4
Case Studies of APHIS Assessments

Chapter 3 provided an overview of the regulations and general procedures used by the Animal and Plant Health Inspection Service (APHIS) to review the large number of transgenic plants coming through the research and development pipeline each year. This chapter examines in detail the specific procedures and judgments made by APHIS in its assessment of a set of cases that have moved through the notification, permitting, and deregulation pathways. The specific set of cases examined was chosen in order to cover a broad array of products, procedures, and potential risks. For each case the types of risks considered by APHIS are noted, and the information and processes used by APHIS in making judgments about these risks are assessed. Risks not considered by APHIS also are pointed out. For all of the case studies, the degree of public and external scientist involvement in the decision-making process also is assessed.

First presented is a case study for a transgenic plant that was field tested through the notification process and one that went through the permitting process. Then four types of transgenic plants that have been deregulated by APHIS are examined. Chapter 5 develops a more general assessment of APHIS oversight and makes recommendations for specific changes.

NOTIFICATION PROCESS CASE STUDY

Notification for Salt- and Drought-Tolerant Bermudagrass

Bermudagrass (Cynodon dactylon and a few related species) is an important grass of lawns and pastures in the United States and elsewhere



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Environmental Effects of Transgenic Plants: The Scope and Adequacy of Regulation 4 Case Studies of APHIS Assessments Chapter 3 provided an overview of the regulations and general procedures used by the Animal and Plant Health Inspection Service (APHIS) to review the large number of transgenic plants coming through the research and development pipeline each year. This chapter examines in detail the specific procedures and judgments made by APHIS in its assessment of a set of cases that have moved through the notification, permitting, and deregulation pathways. The specific set of cases examined was chosen in order to cover a broad array of products, procedures, and potential risks. For each case the types of risks considered by APHIS are noted, and the information and processes used by APHIS in making judgments about these risks are assessed. Risks not considered by APHIS also are pointed out. For all of the case studies, the degree of public and external scientist involvement in the decision-making process also is assessed. First presented is a case study for a transgenic plant that was field tested through the notification process and one that went through the permitting process. Then four types of transgenic plants that have been deregulated by APHIS are examined. Chapter 5 develops a more general assessment of APHIS oversight and makes recommendations for specific changes. NOTIFICATION PROCESS CASE STUDY Notification for Salt- and Drought-Tolerant Bermudagrass Bermudagrass (Cynodon dactylon and a few related species) is an important grass of lawns and pastures in the United States and elsewhere

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Environmental Effects of Transgenic Plants: The Scope and Adequacy of Regulation (Simpson and Ogorzaly 1995, Taliaferro 1995). Consequently, strategies have been sought to improve the performance of this plant under a variety of environmental stresses (Cisar et al. 2000). Cynodon dactylon is also considered one of the world’s worst weeds (Holm et al. 1977), especially in the tropics and subtropics but also in warmer parts of temperate zones. It is an especially important weed of sugarcane, cotton, and corn. It is a troublesome weed in some parts of the United States; for example, in the West it has been described as “posing a serious threat to crop production and turf management” (Ball et al. 2000). The species is wind pollinated and reproduces by seed but more frequently by vegetative spread of plant parts (stolons and rhizomes). Since 1999 APHIS has received and acknowledged the eligibility of five notifications from Rutgers University for New Jersey field tests of salt- and drought-tolerant bermudagrass. One of the notifications (99-308-10n) is discussed here. The notification application names the transformed organism as a bermudagrass hybrid, Cynodon dactylon × C. transvaalensis. The application also describes the mode of transformation (in this case, particle bombardment). The added genes also are detailed. The gene inserted to confer possible drought and salt tolerance was betaine aldehyde dehydrogenase from Atriplex hortensis, a plant species that shows considerable drought and salt tolerance. The promoter for that gene was maize ubiquitin. The terminator was nopaline synthase polyadenylation sequence from Agrobacterium tumefaciens. The plants also are transgenic for a selectable marker, hygromycin B phosphotransferase from the bacterium Streptomyces hygroscopicus with a rice actin promoter and a 35s polyadenylation sequence from cauliflower mosaic virus (CaMV) for a terminator. The specific planned introduction was a field test at Rutgers University Horticulture Farm II. The applicant certified that the regulated article “will be introduced in accordance with the eligibility criteria and performance standards set forth in 7 CFR 340.3.” Because the primary concern for the notification process is containment, adherence to performance standards is important. And because the transformed organism is closely related to an important weed and contains transgenes that might confer an advantage to that weed, that containment is not just an academic exercise. Indeed, the applicant took containment very seriously when reporting his containment procedures to APHIS in a letter dated March 26, 2001. The transformed organism is the variety “TifEagle” (Hanna and Elsner 1999), used primarily as a turfgrass for putting greens in golf courses. The variety is a triploid bermudagrass that is both male and female sterile. Thus, dispersal by pollen and seed does not occur. The field test involved a comparison of the performance

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Environmental Effects of Transgenic Plants: The Scope and Adequacy of Regulation of the transgenic grass to nontransgenic strains in an area considerably less than an acre. Twice weekly mowing maintained the grass at a height of 5/32 inches. The borders of the field test were maintained by application of an herbicide treatment every two weeks. The applicant also states that TifEagle cannot withstand central New Jersey’s cold winters but that the plots would be monitored to see if the transgenic plants had developed winterhardiness. The applicant does not describe other methods for preventing accidental spread by fragments of the grass that may attach to equipment or shoes. But the ultradwarf, dense-growing nature of this particular variety probably makes such fragmentation extremely unlikely—especially compared to the easily broken, rambling runners of the wild type. Environmental Risks Considered by APHIS APHIS does not conduct environmental assessments on notifications, which are assumed to be safe based on meeting the notification criteria and based on using plant-specific performance standards that minimize any chance of plant or gene escape beyond the confines of the field plot. Involvement of Potential Participant Groups There is no public or external scientific involvement for this or any other plant that goes through the notification process. THE PERMITTING PROCESS Permitting of Maize-Expressing Proteins with Pharmaceutical Applications Background This case is a permit application (00-073-01r, dated March 8, 2000) in which the applicant (ProdiGene) requested permission to grow maize transformed with one or more transgene-expressing proteins with pharmaceutical properties (with the date of intended release 60 days later). The specific phenotype is listed as “antibody production in seed.” A description of the transgenic plant was not available because it is confidential business information (CBI). The purpose of the permit was to grow the transformed maize for seed increase and genetic improvement. The test plots, totaling no more than 2 acres, were to be grown in Nebraska.

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Environmental Effects of Transgenic Plants: The Scope and Adequacy of Regulation Environmental Risks Considered by APHIS Although APHIS decided that this application did not require an environmental assessment, the permit application itself provides information that addresses a number of environmental issues (see Chapter 3). The following information was gleaned from the permit application and from APHIS’s letter giving notice of its review to the state of Nebraska. Environmental Impact Related to Donor. Maize was transformed with four genes, one or more of which encode a protein or proteins (one or more antibodies) with pharmaceutical properties. The identities of the genes, their enhancers, their products, and their sources were marked CBI and therefore are not available to the public. The plants were also transformed with the selectable marker, the gene coding for maize-optimized phosphinothricin acetyl transferase (moPAT) derived from the bacterium Streptomyces viridochromogenes, a nonpathogenic soil bacterium. The selectable marker results in tolerance to the herbicide glufosinate. That promotor and terminator for that gene were from CaMV. APHIS concluded that none of these genes contained any inherent plant pest characteristics. APHIS also concluded that the promoter and terminator from CaMV cannot cause plant disease by themselves or in conjunction with any of the genes introduced into the maize plants. Environmental Impact Related to the Vector and Vector Agent. Transformation was facilitated with Agrobacterium tumefaciens. This is a well-characterized transformation system resulting in stably integrated and inherited transgenes. APHIS found no inherent environmental impact. Quarantine of Organism and Final Disposition. The focus of the permitting process is to ensure that the transgene escape does not occur by the dispersal of seed, pollen, or plants and that transgenic plants do not persist after the experiment ends. The applicants stated that an isolation distance of 1,320 feet would be used to minimize transgene flow by pollen (this is double the 660-foot isolation distance recommended in the APHIS 1997 user’s guide). The applicants stated that all seed would be harvested and that the plants would be plowed into the soil. The field would then be monitored for volunteers, which would be destroyed by hand or with an appropriate herbicide. APHIS concluded that these measures were adequate to confine the transgene and the transgenic organism. Supplemental permit conditions included monitoring by the appropriate officials, the reporting of field data to APHIS within six months of the termination of the field test, monitoring the field for one year after the test for volunteers, and notification of any changes in protocols.

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Environmental Effects of Transgenic Plants: The Scope and Adequacy of Regulation An issue taken at face value is the isolation distance requirement for corn, a highly outcrossing, wind-pollinated species. Although corn has no weedy or wild relatives in North America with which it can freely cross, isolation between the transgenic corn and other corn crops remains an issue. Isolation distances are set to prevent some minimum level of contamination but were not set up to provide for zero levels of contamination. And zero levels are what would be needed to absolutely prevent escape of the transgene into the environment. For example, the APHIS-recommended isolation distance of 660 feet for corn is presumably derived from that required by the U.S. Department of Agriculture (USDA 1994a) for producing foundation seed (used for seed increase); the maximum proportion of contamination is 0.1%. There is no reason to assume that absolute isolation should be attained at twice that distance. It is likely there would be some very low level of contamination of any corn grown at or near the 1,320-foot isolation distance from the test plots. If adjacent corn were grown for a purpose such that its seeds were not replanted, there would be no permanent escape into the environment. However, as outlined in Chapter 2, some consider that the risk of these genes entering the food supply should be considered an environmental risk. If contaminated corn were grown such that its seeds were to be replanted, it is possible that the transgenes (for antibody production and glufosinate tolerance) could end up in the genetic stocks. One possible example is adjacent plots of other experimental corn varieties grown for seed increase prior to commercial use. Another example could be nearby plantations of open-pollinated corn grown by a farmer who keeps the seed from year to year. Either of these scenarios could result in perpetuation of the transgenes indefinitely unless they were lost from the breeding stock by random drift. Whether the transgenes for antibody production in seed have an impact in a different corn crop depends on a variety of factors, including the specific pharmaceutical compound created through expression of the transgene; the levels at which that compound is created and stored in tissues that might be consumed by humans, farm animals, or non-target organisms; and the level at which contamination has occurred, and the threshold effects of that compound if used for animal or human food. In this case, because identity of the pharmaceutical compound is not given in the application because it is CBI, it is not possible to judge that impact. Whether the transgene for glufosinate production has an impact in corn-breeding stocks contaminated by pollen would depend on whether the contaminated stocks would find themselves under selection by that herbicide. The environmental impact of glufosinate tolerance in corn is discussed at length in the Bt corn case study below.

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Environmental Effects of Transgenic Plants: The Scope and Adequacy of Regulation Involvement of Potential Participant Groups in Decision Making Because APHIS did not see a need for conducting an environmental assessment before approving this introduction, there was no opportunity for public or external scientific involvement in this permit decision. PETITIONS FOR DEREGULATED STATUS: FOUR CASE STUDIES INVOLVING SIX PETITIONS Two Virus-Resistant Squash Petitions Background Virus-based diseases can sometimes pose important problems for crop production (Hadidi et al. 1998). Virus resistance may be transferred into a crop via conventional breeding methods but only if that resistance already exists in the crop or in a sexually compatible relative. Transgenic virus resistance provides an opportunity for disease resistance in crops whose close relatives are not resistant to the virus in question. Transgenic virus resistance can be obtained by introduction of part of the disease viral genome into the susceptible plant genome; in particular, expression of the viral coat protein (CP) often confers resistance (Powell-Abel et al. 1986, Grumet 1995). Field trials of dozens of crop species with transgenic-based virus resistance have been conducted (see “Field Test Releases in the U.S.,” Information Systems for Biotechnology online database: www.nbiap.vt.edu). As of April 2001, APHIS had approved six petitions for the deregulation of transgenic crops with virus resistance (see “Current Status of Petitions,” APHIS website: www.aphis.usda.gov/biotech/petday.html). The deregulated crops transformed are papaya, potato, and squash. The case of deregulation of Upjohn/Asgrow’s virus-resistant crookneck squash varieties (application numbers 92-204-01p and 95-352-01p) exemplifies how APHIS evaluated a number of different issues associated with the biosafety of a transgenic product. Viral diseases are periodically an important problem for growers of squashes and other cucurbit crops (Desbiez and Lecoq 1997). These diseases include those caused by zucchini yellow mosaic virus (ZYMV), watermelon mosaic virus 2 (WMV2), and cucumber mosaic virus (CMV). Aphids act as the vectors of all three viruses. Interestingly, squash varieties with genetically based virus resistance to WMV2 and ZYMV were developed almost simultaneously by both transgenic and conventional methods. In the same year, 1994, Harris Moran released a conventionally bred virus-resistant zucchini (Tigress)

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Environmental Effects of Transgenic Plants: The Scope and Adequacy of Regulation and Asgrow’s transgenic virus-resistant yellow crookneck squash (ZW-20)—whose resistance was based on expression of viral CP genes—was deregulated (USDA 1994b, Schultheis and Walters 1998). Subsequently, Asgrow’s second transgenic yellow crookneck squash (CZW-3)—containing viral CP genes to confer resistance to WMV2, ZYMV, and CMV—was deregulated in 1996 (USDA 1996). Conventionally created CMV-resistant marrow squash is commercially available from Thompson and Morgan (USDA 1996). As of January 2001, APHIS had received 66 notifications and permit applications for squash varieties with transgenic resistance for as many as five viruses (see “Field Test Releases in the U.S.,” Information Systems for Biotechnology online database: www.nbiap.vt.edu). APHIS’s action on the Upjohn/Asgrow petition for ZW-20 squash received considerable comment from the public, both pro and con. By the time APHIS made its final decision, the agency had published three Federal Register announcements and conducted a number of public meetings. Feedback on the petition is detailed below under “Involvement of Potential Participant Groups.” APHIS provided a detailed response to commenters who disagreed with its ruling in Response to the Upjohn Company/ Asgrow Seed Company Petititon 92-204-01 for Determination of Nonregulated Status for ZW-20 Squash (USDA 1994b). The petition for CZW-3 did not generate controversy. APHIS received only a few comments, all favorable to the petition, in response to a single Federal Register announcement. Nonetheless, APHIS detailed its findings by covering much of the same ground as that for ZW-20 in Response to the Asgrow Seed Company Petition 95-352-01 for Determination of Nonregulated Status for CZW-3 Squash (USDA 1996). Both APHIS response documents on the transgenic virus-resistant squashes considered a number of potential risks in some detail. Below is a highly abstracted overview of APHIS’s arguments for finding “no significant impact.” Environmental Risks Considered by APHIS Disease in the Transgenic Crop and its Progeny Directly Resulting from the Transgenes, Their Products, or Added Regulatory Sequences. Some of the DNA sequences used in transforming these squashes were derived from Agrobacterium tumefaciens (the agent of crown gall disease), but the disease-causing genes were removed. Likewise, the viral CP transgenes and additional viral regulatory sequences to control their expression were all derived from disease-causing organisms, but they and their products do not cause disease. CZW-3 also has a selectable marker for kanamycin resistance; APHIS did not consider whether that transgene and its prod-

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Environmental Effects of Transgenic Plants: The Scope and Adequacy of Regulation ucts will cause disease CZW-3, presumably because the source of this gene is not a pathogenic organism. Thus, APHIS concluded that the transgenes, their products, or added regulatory sequences do not result in plant pathogenic properties (USDA 1994b, 1996). Evolution of New Plant Viruses. For both ZW-20 and CZW-3, APHIS addressed the risks that other viruses would appear with altered host specificities (via transcapsidation, when one virus bears the coat protein of another; also known as “heteroencapsidation,” “genomic masking,” or “masked viruses”) or evolve increased virulence (from recombination with virus-derived transgenes; Matthews 1991). With regard to the first issue, APHIS pointed out that mixed infections by plant viruses are not uncommon, that these viruses are common viruses of squash, and that transcapsidation is already occurring in infected plants. Given that the amount of coat protein in the transgenic squashes is considerably less than that in naturally infected plants, the chances of transcapsidation are lower in transgenic plants than infected ones. Furthermore, APHIS pointed out that even if masked viruses (i.e., viral nucleic acids enrobed with a coat protein of a different virus produced by the transgenes of the plant) were produced, they would have biological properties identical to those produced in naturally infected plants (USDA 1994b, 1996). With regard to evolution of virulence via recombination, APHIS concluded that: because the viral transgene is derived from virus that naturally infects the squash host, is synthesized in the same tissues as in the naturally-infected plants, is produced in less concentration than during natural infections, and if a recombinant was formed would have to be competitive with other squash-infecting viruses. APHIS believes that even if a recombinant virus did occur that [sic] this virus could be managed just like the numerous new viruses that are detected every year in the United States. (USDA 1996; cf. 1994b) APHIS addressed two additional issues for its assessment of CZW-3: the release of subliminally infecting viruses (those unable to move from the initial site of infection) and synergy (the increased severity of symptoms from multiple infections; Matthews 1991). The agency addressed the concern that infection from a different virus may release subliminally infecting viruses. In the case of plants expressing CP genes, the worry is that those genes might facilitate movement out of the transgenic plants. But “since the CP transgenes in CZW-3 are all from viral strains that routinely infect the curcubit family, it is not expected [that] subliminally infecting viruses will present a problem any more serious than can occur

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Environmental Effects of Transgenic Plants: The Scope and Adequacy of Regulation in naturally infected squash plants” (USDA 1996). Synergy was not considered an environmental risk but rather an agronomic problem. “Asgrow inoculated CZW-3 with several common squash-infecting viruses. No synergistic symptoms were seen in infected plants” (USDA 1996). In both response documents, APHIS concluded that the transgenic squashes should pose no greater risk of evolution of new viruses than naturally infected plants. In the second document, that conclusion was extended to cover the case of wild relatives that pick up the transgenic traits through introgression. Increased Weediness in the Transgenic Squash Relative to Convention ally Bred Squash. For both ZW-20 and CZW-3, APHIS addressed the risk that the virus resistance genes would increase the weediness of yellow crookneck squash. Yellow crookneck squash is not listed as a common or troublesome weed anywhere in the United States; for example, it is not on the Weed Science Society of America’s “Composite List of Weeds” (available online at http://ext.agn.uiuc.edu/wssa/). Squash volunteers occur adjacent to squash production fields and, if necessary, are controlled mechanically or with herbicides. They do not readily establish as feral or free-living populations (USDA 1994b, 1996). For ZW-20, Upjohn/Asgrow supplied APHIS with data comparing the transgenic squashes with their nontransgenic counterparts, showing “no major changes in seed germination, cucurbitin levels, seed set viability, susceptibility or resistance to pathogens or insects (except ZYMV and WMV2), and there are no differences in overwintering survivability” (USDA 1994b). For CZW-3 the APHIS response document stated that “Asgrow has reported that there are no major changes in CZW-3 performance characteristics (except for resistance to CMV, ZYMV, and WMV2)” (USDA 1996). Given that the transgenic squashes would be expected to be grown in the same regions as squash is typically grown, APHIS concluded that “there is no evidence to support the conclusion that introduction of virus resistance genes into squash could increase its weediness potential. Many pathogen and insect resistance genes have been introduced into commercial varieties of squash by conventional means in the past without any reports of increased weediness” and noted that conventionally improved cultivars having resistance genes to viruses had already been developed. In both response documents, APHIS concluded that the virus resistance transgenes are unlikely to increase the weediness of yellow crookneck squash (USDA 1994b, 1996). Impact on Non-target Organisms Other Than Wild Relatives. APHIS pointed out that both ZW-20 and CZW-3 transgenic squash plants have

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Environmental Effects of Transgenic Plants: The Scope and Adequacy of Regulation no direct pathogenic properties. The protein products of the transgenes are already present in high concentration in naturally infected plants, and the levels of cucurbitin—a naturally occurring plant defensive compound—are likely to be unchanged. Therefore, APHIS concluded that “there is no reason to believe deleterious effects on beneficial organisms could result specifically from the cultivation of” the new transgenic squashes. APHIS noted Upjohn/Asgrow taste tests for cucurbitin levels, but otherwise its conclusions were based on the fact that the coat proteins present in the transgenic squashes are already present in the environment in virus-infected plants. In the second determination, APHIS briefly examined the issue of whether insecticide usage might be reduced by the introduction of the CZW-3 but did not reach a conclusion. Impacts on Free-Living Relatives of Squash Arising from Interbreeding. Most of the APHIS discussion in the response documents, particularly the 1994 one focuses on whether wild relatives could benefit from virus-resistance alleles, leading to the evolution of increased weediness. The effort was, in part, in response to several negative comments received after the three APHIS Federal Register announcements associated with ZW-20. Many of the comments questioning the decision did so because it marked an important APHIS precedent. As noted in Chapter 2, the sexual transfer of beneficial alleles from a transgenic crop to a wild relative might result in the evolution of a more difficult weed. This issue is perhaps the most widely discussed risk associated with transgenic crops (e.g., Colwell et al. 1985, Goodman and Newell 1985, Snow and Moran-Palma 1997, Hails 2000). This case study has all three elements that could create such a risk— transgenes of a type that could confer a fitness boost in the wild, a sexually compatible wild relative, and the fact that the wild relative has been classified as a weed. If the crop mates with the wild relatives introducing virus resistance into wild populations and if the primary factor limiting the aggressiveness of wild populations is disease caused by the same viruses, introgression of the transgenes could result in increased weediness of the wild relatives. To obtain more information on the relevant biology of the wild relative, APHIS commissioned a report on the risks that might be posed by crop to wild gene flow by Hugh Wilson, an expert on cucurbit taxonomy and ecology. Wilson (1993) concluded that free-living Cucurbita pepo (FLCP) is a significant weed that might benefit from protection from ZYMV and WMV2. Key information on squash and its weedy North American relatives is summarized below with APHIS’s conclusions and the committee’s evaluation of those conclusions.

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Environmental Effects of Transgenic Plants: The Scope and Adequacy of Regulation Yellow crookneck squash belongs to the species Cucurbita pepo. As discussed above, the squash itself is not a weed. However, squash freely crosses with wild weedy plants known as Texas gourd (originally classified as C. texana but now considered a subspecies of C. pepo) and as FLCP. An experiment by Kirkpatrick and Wilson (1988) demonstrated that squash and FLCP naturally hybridize freely; the crop sired 5% of the seed set by FLCP, growing 1,300m from cultivated squash. Hybrids between the crop and FLCP are fully fertile (Whitaker and Bemis 1964). Clearly, if the crop and FLCP grow in the same region, natural hybridization will occur, and crop alleles will readily enter the natural populations. Indeed, cultivated squash and FLCP co-occur in many regions of Texas, Louisiana, Alabama, Mississippi, Missouri, and Arkansas (Wilson 1993). APHIS concludes that natural hybridization will move the virus resistance genes from the transgenic crop to the wild populations (USDA 1994b, 1996). FLCP is an agricultural weed in cotton and soybean fields. At one time it was one of the 10 most important weeds in Arkansas (McCormick 1977). APHIS contacted three weed experts for their opinions on the current status of FLCP as a weed. The three experts noted that FLCP plants appeared to be “less a problem” in 1994 than during the 1980s because of new herbicides not available in the 1980s and suggested that new herbicide-tolerant crops would “further expand the tools for effective control of FLCP plants” (USDA 1994b). APHIS concluded that “FLCP plants are not a serious weed in unmanaged or agricultural ecosystems [because] “the registration of new herbicides now allows effective management of these plants” (USDA 1994b). However, two of the three weed experts reported that FLCP is less of a problem; it is not clear how serious a weed they still consider it to be. The key issue raised by the foregoing data is whether virus resistance genes will provide enough of a benefit that FLCP becomes a more difficult weed. Wild and cultivated C. pepo are susceptible to the same viruses (e.g., Provvidenti et al. 1978). To determine whether viruses limit the population size and number of FLCP, Asgrow conducted a survey in 1993. Fourteen FLCP populations (two in Arkansas, four in Louisiana, and eight in Mississippi—a severe drought precluded sampling in Texas) in nine locations were visited once (when plants were at maturity); no visual symptoms of viral infection were noted. Some of these sites were within a mile of cultivated squash. But it was not reported whether the nearby cultivated plants were infected with virus; that information would have shown whether viruses were present that year. A single plant was sampled from each population. Each plant was subjected to multiple analyses to check for asymptomatic viral infection, and all were found to be virus free. On the basis of these data and qualitative anecdotal reports

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Environmental Effects of Transgenic Plants: The Scope and Adequacy of Regulation might minimize outbreaks of spider mites by allowing their natural enemies to survive. In contrast, minor pests may become more predominant as ECB is controlled and if foliar insecticides are reduced (Ostlie and Hutchison 1997). Community-level tests were restricted to above-ground species; given the persistence of Bt proteins in the soil from root exudates or after incorporation of plant material (Stotzky 2000, 2001), any evidence for no significant effect on communities of soil organisms should also be discussed. Potential for an Adverse Impact on Threatened or Endangered Species. APHIS consulted the list of threatened and endangered species (50 CFR 17.11). None of these species feed on corn, so APHIS concluded that Bt corn would not affect these species. No mention of toxic effects of corn pollen on any sensitive Lepidopteran species was made for either Event 176 or CBH-351. Nor was there mention of any possible effects to nontarget susceptible Lepidopterans that may be dependent on Zea host plants. The latter, though beyond the scope required, may be a proactive consideration for assessing possible environmental consequences should transgenes escape and affect population levels of non-target moths and butterflies, which are also plant pollinators (Letourneau et al. 2001). The possibility that threatened or endangered plants could rely on Bt-susceptible Lepidopteran pollinators was not mentioned. While this possibility may not be high, it is best to be thorough in these assessments. Potential for an Adverse Impact on the Ability to Control Non-target Insect Pests. In the CBH-351 determination, APHIS briefly examined the issue of whether insecticide usage might be reduced by the introduction of Bt corn Event CBH-351 but did not reach a conclusion (USDA 1995, 1998). Perhaps because the APHIS assessments expected no adverse effects on natural enemies and possibly positive effects due to the curtailed usage of broad-spectrum pesticides, the notion of secondary pests is not discussed directly. If, in response to low levels of target pests, non-target insect pests increased and became secondary pests in Bt corn, those species would need to be controlled by alternative measures. Effects of the Cultivation of Bt Corn on the Ability to Control Insects and Weeds in Corn and Other Crops. APHIS considered evolution of pest resistance to Cry proteins for Event 176. These proteins are similar to those used for ECB control in commercially available crystalline powder formations. Based partly on experimental demonstrations of resistance evolution in Lepidoptera to Cry toxins, APHIS predicted that resistant insects would probably evolve in response to Bt corn. However, a resistance management strategy was outside the scope of the determination.

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Environmental Effects of Transgenic Plants: The Scope and Adequacy of Regulation APHIS summarized statements from CIBA Seeds—that Bt corn be used within the scope of an integrated pest management (IPM) strategy, that populations of ECB be monitored for resistance, that high-dose expression coupled with non-Bt corn refugia and development and use of new insect control proteins would delay the evolution of pest resistance, and that farmers be educated about resistance management strategies. APHIS suggested that, should these measures fail, resistant ECB populations might be controlled by agronomic practices such as rotation and alternate insecticides. The determination document concluded that evolution of resistance to Bt-based insecticides is a potential risk associated with Event 176 but that this risk was no greater than that posed by applying insecticides themselves. For Event CBH-351, which expresses a protein different from those in commercial formulations of bacterial sprays, similar arguments were tendered. Cross-resistance was considered unlikely due to separate receptors in some species, so the Cry9C protein was suggested as a useful alternative when resistance evolves to other Bt toxins (such as that in Event 176). APHIS concluded that CBH-351 should pose no greater effects in resistance evolution than the use of ECB-tolerant corn cultivars, chemical insecticides, or biological insecticides (USDA 1998). The possible consequences of herbicide tolerance in affecting weed control, addressed only for Event CBH-351, were predicted to be positive, allowing more choice among postemergent herbicides and no-till options. Although APHIS makes some comments about resistance, the agency has apparently relied on the Environmental Protection Agency (EPA) to formalize and/or enforce resistance management plans, encouraging its consideration. The committee suggests that APHIS either indicate that resistance management is beyond its scope and not discuss it or provide detailed analysis of the practical issues of resistance management efforts in field corn. Otherwise, the outcome can be perceived as different levels of scrutiny between the EPA and APHIS. Environmental Risks Not Considered by APHIS APHIS did not directly consider whether transgenic corn would have a negative impact on corn’s wild relatives in the United States or elsewhere, either in terms of changes in their genetic diversity or in terms of posing an impact that might lead to their extinction. This is likely due to the conclusions that hybrids with Zea or Tripsacum would rarely occur, especially in the United States. Mortality of non-target Lepidoptera should susceptible species ingest toxin-containing pollen on their host plants is not discussed. Although threatened or endangered Lepidoptera were considered, the link between threatened or endangered plants and Bt-susceptible Lepidopteran pollinators was not explored.

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Environmental Effects of Transgenic Plants: The Scope and Adequacy of Regulation Public Involvement The determination documents describe comments received on each of the two petitions (Event 176 and Event CBH-351) during the designated 60-day period after posting in the Federal Register. Most (2,271 of 2,309) were form letters (source not specified), and 2,307 either favored deregulation or endorsed the concept of an ECB-resistant corn variety. One letter pointed out that CBH-351 controls third- and fourth-stage ECB larvae. The two commenters expressing reservations were concerned about resistance management and the establishment of refugia of nontransgenic corn where the 176 Event would be grown. There was no indication from the documents of any other public involvement in APHIS’s decision-making process. Herbicide-Tolerant and Insect-Resistant Cotton Background Cotton production has historically relied on heavy use of both insecticides and herbicides. On a per-acre basis during the 1990s, the number of pounds of insecticide used in cotton was three to eight times more than in corn and about 100 times higher than in soybean (NRC 2000b). A number of key insect pests of cotton such as the tobacco budworm have evolved resistance to many insecticides. In the mid-1990s insecticide resistance threatened the economic viability of cotton farming in a number of areas of the United States (e.g., Luttrell et al. 1994). Herbicide use in conventional cotton has been high and on par with that for other row crops such as soybean and corn. However, the available herbicides have been difficult for farmers to work with because of the limited time period for high efficacy and the limited spectrum of weeds killed by each herbicide. As a result, cotton farmers have often had to use multiple herbicides to control weeds. Any technology that increases the efficiency of weed control is of interest to farmers. As discussed in the prior case study, transgenic cultivars expressing insecticidal proteins derived from the soil bacterium Bacillus thuringiensis (Bt) have been successful in limiting damage by a number of Lepidopteran insect pests (e.g., European corn borer, pink bollworm, tobacco budworm, cotton bollworm; Gould 1998). It was therefore not surprising that many cotton farmers whose livelihood was threatened by insecticide resistance embraced transgenic Bt-cotton, which caused nearly 100% mortality of the tobacco budworm. Transgenic cultivars with herbicide tolerance and/or insect resistance were planted on over 40 million hectares in 2000 (James 2000), making

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Environmental Effects of Transgenic Plants: The Scope and Adequacy of Regulation these two crop traits the most widely used products of agricultural biotechnology in the world. Commercial sale of insect-resistant transgenic cotton began in 1996, and the sale of herbicide-tolerant cotton began in 1997 (USDA 1999; see also Biotech Basics 2001). Increasingly, cotton cultivars are being produced that have both herbicide tolerance and insect resistance. In 2000, one-third of all transgenic cotton in the United States had both traits (USDA-NASS 2001). There are two approaches for gaining regulatory approval of a cotton cultivar that is both herbicide tolerant and insecticidal. The most common approach since 1996 has been to obtain regulatory approval for each trait individually. For example, a herbicide-tolerant cotton genotype is developed and a petition is sent to APHIS asking for deregulated status. A Bt-producing cotton genotype is developed separately and goes through the EPA regulatory process as well as a petition for deregulated status with APHIS. Once the herbicide-tolerant cotton and Bt-producing cotton are granted nonregulated status, APHIS has no authority over those plants. Because the herbicide-tolerant cotton is not in itself a pesticide, EPA has no authority to govern its sale (EPA does regulate the sale of all herbicides). Therefore, anyone with legal access to the deregulated insecticidal cotton germplasm and to the deregulated herbicide-tolerant cotton germplasm can cross the two types of cotton and produce a new cultivar with both traits by this conventional breeding technique. This multitrait (generally referred to as “stacked trait”) cotton can then be commercialized without further regulatory oversight. In the case study examined here (petition 97-013-01p for determination of nonregulated status for Event 31807 and Event 31808), Calgene took a different approach to developing a cotton plant with herbicide tolerance and insect resistance. Although not clearly stated in the APHIS environmental assessment (USDA 1997b), it appears that Calgene developed a single construct for insertion into the cotton genome that contained both a gene for bromoxynil tolerance and the Bt gene, Cry1Ac, for insecticidal activity. The breeding advantage for using a single construct with both genes tightly linked is that the probability of segregation of the two genes during backcrossing to other cotton cultivars is extremely low. Because the two genes are essentially inherited as a unit, Calgene had both traits reviewed simultaneously by APHIS. The committee selected this environmental assessment as a case study of multiple genes because it is a case in which APHIS examined a petition for a plant with two genes, each governing a different agriculturally important trait. Many petitions for deregulation involve plants with multiple transgenes. In most cases one gene produces the phenotype of commercial interest and a second gene acts as a selectable marker. In the case of virus-resistant

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Environmental Effects of Transgenic Plants: The Scope and Adequacy of Regulation squash, multiple genes are present for resistance to a number of viruses. This cotton case stands out because both genes have distinct commercial uses. The environmental assessment and determination documents for this petition were relatively short. The assessment formally considered only two alternative actions: “no action,” which would mean refusal to grant nonregulated status, or a determination of a “finding of no significant impact,” which would result in complete deregulation. These alternatives contrast with other recent environmental assessments. For example, in the environmental assessment of a Bt corn petition that was also reviewed in 1997 (96-317-01p), three alternative actions are stated. The additional action listed is to “approve the petition with geographical limitation.” No explanation was given in this case study’s assessment about why only two options were considered. Environmental Risks Considered by APHIS in Its Environmental Assessments and Determination Documents Disease in the Transgenic Crop and Its Progeny Resulting from the Trans genes. Because the herbicide tolerance and Bt genes were inserted using Agrobacterium tumefaciens, and because a cauliflower mosaic virus 35S promotor and a chimeric 35S promotor were part of the inserted DNA, APHIS examined the potential for risk from these sequences that came from plant pest species. The potential for these sequences to result in risks was dismissed because the disease-causing genes were not present. Potential Environmental Impacts. APHIS recognized the potential for transgenic cotton to cross with wild cottons in some parts of the continental United States but concluded that “none of the relatives of cotton in the United States show any definite weedy tendencies” (USDA 1997b). (APHIS acknowledged that judgment of weediness based on the 12 traits listed by Baker (1965) or subsequent modifications are “imperfect guides to weediness.” (The utility of Baker’s list as a regulatory guide is discussed at length in the previous case study and is not repeated here.) Furthermore, APHIS stated that gene flow to wild relatives would not be a problem because (1) “any potential effects of the trait would not significantly alter the weediness of the wild cotton; and (2) wild cotton populations have not been actively protected, but have in fact been, in some locations such as Florida, subject in the past to Federal eradication campaigns because they serve as potential hosts for the boll weevil” (USDA 1997b). The EPA, which has also reviewed transgenic Bt cotton, came to a different conclusion. EPA allowed the planting of cotton in all areas of the continental United States except southern Florida because of the presence

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Environmental Effects of Transgenic Plants: The Scope and Adequacy of Regulation of feral populations of Gossypium hirsutum that can cross with commercial cultivars. However, the EPA stated that commercial cotton is not presently grown in that area of Florida. The agency also found a risk of transgenic cotton crossing with wild cotton in Hawaii. In this case the agency was specifically concerned with the risk that hybridization of the transgenic cotton with wild G. tomentosum could threaten that species’ biodiversity and put restrictions on all but isolated breeding nurseries. The EPA noted that nontransgenic cotton would pose a similar threat but is not regulated (EPA 2000a). Potential Impacts on Non-target Organisms. APHIS concluded there is no reason to believe that transgenic cotton lines would have deleterious effects on non-target species, based in part on EPA’s finding that “foliar microbial pesticides indicated no unreasonable adverse effects on nontarget insects, birds, and mammals” (EPA 1995). Also, APHIS argued that “invertebrates such as earthworms, and all vertebrate organisms, including non-target birds, mammals and humans, are not expected to be affected by the Btk insect control protein because they would not be expected to contain the receptor protein found in the midgut of target insects” (USDA 1997b; see also BOX 4.1). The comparison of Bt cotton with Btk-sprayed pesticides is not appropriate in this case because the Btk pesticides degrade very rapidly in the field, due in large part to ultraviolet light exposure, while the Bt toxin in cotton is expressed constitutively and is tilled into the soil. Furthermore, no information is given to indicate whether the Cry1Ac toxin produced by the plant is a protoxin, as in the pesticide, or if it is an activated toxin that could have different ecological impacts (see discussion in Chapter 2). Potential Impacts on the Development of Insect Resistance to the Btk Insect Control Protein. APHIS considered the issue of insect pests evolving resistance to the Bt toxin. The environmental assessment indicated that the EPA’s active resistance management program should delay the onset of resistance. APHIS also concluded that if resistance to Bt does evolve in insect pests, the ability to control the insects will not be reduced because conventional insecticides will still be available. At the time this assessment was written, the tobacco budworm had become highly resistant to pyrethroid insecticides in major cotton-growing areas, and the cost of chemically novel replacement pesticides was about triple the cost of pyrethroids. APHIS did not discuss the fact that one of the major pests affected by Bt cotton is the corn earworm (H. zea). That species feeds on many vegetable crops and is treated with Bt sprays by organic farmers. One of the factors that led to developing resistance management programs for Bt crops was concern that in the absence of Bt

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Environmental Effects of Transgenic Plants: The Scope and Adequacy of Regulation BOX 4.1 The Mysterious Ecological Role of Bt Toxins Over 500 scientific journal articles have been published on Bacillus thuringiensis since 1999 (www.WebofScience.com). Of these, only a handful discuss topics related to the natural ecology of this bacterium. An examination of the older literature reveals a similar trend. Most ecological studies tend to examine persistence of the bacterium in the soil (e.g., Addison 1993) or competition between B. thuringiensis and related species (e,g,, Yara et al. 1997). Most of the toxicological testing of B. thuringiensis isolates have pragmatically focused on pest insects (see Schnepf et al. 1998). Some papers examined toxicity to non-target organisms, including collembola, honeybees, and daphnia, as part of the process for regulatory approval (e.g., Sims). Most of these tests indicate that the common B. thuringiensis toxins are specific to small taxonomic groups of insects, and there is therefore a tendency to conclude that insects and B. thuringiensis have coevolved with each other (see Yara et al. 1997). Indeed, there is no basis for such a conclusion. As a case in point, many of the commercialized B. thuringiensis toxins are considered specific to Lepidopteran larvae (Schnepf et al. 1998). However, general knowledge of the ecology of these larvae and this bacterium indicates that they rarely come in contact. B. thuringiensis is considered a soil bacterium and is rapidly killed when exposed to ultraviolet from direct sunlight. In order to be toxic, B. thuringiensis must be ingested. Most Lepidopteran larvae feed on leaves, fruits, flowers and plant stems. The few that feed on plant roots only ingest soil, and the bacteria in it, as a contaminant of their diet. Many Lepidopteran larvae pupate in the soil, but the prepupal stages in the soil do not feed. Furthermore, epizootic of B. thuringiensis in Lepidopteran populations are rare. The only habitat where these larvae and bacteria could commonly come in contact is in grain bins where a small number of Lepidopteran species live. If Lepidopterans are an unlikely natural host for B. thuringiensis, is there some other more likely host? It certainly seems unlikely that this bacterium would use over 10% of its protein to make a toxic crystal of protein unless it had some function. One candidate for a host that has received minimal attention is the bacteriophagous nematode. As the name implies, these nematodes eat bacteria, including B. thuringiensis. It has been known for over 10 years that some B. thuringiensis isolates are toxic to C. elegans (Feitelson et al. 1992) but only recently have studies begun to look at other nematodes (Marroquin et al. 2000, Griffitts et al. 2001). Given that bacteriophagous nematodes are one of the most diverse groups of soil invertebrates, there is at least a reasonable expectation that B. thuringiensis has evolved in interaction with these organisms. Of course, until more studies are done on the ecological interactions of B. thuringiensis and soil-dwelling organisms, it will not be known what is the most common host or food of B. thuringiensis. Without this knowledge, our ability to develop tests to examine non-target effects of B. thuringiensis toxins will at least be inefficient and at most totally misguided. In order for APHIS to develop more rigorous environmental assessments, it would be helpful to accumulate knowledge about the natural ecology of B. thuringiensis.

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Environmental Effects of Transgenic Plants: The Scope and Adequacy of Regulation organic farmers would have no means to control this insect. The environmental assessment does not comment on whether the return to the use of conventional insecticides would cause environmental problems. As indicated in the previous case study on Bt corn, it would seem best for APHIS to consider this issue in more depth or to completely defer to EPA authority. Environmental Risks Not Considered by APHIS in its Environmental Assessments and Determination Documents Potential Impacts on Non-target Organisms. Bacillus thuringiensis is a soil-dwelling organism that would rarely seem to come in contact with foliage- and fruit-feeding insects. Bt protoxin created by this bacteria must be ingested before its insecticidal properties can be activated. Many Lepidoptera pupate in the soil, but Lepidoptera with soil-dwelling feeding phases are very rare. Based on the lack of interaction between the bacteria and Lepidopteran-feeding stages, there is no obvious ecological or evolutionary explanation for B. thuringiensis producing a Lepidopteran-specific toxin. Presumably, the bacteria produce endotoxins for another purpose, but this purpose has not been determined (see BOX 4.1). APHIS presented no data on tests that the applicants might have conducted on impacts of the truncated Bt toxin on organisms in the soil, including microbes and nematodes that could interact ecologically with the Bt bacterium and its toxin. Potential to Cross with Wild Species in Some Geographic Areas in the United States. As stated in the “Background” to this case study section, the environmental assessment presents only two alternative responses to the petition for a finding of nonregulated status. APHIS did not mention the option of approving the petition with geographical limitations, even though this option was presented in other APHIS environmental assessments. In the current assessment, APHIS makes the decision to grant complete approval of the petition. The transgenic cotton lines under consideration were deregulated throughout the United States. Impacts of Commercialization of Transgenic Cotton on Environments Outside the United States. Other APHIS environmental assessments discuss concerns about the impact of a transgenic crop approved for use in the United States being planted in other countries. One example is the squash case study. The environmental assessment of Calgene’s transgenic cotton mentioned the existence of wild cotton in Mexico but does not really assess potential impacts on those species. Furthermore, this environmental assessment did not consider the fact that the specific Bt toxin

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Environmental Effects of Transgenic Plants: The Scope and Adequacy of Regulation gene under review might be useful for resistance management in the United States but might also facilitate resistance in pests that occur beyond U.S. borders. At least one cotton pest, H. zea, is known to move between Mexico and the United States each year (Raulston et al. 1986, Pair et al. 1987). Therefore, inappropriate planting of Bt cotton in Mexico could select for resistant pest individuals that would then migrate to the United States. Interactions among Multiple Transgenic Traits. APHIS treated herbicide tolerance and production of the Bt insecticide as two separate traits. It did not consider that there might be interactions between the two traits that could have a detrimental effect. Integrated pest management (IPM) emerged in the late 1950s as an effort to put pesticide use on a more ecological footing. One of the tenets of IPM is that natural processes can be manipulated to increase their effectiveness, and chemical controls should be used only when and where natural processes of control fail to keep pests below economic-injury levels (NRC 1996). Even with crops that have only Bt toxin genes, it is difficult to follow IPM guidelines because seed must be purchased in the spring before pest abundance can be predicted (Gould 1988). When two traits are combined in a single cultivar and it is impossible to purchase cultivars with only one of the two traits, farmers are forced to buy a cultivar with both traits even if they need only one for their farming operation. In the case of the stacking of herbicide tolerance and Bt toxin production, a farmer who needs herbicide tolerance may end up planting cotton with the Bt trait, even if the densities of the Bt target pests on the farm do not warrant control with the Bt trait. While it is difficult to determine how many farmers have specifically begun to use cotton with the Bt trait based on their desire to use herbicide-tolerant cotton, interviews with North Carolina farmers indicate that it may be over 20% (Bacheler 2000, North Carolina State University Extension, personal communication). This approach to the use of a pest control tool is clearly not an appropriate way to achieve the goals of IPM. In addition to negating progress in adopting IPM farming methods (NRC 2000b), overuse of pesticidal crops due to a lack of seed choice could lead to more intense selection for Bt-resistant pest strains. The EPA has developed regulations to delay the evolution of Bt-resistant pest populations. In 1998 the EPA’s Scientific Advisory Panel recommended that resistance management for Bt crops must include the following two components: (1) the transgenic plants must produce a high enough dose of toxin to kill partially resistant individuals (this dose was set at 25 times the dose needed to kill susceptible individuals) and (2) enough non-

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Environmental Effects of Transgenic Plants: The Scope and Adequacy of Regulation transgenic hosts must be planted on each farm to produce 500 susceptible pest individuals for each resistant individual produced in the Bt crop. In the case of cotton, EPA-registered transgenic plants do not produce a high dose of Bt toxin for the cotton bollworm, so a large proportion of partially resistant individuals could survive on the Bt cotton. This registration was not appropriate according to the EPA Scientific Advisory Panel, and current requirements for on-farm, non-Bt acreage are not expected to produce the desired 500:1 ratio. Only a substantial increase in the refuge acreage could ameliorate this problem. While the EPA may not increase the on-farm refuge requirements, concern over resistance evolution in H. zea due to an inadequate resistance management program was reemphasized by the most recent EPA Scientific Advisory Panel (EPA 2000b). If the stacked trait cottons such as the one approved by the environmental assessment discussed here are commercially successful, they could increase regionwide adoption of Bt cotton, further accentuating the risk of rapid evolution of Bt resistance in H. zea. This case study identifies only two negative environmental effects that could be caused by the interaction of two transgenic traits. If outcrossing to weedy relatives was more of a problem with cotton, the interaction between herbicide tolerance genes and Bt genes could exacerbate an additional risk—the transfer of the Bt trait to noncrop plants. A Bt gene inserted into a crop along with a herbicide tolerance gene could be transferred to wild relative populations much faster than in cases where the Bt gene was inherited separately from herbicide tolerance. A potential scenario is as follows: Pollen for the stacked trait cultivar crosses with a weedy relative in a cotton field. The next year progeny from the cross as well as other individuals of the weed species germinate in the cotton field. The farmer sprays bromoxynil. This kills most of the weeds without the herbicide tolerance gene, but those with the gene increase in frequency. Although the Bt gene confers no direct advantage with regard to survival against herbicide spray, there is a major increase in the frequency of weeds with the Bt gene because it is linked to the herbicide tolerance gene. This results in a large fraction of weeds that are now protected from insect feeding. If APHIS reviews transgenic plants with weedy cross-compatible relatives in the United States, such as canola, with stacked herbicide tolerance and insecticidal genes, it would definitely need to consider this interaction. Public Involvement The APHIS environmental assessment indicated that the agency received no responses to its Federal Register announcement of this petition

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Environmental Effects of Transgenic Plants: The Scope and Adequacy of Regulation for deregulation. The committee is unaware of any other attempt to involve the public in this specific assessment. CONCLUSION This chapter has reviewed case studies of the three primary APHIS regulatory pathways for field release of transgenic organisms as well as a representative sampling of the vast array of transgenic species, phenotypes, and molecular mechanisms designed to obtain those phenotypes. In many cases the committee simply reports, without much comment, how and with what information APHIS made a specific decision. The committee has little to add in those cases. In certain cases, it has pointed out situations in which APHIS might have improved its assessments. The committee has supplied substantial supporting text to explain how those improvements might have been made. While it is recognized that a few of those suggestions benefit from hindsight, most of the suggestions are based on scientific information available, but not utilized, at the time of assessment. The opportunities for improvement of assessment provide a context for the committee’s recommendations in the next chapter.