CO2 emissions, etc.). Recently, good and improving methods to account for unculturable microbes are being developed also caused by an increasing demand of such methods for the Section 3.2 3  68 ecological risk assessment of transgenic organisms released into the environment ( see below " Known impacts of other transgenic organisms in soil ecosystems"). However, depending on the methods used, ' microbial communities' or microbial biomass are investigated which must be cautiously interpreted under full consideration of the constraints of each method.

EPA Response: The USDA and EPA are funding long term effects studies to address these and similar issues.

Commenter Name: Angelika Hilbeck Commenter Organization Name: Greenpeace Comment Number: 30509 113000Part2 Excerpt Number: 19 Excerpt Text: Past knowledge on Bt protein degradation and inactivation stems from research with microbial Btinsecticides Twenty years ago, using liquid formulations of the commercial Btinsecticide ' Thuricide', Pruett et al. ( 1980) showed that the activity of crystals and spores declined relatively fast, with crystals loosing their activity faster than spores. Although the majority of spores could still be recovered after one month, 50% of activity was lost within the first week arid 95% within one month ( 31 days). The authors speculated that microorganisms may not alone be responsible for degradation and hypothesized that other soil bacteria could also impact the activity of Bt. For instance, Kiselek ( 1974) found that of 50 species of soil bacteria tested, 24% were found to be potentially antagonistic to Bt. In another study, West ( 1984) found a reduction of Bt insecticidal activity of 93 .4 to 99.7% after 23 days and a half life of 2.7 5.8 days depending on the supplements added to the test soils ( CaCO3 and/ or organic material). The author concluded from his experiments that protein crystals were decomposed primarily by soil microorganisms. West et al. ( 1984) reported that loss of crystal insecticidal activity declined in a natural soil at a complex, non exponential rate. Initial rapid decrease gradually slowed, and the level of activity stabilized at 10% of the initial inocculum after 250 days ( sampling was terminated then). In autoclaved soil, they did not find a loss of crystal activity, suggesting again that microorganisms were primarily responsible for degradation.

The underlying pattern in the decline of insecticidal activity seems to be an initial, rapid reduction followed by a gradually slower inactivation to a low level, which remains constant for a long time. However, there is no clear picture as to what factors were involved in inactivation of Bt crystals. Clearly, microorganisms were of primary importance. However, there also seem to be other factors that influence the fate of Bt spores and crystals in soil. Competition by other microorganisms was suggested. Different soil types may also be responsible for variability of inactivation.

EPA Response: The USDA and EPA are funding long term effects studies to address various aspects of the degradation of Cry proteins in agricultural soils.

Commenter Name: Angelika Hilbeck Commenter Organization Name: Greenpeace Comment Number: 30509 113000Part2 Excerpt Number: 20 Excerpt Text: Section 3.2 3  69 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: There is no evidence that Bt toxins are released into the soil in " enormous quantities". Data reviewed by the Agency show that the degradation in soil occurs within days to a barely detectable background levels. ( USEPA. 2002. MON 863 BRAD Section C. I. B. 4). Data available to date also do not show any adverse effects on the soil biota. ( USEPA. 2002. MON 863 BRAD Section C. I. B. 3). These aspects will, however, be investigated over several years to provide confirmatory long term effects information.

Commenter Name: Angelika Hilbeck Commenter Organization Name: Greenpeace Comment Number: 30509 113000Part2 Excerpt Number: 23 Excerpt Text: 1) Input of Bt proteins from transgenic plants  Root exudation Only one study investigated an input route ( Table 1) by studying Bt proteins exuded from roots into the rhizosphere. It is well known that roots exudate carbohydrates and amino acids ( see below ' Introduction'). However, only few plants are known that excrete large molecules like proteins via their roots. This paper was the first demonstrating exudation of Bt proteins from roots of transgenic Bt com seedlings. It was shown in a soil free nutrient solution that Bt toxin is released as exudate from roots. In the soil, it was not clear from the experimental description how discrimination between leakage from injured roots and exudated toxin was achieved. However, the ecologically meaningful new outcome of this study was that Bt proteins are continuously released into the rhizosphere. Therefore, the microorganisms living in or nearby the root rhizosphere are exposed to Bt toxins throughout the plants' lifespan. Because rhizosphere microorganisms are very important for plant health and development, it is essential that proper testing of these microorganisms with the respective released insecticidal compound is undertaken prior to the largescale production of such insecticidal transgenic plants.

2) Fate of Bt proteins from transgenic plants: Mechanisms of persistence and degradation Section 3.2 3  70  Mechanisms of persistence Adsorption. Laboratory studies with protoxins and toxins of Bacillus thuringiensis isolated from microbial cultures or commercial formulations showed that the toxins bind with varying affinity to different soil components. The clay size fraction of soil ( studies 3 5) and humic acids ( study 7) are especially effective in binding the toxins, whereas the silt and sand size fraction of soil have a minor role ( study 5).

Clay minerals. In study 3, more Btk toxin ( Bacillus thuringiensis subsp. kurstaki) adsorbed on the clay mineral montmorillonite than on the clay mineral kaolinite ( with a mixed cation complement). Maximum adsorption on the clay minerals occurred within 30 minutes ( studies 3 and 4). The pH had a significant role in binding: In one study ( study 3), adsorption of the toxin ( Btk) on montmorillonite decreased as the pH increased from 4.4 to 10.0. In another study ( study 4) adsorption of Btk and Btt proteins ( Bacillus thuringiensis subsp. Tenebrionis) on montmorillonite and kaolinite decreased as pH increased from 6.0 to 11.0. Below pH 60. adsorption decreased also ( study 4). On clays coated with hydrous ferric oxides ( HFO coated;. in the study called ' dirty' clays) maximal adsorption occurred at pH 5.0 ( Btk) ( study 4).

EPA Response: EPA receives data on soil degradation of Cry proteins performed at high Cry protein doses that would account for the amount of Cry protein released by continuous exudation by plant roots. In addition, it is general knowledge that microbial degradation of proteins in soil is ubiquitous. This observation was reaffirmed by the SAP ( SAP Report No. 99 06, February 4, 2000). The authors of the studies quoted above have also shown that Cry proteins adsorbed to soil minerals are quickly metabolized by soil microbes upon elution. The data reviewed by the Agency show that the degradation in soil occurs within days to a barely detectable background levels. Data available to date also do not show any detrimental effects on the soil biota ( USEPA. 2002. MON 863 BRAD Section C. I. B. 3). These aspects will, however, be investigated in greater detail over several years to provide confirmatory long term effects information.

Commenter Name: Angelika Hilbeck Commenter Organization Name: Greenpeace Comment Number: 30509 113000Part2 Excerpt Number: 24 Excerpt Text: The Bt proteins did not significantly intercalate the clay minerals montmorillonite and kaulinite when bound. Therefore, it can be assumed that the proteins bind primarily on the external surface of these clay minerals.

Humic acids. As for the clay minerals, pH and, additionally, the content of phenolic groups were primarily responsible for differences in the amount of Bt proteins bound ( study 7). Lower pH and higher content of phenolic groups resulted in higher amounts of proteins bound. Maximum adsorption occurred within I to 2 hours on humic acids from forest soils and within 4 to 8 hours on humic acids from cultivated soils.

Desorption of clay minerals. In study 3, most of the adsorbed toxin ( Btk) was desorbed from kaolinite by two washes with double distilled water. In the same study, toxins bound on montmorillonite were not desorbed with double distilled water. In study 4, larger amounts of Btt were desorbed by one or two washes with double distilled water than of Btk, indicating that the Btk proteins had a higher affinity for the clays Section 3.2 3  71 tested.

Desorption of humic acids. Larger amounts of Bt proteins could be desorbed with double distilled water from huniic acids at higher pH and lower content of phenolic groups ( study 7).

All toxins, whether bound to clay minerals or humic acids, retained their insecticidal activity ( studies 3, 5 8).

EPA Response: This comment is a summary of soil binding findings which were considered during the EPA risk assessment process. Additional research on the long term accumulation and effects of Bt proteins in soils are in progress.

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 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: This comment is a summary of studies of the effects of Cry protein containing plant tissue on soil microbial composition. These findings were considered during the EPA risk assessment process. Additional research Section 3.2 3  72 is ongoing on the long term accumulation and effects of Bt proteins in soils.

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; 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 above comment is a discussion of a transgenic potato experiment recommending use of untreated control plots in assessing effects of Cry proteins. This comment is not relevant to the MON 863 registration. The field studies submitted for Cry3Bb MON863 corn did include data from untreated control plots.

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 Section 3.2 3  73 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 issue of actual ingestion of the test material by test species has been presented by the EPA to the August 2000 SAP. As a result, new testing protocols are being developed to directly address this issue.

Commenter Name: Angelika Hilbeck Commenter Organization Name: Greenpeace Comment Number: 30509 113000Part2 Excerpt Number: 28 Excerpt Text: Where plant meets soil In agroecosystems, living, plants and dead plant material enter the soil ecosystem in numerous ways, as roots, tubers, etc., and plant residues/ debris entering the soil ecosystem constantly throughout the season, and with distinct cyclic peaks upon harvest when great amounts of plant residues are left behind. Another route that plant compounds can enter the soil are root exudates.

Root exudation The rhizosphere comprises the outer tissues of the root ( cortex and epidermis), the root surface, and the soil around the root. It can be thought of as the microbial continuum that stretches out from the endoderniils of the root ( a layer of cells within the root) into the soil. The plants stimulate microbial colonization by secreting carbohydrates and other compounds that can be used by the microorganisms as a source of carbon and energy. In return, the plants benefit from some of these associations through a greater availability of mineral nutrients. Beneficial, neutral, and harmful bacteria, fungi, algae, protozoa, nematodes, and microarthropods live in the rhizosphere in an intricate food web ( see below). This entire underground ecosystem depends for its food primarily on carbohydrates and other molecules secreted by the roots into the soil, collectively called ' exudates'. The health of this underground ecosystem can be profoundly affected by agricultural practices, tipping the balance in favor of beneficial or harmful organisms. The interactions are enormously complex, and we know only little about them ( Chrispeels and Sadava 1994). Section 3.2 3  74 A number of microorganisms in the rhizosphere have specific functions that benefit the plant, such as the nitrogen fixation in low input agricultural systems or phosphate uptake ( see below). Others simply aid in decomposition of organic matter and, consequently, the release and recycling of important nutrients.

EPA Response: The above comment is a statement on the role of microbes in the root zone of plants and on the symbiotic relationship between the plant and the soil biota. The effects on soil biota were considered by the EPA during the MON 863 corn risk assessment process. ( USEPA. 2002. MON 863 BRAD Section C. I. B. 5,6).

Commenter Name: Angelika Hilbeck Commenter Organization Name: Greenpeace Comment Number: 30509 113000Part2 Excerpt Number: 29 Excerpt Text: Every root tip is covered by a root cap, a specialized tissue that forms a cap like structure at the end of the root tip. Cell divisions in this meristem assure that root cap cells are continuously produced. As the cells mature, they are shed or abraded from the cap surface between the soil particles as the root grows. For instance, the cap of corn roots produces up to 20' 000 cells during the plants life. These root cap cells are differentiated cells whose main function is to produce mucilage or slime that serves as protection or a lubricant for the delicate young root tissue. Corn roots, for example, produce so much of this slime that it forms small droplets at the root tips.

In addition to these slimy secretions, the root itself secretes soluble molecules, mainly low molecular organic acids, amino acids, and sugars. This is an active process and not a passive leakage. As roots grow, they leave behind debris consisting of broken off root hairs, mucilage, root cap cells, and other molecules, all of which provide food for microorganisms that, in turn, make certain nutrients available to plants.

Symbiotic relations between plant roots and microorganisms Mycorrhiza. Most higher plants have fungal hyphae closely associated with their actively growing roots. Such an association is called ' mycorrhiza'. It is a true symbiotic relationship. The flow of photosynthetic products from the shoot to the roots benefits the fungus, which uses these products as a source carbon. The plant, in turn, benefits from the capability of the fungus to take up, for example, phosphate or other nutrients in otherwise nutrient poor soils and make them available to the plant. Without mycorrhizae, these plants grow very poorly. One reason is that fungi are able to ramify in the soil with their delicate hyphae much farther than plant roots can. Therefore, they can draw nutrients from a much larger volume of soil. Another reason is that mycorrhizae produce acids that solubilize inorganic soil compounds making them available to the plants.

EPA Response: The above comment is a statement on the role of mycorrhizal fungi in the root zone of plants and on the symbiotic relationship between the plant and the soil biota. The effects on soil biota were considered by the EPA during the MON 863 corn risk assessment process. ( USEPA. 2002. MON 863 BRAD Section C. I. B. 5,6). Section 3.2 3  75 Commenter Name: Angelika Hilbeck Commenter Organization Name: Greenpeace Comment Number: 30509 113000Part2 Excerpt Number: 30 Excerpt Text: Nitrogen fixation. Besides photosynthesis, biological nitrogen fixation is the single most important biological process that supports life on Earth ( Chrispeels and Sadava 1994). Crucial in nitrogen fixation are Rhizobium, bacteria ( belonging to several species) that live symbiotically with leguminous plants. They live in nodules of plant roots where they are able to fix, i. e.,, reduce, molecular nitrogen to ammonia, which they transfer to plants. Rhizobium spp. are free living bacteria that attach themselves to the surface of the root hairs of legume plants and cause the root hairs to engulf them. The interaction between Rhizobium and its legume host is very specific. Usually one species of Rhizobium can infect only one or a few closely related plant species.

Soil Ecosystems and Soil Food Webs As soil ecosystems are poorly understood, it is difficult to assess and interpret potential impacts of a steady input of an insecticidal protein like Bt from transgenic plants on populations and communities of soil organisms. Via direct and indirect exposure of soil micro and macro organisms to the Bt toxins, vital processes like decomposition of organic matter and nutrient cycling can potentially be affected in the long run. In undisturbed ecosystems, these processes are tightly coupled with plant growth, and in disturbed ecosystems, this coupling is lost or reduced and, hence, human intervention becomes increasingly necessary for their compensation. Lal and Stewart ( 1992) suggest that soil organism losses correlate with detrimental ecosystem changes. Therefore, monitoring the development of structure and function of soil food webs and assessment of potential toxic impact should be mandatory for assessing ecosystem health after introduction of great amounts of bioactive compounds like Bt toxins.

Soil food web research is a fairly new field, and we are just at the beginning to understand how it influences plant communities and how in turn plant communities influence soil food web structure ( Hendrix et al. 1986). However, it can be safely stated that their importance for plant health and ecosystem functioning can hardly be overestimated.

EPA Response: This comment is a summary of the beneficial effects of nitrogen fixing bacteria and the need to ensure that introduction of Cry protein containing plant tissue does not disrupt these processes. These issues were considered by the EPA during the risk assessment process. To date there is no evidence that such disruption is taking place by Cry proteins, especially since corn is not a legume. Nevertheless, additional research on the long term accumulation and effects of Bt proteins on soil processes are in progress.

Commenter Name: Angelika Hilbeck Commenter Organization Name: Greenpeace Comment Number: 30509 113000Part2 Excerpt Number: 31 Excerpt Text: Organic matter in soil is derived from the remains of plants, animals, and microorganisms that live in or on the soil. Leaf litter accumulates on the soil surface, and as it decays, it is gradually mixed in the top layer Section 3.2 3  76 of soil. Soil surface inhabiting animals such as earthworms, centipedes, millipedes, etc., ingest large parts of plant material or chew 1arge~ and tough plant parts tQ smaller pieces, thereby preparing the plant material for further degradation by soil microorganisms. Some, e. g. earthworms, also actively incorporate plant material from the soil surface into the soil by pulling the plant material downwards through small soil channels. Earthworms are major agents in the process of soil creation through the formation of clay humus complexes. The eaten portions of the plant material are excreted by the soil organisms as a black organic residue. This process of transforming organic residues into humus is completed by soil microorganisms that secrete cell wall degrading enzymes that convert insoluble complex carbohydrates and other compounds into soluble products that they can take up and further transform ( Chrispeels and Sadava 1994). Primary and secondary minerals derived from weathered rocks and organic particles are intimately mixed in soil, forming various types of soil aggregates, which are found in association with air ( i. e., the soil atmosphere) and soil water ( i. e., the soil solution) in the pores that exist between particles and/ or aggregates. Other soil arthropods living below the soil surface include, for example, the larvae of several lepidopteran, coleopteran, and dipteran species, collembolans, and mites. Among these are also a number of pest species such as Diabrotica spp. ( corn root worms), Melolontha spp. ( chafer larvae), Agrotis spp. ( cutworms), or Agriotes spp. ( wireworms).

Viruses and microorganisms, including bacteria, fungi, algae, and protozoa, are found in large numbers in soil  usually between one and ten million microorganisms are present per gram of soil  with bacteria and fungi being the most prevalent in agricultural soils. Microorganisms are important as almost every chemical transformation taking place in soil involves active contributions from soil microorganisms. In particular, they have an active role in soil fertility, as a result of their involvement in the cycle of nutrients, such as carbon and nitrogen, which are required for plant growth. Saprophytic fungi and bacteria are the base of the detrital food web ( Figure 1). Other soil microorganisms increase the availability of mineral nutrients ( e. g. phosphorus) t6 plants ( e. g. mycorrhizal fungi, see above) or can transform mtrogen gas present in the atmosphere into soluble nitrogenous compounds that plants can utilize for growth ( e. g. nitrogen fixing bacteria, see above).

EPA Response: This comment is a summary of the beneficial effects of soil biota on the formation of fertile agricultural soil. These issues were considered by the EPA during the MON863 corn risk assessment process. Additional research on the long term accumulation and effects of Bt proteins in soils is in progress.

Commenter Name: Angelika Hilbeck Commenter Organization Name: Greenpeace Comment Number: 30509 113000Part2 Excerpt Number: 32 Excerpt Text: Protozoa are important in maintaining plant available N and mineralization processes, and as bacteria feeders they are important in controlling bacteria populations ( Coleman 1985, Foissner 1986). Protozoa can act as indicators of the presence of certain hazardous wastes and, therefore, may be highly useful indicators of certain types of environmental impacts ( Foissner 1986). Collembolans feeding on saprophytic fungi are therefore also important regulators of these processes ( Figure 1).

[ see PDF for " Figure 1. Schematic soil food web. Arrows indicate the direction of flows of nutrients"] Section 3.2 3  77 Nematodes are one of the most ecologically diverse groups of animals and grouped in various trophic categories, most importantly plant feeding and predatory nematodes, but there are also bacteria feeders or those that eat protozoa and fungi.

Nematodes and protozoa function as main regulators of mineralization processes in soil ( Coleman 1985). They release a large portion of N when feeding on their various prey groups. On the other hand, plantfeeding nematodes are among the worst pest species in agriculture and with many of them becoming increasingly resistant against the synthetic nematicides, they may increase in meaning in the future. However, the activities of these groups and predator prey interactions, ( which determine the rate at which mineralization occurs) are in turn affected, and perhaps controlled by, higher level predators such as centipedes, ground beetles, spiders, and small mammals.

Known impacts of other transgenic organisms in soil ecosystems EPA Response: This comment is a discussion of the role played by nematodes in the soil and the agroecosystem. This issue was considered by the EPA during the MON863 corn risk assessment process. Preliminary data were presented that show possible beneficial effects of MON 863 by controlling some pathogenic nematodes. ( USEPA. 2002. MON 863 BRAD Section C. I. B. 3. b). Conclusive data on the effects of Cry3Bb on nematodes was requested by EPA. ( USEPA. 2002. MON 863 BRAD Section C. I. F).

Commenter Name: Angelika Hilbeck Commenter Organization Name: Greenpeace Comment Number: 30509 113000Part2 Excerpt Number: 33 Excerpt Text: In a field study conducted by Donegan et al. ( 1999), three genotypes of alfalfa ( 2 transgenic and one parental) were planted in an agricultural field. Immediately prior to planting, the roots of these plants were either left uninoculated or were inoculated with a wild type strain, a recombinant strain with antibiotic resistance, or a recombinant strain with antibiotic resistance and enhanced nitrogen fixation capability ( Sinorhizobium meliloti). Effects on the soil micro organisms were measured ( metabolic and DNA fingerprinting, soil microbial respiration; population counts, nematode and micro arthropod diversity) as well as plant and shoot growth, enzyme activities and soil chemistry. The researchers found significant differences in the soil bacterial communities associated with these alfalfa genotypes. Most distinct differences were observed for one transgenic, lignin peroxidase producing alfalfa genotype. Significant differences in the microbial community composition were observed that were associated with differences in the respective microbial activities, e. g. soil enzyme activities and pH levels. This resulted also in altered plant growth. To evaluate the effects regarding their overall effect on soil fertility and soil productivity requires further experimentation.

In an earlier study, researchers from the same laboratory studied the decomposition of genetically engineered proteinase inhibitor producing tobacco plants under field conditions ( Donegan et al. 1997). They buried litterbags containing transgenic and parental tobacco plants m field plots over a 5 month period. Proteinase inhibitor I protein was detectable and immunologically active for at least 57 days. Further, decomposing parental and transgenic plant material differed in quality ( carbon content) and in the Section 3.2 3  78 response of exposed soil organisms ( collembola and nematodes). Collembola populations in the soil surrounding the transgenic plant litterbags were significantly lower than in the non transgenic control. Nematode populations in the soil surrounding the litterbags of transgenic plants were greater than m those of the non transgenic control and had a different trophic composition ( higher ratio of ftingal feeding vs. bacterial feeding nematodes). Again, an evaluation of these effects regarding their significance for soil health and fertility requires further experimentation.