WILDLIFE
TOXICOLOGY
^BRIEFING BOOK
ENVIRONMENTAL RESEARCH LABORATORY- CORVALLIS, OREGON
&EPA
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WILDLIFE TOXICOLOGY
BRIEFING BOOK
November 1986
Compiled by
S. Mark Meyers
Environmental Research Laboratory -- Corvallis
Toxics/Pesticides Branch
Corvallis, Oregon
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Table of Contents
Introduction 1
Overview 2
Goals and Objectives of the Wildlife Toxicology Team 2
Technical needs of the Agency 4
Accomplishments 8
Current Research 11
Facilities 15
Avian Toxicology Facility (Greenhouse 8) 17
Wildlife Toxicology Facility (New Building) 18
Western Fish Toxicology Station (WFTS) 19
WFTS Main Building 20
Personnel 21
Wildlife Toxicology Team 22
Resume's 23
EPA 23
Visiting Scientists 31
NSI 34
Appendices
A. Experimental Synopses 42
Inhouse
Completed 42
Ongoing 48
Future 57
Extramural 62
B. Chemistry Capability and Laboratory Analysis 70
C. Publications 73
Inhouse 74
Extramural 79
D. Quality Assurance Program 82
E. CERL Institutional Animal Care and Use Committee
Interagency Research Animal Committee Proposed 87
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INTRODUCTION
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OVERVIEW
This publication summarizes the goals and objectives of the Wildlife
Toxicology Research Team within the Toxics and Pesticides Branch of the
Corvallis Environmental Research Laboratory (CERL). In accord with the
objectives of the Agency, it is important to develop an in-house capability to
coordinate and conduct research in terrestrial wildlife toxicology. This
capability serves several administrative and technical functions, and provides
data needed to verify and evaluate effects of toxic substances in support of
registration and use.
Preliminary information about the biological effects of toxic substances
can initially be answered in a laboratory environment. Ultimately, however, it
is necessary to determine the relationship between standard EPA laboratory test
protocols and toxic effects observed in actual field exposures. Only through
carefully coordinated laboratory and field investigations can this goal be
achieved.
A primary goal of the Wildlife Toxicology Research Group is to compare
both laboratory and actual field toxicity data for chemicals of interest to
Office of Toxic Substances (OTS) and Office of Pesticide Programs (OPP). Acute
toxicity and the coincident tissue residue levels are determined in the labora-
tory and compared to those observed in the field, to evaluate actual field
exposures.
GOALS AND OBJECTIVES OF THE WILDLIFE TOXICOLOGY TEAM
One of the primary functions of the U.S. Environmental Protection Agency
is to evaluate the impact of chemicals and toxic substances on the environment.
Under the terms outlined in the Toxic Substances Control Act (TSCA), the EPA is
charged with the regulation of the manufacture, commercial distribution, use,
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and disposal of chemical substances and mixtures shown to present an unreason-
able risk to human health or to the environment (Public Law 94-469, 1976). In
response to this law, the EPA must assess the magnitude of expected exposures
and the potential effects of new chemicals (or of old chemicals used in new
ways) on ecosystems and their components. The toxic or deleterious effects of
chemicals on biological systems is one of the most important considerations in
determining the effects of a chemical introduced into an ecosystem. An eco-
system is a complex, stratified structure which is made up of both biotic and
abiotic components, and assessment of the impact of a chemical introduced into
an ecosystem should ultimately account for as many interactions of the system
as necessary. The final measure of the impact on an ecosystem is the survival
of its components, but other subtle alterations in the system may be less
dramatic. The impact of some sublethal effects are not obvious until long
after the exposure occurs and thus may be masked by a significant time delay.
In the overall assessment of the impact of any toxic substances, it is
necessary to determine both the acute (lethal) and subacute (sublethal) effects
on the species in the exposed ecosystem. The impact of toxic substances on
terrestrial wildlife in an ecosystem is thus very complex.
Concern about the effects of toxics on terrestrial wildlife was greatly
stimulated by the passage of the Federal Insecticide, Fungicide, and Rodenti-
cide Act (FIFRA), which specifically and implicitly requires wildlife toxico-
logical testing. In response to the FIFRA, guidelines for the test and evalua-
tion of toxic chemicals which impact terrestrial wildlife were published in the
Federal Register on July 10, 1978. The guidelines are specific and comprehen-
sive in scope, and form the basis for most of the current interest in toxico-
logical testing. The testing of potentially toxic substances under these
guidelines emphasizes the acute (short term, lethal) effects, as determined by
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LC50 and LC50 tests (FIFRA Section 163.71-1 and 163.71-2). Less understood are
the chronic (sublethal) toxic effects. Acute tests, such as the LD50 and LC50
tests, determine those levels of a chemical which, when ingested (in a single
dose or in the diet), cause mortality in 50% of the treated animals. LD50
tests provide data which can be used to estimate levels of chemical which, if
released into the environment, would produce significant effects on the
animal s.
Results from these rather basic laboratory tests cannot be easily used to
predict or evaluate effects beyond the single-species level of organization.
Sublethal effects of a chemical may be manifest in the exposed population only
after significant elapsed time. Thus, in the review or registration of poten-
tially toxic substances, it is necessary to identify both the acute and chronic
effects.
Technical Needs of the Agency
To refine the Agency's ability to assess the potential toxicity of chem-
icals, the Agnecy needs support from the CERL in several technical and research
areas, including:
1. Technical Assistance. The Wildlife Toxicology Research Team provides
technical assistance for questions pertaining to effects of toxic chem-
icals in several scenarios, including interpretation of monitoring or test
results, support for administrative decisions, and evaluation of long-term
hazards (assessment). The Wildlife Toxicology Research Team forms a
nucleus of toxics expertise upon which the Agency can draw for technical
support in questions of registration or validation of guidelines.
2. Test Protocol Guidelines. The effort to validate test methods must be
directed towards identification and modification of those procedures in
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the current guidelines which can produce inconclusive or spurious test
results. New (or modifications to old) tests must be developed which
account for the variations in exposure experienced in natural systems.
3. Coordination of Comparative Toxicology Research. Coordination is needed
in the EPA research efforts in comparative toxicology, especially in the
context of comparing chemical effects across terrestrial wildlife species.
The applicability of laboratory test data to actual field conditions and
to wild species must be considered to determine whether or not test
results on selected laboratory species can be used to estimate toxicity in
other species.
4. Field Validation of Laboratory Test Protocols. Toxicity tests must be
performed in actual field conditions to determine the effects of toxics on
species in the field. The field validation effort provides direct compar-
ison of chemical toxicity between laboratory tests and in actual field
conditions. Coordination of both laboratory and field test protocols
provides synchronization of test data which allows direct comparison of
toxic effects between tests. If the laboratory and field test protocols
(and the subsequent data produced) are properly formatted, the test
results can be compared directly.
5. Development of a Wildlife Toxicology Data Base. An important function in
the Terrestrial Wildlife Toxicology Research Team is to coordinate and
develop a comparative data base using available research and test data
relating toxicity of chemicals to terrestrial wildlife. These data
include results from different types of tests, effects on different
species, relationships between single-species and multiple-species
toxicity tests, microcosms, and natural ecosystem (field) tests. This
data base serves two important functions; (a) it provides a central
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information storage and retrieval network which can be used to evaluate
existing test results and to design new experimental protocols; and (b)
the data base provides a means of assessing toxicity data quickly and
efficiently for evaluations of structure-activity relationships (SAR) and
common mechanisms of action for selected classes of chemicals. The data
base will become the central repository for all EPA terrestrial wildlife
toxicology data and will be a valuable tool in the assessment of effects
of specific chemicals and classes of chemicals. In addition, the data
base can be used to determine inter- and intra-species responses to
selected chemicals, and provides a valuable tool in the development of
computer simulation models of toxic effects of chemicals on physiological
systems for risk assessment.
Development of Sublethal Tests. Toxicity tests are conducted in-house to
validate acute and chronic effects of chemicals on terrestrial wildlife.
Research in sublethal effects include relationships between body burden of
chemical and observed physiological/behavioral effects. There are several
techniques which can be used to identify and measure sublethal effects,
especially effects on metabolic and enzymatic systems, and effects on the
immunological system. Another important indicator of sublethal toxicity
is evidenced in the decreased ability to survive stress and stressful
environments. This is of particular importance to the continuation of any
species, since individual organisms have been shown to be more susceptible
to disease or other stresses following their exposure to environmental
contaminants. Alteration in basal metabolic rates for wildlife exposed to
chemicals has also been demonstrated. Under additional stressful condi-
tions, such reduced rates may result in altered behavioral responses or
diminished energy available for food collection, and may lower the overall
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potential for survival. If increased chemical sensitivity is extrapolated
to the population, the impact of stress on a system becomes very impor-
tant. Many simple assays which are good indicators of stress or trauma
can be determined from blood or tissue samples. Alterations in stress and
other physiological responses to exposure of toxic substances is an
important element of the research program of the Wildlife Toxicology
Research Team at CERL.
The laboratory thus fulfills a functional role, but at the same time
provides the basis for in-depth research into the complex mechanisms and
interactions of toxic substances.
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ACCOMPLISHMENTS
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WILDLIFE TOXICOLOGY ACCOMPLISHMENTS
Developed a multiple regression technique for evaluating the response of
birds to treated and untreated food in dietary choice tests. Birds could
detect the presence of many chemicals on food at sublethal levels, but
their response was affected by the abundance and availability of food
choices. Results will be used to determine the potential for birds to
modify their exposure to chemicals by changing foraging patterns.
Demonstrated that short-term, dietary exposure (400 ppm) to methyl
parathion can affect incubation behavior (nest attentiveness and abandon-
ment) and duckling production in mallards. Similar effects on nest
abandonment and reduced hatchling production have also been observed in
the field. This test demonstrated the types of pesticide-related effects
which are not now considered in the avian reproduction test.
Developed a technique for evaluating egg shell quality which is more
sensitive than the current egg shell thickness measurement. A test using
bobwhites and mallards demonstrated a more sensitive response to shell
breakage force than to thickness.
Determined that the accumulation of hexachlorobenzene residue in bobwhite
egg yolks plateaus at approximately five times the dietary concentration
(in ppm).
Determined that the toxic effects of organophosphate chemicals to bobwhite
are more pronounced at lowered environmental temperatures. Decreasing the
test temperature from 35C to 28C decreases the LC50 of chlorpyrifos from
590 ppm to 280 ppm. This increased sensitivity to chemical at low temper-
atures indicates that exposure to toxic chemicals in the field (where
environmental temperatures are often quite low) will produce more mortal-
ity than indicated in standard laboratory tests.
Determined that the LC50 of chlorpyrifos to mallard ducks is more sensi-
tive to size (weight) than to age. Calculated LC50 values varied by an
order of magnitude in birds of the same age, but of different weights
(> 100 g difference). This variation in size of birds can result from
shipping or dietary differences. This illustrates the importance of
selecting test birds of specific size and age. Current OTS test guide-
lines allow birds from 10-17 days of age. This range can encompass birds
with over 250 grams variation in weight.
Demonstrated the importance of longer dietary tests (longer than 5 days)
for the chemicals which cause delayed toxicity and those which produce
food avoidance. Chemicals which cause delayed toxicity (e.g., anti-
coagulants) often show little or no toxicity within the time frame of
standard test guidelines. Many chemicals produce food avoidance and some
animals can survive the 5 days of dietary exposure (LC50) by not eating.
The toxicity of the test chemical is thus not appropriately measured.
Longer-term tests alleviate these confounding problems.
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Determine that the accumulation of hexachlorobenzene residue in egg yolks
of bobwhite hens fed HCB plateaus to approximately five times the dietary
concentration within 28 days. Accumulation of HCB in eggs is in addition
to the accumulation of chemical in liver, fat, and other body tissues of
the hens. This relationship will be used to evaluate the capability of
estimating hen dietary uptake from egg residues.
The reproductive impact of an organophosphate on nesting mallards was
evaluated in both a standard laboratory test and a natural (pond) setting.
In contrast to the caged birds in the laboratory setting, the birds on the
ponds were able to build nests and incubate eggs. These conditions
provided a more realistic test scenario to evaluate the impact of chemi-
cals on "natural" reproductive processes. Treated birds in both environ-
ments exhibited reproductive impairments typical of OP exposure. Similar
results in both environments illustrates the potential utility of pond
tests as a realistic link between laboratory testing and field conditions.
Dr. John Emlen, population ecologist at Oregon State University, was
selected as a Visiting Distinguished Scientist to spend one year doing
population modeling in the Wildlife Toxicology Research Group. He is
active in determination of Risk Assessment Models for EPA and is currently
leading the CERL Risk Assessment Team.
Dr. Robert Ringer, distinguished Avian Physiologist from Michigan State
University, spent one year in the Wildlife Toxicology Research Group
conducting avian researches. Dr. Ringer was at CERL as a part of a
cooperative agreement and continues collaborative research with the
wildlife toxicology scientists at CERL.
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CURRENT RESEARCH
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WILDLIFE TOXICOLOGY CURRENT RESEARCH
I. EXPOSURE
A. How does an animal's behavioral response to chemicals modify its
subsequent exposure?
1. Dietary discrimination tests (R. Bennett)
a. Measurement of detectability
b. Role of choices in discrimination
2. Response of valley quail to contaminated habitats (Crawford)
3. Response of sage grouse to contaminated habitats (Hal ford, Blus)
B. How is chemical exposure modified through the food chain?
1. Secondary exposure to mink and ferrets (Ringer)
2. Dioxin in large animals (Texas A&M)
C. What are the relative sensitivities of various routes of exposure?
1. Oral (food and/or water) vs. dermal toxicity (Williams/Cairns)
2. Inhalation vs. oral toxicity (Williams/Gile)
D. What period in the nesting cycle is most sensitive to chemical
exposure? Effects on reproductive success.
1. Time of OP exposure related to nest success (R. Bennett)
E. How does the duration of dietary chemical exposure affect toxicity
and sublethal endpoints?
1. Five vs. twenty-eight day LC50 tests (Ringer)
F. How do bioassay measures of exposure relate to chemical availability
in the environment?
1. Acetylcholinesterase assays from alfalfa and potatoe field
studies (Blus, Halford)
2. Acetylcholinesterase assays from nesting habitat studies
(Kendall)
3. Acetylcholinesterase assays from laboratory exposure (Maguire,
Williams)
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II. EFFECTS
A. What biologically relevant endpoints could be refined or revised for
better determination of chemical effects on reproduction?
1. What parameters or methods of the current reproductive tests
need to be evaluated?
a. HCB reproductive test, bobwhite (Shiroyama)
2. Is the current test the most realistic treatment schedule for
OPs and carbamates (timing, duration, and route of exposure)?
a. Effects of short-term OP exposure on nest abandonment and
success (R. Bennett)
3. Do AChE inhibitors affect levels of reproductive hormones?
a. Prolactin/OP interactions (R. Bennett, J. Bennett)
4. What is the relationship between chemical residues in the egg,
dose, and duration of exposure?
a. HCB residue movement into eggs (Williams)
5. What is the best measure of eggshell quality?
a. Shell thickness vs. breaking strength (J. Bennett)
6. Do laboratory reproductive tests predict field effects?
a. Field reproductive effects with waterfowl (Kendall)
7. What are the best techniques for measuring reproductive success
in passerines?
a. Nest box technique development (Kendall, Blus, and Halford)
b. Red-winged blackbirds (Meyers)
III. HAZARD
A. What factors affect dietary toxicity?
1. How do alternative dietary choices affect response?
a. Discrimination tests (R. Bennett)
2. How does duration of the test and age of bird affect response?
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a. Five vs. twenty-eight day dietary test (Ringer)
3. How do environmental stresses affect response?
a. Temperature effects on LC50/LD50 (Maguire, Williams)
4. How does previous chemical exposure affect response?
a. Pre-exposure experiments (Williams, Bennett)
5. What is the relationship between amount of chemical ingested,
consumption, food passage rate, and response?
a. Dose tests (Bennett)
B. Population Level Effects
1. What methods are best for measuring chemical exposure and
population effects?
a. Methods for sage grouse in Idaho (Blus, Hal ford)
b. Methods for valley quail (Crawford)
2. Can complex population effects be predicted with models of
population dynamics?
a. Valley quail methodology/lab interactions (Emlen)
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FACILITIES
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FACILITIES:
The Wildlife Toxicology Research Team conducts investigations using the
mallard duck and bobwhite quail as the test species in accord with the guidance
suggested in the FIFRA Guidelines for acute toxicity test protocols (Section
163.70-1). It is necessary to provide housing and test areas for animals used
in these investigations according to good labortory practices (GLP) and the
general protocol guidelines of the FIFRA. In addition, animal housing will
conform to Public Laws 89-544, 91-579, and 94-279 (Animal Welfare Acts of 1966,
1970, and 1976, respectively) which requires strict adherence to general
procedures for the well-being of test animals.
The husbandry support and test cages for the experimental animals are
situated in a sophisticated test facility of approximately 4,000 square feet
(page 18). This facility is adequate for each of the approved toxicity labora-
tory tests such as the LD50, LC50, and other variations of these tests. In
support of these investigations, a laboratory preparation area, a food prepara-
tion area, a residue analysis laboratory, and a data anaysis area are also
located within the test area. Large-pen (field simulation) testing is con-
ducted at Western Fish Toxicology Station (WFTS, page 19), utilizing the
existing ponds to provide a more natural environment for testing. Investiga-
tions at this site are conducted in conjunction with tests using similar
protocols in the laboratory. The combination of laboratory tests and large-pen
simulations provide an important link in verifying the relationship between
laboratory data and field data.
The test area at the WFTS site is large enough to support additional
laboratory or test space as required. A block building (approximately 3.0 m x
3.7 m) which is located on the test area is partially equipped for laboratory
testing.
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PERSONNEL
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WILDLIFE TOXICOLOGY TEAM
EPA Personnel
Team Leader
Bill A. Williams
Research Biologist
Richard S. Bennett
Research Ecologist
Michael A. Cairns
Biologist
Stephen E. Dominguez
Biologist
Anne Fairbrother
Research Biologist
Jay D. Gile
Toxicologist
Tamotsu Shiroyama
Microbiologist
Simon Wray
Student (SIS)
NORTHROP SERVICES, INC.
PERSONNEL
Section Supervisor
David W. Schmedding
Chemist
Jewel Bennett
Scientist
Ray Bentley
Associate Scientist
Douglas Bonfoey
Chemist
Melissa A. Fix
Laboratory Technician
S. Mark Meyers
Scientist
Susan M. Schiller
Laboratory Analyst
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RESUMES
EPA
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EDUCATION
EXPERIENCE
Richard S. Bennett
Research Ecologlst -- Wildlife
Ph.D. Iowa State University, Animal Ecology/Environmental 1983
Toxicology
M.S. -- Michigan State University, Wildlife Ecology
B.S. -- Michigan State University, Zoology
1979
1976
1985-Present
Research Ecologist
Corvallis Environmental Research Laboratory
Relationships between patterns of chemical exposure and
effects; effects of sublethal chemical exposure on avian
nesting success.
Research Associate, National Research Council 1983-1985
Corvallis Environmental Research Laboratory
Avian dietary toxicity testing; ability of avian species to
detect pesticide-treated food.
Research Associate
Iowa Cooperative Wildlife Research Unit
Snow goose winter and spring migratory ecology.
1983
1979-1982
Graduate Research Assistant
Iowa State University
Effects of synthetic pyrethroids on field populations of
invertebrates and small mammals.
Graduate Assistant 1977-1979
Michigan State University
Response of ring-necked pheasants to pesticide-treated
preferred foods; land owner surveys.
Research Technician 1976
Michigan State University
Upland game bird population ecology and habitat management;
census technique development.
PROFESSIONAL
AFFILIATIONS Society of Environmental Toxicology and Chemistry
The Wildlife Society
The American Ornithologists' Union
American Society of Mammalogy
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Michael A. Cairns
Biologist
EDUCATION M.S. Oregon State University, Fisheries 1980
B.S. -- San Jose State University, Zoology (Wildlife) 1974
EXPERIENCE Biologist 1985-Present
Corvallis Environmental Research Laboratory
Wildlife Toxicology Group
Water route of exposure of waterfowl to toxicants; age and
size effects on avian sensitivity to pesticides.
Biologist 1981-1985
Corvallis Environmental Research Laboratory
Toxic Sediments Group
Freshwater fish and invertebrate sediment toxicity test
development; bioassessment of acute and chronic toxicity of
contaminated sediment.
Biologist, Lab Technician 1976-1981
Corvallis Environmental Research Laboratory
Freshwater Division
Freshwater toxicity testing; developed toxicity test based
on fish respiration measurements; methods development;
sublethal stress/fish health.
Biological Aide 1974-1976
Corvallis Environmental Research Laboratory
National Eutrophication Survey
Freshwater algal assays; nutrient limitation studies.
Widlife Conservation Aide 1974
Oregon Department of Fish and Wildlife
Research Division
Conducted angler interviews, creel censuses, ear counts to
obtain data on trout and steel head movement and catch.
PROFESSIONAL
AFFILIATIONS The Wildlife Society
American Fisheries Society
North American Benthological Society
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Stephen E. Dominguez
Biologist
EDUCATION B.S. Oregon State University, Microbiology 1972
EXPERIENCE Biologist 1986-Present
Corvallis Environmental Research Laboratory
Effects of sublethal chemical exposure on avian physiology.
Aquatic Biologist 1980-1986
Corvallis Environmental Research Laboratory
Lethality of heavy metals, organic chemicals, and hypoxia to
freshwater fish and invertebrates.
Biologist 1972-1979
Corvallis Environmental Research Laboratory
Effects of sedimentation on biotic communities and coho
salmon production in streams; assessment of the extent of
culturally accelerated eutrophication of lakes within the 48
contiguous states (EPA's National Eutrophication Survey).
Physical Science Aide 1969-1972
Corvallis Environmental Research Laboratory
Limnological research related to causes and effecs of
eutrophication.
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EDUCATION
EXPERIENCE
Anne Fair-brother
Research Biologist
Ph.D. University of Wisconsin, Madison, Veterinary
Science/Zoology
M.S. University of Wisconsin, Madison, Veterinary
Science/Zoology
1985
1982
D.V.M. University of California, Davis 1980
B.S. University of California, Davis, Wildlife Ecology 1976
Research Biologist 1986-Present
Corvallis Environmental Research Laboratory
Determine effects of sublethal chemical exposure on avian
physiological parameters, such as blood chemistries and
immune competence.
Graduate Research Assistant 1980-1985
University of Wisconsin, Madison
Determined interactive effects of pathogens, toxicants, and
temperature on immune function and bioenergetics of deer
mice.
Research Assistant
University of California, Davis
Ectoparasite reinfestation rates on deer mice.
cycles of numbers of deer mouse ectoparasites.
1978-1980
Annual
1978-1979
Teaching Assistant
University of California, Davis
Department of Wildlife and Fisheries Biology
Mammalogy laboratory, teaching field identification of North
American mammals. Summer field course for wildlife students
to learn field techniques.
Intern
U.S. Forest Service, Inyo National Forest
Conducted a survey of native fauna in White and Inyo
Mountains, California.
PROFESSIONAL
AFFILIATIONS American Society of Mammalogists
American Veterinary Medical Association
Association of Wildlife Veterinarians
Wildlife Disease Association
1974
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Jay D. Gile
Toxicologist
EDUCATION Graduate Study Oregon State University 1983
Phys i ology/Toxi cology
M.S. -- Oregon State University, Marine Biology 1971
B.S. Central Washington State University, Biology 1969
EXPERIENCE Toxicologist 1982-Present
Corvallis Environmental Research Laboratory
Toxics/Pesticides Branch
Development of mallard research facility. Conduct of avian
toxicity tests in support of FIFRA and TSCA guidelines.
Project officer for extramural wildlife research projects.
CERL Radiation Safety Officer.
Research Biologist 1973-1982
Corvallis Environmental Research Laboratory
Development of Terrestrial Microcosm Chamber (TMC) for
evaluation of chemical transport and fate. Project Officer
for extramural chemical transport and fate projects.
Research Biologist 1972-1973
Research Triangle Park (EPA)
Synergistic effects of radiation and heavy metals on mammal-
ian reproduction.
Research Biologist 1971-1972
Twinbrook Research Laboratory (EPA)
Synergistic effects of radiation and heavy metals on the
hematopoidic system in rodents.
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Tamotsu Shiroyama
Microbiologist
EDUCATION B.S. ~ Oregon State University, Microbiology 1969
EXPERIENCE Microbiologist 1984-Present
Corvallis Environmental Research Laboratory
Toxics/Pesticide Branch
Wildlife Toxicology Program
Oversee and coordinate all the activities in the avian
(bobwhite) facility. Also provide assistance to ongoing
research projects.
Microbiologist 1982-1984
Freshwater Division
Corvallis Environmental Research Laboratory
Determined the LC50 responses of selected freshwater phyto-
planktons to organic toxicants that were used in ongoing
field and laboratory microcosm studies.
1980-1982
Malheur National Wildlife Refuge: Studied the bioavail-
ability of nitrogen and phosphorus to phytoplankton.
1978-1980
Cooperative study on "Textile Plant Wastewater Toxics
Study." Provided toxicological data on freshwater algae
from wastewater samples collected from selected textile
plants.
1969-1977
Assisted in the development and refinement of the
Algal Assay Procedure: Bottle Test.
1974-1976
Cooperative study with Western Washington State College on
biological field-laboratory correlation of primary producers.
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EDUCATION
EXPERIENCE
Bill A. Williams
Research Biologist
Ph.D. University of Illinois, Neurophysiology
M.S. University of Illinois, Physiology
B.A. University of California, Physiology
1968
1965
1963
1982-Present
Research Biologist
Corvallis Environmental Research Laboratory
Toxics/Pesticides Branch
Serves as project leader of Wildlife Toxicology Group at
CERL.
Research Scientist 1977-1982
NASA-Ames Research Center
Biosystems Division
Project Scientist in charge of biomedical experiments for
Spacelab/Space Shuttle program. Provided scientific support
for zero-gravity biomedical experiments for NASA.
Research Physiologist 1972-1977
NASA-Ames Research Center
Environmental Research Center
Environmental Control Research Branch
Project Scientist for NASA ground-based biomedical experi-
ments in temperature regulation and metabolism. Principle
investigator for metabolic experiments for Spacelab. Served
as crewman for Spacelab missions for NASA.
Research Physiologist 1970-1972
NASA-Ames Research Center
Man-Machine Integration Branch
Research Scientist specializing in determination of chemical
interactions and control of temperature metabolism and
hibernation in mammals. Designed, tested, and evaluated
liquidcooling techniques for astronaut space suits.
National Academy of Science 1969-1970
Post-Doctoral Fellow
NASA-Ames Research Center
Instructor in Endocrinology 1965-1966
University of Illinois
Instructor of Physiology 1965
University of Illinois
PROFESSIONAL
AFFILIATIONS Society of Environmental Toxicology and Chemistry
Sigma Xi
American Physiological Society
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RESUMES
Visiting Scientists
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John Merritt Emlen
Senior Scientist
EDUCATION Ph.D. University of Washington 1966
B.A. University of Wisconsin (Madison) 1961
Haverford College 1956-1958
EXPERIENCE Distinguished Visiting Senior Scientist 1985-Present
Corvallis Environmental Research Laboratory
Full Professor (Courtesy) 1984-Present
Oregon State University
The evolution of patterns of population density dependence
and interspecific interactions; development of models and
methods for determining density-dependence and species
interactions in the field; modeling of impact of toxins on
spatial, dietary behavior.
Associate Professor 1971-1984
Indiana University
Trustee, Environmental Defense Fund (EOF). Laboratory and
theoretical research on foraging and dietary behavior,
microhabitat associations, reproductive and territorial
behavior, population genetics, and the evolution of ecolog-
ical characteristics; population consequences of life
history evolution.
Assistant Professor 1966-1968
State University of New York, Stony Brook
32
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EDUCATION
EXPERIENCE
Robert K. Ringer
Professor
Ph.D. Rutgers University, Physiology and Biochemistry 1955
M.S. Rutgers University, Agriculture
B.S. Rutgers University, Poultry Science
1952
1950
1985-Present
Professor
Department of Animal Science
Department of Physiology
Michigan State University
Coordinator of Toxicology, Pesticide Research Center
Research Involvement: Effect of polybrominated biphenyl and
polychlorinated biphenyls on avian and mammalian species
(mink and ferret), Refinement and development of wildlife
toxicology protocols. Effect of various toxicants on
genesis of lymphoblasts.
Professor 1966-1978
Department of Poultry Science
Department of Physiology
Michigan State University
Research Involvement: Development of protocols for wildlife
toxicology influence of polychlorinated and polybrominated
biphenyls on avian and mammalian species; toxicology of
methylmercury in mink; cardiovascular and reproductive
physiology of domestic avian species.
Professor
1964-1966
Department of Poultry Science, Michigan State University
Associate Professor 1961-1964
Department of Poultry Science, Michigan State University
Assistant Professor 1957-1961
Department of Poultry Science, Michigan State University
Assistant Professor
Department of Poultry Science, Rutgers University
PROFESSIONAL
AFFILIATIONS Society of Environmental Toxicology and Chemistry
The Poultry Science Association
American Physiological Society
American Association of Avian Pathologists
World's Poultry Science Association
1955-1957
33
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RESUMES
NSI
34
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EDUCATION
EXPERIENCE
Jewel Bennett
Scientist
M.S. Iowa State University, Wildlife Biology
B.S. University of WisconsinMadison, Biology
1981
1978
1983-Present
Scientist
Northrop Services, Inc.
Conducting research on the effects of environmental contam-
inants on avian reproduction; perform cholinesterase assays
on avian tissue.
Wildlife Extension Writer 1982
Iowa State University
Produced extension bulletins and magazine articles about
Iowa's threatened and endangered wildlife.
Wildlife Research Assistant 1978-1981
Iowa State University
Conducted master's degree research on the seasonal range
ecology of bison living in the mixed grass prairie at Wind
Cave National Park, South Dakota.
Biological Aide 1979-1980
National Veterinary Services Lab, USDA
Produced and conducted quality control tests on diagnostic
reagents.
Wildlife Technician 1976-1978
University of Wisconsin
Field assistant on a white-tail deer study in Wisconsin and
a songbird censusing study in Montana.
PROFESSIONAL
AFFILIATIONS Sigma Xi
The Wildlife Society
35
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Ray Bentley
Associate Scientist
EDUCATION B.S. ~ Oregon State University, Wildlife Science 1980
EXPERIENCE Associate Scientist 1984-Present
Northrop Services, Inc.
Assists in experiments on bobwhite involved with environ-
mental hazards risk assessment. Conducts LCsg testing and
in charge of test facility preparation and maintenance.
Responsible for quail colony production,dispersal, and
maintenance. Assists in data analysis, necropsy, and tissue
preparation for subsequent residue analysis. Maintains
colony production records and QA records on computer disk.
Biological Aide 1983-1984
Oregon Department of Fish and Wildlife
Assisted in data collection and analysis for Tillamook field
office. Conducted census and demographic data on coastal
black-tailed deer herds. Conducted district 1984 waterfowl
inventory. Conducted Creek survey and scale sampling of
district river systems. Participated as recorder during
public elk workshop sessions.
Culturist 1983
Anadromous, Inc.
Assisted in general fish hatchery duties related to salmon
production including spawning, scale sampling, vaccination,
disease treatment, loading smelts for release, measurement
and control of 02 levels and H20 flow/temp.
Biologist Aide (Volunteer) 1983
Oregon Department of Fish and Wildlife
Assisted in marking, attachment of transmitters, and subse-
quent monitoring of spring released pheasants.
36
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Douglas B. Bonfoey
Chemist
EDUCATION M.S. Michigan State University, Chemistry 1973
B.S. -- Western Michigan University, Chemistry 1967
EXPERIENCE Analytical Chemist 1983-Present
Corvallis Environmental Research Laboratory
Analysis of avian tissue for trace amounts of pesticides.
Methods development utilizing solvent extraction, sample
cleanup, and capillary column gas chromatography. Computer-
ized data handling and data analysis.
Analytical Chemist 1981-1983
University of Utah Research Institute
Analysis of water and soil samples for trace amounts of
pesticides and PCBs. Analysis of hazardous waste samples.
Research Chemist 1973-1976
The Dow Chemical Company
Process research: Experimentally determined the optimum
reaction conditions and purification methods for a chemical
manufacturing process. New product research: synthesized
and evaluated experimental formulations.
Analytical Chemist 1968-1973
The Dow Chemical Company
Provided analytical chemistry support to technical service
groups. Analyzed chemical formulations using infrared
spectroscopy, gas chromatography, thin layer chromatography,
UV-VIS and wet methods.
37
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Melissa A. Fix
Laboratory Technician
EDUCATION A.S. Linn-Benton Community College, Liberal Arts 1983
A.S. Linn-Benton Community College, General Science 1981
Certificate -- Walter Reed Research Institute, Animal Care
Specialist 1981
EXPERIENCE Laboratory Technician 1985-Present
Northrop Services, Inc.
Analysis of avian tissue, egg, and fecal material with a gas
chromatograph. Assisting scientist in data and sample
collecting, necropsies, routine animal care, data charting
and entry. Collect mallard blood and run PCV and protein
analysis. Run analysis of blood and tissue on SBA 300.
Biological Aide 1985
Corvallis Environmental Research Laboratory
Analysis of avian egg samples, routine animal care, assist-
ing scientist in measuring egg shell thickness, literature
search, data entry and editing.
Biological Aide 1984-1985
Western Fish Toxicology Station
Routine care and analysis of water microcosms measuring
frequency of microorganisms within microcosms. Collecting
various pond waters. Data entry and editing.
Animal Care Specialist 1981-1983
U.S. Army
Acting non-commission officer of biomedical facility.
Assisting in gastro-intestinal, cardiac-pulmonary, and
reproductive surgeries. Anesthetic and x-ray operator, pre
and post operative animal care, necropsy, tissue collection
and analysis. Narcotic drug control officer.
38
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EDUCATION
EXPERIENCE
S. Mark Meyers
Scientist (Wildlife Biologist)
M.S. -- Oregon State University, Wildlife Science
B.S. California State University, Biological Sciences
1983
1978
PROFESSIONAL
AFFILIATIONS
Scientist (Wildlife Biologist) 1982-Present
Northrop Services, Inc.
Conduct and support avian toxicological research for the
Wildlife Toxicology Program at CERL. Development of a
terrestrial toxicology data base (TERRE-TOX) for EPA, CERL.
Graduate Research Assistant 1980-1982
Oregon State University
Conducted field studies on survival, reproduction, and
habitat use of the ring-neck pheasant. Completed Masters
thesis and received M.S. degree from Oregon State University.
Biological Technician 1978-1979
Bureau of Land Management
Recorded and evaluated physical and biotic stream character-
istics. Determined condition of riparian zones. Evaluated
human impacts on stream ecosystems. Classified, rated, and
inventoried public lands to determine suitability of habitat
for wildlife. Evaluated human impacts on wildlife habitats.
Biological Intern 1977-1978
California Department of Fish and Wildlife
Assisted in the food habits study of the San Joaquin kit fox
and bobcat. Responsible for updating the reference
herbarium for the Food Habits Laboratory.
The Wildlife Society
American Ornithologists' Union
Northwest Scientific Association
39
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EDUCATION
EXPERIENCE
Susan M. Schiller
Laboratory Analyst
B.A. ~ Oregon State University, German
1979
1985-Present
PROFESSIONAL
AFFILIATIONS
Laboratory Analyst
Northrop Services, Inc.
In support of the Wildlife Toxicology Group, EPA. Performs
cholinesterase assays on avian tissue using Perkin-Elmer
spectrophotometer or Gilford SBA 300. Provide general
biochemistry and chemistry support. Assist in necropsies
and collect data in support of eggshell quality research.
Laboratory Analyst 1982-1985
Northrop Services, Inc.
In support of the development of a wildlife toxicology
database (TERRE-TOX). Major duties include the acquisition
and archiving of literature, quality assurance, information
dissemination, and data entry.
Library Technician 1979-1981
Northrop Services, Inc.
In support of the Corvallis Environmental Research Labora-
tory, EPA. Responsible for literature acquisition, search-
ing, recordkeeping, current awareness.
Library Aide 1976-1977
Oregon State University
Wililam Jasper Kerr Library
In support of Business Records Department, processed contin-
uations, typed vouchers, ordered missing journal issues,
general office work.
Phi Kappa Phi
40
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David W. Schmedding
Analytical Chemist
EDUCATION B.A. ~ California State University, Chemistry 1969
EXPERIENCE Analytical Chemist 1983-Present
Northrop Services, Inc.
Presently involved in analytical methods development and
feed preparation and treatment level verification for
toxicants used in IC^Q, LDsQ, induced tolerance, and repro-
ductive tests both at CERL and WFTS.
Research Chemist 1970-1983
Department of Agricultural Chemistry
Oregon State University
With a variety of pesticide and industrial chemical research
projects. Areas of research include nuclear magnetic
resonance studies on the binding of toxicants to biomem-
branes, physical property determinations for chemicals of
environmental concern, determination of adsorption isotherms
for various PCBs and pesticides on sand, soil, and clay
surfaces, and correlations of physical properties with
bioaccumulation. Developed a method to measure the evapora-
tive loss rate of aerial agricultural sprays as modified by
various adjuvants. Work with toxicants led to an involve-
ment in a U.S.-USSR joint research project on substituted
aniline compounds and their mammalian toxicity as related to
physical constants. A cooperative research project with the
University of Miami School of Medicine led to the solution
of the delayed neurotoxicological symptomology and resultant
deaths of victims of accidental ingestions of high partition
coefficient organophosphate insecticides. The interest in
organophosphates led to the development of a Knudsen effusion
apparatus for determining vapor pressures of "non-volatile"
compounds of interest for use in environmental models on
bioaccumulation.
Research Chemist 1969-1970
Frideh Research
Where work involved computerized formulation and preselec-
tion of fluorescent inks, intrinsic viscosity, UV quenching,
and volatility determinations with subsequent plant produc-
tion and screening.
41
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APPENDIX A. EXPERIMENTAL SYNOPSES
Inhouse
Completed
42
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Title: Effects of Adult Mallard Age on Avian Reproductive Tests
Introduction; Avian reproduction tests are included in the hazard evaluation
process under the Toxic Substances Control Act (TSCA). Recommended test
species are the bobwhite (Cplinus virglnianus) and the mallard (Anas
platyrhynchos). TSCA guidelines specify several parameters for selection of
test birds:(1) the test birds should be pen reared either in the test labora-
tory or purchased from a commercial breeder; (2) the source should have known
breeding history; (3) the sources of the test birds should be outbred with
genetically wild stock to maintain the genetic heterogeneity of wild birds; (4)
the test birds should be phenotypically indistinguishable from wild birds. The
guidelines further specify that the test birds be approaching their first
breeding season and at least 7 months old at the beginning of the test.~After
these guidelines had been implemented, questions arose on the potential
variability of test results that might result from using young (7 month) birds.
Methods: Two age groups of mallards were selected at the age extremes of birds
approaching their first breeding season. Birds 7 months old at test initiation
represented the youngest birds that EPA would accept in a reproduction test.
Birds 11 months old were used to represent the oldest birds which might be used
in this type of study. The organophosphate insecticide, chlorpyrifos was
selected as the test chemical because of its widespread use on a variety of
crops and to compare with previous mallard reproductive tests conducted at the
Corvallis Environmental Research Laboratory (CERL). Parameters measured
included adult food consumption and body weight, brain acetylcholinesterase,
onset of laying, egg production, fertility, hatchability, shell thickness, egg
weight, and duckling weight and survival. Pairs from each age group were
randomly assigned to reproductive pens housed in a plastic covered greenhouse.
The birds were allowed to acclimate for 2 weeks, then placed on the test diet
(80 ppm chlorpyrifos or corn oil control mixed with feed). The birds were
weighed at 2-week intervals until the onset of laying and then at the end of
the study (12 weeks after laying began). Eggs were collected daily and set
weekly. Upon hatching each duckling received a wing tag providing individual
identity and pen or parent identity. The ducklings were weighed on day zero
and maintained.
Status: Duckling day 14 weight was the only measured parameter which appeared
to be effected by adult age. However, three of the 7-month old hens produced
atypically colored ducklings indicating a genotype different from the typical
mallard. These ducklings were larger at day 14 than the other ducklings. It
is possible that this phenotypical difference, although not evident in their
offsprings from all 7-month old hens, was not just restricted to the three pens
identified by the atypically colored ducklings.
Since 11 of the 12 parameters tested were not affected by age, we conclude
that breeder age was probably not a factor in this test.
This project was completed 12/85, and the manuscript has been accepted by
Archives of Environmental Contamination and Toxicology.
Investigators: J. Gile, M. Meyers
43
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Title: Chlorpyrifos Toxicity to Bobwhite at Lowered Test Temperatures
Introduction: It is necessary to determine the effect of environmental
stresses on susceptibility to chemicals in avians.Juvenile "homeothermic"
birds with developing thermoregularity capabilities may be adversely affected
by environmental temperatures which are thermoneutral to adults. Such
responses may be exacerbated by pesticide exposure. To evaluate the inter-
active effects of temperture stress and organophosphate exposure, 14-day-old
bobwhite quail were fed chlorpyrifos at four environment (test) temperatures
(35, 32.5, 30, nad 27.5°C). A standard LC50 (dietary) test design was followed
(test birds were fed dietary chlorpyrifos for five days, followed by three days
of clean feed). Results indicate that exposure of the test birds to lowered
environmental temperature during the dietary exposure to this organophosphate
produced LC50 values significantly lower than control values (35°C test temper-
ature). A significant (45%) drop in the LC50 occurred between 35°C (optimal
incubator temperature) and 27.5°C. Brain cholinesterase (ChE) activity
measured in brains of non-surviving quail was significantly greater at low
temperatures. This difference was as great as 70% between 35 and 27.5°C.
Juveniel bobwhite exposed to chlorpyrifos were significantly more susceptible
to low test temperatures as evidenced by earlier mortality in the treated
birds.
Methods and Materials: Juvenile bobwhite were exposed to dietary concentra-
tions of chlorpyrifos which bracketed the LC50 value for the 14-day-old
bobwhite at four test (environmental) temperatures. The control birds were
housed at 35°C (optimal brooder temperature), while similar groups of birds
were tested at temperatures of 27.5, 30, and 32.5°C. Values for the LC50 were
calculated using the probit method. Results were reported as absolute LC50
values and as relative to control values.
Status: The investigations completed were designed to evaluate the sensitivity
of juvenile bobwhite in order to follow the standard test protocol. These data
are representative of the effects of environmental cold stress on young
bobwhite which are exposed to chemical. Additional testing is planned to
evaluate the effects of cold stress on the toxicity of several organophosphate
chemicals in adult bobwhite. Two manuscripts are in review for submission to
Environmental Toxicology and Chemistry.
Investigators: C. Maguire, B. Williams
44
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Title: Mallard Reproductive Testing in a Pond Environment: A Preliminary
Study
Introduction: The Toxic Substance Control Act (TSCA) and the Federal Insecti-
cide, Fungicide, and Rodenticide Act (FIFRA) require standard lethality studies
(i.e., median lethal dose, 1059, or median lethal concentration,
be performed as part of the registration process for new chemicals. In addi-
tion, TSCA and FIFRA have provisions which permit the U.S. Environmental
Protection Agency (EPA) to require in-depth studies into other potential
environmental hazards. One such recommended study is a reproductive test
conducted with bobwhite quail (Colinus virginianus) and mallard duck (Anas
platyrhynchos). Typically these studies are conducted in indoor laboratory
situations. Th i s is desirable for both environmental control and convenience,
but there is little evidence that these studies provide information comparable
to exposure situations in the wild. A 2-year study was initiated to determine
the feasibility of using outdoor pond enclosures for reproductive testing on
mallards.
Methods: A 2-year reproductive study was conducted on mallard ducks using 3-5
0.1 ha fenced ponds. Ducks received feed treated with 0, 8, and 80 ppm chlor-
pyrifos for 18 weeks in 1984 and 20 weeks in 1985. Opportunity was given to
each hen to nest, incubate, and hatch out broods with minimal disturbance from
investigators. The study was terminated for both years when the last clutch on
each pond hatched and ducklings were 7 days old.
Results: No negative effects were observed for mallards receiving 8 ppm
chlorpyrifos in their diet. Birds receiving 80 ppm chlorpyrifos hatched
significantly (p < 0.05) fewer ducklings per successful nest (5.8) than
controls (10.2). None of the ducklings on treatment ponds survived to 7 days.
Control birds produced 8.4 ducklings per successful nest surviving to 7 days or
longer. Birds in the 80 ppm treatment group consumed less feed than did
controls (p < 0.01). Weight loss from reduced feed consumption did not occur
to the extent we expected, indicating that birds supplemented their diets with
natural foods found in and around the ponds. In spite of relatively low
treated feed consumption, brain acetylcholinesterase was significantly (p <
0.05) depressed (57% of controls) for 80 ppm treated birds. Studies on indoor
penned mallards fed 80 ppm chlorpyrifos in their diet also resulted in acetyl-
cholinesterase depression to the same extent, but at much higher feed consump-
tion levels. The study described in this paper demonstrated the potential of
using outdoor pond enclosures to evaluate chemical effects on food consumption,
brain acetylcholinesterase, and reproduction in mallards. Furthermore, repro-
ductive effects could be observed during a much shorter treatment period than
currently required for avian reproductive testing (approximately 20 weeks).
Status: This project was completed 12/85, and the manuscript has been pub-
lished in the Archives of Environmental Contamination and Toxicology
(15:757-761).
Investigators: M. Meyers, J. Gile
45
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Title: Terre-Tox: A Database for Effects of Anthropogenic Substances on
Terrestrial Animals
Introduction: Terre-Tox is a new database developed for the U.S. Environmental
Protection Agency to aid In Premanufacturing Notices and research.It is
designed to become part of SPHERE (Scientific Parameters for Health and Environ-
ment, Retrieval and Estimation) within the Chemical Information System (CIS).
Terre-Tox contains published (1970 to present) information on the toxicity of
anthropogenic substances to terrestrial animals. It is comprised of a biblio-
graphic file and a data file.
Methods: The bibliographic file is a listing of all articles collected,
reviewed, and accepted for Terre-Tox. Articles on the effects of chemicals on
birds, mammals, reptiles, terrestrial stages of amphibians, and selected
invertebrates are collected. These include laboratory, pen, and field studies.
After articles are obtained and entered into the bibliographic file,
acceptable article are coded and entered into the data file. The material in
the data file includes chemical information, test organism information, test
conditions, calculated endpoints (i.e., LDso, LCso), and other results (signs
of toxicity, behavior, avoidance, physiological, etc.), and comments.
Status and Results: The bibliographic file contains 4,962 citations of pub-
lished articles from about 480 journals. The data file consists of test data
of more than 150 species and 800 chemicals. At present there is a total of
3,905 studies entered into the data file. Both files can be word-searched on
any of their fields. The databases are updated on a regular basis as new
articles are obtained and coded. A tape of Terre-Tox has been sent to NTIS
(National Technical Information Service). It will be available for use by the
general public (CSI, vendor).
Investigators: M. Meyers, S. Schiller
46
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Title: Toxicity of Contaminated Prey to Carnivores
Introduction: Previous studies have suggested there may be significant differ-
ences in mammalian carnivores in the toxicity of xenobiotics derived vTa
primary and secondary routes of contamination. This study was designed to
assess the toxicity to carnivorous mammalian wildlife of selected potentially
hazardous xenobiotics derived via the consumption of contaminated prey. The
mink was used as a test animal representative of mammalian carnivores. Compar-
ison of the toxicity of the metabolized forms of Aroclor 1254, Compound 1080,
Warfarin and Methrl Parathion (as contaminated natural [live] food) were made
with comparable concentrations of the unmetabolized forms of the chemicals
added to the feed of the test species.
Methods:
1. Range-Finding Tests. Each chemical was first fed to mink in a range-
find i?ig~TelT~fo~9etermTne appropriate concentrations for use in the definitive
tests (LCso). Procedures for range-finding tests consisted of dosing via the
feed several mink with three to five widely-spaced concentrations of the
chemical. A similar preliminary feeding trial with the prey species (rabbit)
was also conducted to ascertain the rate of bioaccumulation of the test sub-
stance in the whole body mass of these animals so that appropriate chemical
concentrations could be obtained, via the contaminated prey species, for
incorporation into the LC5Q test diets fed to the mink.
2. Subacute Tests (LCsp). A subacute (LCso) test was conducted with mink,
using proposed OTS mink toxicity guidelines, for each selected chemical. The
LCso test consists of a 7-day acclimation period followed by a 28-day treatment
period. One hundred twenty (120) mink were employed for each LCso test. The
test animals were randomly assigned to cages. In one test room, the animals in
the treatment groups were fed a basal diet in which the chemical being tested
was added to the diet in a geometric progression of concentrations designed to
produce from 10% to 90% mortality. In another room, the diets fed to the test
animals in the treatment groups contained xenobiotic-contaminated prey, to
simulate secondary toxicity. The concentrations of the test substance in the
diets of the treated animals in both rooms were comparable and all diets fed to
the animals in the controls and treatment groups in both rooms contained the
same percentage of prey species. Feed and water were provided jid libitum
throughout the trial.
Body weights of the test animals were recorded and reported at the initia-
tion of the acclimatization period, initiation of the test period, and at days
7, 14, 21, and 28 of the test period. Food consumption was measured and
reported for all groups during the acclimation and test period. Daily observa-
tions were made throughout the test period for any signs of intoxication or
other abnormal behavior and mortality. At the termination of a test, LCso
values and 95% CI were determined by Probit analysis and comparisons made to
determine the comparative toxicity between primary and secondary exposure.
Status: This project was requested by OTS. All experimental work and analysis
is complete. Journal articles have been submitted and a draft Secondary
Toxicity Test Protocol has been received by CERL.
Investigator: R. Ringer
47
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Inhouse
Ongoing Experiments
48
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Title: The Relationship Between Organophosphate Exposure, Reproductive
Hormones, and Nesting Success
Introduction: Research at CERL has shown that sublethal exposure of nesting
waterfowl to Organophosphate (OP) compounds may lead to reproductive failures
by causing nest abandonment and an abrupt loss of incubation behavior from
brooding hens. There is evidence that OPs can cause a reduction in egg produc-
tion by disrupting the production or release of lutenizing hormone (LH), one of
the hormones responsible for ovulation. This research was initiated to deter-
mine if OPs also affect the release of prolactin, the hormone reponsible for
incubation behavior in birds. A disruption in the release of prolactin in wild
incubating birds could lead to nest abandonment or reduced nest attentiveness,
which would adversely impact hatchability and chick production.
If the relationship between OP exposure and hormone levels can be deter-
mined, it may be possible to screen for potential adverse effects of chemicals
on reproduction with a blood plasma assay that determines the exposure level
responsible for disruption of egg production and incubation behavior.
Methods: Preliminary research at CERL in 1985 exposed incubating mallard hens
to methyl parathion-treated feed for 6 days. Hens were allowed to set and
incubate their own clutches in indoor reproduction cages. Blood serum samples
were collected routinely and analyzed for prolactin and LH. The nest attentive-
ness of each hen was also determined with thermocouple probes that recorded the
temperature of each nest every 30 minutes.
It was demonstrated, based on a small sample size, that sublethal methyl
parathion exposure to incubating hens produced a significant reduction in blood
serum prolactin. Hens with prolactin depression also exhibited poor incubation
behavior as detected by a reduction in nest attentiveness.
An expanded test was conducted in 1986 to further evaluate the relation-
ship between dietary OP exposure and reproductive hormones as it affects
mallard nesting success. This test monitored plasma prolactin concentrations,
plasma cholinesterase, blood chemistry parameters, corticosterone levels, food
consumption, and nesting behavior throughout incubation for birds in three
treatment groups: control, 4000 ppm dietary methyl parathion, and paired-fed
groups. The paired-fed group matched the reduced level of daily food consump-
tion measured in the methyl parathion group.
Status: Prolactin and blood chemistry assays are in progress. The continua-
tion of this project in 1987 will be directed by the questions arising from
this year's findings, although further research into the causes and ecological
importance of nest abandonment will continue in order to develop a better
capability of predicting chemical effects on nesting success.
Investigators: R. Bennett, A. Fairbrother, J. Bennett, R. Ringer, M. El-Halawani
49
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Title: Time Course and Level of HCB Transfer From Hen to Egg in Bobwhite
Introduction: It is necessary to determine the timing and amount of dietary
HCB which reaches'eggs and embryos in aviansThexachlorobenzene (HCB) is a
chlorine substituted aromatic fungicide which accumulates in body tissue and is
transferred to the egg by laying hens. The amount of HCB in feed which is
necessary to produce transfer and accumulation of chemical in bobwhite quail
eggs has been determined. Five levels of HCB were fed to paired and isolated
laying bobwhite quail. Total egg HCB residue was measured for each egg in
order to determine the time course of transfer of HCB from the food to the egg.
The total chemical level in food necessary to produce uptake of HCB in the egg
is related to levels which cause mortality (from LC50 data) and deficiencies in
reproductive capability. Total active ingredient consumed is compared to egg
and hen liver residue at the end of the study to evaluate the exposure to HCB
which is necessary to produce accumulation of chemical in eggs of bobwhite
quail.
Methods: Adult laying bobwhite hens were exposed to dietary HCB at 1.5, 3.0,
4.5, b.O, and 12 ppm and eggs were collected for 48 days after the initial
exposure to chemical. Egg yolks were separated and analyzed for HCB residue
using a gas chromatograph. Birds were placed back on clean feed after 48 days
and eggs were analyzed for HCB. Residues of livers were determined in some
hens at selected intervals and for all the remaining hens at the end of the
test.
Status: HCB residues have been traced into the eggs of bobwhite hens at each
feeding levels of HCB in diet. The residues begin to show up in significant
levels within 3 days (greater than 1 ppm) and accumulate in egg yolks at a rate
of approximately 1.75 ppm/day. After approximately 30 days of dietary expo-
sure, both treatment levels of HCB reached plateau levels approximately 5 times
(5x) the dietary concentration. Research is continuing in an effort to deter-
mine the movement of HCB residue into embryos of eggs produced by hens fed
dietary HCB.
Investigators: B. Williams, R. Ringer, T. Shiroyama, R. Bennett, Doug Bonfoey
50
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Title: Egg Shell Quality Evaluation
Introduction: There is concern that environmental contaminants may affect the
quality of egg shells produced by wild birds leading to egg breakage, nest
failures and, ultimately, effects on certain wildlife populations. Conse-
quently, EPA requires that egg shell thickness be measured during reproduction
testing. However, there are reports of chemical-associated egg breakage in
which significant shell thinning was not demonstrated. Research at CERL found
many eggs produced by bobwhite dosed with sulfonilamide, a drug known to cause
shell thinning, retained normal thickness but were significantly weaker,
suggesting the ultrastructure of the shell had been affected.
Approach and Methods: Research is currently underway to evaluate a compression
test to determine egg shell strength as a potential additional evaluator of egg
shell quality for EPA chemical registration tests. A breaking strength test
may prove to be a method that can provide an especially relevant assessment of
the potential for a chemical to cause broken eggs in wild birds.
The ultrastructure of the egg shells are also being examined by scanning
electron microscopy to determine if the chemical exposure affects the shell
structure and how that relates to shell thickness and shell strength.
Status: In 1986, two experiments were performed to compare the shell thick-
ness, shell strength, and ultrastucture of eggs produced by bobwhite exposed to
chemical substances. One experiment involved a 9-week exposure to DDE, the
stable breakdown product of DDT frequently linked to egg shell thinning
problems in wild birds. The other experiment exposed bobwhites to an organo-
phosphate insecticide for a 7-day exposure period. The results of both experi-
mentes are currently being compiled and analyzed. Scanning electron microscope
examination of shells produced in those experiments is currently underway.
Research in 1987 will compare the shell quality evaluation methods on
mallards exposed to various chemical substances.
Investigators: J. Bennett, R. Ringer, R. Bennett, B. Williams, P. Humphrey
(OSU)
51
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Title; Determination of the Ability of Avian Species to Discriminate Between
Treated and Untreated Food
Introduction: One of the complicating factors in interpreting data from the
subacute dietary toxicity test (LC50) is the significant reduction in food
consumption associated with many chemicals at the higher dietary concentra-
tions. For these chemicals the toxic effects are often confounded by chemic-
ally-induced starvation, which may or may not be indicative of the patterns of
field exposure. Many avian species are able to detect and avoid chemically-
contaminated food in the lab when untreated or less toxic alternative food
exists. But under what circumstances can birds respond to contaminated food in
the field by switching to less contaminated foods or feeding sites? Our
research has shown that this ability is affected by the number of choices
available, the proportion of treated and untreated choices, and the detect-
ability of the chemical. Other factors which will be investigated include
total food abundance and physiological status of the birds.
The objectives of this ongoing project are: (1) to determine the factors
influencing food avoidance behavior; and (2) to identify conditions under which
birds are able to respond to chemically-contaminated foods or habitats by
avoiding lethal or harmful exposure levels. The results from this research
should aid in the interpretation of data from dietary toxicity tests, as well
as help to identify parameters that are important in realistic models of
chemical exposure for risk assessment.
Methods: Thus far, the ability of bobwhite to detect and avoid food treated
with various pesticides has been determined in a series of food choice tests.
These tests consist of a 5-day treatment period followed by a 3-day recovery
period so that the results can be directly compared to the LC50 test. The
tests range from a simple 2-choice test, with one food tray containing treated
food and the other untreated, to 10-choice tests where the proportion of
treated and untreated food trays can be varied. The dietary concentration
above which birds exhibit a preference for the untreated food is defined as the
discrimination threshold (DT), and is calculated using a multiple regression
method developed at CERL. An avoidance index can be calculated from LC50/DT
such that the higher the index value, the greater the potential to reduce
chemical exposure.
Status: Laboratory tests have been conducted on 7 pesticides and manuscripts
are in preparation. Further laboratory tests are planned to evaluate the role
of other factors on avoidance behavior and to test models of chemical exposure.
Future field studies are being planned to validate lab results and to further
identify field conditions under which chemical exposure is influenced by
behavioral responses.
Investigator: R. Bennett
52
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Title: Normal Mallard Blood Enzyme Values for All Age, Sex, and Reproductive
Classes
Introduction: Blood enzyme values are used routinely in clinical medicine to
diagnose organ malfunctions. It would be equally useful to use them as biolog-
ical markers indicating exposure to pesticides or toxic substances. Unfortu-
nately, there is little information in the literature about normal values in
mallard ducks, one of the avian species commonly used in toxicological experi-
ments. What information is available is applicable only to adult, nonbreeding
birds. Since birds exhibit marked physiological changes during growth and
during the reproductive period, it is necessary to generate normal values
throughout the mallard life cycle to evaluate subsequent data on enzyme changes
following chemical exposure.
Methods and Materials: Forty-eight adult mallard pairs will be purchased in
the spring in a nonreproductive state. Birds will be housed in outdoor ground
pens provided with nest boxes and materials. Serum samples will be collected
via jugular puncture at selected intervals during the nonbreeding, egg laying,
and brooding periods. Samples also will be collected from chicks until they
reach young adult age. A panel of ten blood enzymes indicative of liver,
kidney, heart, pancrease, and muscle function will be run, utilzing a Gilford
Impact 400 autoanalyzer, in cooperation with Dr. M. Craig, Oregon State Uni-
versity School of Veterinary Medicine.
Status: The experiment will be conducted from April 1986-March 1987.
Investigator: A. Fairbrother, M. Craig, R. Bennett
53
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Title: The Role of Modeling in Risk Assessment at the Individual and Popula-
tion Levels
Introduction: The construction of models enables us to generalize from basic
principles to the predicting and understanding of novel situations.In the
case of chemical risks assessment, it makes possible the prediction of a
specific chemical's impact on an ecosystem never before tested for sensitivity
to any chemical at all. Given the enormous array of species, both chemical and
biological, models offer us the only hope for far-reaching, reliable risk
assessment.
Methods and Materials: Models can be classified by types; it is our intention
to pursue three types:
(1) We intend to extend existing, species-specific models to incorporate the
effects of chemical insult. Such models are not ideal because they are general-
izeable only to the array of chemical, not biological types. They have the
advantage, however, of having been tested, are known to work, and so fill a
short-term need for risk assessment in limited situations.
(2) We will construct "holistic" models driven by ecological principles and
empirically measured patterns that provide direct descriptions of chemical
effects on individuals or on populations. Such modeling will be extremely
flexible in that appropriate parameterization will permit application to almost
any chemical and biological species combination.
(3) We will construct "reductionist" models, based on first (physical and
chemical) principles and general, ecological laws. These models use chemical
uptake data and knowledge of physiology to predict toxic impacts on indi-
viduals, or use toxicity information and laws of population processes to
predict population-level impacts. Unfortunately, because our understanding of
toxicological and population processes are extremely poor, such models, while
worth pursuing for their comprehensive, explanatory nature, are unlikely to
bear fruit in the foreseeable future.
Status: Underway. Modeling effort will be coordinated with work done on
contract to Dr. Warren Porter and Ron Hindsell, University of Wisconsin.
Investigators: J. Emlen
54
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Title: Effects of an Acetylcholinesterase Inhibitor on Red-Winged Blackbird
(Agelaius phoem'ceus) Reproduction
Introduction; To evaluate chemicals as potential avian toxicants, the Environ-
mental Protection Agency requires chemical manufacturers to perform acute,
subacute, and/or reproductive tests on mallard ducks (Anas platyrhynchos) and
bobwhite quail (Colinus virgianus). In some cases the nng-necked pheasant
(Phasianus colchicus) may be used. As indicator species, mallards, bobwhites,
and pheasants only represent about 5 percent of the total avifauna of North
America. Passerine birds may constitute more than 50 percent of the total
post-breeding population. It appears apppropriate for future avian toxicity
testing that a passerine bird(s) be included as an indicator (test) species in
TSCA and FIFRA guidelines.Research by the U.S. Fish and Widlife Service has
indicated that the red-winged blackbird is more sensitive to toxic chemicals
than other passerines tested and more sensitive than the mallard, quail, or
pheasant. The red-winged blackbird is both abundant and ubiquitous in the
United States. The objectives of our study are to determine the feasibility of
using the Corvallis facilities (WFTS, duck ponds) to conduct reproductive
studies on free-living birds and to determine the effects of an acetylcholin-
esterase inhibitor on red-winged blackbird hatching process, fledgling, and
female behavior. In addition, It is the goal of this project to determine the
suitability of red-winged blackbirds as indicator species as related to TSCA
and FIFRA guidelines.:
Methods: Conduct a 2-year study using 6 0.1 ha fenced ponds at WFTS. Each
pond is surrounded by a band of emergent vegetation that supports nesting
red-winged blackbirds. Females will be captured and marked with color-coded
leg bands for future identification. After a female completes her clutch
(approx. 4 eggs) she will be captured and gavaged with either a carrier or
carrier plus test chemical in a random manner. Female attentiveness to her
young will be monitored as well as hatching and fledgling success.
Status: The first season was completed in June 1986. Methods were developed
for capture, tagging, and dosing blackbirds. The second season will begin
April 1987 with the assistance of the Denver Wildlife Center (Ed Schafer and
John Cummings).
Investigators: M. Meyers, E. Schafer, J. Gile
55
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Title: Changes in Mallard Hen Behaviors in Response to Methyl Parathion-
Induced Illness of Chicks
Introduction: Waterfowl chicks may become ill following exposure to organo-
phosphates. The hen must then choose to alter the behaviors of herself and the
remainder of the brood to accommodate the sick chicks or to maintain normal
behaviors and leave the sick young to follow as best they can. If the former
course is taken, the hen may increase the risk of predation to the entire brood
and decrease their ability to forage optimally. If the latter course if
followed, the sick chicks will most likely die. Either way, the effects of
low-level exposure to the organophosphate may be different than predicted by
LC50 or LD50 trials.
Methods and Materials: The methyl parathion LD50 for five-day old ducklings
will be determined in a pilot study. Hen and drake mallards will be housed in
ground pens with nest boxes and allowed to come into egg production and estab-
lish and incubate a clutch on their own schedule. When ducklings in a brood
are five days old, half of them will be dosed by oral gavage with the pre-
determined dose of methyl parathion and the remainder will be dosed with
diluent (corn oil) only. Numbered back tags will be put on all ducklings for
identification of individuals. After dosing, the hen and her brood will be
released on one of the 4.9 meters deep ponds at WFTS and observed continuously
for 4 hours. Observations will be made at selected intervals for the next 20
hours. Observations will consist of noting the length of time the hen spends
with the brood, foraging time, and behaviors of hen and ducklings, time spent on
land and on water, whether ducklings stay together in a group or if some spend
time alone, and any other behavioral observations considered significant.
After 24 hours postinoculation, broods and hens will be captured and removed
from the pond.
Status: Small scale study was conducted in May-August 1986. Expanded study is
under consideration for Spring-Summer 1987.
Investigators: A. Fairbrother, M. Meyers, R. Bennett
56
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Inhouse
Future Experiments
57
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Effects of Age on Mallard Sensitivity to Pesticides in Avian Dietary
Introduction: Office of Toxic Substances (OTS) guidelines for the conduct of
avian dietary tests prescribe birds between ten and seventeen days of age at
the start of exposure. Office of Pesticide Programs (OPP) guidelines prescribe
ducks between five and ten days of age. Literature perusal indicates that a
fairly significant variation in LC5Q values obtained from birds in this age
range (5-17 days) occurs for a given toxicant. The standard test protocol may
need to be re-examined in terms of test organism procurement and age at the
time of starting dietary exposures. Differences in sensitivity at different
ages may be a function of size and not actual age.
Methods and Materials: Mallards of at least three different ages (i.e., 5, 10,
14-d) will also be exposed to several toxicants using the standard protocol to
determine age-related differences in sensitivity. To determine whether differ-
ences in sensitivity with age is due to development of detoxification mechan-
isms, liver enzymes may be measured as well. These two experimental strategies
should give insight into whether sensitivity is age and/or size related.
Status: The age sensitivity investigations began in the Summer of 1986 and
extend into and through 1987.
Investigators: J. Gile, M. Meyers
58
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Title: Water as a Route of Exposure of Waterfowl to Pesticides
Introduction: Because the aquatic environment is usually the eventual recipi-
ent of most chemical contaminants, and because waterfowl are so intimately
associated with that environment, we have initiated a study of waterfowl
exposure to toxicants via water.In initial experiments with mallard ducklings
exposed to methyl parathion in their drinking water, it was observed that
within 24 hours the concentration of chemical in the water column had decreased
to practically zero. Our immediate objective is to determine the physical/
chemical fate of methyl parathion, as well as several other pesticides, when
presented to mallard ducks via drinking water. The longer-term objectives are:
(1) to experimentally determine water LCso's for mallards exposed to various
pesticides; and (2) to experimentally determine the relative importance of
water vs. food as routes of exposure to various pesticides.
Methods and Materials: Physical/chemical fate of pesticides is to be examined
by short-term (24-48 hr) measurements of soluble chemical remaining with and
without the presence of organic matter (i.e., feed, feces), as well as in
various types of water containers (i.e., glass, PVC, galvanized metal). Water
LCso's will be determined by using a modification of the OTS Avian Dietary Test
(EG-15). Relative importance of food vs. water as routes of exposure will be
examined by exposing mallards to contaminated food, contaminated water, both,
or clean food and water and statistically analyzing the resultant mortality,
growth, and cholinesterase activity using a factorial design.
Status: Currently we are beginning to determine the chemical/physical fate of
toxicants in the water matrix and will complete this investigation in FY87. We
will also begin the determination of water LCso's for various pesticides in
FY87 and continue in FY88.
Investigators: M. Cairns, B. Williams, R. Bennett
59
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Title: Immune Function Panel for Avian Species
Introduction: There are several immune function tests developed for avian
species, with primary emphasis on the chicken. The objective of this study
are: (1) to determine which ones are feasible for use with bobwhite quail and
mallard ducks; and (2) to select a panel of tests that can be performed in
field laboratories with a minimum of specialized equipment yet still provide
information about both cellular and humoral immune functions. Ultimately,
these tests will be used to determine if pesticides and toxic substances in the
environment cause immunosuppression in avian species, making them more vulner-
able to endemic pathogens.
Methods and Materials: Standard operating protocols gleaned from the litera-
ture or provided by other investigators will be followed, making changes
necessary to adapt them to quail and ducks. The following tests are being
considered for use: macrophage migration inhibition; delayed type hyper-
sensitivity; graft versus host reaction; lymphocyte growth on chorioallantoic
membrane; natural killer cell activity; lymphocyte blastogenesis; splenic
plaque forming assay (direct and indirect); ELISA for IgB and IgM; total and
differential lymphocyte counts.
Status: These experiments are currently in the planning stage. Actual experi-
mentation should begin in the Fall of 1986.
Investigators: A. Fairbrother
60
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Title: Effects of Environmental Stresses on Avian Thermoregulation and
Metabolism
Introduction: The objective of this test is to determine the effects of
environmental stress on the metabolism, thermoregulation, and sensitivity of
birds to toxic chemicals. This project will provide information about the
ability of standard OTS toxicity tests to evaluate toxic effects in animals
subjected to stress conditions representative of those likely to be encountered
in the field.
Methods; Standard OTS toxicity tests will be used to determine normal acute
toxicity values in bobwhite quail and the results compared with similar tests
in which environmental stresses are imposed. Brain cholinesterase activity,
metabolic efficiency, and survivorship will be monitored and evaluated in
relation to the interaction between chemical and temperature stresses.
Status: This project will begin summer of 1987 if funding is approved at an
appropriate level.
Investigators: B. Williams, A. Fairbrother
61
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Extramural
62
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Title: Effects of Methyl Parathion on Wild Avian Species in Agricultural Areas
of Skagit Valley, Washington
Introduction: The effects of organophosphate (OP) chemicals on non-target
avians which utilize sprayed habitats needs to be evaluated. This project was
designed to assess the effects of methyl parathion (0,0-dimethyl o,p-nitro-
phenyl phosphorothioate) on wild avian species in agricultural areas of Skagit
Valley, Washington. The study was designed to investigate the following two
objectives: (1) to determine any disturbances in populations of non-target
avian species that are utilizing habitats that receive agricultural spraying of
methyl parathion; and (2) to assess reproductive and biochemical (i.e77
cholinesterase inhibition) impacts of exposure of these bird species to methyl
parathion.
The production of teal and mallard broods and the survival of teal and
mallard ducklings were reduced by the application of methyl parathion. In
addition, four independent occurrences of abnormal bird behavior were observed
on the study fields following treatment with methyl parathion. These observa-
tions included abandonment of nests by mallard hens incubating eggs. Other
researchers have noted disturbances in nesting behavior of birds exposed to
organophosphate pesticides.
Occupancy of nest boxes by starlings was 73% on the treatment field and
82% on the control field. Starlings began using the nest boxes within one hour
after their placement in the fields. Nest-building activity was evident in
many of the nest boxes within 24 hours.
Methods and Materials: The experimental design included the identification of
six agricultural areas that averaged approximately 28.3 hectares (70 acres) in
size and were approximately 60-70% bordered by waterways. Three of these
treatment areas receive aerial application of methyl parathion at 0.7 kg/ha.
Data collected included: (1) avian censusing; (2) carcass search; (3) repro-
ductive study on starlings (Sturnus vulgaris); (4) residue analysis on environ-
mental samples (i.e., water, soil, and vegetation); and (5) brain cholinester-
ase levels on birds.
Status: This project was completed in 1985 and several manuscripts are in
preparation. (1) No significant difference was observed in the number of young
starlings which fledged from nests on either field. However, overall a lower
proportion of starling nestlings fledged from the methyl parathion treated
field (64.5%) when compared to the control field (89%). (2) Hatchability of
starling eggs in exposed nest boxes was not significantly affected by applica-
tion of methyl parathion (1.4 kg AI/ha). (3) Nestling mortality tended to be
higher on the treated field. (4) Under the conditions of this study, aerial
application of 1.4 kg AI/ha methyl parathion did not affect reproduction in
breeding starling populations on the treated field. Exposure via food chain
contamination was probably very low due to the apparent low density of insects
with associated low foraging activity on the field at the time of spray.
Investigators: R. Kendall £t jiK, Western Washington University
63
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Title: Primary Hazards to Game Birds Associated with the Use of Ramik Brown
(Diphacinone Bait) for Controlling Voles in Orchards
Introduction; EPA, Washington State Department of Game and Agriculture, as
well as the U.S. Fish and Wildlife Service, have been concerned about the
otential hazards associated with rodenticides that have been registered in
asrnngton to replace endrin use for controlling orchard mice.Preliminary
information indicated that chukar partridge may be more susceptible than quail
to anticoagulant poisoning (C. Henny, personal communication). Little is known
about the primary hazards of anticoagulants to game birds under field condi-
tions.
This study was conducted to evaluate the potential primary hazards to game
birds resulting from the use of diphacinone bate to control mice in orchards.
The two study areas were located in the Yakima Valley in south-central
Washington, one was near Moxee, and the other near Sunnyside.
In general, it appears that the diphacinone bait caused little if any
direct mortality to game birds. However, pheasants and quail consumed large
quantities of the bait. In some birds collected post-treatment, over 90% of
the crop contents was bait. While these birds appeared normal, the long-term
effect of feeding on bait are unknown. Some birds contained substantial
amounts of bait in their crop a month after treatment.
While primary hazards appear to be low, secondary hazards may be a major
concern with the diphacinone bait. Certainly the bait being consumed by the
target and by game birds (even if they survive) presents a hazard to predators
that may consume them. Some of the radio-equipped birds were killed and at
least partially consumed by both mammalian and avian predators. In one case, a
weasel had partially consumed a radio-equipped quail.
Methods and Materials: Sixty-eight wild ring-necked pheasants and 15 wild
California quail were captured, radio-equipped, and released in or adjacent to
orchards scheduled for treatment. In addition, because of difficulties in
capturing, 15 game farm-raised California quail and 41 game farm-raised chukar
partridge were released in or adjacent to orchards scheduled for treatment.
These 139 birds were radio-tracked before, during, and after the rodenticide
treatment. Any birds found dead were necropsied and tissues were preserved
(frozen) for later residue analysis. All birds with operational transmitters
at the end of the study were collected for necropsy and tissues were preserved
for residue analysis.
Status: Tests completed and final report and manuscript are in re-write stage.
Anticipated publication as both an EPA document and a journal article.
Investigators: P. Hegdahl, U.S. Fish and Wildllife Service, Denver
64
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Title: Comparative Toxicity of Methyl Parathion and Dieldrin in Laboratory
Rats and Mice, vs. Captive Feral Cotton Rats and White-Footed Deer Mice
Introduction: It is often necessary to utilize laboratory animals to predict
hazard to terrestrial wildlife. We are unclear, however, as to the appropriate-
ness of using laboratory (surrogate) animals to extrapolate hazard data to wild
species. This project was initiated to evaluate the similarities of response
of wild and laboratory mammals (rodents) to toxic chemicals.Acute oral LDso
values for methyl parathion and dieldrin were determined in both sexes of four
rodent species. Two laboratory species, albino rats and albino mice, and two
captive feral species, Sigmodon hispidus and Peromyscus maniculatus, were
chosen. For methyl parathion, the males were found to be more sensitive than
females and laboratory rodents more sensitive than captive feral rodents. The
response of brain acetylcholinesterase (AChE) with respect to mortality was
also determined for the animals in the methyl parathion acute toxicity study.
A close agreement for brain AChE activity level was observed for both sexes in
both mice species. A similar agreement was noticed for both sexes of both rat
species. Brain acetylcholinesterase recovery (BAAR) was determined at day 28
for the feral rodent survivors, Sigmodon hispidus rats failed to recover,
whereas male Peromyscus maniculatus exhibited recovery and female Peromyscus
mam'culatus exhibited over-recovery when compared to brain AChE activity in
control animals.
Materials and Methods: Sigmodon hispidus (cotton rats) and Peromyscus
maniculatus (white-footed deer mice) and domestic albino laboratory mice (Mus
musculatus) and the domestic albino laboratory rat (Rattus nprvegicus) were
compared in this study. Acute oral toxicity of methyl parathion and dieldrin
was determined using standard oral gavage techniques.
Status: The appearance of signs of intoxication, mortality, and apparent
recovery from intoxication were dose-dependent in the domestic rodents. This
relationship was less apparent in the feral species. Values for laboratory
rats and mice correlate closely with literature values. The laboratory rodents
were found to be more sensitive (p = 0.05) to acute MP intoxication with the
exception of the male laboratory mice vs. male white-footed deer mice.
This paper has been accepted for publication in Environmental Contamina-
tion and Toxicology.
Investigators: D. Roberts, N. Silvy, E. Bailey, Texas A&M University
65
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Title: Uptake of 2,3,7,8-Tetrachlorodibenzo-p-dioxin by Dairy Cows
Introduction: 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a highly toxic
byproduct formed during the commercial preparation of chlorinated phenol -
derived products such as the phenoxy acid herbicides, and during the combustion
of diverse organics and municipal waste. Once in the environment, TCDD binds
tightly to the soil and usually remains on or near the soil surface where it is
resistant to both photochemical and biochemical degradation. TCDD appears to
be particularly persistant in deeper and more protected soils with an estimated
half-life of more than 10 years.
Absorption of TCDD from the gastrointestinal tract has been extensively
studied in the rodent. Relatively little is known regarding the fate of TCDD
in ruminants. Recent studies suggest that the distribution of TCDD in cattle
is similar to that seen in rodents with the liver or fat containing the highest
residue levels. Given the highly toxic nature of TCDD. its environmental
stability, its widespread occurrence as an environmental pollutant, and the
fact that grazing livestock are surely exposed to TCDD through incidental
consumption of soil and perhaps through other routes, it is apparent tha"t a
thorough understanding of the interactions of TCDD with ruminants needs to be
obtained.
Methods: The research will focus on the following interrelated objectives:
(1) Isolation and identification of the metabolities of 2,3,7,8-TCDD in the
ruminant.
(2) Determination of the pharmacokinetics, metabolism and biological effects of
TCDD in the ruminant. Both the parent compound and metabolites in the milk,
urine, feces, and various tissues will be determined. This will permit the
comparison of the fate of non-bound TCDD with the effects of aged, soil bound
TCDD in the ruminant.
(3) Investigation of the bioavailability of TCDD from soil. This study will
use a soil type to be approved by the EPA. Cattle will be treated orally with
tritiated TCDD which has been allowed to age on the soil for four weeks. The
level of TCDD exposure will be based on the best estimates of incidental soil
consumption by grazing cattle. Pharmacokinetic data, parent compound and
metabolite concentrations as well as biological effects on hepatic enzyme
activity will be determined.
Status: This study is being conducted as a Cooperative Agreement with Texas A&M
University and is in response to a request by ORD.
Investigators: Texas A&M
66
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Title: Relative Importance of Pathways of Exposure and Influence of Environ-
mental Variables on Dose and Impacts of Parathion on Wild Game Birds
Introduction: Pesticides' impacts on wildlife, and in particular wild game
birds, is an important issue associated with registration and permit review for
use of a chemical in agriculture. Data requirements include estimation of
effects on survival and reproductive potential as well as tissue residue
analysis from oral laboratory feeding studies. Other pathways of exposure,
such as; (1) inhalation; (2) surface adsorption through feather and dermal1y
deposited pesticide; (3) indirect oral ingestion through preening; and (4T
percutaneous adsorption through the horny epithelial scales of the feetlmd
legs, have received little attention and are generally overlooked.
Attempts to estimate relative importance of each pathway by simply expos-
ing wild game birds to spray of a pesticide in animal aerosol exposure chambers
will not allow for accurate estimation of field exposures and potential
impacts. Environmental variables, such as: (1) time of day; (2) ground cover
type, density, and height; (3) wind speed; (4) temperature; and (5) relative
humidity influence the exposure concentration of pesticides reaching the bird.
Ground cover influences exposure by providing a physical shield for pesticide
interception along with a microclimate for the birds. Several of the other
environmental variables influence exposure by altering particle coagulation,
condensation and subsequently deposition velocity. Additionally, time of day,
temperature, wind speed and relative humidity influence the physiologic state
of the bird which clearly influence the concentration necessary to produce a
given effect. For example, the influence of temperature extremes on the bird's
metabolic rate could be the key variable that influences survival to a given
exposure.
Methods: The primary objective of this proposed two year research project will
be to provide a first order estimate of the relative importance of these
potential routes of exposure on dose (i.e., tissue concentration) and impacts
of the pesticide parathion on bobwhite quail. Within the confines of the
experimental portion of this project we will determine the relative importance
of extremes of key environmental variables on both route of exposure and impact
of parathion to quail.
During the first year of this study efforts will concentrate on examining
the relative importance of the routes of exposure, with limited time and funds
being focused on the influence of environmental variables. Special emphasis
will be placed on examining inhalation of pesticides through nostrils, bronchi,
and into the alveoli of the lungs of the quail because so little is known about
this route of exposure. Then in the second year, if warranted, the focus would
be on the influence of critical environmental variables on exposure and
effects. The final output of this project would be a simple mathametical
computer simulation model that would allow one to change certain key parameters
influencing concentration of exposure and estimate the dominant route of
exposure that needs to be examined in detail. Parathion was selected for study
based on providing timely data for evaluating reregistration of this pesticide
as well as on the pesticide's physical and chemical characteristics.
Status: This is a new project which will be conducted extramurally as an
interagency agreement with the U.S. Department of Energy, Battelle Northwest.
Investigators: Department of Energy
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Title: Risks of Chemical Contamination to Mammalian and Avian Populations
Introduction: Models have not yet been developed which assess population level
effects of chemicals.The intent of this work is to develop and test models
for predicting population-level impacts of chemical contamination on mammalian
and avian populations.
Methods and Materials: Because of the vast array of chemical and biological
species it is impossible, by performing laboratory or field tests on a species-
and chemical-specific basis, ever to achieve a general capability for chemical
risk assessment. On the other hand, predictive population models, by allowing
us to extrapolate from general principles, permit the assessment of risks
arising from novel combinations of chemical and biological species. We will
expand and develop population models, and gradually incorporate existing,
species-specific models into more general ones capable of predicting hazards
for a wide variety of situations. Consideration will be given to avoidance
behavior, age (stage)-specific sensitivities, population density feedback,
inter-specific interactions (competitive and predative) and random fluctuations
of the natural, physical environment.
Status: Several models have already been developed; others are under consider-
ation. Field studies for evaluating models of spatial avoidance of toxicants
and the importance of species interactions to risk assessment have been
designed in detail for mammals, and are in an active planning stage for birds.
Investigator(s): TBD
68
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Title: Tracking of Avians in the Field Using Telemetry
Introduction: The objectives of this task include: (1) to utilize telemetry
to develop the capability to accurately measure the movements of quail into and
out of fields which have been treated with pesticide; and (2) to document the
movement patterns of birds which reflect their ability to detect (and avoid)
chemically treated areas. This project will provide information about the
exposure of avians to chemically treated fields by documenting the movement of
avians into and out of the treated fields.
Methods and Materials: The approach will be to set up a field test of sophisti-
cated radio telemetry tracking equipment and to utilize wild quail trapped at
the 650-ha Wilson Game Farm south of Corvallis. Each bird will be tagged,
weighed, and equipped with a small radio transmitter coded to transmit a unique
signal. The system will be automated to provide precise time, location, and
movement information on each of the test birds. The precise location and
movements of each bird will be determined and stored in the computer for
subsequent analysis of home ranges, habitats, breeding activities, and response
to chemical.
Status: A cooperative agreement has been awarded to Oregon State University
for the conduct of this project. A close scientific and working relationship
with the Principal Investigator, Dr. John Crawford, has begun. The first field
demonstration of this new technique will begin in early 1987.
Investigators: J. Crawford, B. Williams, R. Bennett
69
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APPENDIX B. CHEMISTRY CAPABILITY AND LABORATORY ANALYSES
70
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AVIAN TOXICOLOGY ANALYTICAL CHEMISTRY CAPABILITIES
The avian toxicology program has a requirement for feed and residue
analysis in the ongoing LC5Q, LDsg, reproduction, discrimination, and induced
tolerance tests currently being conducted both at CERL and WFTS. The ability
to verify toxicant concentrations in avian food allows confirmation of levels
as well as meeting the required protocols of reproductive studies. The track-
ing of toxicants and their metabolites permits resolution of the more subtle
effects commonly occurring in a reproductive study. The fate of ingested
chemicals varies with chemical type. They can accumulate in the body, undergo
chemical breakdown (metabolize), be rapidly eliminated with no adverse effects
or, in the case of avian species, they may be eliminated through the egg with
the resultant adverse effects on the offspring. Chemical analysis of body
tissues aids the researcher designing studies to correlate exposure with
toxicological effects. After determination of the body burden, it is possible
to estimate the dietary intake responsible for those toxicological effects.
Comparison of tissue residues and their effects found in laboratory tests can
be used to estimate the effects from actual field exposures and design more
appropriate studies.
The Wildlife Toxicology Research Team has recently acquired a state of the
art HP 5880 dual capillary gas chromatograph outfitted with several different
detectors to allow the quantitation of many classes of environmentally encount-
ered toxicants and their metabolites. This instrument is capable of a high
degree of sample resolution at very low levels and, coupled with the high
sample capacity of the auto sampler, is a powerful analytical tool.
Two additional gas chromatographs (HP 5830 and Varian 2700) are presently
used for backup and for methods development. A Waters 440 HPLC with an auto
sampler is used for high level feed analysis and for heat labile compounds.
Also available for use within the branch are a Varian 5000 HPLC, a Perkin-Elmer
2000 GC with auto sampler, and a new Finnigan 5100 GC/MS for chemical variation
of unknown toxicants and their metabolites.
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LABORATORY ANALYSES
Blood Analyses
One means of accurately determining the level of toxicity exposure of an
animal is to measure several blood constituents associated with the stress or
toxic response. Although several new blood-related tests have been developed
in the last few years, initial indications of stress are exhibited in portions
of the differential blood count. The lymphycote count can be a direct indica-
tion of previous stress (e.g., the relationship between "T" lymphocytes and "B"
lymphocytes). In addition to the differential count and an analysis of popula-
tion and types of cells present, several clinical enzyme and electrolyte assays
are now available which will identify recent stress-related responses. The
tests are an important component of the CERL in-house testing capability. A
sophisticated Gilford SBA 300 Analyzer is on-line and operational for these
tests. A blood sample can also be used to determine cholinesterase activity as
an indication of complications of anticholinesterases or specific clotting time
as an indication of complications of anticoagulants. The battery of tests
available from a blood sample and the relative ease by which it is obtained
make it a valuable tool in the assessment of the well-being of test animals.
Cholinesterase Determination
The Wildlife Toxicology Research Team has developed standard tests for
acetylcholinesterase (AChE) activity in brain tissue and blood. These tests
are important because many pesticides (organophosphates and carbamates) reduce
the AChE levels in the brain and blood resulting in dysfunction of nerve pulse
transmission. Current methods include the use of the Gilford SBA 300 and
Perkin-Elmer Lambda One Spectrophotometer, which are on-line at CERL.
Resi due Analysi s
The analysis of chemical residue in selected tissues are determined using
GCMS and HPLC techniques. These assays are important to quantify the level of
toxic chemical exposure and the uptake of these pesticides in tissue after
exposure.
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APPENDIX C. PUBLICATIONS
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INHOUSE
Articles Published or Submitted
Aulerich, R. J., R. K. Ringer, and S. Safronoff. Assessment of Primary vs.
Secondary Toxicity of Aroclor 1254 to Mink. Arch. Environ. Contain.
Toxicol. (1986). Submitted.
Bennett, R. S. Role of Dietary Choices on the Ability of Bobwhite to Discrim-
inate Between Treated and Untreated Foods. Environ. Toxicol. Chem.
(1986). In press.
Bennett, R. S., and D. W. Schaefer. Procedure for Evaluating the Potential of
Birds to Avoid Chemically-Contaminated Food. Environ. Toxicol. Chem.
Submitted.
Gile, J. D., J. B. Beavers, and R. Fink. The Effect of Chemical Carriers on
Avian LCso Toxicity Tests. Bull. Environ. Contam. Toxicol. (1983).
V.31:195-202.
Gile, J. D., and S. M. Meyers. Effect of Adult Mallard Age on Avian Reproduc-
tive Tests. Arch. Environ. Contam. Toxicol. (1986). In press.
Kononen, D. W., J. R. Hochstein, and R. K. Ringer. Avoidance Behavior of
Mallards and Bobwhite Exposed to Carbofuran-Contaminated Food and Water.
Environ. Toxicol. Chem. (1986). Submitted.
Kononen, D. W., J. R. Hochstein, and R. K. Ringer. A Graphical Method for
Evaluating Avian Food Avoidance Behavior. Environ. Toxicol. Chem. (1986).
Submitted.
Meyers, S. M., and J. D. Gile. Mallard Reproductive Testing in a Pond Environ-
ment: A Preliminary Study (1986). V. 15:757-761.
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Meyers, S. M., and S. M. Schiller. Terre-Tox: A Data Base for Effects of
Anthropogenic Substances on Terrestrial Animals. J. Chem. Info. Comput.
Sci. (1986). V. 26:33-36.
Presented at the Fifth Annual Meeting of Society of Environmental
Toxicology and Chemistry, Arlington, Virginia, November 4-7, 1984
Bennett, R. S. Role of Dietary Choices on Pesticide Discrimination in Bob-
white. Poster.
Gile, J. D. Comparative Reproductive Effects of Chlorpyrifos on Mallard Ducks:
Indoor Pens vs. Natural Habitat. Poster.
Maguire, C. C. Cholinesterase Activity: Depression and Recovery Rates in
Bobwhite Quail Exposed to an Organophosphorus Insecticide. Presentation.
Presented at the Sixth Annual Meeting of Society of Environmental
Toxicology and Chemistry, St. Louis, Missouri, November 10-13, 1985
Bennett, J. K., R. K. Ringer, and B. A. Williams. Evaluation of Methods to
Determine Eggshell Quality. Presentation.
Bennett, R. S. Comparison of Pesticide Effects on Food Avoidance Behavior in
Bobwhite. Presentation.
Gile, J. D., B. A. Williams, and S. M. Meyers. The Dietary Toxicity of Methyl
Parathion to 14-Day-Old Mallards. Poster.
Maguire, C. C. and B. A. Williams. Chlorpyrifos Toxicity to Bobwhite at
Lowered Test Temperatures. Poster.
Meyers, S. M. and S. M. Schiller. Terre-Tox: A New Data Base for Effects of
Anthropogenic Substances on Terrestrial Animals. Poster.
/ O
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Ringer, R. K. The Future of a Mammalian Wildlife Toxicology Test. Presenta-
tion.
Ringer, R. K., T. Shiroyama, and R. Bentley. Hexachlorobenzene on Reproduction
of Bobwhite. Presentation.
Williams, B. A. Wildlife Toxicology at CERL: A Look to the Future. Presenta-
tion.
Williams, B. A., R. K. Ringer, and T. Shiroyama. Time Course and Level of HCB
Transfer from Hen to Egg in Bobwhite Quail. Presentation.
Presented at the Seventh Annual Meetings of the Society of Environmental
Toxicology and Chemistry, Alexandria, Virginia, November 2-5, 1986
Bennett, R. S., J. K. Bennett, M. El Halawani, and R. K. Ringer. Relationship
Between Dietary Organophosphate Exposure, Prolactin Levels, and Incubation
Behavior in Mallards. Poster.
Bennett, R. S., B. A. Williams, D. W. Schmedding, and J. K. Bennett. Effects
of Short-Term, Dietary Exposure to Methyl Parathion on Nesting Success in
Mallards. Presentation.
Williams, B. A., T. Shiroyama, D. Bonfoey, and R. Bentley. Hexachlorobenzene
Uptake in the Eggs and Embryos of Bobwhite. Presentation.
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Articles in Preparation
Bennett, J. K., R. K. Ringer, R. S. Bennett, and B. A. Williams. Comparison of
Breaking Force and Shell Thickness of Sulfanilamide-Influenced Bobwhite
Eggshells.
Bennett, R. S. Comparison of Pesticide Effects on Food Avoidance Behavior in
Bobwhite.
Bennett, R. S., B. A. Williams, J. K. Bennett, and D. W. Schmedding. Effects
of Sublethal Methyl Parathion Exposure on Nesting Success in Mallards.
Emlen, J. M. Competition, Density Feedback, and Dispersion Patterns in
Atriplex confertifolia.
Emlen, J. M. Interaction Assessment: An Appplication and Test with Orzyopsis
hymenoides.
Maguire, C. C. and B. A. Williams. Acute Temperature Stress and Organophos-
phate Effects on Juvenile Bobwhite.
Maguire, C. C. and B. A. Williams. Chlorpyrifos Toxicity to Bobwhite at
Lowered Test Temperatures.
Williams, B. A., T. A. Shiroyama, and R. K. Ringer. HCB Uptake in Egg and
Embryo of Bobwhite.
In-House Report
Bennett, J. K. and B. A. Williams. Acetylcholinesterase Determination Proce-
dure. Corvallis Environmental Research Laboratory, U.S. EPA (1985).
28 pp.
Bennett, R. S. and D. W. Schafer. Procedure for Determining the Potential of
Birds to Avoid Chemically Contaminated Food.
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Bennett, R. S. Effect of Methylparathion on Mallard Nesting Success in Outdoor
Pens. 1985. 4 pp.
Bennett, J. K., and B. A. Williams. Use of the Assay for Acetylcholinesterase
to Determine Exposure to Organophosphates or Carbamates in the Field. EPA
Special Report. 1986.
Emlen, J. M., and R. S. Bennett. Review of Effects of Pesticides on Avian
Foraging Strategies. 1985. 20 pp.
Ringer, R. K. Protocol for Dietary LC5Q Test to Assess Primary and Secondary
Toxicity to Mammalian Wildlife. Michigan State University. 1986. 14 pp.
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EXTRAMURAL
(Supported All or in Part by CERL)
Berry, S. C., C. J. Driver, R. J. Kendall, and T. E. Lacher. Effects of
Agricultural Spraying of Methyl Parathion on Reproduction and Cholin-
esterase Activity in Starlings (Sturnus vulgaris) in Skagit Valley,
Washington. Sixth Annual Meeting of Society of Environmental Toxicology
and Chemistry, St. Louis, Missouri. November 10-13, 1985.
Blus, L. J., D. K. Halford, C. J. Henny, T. Craig, E. Craig, and C. M. Bunck.
Relations of Organophosphate Insecticides to Sage Grouse in Idaho. 56th
Annual Meetings of the Cooper Ornithological Society. Davis, California.
September 1986.
Brewer, L. and R. J. Kendall. Effects of Agricultural Spraying of Methyl
Parathion on Nesting Ducks and Duck Broods in Skagit Valley, Washington.
Sixth Annual Meeting of Society of Environmental Toxicology and Chemistry,
St. Louis, Missouri. November 10-13, 1985.
Galino, J. D., R. J. Kendall, C. J. Driver, and T. E. Lacher, Jr. The Effect
of Methyl Parathion on the Susceptibility of Bobwhite quail (Colinus
virgim'anus) to Domestic Cat Predation. Pacific Northwest Association of
Toxicologists Meeting, Seattle, Washington. September 1984.
Galino, J. D., R. J. Kendall, C. J. Driver, and T. E. Lacher. The Effect of
Methyl Parathion on Susceptibility of Bobwhite Quail (Colinus virgim'anus)
to Domestic Cat Predation. Behavioral and Neural Biology (1985).
V.43:21-36.
Hill, E. F., and M. B. Camardese. Toxicity of Anticholinesterase Insecticides
to Birds: Technical Grade Versus Granular Formulations. Ecotoxicol.
Environ. Safety (1984). V.8:551-563.
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McAlpine, C., R. Kendall, C. J. Driver, and R. Thompson. Effects of Methyl
Parathion on Tonic Immobility and Brain Acetylcholinesterase Activity in
Bobwhite Quail (Colinus virgim'anus). Pacific Northwest Association of
Toxicologists Meeting, Seattle, Washington. September 1984.
Roberts, D. K., N. J. Silvy, and E. M. Bailey. Comparative Toxicity of Methyl
Parathion and Dieldrin in Laboratory Rats and Mice vs. Captive Feral
Cotton Rats and White-Footed Deer Mice. Arch. Environ. Contam. Toxicol.
In press.
, E. M. Bailey, and N. J. Silvey. Recovery Patterns of Brain
Acetylcholinesterase Activity Following Non-Lethal Acute Methyl Parathion
Intoxication in Sigmodon^ histidus, Mus musculatus, and Their Laboratory
Animal Counterparts. Arch. Environ. Contam. Toxicol. (1986). Submitted.
, E. M. Bailey, and N. J. Silvey. Comparative Acute Toxicity of
Methyl Parathion and Dieldrin Between Three Indigenous Texas Rodent
Species and Their Laboratory Animal Counterparts. Bull. Environ. Contam.
Toxicol. (1986). Submitted.
, E. M. Bailey, and N. J. Silvey. A Report of the Animal Hus-
bandry Experiences in the Production of Colony-Reared Omnivorous Rodents
From Captive Feral Stock. Lab. Anim. Med. (1986). Submitted.
, E. M. Bailey, and N. J. Silvey. Brain Acetylcholineterase
Activity Following Lethal Methyl Parathion Intoxication in Various Captive
Feral Colony-Reared and Laboratory Rodent Species. Bull. Environ. Contam.
Toxicol. (1986). Submitted.
, E. M. Bailey, and N. J. Silvey. Use of Colony-Reared Rodents
From Captive Feral Stock as an Animal Model for the Assessment of Environ-
mental Pesticide Toxicity. Ecotoxicol. Environ. Safety (1986). Submitted,
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Roylance, K. J., C. D. Jorgensen, G. M. Booth, and M. W. Carter. Effects of
Dietary Endrin on Reproduction of Mallard Ducks (Anas platyrhynchos).
Arch. Environ. Contam. Toxicol. (1985). V.14:705-711.
Smith, D. E., and R. J. Kendall. Assessing the Effects of Pesticides on
Wildlife. J. Pesticide Reform. (1985). V.5:5-7.
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APPENDIX D. QUALITY ASSURANCE PROGRAM
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OUTLINE OF
QUALITY ASSURANCE PROGRAM PLAN
FOR THE
CORVALLIS ENVIRONMENTAL RESEARCH LABORATORY
U.S. Environmental Protection Agency
Office of Research and Development
200 S.W. 35th Street
Corvallis, Oregon 97333
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TABLE OF CONTENTS
Section
1.0 QUALITY ASSURANCE POLICIES, PROCEDURES, AND MANAGEMENT SYSTEMS
1.1 Introduction
1.2 QA Policy Statement
1.3 Laboratory Mission
1.4 Administrative Sample Handling Procedures
1.5 Data Generation
1.6 Organism Care
1.7 The Audit Program
1.8 QA Responsibilities
1.9 Resources for the QA Program
1.10 Annual Planning
1.11 Training
2.0 RESEARCH SUPPORT SERVICES
2.1 Introduction
2.2 Organization of the Corvallis Environmental Research Laboratory
Analytical Support
2.3 Facilities, Equipment, and Support Services
2.4 Maintenance Contract
2.5 Automated Data Processing (ADP)
3.0 ANNUAL REPORT AND QA WORK PLAN
3.1 Introduction
3.2 Training
3.3 Data Quality Objectives (DQOs)
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Section
3.4 QA Project Plans
3.5 Analytical Laboratory QA Plans
3.6 QA Audits
3.7 Peer Review
3.8 Publications and Reports
3.9 Performance Evaluation Program
3.10 QA Resources
3.11 QA Operating Constraints
Appendix A. Guidelines for Development of Quality Assurance Project Plans
Appendix B. Quality Assurance Review Form for CERL Extramural Projects
Appendix C. Quality Assurance in Organism Care and Testing
Appendix D. Quality Assurance Project Review for the Corvallis Environmental
Research Laboratory
Appendix E. Laboratory QA Organization
Appendix F. Research Notebook Policy
Appendix 6. Annual Audit Summary Report for FY 1985
Appendix H. Problems Discovered During Audits
Appendix I. Peer Review Report Findings
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QUALITY ASSURANCE
(A Process of Obtaining Data of Known Quality)
I
Data Collection and Use
\
Determine Criteria
for Decision
\
Identify Data and Data
Qualilty Requirements
Design Study and
Prepare QA Project Plan
I
Develop Sampling Plan
I
Choose Appropriate
Analytical Methods
Perform Data Collection
with Quality Control
Assess and Report Data
With Its Quality
Peer Review of Data
and Conclusions
86
Audit
Project
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APPENDIX E. CERL INSTITUTIONAL ANIMAL CARE AND USE COMMITTEE
INTERAGENCY RESEARCH ANIMAL COMMITTEE PROPOSED
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CERL INSTITUTIONAL ANIMAL CARE AND USE COMMITTEE
General Guidelines:
A. CERL Commitment to Comply to Laboratory Animal Welfare
The Corvallis Environmental Research Laboratory (CERL) of the U.S.
Environmental Protection Agency (EPA) is committed to, adheres to, and
will continue to adhere to the principles and recommendations set forth in
the NIH Guide for the Care and Use of Laboratory Animals and the princi-
ples set forth by the "U.S. Government Principles for the Utilization and
Care of Vertebrate Animals Used in Testing, Research, and Training." CERL
is also committed to complying with the Animal Welfare Act passed by
Congress in 1966 as P.L. 89-544 and amended in 1970 and again in 1976
(P.L. 94-279) and all-other applicable Federal statutes and regulations.
B. Committee Charge and Composition
In order to promulgate this commitment to comply, CERL has established
an Institutional Animal Care and Use Committee (IACUC) to advise the
Director on oversight of the Laboratory's animal care program and to
review research activities involving animals conducted at the Laboratory.
This committee is composed of at least five members, sufficiently quali-
fied through experience and expertise to advise the Director on humane
animal care and use. Of these five members, at least one is a Doctor of
Veterinary Medicine; at least one is a practicing scientist experienced in
research involving animals; at least one is from a nonscientific area as
his/her primary vocation; and at least one is an individual who is not
otherwise affiliated with CERL and is not a member of the immediate family
of a person who is affiliated with CERL. The names, position titles and
credentials of the IACUC members, the IACUC chairperson and the respons-
ible institutional official (one who has the authority to sign commitments
on behalf of CERL to EPA Headquarters) are on file in the CERL Director's
office.
C. Function of the Committee
(1) The IACUC is the principal advisory body on humane care and use of
animals to the Laboratory and to researchers who use animals. (2) The
IACUC will resolve concerns within the Laboratory involving the care and
treatment of animals brought to the attention of the committee. (3) The
IACUC will recommend to the Laboratory Director termination a project
when, in its judgment, the conduct of the research is such that compliance
with the policy of humane treatment to animals is jeopardized but only
after suggested changes are made in writing to the researcher, with a copy
to the Branch Chief, and the researcher then fails to comply or satisfy
the IACUC that altering the protocol will compromise the research. (4)
The IACUC will recommend to the Director of CERL, or his/her designated
representative, changes and improvements regarding the Laboratory's animal
care program or its animal facilities.
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D. Conduct of the Committee
In the conduct of meeting the functions of the IACUC the committee
shall:
1. Review and approve the care and use of all animals in proposals
submitted by a researcher for CERL approval (i.e., fiscal support or
safety approval). Proposals that reflect only a minor change in use
(such as a different number of animals or length of test duration) in
previously approved protocols may be approved by the chairperson of
the IACUC without full committee consideration. The exception is
when the IACUC chairperson is in direct conflict; i.e., serves as
principal investigator on protocol under consideration. In such
cases, another IACUC member may be delegated to make a decision.
Records of such actions are to be maintained in the committee files.
2. Meet at least semiannually or when protocols are to be reviewed. All
decisions must be based on a majority vote of those members convened
at a duly announced meeting. The IACUC chairperson may poll members
of the IACUC on project proposals when a delay in decision on
approach or rejection would jeopardize the initiation of a project.
A majority vote must be recorded.
3. Conduct inspections of animal facilities and report the findings of
the inspections to the Branch Chief, the Director, and the researcher
in charge of the animal facility inspected. Such inspections shall
be made at least semiannually.
4. Prepare an annual report on CERL animal care and use to be submitted
to the Director and/or EPA Headquarters.
Review of all protocols to IACUC must include consideration of:
1. Species
2. Cage size adequacy
(a) animals per cage or per unit of size
(b) height
3. Chemical or biological administration
(a) radioactive compound
(b) potential carcinogen
(c) biological
(d) toxic substance
4. Anesthetic, analgesic, or tranquilizer to be used and dosage.
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5. Euthanasia methodology
(a) must comply with recommendations of the American Veterinary
Medicine Association Panel on Euthanasia (JAVMA, 1978,
173:59-72); or
(b) approved by a veterinarian
6. Degree of pain inflicted by protocol methodology
(a) restraint duration
(b) invasive procedures
(c) adequacy of diet
Status:
The CERL Institutional Animal Care and Use Committee has been selected, has
met for introductory discussions, and has been briefed on the operation and
responsibilities of the Committee. The membership selected Dr. Dean Bauman as
the Chairman.
The Committee has agreed to meet semiannually for general reviews, and to
meet as needed to discuss and review new protocols.
The Committee membership includes the following:
Chairman
Dean Bauman, D.V.M.: Owner of Alpine Veterinarian Hospital
5120 N.W. Highland Avenue
Corvallis, Oregon 97330
John Crawford, Ph.D.: Associate Professor
Department of Wildlife and Fisheries
Oregon State University
Corvallis, Oregon 97331
Anne Fairbrother, D.V. M., Ph.D.: Research Biologist and Staff Veterinarian
USEPA
Corvallis, Oregon 97333
Eva Pendleton, D.V.M.: Veterinarian specializing in avian diseases
Department of Poultry Science
Oregon State University
Corvallis, Oregon 97331
Safa Shirazi, Ph.D.: Systems Analyst in Environmental Sciences
USEPA
Corvallis, Oregon 97333
Roger Zimmerman, Ph.D.: Minister, First Christian Church
4550 N.W. Queens
Corvallis, Oregon 97330
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INTERAGENCY RESEARCH ANIMAL COMMITTEE PROPOSED
U.S. GOVERNMENT PRINCIPLES FOR THE UTILIZATION AND CARE OF
VERTEBRATE ANIMALS USED IN TESTING, RESEARCH, AND TRAINING
The development of knowledge necessary for the improvement of the health
and well-being both of man and of animals requires recourse to HI vivo experi-
mentation with a wide variety of animal species. Methods such as mathematical
models, computer simulation, and in vitro biological systems should be used
wherever appropriate. Whenever U.S. Government agencies develop requirements
for testing, research, or training procedures involving the use of vertebrate
animals, the following principles shall be considered; and whenever these
agencies actually perform or sponsor such procedures, the responsible institu-
tional official shall ensure that these principles are adhered to:
1. The transportation, care, and use of animals shall be in accordance with
the Animal Welfare Act (7 U.S.C. 2131 et. seq.) and other applicable
federal, state, and local laws and prescribed policies.1
2. Procedures involving animals should be designed and performed with due
consideration of their relevance to human or animal health, the advance-
ment of biological knowledge, or the good of society.
3. The animals selected for a procedure should be of an appropriate species
and quality, and the minimum number required to obtain scientifically
valid results.
4. Proper care of animals, including the avoidance or minimization of discom-
fort, distress, or pain is a moral imperative. Lacking evidence to the
contrary, investigators should consider that procedures that cause pain in
human beings cause pain in other animals.
5. Procedures with animals that may cause more than momentary or slight pain
or distress should be performed with appropriate sedation, analgesia, or
anesthesia. Surgical or other painful procedures should not be performed
on unanesthetized animals paralyzed by chemical agents.
6. Animals that would otherwise suffer severe or chronic pain or distress
that cannot be relieved should be painlessly killed at the end of the
experiment or, if appropriate, during the experiment.
7. The living conditions of animals kept for biomedical purposes should
contribute to their health and comfort. Normally, the housing, care, and
feeding of all animals used for these purposes must be supervised by a
properly qualified veterinarian. In any case, veterinary care shall be
provided as indicated.
8. Investigators and other personnel shall be appropriately qualified and
experienced for conducting procedures on living animals. Adequate arrange-
ments shall be made for their in-service training, including the proper
and humane care and use of laboratory animals.
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If it is deemed necessary to waive one of the foregoing principles, the
decision should be made, with due regard to the provisions of Principle 2, by
an appropriate review board, such as an institutional animal research committee.
Such waivers should not be made where the primary purpose is teaching or
demonstration.
1 For guidance throughout these Principles, the reader is referred to Guide for
the Care and Use of Laboratory Animals prepared by the Institute of Labora-
tory Animal Resources, National Academy of Sciences.
Date
Director, National Institutes of Health
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