PROCEDURES AND CONCEPTS FOR EVALUATING EFFECTS OF MICROBIAL PEST CONTROL AGENTS: A RESEARCH COORDINATION WORKSHOP APRIL 1992 PREPARED BY BIOTECHNOLOGY / MICROBIAL PEST CONTROL AGENT RISK ASSESSMENT RESEARCH PROGRAM, OFFICE OF RESEARCH AND DEVELOPMENT U.S. ENVIRONMENTAL PROTECTION AGENCY ------- PROCEDURES AND CONCEPTS FOR EVALUATING EFFECTS OF MICROBIAL PEST CONTROL AGENTS: A RESEARCH COORDINATION WORKSHOP Richard Anderson1 and James Harvey2 U.S. Environmental Protection Agency1 Environmental Research Laboratory Duluth, MN Technical Resources, Inc.2 Environmental Research Laboratory Gulf Breeze, FL Project Officer Richard B. Coffin Office of Research and Development Matrix Manager for Biotechnology/Biological Control Agent Risk Assessment Research Program Gulf Breeze, FL April 1992 ------- DISCLAIMER This document has not been peer and administratively reviewed within the Environmental Protection Agency and is for Agency use/distribution only. Mention of trade names or commercial products does not constitute endorsement or recommendation for use. ------- ACKNOWLEDGEMENTS We wish to acknowledge the diligent efforts of Ms. Valerie Coseo and Ms. Maureen Stubbs, Word Processing Staff; and Ms. Barbara Wireman and Ms. Nancy Padgett, Meeting Coordination. We especially wish to thank Office of Pesticide Programs and Office of Research and Development research staff and the scientists engaged in collaborative projects whose contributions made this workshop possible. ------- ABSTRACT The Environmental Protection Agency's Office of Pesticide Programs (OPP) has responsibility to register microbial pest control agents (MPCAs) under the authority from the Federal Insecticide, Fungicide and Rodenticide Act (FIFRA). EPA has authority to prescribe tests and data-reporting requirements for both genetically engineered microorganisms or naturally-derived MPCAs. EPA's Office of Research and Development (ORD) provides scientific information to OPP on which their requirements and decisions are based. Through the Biotechnology/MPCA Risk Assessment Program, ORD conducts research on the effects of microorganisms introduced as MPCAs on ecosystems, specifically including non-target organisms and on human health. Protocols and methods to determine these effects are being developed and continually refined. Recognizing the importance of information exchange between the research and regulatory groups, the EPA Biotechnology/MPCA Risk Assessment research group of ORD and OPP held a workshop to discuss issues and develop plans for MPCA research registration needs. The workshop provided a forum for EPA personnel to exchange research and regulatory philosophies, approaches, methods, and the data developed in response to the challenges of MPCA registration. This report follows the progress of the meeting; 1) an introductory section discusses, in broad terms, EPA's mandate to regulate environmental releases of MPCAs and research issues stemming from that mandate, 2) presentations from the Office of Pesticide Programs covering regulatory issues, 3) ORD's Office of Environmental Processes and Effects Research and Office of Health Research laboratories' accomplishments, current and future research directions and publications, 4) research needs for ecological and human health protocols and methods, and 5) a discussion of ecological significance and human health considerations. Appendices containing a listing of OPP research needs, the meeting agenda, and meeting attendees are provided. ------- TABLE OF CONTENTS Abstract 1. Introduction 1 2. Office of Pesticide Programs Presentations 3 3. Office of Research and Development Presentations Duluth Environmental Research Laboratory 11 Corvallis Environmental Research Laboratory 18 Gulf Breeze Environmental Research Laboratory 23 Research Triangle Park Health Effects Research Laboratory 28 4. Standardized Protocols/Methods Ecological Testing 35 Human Health Testing 40 5. Ecological Significance and Human Health Considerations Ecological Significance 41 Human Health 46 Appendix A. Research needs from Office of Pesticide Programs 48 Appendix B. Meeting Agenda 54 Appendix C. List of Attendees 56 ------- SECTION 1 INTRODUCTION The Environmental Protection Agency has responsibility for regulating distinct aspects of the production and use of either genetically engineered microorganisms (GEMs) or naturally derived microorganisms under two legislative Acts. The Federal Insecticide, Fungicide and Rodenticide Act (FIFRA) directs EPA to register microbial pest control agents (MPCAs). The Toxic Substances Control Act (TSCA) provides EPA with jurisdiction over all chemicals already in commerce or new chemicals intended for commercial use that are not specifically covered by other regulatory authorities1. FIFRA gives EPA authority to prescribe tests and data-reporting requirements for both GEMs or naturally-derived MPCAs. The emphasis in this workshop will be on those microorganisms which are used as pest control agents. Office of Pesticide Programs (OPP) has the responsibility to register MPCAs. Exchange of information between laboratory and regulatory scientists is crucial and must be continually encouraged as regulations change and "state-of-the-art" microbial pest control is advanced. This workshop is the most recent gathering of an irregular series of meetings that started in 1982. OPP requested the Office of Research and Development (ORD) to develop testing protocols in support of Subdivision M of the Pesticide Registration Guidelines for fate and effects testing of MPCAs. Two workshops, convened in Gulf Breeze, resulted in describing freshwater and marine toxicity and pathogenicity testing protocols as well as freshwater and marine multi-species testing systems2. FIFRA regulates through ecological and health assessments of the risk of applying an MPCA. Risk assessment includes a measure of hazard determined through a laboratory testing process and an estimation of the potential environmental exposure of non-target populations3. Producing data for risk assessment requires a family of assay methods. These methods are used in a tier system, the first tier being a direct challenge to selected species. If effects are found, more exposures of increasing complexity are conducted. The next tiers may include life cycle exposures, laboratory microcosms and tests in outdoor systems. 1 Stern, A.M. 1986. Potential impacts of environmental release of biotechnology products: Assessment regulation and research need. 3. Regulatory Aspects. Environmental Management 10:453-462. 2 Couch, J. A. and K. Ranga Rao. 1983. Biorational Workshop. U.S. Environmental Protection Agency. EPA-600/X-83-054, Gulf Breeze ERL, Gulf Breeze FL Couch, J. A., T. W. Duke, S. S. Foss, and K. T. Perez. 1986. Enclosed systems for testing microbial pest control agents. Proc. workshop at ERUGulf Breeze, sponsored by U. S. EPA, Office of Pesticides Programs, Washington, D. C. 3 Urban, D.J. and N.J. Cook. 1986. Hazard Evaluation Division Standard Evaluation Procedure Ecological Risk Assessment. U.S. Environmental Protection Agency. EPA 540/9-85-001. 1 ------- Data for both human health and ecological hazards are developed through laboratory tests that produce acute exposure data usually in the form of LC50, LD50 or no effect values for some maximum exposure concentration. Predicting the environmental exposure of non-target organisms is a process which includes estimates of the quantity available to the organisms and the number, type, distribution, abundance, dynamics and natural history of organisms that may be exposed. Because MPCAs are live organisms estimating environmental concentrations and predicting the effects of microorganisms on non-target population structure and functions are a challenge. ORD has supported research on measurement of effects of MPCAs on human health and on non-target organisms. Much emphasis has been placed on single species exposures and on how complex laboratory microecosystems respond to an MPCA. An emerging research topic is the validation of laboratory test data in natural systems. Determining potential human health effects has required development of identification methods for MPCAs, tests for assessing the consequences of exposure through likely natural routes such as pulmonary and oral, genetic effects of GEMs, and establishing the mode of action of model MPCAs. The purpose of this workshop was to have EPA personnel exchange research and regulatory philosophies, approaches, methods, and data developed in response to the challenges of MPCA registration. The workshop proceedings report describes: Current methods used in MPCA registration. Current research on single species exposures of non-target organisms. - Progress in conducting successful exposures of non-target organisms in complex laboratory systems such as microcosms. Progress in ecological effects testing. Research needs on how microorganisms interact with the components of specific ecosystems. Research needs in MPCA registration. Progress in human health effects testing. Research needs in health effects testing. ------- SECTION 2 OFFICE OF PESTICIDES PROGRAMS PRESENTATIONS ENVIRONMENTAL FATE AND EFFECTS DIVISION William Schneider The Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) establishes EPA's authority over the distribution and use of pesticide products. It is the role of EPA's Office of Pesticides Programs (OPP) to determine that "the pesticide, when used in accordance with widespread and commonly recognized practice, will not cause (or significantly increase the risk of) unreasonable adverse effects to humans or the environment". Figure 2.1 references several applicable regulations governing pesticide registration. Part 158 of Title 40 of the Code of Federal Regulations (CFR) specifies information that must be submitted to EPA to support pesticide registration. Subdivision M of the Pesticide Testing Guidelines covers biological and biologically-derived pesticides; microbial pest control agents (MPCAs) such as bacteria, algae, fungi and viruses, and biochemical pest control agents (BPCAs) such as pheromones, hormones, natural insect and plant growth regulators and enzymes. The Office of Prevention, Pesticides, and Toxic Substances (OPPT) is divided into the Office of Pesticides Programs and the Office of Toxic Substances (OTS). OPP is organized into several divisions (Figure 2.2). The three divisions are the registration division, environmental fate and effects division, and the health effects division. The registration division serves as an interface between potential registrants and the two aforementioned science branches. The science branches evaluate the risk of the use of pesticides. OPP determines, on a case-by-case basis, whether a pesticide is regulated as a conventional chemical pesticide or as a biological control agent. Criteria on which the decision is based include mode of action, low use volume, specificity to the target species, and natural occurrence. Microbial pest control agents are unlike chemical or biochemical control agents in that microorganisms have the capacity to survive and reproduce once they have been released to the environment, perhaps to exert unwanted effects (infectivity, pathogenicity) on non-target organisms. A tiered testing system is used to minimize uncertainty about the toxicology of these biological pesticides. Tier 1 consists of short-term tests to evaluate toxicity, infectivity, and pathogenicity. If toxicity or infectivity is shown in Tier 1, Tier II tests are designed to further evaluate pathogenicity. Tier III resolves issues of suspected human pathogenicity, and provides tests for particular adverse effects of intracellular parasites of mammalian cells. Ecological effects and environmental expression of biological pest control agents are similarly examined in a tiered testing system. Tier I is comprised of maximum dose single species hazard tests on non-target organisms. Tier II tests examine the potential for exposure of the pesticide ------- to the non-target organisms, if adverse effects are seen in Tier I. If the potential for exposure is determined to be significant, Tier III studies establish dose response effects, or investigate chronic effects. Tier IV tests, under simulated or actual environmental conditions, are designed on a case-by-case basis to evaluate any specific problem that cannot be resolved by lower tiered testing. ------- MICROBIAL PESTICIDE REGULATION The Federal Insecticide, Fungicide, and Rodenticide Act Pesticide: (1) Any substance or mixture of substances intended for preventing, destroying, repelling, or mitigating any pest, and (2) any substance or mixture of substances intended for use as a plant regulator, defoliant, or desiccant. 40 CFR Part 152.20: Federal Register May 4, 1988 All biological control agents are exempt from FIFRA except: (1). Eucaryotic microorganisms including protozoa, algae and fungi; (2). Procaryotic microorganisms, including bacteria; and (3). Viruses f 40 CFR 158.740 - Data Requirements for Registration Subdivision M of the Pesticide Assessment Guidelines October 1982 1989 revision being signed for publication by NTIS 40 CFR Part 172: Environmental Use Permit Regulations Draft proposed amendment to Part 172 available for comment Small Scale Field Test Notification Policy Coordinated Framework for Regulation of Biotechnology, June 26, 1986 Federal Register; FR23302. Figure 2.1 Applicable regulations governing pesticide registration. ------- OFFICE Cf PESTICIDE Regirtraiioi Environmental Fate and Efleet* DrvLuoD EEE CEANCH EfGWE Health Effeeto DEC Figure 2.2 OPP organizational chart. 6 ------- OPP has identified Subdivision M protocol development and evaluation as its primary research need. Some specific problems with the ecological effects testing are: (1) We generally test adult honeybees since larvae are difficult to keep alive yet larvae are the most likely stage to be susceptible to the biological insecticidal toxins. (2) It has been difficult to maintain Daphnia for a sufficient time to evaluate potential pathogenicity. (3) Very few species of beneficial insects are available for testing purposes. (4) Which aquatic species are most at risk and are we adequately protecting them? (5) Are the protocols adequately reflecting a maximum hazard approach? In addition to the actual testing protocol development, there is a need for information that allows us to evaluate the study results. In many cases, we do not have much information on the role of the pesticidal gene products in the environment, even for the most prevalent ones, the Bacillus thuringiensis toxins. Bacillus thuringiensis is ubiquitous in the soil but not at levels that appear to affect insects. Microcosm, mesocosm, and/or field studies could be utilized to study the relationship of Bacillus thuringiensis and/or-its many toxins to the soil and aquatic ecosystems. Are there alternative soil hosts? Do the toxins give Bacillus thuringiensis a competitive advantage over other soil microorganisms? The potential human health effects from pulmonary exposure to microbial pesticides are among our primary concerns, since bacterial, fungal, and viral pesticides are frequently broadcast over large areas by aerial applications. These extensive uses present possibilities for human epidemiology studies and provide a basis for relating experimental test results to actual effects. Health research has centered on the Tier 1 acute pulmonary exposure tests for Subdivision M of the Pesticide Assessment Guidelines. These tests were developed, standardized and evaluated for the assessment of the infectivity primarily of naturally- occurring agents. In these studies, intratracheal and intranasal challenges were demonstrated to be equivalent to, if not better than, aerosol inhalation for the assessment of pulmonary exposure to infectious bacterial and viral agents. In addition, results from EPA sponsored studies have shown that pulmonary exposure of laboratory rodents to Bacillus thuringiensis resulted in adverse toxicological effects at sufficiently high dose levels. The extent of toxicity observed appears related to differences in the Bacillus thuringiensis strain administered. While this is a significant first step in methodology and protocol development for testing guidelines, relevant new technologies and innovative methods must be incorporated to improve testing to increase the breadth of coverage of these tests for a wider range of agents that will be utilized in the future. This can be attained by obtaining, through research, a better understanding of the basis of these empirical tests. Areas of additional inquiry should address the microbial agent/cellular interactions, factors influencing the reisolation and identification of the microbial pesticides, the utility of these tests for repeated pulmonary exposures, the equivalence of intratracheal and intranasal challenge results with aerosol inhalation results when toxicity instead of infectivity is the endpoint. New approaches and appropriate tests must be developed for addressing problems such as assessment of the potential for transfer ------- of deleterious genes to mammalian tissues and cells by the innovative and complex genetically altered agents expected to be developed in the future. Microbial agents of greater anticipated use should be identified and studied to better understand any adverse cellular responses or effects that might be produced in pulmonary tests. In particular, Bacillus thuringiensis, baculoviruses, and Beauvaria bassiana have high pulmonary exposure potential. Additionally, as successful agents are utilized more heavily and exposure becomes more frequent, components of microbial pesticides of minor relevance to the human health effects must be reassessed. Table 2.1 displays registered microbial pesticides. Two notable aspects of this table are the diversity of the microbial pesticide products and the increase in registered products after 1988. Advancements in biotechnology are contributing to the development of pesticide products. Agriculturally important crops can now be genetically transformed (for example, Bt gene incorporation) and increasing numbers of natural and man-made pesticidal substances are being isolated (Table 2.2). An area expected to become markedly active is transgenic plant pesticides. These are pesticides produced by genetically altered plants. Transgenic plants create several specific risk issues: the fate and effects of the pesticidal product made by the plant, and the fate and effects of the plant's genetic insert. OPP must be concerned with environmental fate issues such as persistence in soil, mobility, degradation, and ecological effects issues such as toxicity to non-target and endangered species, adaptation and resistance and the potential for adverse human health effects. Fate and effects issues addressing the plant's genetic insert are less direct but equally important; these issues are dispersal of seeds, vegetative reproduction, and pollen transfer. To prepare to effectively regulate transgenic plant pesticides, information must be gathered in these issue areas. 8 ------- Microorganism Year Reqistered Pest Controlled Bacteria Bacillus popilliae + 1948 B.. lentimorbus B.. thurinqiensis "Berliner" 1961 Aqrobacterium radiobacter 1979 B.. thurinaiensis israeliensis 1981 B.. thurinqiensis aizawai 1981 Pseudomonas fluorescens 1988 B.. thurinqiensis San Diego 1988 B.. thurinqiensis tenebrionis 1988 B.. thurinqiensis EG2348 1989 B.. thurinqiensis EG2371 1989 JL thurinqiensis EG2424 1990 Japanese Beetle larvae Moth larvae Ai tumefaciens (crown gall disease) Mosquito larvae Wax Moth larvae Pythium. Rhizoctonia coleopterans coleopterans gypsy moth lepidopterans lepidopterans/coleopterans Viruses Heliothis Nuclear Poly- hedrosis Virus (NPV) Tussock Moth NPV Gypsy Moth NPV Pine Sawfly NPV 1975 Cotton Bollworm, Budworm 1976 Douglas Fir Tussock Moth larvae 1978 Gypsy Moth larvae 1983 Pine Sawfly larvae Fungi Phytophthora palmivora 1981 Colletotrichum gloeosporioides 1982 Trichoderroa harzianum ATCC20476 1989 + T.polysporum ATCC20475 Gliocladium virens GI21 1990 Trichoderma harzianuro KRIAG2 1990 Protozoa Nosema locustae 1980 Citrus Strangler Vine Northern Joint Vetch wood rot Pythium. Rhizoctonia Pvthium Grasshoppers Table 2.1 EPA list of registered microbial pesticides. ------- INSERTED GENE BACILLUS THURINGIENSIS TOXIN PLANT TOMATO POTATO COTTON TOBACCO WALNUT TREES VIRAL COAT PROTEIN GENES CANTALOUPE SQUASH ALFALFA TOBACCO TOMATO CUCUMBER POTATO CfflTINASE GENE TOBACCO DISEASE RESISTANCE RESPONSE GENES FROM PEA POTATO ANTI-BACTERIAL GENE FROM SILK MOTH POTATO CYTOKININ BIOSYNTHESIS GENE TOMATO GALACTURONASE ANTISENSE GENE TOMATO Table 2.2 Examples of pesticidal transgenic plants. 10 ------- SECTION 3 OFFICE OF RESEARCH AND DEVELOPMENT PRESENTATIONS OFFICE OF ENVIRONMENTAL PROCESSES AND EFFECTS DULUTH ENVIRONMENTAL RESEARCH LABORATORY Richard L. Anderson I. BACKGROUND The aquatic research program at Duluth Environmental Research Laboratory is providing protocols and scientific data to support Program office needs in MPCA registration. The Duluth research program is based on the premise that results from laboratory tests of increasing ecological complexity will increase confidence in extrapolation of the data to natural systems. Our research has been separated into two areas. The first develops single species test methods to measure direct effects. The second develops methods that assess the impact of direct effects on ecosystems, including assessing the fate and distribution of MPCAs in natural systems. ON-GOING PROJECTS: SINGLE SPECIES TESTING The goal in single species test protocols is to optimize the exposure conditions during a test. For MPCAs, the goal is complicated by the necessity of optimizing conditions for the host and the MPCA. Techniques for exposing aquatic animals to chemicals are advanced when compared to MPCA exposure techniques, so a dual strategy was developed. The first strategy is to have specialists in laboratory testing of chemicals adapt their methods to MPCA testing. The second strategy is to conduct exploratory laboratory research for species that are not usually tested and to begin evaluation of new test endpoints. Both approaches have resulted in test procedure information for many aquatic species that have never been exposed to MPCAs. The exploratory research also describes how the target and non-target invertebrates may affect expression and distribution of an MPCA. Two sets of test guidelines resulted from our first strategy. These methods were for two species of entomopathogenic bacteria and an entomopathogenic fungus. 11 ------- BACTERIA SPORES (Bti} Single species tests with the spore form of the registered mosquito larvicide Bacillus thuringiensis subsp. israelensis (Bti) showed that the physical handling and rearing conditions sections of chemical-based protocols can be used with spore-based MPCAs. However, the biological portions required modification. Suggested modifications include: A post-exposure observation period to allow pathogen expression, Consideration of the formulation of the agent, Careful monitoring of the water quality conditions during the test and Verification of exposure through uptake studies. The post-exposure time cannot be definitively stated because of different life-cycles for the test species. No non-target effects were observed, so assessing the sensitivity of the procedures for infective/pathogenic endpoints was difficult. The tested product's form and formulation are important because the active agent may settle from the water and not be available to the water column animals. Alternatively, the formulation could be attractive so the agent could be selectively ingested and those species may receive a high exposure. Formulated mixtures at high concentrations may affect water quality. For example, at >106 CFU/mf, a formulated product reduced the oxygen content, killing the test animals. FUNGUS Test guidelines were developed for exposing and evaluating effects of a fungus (Lagenidium) on freshwater invertebrates and fish exposed in single species systems. Twelve protocols that describe methods to expose three Cladocera species, two insect species, a copepod, ostracod, oligochaete, snail and a fish, Pimephales promelas are available. The exposure system sections of the chemical-based protocols could be, in general, directly applied for use with this type of MPCA. However, performing acceptable Lagenidium exposures was more difficult than performing those with the spore/toxin complex of Bti. Lagenidium zoospores are sensitive and changes in their environment affect their ability to attach and infect a host. 12 ------- Lagenidium zoospores must be alive and infective, so conducting a successful test requires special attention to the zoospores fitness. We recommend a static, daily renewal exposure method and the addition of a "positive" control (i.e. a control with the agent and the natural host animal) for all fungus tests. Other conclusions include: Maintaining viable infective stages in the test system is difficult. Zoospores may germinate and lose their invasive capability at any time. Avoid rapid transfers from one container to another or swirling of the test water. Zoospores are sensitive to water quality changes and physical disturbance. Monitor zoospore viability during the test or use frequent additions of viable agent to the system. Include a post-exposure observation period in all tests to allow fungi to grow in the animals. The length of the post-exposure time cannot be definitively stated because the fungus may grow at different rates in different animals. We arbitrarily selected 3 days. Conduct monitoring of the water quality conditions frequently during the test. We found that zoospore concentrations > 50,000/mC may reduce the dissolved oxygen concentrations in the test chambers. Laboratory cultures of Lagenidium are sensitive to temperatures below 20ฐC and above 28ฐC. Zoospore viability is also linked to water quality. In our tests, Lagenidium did not survive well in total water hardness > 100 mg/C CaC03. Because of batch dependent variability in the viability of the zoospores, tests should be repeated. Expose bottom dwelling animals in shallow containers because Lagenidium zoospores accumulate near the water surface. Documented and verified measurement procedures for viable zoospores should be established before a test is started. EXPLORATION TESTS: INVERTEBRATES In our exploratory research we exposed a range of invertebrate species. In total, 9 species, including snails, oligochaetes, amphipods, stoneflies, caddisflies, dipterans and cladocerans were exposed to Bii. Our tests have included exposing, determining the extent of uptake and retention by the animal, verification of passage through the animal and effects due to the exposure. The ingestion experiments show that bacterial spores are ingested in laboratory exposures by either filtering the microorganism from water, ingestion of settled particles or through predator-prey activities. 13 ------- FISH Many studies have been conducted to determine the direct effects of MPCAs, and in particular Bti, on fish. Most studies consist of exposure of the fish to an MPCA in the laboratory or, less commonly, in the field, followed by measurements of the fishes' responses: i.e., their survival, growth, and incidence of infection. Much less has been investigated and is understood about the extent and significance of other, less apparent, interactions between MPCAs and fish. However, a basic understanding of these interactions could enhance our ability to predict both the direct effects and the ecological fate of an environmental application of an agent. Basic questions that need to be addressed include: How much contact will occur between the MPCA and fish? How important are different routes of exposure? What defense mechanisms are responsible for protecting a fish (or any nontarget species) against a particular MPCA? Will stressors of environmental or physiological origin adversely affect these defenses (that is, could opportunistic infections with the MPCA result)? What influence does the fish have on the survival, multiplication, and distribution of the agent in the environment? To begin addressing these questions, we have used single species laboratory exposures to several commercial formulations of the bacterial spore-crystal complex, Bti. Experiments conducted thus far have focused on measuring the uptake and retention of an MPCA in three freshwater fishes - fathead minnows, bluegill sunfish, and brook trout. These species were selected because of their differing habitat preferences and feeding habits. These studies show that fish rapidly accumulate bacterial spores which have been added to the water. Differences in uptake between species were related to the feeding habits of the fish. Omnivorous fathead minnows actively ingested the particles. Brook trout, which are more predaceous and prefer much larger food, displayed a much lower spore intake. Clearance of the spores was fairly rapid through fecal elimination, with gut clearance times of 4 to 7 days. Spore germination in the gut was observed; however, no evidence of vegetative cell multiplication or colonization of the gut was found. These findings suggest that fish could contribute to dissemination of a spore-based agent in the environment. The rapid clearance and lack of gut colonization supports the 14 ------- observed lack of direct effects. MULTISPECIES TESTS Single species laboratory tests cannot provide all the information needed to assess the risk of a MPCA. Laboratory microcosms offer a multispecies exposure system that includes ecologically relevant conditions. Many types of laboratory microcosms have been used for chemical effects evaluations but their suitability for both effects and fate research on microorganisms was unknown. To begin our test evaluation, we developed a list of "freshwater microcosm characteristics" to describe a system for testing effects, distribution and fate of MPCAs. Our major selection criterion was that a suitable microcosm should contain a community capable of performing trophic interactions seen in natural systems. It should be ecologically complex, allow for decomposition, nutrient cycling, primary production, and contain at least one level of consumers. We have experimented with several aquatic microcosm systems that possess these characteristics. Our goal is to establish protocols for predicting direct effects, survival and ecological effects of a variety of microorganisms. After tests with Bti showed where changes were needed in systems, we began to modify our old protocols and to develop new ones. We have settled on two systems which rely on either generic or site specific models of small ponds. The first of these is the Mixed Flask Culture (MFC) protocol. This method has been thoroughly evaluated and standardized for chemical testing. A core-based microcosm containing a complex sediment which supports a diverse microbial community and a complex aquatic community is under development. We have completed preliminary field tests and are now calibrating both microcosm systems to the field (i.e. relating structure and function in microcosms with pond sites). The initial results are encouraging. While physical conditions, (e.g. pH, dissolved oxygen, temperature) vary considerably between lab and field, we have found that the same processes occurr and similar populations of decomposers, protozoans and zooplankton develop in both lab and field. At this stage, the core microcosms show a higher correlation with the field than the MFC system. PUBLICATIONS Anderson, R.L., and E. Mead. 1990. pp 186-188. Test protocols to determine the toxicity of microbes to aquatic invertebrates. In Review of Progress in the Biotechnoloqy-Microbial Pest Control Agent Risk Assessment Program. EPA/600/9-90/029 Brazner, J. C. and R. L Anderson. 1986. Ingestion and adsorption of Bacillus thuringiensis subsp. israelensis by Gammarus lacustrus in the laboratory. Appl. Environ. Microbiol. 52 (6): 13386-1390. 15 ------- Flum, T. E. 1988. Ingestion of the bacterial mosquito and blackfly larvicide Bacillus thuringiensis var. israelensis, by nymphs of the stonefly Pteronarcys dorsata. Master of Science, University of Minnesota-Duluth. Nestrud, L.B., and R.L. Anderson. 1990. Protocols for exposing freshwater fish and invertebrates to fungi used as insect control agents. Report, Environmental Research Laboratory-Duluth (report contains 12 separate protocols for exposing common and uncommonly tested invertebrates and fish). Shannon, L.J, I.E. Flum, R.E. Anderson, and J.D. Yount. 1989. Adaptation of the mixed flask culture microcosm for testing the survival and effects of introduced microorganisms. pp. 224-242. In. U.M. Cowgill and LR. Williams (eds.), Aquatic Toxicology and Hazard Assessment: 12th Volume, ASTM STP 1027, Amer. Soc. for Testing and Materials, Philadelphia, PA. Shannon, L. J. and R. L Anderson. 1989. Use of the mixed flask culture (MFC) microcosm protocol to estimate the survival and effects of microorganisms added to freshwater ecosystems. American Society of Microbiologists. NTIS EPA-600/D-89/058. Snarski, V. M. 1990. Interactions between Bacillus thuringiensis subsp. israelensis and fathead minnows, Pimephales promelas Rafinesque, under laboratory conditions. Appl. Environ. Microbiol. 56:2622. Stay, F.S., I.E. Flum, L.J. Shannon, and J.D. Yount. 1989. An assessment of the precision and accuracy of SAM and MFC microcosms exposed to toxicants, pp. 189-203. hi U.M. Cowgill and LR. Williams (eds.). Aquatic Toxicology and Hazard Assessment: 12th Volume, ASTM STP 1027, Am. Soc. for Testing and Materials, Philadelphia, PA. Vaishnav, D. D., L T. Brooke, S. H. Poirier, and E. T. Korthals. 1988. Protocol for an acute toxicity test with the cladoceran Daphnia dubia exposed to bacteria used as pest control agents. EPA- 600/X-88-370. U.S. Environmental Protection Agency, Duluth, MN. Vaishnav, D. D., L. T. Brooke, S. H. Poirier, and E. T. Korthals. 1988. Protocol for a 7- day larval growth and survival test with the fathead minnow exposed to bacteria used as pest control agents. EPA-600/X-88-369. U.S. Environmental Protection Agency, Duluth, MN. Vaishnav, D. D., L T. Brooke, S. H. Poirier, D. J. McCauley, and E. T. Korthals. 1988. Protocol for an acute toxicity test with cyclopoid copepods exposed to bacteria used as pest control agents. EPA-600/X-88-369. U.S. Environmental Protection Agency, Duluth, MN. Vaishnav, D. D., L. T. Brooke, S. H. Poirier, D. J. McCauley, and E. T. Korthals. 1988. Protocol for an acute toxicity test with the cladocerans Daphnia magna and Daphnia pulex 16 ------- exposed to bacteria used as pest control agents. EPA/600/X-88/370. U.S. Environmental Protection Agency, Duluth, MN. Vaishnav, D. D., L T. Brooke, S. H. Poirier, D. J. McCauley, and E. T. Korthals. 1988. Protocol for an acute toxicity test with the fathead minnow exposed to bacteria used as pest control agents. EPA-600/X-88-369. U.S. Environmental Protection Agency, Duluth, MN. 17 ------- OFFICE OF ENVIRONMENTAL PROCESSES AND EFFECTS CORVALLIS ENVIRONMENTAL RESEARCH LABORATORY Bruce Lighthart I. BACKGROUND The MPCA program at the Corvallis Environmental Research Laboratory has developed in two areas: the effects of MPCAs on terrestrial avian species and on beneficial terrestrial arthropods. It is anticipated that a third area concerning the effects of MPCAs on non-target plants will soon be active. The objectives of the first two areas are to prepare test protocols to evaluate effects of MPCAs, including GEMs, on non- target, beneficial insects and birds, and to develop MPCA field monitoring and mitigation procedures. The approach used to evaluate the effects of MPCAs on birds has been to prepare protocols describing pathogenicity and LD50 tests using oral, intravenous, and respiratory exposure of bobwhite quail and mallard ducks. Tests have been validated for exposure by oral and intravenous routes to viral, bacterial, and fungal MPCAs. An interim protocol for respiratory exposure has been written. An ELISA test has been developed to assess exposure of birds to Bacillus thuringiensis through detection of antibody production. Future avian research directions are to: Validate protocol of respiratory exposure of birds to MPCAs, Continue ELISA assay development and application protocol, Determine effects of MPCAs on avian reproduction; a new assay endpoint, Determine methods of testing chronic exposure of birds to MPCAs on natural food, and Determine effects of MPCA use on bird communities due to food web depletion. The approach used to evaluate the effects of MPCAs on arthropods is similar to those used for birds except that ELISA-type tests have not been developed, and the environmental testing conditions were varied to optimize susceptibility of the arthropods to the infectious agent (i.e., MPCAs). Accomplishments to date include: Review of MPCA effects on non-target insects. Detection of large-scale loss of non-target insects due to MPCA application. 18 ------- The observed loss of biocontrol insects by MPCA infection. Detection of dramatic effects of numerous modulators to effect the susceptibility of insects to MPCAs. Development of artificial intelligence software to evaluate MPCA risk. Preparation of ten (10) interim protocols for MPCA effects on representative beneficial arthropods. Prepararation of five (5) final protocols for MPCA effects on representative beneficial arthropods. Our present research includes: Preparation of two (2) final protocols for MPCA effects on representatives of certain insect orders. Development of a field monitoring protocol for MPCA effects on non-target insects. It is anticipated that future areas of research will include: Evaluation of airborne survival of MPCAs. Preparation of a technological forecast for future MPCA problem areas. Preparation of protocols to monitor, model, and mitigate MPCAs. Comparison of laboratory and field MPCA effects tests for similarity. Compilation of protocols for effects of MPCAs on representative beneficial arthropods. Preparation of five (5) final protocols for MPCA effects on certain insect orders. Preparation of multi-insect species/MPCA exposure protocol if practical. Development of a model to predict insect susceptibility to MPCA under dynamic modulators (e.g., stress) over time. The protocols to monitor, measure, and mitigate MPCAs effects on non-target arthropods will be developed to support the detection and correction scheme for unwanted MPCA effects in application areas (Figure 3.1). Honey bees, spiders, ants, and termites are the focus for MPCA-arthropod effects protocol development. 19 ------- Finally, with the advent of pest plant (weed) MPCAs, a program to prepare protocols to evaluate MPCA effects on non-target plant species will be studied and, if possible, initiated. PUBLICATIONS Croft, B.A., ed. 1989. Arthropod Biological Control Agents and Pesticides. Wiley Intersci. J. Wiley and Sons, New York, N.Y. 736 pp. Croft, B.A. 1989. Standardized assessment methods. Chap. 5. p. 101-126. ]n B.A. Croft, (ed.), Arthropod Biological Control Agents and Pesticides. J. Wiley and Sons, New York, N.Y. 736 pp. Croft, B.A., and J.L. Flexner. 1989. Microbial pesticides. Chap. 11. p. 269-303. ]n B.A. Croft, (ed.), Arthropod Biological Control Agents and Pesticides. J. Wiley and Sons, New York, N.Y. 736 pp. Croft, B.A., R.H. Messing, and J. Hendricks. 1989. Data integration for risk assessment: A case study of microbial pathogen impact on terrestrial arthropods. In The Integration of Research and Predictive Model Development in Biotechnology Risk Assessment. EPA 600/X-89/366, Washington, D.C. 152 pp. Croft, B.A., and Karen Theiling. 1989. Pesticide effects on arthropod natural enemies: a database summary. Chap. 2. p. 17-46. ]ri B.A. Croft (ed.), Arthropod Biological Control Agents and Pesticides. J. Wiley and Sons, New York, N.Y. 736 pp. Donegan, K., and B. Lighthart. 1989. Effect of several stress factors on the susceptibility of the predatory insect, Chrysoperla carnea (Neuroptera: Chrysopidae), to the fungal pathogen Beauveria bassiana. Journal of Invertebrate Pathology 53:79-84. 20 ------- START mitigation procedure MONITOR non-target populations in field ecosystem Changes in non-target populations survival time >yes-W STOP DIRECT bioassay on non-target insects RESIDUAL bioassay of environment DIRECT < RESIDUAL Positive DIRECT assay MITIGATE MPCA INDIRECT bioassay of Target/MPC A/non-target dynamics using microcosm, and simulation . Figure 3.1 MPCA mitigation procedure flow diagram: Detection and correction. 21 ------- Fairbrother, A., M. Craig, K. Walker, and D. O'Loughlin. 1989. Effects of age, sex, and reproductive status on normal serum chemistry of mallards (Anas platyrhynchos). J. Wildl. Dis. 26(1) In Press. Flexner, J.L., B.A. Croft, and B. Lighthart. 1986. The effects of microbial pesticides on non-target, beneficial arthropods. Agric., Ecosyst. and Environ. 16:203-254. Lighthart, B., D. Sewall, and D.R. Thomas. 1988. Effect of several stress factors on the susceptibility of the predatory mite, Metaseiulus occidentalis (Acari: Phytoseiidae), to the weak bacterial pathogen Serratia marcescens. Journal of Invertebrate Pathology. 52:33- 42. Messing, R.H., and B.A. Croft. 1990a. NERISK: An expert system for predicting the effects of pesticides on arthropod natural enemies. Acta Hort. In Press. Messing, R.H., and B.A. Croft. 1990b. NERISK users manual. Public. Oregon State University, Dept of Entomology. 40 pp. (Contact B. Croft for copies) Messing, R.H., B.A. Croft, and K. Currans. 1989. Assessing pesticide risk to arthropod natural enemies using expert system technology. A.I. Applic. Nat. Res. Mngmt. 3:1-12. Theiling, K. 1987. The SELCTV Database: The Susceptibility of Arthropod Natural Enemies of Agricultural Pests to Pesticides. M.S. Thesis. Oregon State University, Corvallis, OR. 170 pp. Theiling, K., and B.A. Croft. 1987. The SELCTV Database. Version 1.1. Copyright O.S.U., Software Product., Dept. Entomol., O.S.U. Corvallis, OR. Theiling, K., and B.A. Croft. 1988a. Pesticide effects on arthropod natural enemies: A database summary. Agric., Ecosys. and Environ. 21:191-218. Theiling, K., and B.A. Croft. 1988b. Toxicity, selectivity and sublethal effects of pesticides on arthropod natural enemies: A database summary. Abst. Symp. Evaluation of Pesticide Side-Effects on Non-target Invertebrates. Proc. XVIII Intern. Cong. Entomol. Vancouver, B.C. p. 464. Theiling, K.M., and B.A. Croft. 1989. Toxicity, selectivity and sublethal effects of pesticides on arthropod natural enemies: A database summary. Chapter 11. p. 213-232. In P.C. Jepson (ed.), Pesticides and Non-target Invertebrates. Intecept Publ. Dorset, U.K. 240 pp. Theiling, K.M., and B.A. Croft. 1990. Systems manual for SELCTV and REFER databases and SELCTV data system program. Public. O.S.U. Dept. Entomol. 60 pp. 22 ------- OFFICE OF ENVIRONMENTAL PROCESSES AND EFFECTS GULF BREEZE ENVIRONMENTAL RESEARCH LABORATORY John W. Fournie and William S. Fisher NONTARGET TESTING OF MICROBIAL PEST CONTROL AGENTS I. Background Gulf Breeze Laboratory has a long history of research on effects of MPCAs. Early results were the recognition that MPCA test systems must be capable of maintaining nontarget test organisms for at least 28 days, must be inexpensive and simple to construct, and should represent as closely as possible the conditions of a natural system. Additionally, the test systems should be capable of completely containing an MPCA so that there is little risk associated with testing exotic or genetically engineered microorganisms. Single species test systems used at Gulf Breeze quickly evolved into the present totally-enclosed aquarium that incorporates an undergravel filter system using artificial substratum and an external ultraviolet light to decontaminate recirculating water after exposure and depuration studies. Using this system, representatives of all four MPCA groups have been tested, including a baculovirus Autographa califomica, a bacterium Bacillus thuringiensis, a protozoan Nosema cuneatum and a fungus Lagenidium giganteum (Couch et al. 1984; Couch et al. 1985). Positive controls demonstrated characteristic infections and pathogenicity in target species; however, no signs of infection or pathogenicity were found in nontarget species using histology, serology, and electron microscopy. Nontarget test organisms in the early single species tests included grass shrimp and bivalve molluscs. Multiple species test systems were developed to enable concurrent exposure of plants and animals from different phyletic groups and to investigate potential-interactions among the test species and MPCAs (Fournie et al. 1988). Initially, four compatible nontarget organisms (fish, bivalve, crustacean and plant) were selected for both a freshwater and a marine assay system. The same totally-enclosed aquarium system for single species tests was used except that individual containers were provided for grass shrimp (to prevent cannibalism) and for burrowing bivalves (for accessibility). The first multi-species tests were conducted with Colletotrichum gloeosporioides (Collego), a registered fungal herbicide. Both the commercial formulation and the spores were tested and no signs of infectivity or pathogenicity were noted. One innovation employed with Collego was placement of the positive control (northern jointvetch weed) into the same assay system with the nontarget organisms. Not only are the contrasting results (i.e. target vs. nontarget) easily visible with this technique, but deactivation of the MPCA by nontarget organisms could also be detected. 23 ------- Preliminary fate and survival studies were conducted in the single species test system using spores of the bacterium Bacillus sphaericus. Results indicated that the spores could persist in the underlying artificial substratum and in test oysters for several weeks. Depuration of spores by oysters was examined by exposing the oysters, removing them from the system and intermittently examining them for spore content using standard microbiological techniques. II. Current Research Research at Gulf Breeze is continuing to concentrate on effects and fate of MPCAs on nontarget aquatic organisms. Current efforts on MPCA effects have focused on microsporidians (Nosema algerae, Edhazardia aedis) and a fungus (Beauveria bassiana). Studies on fate and survival of MPCAs have related the depuration rates of oysters held in the totally-enclosed system with rates of oysters in nature. Nontarqet testing of insect microsporidians There is one registered microsporidian, Nosema locustae, used for control of rangeland grasshoppers and other requests for registration have been received by the Office of Pesticides Programs. There is reason to believe that registration requests for microsporidians will increase. In particular, an abundance of new information on the biology of microsporidians has recently become available; many are now known to have indirect life cycles, multiple sporulation sequences, vertical as well as horizontal transmission and a broader host specificity than previously believed. The first microsporidian tested at Gulf Breeze was the mosquito pathogen Nosema algerae, which has a relatively simple direct life cycle. Development of this MPCA was originated in the early and mid-1970's for use as a biological control agent and limited field tests were performed. In current research using the laboratory test systems described above, a series of single species tests were conducted on freshwater and estuarine grass shrimp, marine rotifers, and a fish, the inland silverside. The nontarget organisms were exposed by intrahemocoelic injection, gavage, or ingestion. Infections did not develop in either of the gavaged grass shrimp, but did develop in those subjects that received injections. Infected tissues included the gills, antennal gland, eyes, skeletal muscle, heart, and gonads. Proof of infection was demonstrated ultrastructurally by the presence of mature spores and developmental stages in infected tissues. Infections did not occur in the marine rotifer after ingestion of spores or in inland silversides fed marine rotifers containing ingested spores (Fournie et al. 1990). The microsporidian Edhazardia aedis is now being investigated by the Gulf Breeze ERL in collaboration with the USDA's Insects Affecting Man and Animals Research Laboratory for possible use in the control of container-breeding mosquitos. This microsporidian is an interesting model because it has a more complex life cycle, exhibiting 24 ------- multiple sporulation sequences and the capability of both vertical and horizontal transmission. Initial research with E. aedis has included single species testing of grass shrimp with gavage, intrahemocoelic injection and water-borne exposures; single species testing of mysid shrimp with water-borne exposure under optimal and stressful laboratory conditions; single species testing of inland silverside embryos and larvae (an innovative adaptation of toxicity assays) via water-borne exposures; and preliminary research with feeding of spore-containing rotifers to adult inland silversides. Studies were also recently initiated to investigate ingestion of E. aetf/s-infected mosquito larvae by mosquito predator fish, Gambusia affinis. At present, there is preliminary indication that E. aedis does not disrupt fish larval development or cause any infection in Gambusia. Nontarqet testing of Beauveria bassiana The mycopesticide B. bassiana is being developed for potential use against fire ants. In preliminary tests, a virulent strain of the fungus was exposed to mysid shrimp in fingerbowls and caused significant mortalities. Upon further investigation, mortalities were believed to be mechanical, i.e. some physical effect of fungal spores on mysid respiration or other physiological process. Water-borne exposures to inland silverside in fish embryo/larval assays have clearly demonstrated embryonic disorders. A bleb, or bubble, of tissue forms under the chorion of the fish embryo and causes mortality prior to or soon after hatching. The fungus can be re-isolated from affected embryos, but it is not yet clear whether fungal spores have become vegetative and are invasive to embryonic tissue. Fate and survival of MPCAs in enclosed test systems It is relevant to MPCA research to know that depuration rates in enclosed test systems reflect depuration in natural conditions. If so, then exotic MPCAs and GEMs can be tested reliably prior to release in field trials. For this comparison, indigenous microorganisms are being used as models to compare oyster depuration rates in the laboratory system and in the natural waters of the Santa Rosa Sound near Gulf Breeze. To date, oysters have been exposed for two weeks to endospores of Bacillus sphaericus, vegetative stages of Pseudomonas fluorescens and spores of the fungus Colletotrichum gloeosporiodes, removed from the exposure system and divided between the totally- enclosed laboratory system and natural waters. In all three cases, the laboratory test system has demonstrated depuration rates nearly identical to those in nature. III. Future Directions Research at Gulf Breeze ERL will continue to develop test systems, expand the number of MPCAs tested and determine toxicity, infectivity and pathogenicity of MPCAs on nontarget aquatic organisms. Emphasis will be placed on evaluation of a broader spectrum of MPCA microorganisms with different and more complex life cycles. Additional research on microsporidians, for example, will include species of Amblyospora 25 ------- and Parathelohania, which have intermediate hosts in their life cycles. Research on MPCA effects to nontarget organisms will begin to explore immunological, biochemical and physiological endpoints in addition to those already measured. Continued research on the effects of E. aedis and B. bassiana on fish embryos and mysids will emphasize mechanisms of action. Exposure conditions for MPCA test systems will be altered to determine infectivity and pathogenicity under stressful environments for the nontarget species. Stressors will include both natural environmental factors (salinity and temperature) and chemical pollutants. It is expected that these types of stress are prevalent in nature and more accurately reflect conditions of susceptibility. IV. PUBLICATIONS Couch, J. A., T. W. Duke, S. S. Foss, and K. T. Perez. 1986. Enclosed systems for testing microbial pest control agents. Proc. workshop at ERL/Gulf Breeze, sponsored by U. S. EPA, Office of Pesticides Programs, Washington, D. C. Couch, J. A., S. S. Foss, and L A. Courtney. 1985. Evaluation for risks of an insect virus, bacterium, and protozoan to a nontarget, estuarine crustacean. Report EPA 600/X- 85/290, U. S. EPA, Gulf Breeze FL. Couch, J. A., S. M. Martin, G. Tompkins, and J. Kinney. 1984. A simple system for the preliminary evaluation of infectivity and pathogenesis of insect virus in a nontarget estuarine shrimp. J. Invertebr. Pathol. 43:351-357. Couch, J. A. and K. Ranga Rao. 1983. Biorational Workshop. Report EPA-600/X-83-054, Gulf Breeze ERL, Gulf Breeze FL Gripe, G.M., and F.J. Genthner. Application of standard toxicity test methods for exposure of Mvsidopsis bahia to the insect pathogen Beauveria bassiana. In Preparation. Foss, S.S., LA. Courtney, J.W. Fournie, and D.V. Lightner. 1989. Nontarget testing of microbial pest control agents in aquatic systems. EPA/600/X-89/387, U.S. Environmental Protection Agency, Environmental Research Laboratory, Gulf Breeze, FL. 21 pp. Fournie, J.W., S.S. Foss, and J.A. Couch. 1987. Effects of a fungal mycoherbicide in enclosed multi-species freshwater and estuarine systems. EPA/600/X-87/324, U.S. Environmental Protection Agency, Environmental Research Laboratory, Gulf Breeze, FL. 15pp. 26 ------- Fournie, J.W., S.S. Foss, and J.A. Couch. 1988. A multispecies system for evaluation of infectivity and pathogenicity of microbial pest control agents in nontarget aquatic species. Diseases of Aquatic Organisms 5:63-70. Fournie, J.W., S.S. Foss, and LA. Courtney. 1988. Single -species tests of the mosquito microsporidian Nosema alqerae in nontarget aquatic species. EPA/600/X-88/415, U.S. Environmental Protection Agency, Environmental Research Laboratory, Gulf Breeze, FL 15p. Fournie, J.W., S.S. Foss, L.A. Courtney, and A.M. Undeen. 1990. Testing of insect microsporidians (Microspora: Nosematidae) in nontarget aquatic species. Diseases of Aquatic Organisms. 8:137-144. Genthner, F.J., S.S. Foss, and P.P. Campbell. An enclosed system for testing effects of microbial pest control agents on nontarget aquatic invertebrates. In Preparation. Genthner, F.J., and D.P. Middaugh. Effects of Beauveria bassiana on embryos of inland silverside fish Menidia beryllina. Submitted for Publication. McKenney, C.L, Jr.. 1986. Methods for determining the influence of biochemical biological control agents on metamorphosis of marine Crustacea. EPA.600/X-86/234, U.S. Environmental Protection Agency, Environmental Research Laboratory, Gulf Breeze, FL 18pp. McKenney, C.L., Jr., and E. Matthews. 1988. Influence of an insect growth regulator on larval development of a marine crustacean. EPA Environmental Research Brief. EPA/600/M-88/003, U.S. Environmental Protection Agency, Environmental Research Laboratory, Gulf Breeze, FL. 6 pp. McKenney, C.L, Jr., and E. Matthews. 1990. Influence of an insect growth regulator on the larval development of an estuarine shrimp. Environmental Pollution 64:169-178. Yousten, A.A., E.F. Benfield, R.P. Campbell, S.S. Foss, and F.J. Genthner. Fate of Bacillus sphaericus 2362 spores following ingestion by nontarget invertebrates. J. Invert. Pathol. 58:427-435. Yousten, A.A., E.F. Benfield, and F.J. Genthner. Fate of Bacillus sphaericus 2362 spores in nontarget invertebrates. Submitted for Publication. 27 ------- OFFICE OF HEALTH RESEARCH HEALTH EFFECTS RESEARCH LABORATORY Clinton Y. Kawanishi I. BACKGROUND Microbial pest control agents (MPCAs) research was initiated at the Health Effects Research Laboratory, Research Triangle Park, North Carolina in 1975. The goal of the project was to assess the potential of MPCAs to cause detrimental health effects in humans. While the goal was straightforward, deficiencies in ancillary methodology for reliable identification, classification and detection of certain novel classes of MPCAs precluded definitive hazard evaluation. The program therefore, focused on: 1) providing a database on physical and biochemical properties of model agents that were representative of specific classes of MPCAs, and 2) developing methods for their identification, classification and detection. These studies showed that technologies associated with molecular biology such as restriction endonuclease analysis, monoclonal antibodies and immunoblotting provided information on the taxonomic relatedness of MPCAs to known human pathogens, identified with some certainty the agent that was registered, and permitted more definitive testing of their potential health effects. The early program provided a foundation for our present research which focuses strongly on the health concerns about genetically altered MPCAs and on-the development of appropriate test methods based on our basic knowledge of MPCA properties. Potential genetic effects such as gene transfer and perturbation of normal gene regulation in human cells by MPCAs are being investigated. Projects to examine baculovirus expression vector transactivation of nontarget genes and the fate of foreign genes carried into human cells utilize a well established baculovirus model initially characterized at the molecular level earlier in our program. Toxicology studies on a bacterial MPCA (Bti) mammalian toxic protein (28 kDa) utilizes a monoclonal antibody and methods developed previously in our program. Research to characterize Bti and studies of plasmid curing led to the discovery of a Bti mutant currently utilized to investigate the mechanism of plasmid movement between genetic components of the MPCA. Test methods for detecting health effects (i.e. Acute Pulmonary Toxicity/ Pathogenicity Tests) were developed and standardized with known viral and bacterial 28 ------- pathogens of laboratory animals, and evaluated with registered MPCAs. Standards to aid in interpretation of results and internal controls for performance assessment were provided with test protocols to enhance practical utility. These tests are being refined for use in determining the mode of action of the test substance, for assessing other endpoints, and for utilization with other types of MPCAs. Figure 3.2 shows the conceptual model on which MPCA health research is organized. MPCAs can cause human health effects through a toxic or infectious mode or through genetic perturbations such as mutations (structural) or disruption of normal gene regulation (functional). The hazard identification information then serves as the basis for test method development. The research is basically organized according to the mode of action of MPCAs. Our research addresses the following questions: What diseases or detrimental health effects can the MPCA cause? Can MPCAs produce detrimental genetic effects? What is the molecular mechanism by which the MPCA causes the effect? How can we use this information to test MPCAs for their potential health effects? II. ON-GOING PROJECTS Current projects are grouped under broad research areas of toxicity, genetic effects, functional perturbations, infectivity, and test method development. Descriptions of each project are provided under the appropriate research area in the following paragraphs. TOXICITY 1. Microbial Pesticide Interaction with Mammals. This project provides information on the identification of potential hazard related to the use of microbial pesticides. Specifically, we are characterizing the effects of a protein produced by an Agency-registered bacterial pesticide, Bacillus thuringiensis subsp. israelensis (Bti), which we have shown to be toxic to laboratory animals. The intent is to obtain enough information about it to assess its human health potential. A toxic 28 kDa crystal polypeptide has been identified and investigations are determining the mode of action of this protein toxin. Research elucidating the mode of action by determining sites of action, physiological effects and routes by which it is toxic is proceeding. 29 ------- HEALTH EFFECTS RESEARCH WITH MICROBIAL AGENTS TESTS AGENT r^VJ ^ 1 'I 1 xxxx 1 1 1 1 1 HUMAN CELL NUCLEUS GENETIC EFFECTS Figure 3.2 Conceptual model underlying human health effects research.Our progress has been marked by several important accomplishments: 30 ------- The Sf/28 kDa protein causes bradycardia, hypothermia and possibly hypotension in exposed mice and rats. Gross necropsy reveals major lesions in the jejunum. Histopathology indicates effects in the jejunum and liver. GENETIC EFFECTS 1. Mobility of Bacillus thuringiensis subsp. israelensis Chromosomal and Extrachromosomal Genetic Elements Microbial pesticide hazard identification associated with the transfer or movement of genes with detrimental health effects potential is examined. Determination of the occurrence and mechanisms of gene mobility contribute to understanding and aid in health risk assessment. The mechanism of movement of the gene for the Bti28 kDa crystal polypeptide from plasmid to the bacterial chromosome is the primary focus of this project. By utilizing a mutant of Bti\r\ which the 28 kDa gene appears to have moved into the chromosome, we are determining how much of the plasmid has moved into the chromosome and if the markers for the activity of transposon (jumping genes) or other mechanisms of DMA transposition are present. The possible site of chromosome integration of the 28 kDa DMA has been identified and evidence indicates that most of the plasmid is present within the chromosome (Held et al. submitted). 2. Refinement and Standardization of Mammalian Cell Culture Tests for Viral Pesticidal Agents Potential genetic health hazards associated with biotechnology and microbial pesticidal agents are being identified by assessing the fate and impact of genes present in a baculovirus expression vector when it enters human cells. The fate of a marker resistance gene engineered into a baculovirus expression vector is being followed in human and other vertebrate cells in culture. A baculovirus expression vector was generated with a selectable marker with primate regulatory sequences. A test system was developed and standardized, the baculovirus expression vector constructed, cells treated and cell clones expressing the resistant phenotype have been obtained (Hartig et al. 1989, Hartig et al. 1991). 31 ------- FUNCTIONAL PERTURBATIONS 1. Effect of Genetically Engineered Agents on Mammalian Cells Potential genetic health hazards associated with biotechnology and microbial pesticidal agents are being investigated. This project examines if baculoviruses, natural or genetically engineered, can affect gene function in human cells. The information provided from this project examines if detrimental health effects can result from exposure to these agents by perturbing gene regulation in human cells. This project's primary objective is to determine whether immediate-early genes (genes that are active immediately upon entering a cell and activate other viral genes) of baculovirus and human viruses that reside in human cells are capable of transactivating one another. Using cloned immediate early genes of baculovirus, human cytomegalovirus, herpesvirus and AIDS virus to which reporter genes have been genetically engineered, researchers are determining if transactivation can occur in a variety of human, primate and insect cells. The transactivation systems have been standardized in their respective host cells, and have begun in human cells. Competition between progenitor and genetically engineered baculovirus expression vector strains have been completed (Huang et al., submitted). INFECTIVITY 1. Invertebrate Expression Vector Function and Stability in Vertebrate Cells Can baculoviral expression vectors utilized experimentally or commercially tov synthesize large quantities of human proteins that possess potent biological effects have the potential to cause human health effects in exposed personnel? This question underlies this project's objective of determining whether baculovirus expression vectors that synthesize rat preproenkephalin can enter experimentally exposed laboratory animals and affect them. After a baculovirus expression vector bearing the rat preproenkephalin gene is constructed, researchers will determine that it produces the product in its normal expression system (insect cells), determine its capability to enter the cells of dosed mice or rats, and ascertain if there are detectable changes in either the behavior or function of treated individuals. The preproenkephalin gene has been prepared for insertion into the plasmid transfer vector and amplified by the polymerase chain reaction. This constitutes the first phase of the project. 32 ------- TEST METHOD DEVELOPMENT 1. Mechanism of Microbial Pesticide Pulmonary Toxicity/Pathogenicity When Agency-registered bacterial pesticides caused dose-related deaths in mice challenged at high doses by newly developed protocols, questions concerning the limitations of the tests were raised. Thus, research was planned to determine whether the mortality was due to nonspecific factors or specific effects caused by the bacteria. Experiments focus on determining the mechanism of bacterial pesticide pulmonary toxicity/pathogenicity and the components responsible for the effects. Our approach has been to conduct organ clearance studies in animals challenged with bacteria by intranasal or intratracheal instillation for evidence of bacterial growth. Physical blockage of the lungs is also assessed as a possible factor in the observed mortality of the challenged animals. We are attempting to determine the components responsible by challenging animals with different forms of the bacterium, with mutants lacking specific components, and by challenging with closely-related and distantly-related species of bacteria. Earlier studies demonstrated that intranasal and intratracheal instillation procedures can be used in place of aerosol inhalation studies for assessing infectivity after pulmonary exposure challenge with viral and bacterial MPCAs (Sherwood et al., 1988). Using these protocols it was demonstrated that mortality can be caused by high doses of certain bacterial MPCAs. This has been more recently shown to be due to pulmonary toxicity of the bacterial preparation. Toxicity is associated with the vegetative bacilli of the bacterium. Phylogenetically and phenotypically closely-related but not distantly-related species are similarly toxic. Different subspecies of the bacteria differ in the degree of toxicity. III. NOVEL RESEARCH Immense amounts of academic and industrial research effort is being directed to cloning and shuffling different delta endotoxin genes of Bacillus thuringiensis (Bt) between subspecies of this MPCA. Thus, it is anticipated that a large variety of permutations of bacterial subspecies delta endotoxins combinations will be submitted for MPCA registration in the future. Recent studies have shown that different properties of relevance to health effects potential (different toxicity to laboratory animals, different potential fortranslocation to different organs, etc.) are possessed by the different bacterial subspecies irrespective of the type of delta endotoxin it produces. Research will, therefore, be initiated to utilize chromosomal DNA restriction-fragment length polymorphism and bacterial chromosomal maps to identify more precisely the bacterial subspecies. Areas of future research relevant to concerns about baculovirus expression vector health effects are (1) studies on the activation of representative baculovirus immediate- 33 ------- early genes (first genes to be activated in a cell) in a number of human cells, (2) the determination of the efficiency with which these agents enter mammalian epithelial cells, etc. These data will facilitate risk assessment with baculoviral recombinants. IV. PUBLICATIONS Hartig, P.C. 1989. Evaluation and Standardization of Mammalian Cell Culture Test Protocols for Viral Pesticidal Agents Required in Subdivision M of the Pesticide Assessment Guidelines. PRIORITY RESEARCH PRODUCT: Task 82941E104. Hartig, P.C., M.A. Chapman, G.G. Hatch, and C.Y. Kawanishi. 1989. Insect Virus: Assays for Toxic Effects and Transformation Potential in Mammalian Cells. Appl. Environ. Microbiol. 55:1916-1920. Hartig, P.C., M.C. Cardon, and C.Y. Kawanishi. 1991. Generation of recombinant baculovirus via liposome mediated transfection. Biotechniques. 11:310-313. Hartig, P.C., M.C. Cardon, and C.Y. Kawanishi. 1991. Insect Virus: Assays for viral replication and persistence in mammalian cells. J. Virological Methods. 11:335-344. Held, G.A., Y-S. Huang, and C.Y. Kawanishi. 1986. Effect of removal of the cytolytic factor of Bacillus thuringiensis subsp. israelensis on mosquito toxicity. Biochem. Biophys. Res. Commun. 126:961-965. Held, G.A., Y-S Huang, and C.Y. Kawanishi. 1990. Characterization of the parasporal inclusion of Bacillus thuringiensis subsp. kyushuensis. J. Bacteriol. 172:481-483. Kawanishi, C. Y., Y.S. Huang, K.L. Bobseine, and W. Setzer. 1991. Selection kinetics during serial cell culture passage of mixtures of wildtype Autographa califomica nuclear polyhedrosis virus E2 and its recombinant Ac360-(3-gal". J. Gen. Virology. 72:2653-2660. Mayes, M.E., G.A. Held, C. Lau, J.C. Seely, R.M. Roe, W.C. Dauterman, and C.Y. Kawanishi. 1989. Characterization of the mammalian toxicity of the crystal polypeptides of Bacillus thuringiensis subsp. israelensis. Fund. Appl. Toxicology 13:310-322. Sherwood, R.L, Mega, W.M., Kawanishi, C.Y., and Sjoblad, R. 1989. Microbial size and concentration effects on the pulmonary toxicity/pathogenicity test for microbial pesticides. Abstract for the American Society of Microbiology, New Orleans, LA. Sherwood, R.L, Thomas, P.T., Kawanishi, C.Y., and Renters, J.D. 1988. Comparison of Streptococcus zooepidemicus and influenza virus pathogenicity in mice by three pulmonary exposure routes. Appl. and Environ. Microbiol. 54:1744-1751. 34 ------- SECTION 4 RESEARCH NEEDS FOR ECOLOGICAL AND HUMAN HEALTH PROTOCOLS AND METHODS ECOLOGICAL PROTOCOLS AND METHODS NEEDS Panel: F. Genthner, C. Easterly, V. Snarski and R. James Single Species Protocol Research Needs The discussion panel focused attention on research needs for single species, Tier 1 tests because results of those tests often determine the need for advancement to the more complex Tiers 2, 3, and 4. Information gaps in Tier 1 tests may not present an adequate or relevant interpretation on which to recommend more complex tests. Discussion identified both immediate and longer-term research needs, primarily for single species tests. Immediate needs can be divided into two sections; protocol development for species identified by OPP, and protocol development for species not requested by OPP, but recognized as important by ORD researchers. The list of Office of Pesticide Programs immediate protocol needs is provided in Appendix A. The Office of Research and Development Biotechnology/ Biological Control Agent Program Matrix Manager and Team Leaders encourage scientists to address these important needs through in-house, contract and cooperative agreement research. ORD scientists feel protocol development for other species is important because i) of its potential commercial value, ii) relatedness to OPP identified species, iii) importance in ecosystem stability and function, iv) the need to define host range, and v) the potential to eventually become of interest to OPP. The need for new protocols was emphasized by OPP discussions on the difficulty of interpreting test results submitted by prospective registrants. Data generated by prospective registrants are only as good or thorough as the current test procedures. Examples of factors complicating interpretation of test results are: fundamental lack of biological information on the test animals (including test animal rearing, maintenance and health assessment) and undefined operational procedures in the tests (endpoint definition, post-exposure observational period, dosage, formulation and experimental administration procedures). At this time, equivocal results are often presented, making risk assessments based on these data more difficult. It is well known that many factors, such as those previously mentioned, can affect the results of a test but it was agreed that validated test protocols may reduce uncertainty. The process of protocol validation will, by necessity, refine a test procedure and increase its precision and perhaps its accuracy. Some protocols have been tested with a few agents but none have been tested with representatives of the major control agent groups. Two insect orders (Hemiptera and Diptera) are not validated nor has an adequate assay been developed for the honey bee. In addition, protocols for exposing terrestrial and aquatic invertebrates to viruses and protozoa are needed in anticipation of their use as microbial pest control agents in the near future. The solution identified at the workshop is to emphasize the validation of existing Tier 1 protocols and to continue development of protocols to provide exposure 35 ------- methods for new non-target species. The addition of newspecies to the testing regime will allow an evaluation of the host range of a prospective control agent. Host range is the fundamental question underlying all non-target organism testing. New microbial pesticides will be altered in ways to increase virulence (to elicit control effect, i.e. lethality, reproductive disruption, more quickly) or to increase stability and longevity of the agent in the environment allowing a longer time for the desired effect. Any altered agent may result in changes in the relationship between the original, unaltered organism and its environment, including other animals. Determining changes in host range is an important component of Tier 1 testing. Interpretation of nonspecific toxicity due to exposure to massive quantities of any substance also needs investigation. Under Subdivision M, a single maximum hazard dose approach is recommended for Tier I testing. Research is needed to address ways in which to separate nonspecific toxicity from specific effects. Tier 1 tests for chemicals have traditionally used death of the exposed organism as the endpoint. The possibility of subtler, sublethal effects of MPCAs on non-target organism fitness, fecundity, competitiveness, etc. cannot be ignored. Recognition and incorporation of endpoints that indicate these subtle effects should be developed. Molecular biological techniques could provide simple, rapid assessments for these endpoints. Research questions which should be addressed to improve interpretation of the Tier 1 results/validations include: What is the maximum dosage concentration that can be used before nonspecific effects occur? What is an appropriate exposure method for each type of MPCA and for each category of non-target? This includes needed research to determine appropriate exposure times. Is one large dose or several small doses more representative? What endpoints should be used? Preparations of B. thuringiensis were of particular concern to the discussion group; these preparations constitute the majority of microbial pesticides that have been submitted for review. The results of nontarget testing of B. thuringiensis preparations are difficult to interpret because the active ingredients in many preparations are unknown. Research is needed to best identify and quantitate the active ingredients present, i.e., number of spores, presence of crystal toxins, heat-tolerant exotoxin(s), and heat-label exotoxin(s). When a large number of crystal toxins are present, what is the best method of identification; serology, SDS-PAGE, HPLC, or is it more appropriate to identify the toxin 36 ------- genes themselves? The presence of heat-labile exotoxin has been established by bioassay, but efforts at chemical identification have not been successful. Methods to chemically identify and quantitate these toxins must b'e developed. The panel also discussed the difficulty of interpreting effects due to inactive ingredients in commercial MPCA formulations. The effects of fermentation media, carrying agents and environmental conditions on the activity of the recovered B. thuringiensis preparations to nontarget organisms require investigation. Any synergistic effects of formulation components also must be determined. The very different effects on Daphnia by nonformulated subsp. B.t. tenebrionis preparations, produced by several different manufacturers, is an example of how different fermentation media and conditions may affect the results of nontarget testing. Other immediate research needs were identified in the discussions. These needs are not part of the tier 1 validation but are specific questions to assist OPP in current registrations of MPCAs. How to identify and quantitate the active ingredients present in the test material. How to measure effects of fermentation media and conditions on the activity of the test preparations. How to select non-target organisms for testing effects. Sufficient knowledge of lethal mechanisms and modes of action is limited to only a few MPCAs. Research is needed in molecular pathology of insect diseases to improve our basic etiological understanding and predictive capabilities for new MPCAs. Information is needed for design, selection of exposure methods and endpoint determinations. Accurate, sensitive and reliable methods for enumeration and identification must be developed for all classes of MPCAs, particularly the protozoa, virus and fungi. Currently there are no validated methods that can mark and identify these microorganisms to allow tracking and separation from indigenous organisms of the same species. Research to expand knowledge of host range is also required. Conditions affecting host range are not well-known. These data will assist in the selection of appropriate non-target test plants or animals. It is hoped that knowledge gained through studies on host range and mechanisms of action will produce better controls (e.g., inactivated or nonpathogenic surrogates). Research to develop a positive control methodology is needed. Use of positive controls would demonstrate that the test duration is adequate to produce infection and expression of pathogenicity. It would provide confidence that the experimental design is adequate to allow detection of the infectivity/pathogenicity of the agent being registered. Two types of positive controls were identified. One type acts to determine if the target is susceptible to the MPCA in the test system. The other is to determine if the non-target 37 ------- organism (NTO) is sensitive to a pathogen in the test system. This would usually be a known pathogen of the nontarget organism, ideally, an organism that is related to the MPCA. However, identification and culture procedures for pathogens of animals of low economic value (often the NTO) are rare. This information should be developed if test protocols are to be truly validated and if positive controls are to be included in the Tier test system. In natural habitats, NTOs will interact with the MPCA in conditions that are less optimal than those used in the Tier 1 laboratory evaluations. A greater confidence in the Tier 1 data would be realized if tests that include exposure of both healthy and physiologically-stressed NTOs were required. Such tests would address the potential for opportunistic attacks by the MPCA. Research should focus on environmentally- and physiologically- relevant sublethal stressors, i.e., those stressors to which an NTO would be subject to its natural environment. Examples of these stressors are temperatures or salinities which fall outside the preferred range for the species, reduced dissolved oxygen for aquatic organisms, inadequate nutrition, and exposure to sublethal levels of chemical pollutants. Altered disease susceptibility in fish and other vertebrates inhabiting (xenobiotic-chemical) contaminated environments have been documented. Research is needed to develop and validate protocols for stressed and nonstressed NTOs. These research needs can be summarized as follows: methods for having control treatments in all protocols and including positive controls to assure the exposure protocol is adequate an understanding of mechanisms and modes of action of representative MPCA taxa reliable methods by which to mark or identify viruses, protozoa or fungi an incorporation of the MPCA life cycle characteristics into risk assessment protocols which include exposure of both healthy and physiologically-stressed NTOs to address the potential for opportunistic infections by the MPCA. This includes evaluations of how the operation of the test can affect results and life- stage susceptibility protocols for species that have not been commonly tested As mankind's biotechnological ingenuity increases, it becomes more reasonable to engineer pesticidal plants; incorporating the pesticide-producing genes directly into the plant tissues rather than developing pesticides to be applied to the plant. Tier testing protocols must be examined to assess their applicability to these transgenic plants. Similarly, an emerging research challenge is the development of methods that will evaluate effects of MPCAs on non-target plants. Plants have not been emphasized in 38 ------- prior research but an increase in the registration requests for the use of fungi as herbicides will require additional forms of plant bioassays. 39 ------- HUMAN HEALTH PROTOCOLS AND METHODS NEEDS Panel: Z. Vaituzis, R. Sjoblad, P. Hartig, Y. Huang, R. Sherwood The research efforts of the past 5 years have led to a useful protocol for pulmonary infectivity testing. Two key areas requiring further research are pulmonary and intraperitoneal exposure methods. Pulmonary exposure methods (intranasal/intratracheal) have been validated for rodent infectivity testing. Further research is needed to evaluate the pulmonary exposure method as a useful model for toxicity assessment of microbial pesticides, (i.e. studies to include histopathology, pulmonary immunology, assay limitations, rodent/human extrapolations, and evaluations of formulated products). The intraperitoneal (i.p.) exposure model for rodents should be evaluated and validated for infectivity and toxicity assessment of microbial pesticides. The intraperitoneal and subcutaneous (s.c.) exposure methods should be compared and evaluated as to their usefulness in toxicity tests for the purpose of validating the current tolerance exemption requirement (40 CFR 180.1011). Studies are also needed to validate protocols which address temperature and growth parameters of microbial pesticides, and in some instances, methods for enumeration of microbes requiring unique propagation conditions. 40 ------- SECTION 5 ECOLOGICAL SIGNIFICANCE AND HUMAN HEALTH CONSIDERATIONS ECOLOGICAL SIGNIFICANCE - PREDICTING EFFECT IN THE FIELD Panel: R. Anderson, J. Fournie, W. Fisher, L. Shannon This section defines research needs that will improve confidence in predicting effects in natural systems from data obtained in laboratory studies. The panel grouped discussions into single species tests, multi-species tests, and significance of tests for hazard assessment. Research questions and salient points of discussion are presented below: 1. Do single species tests reflect the response of organisms exposed in nature? Non-target organism tests are conducted to produce data needed to assess the potential hazard of MPCAs to these organisms (Subdivision M, Series 154A-1). Thirty day exposures are recommended in Subdivision M Guidelines and these single species exposures can provide information on lethal effects and less than lethal changes in the animals' health. If test animals have a short life-cycle (days or weeks) the 30 day exposure could include several generations and the results become better predictors of an effect. If the animal has a longer life cycle (months or years), then a thirty day test may not be appropriate. The difference in the length of the life-cycle also requires an evaluation of the endpoints used in the tests. For example, a complete assessment for those animals with longer life cycles may require exposure of specific parts of the life cycle and include measures of growth or reproduction changes. Endpoints Endpoints such as death, behavioral change, growth and reproduction are classical measures of effect for chemical tests, but most current protocols for MPCAs have centered only on death. The unique MPCA features of reproduction, host specificity and disease production, coupled with the typical lack of information on the life cycles and biology of potential hosts calls for further study of endpoints. Techniques are available for measuring some sublethal endpoints such as changes in reproduction, growth, and behavior. However, not all have been evaluated in nontarget animals. Reproductive effort of zooplankton has recently been used to assay a protozoan MPCA, but growth and behavior assays have not been employed for marine organisms. Another potential sublethal indicator that has not been investigated is immunosuppression. Immunosuppression could increase susceptibility to natural pathogens or change the quantity or diversity of natural parasites. There is little information on how the biochemical or physiological condition of the non-target or expected host may affect the expression of the agent in that animal. This work can be started with studies that include pathogens known to affect the specific non-target animal being evaluated. Without the known-pathogen tests it is not possible 41 ------- to assess questions of how the condition of the animal may mediate its response to doses of MPCA, form or formulation of the exposing agent and environmental exposure conditions. An endpoint that is now being evaluated is the effect of the presence of the MPCA in or on the organism. This measurement is accomplished by tissue squashes, gut dissections and histology, and can be performed on living, dead or moribund animals. These methods provide information on whether the MPCA-host association is due to adherence, ingestion or infection. Another endpoint being evaluated is the fate and survival of the MPCA in the test system (usually considered a Tier 2 issue) to assure that the organism is viable during the test. Exposure Conditions Underlying the basic question "Do single species tests reflect the response of organisms exposed in nature?" is whether responses of organisms in the laboratory are analogous to responses in natural systems. One goal of single species tests is to select conditions which optimize an animal's survival during the exposure. The controlled physical and biological conditions found in the laboratory are not found in natural systems, where the organisms survive in a variety of environmental conditions, many of which may be near the extent of their physiological survival range. Thus, optimal rearing conditions for the test organisms do not necessarily reflect the natural condition. A research need is to test single species under conditions that approach conditions found in natural systems. Such an evaluation should begin with a description of how the organism will respond when exposed to variations of the conditions found in the systems that would receive the MPCA. Recent experiments have initiated exposures under various salinity and temperature regimes. Physiological (metabolic) parameters are measured to determine the extent of stress on the organisms. This approach increases the amount of research to be performed on a MPCA, but also increases the value of the laboratory studies in relation to natural exposures. Laboratory tests may investigate other physical factors such as temperature and variations in water quality for aquatic systems or temperature and humidity for terrestrial systems. Biological variables such as age, reproductive condition and physiological and immunological state may also be evaluated. New Protocols There are continuing needs for single, multiple species and microcosm test protocols. A single species need is to develop methods for species that are closely related to the targeted pest. Exposures of these animals would address the question of host range. Test methods that address the many potential exposure methods and routes and an evaluation of the concept of maximum exposure should be conducted. Routes 42 ------- tested for marine freshwater and terrestrial organisms have included exposure in either the water column or air and gavage and injection. The latter two routes insure maximum exposure. Exposures in water or air can be at an expected "maximum" concentration or at a concentration expected in nature with the highest MPCA application; however, the agent's interaction with the natural system will affect the actual dose in nature. Some dose-response research has been conducted, but research is limited because the MPCAs tested to date appear to have a narrow host range. Chronic exposures have rarely been investigated. 2. Do multispecies tests simulate interactions between species in nature? Multiple species laboratory tests can have many forms. For aquatic ecosystems, designs include assemblages of species in separate containers but sharing the same water to systems that mimic natural systems. Each system has unique characteristics and each were designed to provide data for specific questions. Multispecies tests to evaluate the effects of MPCAs on beneficial terrestrial arthropods (mostly insects) have not been developed. In containered multi-species tests, the organisms are usually maintained within the same exposure tank; thus interaction (other than exposure to the agent) is not intended to be a major consideration. Most organisms used in multi-species tests can inhabit the same environment (i.e. withstand the same conditions) but separation allows better management of the animals and increased accuracy in effects measurements. The test systems that mimic natural processes have been known by different names, including laboratory microecosystem and laboratory microcosm. Two types are being evaluated by ORD. The mixed flask culture (MFC) protocol is a generic system that develops a phytoplankton/periphyton/invertebrate community beginning with a nutrient solution and a naturally derived population of phytoplankton/periphyton and invertebrates. The second type is an environment core-based protocol that allows testing that can be site-specific. Soil cores are collected from temporary pools and rehydrated in the laboratory. Both microcosms contain self-sustaining aquatic communities including decomposers (bacteria and fungi), primary producers (diatoms, blue-green and green algae, and aquatic moss), grazers (e.g., amphipods), filter feeders (e.g., cladocerans), scrapers (e.g. snails), and predators (e.g., predaceous copepods and rotifers). They have also supported insect target organisms such as mosquito larvae. Multi-species tests and microcosm protocols used today usually are limited to the same endpoints as single species tests, but other measures are being developed. Measures of how the MPCA may be accumulated or altered through interaction with components of the system are available. One species can affect the MPCA and change the MPCA's association with other test species. For example, filter-feeding bivalve molluscs accumulate the agent and may reduce exposure densities. Other animals ingest and repackage the spore-toxin combination of Bacillus thuringiensis, thereby increasing 43 ------- its distribution or decreasing its toxicity. Other effects which warrant consideration include how to measure activation or de-activation of the agent, whether a "reservoir" species exists, or does the system provide a refuge for the MPCA. There is a potential for different routes of exposure due to transport of material between species. Research to define these and other interactions has started and continued support is needed. Underlying the basic question: "Do multispecies tests simulate interactions between species in nature?" is whether the responses of organisms in the multispecies laboratory exposures are analogous to responses in natural systems. Continuing the comparative studies in natural and laboratory systems is necessary if laboratory and field risk assessments are to be made with confidence. There are two aspects to comparing laboratory to field results. The first, calibration, is the process of correlating the biological structure and functions in unperturbed microcosms with field sites. The second step is validation, a determination of how closely survival and other effects seen in the microcosms match those measured in field tests. The value of laboratory and field calibration has been shown for both multiple species and microcosm exposures. Multiple species tests have shown that depuration of bacteria and fungal spores by oysters occurs at the same rate in both laboratory and field conditions. In the case of an unknown (potentially harmful) MPCA, the laboratory system could be used with some adjustments. Many of the marine organisms have been used extensively in chemical toxicity studies, and methods to hold and expose organisms have reduced potential problems such as handling and acclimation stresses. There are no field verifications, no testing of physical/chemical ranges and no testing of different routes, times or doses found with the multi-species system. Calibration studies show that core microcosms are much closer surrogates of natural ponds than MFC microcosms. MFC microcosms are constructed with a nutrient medium and are highly eutrophic with higher nutrient concentrations, pH, and primary productivity than natural systems. The core microcosms are much less eutrophic and more similar to natural systems. Both natural and core systems have pH values from 6 to 6.5 and show a diel ("pre-dawn" to "late afternoon") oxygen shift of approximately 25% reflecting similar levels of primary production. Nutrient concentrations (nitrogen and phosphorus) are also similar to field values. Zooplankton populations in the core systems readily reach densities comparable to those seen in the field, and continue to increase for several months, presumably due to an absence of predators. More validation studies are needed. Studies with Bacillus thuringiensis var. israelensis (Bti) were conducted in divided temporary pools. Comparisons of temporary pool data to core microcosm data found similar patterns of survival and distribution of the organism. Effects were difficult to compare since no significant effects were seen on animals in either system. True evaluation of a test system can occur only if effects are seen, so tests with an agent that does affect non-target animals are needed. One candidate is the zoospore producing fungi, Lagenidium. In single species tests, cladocerans, Daphnia pulex and Ceriodaphnia dubia and the insect Chironomus were 44 ------- killed by the Lagenidium zoospores. The effect concentrations were 10 to 100 times greater than the LC50 of the targeted mosquitoes. Another feature of microcosms that should be evaluated relative to other test systems is perturbation resiliency. Microcosm protocols include a reinoculation procedure which models the presence of a refuge in natural systems and allows measurements of the time it takes for a disturbed system to return to control levels. Since effects have not been recorded in the tests completed to date, a comparision of the resiliency found in the microcosm to that found in natural systems has not been done. 3. What is the significance of the tests for hazard assessment? The Tier test system provides a progression of tests with increased complexity at each tier. Each tier produces data on different responses of the animals or the system. Bioassay tests are being used and more are being developed to evaluate the effects of applications on non-target animals. However, the single species laboratory tests do not determine 1) if there is an effect of MPCA application toward untested non-target animals, 2) if there is an indirect effect, or 3) if the residual MPCA in the application area remains above a deleterious threshold level or how it may be reduced below that threshold level. The questions arising from the single species tests can be addressed in the multiple species and microcosm tests, but the interpretation of results from those tests cannot be fully assessed until field verification of the laboratory data is complete. At this time, there are no complete evaluations of an MPCA through all Tiers. Microcosms that mimic some responses of the environment can be constructed and calibration and validation have begun. The next step is to move beyond the measuring of effects to interpretation of the ecological significance of any effect. This will require a higher level of understanding of how natural systems operate. One requirement will be to determine the variability of ecosystem structure and function measurments. Direct or subtle indirect effects can be detected only if the variability of the receiving system is known. Some information on variability of animal, microorganism or ecosystem function measurements is available from studies done for other purposes including those conducted for chemical pesticide registration, but these are not readily available to the MPCA researcher. Developing an understanding of measures of variability can best be accomplished with long-term field studies. 45 ------- HUMAN HEALTH CONSIDERATIONS Panel: C. Kawanishi, W. Schneider, G. Held Test parameters that influence the extrapolation of experimental results from exposure to MPCAs are important considerations for human health effects testing. The test organisms most frequently utilized are cells in culture, including human cells and laboratory animals such as mice, rats, or hamsters. While results obtained in human cell culture tests bypass the problem of interspecies extrapolation, there still remains the difficulty of estimating the role of normal defense mechanisms present in the whole animal. However, this situation, along with the finding that many cells in culture are susceptible to a wider range of agents than in the intact organisms, comprises a maximum hazard situation consistent with the basic testing principles for MPCAs. Nevertheless, the problem of realistic extrapolation to the intact human remains. Newer approaches to examine this problem would be important to risk assessment with MPCAs. Endpoints normally utilized in MPCA testing are toxicity and infectivity. The transfer and/or functional perturbation of genetic information into human cells is a more recent consideration. Research into whether model MPCAs are capable of causing genetic effects and the mechanisms involved are essential to more complete health effects assessment especially with genetically mobile or altered agents. However, research on the mechanisms of infectivity and toxicity with MPCAs would greatly enhance extrapolation of the results to humans and facilitate the development of more definitive tests. Test conditions can also affect extrapolation of results. Route of exposure is an extremely important consideration. Most MPCA human exposures are believed to occur by the pulmonary route. Traditional aerosol inhalation studies are plagued by numerous shortcomings including inability to deliver high dose rates most appropriate for relevant human studies, difficulty of measuring delivered dose, inactivation of agents and cost. Results to date demonstrate that simpler intratracheal/intranasal dosing methods are more reliable and applicable to MPCAs. Consequently, mechanisms of action of representative agents of different MPCA classes should be examined to facilitate extrapolation of findings to human exposure scenarios. The importance of other routes of exposure is highly dependent upon the nature of MPCA, including the physical nature of the inoculum and its intended use. These relationships should be examined in greater detail. The influence of dosing regimen and stress effects on test organism susceptibility are factors that should also be examined. Epidemiological aspects such as studies on MPCA dispersal mechanisms in the environment, including bioaccumulation by organisms such as filter feeders, transmission by biological vectors, fomites and physical factors such as wind, should be examined. The role of susceptibility factors such as altered immune responses could be evaluated for their importance in the effects of MPCAs on humans. There are a number of areas of MPCA research that are critically significant for 46 ------- interpretation of test results and their extrapolation to real-world human health effects. These research areas are: (1) effects and mechanisms of action of MPCAs such as Bacillus thuringiensison mammalian systems, (2) biochemical, physiological degradation interaction of mammalian systems with intracellular parasitic MPCAs to facilitate understanding of the significance of persistence of agents in test organisms and (3) development (on a case-by-case basis) of identification methods for new, uncharacterized MPCAs. Identification is a basic component of clinical microbiology and MPCA registration. Basic research in this area will aid program offices in recommending identification methodologies for unique agents belonging to poorly characterized taxonomic groups. 47 ------- APPENDIX A RESEARCH NEEDS AND FUTURE DIRECTIONS (ECOLOGICAL EFFECTS) OPP RESEARCH/DEVELOPMENT NEEDS I. BACKGROUND CFR Part 158.170 lists the testing required for Microbial Pest Control Agent registration. Subdivision M of the Pesticide Testing Guidelines provides the basic testing parameters. Specific protocols for required testing, however, are lacking. In 1982, OPP asked ORD to develop protocols for environmental nontarget effects testing of MPCA's and GEMP's. Ramon Seidler at the Corvallis laboratory, John Couch of Gulf Breeze and Richard Anderson of Duluth have been responsible for providing much of the necessary testing protocols. These efforts, however, have not been at a the program level. II. OPP NEEDS A. LABORATORY BIOASSAY PROTOCOLS The above initiatives need to continue and need to be upgraded to a priority status. They need to be comprehensive enough to include all terrestrial (soil, vegetation, insect, avian) requirements in CFR 158.170 and they need to be validated. B. MULTISPECIES SYSTEMS AND MICROCOSMS Research/development needs to concentrate on ecological effects methods, including multispecies systems and microcosms wherever possible. This includes "fate" determination (colonization) protocols utilizing currently available microbe detection methodology. C. PROTOCOL VALIDATION Gene probe, plasmid and other molecular research can be combined with the above, but the protocol development work is of primary importance to the Program Office, followed by: - protocol validation with microorganisms of known effects, - protocol refinement, incorporation of new microbe detection methods as they become available (including stressed microbe detection), etc. Protocols for DNA exchange on plants, in soil and insects are certainly also useful, but they are of secondary importance to the above-mentioned direct species effects testing protocols. 48 ------- The following is a listing of QPP protocol needs for ecological effects testing in support of Subdivision M guidelines: 1. Avian a. Mallard duck oral pathogenicity single dose 30-day test. b. Bobwhite quail oral pathogenicity and single dose 30-day test. c. Injection pathogenicity test for mallard and quail. d. Inhalation pathogenicity test for mallard and quail (LD50 procedures for pathogens). e. Long-term avian pathogenicity and reproduction test. 2. Wild mammal a. Wild mammal pathogenicity test chronic exposure (oral and inhalation). b. Single dose injection 30 day test (LD50 procedure for pathogens). 3. Simulated field testing for mammals and birds: Effects testing on confined bird and mammal populations (growing and reproducing). 4. Terrestrial environmental expression tests Greenhouse (contained) tests to determine: a. Colonization of soil(s). b. Colonization of plants. c. Colonization of nontarget animals (including substrate enrichment, as applicable). 5. Freshwater fish a. Rainbow trout and Bluegill sunfish single dose 30-day toxicity test. b. Rainbow trout and Bluegill sunfish pathogenicity test - chronic exposure (LC50 procedure for pathogens). 6. Freshwater aquatic invertebrate a. 7-day Daphnia single dose toxicity test. b. 21-day Daphnia chronic exposure pathogenicity test (LC50 procedure for pathogens). 7. Estuarine and marine (shrimp, fish and oyster): a. Single dose toxicity test. b. Chronic exposure pathogenicity test (LC50 procedure for pathogens). 49 ------- listing of OPP protocol needs for ecological effects testing, continued 8. Definitive aquatic animal pathoqenicitv tests: Single species protocols for LC50 determinations on representative species in the aquatic food chain groups - - to determine the spectrum of susceptible nontarget species. to determine susceptible routes of exposure and dose-response relationships. 9. Embryo/larvae studies and life-cycle studies of fish and aquatic invertebrates: a. Fish early life stage pathogenicity (LC50). b. Fish life-cycle (LC50). c. Invertebrate life-cycle (LC50). 10. Simulated field testing (microcosm) for aquatic organisms: Long-term simulated testing of effects on reproduction and growth of a confined population of aquatic vertebrates and invertebrates. 11. Aquatic ecosystem tests: Acute effects on aquatic nontarget populations (ecosystem effects testing). 12. Simulated field testing (microcosm) to determine MPCA fate in freshwater and marine/estuarine environment: Aquarium (contained) tests to determine: a. Colonization of water column. b. Colonization of bottom sediments. c. Colonization of nontarget plants and animals (include: substrate or target enrichment, where applicable; conditions to mimic seasonal variations). 13. Terrestrial wildlife and aquatic organism multispecies testing: Terrestrial and aquatic invertebrate and fish multispecies test systems containing representatives of several taxonomic groups of nontarget organisms. Pesticidal agents to be applied as per label use instructions. (The pesticidal agents equilibrate to their "niches" and population number levels. The nontargets are given the opportunity to avoid microbial pesticide colonized areas). 14. Nontarqet plant studies a. Plant pathogenicity test methods. b. Terrestrial plant toxicity. c. Terrestrial plant growth and reproduction. d. Aquatic plant pathogenicity, toxicity, growth and reproduction. 50 ------- 15. Nontarqet insect testing (insect predators and parasites) Chronic exposure and single dose pathogenicity tests (LD50 procedures for pathogens) on adult and larval stages of: a. Predaceous hemipterans (true bugs). b. Predaceous coleopterans (beetles). c. Predaceous mites. d. Predaceous neuropterans (lacewings). e. Parasitic hymenopterans (wasps). f. Parasitic dipterans (flies). g. Dose response relationship protocols for lepidopteran nontargets. 16. Honey bee toxicitv/pathoqenicitv a. Single dose pathogenicity test on adults and honey bee larvae. b. Chronic exposure pathogenicity test (LD50 procedures for pathogens). 17. Simulated (microcosm) field testing for insect predators and parasites: Simulated testing of effects on reproduction and growth of a confined population of insect predators and parasites. 18. Simulated (microcosm) field testing for insect pollinators: Simulated testing of effects on reproduction and growth of a confined population of insect pollinators. 19. Dissemination of microbial pesticides by insects. 51 ------- AN EXAMPLE OF RESEARCH USEFUL TO OPP: Clarify the "strain" issue: 1. Are there different strains of, say, P. fluorescens and/or P. synngael We all know that there are, however: Do some grow only in soil and other strains only on vegetation? Do certain strains have affinity for only select plant species? (i.e., when the registrant says that their P. fluorescens grows only on corn roots, or only on strawberry blossoms, and that the P. fluorescens found on other plants are different strains - is there any validity to these claims?) Will strains found in Montana on trees also colonize trees in Florida, etc.? Do some strains colonize only specific plant families? Assuming the answers to the above are affirmative, can these "strains" be characterized/described and can criteria for their identification/differentiation be developed? Plant colonization studies: There is a need to define the "sampling strategy" during leaf colonization studies -i.e. define effects of temperature, rainfall, plant maturation cycle stage, dew, drying, sunlight UV, windblown soil bacteria, seasonal effects, etc., on population fluctuations on leaves. The output should be a protocol for determining plant colonization which defines all variables including duration of sampling for a definitive colonization determination. 52 ------- OPP PRIORITIZED SHORT AND LONG TERM PROTOCOL NEEDS FOR ECOLOGICAL EFFECTS TESTING Short-Term Develop single dose and chronic exposure toxicity/pathogenicity protocol for honeybee larvae. Develop aquatic benthic and planktonic invertebrate chronic toxicity/pathogenicity testing protocols. Species selection to include representatives from aquatic food chain groups and routes of exposure and dose/response relationships. Also fish and invertebrate life cycle testing protocols. Long-Term Develop and validate freshwater fish chronic exposure toxicity/pathogenicity protocols. Need to perform avian and wild mammal intraperitoneal testing with various Bacillus thuringiensis strains and commercial products, especially Dipelฎ and Javelinฎ, to determine what, the dosage and conditions which are causing mortality. The same for nontarget insects and freshwater and estuarine and marine invertebrates exposed to the Bt strains. Need to refine and validate the existing avian, aquatic and nontarget insect protocols ( to include definition of how to "inactivate" test materials such as Bt preparations). Develop long-term avian pathogenicity and reproduction test protocol. Develop wild mammal acute and chronic exposure pathogenicity/toxicity test protocols. 53 ------- APPENDIX B Agenda "Procedures and Concepts for Evaluating Effects of MPCAs" February 5-7, 1991 Annapolis, Maryland Tuesday, February 5th 8:30 - 8:45 Workshop Introduction - R.B. Coffin, ORD Matrix Manager 8:45 - 10:00 OPP Presentations 8:45 - 9:00 Introduction to Regulation of MPCAs - Amy Rispin 9:00 - 9:30 Regulatory Requirements Fred Betz 9:30 - 10:00 Testing Guidelines Bill Schneider 10:00-10:30 BREAK 10:30 -10:50 Kinds of Products Reviewed - Bernice Slutzky 10:50 -11:10 Ecological Studies: Effectiveness and Development Needs - Zig Vaituzis 11:10 -11:30 Health Effects Studies: Effectiveness and Development Needs - Roy Sjoblad 11:30-1:00 LUNCH 1:00 - 2:15 ORD Presentations 1:00 - 1:30 Duluth Program Presentation - Dick Anderson 1:30 - 2:00 Corvallis Program Presentation - Bruce Lighthart 2:00 - 2:30 BREAK ป 2:30 - 3:00 Gulf Breeze Program Presentation - Jack Fournie 3:00 - 3:30 RTP Program Presentation - Clint Kawanishi 3:30 - 5:00 Questions/Comments on presentations Open Discussion followed by identification of topics for discussion the next day 5:00 - Meeting of Steering Group' to summarize and develop an agendad the next day 54 ------- Wednesday, February 6th 8:30 - 9:30 9:30- 10:00 10:00- 12:00 Summary of yesterday's events. Outline of this days activities. Selection of topics for group/subgroup discussion and writing assignments. BREAK Discussions and Writing Assignments 12:00-2:00 LUNCH 2:00 - 4:00 Review and continue discussions and writing Th ursday, February 7th 8:30- 10:00 10:00- 10:30 10:30- 12:30- Summarizing and consensus of report information - discuss finalization of report BREAK Continuation to completion Team Leaders or Designees leave for Headquarters, remaining participants continue to completion 55 ------- APPENDIX C LIST OF ATTENDEES Dr. Beth Anderson U.S. Environmental Protection Agency Office of Toxic Substances Chemical Control Division 401 "M" Street, S.W. Washington, D.C. 20460 Dr. Richard L Anderson U.S. Environmental Protection Agency Environmental Research Laboratory 6201 Congdon Blvd. Duluth, MN 55804 Dr. H. Kay Austin U.S. Environmental Protection Agency Office of Toxic Substances 401 "M" Street, S.W. Washington, D.C. 20460 Dr. David Bays U.S. Environmental Protection Agency Office of Toxic Substances 401 "M" Street, S.W. Washington, D.C. 20460 Dr. Clay Beegle U.S. Environmental Protection Agency Office of Pesticide Programs 401 "M" Street, S.W. Washington, D.C. 20460 Dr. Rita Briggs U.S. Environmental Protection Agency Office of Pesticide Programs 401 "M" Street, S.W. Washington, D.C. 20460 Dr. Ellie Clark U.S. Environmental Protection Agency Office of Toxic Substances Chemical Control Division 401 "M" Street, S.W. Washington, D.C. 20460 Dr. Rick Coffin U.S. Environmental Protection Agency Environmental Research Laboratory Sabine Island Gulf Breeze, FL 32561 Dr. William Fisher U.S. Environmental Protection Agency Environmental Research Laboratory Sabine Island Gulf Breeze, FL 32561 Dr. John Fournie U.S. Environmental Protection Agency Environmental Research Laboratory Sabine Island Gulf Breeze, FL 32561 Dr. Frederick Genthner U.S. Environmental Protection Agency Environmental Research Laboratory Sabine Island Gulf Breeze, FL 32561 Dr. Christina Good U.S. Environmental Protection Agency Office of Toxic Substances Chemical Control Division 401 "M" Street, S.W. Washington, D.C. 20460 56 ------- Mr. James E. Harvey Technical Resources, Inc. Sabine Island Gulf Breeze, FL 32561 Dr. Phillip C. Hartig METI P.O. Box 12313 RTP, NC 27709 Dr. Gary Held U.S. Environmental Protection Agency Health Effects Research Laboratory RTP, NC 27711 Dr. Yuan-Shen Huang U.S. Environmental Protection Agency Health and Effects Research Laboratory RTP, NC 27711 Ms. Rosalind James NSI Technology Services Corp. 200 S.W. 35th Street Corvallis, OR 97333 Dr. Clint Kawanishi U.S. Environmental Protection Agency Health Effects Research Laboratory RTP, NC 27711 Dr. John Kough U.S. Environmental Protection Agency Office of Toxics Substance Health and Environmental Review Division 401 M Street, S.W. Washington, DC 20460 Dr. Bruce Lighthart U.S. Environmental Protection Agency Office of Research and Development 200 S.W. 35th Street Corvallis, OR 97333 Dr. Gerald D. Laveck U.S. Environmental Protection Agency Office of Toxic Substances Exposure Evaluation Division 401 "M" Street, S.W. Washington, D.C. 20460 Dr. Thomas McClintock U.S. Environmental Protection Agency Office of Pesticide Programs 401 "M" Street, S.W. Washington, D.C. 20460 Dr. Gwen McClung U.S. Environmental Protection Agency Office of Toxic Substances Health and Effects Research Laboratory 401 "M" Street, S.W. Washington, D.C. 20460 Dr. Susan McMaster U.S. Environmental Protection Agency Office of Pesticides and Toxic Substances 401 "M" Street, S.W. Washington, D.C. 20460 Ms. Nancy Padgett Technical Resources, Inc. Sabine Island Gulf Breeze, FL 32561 Dr. Delaos Powell U.S. Environmental Protection Agency Office of Toxic Substances 401 "M" Street, S.W. Washington, D.C. 20460 Dr. Martha Saga U.S. Environmental Protection Agency Office of Pesticide Programs 401 "M" Street, S.W. Washington, D.C. 20460 57 ------- Dr. Phillip G. Sayre U.S. Environmental Protection Agency Office of Toxic Substances 401 "M" Street, S.W. Washington D.C. 20460 Dr. William Schneider U.S. Environmental Protection Agency Office of Pesticide Programs 401 "M" Street, S.W. Washington, D.C. 20460 Dr. Mark Segal U.S. Environmental Protection Agency Office of Toxic Substances 401 "M" Street, S.W. Washington, D.C. 20460 Mr. Lyle Shannon University of Minnesota, Duluth Biology Department Duluth, MN 55811 Dr. Robert Sherwood NT Research Institute 10 West 35th Street Chicago, IL 60616-3799 Dr. Roy Sjoblad U.S. Environmental Protection Agency Office of Pesticide Programs 401 "M" Street, S.W. Washington, D.C. 20460 Ms. Virginia Snarski U.S. Environmental Protection Agency Environmental Research Laboratory 6201 Congdon Blvd. Duluth, MN 55804 Dr. Bernice Slutsky U.S. Environmental Protection Agency Office of Pesticide Programs 401 "M" Street, S.W. Washington, D.C. 20460 Dr. Zigfridas Vaituzis U.S. Environmental Protection Agency Office of Pesticide Programs 401 "M" Street, S.W. Washington, D.C. 20460 Ms. Barbara Wireman Technical Resources, Inc. Sabine Island Gulf Breeze, FL 32561 Dr. Larry R. Zeph U.S. Environmental Protection Agency Office of Pesticides and Toxic Substances 401 "M" Street, S.W. Washington, D.C. 20460 58 ------- |