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
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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
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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.
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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.
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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.
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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
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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.
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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.
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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
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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.
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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.
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OFFICE Cf PESTICIDE
Regirtraiioi
Environmental
Fate and
Efleet* DrvLuoD
EEE
CEANCH
EfGWE
Health
Effeeto
DEC
Figure 2.2 OPP organizational chart.
6
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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
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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.
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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.
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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.
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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.
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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.
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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.
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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
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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.
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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
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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.
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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
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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.
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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.
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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.
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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
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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.
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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
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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
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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.
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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.
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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
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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.
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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
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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).
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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.
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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-
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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.
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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
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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
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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
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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
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prior research but an increase in the registration requests for the use of fungi as
herbicides will require additional forms of plant bioassays.
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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.
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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
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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
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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
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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
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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.
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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
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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.
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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.
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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).
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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.
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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.
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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.
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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.
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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
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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
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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
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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
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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
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